AU2016204172A1

AU2016204172A1 - Allosteric regulation of haemostatic activity

Info

Publication number: AU2016204172A1
Application number: AU2016204172A
Authority: AU
Inventors: Philip John Hogg
Original assignee: University of Sydney
Current assignee: University of Sydney
Priority date: 2016-06-20
Filing date: 2016-06-20
Publication date: 2018-01-18
Also published as: WO2017219078A1

Abstract

)01489620 Peptides and compositions for treating von Willebrand disease or acquired von Willebrand syndrome.

Description

invention

The invention relates to von Willebrand factor, to recombinant and plasma sources of same, to von Willebrand disease and to acquired von Willebrand syndrome.

Background of the invention von Willebrand factor (VWF) is a plasma protein produced by vascular endothelial cells and megakaryocytes that chaperones blood coagulation cofactor Factor VIII and tethers platelets to the injured blood vessel wall (1). It is a large glycoprotein that circulates as a series of multimers containing variable numbers of 500 kDa dimeric units. Pro-VWF 0 dimers are assembled in the endoplasmic reticulum via disulphide bridges between cysteine residues located in the C-terminal domains of the 250 kDa monomers. These tail-to-tail linked homodimers are subsequently variably multimerized within the Golgi apparatus by formation of head-to-head disulphide bonds near the N-termini.

The multimers can range in size from 500 to 20,000 kDa and the largest multimers are more effective at capturing platelets in the shear forces of flowing blood. This is due to the polyvalent nature of the protein as each monomer contains binding sites for collagen and for platelet glycoproteins lb (GPIb) and integrin allbp3 (1). VWF multimer size is regulated in the circulation by ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type-1 motifs) proteolysis of the Tyr1605-Met1606 peptide bond in the

A2 domain (2, 3). This is important as excess of large multimers can lead to pathological thrombosis in thrombotic thrombocytopenic purpura (4). Conversely, paucity of large multimers is associated with bleeding in von Willebrand disease (1). ADAMTS13 cannot access the Tyr1605-Met1606 peptide bond in the A2 domain until it is unfolded by the shear forces encountered in flowing blood (5-7).

Each of the three VWF A domains contain a single disulphide bond that links a pair of cysteine residues (Fig. 1). The disulphide bond in the A1 and A3 domains link cysteines located at either end of the polypeptide. In contrast, the A2 domain disulphide links adjacent cysteines (Cys1669 and Cys1670) at the C-terminal end of the domain (5, 8). The Cys1669-Cys1670 disulphide bond has a +/-LHStaple configuration (9, 10) in both crystal structures of the protein (5, 11) (Table S1). The distance between the a-carbon atoms of the cysteine residues is very short (3.76 A; average for all cystines is 5.63 A)

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2016204172 20 Jun 2016 (12-14).

von Willebrand disease and acquired von Willebrand syndrome remain of concern. It would be advantageous to provide improved and/or alternative treatments for these conditions.

Summary of the invention

In one embodiment there is provided a recombinant or synthetic peptide having an amino acid sequence of a von Willebrand factor (VWF), the amino acid sequence including a VWF A2 region in the form of an amino acid sequence for formation of a VWF domain A2, the VWF A2 region including adjacent selenocysteine residues, the 0 adjacent selenocysteine residues being positioned in the VWF A2 region to enable the formation of a covalent bond between the selenium atoms of the selenocysteine residues.

In another embodiment there is provided a composition including the above described peptide.

In another embodiment there is provided a method for increasing the relative abundance of oxidised VWF in a composition including reduced VWF, the method including:

- providing a composition including reduced VWF;

- contacting the composition with an oxidising agent for selectively oxidising adjacent cysteine residues in domain A2 of reduced VWF, thereby forming a covalent bond between the sulfur atoms of the adjacent cysteine residues;

wherein the formation of a covalent bond between the sulfur atoms of the adjacent cysteine residues in domain A2 of reduced VWF increases the relative abundance of oxidised VWF in the composition;

thereby increasing the relative abundance of oxidised VWF in the composition.

In another embodiment there is provided a composition produced by the above described method.

- providing a composition including reduced VWF;

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- adding a composition or a peptide as described above to the composition;

wherein the addition of the composition or peptide increases the relative abundance of oxidised VWF in the composition.

In another embodiment there is provided a method for determining the likelihood of a 5 VWF composition to induce the formation of a thrombus in an individual;

- providing a VWF composition;

- measuring the relative abundance of oxidised VWF in the composition, the oxidised VWF being a form of VWF in which adjacent cysteine residues in the A2 domain of VWF are linked by a covalent bond between sulfur atoms of the adjacent cysteine resides

- determining that the VWF composition has a high likelihood for inducing formation of a thrombus in the individual where the amount of oxidised VWF in the composition is measured to be greater than about 25% of the total amount of VWF in the composition.

In another embodiment there is provided method for treating an individual for von 5 Willebrand disease (VWD), the method including the following steps:

- providing an individual having VWD;

- administering a peptide or composition as described above to the individual, thereby treating the individual for VWD.

In another embodiment there is provided a recombinant or synthetic peptide having an amino acid sequence of a von Willebrand factor (VWF),

- the amino acid sequence including a VWF A2 region in the form of an amino acid sequence for formation of a VWF domain A2

- the VWF A2 region being devoid of adjacent cysteine residues or adjacent selenocysteine residues, a residue of the region thereby being incapable of forming a covalent bond with another residue in the region.

In another embodiment there is provided a composition including the aforementioned peptide.

In another embodiment there is provided a method for increasing the relative abundance of a reduced VWF in a composition including oxidised VWF, the method including:

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- providing a composition including oxidised VWF;

- contacting the composition with a reducing agent in conditions enabling selective reduction of adjacent cysteine residues in domain A2 of oxidised VWF, thereby breaking a covalent bond between the sulfur atoms of the adjacent cysteine residues in oxidised VWF;

wherein the breakage of a covalent bond between the sulfur atoms of the adjacent cysteine residues in domain 2A of oxidised VWF increases the relative abundance of reduced VWF in the composition;

thereby increasing the relative abundance of reduced VWF in the composition.

In another embodiment there is provided a method for increasing the relative abundance of reduced VWF in a composition including oxidised VWF, the method including:

- providing a composition including oxidised VWF;

- adding a composition or a peptide described above to the composition;

wherein the addition of the composition or peptide increases the relative abundance of reduced VWF in the composition.

In another embodiment, there is provided a method for treating for or preventing an individual from developing AVWS, the method including the following steps:

- providing an individual having, or at risk of developing AVWS;

- administering a peptide or composition described above to the individual, thereby treating the individual for AVWS, or preventing the individual from developing AVWS.

Brief description of the drawings

Figure 1. Domain structure of the VWF subunit and the unusual disulphide bond in the

A2 domain (Cys1669-Cys1670). The A1 domain binds GPIba receptor on platelets, the A2 domain Tyr1605-Met1606 peptide bond is cleaved by ADAMTS13, and the A3 domain binds collagen exposed during vascular damage. The oxidised A2 crystal structure is of Protein Data Bank (PDB) identifier 3GXB.

Figure 2. The VWF A2 domain disulphide bond exists in oxidised and reduced states in healthy donors. A. Reduced disulphide bond cysteines in VWF A2 domain are alkylated

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2016204172 20 Jun 2016 with 12C-IPA and the oxidised cysteine thiols with 13C-IPA following reduction with DTT. One or both of the reduced disulphide bond cysteines can be labeled with either alkylator. The ratio of 12C-IPA to 13C-IPA alkylation represents the proportion of the disulphide bond in the population that is in the reduced state. B. In healthy donors, a 5 mean of -75% of the VWF A2 domains are reduced and -25% are oxidised (n = 23, s.d. = 6%, coefficient of variation = 8%). C. VWF from normal pooled plasma (4 units) was collected by cryoprecipitation and gel filtered on Sepharose CL-2B. Fractions across the VWF peak were collected and analysed for multimer size by agarose gel electrophoresis and immunoblotting (upper panel), and redox state of the A2 domain 0 disulphide bond (lower graph). The higher molecular multimers are more oxidised than the smaller multimers.

Figure 3. Oxidised VWF is more effective at capturing platelets in flowing blood than reduced VWF. A. Representative images of platelet adhesion on channels coated with wild type or reduced (mutant) VWF at fluid shear rates of 40, 80 and 150 dyn/cm2. B.

Surface coverage of platelets (expressed as a percentage of total area) on channels coated with wild type or reduced (mutant) VWF at fluid shear rates of 40, 80 and 150 dyn/cm2. C. Mean diameter of platelet aggregates on channels coated with wild type or reduced (mutant) VWF at fluid shear rates of 40, 80 and 150 dyn/cm2. Bars and errors are mean ± s.e.m. of 3 technical replicates of platelets from 4 healthy donors (n=12 measurements). *, p<0.05; **, p<0.01; ***, p<0.001. D. BFP brightfield scheme (upper panel) and protein functionalization (lower panel). A micropipette-aspirated RBC with a probe bead attached to the apex (left) was aligned against a human platelet held by an aposing micropipette (right). The probe bead was covalently linked with polyclonal antibody (pAb) for capture of VWF and streptavidin (SA) for attachment of the bead to biotinylated RBC (left). VWF is the focus for interaction with the GPIb on an aspired platelet (right). E. Force versus time traces from two representative test cycles. A target was driven to approach a probe, contacted and retracted. In a no bond event (black), the cycle ended after the probe-target separation. In a bond event (blue), the target was held (marked by *) at a preset force until dissociation. Lifetime was measured from the point when the clamped force (30 pN) was reached to the point when the bond dissociated, signified by a force drop to zero. F. Adhesion frequencies of 5 beadplatelet pairs with 50 touches for each pair. Errors are mean ± s.e.m. G. Lifetime of VWF-platelet bonds as a function of force. Errors are mean ± s.e.m of > 50 measurements per point.

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Figure 4. Reduced VWF is more efficiently cleaved by ADAMTS13 but has the same affinity for collagen as oxidised protein. A. Reduced VWF is more readily cleaved by ADAMTS13 than oxidised VWF. Wildtype (wt) and reduced mutant (mut) VWF were incubated with ADAMTS13 for discreet times and the reaction quenched with EDTA. 5 Samples were resolved on SDS-PAGE under reducing conditions and blotted with a polyclonal VWF antibody to measure intact VWF and proteolytic fragments. B. Reduced and oxidised VWF have the same affinity for collagen under static and shear conditions. Various concentrations of wt and reduced (mutant) VWF were incubated on a plate coated with collagen in the absence (left hand graph) or presence (right hand graph) of 0 32 dynes/cm2 fluid shear stress. VWF binding was detected using a peroxidaseconjugated polyclonal anti-VWF antibody.

Figure 5. Reduction of the A2 domain disulphide bond has a pronounced effect on domain structure as revealed by molecular dynamics simulations. A. Overall structure of the VWF A2 domain (from the X-ray structure, PDB identifier 3GXB), used as starting conformation for the MD simulations. The structure is shown in cartoon representation, highlighting the Cys1669-Cys1670 disulphide bond in yellow. B. Dynamics of the oxidised (green) and reduced (orange) VWF A2 domain projected onto the two first eigenvectors (main modes of motion) obtained from principal component analysis. Each dot represents a conformation observed in the MD simulations. The black dot corresponds to the starting structure for both simulations. C. Interpolation of the structures along the first PCA eigenvector (main mode of motion), ranging from the conformations sampled in the oxidised state (green) to the reduced state (orange) (going from the extreme left to the extreme right). D. Per-residue difference of the Root Mean Square Fluctuations (RMSF) between the reduced (red) and oxidised (oxi) forms of VWF A2 mapped on the protein backbone. Blue (red) color reflects less (more) positional fluctuations in the reduced than in the oxidised state. E. Force distribution analysis. (Left) Residue pairs (i,j) with the time-averaged pair-wise force of the reduced minus the one of the oxidised state larger than a cutoff value of 90 pN, |<Fij(red)> <Fij(oxi)>| > 90 pN. Here, | | indicates absolute values. For the dependence on the cutoff value, see Fig. S4. Secondary structure elements of A2 are shown on both axes. To guide the eye, the regions corresponding to the beta strands B4, B5 and B6 are displayed with the gray areas, and the one to the Cys1669-Cys1670 bond in orange. (Right) The three groups of residue pairs enclosed by circles, showing interactions between the beta strands and their surrounding helices, loops or the C-terminus, are

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2016204172 20 Jun 2016 explicitly shown as lines connecting points in the A2 structure.

Figure 6. Treatment of heart failure patients with mechanical circulatory assist devices results in marked depletion of reduced VWF that is consistent with mechanical shearinduced oxidation of the A2 domain disulphide. A. Plasma VWF in LVAD (n = 13) and 5 ECMO patients (n = 11) is significantly more oxidised than in heart failure patients not implanted with these devices (n = 9). B. Redox potential of the Cys1669 and Cys1670 disulphide bond in plasma VWF. Plot of fraction of reduced VWF as a function of the ratio of reduced to oxidised dithiothreitol (DTT). The line represents the best non-linear least squares fit of the data to supplementary equation 1. The calculated equilibrium 0 constant (Keq) was used to determine the standard redox potential (Eo’) from supplementary equation 2. C. The plasma oxidoreductase, thioredoxin-1, oxidises and reduces VWF under fluid shear stress. Purified plasma VWF was indicated with 5 μΜ oxidised or reduced thioredoxin-1 for 60 min in the absence or presence of 32 dyn/cm2 fluid shear stress and the redox state of the A2 disulphide bond measured. Data points 5 are from biological replicates. D. The oxidation state of VWF Met1606 is unchanged in the heart failure, ECMO and LVAD patient groups. E and F. There is a significant depletion of plasma VWF in ECMO patients compared to LVAD patients or heart failure patients not implanted with these devices (part E), which corresponds to a significant reduction in average VWF multimer size (part F, measured using the collagen-binding 0 assay). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

Figure 7. Amino acid sequence of human VWF (SEQ ID No: 1).

Figure 8. Amino acid sequence of a naturally occurring A2 domain of human VWF (SEQ ID No: 2).

Figure 9. Amino acid sequence of human VWF comprising mutation of both Cys residues in A2 domain to Gly (SEQ ID No: 3).

Figure S1. Differential cysteine alkylation and mass spectrometry analysis of the VWF Cys1669-Cys1670 disulphide bond. a. Plasma VWF collected on antibody-coated beads, resolved on reducing SDS-PAGE and stained with colloidal coomassie. The positions of molecular weight standards in kDa are shown at the left. b. and c. Tandem mass spectrum of the VWF LVLQRCCSGE peptide containing the Cys1669 and Cys1670 dithiol/disulphide. Part b is an example of the 12C-IPA-alkylated peptide and part c the 13C-IPA-alkylated peptide. Both Cys1669 and Cys1670 are alkylated in these examples. The accurate mass spectrum of the peptide is shown in the inset (part b

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2016204172 20 Jun 2016 observed [M+2H]2+ = 687.3203 m/z and expected [M+2H]2+ = 687.3207 m/z; part c observed [M+2H]3+ = 693.3413 m/z and expected [M+2H]2+ = 693.3408 m/z).

Figure S2. Wild-type and reduced (mutant) VWF. HEK293 cells were transfected with wild-type or reduced (mutant) VWF plasmids. Conditioned medium was collected after 3 5 days and concentrated using a 100 kDa cut-off centrifugal filter. Equal volumes were resolved by reducing SDS-PAGE (part a) or agarose gel electrophoresis (part b) and blotted with anti-VWF polyclonal antibodies.

Figure S3. Force distribution analysis. Residue pairs (i,j) with a time-averaged pair-wise force of the reduced minus the one of the oxidised state, |AFij|=|<Fij(red)> - <Fij(oxi)>|, 0 larger than the specified cutoff values. Here, | | indicates absolute values. Secondary structure of A2 is shown on both axes. To guide the eye, the regions corresponding to the beta strands B4, B5 and B6 are displayed with gray areas, and the one to the Cys1669-Cys1670 bond in orange.

Figure S4. Mass spectrometry analysis of oxidation of Met1606 of VWF. Tandem mass spectrum of the QAPNLVYMVTGNPASDE peptide containing oxidised Met1606 (mass addition of 15.9949). The accurate mass spectrum of the peptide is shown in the inset (observed [M+2H]2+ = 911.4208 m/z and expected [M+2H]2+ = 911.4198 m/z).

Detailed description of the embodiments

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

As described herein the inventors have identified two forms of VWF in human blood defined by the redox state of the A2 domain Cys1669-Cys1670 disulphide bond. The two redox forms have different haemostatic activity. The minor (oxidised) VWF is much more effective at engaging platelet glycoprotein lb in flowing blood due to higher collision frequency and longer bond lifetimes, while the major (reduced) VWF is more efficiently cleaved by the metalloprotease ADAMTS13 that regulates multimer size.

We have found that the A2 domain responds to the shear of flowing blood to control these VWF interactions. The minor form appears to arise from the major form in high shear conditions. Molecular dynamics simulations reveal that cleavage of the disulphide bond has a pronounced effect on A2 domain structure that will influence its force8

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2016204172 20 Jun 2016 sensing.

Patients on mechanical assists devices for heart failure are prone to acquired von Willebrand syndrome (AVWS). We have found that these patients have a much higher proportion of pro-thrombotic oxidised VWF in their blood due to mechanical shear5 mediated oxidation of the A2 domain. These findings indicate that the A2 domain disulphide bond mediates an allosteric conformational and functional switch in VWF that is relevant to individuals at risk of thrombosis (for example individuals with or at risk of AVWS) or haemorrhage (for example, individuals with von Willebrand disease (VWD)). On the basis of these surprising findings the inventors have developed:

· new forms of VWF and compositions containing same, • methods of production of those forms and compositions, • methods of using the VWF forms and compositions for treatment of VWD or for treatment of those at risk of or having AVWS, • methods for standardising blood and plasma products and recombinant products to adapt them for treatment of VWD or for treatment of those at risk of or having

AVWS, • methods for determining whether a given blood, plasma or recombinant product is suitable for treatment of VWD or for treatment of those at risk of or having AVWS.

A. Definitions

As is well understood in the art, von Willebrand Factor (VWF) is generally found in the body as a multimer of dimers, each dimer being formed from 2 peptide monomers. Each peptide monomer includes 3 domains, known as domains A1, A2 and A3. Within each domain are certain important cysteine residues as follows (numbering is for the human protein including signal sequence):

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Name Domain Position (with reference May form

	to SEQ ID No. 1)	disulphide bond with
Cys A1-1	A1	1272	Cys A1-2
Cys A1-2	A1	1458	Cys A1-1
Cys A2-1	A2	1669	Cys A2-2
Cys A2-2	A2	1670	Cys A2-1
Cys A3-1	A3	1686	Cys A3-2
CysA3-2	A3	1872	CysA3-1

These cysteine residues and their role in forming disulphide bonds is generally described in Figure 1 herein and also in references (5) and (8) herein.

The numbering of the cysteine residues as in the above table is based on the amino acid sequence shown in SEQ ID No: 1. This is the widely accepted amino sequence for human VWF. It is found at UniProtKB - P04275 (VWF_HUMAN).

As per the above table, SEQ ID No:1 describes the critical cysteine residues of domain A2 as being located at positions 1669 and 1670. However, given that VWF exists in polymorphic forms, some arising from deletion or insertion of residues, it will be understood that these domain A2 cysteine residues may have slightly different numbering with respect to a particular VWF form, i.e. a form other than that described by SEQ ID No. 1.

The cysteine residues in the above referenced tables are referred to in the art as “invariant” residues. This is because they are essential for formation of disulphide bonds and, when a disulphide bond has formed, essential for the maintenance of the relevant allosteric structure and/or conformation of VWF as it exists in nature. In particular, while there may be some variance as between the sequences of VWF forms, these cysteine

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2016204172 20 Jun 2016 residues responsible for structure and maintenance of VWF conformation are always found in naturally occurring VWF forms.

The only cysteine residues known to occur adjacent one another (i.e. as a Cys-Cys dipeptide sequence) in domain A2 of all VWF forms characterised to date are those 5 described above as Cys A2-1 and Cys A2-2. In this context, CysA2-1 and CysA2-2 are “adjacent cysteine residues” as referred to herein.

As mentioned above, a disulphide bond may occur between these cysteine residues. The disulphide bond has a +/-LHStaple configuration in both crystal structures of the protein and the distance between the α-carbon atoms of the cysteine residues is very 0 short (3.76 A; average for all cysteines is 5.63 A) and both cysteine residues of the disulphide bond are exposed to solvent.

Reference to “Cys Α2-Γ and “Cys A2-2’ is a reference:

- to those cysteine residues that are located at positions 1669 and 1670 of the sequence in SEQ ID No: 1 and/or

- to those cysteine residues, which when oxidised form a disulphide bond within domain

A2 having a +/-LHStaple configuration in both crystal structures of naturally occurring VWF (including VWF having sequence of SEQ ID No: 1 and all other naturally occurring variants), and wherein the distance of the α-carbon atoms involved in the bond between the residues is about 3.76A.

As described herein, it has been found that the formation or loss of a disulphide bond between Cys A2-1 and Cys A2-2 confers allosteric changes on domain A2 which have profound functional implications for the capacity of the particular resulting conformation to influence clot formation.

In more detail, where there is no disulfphide bond, i.e. where Cys A2-1 and/ or Cys A2-2 are reduced, the resultant VWF A2 domain changes conformation to enable ADAMST13 to cleave the domain at position Tyr1605-Met1606 (numbering with reference to SEQ ID No: 1) which ostensibly reduces the valency of multimeric forms. Further, there is a reduced affinity for GPIb. These effects lead to reduced capacity to form thrombi, and as recognised herein, a therapeutic application in those indications arising from unwanted thrombogenesis, such as acquired von Willebrand syndrome.

In contrast, where there is a disulfphide bond, i.e. where Cys A2-1 and Cys A2-2 are oxidised, the resultant VWF A2 domain changes conformation to preclude ADAMST13

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2016204172 20 Jun 2016 from cleaving the domain at position Tyr1605-Met1606 (with reference to SEQ ID No: 1) which ostensibly maintains the valency of multimeric forms. Further, there is no reduced affinity for GPIb. These effects lead to increased capacity to form thrombi, and as recognised herein, a therapeutic application in those indications arising from insufficient 5 thrombogenesis, such as von Willebrand disease.

The invention described herein is particularly concerned with the manipulation of Cys A2-1 and/ or Cys A2-2, either by:

(i) reducing or oxidising these residues, so as to provide for the desired allosteric change, (ii) replacing them with selenocysteine residues so as to provide a stronger intra A2 domain bond at the location where Cys A2-1 and Cys A2-2 naturally occur, or (iii) replacing one or both of them with a residue that does not contain selenium or sulphur and that therefore precludes the formation of the intra A2 domain bond at the location where Cys A2-1 and Cys A2-2 naturally occur.

Reference to a “reduced VWF’ as used herein will be understood as meaning a form of VWF where there is no covalent bond at the location where Cys A2-1 and Cys A2-2 naturally occur. A “reduced VWF’ may have an amino acid other than cysteine or selenocysteine at the position where Cys A2-1 is found in naturally occurring VWF, or an amino acid other than cysteine or selenocysteine at the position where Cys A2-2 is found in naturally occurring VWF. A “reduced VWF’ generally has a greater susceptibility for cleavage at domain A2 by ADAMST13 than does a VWF which has a disulfide bond between Cys A2-1 and Cys A2-2. A “reduced VWF generally has a lesser affinity for binding to platelet GPIb than does a VWF which has a disulfide bond between Cys A2-1 and Cys A2-2.

Reference to an “oxidised VWF as used herein will be understood a meaning a form of VWF where there is a covalent bond at the location where Cys A2-1 and Cys A2-2 naturally occur. An “oxidised VWF’ may have Cys A2-1 and Cys A2-2, or it may have selenocysteines at the positions where Cys A2-1 and Cys A2-2 is found in naturally occurring VWF. An “oxidised VWF’ generally has a lesser susceptibility for cleavage at domain A2 by ADAMST13 than does a VWF which does not have a disulphide bond between Cys A2-1 and Cys A2-2. An “oxidised VWF’ generally has a greater affinity for

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2016204172 20 Jun 2016 binding to platelet GPIb than does a VWF which does not have a disulphide bond between Cys A2-1 and Cys A2-2.

Reference to “adjacent selenocysteine residues positioned in VWF A2 region to enable the formation of a covalent bond between the selenium atoms of the selenocysteine 5 residues means that the selenocysteine residues are positioned at the locations that are otherwise occupied by Cys A2-1 and Cys A2-2 in naturally occurring VWF forms. An isoform having said adjacent selenocysteine residues with a covalent bond between the selenium atoms is otherwise known herein as an “oxidised VWF.

Reference to “the VWF A2 region being devoid of adjacent cysteine residues or adjacent selenocysteine residues refers to the region being devoid of either or both of Cys A2-1 and Cys A2-2, or devoid of selenocysteine residues that are positioned at the locations that are otherwise occupied by Cys A2-1 and Cys A2-2 in naturally occurring VWF forms.

Typically, the peptides “having an amino acid sequence of a von Willebrand factor (VWF}’ described herein have potential for platelet capture capacity and/or Factor VIII carrier capacity akin to natural VWF.

“Recombinant’ peptides may generally be produced by translation from a nucleic acid. “Synthetic peptides may generally be produced by chemical synthesis, for example solid phase peptide synthesis.

B. Recombinant or synthetic forms of oxidised VWF

In one embodiment there is provided a recombinant or synthetic peptide having an amino acid sequence of a von Willebrand factor (VWF), the amino acid sequence including a VWF A2 region, the region being an amino acid sequence for formation of a VWF domain A2 the VWF A2 region including adjacent selenocysteine residues, the adjacent selenocysteine residues being positioned in the VWF A2 region to enable the formation of a covalent bond between the selenium atoms of the selenocysteine residues.

Relevantly, the inventors have recognised that the allosteric conformation conferred by oxidised VWF may be particularly useful for those indications where there is a deficit of thrombogenic potential, VWD being one example. Further the inventors have recognised that the oxidised form of VWF could be further improved by replacing Cys A2-1 and Cys A2-2 with selenocysteine residues. The effect of this design is to increase

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2016204172 20 Jun 2016 the strength of the covalent bond that is otherwise provided via Cys A2-1 and Cys A2-2. By providing a stronger bond, this minimises the likelihood of the return of the conformation and low thrombogenic potential conferred by reduced VWF that otherwise occurs when oxidised VWF is converted to reduced VWF.

Methods for the production of recombinant constructs enabling the incorporation of selenocysteine residues is generally known in the art. The methods generally require the use of selenocysteine codons at the relevant position (in this case in place of codons that would normally encode Cys A2-1 and Cys A2 -2), and transformation of the resulting construct into an host having an enzyme system enabling synthesis of selencysteine and incorporation into a translation product. See for example reference: Guo X, Song J, Yu Y, Wei J. Can recombinant human glutathione peroxidase 1 with high activity be efficiently produced in Escherichia coli? Antioxid Redox Signal. 2014 Mar 20;20(9):1524-30. doi: 10.1089/ars.2013.5617.

According to this embodiment, the covalent bond that is formed between the selenium atoms of the adjacent selenocysteine residues ostensibly models the bond that is otherwise formed between Cys A2-1 and Cys A2-2 in naturally occurring VWF isoforms, and this confers on the peptide a reduced susceptibility to cleavage of the peptide by ADAMTS13 and/or an increased affinity for binding of the peptide with glycoprotein lb.

It will be understood that the peptide may have a sequence of any of the naturally occurring isoforms of VWF provided that where Cys A2-1 and Cys A2 -2 are normally found in naturally occurring isoforms, the residues are selenocysteine instead of cysteine.

SEQ ID No:2 shows the sequence of a naturally occurring A2 domain of VWF. In one embodiment, the peptide has an amino acid sequence having at least 80%, preferably

85% preferably 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 2., provided that the peptide has selenocysteine where Cys A2-1 and Cys A2-2 are generally located in naturally occurring VWF isoforms i.e at position 172 and 173 of SEQ ID No: 2.

Percent sequence identity is determined by conventional methods, by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) as disclosed in Needleman, S. B. and Wunsch, CD., (1970), Journal of Molecular Biology,

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48, 443-453, which is hereby incorporated by reference in its entirety. GAP is used with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.

In another embodiment, the peptide may have an amino acid sequence having at least

80%, preferably 85%, preferably 90%, preferably 95% identity, preferably identity with the sequence shown in SEQ ID No: 1 provided that the peptide has selenocysteine where Cys A2-1 and Cys A2-2 are generally located in naturally occurring VWF isoforms i.e. at position 1669 and 1670 of SEQ ID No. 1.

C. Compositions including recombinant forms of oxidised VWF

In another embodiment there is provided a composition including a peptide having selenocysteine residues in place of cysteine residues where Cys A2-1 and CysA2-2 are located in naturally occurring VWF forms, as described above. The VWF isoforms in the composition may consist solely of this peptide, or the composition may include other isoforms.

In more detail, it is envisaged by the inventors that the selenocysteine -containing peptide described above may be utilised alone in the treatment of VWD. In other embodiments, the selenocysteine -containing peptide may be added to another recombinant source of VWF which may contain reduced VWF, so as to ostensibly increase the amount or relative abundance of oxidised VWF forms in the composition, thereby increasing the suitability of the recombinant source for treatment of VWD individuals. Alternatively, the selenocysteine -containing peptide may be added to a naturally occurring source of VWF, such as blood, plasma or product derived therefrom, which may contain reduced VWF, so as to ostensibly increase the amount or relative abundance of oxidised VWF forms in the composition. Again, this would increase the suitability of such a naturally derived VWF source for use in treatment of VWD.

Thus in one embodiment, the composition including a peptide having selenocysteine residues in place of Cys A2-1 and CysA2-2 includes a recombinant form of oxidised VWF (ie. in which adjacent cysteine residues in domain A2 of the VWF are linked by a covalent bond between the sulfur atoms of the adjacent cysteine residues), and/or including a recombinant form of reduced VWF (i.e. in which adjacent cysteine residues in domain A2 of the VWF are not linked by a covalent bond between the sulfur atoms of the adjacent cysteine residues).

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In another embodiment, the composition including a peptide having selenocysteine residues in place of Cys A2-1 and CysA2-2 includes a blood or plasma -derived form of oxidised VWF and/or includes a reduced blood or plasma -derived form of reduced VWF.

The above described composition may further including cells and/or platelets. Particular cells of interest may be any cells typically contained in whole blood, including red blood cells, leukocytes etc. The composition may further include blood or plasma -derived proteins such as immunoglobulin and other globulins, albumin etc. It will be understood that in these embodiments, the inventor has recognised that the peptide having selenocysteine residues in place of Cys A2-1 and CysA2-2 could be used to “spike” a very large range of blood or plasma products so as to increase the amount of oxidised VWF in them, thereby adapting those products for use in individuals having low thrombogenic potential including those having VWD.

D. Formation of oxidised VWF

An extension of the work described herein wherein the inventor has identified two conformations of domain A2 conferred by the redox potential of the bond between Cys A2-1 and Cys A2-2 is that the inventor has determined how to chemically convert reduced VWF to oxidised VWF without impacting on the other critical invariant cysteine residues in domains A1 or A3 described in the above tables. This enables the formation of compositions having increased amounts of natural or recombinant sources of oxidised VWF. Thus in one embodiment there is provided a method for increasing the relative abundance of oxidised VWF in a composition. Typically the composition provided for such treatment includes reduced VWF and the method includes contacting the composition with an oxidising agent for selectively oxidising the adjacent cysteine residues in domain A2 of reduced VWF, thereby forming a covalent bond between the sulfur atoms of the adjacent cysteine residues; wherein the formation of a covalent bond between the sulfur atoms of the adjacent cysteine residues in domain A2 of reduced VWF increases the relative abundance of oxidised VWF in the composition; thereby increasing the relative abundance of oxidised VWF in the composition.

According to the method the oxidising agent selectively oxidises the Cys A2-1 and Cys A2-2 residues in reduced VWF thereby forming a disulphide bond between these residues and increasing the amount of oxidised VWF in the composition relative to reduced VWF in the composition.

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The inventor has found that 4,5-dihydroxy-1,2-dithiane (otherwise known as oxoDTT) is a useful selective oxidant for this purpose. Other oxidants may be chemical reagents (such as oxidised glutathione, HgCb, trivalent arsenicals, trans-3,4-dihydroxyselenolane oxide) or enzymes (the oxidised forms of oxidoreductases, including thioredoxin, PDI, 5 ERp5, ERp57, ERp72).

It will be understood that the method is particularly useful for adapting or standardising VWF - containing products so that they are useful for the treatment of individuals having clotting disorders arising from insufficient oxidised VWF, such as individuals having VWD. In one embodiment, the relative abundance of oxidised VWF in the 0 composition is increased by at least 10%, preferably 50%, preferably 100% or more.

In another embodiment, at least 75% of VWF in the composition selected for treatment with the oxidising agent is reduced VWF.

In another embodiment there is provided a composition produced by the method. The composition may include cells or other proteins.

E. Standardising VWF compositions

A further extension of the inventor’s work described herein is the recognition that there is variance between VWF sources in terms of their ratio of oxidised to reduced VWF forms. The variance is particularly pronounced from batch to batch of naturally derived compositions (i.e. compositions pooled from individual donors) because different pools may have different ratios of oxidised to reduced VWF forms. From this variance arises the risk that some batches of VWF compositions may be inherently unsuitable for the treatment of individuals with low thrombogenic potential because those batches have an unacceptable high amount of reduced VWF relative to oxidised VWF. Accordingly there is a need to standardise VWF compositions, be they compositions formed from recombinant DNA technology, or sources formed from donor pools so that the practitioner can have some confidence that the source of VWF that he is to use to treat an individual with low thrombogenic potentially will not exacerbate the condition.

Thus in one embodiment there is provided a method for increasing the relative abundance of oxidised VWF in a composition including reduced VWF, the method including providing a composition including reduced VWF, adding a composition consisting essentially of oxidised VWF to the composition, wherein the addition of the composition increases the relative abundance of oxidised VWF in the composition.

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According to this embodiment, the composition which is added may be one including selenocysteine residues as described above, or one which has been treated with a oxidising agent to selective oxidise Cys A2-1 and CysA2-2 as described above.

F. VWD treatments

It is understood that the peptides and compositions having increased abundance of oxidised VWF may find application in the treatment of individuals having low thrombogenic potential, and especially those individuals having either a paucity of VWF or oxidised VWF or defective VWF. Particular examples of these individuals include those having VWD.

In another embodiment the insufficient thrombogenesis may be syndrome such as a consequence of autoimmune disease or hypothryroidism. Other forms of insufficient thrombogenesis may arise as a side effect of therapy including heart failure patients treated with mechanical circulatory assist devices (as observed in some forms of acquired von Willebrand syndrome), the anti-seizure medication valproic acid (Depakene) or the antibiotic ciprofloxacin (Cipro).

Thus in one embodiment there is provided a method for treating an individual having low thrombogenic potential, the method including providing an individual having low thrombogenic potential, administering a peptide or composition having increased abundance of oxidised VWF to the individual, thereby treating the individual for low thrombogenic potential. Preferably the individual has VWD.

G. Recombinant or synthetic forms of reduced VWF

As described above, in certain embodiments, the invention described herein is particularly concerned with the manipulation of Cys A2-1 and/ or Cys A2-2 by replacing one or both of these residues with a residue that does not contain selenium or sulphur and that therefore precludes the formation of the intra A2 domain bond at the location where Cys A2-1 and Cys A2-2 naturally occur. It is believed that these forms may find application in preparation of VWF and related products for treatment of individuals having an acquired von Willebrand syndrome and other haemostatic abnormalities arising from dysregulation of VWF function. Specifically, the concern in such applications is that reduced VWF as may be given in a proposed therapeutic application, under high shear conditions found in some of these conditions, converts to oxidised VWF, increasing the amount of the thrombogenic oxidised VWF in the

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2016204172 20 Jun 2016 individual and hence increasing the likelihood of clotting. The principle underlying these recombinant forms of reduced VWF is to deliver VWF that is unable to take the conformation of oxidised VWF and therefore be less likely to form large multivalent multimers that have a high affinity for prothrombogenic interaction with platelet GPb1.

Thus in one embodiment there is provided a recombinant or synthetic peptide having an amino acid sequence of VWF, the amino acid sequence including a VWF A2 region, said region being an amino acid sequence for formation of a VWF domain A2, the VWF A2 region being devoid of adjacent cysteine residues or adjacent selenocysteine residues, a residue of the region thereby being incapable of forming a covalent bond with another residue in the region. According to this embodiment of the invention, a polynucleotide sequence may be prepared in which codons for Cys A2-1 and Cys A2-2 are replaced or substituted with codons for other than cysteine, and for other than selenocysteine. The translation product therefore contains amino acid residues other than cysteine or selenocysteine where Cys A2-1 and CysA2-2 are normally found in naturally occurring VWF isoforms. This is exemplified in the examples that following whereby the adjacent cysteine residues (Cys A2-1 and Cys A2-2) are replaced with a Gly-Gly dipeptide.

In one embodiment, in the absence of cysteine or selenocysteine residues, and the consequential absence of an intra A2 domain covalent bond normally conferred by Cys

A2-1 and CysA2-2, the A2 region confers on the peptide an increased susceptibility to cleavage of the peptide by ADAMTS13.

In one embodiment, in the absence of cysteine or selenocysteine residues, and the consequential absence of an intra A2 domain covalent bond normally conferred by Cys A2-1 and CysA2-2, the A2 region confers on the peptide a decreased affinity for binding of the peptide with glycoprotein lb.

In one embodiment, the peptide has an amino acid sequence having at least 80%, preferably 85%, preferably 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 2, provided that the peptide has a residue other than cysteine or selenocysteine at position 172. Preferably the peptide has glycine, alanine or serine at position 172. In this embodiment for, example, the Cys A2-1 may be replaced so that CysA2-1 may become Gly, Ala or Ser.

In one embodiment, the peptide has an amino acid sequence having at least 80%, preferably 85%, preferably 90%, preferably 95% identity, preferably identity to the

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2016204172 20 Jun 2016 sequence shown in SEQ ID No: 2, provided that the peptide has a residue other than cysteine or selenocysteine at position 173. In this embodiment for, example, the Cys A2-2 may be replaced so that CysA2-2 may become Gly, Ala or Ser.

In certain embodiments it is not necessary that substitutions or replacements occur at both Cys A2-1 and Cys A2-2. For example in one embodiment, Cys A2-1 may be Gly, Ala or Ser and Cys A2-2 may be cysteine or selenocysteine. In another embodiment, Cys A2-1 may be cysteine or selenocystein and Cys A2-2 may be Gly, Ala or Ser.

Further in certain embodiments it is not necessary that Cys A2-1 and Cys A2-2 are replaced with the same amino acid. For example Cys A2-1 could be Ala and Cys A2-2 0 could be Ser.

In one embodiment, the peptide has an amino acid sequence at least 80%, preferably 85%, preferably 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 1, provided that the peptide has an amino acid other than cysteine and selenocysteine at position 1699 or at position 1670.

H. Compositions including recombinant forms of reduced VWF

In another embodiment there is provided a composition including a peptide having residues other than selenocysteine or cysteine residues where Cys A2-1 and CysA2-2 are located in naturally occurring VWF forms, as described above. The VWF isoforms in the composition may consist solely of this peptide, or the composition may include other isoforms.

In more detail, it is envisaged by the inventors that the above peptides may be utilised alone in the treatment of conditions characterised by abnormal haemostasis such as AVWS. In other embodiments, the peptide may be added to another recombinant source of VWF which may contain oxidised VWF, so as to ostensibly increase the amount or relative abundance of reduced VWF forms in the composition, thereby increasing the suitability of the recombinant source for treatment of AVWS individuals. Alternatively, the peptides may be added to naturally occurring source of VWF, such as blood, plasma or product derived therefrom, which may contain oxidised VWF, so as to ostensibly increase the amount or relative abundance of reduced VWF forms in the composition. Again, this would increase the suitability of such a naturally derived VWF source for use in treatment of AVWS.

Thus in one embodiment, the composition including a peptide having residues other

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2016204172 20 Jun 2016 than cysteine and selenocysteine residues in place of Cys A2-1 and CysA2-2 includes a recombinant form of oxidised VWF, and/or includes a recombinant form of reduced VWF (i.e. in which adjacent cysteine residues in domain A2 of the VWF are not linked by a covalent bond between the sulfur atoms of the adjacent cysteine residues).

In another embodiment, the composition including a peptide having residues other than cysteine and selenocysteine residues in place of Cys A2-1 and CysA2-2 includes a blood or plasma -derived form of oxidised VWF and/or includes a blood or plasma derived form of reduced VWF.

The above described composition may further including cells and/or platelets. Particular cells of interest may be any cells typically contained in whole blood, including red blood cells, leukocytes etc. The composition may further include blood or plasma -derived proteins such as immunoglobulin and other globulins, albumin etc. It will be understood that in these embodiments, the inventor has recognised that the peptide having residues in place of Cys A2-1 and CysA2-2 could be used to “spike” a very large range of blood or plasma products so as to increase the amount of reduced VWF in them, thereby adapting those products for use in individuals having high thrombogenic potential including those having AVWS.

I. Formation of reduced VWF

It will be understood that compositions enriched for reduced VWF may be obtained by chemical treatment of VWF -containing compositions so as to convert oxidised VWF to reduced VWF without impacting on the other critical invariant cysteine residues in domains A1 or A3 described in the above tables. This enables the formation of compositions having increased amounts of natural or recombinant sources of reduced VWF, and in particular compositions that have a greater propensity for cleavage by

ADAMST13, a decreased propensity for mulimerisation into larger multimers and a decreased affinity for GPbl receptors.

Thus in one embodiment there is described a method for increasing the relative abundance of a reduced VWF in a composition. The method includes providing a composition including oxidised VWF, contacting the composition with an reducing agent in conditions enabling selective reduction of adjacent cysteine residues in domain A2 of oxidised VWF, thereby breaking a covalent bond between the sulfur atoms of the adjacent cysteine residues in oxidised VWF; wherein the breakage of a covalent bond between the sulfur atoms of the adjacent cysteine residues in domain 2A of oxidised

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VWF increases the relative abundance of reduced VWF in the composition; thereby increasing the relative abundance of reduced VWF in the composition.

According to the method, the reduction is undertaken in conditions selective for reduction of cysteine residues Cys A2-1 and CysA2-2. A variety of reducing agents may 5 be selected for the reduction including dithiothreitol (DTT), 2 mercapto-ethanol and an oxidoreductase.

It will be understood that the method is particularly useful for adapting or standardising VWF - containing products so that they are useful for the treatment of individuals having clotting disorders arising from an over-abundance of oxidised VWF, such as 0 individuals having AVWS. In one embodiment, the relative abundance of reduced VWF in the composition is increased by at least 10%, preferably 50%, preferably 100% or more.

In one embodiment at least 25%, preferably 30 or 35% of VWF in the composition prior to contact with the reducing agent is oxidised VWF.

- providing a composition including oxidised VWF;

- adding a composition under sub-heading H or peptide of sub heading G to the composition;

J. AVWS treatments

Individuals on mechanical assist devices for heart failure are at risk for pro-thrombotic evens because the high mechanical shear associated with these devices induces conformation change in the A2 domain of VWF leading to an increased affinity for platelet GP1b. These individuals are said to have or be at risk of an acquired von

Willebrand syndrome.

One problem with the treatment of these individuals with recombinant and naturally 22

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2016204172 20 Jun 2016 derived blood or plasma products is that, as established herein, there is variance in the relative amount of oxidised VWF in in these products, and some have an unacceptably high level of oxidised VWF. In certain embodiments, there is provided a method for treating for or preventing an individual from developing AVWS, the method including 5 providing an individual having, or at risk of developing AVWS; administering a peptide or composition as described under sub headings G to I to the individual, thereby treating the individual for AVWS, or preventing the individual from developing AVWS.

K. Assessing VWF containing products

As exemplified in the examples herein, the inventor has developed a technology that enables one to determine the relative amount of oxidised VWF or reduced VWF in a sample. In summary, the method alkylates reduced Cys A2-1 and Cys A2-2 with ¹²CIPA and the oxidised Cys A2-1 and Cys A2-2 with ¹³C-IPA and determines the ratio of ¹²C-IPA to ¹³C-IPA.

The technology is important for enabling the screening of recombinantly- or naturally5 sourced VWF products for suitability for use in the treatment of individuals with VWD or other low thrombogenic potential individuals and/or to determine the potential of a VWF composition to induce the formation of a thrombus in an individual. That is, it enables one to determine whether a composition has an amount of reduced VWF that makes it suitable for use in treatment of individuals having, or at risk of having AVWS, or an amount of oxidised VWF that makes it useful in treatment of individuals having VWD.

The method includes the step of providing a VWF composition, measuring the relative abundance of oxidised VWF in the composition, (the oxidised VWF being a form of VWF in which adjacent cysteine residues in the A2 domain of VWF are linked by a covalent bond between sulfur atoms of the adjacent cysteine resides) and determining that the VWF composition has a high likelihood for inducing formation of a thrombus in the individual where the amount of oxidised VWF in the composition is measured to be greater than about 25% of the total amount of VWF in the composition; and determining that the composition has a low likelihood for inducing formation of a thrombus in the individual where the amount of oxidised VWF in the composition is measure to be less than about 25% of the total amount of VWF in the composition.

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Examples

Example 1 The VWF A2 domain disulphide bond exists in oxidised and reduced states in healthy donors

VWF was isolated from the plasma of healthy donors (n=23) and the redox state of the

A2 domain Cys1669-Cys1670 disulphide bond determined by differential cysteine alkylation and mass spectrometry. Briefly, blood was collected by venipuncture into evacuated tubes containing EDTA as anticoagulant and plasma prepared by centrifugation. The VWF was immunoprecipitated from plasma, reduced Cys1669Cys1670 disulphide bond cysteines alkylated with 2-iodo-N-phenylacetamide (12C-IPA), and the oxidised disulphide bond cysteines with a stable carbon-13 isotope of IPA (13CIPA) following reduction with dithiothreitol (Fig. 2A). Two peptides encompassing the Cys1669-Cys1670 disulphide bond cysteines (LVLQRCCSGE and TLPREAPDLVLQRCCSGE) were resolved by mass spectrometry and quantified (Fig. S1). The ratio of peptides containing the disulphide bond cysteines alkylated with 12C5 IPA compared to 13C-IPA represent the fraction of the disulphide in the population that is in the reduced state. The advantage of this pair of cysteine alkylators is that they have the same chemical reactivity and the same structure, which enhances the reliability of alkylation, resolution of the alkylated peptides by liquid chromatography and their detection by mass spectrometry (15). A mass difference of 6 Da is the only change in a cysteine labeled with 12C-IPA or 13C-IPA.

The measure of VWF redox state is independent of whether blood is collected into EDTA or citrate as anticoagulant, freeze/thawing of plasma (at least three times) and plasma storage time (for at least 3 years). On some occasions, blood was collected into anticoagulant containing 12C-IPA to freeze the redox state of VWF upon collection. The redox state of plasma VWF, though, was the same whether blood was collected into 12C-IPA or not. These results indicate that the VWF redox state is not appreciably influenced by the collection or processing of blood.

In 23 healthy donors, -75% of the VWF A2 domains are reduced and -25% are oxidised (s.d. = 6%, coefficient of variation = 8%; 5 males, 18 females) (Fig. 2B).

Circulating VWF is composed of 500 kDa dimeric units (2 A2 domains) and multiples thereof. The largest multimers are in the 20,000 kDa range (~80 A2 domains). As multimer size is an important aspect of VWF function, we determined whether the redox state of the A2 domain disulphide bond is influenced by this parameter. VWF from

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2016204172 20 Jun 2016 pooled normal plasma was isolated and fractions of decreasing average multimer size resolved by gel filtration. The A2 domains of higher molecular multimers are more oxidised than in the smaller multimers (Fig. 2C).

We anticipated that the redox state of the A2 domain disulphide bond would influence the domain’s response to mechanical force and by association the binding of platelet GPIba to the A1 domain, proteolysis of the A2 domain Tyr1605-Met1606 peptide bond by ADAMTS13 and binding of collagen to the A3 domain (see Fig. 1). These interactions with oxidised versus reduced VWF were examined.

Example 2 Oxidised VWF is more effective at capturing platelets in flowing blood than reduced VWF

A primary haemostatic function of VWF is to capture platelets from flowing blood to an injured vessel wall. To test the effect of the redox state of the A2 domain disulphide bond on this function, full-length recombinant wild-type (wt) or disulphide bond mutant (both A2 domain Cys changed to Gly) VWF was expressed in mammalian cells and examined for platelet binding as a function of fluid shear stress. Wild-type protein is a mixture of reduced (48%) and oxidised (52%) forms as for plasma VWF (Fig. 2B), although the recombinant protein is more oxidised than the plasma protein. The disulphide bond mutant VWF represents the ‘fully reduced’ VWF and was expressed and secreted from HEK293 cells at comparable levels as wild-type protein and has comparable multimer size (Fig. S2), indicating that ablation of the A2 disulphide bond does not appreciable affect maturation of VWF.

Platelet adhesion to wild-type and reduced (mutant) VWF was measured at fluid shear rates of 40, 80 and 150 dyn/cm2, which is in the range found in arterioles and stenosed vessels (16). At high fluid shear rates, platelet coverage on reduced VWF (Fig. 3A and

B) and mean platelet aggregate size (Fig. 3C) was significantly reduced compared to wild-type protein. Platelet adhesion and thrombus formation is strictly dependent on VWF interaction with platelet GPIba at these shear rates (17). The nature of the impaired binding of platelet GPIba to reduced VWF was examined using singlemolecule force spectroscopy incorporating a biomembrane force probe (18-20).

The biomembrane force probe consists of a streptavidin and wild-type or reduced (mutant) VWF coated bead (Fig. 3D, left) glued to the apex of a micropipette-aspirated human red blood cell (RBC) to form a picoforce transducer (spring constant ~0.3 pN/nm), facing a human platelet (Fig. 3D, right) aspired by another apposing

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2016204172 20 Jun 2016 micropipette. In a BFP test cycle, the target is driven with subnanometer precision to touch the probe bead, allowing proteins to interact. Upon target retraction, whether a bond is absent (Fig. 3E, black) or present (Fig. 3E, blue) is detected by the bead displacement, monitored at a temporal and spatial precision of 0.7 ms and 3 nm, 5 respectively. The binding parameters were calculated from the RBC-bead displacement (18-20).

Platelet adhesion frequency to reduced VWF was significantly lower than to wild-type protein (Fig. 3F). Adhesion frequency reflects the zero-force 2D cellular avidity and is the number of test cycles with a bond divided by the number of total cycles. To 0 investigate the force-dependent dissociation, we measured VWF-platelet bond lifetimes at multiple clamped forces. Both wild-type and reduced (mutant) VWF displayed catch bonds in binding to platelets, in accordance with previous studies of this interaction (1820). Although, from 10 to 60 pN, the reduced (mutant) VWF-platelet bond lifetimes were globally suppressed compared to wild-type VWF (Fig. 3G). These findings indicate that 5 reduced VWF has a significantly lower affinity for platelet GPIba, which correlates with the impaired capture of platelets at high fluid shear stress (Figs. 3a-c).

Example 3 Reduced VWF is more efficiently cleaved by ADAMTS13

It is important that VWF multimer size is controlled in the circulation. Too many large multimers can lead to unwanted thrombosis (4), while not enough large multimers is associated with bleeding (1). Reduced (mutant) VWF is more efficiently cleaved by ADAMTS13 than oxidised VWF (Fig. 4A). After 5 h incubation there was ~40% cleavage of reduced VWF and < 10% cleavage of wild-type VWF. This finding is in accordance with functional studies of recombinant full-length VWF and A2 domain, where ablation of the A2 domain disulphide bond similarly increased ADAMTS13 cleavage (21).

Example 4 Oxidised and reduced VWF bind equally well to collagen

VWF binds to basement membrane collagen exposed at sites of vascular injury via the A3 domain. Wild type and reduced (mutant) VWF bound equally to collagen under both static and fluid shear (32 dynes/cm2) conditions (Fig. 4B). The apparent dissociation constants for binding of both forms of VWF to immobilised collagen were 1.7 pg/mL under static conditions and 0.3 pg/mL under shear conditions.

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Example 5 Reduction of the A2 domain disulphide bond has a pronounced effect on domain structure as revealed by molecular dynamics simulations

We performed MD simulations of the VWF A2 domain to investigate variations induced by the redox state of the Cys1669-Cys1670 disulphide bond (Figs. 5 and S3). The 5 oxidised and reduced A2 domains displayed highly divergent global collective motions (Figs. 5b-c). As seen in the projection onto the first two major collective mode of motion, obtained from a principal component analysis, the oxidised A2 domain (green) adopted structures similar to the starting structure (black), whereas the reduced domain (orange) substantially changed its conformation and stabilised around a structure dissimilar from 0 the starting structure (Fig. 5B). In particular, the L4 loop, helix H5, loop L3-4 and the Cterminus displayed the largest conformational changes upon reduction (Fig. 5C).

Further local dynamical changes upon reduction of the A2 domain were quantified by the difference in their root mean square fluctuations (RMSF), RMSF(reduced)RMSF(oxidised) (Fig. 5D). The C-terminal (Ct) region close to the Cys1669-Cys1670 disulphide bond was found less flexible in the reduced than in the oxidised state (blue). The differences in RMSF were not only concentrated in this region, but also spread over the entire A2 domain. Intriguingly, the L4 loop and helices H3 and H5, motifs with high conformational displacement upon reduction (Fig. 5C), also presented less positional fluctuations in the reduced than in the oxidised state (blue), opposite to the loops L2 and

L3-4, which were found more mobile in the reduced form (red).

Contrary to the helical elements and the loops, the beta strands (in particular B4 where the peptide bond cleaved by ADAMTS13 is located) did not present appreciable dynamical changes depending on the redox state of the bond Cys1669-Cys1670. However, a force distribution analysis is more sensitive in revealing allosteric communication through protein cores (22) and was used here to reveal residues drastically changing their time-averaged pair-wise force in the reduced state compared to the oxidised state (Fig. 5E). Many of these pairs involved the beta strands, thus implying that the internal stress between beta strands and their surrounding elements is altered upon reduction. Taken together, these results indicate a change in redox state of the VWF A2 domain leads to subtle yet significant changes in dynamics and stress throughout the domain.

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Example 6 Treatment of heart failure patients with mechanical assist devices results in marked depletion of reduced VWF that is consistent with mechanical shearinduced oxidation of the A2 domain disulphide

Implantation of left ventricular assist devices (LVAD) is an established therapy in patients with end-stage heart failure, while extracorporeal membrane oxygenation (ECMO) is used for patients with refractory heart failure with or without respiratory failure. Haemostatic complications are a major problem with use of these devices and has been associated with an acquired von Willebrand syndrome (23, 24).

The circulating VWF in LVAD (n = 13) and ECMO patients (n = 11) is significantly more oxidised than in heart failure patients not implanted with these devices (n = 9) (p < 0.0001) (Fig. 6A). Despite the variability in disease severity and also anticoagulation or antiplatelet treatment in these patients, the coefficient of variation for VWF redox state measurements is only 2.6% (heart failure), 10.8% (LVAD) and 15.9% (ECMO), respectively. This implies that the loss of reduced VWF in the patients is a fundamental result of the circulatory assist device and not noticeably influenced by disease or therapy variations.

The mechanism of this effect was explored by determining the redox potential of the VWF disulphide bond and the influence of a plasma oxidoreductase that can manipulate the redox state of the bond. Oxidoreductase active sites contain a reactive dithiol/disulphide that can reduce or oxidize a substrate disulphide bond. The A2 domain disulphide bond has a standard redox potential of -287 mV (Fig. 6B). This is in the range of the redox potential of the active site catalytic disulphide of thioredoxin-1, -270 mV (25). The other circulating oxidoreductases (12) have higher redox potentials. Thioredoxin gene expression is upregulated and the protein is secreted by immune cells during inflammation, leading to a high local concentration and also elevated blood levels (26). High serum levels of thioredoxin have been observed in patients with asthma, rheumatoid arthritis and heart failure (27, 28), and serum levels correlate with disease activity in rheumatoid arthritis (29, 30). Incubation of oxidised or reduced thioredoxin-1 with plasma VWF resulted in oxidation or reduction, respectively, of the A2 domain disulphide bond, but only under fluid shear conditions (Fig. 6C). Notably, the extent of oxidation of VWF by oxidised thioredoxin-1 is in the range observed in the ECMO patients (Fig. 6A). The shear-dependent manipulation of the VWF disulphide bond by thioredoxin is not unexpected. Mechanical stretching, twisting or pulling of proteins

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2016204172 20 Jun 2016 makes their disulphide bonds easier or harder to cleave by changing the alignment of the three sulfur atoms involved in the cleavage (the sulfur ion nucleophile of the oxidoreductase and the two sulfur atoms of the disulphide bond) (31 -34).

It was possible that neutrophil reactive oxygen species associated with an increased inflammatory state were responsible for oxidizing the VWF disulphide in mechanical assist device patients. Neutrophil-derived hypochlorous acid can oxidise Met1606 of the Tyr1605-Met1606 peptide bond cleaved by ADAMTS13, which impairs ADAMTS13 cleavage of VWF and may contribute to a pro-thrombotic inflammatory environment (35). Other neutrophil species such as H2O2 have been shown to mediate oxidation of cysteine thiols to a disulphide bond (13). A peptide encompassing Met1606 (QAPNLVYMVTGNPASDE) in the patients’ VWF was resolved by mass spectrometry and quantified for unmodified and oxidised residue (Fig. S4). Only a few percent (range of 1-13%) of Met1606 was found to be oxidised in control, LVAD and ECMO groups and there was no difference between the groups (Fig. 6D). This result indicates that VWF is not being ‘damaged’ by reactive oxygen species from activated neutrophils as a result of the devices.

Finally, in accordance with published observations (24), there is a significant depletion of plasma VWF in the ECMO patients compared to control patients (p < 0.01) (Fig. 6E), and the VWF is of smaller average multimer size measured using the collagen binding assay (p < 0.05) (Fig. 6F). The marked differences in oxidation status (Fig. 6A) provide a clear mechanistic advance for understanding the haemostatic dysfunction of VWF in circulatory support patients and potentially other human disease.

VWF tethers platelets to the injured blood vessel wall via interactions with basement membrane collagen and the platelet receptors, activated allbp3 and GPIb. Interaction with platelet GPIb dominates under the high fluid shear of arterioles and stenosed vessels (17). The findings presented herein indicate that only the minor circulating form of VWF, oxidised VWF, is effective at capturing platelets via GPIb at high shear. The major form of VWF in blood, reduced VWF, has the same affinity for collagen as oxidised VWF but much lower affinity for GPIb under both low and high shear conditions. The shear forces of flowing blood unfold the A2 domain allowing access of ADAMTS13 to the Tyr1605-Met1606 peptide bond (5, 6). In contrast to platelet adhesion, the major form of VWF is more efficiently cleaved by the metalloprotease than the minor form. These functional differences are likely a result of the redox switch

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2016204172 20 Jun 2016 mediating allosteric conformational transitions in the A2 domain and its vicinity.

From our MD simulations, reduction of the Cys1669-Cys1670 disulphide bond markedly alters A2 domain dynamics. The changes were not only observed near the two cysteines, but they also propagated to other distant regions of the protein, such as 5 helices H3 and H5, and the loops L2, L3-4, and L4. The beta strand containing the ADAMTS13 cleavage site, as well as the other strands constituting the internal beta sheet, were only marginally affected by the redox state, namely through an allosteric propagation of internal stresses originating from the cysteine pair. Our simulations thus suggests that oxidation/reduction of the disulphide bond influences the behavior of the 0 A2 domain through a dynamic allosteric mechanism. The oxidised and reduced domains, therefore, are predicted to respond differently to mechanical force and so influence binding of platelet GPIb to the A1 domain and ADAMTS13 proteolysis of the A2 domain.

Large VWF multimers are more effective than small multimers at capturing platelets in the shear forces of flowing blood. This has been attributed to the polyvalency of the large multimers for platelets and collagen(1). Our findings that large multimers are more oxidised than small multimers and oxidised VWF more effectively captures platelets, indicates that both polyvalency and redox state are important for VWF pro-thrombotic activity. This balance is shifted in patients on mechanical assist devices for heart failure.

The high mechanical shear associated with these devices results in more prothrombotic oxidised VWF.

Mutations in VWF resulting in von Willebrand disease (VWD) is the most common hereditary coagulation abnormality described in humans and can be acquired as a result of other medical conditions. VWD is due to a qualitative or quantitative deficiency of VWF. For milder VWD (type 1 and some type 2), treatment with desmopressin, which stimulates secretion of endogenous stores of VWF from endothelium, can be effective. In severe type 1 VWD, type 3 VWD or when desmopressin is contraindicated, replacement VWF concentrate is used in many clinical scenarios (36). The VWF concentrates in use involve plasma-derived VWF from pools of donors and a recombinant VWF has recently been approved for use (37). The VWF 'activity' in concentrates is poorly defined and varies from batch to batch. Both pooled plasma and recombinant VWF contain the two forms of VWF in variable ratios. Defining the proportion of the two VWF forms is predicted to improve standardisation of their clinical

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The finding that the A2 domain disulphide bond mediates a functional switch in VWF led us to investigate if disulphide bonds linking adjacent cysteines are found in other proteins. A search of protein crystal structures using the Disulphide Bond Analysis tool 5 (38) uncovered 37 other disulphide bonds linking adjacent cysteines (Table S2). A number of different types of enzymes and receptor proteins contain disulphide bonds of this type. 27 of the 37 disulphide bonds have the same +/-LHStaple configuration as the VWF A2 domain bond and all of the cystines have a very short α-carbon-a-carbon atom distance. Interestingly, the next most common configuration with 6 examples is the 0 RHStaple, which is the archetypal allosteric configuration (13, 14).

Example 7 Materials and Methods:

A. Plasma and recombinant VWF

Blood was collected by venipuncture into evacuated tubes containing EDTA or citrate as anticoagulant and plasma prepared by centrifugation. VWF was purified from four units of fresh frozen plasma as described (39, 40). Briefly, cryoprecipitate was collected and the VWF precipitated with PEG4000. The pellet was resuspended in Hepes-buffered saline containing EDTA and benzamidine, and gel filtered on Sepharose CL-2B. Discrete fractions were collected over the VWF peak. Recombinant full length human wild-type and reduced (mutant) VWF (C1669G,C1670G)(21) were transiently expressed in HEK293 cells using polyethylenimine HCI MAX (PEI MAX, MW 4,000) with a PELDNA w/w ratio of 4:1 (41). Three days post-transfection, culture media was collected and concentrated using centrifugal filters with a 100 kDa molecular weight cut-off. VWF was resolved by SDS-PAGE or agarose gel electrophoresis and Western blotted as described (42, 43).

B. Redox state of the VWF Cys1669-Cys1670 disulphide bond

VWF was purified from ~0.5-1 mL of fresh or fresh frozen plasma. Briefly, VWF was precipitated using a plasma:16% PEG 4000 w/w ratio of 1:8, the pellet resuspended in phosphate-buffered saline and the VWF collected on polyclonal anti-VWF antibody (Dako) coated Dynabeads (MyOne Streptavidin T1, Life technologies). Unpaired cysteine thiols in the bead-bound VWF were alkylated with 5 mM 2-iodo-Nphenylacetamide (12C-IPA, Cambridge Isotopes) for 1 h at room temperature, the protein resolved on reducing SDS-PAGE and stained with Coomassie (Sigma). The

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VWF band was excised, destained, dried, incubated with 40 mM dithiothreitol and washed. The fully reduced protein was alkylated with 5 mM 2-iodo-N-phenylacetamide where all 6 carbon atoms of the phenyl ring have a mass of 13 (13C-IPA, Cambridge Isotopes). The gel slice was washed and dried before digestion of VWF with 14 ng/pL of 5 Glu-C (Roche) in 25 mM NH4CO2 overnight at 25 °C. Peptides were eluted from the slices with 5% formic acid, 50% acetonitrile. Liquid chromatography, mass spectrometry and data analysis were performed as described (44, 45). Two peptides encompassing the Cys1669-Cys1670 disulphide bond cysteines (LVLORCCSGE and TLPREAPDLVLORCCSGE) were resolved and quantified. The fraction of reduced 0 disulphide bond was measured from the relative ion abundance of peptides containing 12C-IPA and 13C-IPA. To calculate ion abundance of peptides, extracted ion chromatograms were generated using the XCalibur Oual Browser software (v2.1.0; Thermo Scientific). The area was calculated using the automated peak detection function built into the software.

C. Platelet adhesion

Microchannels of Vena8 Fluor Biochip (Cellix Ltd) were coated with wild-type or reduced (mutant) VWF (5 pg/mL) or bovine serum albumin (10 pg/mL in phosphate-buffered saline as control) overnight at 4^QC in a humidified box. Microchannels were blocked with 10 pL of 10 pg/mL bovine serum albumin for 1 h at room temperature and washed with

40 pL of phosphate-buffered saline. Whole blood from healthy donors was drawn into

ACD-A tubes (BD Vacutainer) and labeled with 1 pg/mL calcein (Thermo Fisher). Blood was injected by Mirus NanoPump into the channel at fluid shear stresses of 40, 80, 120 or 150 dynes/cm2 (flow rate 592, 1185 and 2222 nL/sec, respectively) within 3 h of collection. Adhesion of platelets was monitored in real time with images captured via an

ExiBlu CCD camera (Q imaging) connected to an AxioObserver A1 Inverted EpiFluorescence microscope (Zeiss). Images were captured using VenaFlux 2.3 imaging software (1 frame per second). Images were analysed at positions 2, 4 and 6 of the microchannels over time using ImagePro Premier software. Area coverage by platelets (expressed as % of total surface area) and mean diameter of platelet aggregates was calculated for each time point. Results are presented for the 3 min time point and expressed as mean ± s.d. (n=4). Statistical analysis used non-parametric T test (Prism 6.0).

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D. Single-molecule force spectroscopy

All procedures involving collection of mouse and human blood were in accordance with the Human Research Ethics Committee (HREC) (Project number 2014/244) at University of Sydney. Human blood was obtained from healthy volunteers who had not 5 received any anti-platelet medication in the preceding 2 weeks. Blood was anticoagulated with acid-citrate-dextrose (ACD) anticoagulant (13 mM sodium citrate, 1 mM citric acid, 20 mM dextrose, and 10 mM theophylline). Washed human platelets and red blood cells were prepared as previously described(46). To avoid preactivation, human washed platelets were kept at 37 ^QC in platelet-washing buffer (PWB) (4.3 mM 0 K2HPO4, 4.3 mM Na2HPO4, 24.3 mM NaH2PO4, pH 6.5, 113 mM NaCI, 5.5 mM glucose, 0.5% bovine serum albumin) containing theophylline (10 mM) and apyrase (0.04 units/mL), and then resuspended in Tyrode’s buffer (10 mM Hepes, 12 mM NaHCO3, pH 7.4, 137 mM NaCI, 2.7 mM KCI, 5 mM glucose) prior to use in adhesion studies.

To make the biomembrane force probe (BFP), human RBCs (3 μΙ_) were resuspended in a carbonate/bicarbonate buffer (0.1 M NaHCO3 and Na2CO3, pH 8.5) and then biotinylated by covalently linking polymer biotin-PEG3500-SGA (JenKem) with a 30 min incubation at room temperature(18, 47-49). To balloon RBCs for the force probe use in the Tyrode buffer of physiological osmolarity, RBCs were further incubated with nystatin (Sigma-Aldrich) in an N2-5% buffer (265.2 mM KCI, 38.8 mM NaCI, 0.94 mM KH2PO4, 4.74 mM Na2HPO4, 27 mM sucrose; pH 7.2, 588 mOsm) for 30 min at 0^Q C. The modified RBCs were washed twice with the N2 buffer and resuspended in the N2 buffer for the BFP experiments (50).

To functionalize the glass beads, anti-VWF (ab6994; Abeam) was pre-coupled with maleimide-PEG3500-NHS (MW -3500; JenKem) in carbonate/bicarbonate buffer (pH 8.5). To coat proteins on glass beads, 2-pm (diameter) borosilicate beads (Thermo Scientific) were first silanized with mercapto-propyl-trimethoxy silane, then covalently linked to maleimide modified streptavidin (SA-maleimide, Sigma-Aldrich) and anti-VWF in phosphate buffer (pH 6.8). After overnight incubation, the beads were resuspended in phosphate buffer with 1% BSA. To capture VWF, beads were mixed with VWF at volume ratio 10:1 and incubated for 3 hours at room temperature. After resuspending in phosphate buffer with 1% BSA, beads were ready for immediate use in BFP experiments.

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The binding parameters were calculated from the RBC-bead displacement as previously described (18-20). Bond formation/dissociation and force application were enabled and monitored in controlled BFP cycles of a few seconds each. In each BFP cycle, an aspired platelet was driven to approach and contact the probe (a VWF-bearing bead) 5 with a 25-pN compressive force for a certain contact time (0.2 s) that allowed for bond formation and then retracted at a constant speed (3.3 pm/s) for bond detection. During the retraction phase, a bond event was signified by tensile force. No tensile force was detected in a no-bond event. For the adhesion frequency assay, bond and no-bond events were enumerated to calculate an adhesion frequency in 50 repeated cycles for 0 each probe-target pair. The force-clamp assay was used to measure bond lifetimes. In a similar BFP cycle as to the adhesion frequency assay, upon detection of adhesion event, a feedback loop controls the retraction so that it would be paused at a desired clamping force (5-50 pN) to wait for bond dissociation. After that the target pipette returns to the original position to complete the cycle. Lifetimes were measured from the 5 instant when the force reached the desired level to the instant of bond dissociation.

E. ADAMTS13 proteolysis

ADAMTS13 was expressed in HEK293 cells and collected from conditioned medium as described (51). ADAMTS13 proteolysis of VWF was performed as described (52). Briefly, wild-type or reduced (mutant) VWF (~5 pg/mL) was denatured with urea, incubated with Ca2+-activated ADAMTS13 (2 nM) and aliquots of the reaction quenched a discrete times with EDTA.

F. Collagen binding

Nunc Maxisorp plates were coated with 100 pL of 5 pg/mL equine type 1 collagen (Kollagen Reagens Horm Suspension, Takeda) in phosphate-buffed saline overnight with shaking. Wells were blocked with 200 pL of Superblock (Thermo Fisher Scientific) and incubated with wild-type or reduced (mutant) VWF in Hepes-buffered saline containing 2 mM CaCI2, 0.05% Tween 20, 1% bovine serum albumin and incubated for 90 min at room temperature. One some occasions the incubations were subjected to 32 dynes/cm2 of fluid shear stress by shaking the plate at 2,500 rpm in a MixMate orbital shaker (Eppendorf)(53). VWF binding was measured using peroxidase-conjugated polyclonal anti-VWF antibodies (1:1000, Dako).

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G. Thioredoxin-1 manipulation of the VWF Cys1669-Cys1670 disulphide bond

Wild type human thioredoxin-1 was produced in E. coli (45). Oxidised thioredoxin-1 was prepared by incubating with 200 μΜ oxidised glutathione in phosphate-buffered saline at 25°C for 18 h. Reduced thioredoxin-1 was prepared by incubating with 10 mM 5 dithiothreitol for 30 min at 25°C. The small compounds were removed by using Zeba spin desalting columns equilibrated with phosphate-buffered saline (Thermo Scientific). Purified plasma VWF (12 pg/mL) was incubated without or with 5 μΜ thioredoxin-1 for 1 h at 22°C in the absence or presence of 32 dynes/cm2 of fluid shear stress using a MixMate orbital shaker (see above).

H. Molecular dynamics simulations

We used the Amber99sb-ildn* force field(54-56) for all energy minimizations and molecular dynamics (MD) simulations of the VWF A2 domain in both oxidised and reduced forms. All simulations were carried out using the Gromacs 4.5.5 package (57, 58). Initial structures of the protein in both redox states were taken from the crystal structure of the human VWF A2 domain (PDB identifier 3GXB)(5). For the reduced state, hydrogens were added to the cysteines. Acetyl and N-methyl groups were placed on N- and C-termini, respectively, in order to cap the termini. Both structures, oxidised and reduced, were immersed in a cubic box containing approximately 23600 TIP3P(59) water molecules, with sodium and chloride ions added at a concentration of 150 mM, and few sodium counterions to maintain neutrality of the simulation box. Parameters for the ions were taken from Joung and Cheatham (60). Prior to MD simulations, both systems were minimized using the steepest descent method for 10000 steps, which was followed by 2 ns of MD simulations, during which harmonic positional restraints were applied on protein heavy atoms (harmonic elastic constant of 1000 kJmol-1 nm-2).

Finally, unrestrained equilibrium MD simulations were carried out for 200 ns, out of which the last 150 ns was used for subsequent analysis. The temperature was kept constant at 300 K by using a velocity rescaling thermostat (61,62) with a coupling time of 0.5 ps. The pressure was kept constant at 1 bar using an isotropic coupling to a Parrinello-Rahman barostat (63) with a coupling time of 5.0 ps. An integration time step of 4 fs was used. In all simulations, the long-range electrostatic interactions were treated with the Particle Mesh Ewald method (64, 65). All bonds were constrained using the LINCS (66) algorithm, and angular degrees of freedom of hydrogen atoms were replaced by using virtual interaction sites (67). For the water molecules, both bond

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2016204172 20 Jun 2016 lengths and angular vibrations were constrained using the SETTLE algorithm(68). In order to investigate the conformational dynamics of both redox states of the VWF A2 domain, the MD trajectories were subjected to a principal component analysis (69). This analysis consists in the calculation and diagonalization of the covariance matrix of the 5 atomic positions (here of the backbone atoms), enabling filtering relevant largeamplitude collective motions from local fluctuations. The eigenvalues and eigenvectors of the covariance matrix were calculated for a concatenated trajectory of both redox states. Subsequently, MD trajectories were projected onto the first two eigenvectors, representing the two collective motions with largest variance, with each protein 0 conformation represented by a point in this 2-dimensional projection. These two motional modes accounted for almost 50 percent of the total motion of the protein backbone. Protein structures were visualized with PyMOL (70) and UCSF Chimera (71). Changes in the internal stress of the protein upon reduction were quantified by carrying out a force distribution analysis (72). Time-averaged pair-wise forces <Fij> were 5 computed for all residue pairs (i,j) of the A2 domain, in the oxidised (<Fij(oxidised)>) and the reduced (<Fij(reduced)>) states. The absolute value of the difference |DFij|=|<Fij(reduced)> - <Fij(oxidised)>| was subsequently calculated as a measure of the change in internal stress upon reduction. Here | | indicates absolute values. Residue pairs showing differences larger than 90 pN were selected.

I. Aquired von Willebrand syndrome

Samples were collected in accordance with the Alfred Hospital Ethics and the Monash University Standing Committee for Research in Humans, and the Helsinki Declaration of 1983. Blood was collected from patients at a single centre who had mechanical circulatory support (LVAD or ECMO) or patients with cardiac comorbidities including severe congestive cardiac failure. Patients with mechanical circulatory support received anti-coagulation and/or anti-platelet medications based on at clinicians’ discretion or institutional guideline where patients typically commenced on warfarin, target international normalised ratio (INR) 2-3, with bridging heparin infusion and aspirin therapy, as well as dipyridamole for those who are considered high risk for thrombosis.

Blood was drawn into vacutainer® tubes containing sodium citrate or EDTA, and routine blood tests determined using standard laboratory procedures. VWF antigen (VWF:Ag) was measured using latex immunoassay (LIA) method and collagen binding assay (VWF:CB) was performed by ELISA (Diagnostica STAGO).

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J. Statistics

Parametric unpaired two-tailed t test was used to evaluate differences between groups. Statistical significance was defined as p values < 0.05.

Example 8 Materials and Methods

A. Redox potential of the Cys1669-Cys1670 disulphide bond.

Plasma VWF was incubated in argon-flushed phosphate-buffered saline containing 0.1 mM EDTA, 0.5 mM DTTox (oxidised DTT or 4,5-dihydroxy-1,2-dithiane; Sigma) and various concentrations of DTTred (reduced DTT or dithiothreitol; Sigma) for 18 h at room temperature to allow equilibrium to be reached. Microcentrifuge tubes were flushed with argon prior to sealing to prevent ambient air oxidation during the incubation period. Reduced disulphide bond cysteines were alkylated with 2-iodo-Nphenylacetamide (12C-IPA) and the oxidised disulphide bond cysteines with a stable carbon-13 isotope of IPA (13C-IPA) following reduction with dithiothreitol (see Fig. 1). The ratio of 12C-IPA and 13C-IPA alkylation represents the fraction of the cysteine in the population that is in the reduced state.

The results were expressed as the ratio of reduced to oxidised protein and fitted to equation 1:

/[DTTredh I [DTT_0X] J

L[DTT_reci\\ I [DTT_0X] J (1) where R is the fraction of reduced protein at equilibrium and Keq is the equilibrium 20 constant. The standard redox potential (E°') of the Cys1669-Cys1670 disulphide bond was calculated using the Nernst equation (equation 2):

E°' =E°_D'_TT-^\nK_eq (2) using a value of -307 mV for the redox potential of the DTT disulphide bond (73).

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Table S1. Structural features of the VWF A2 domain Cys1669-Cys1670 disulphide bond.

PDB identifier (reference)	Chain	Cvs1669 solvent accessibility (~A²)	Cvs1670 solvent accessibility (~A²)	a-carbon atoms distance (A)	Confiauration
3GXB (5)	A	18	28	3.74	+/-LHStaple
B	23	29	3.73	+/-LHStaple
3ZQK(7 7)	A	18	2	3.73	+/-LHStaple
B	35	35	3.80	+/-LHStaple
C	51	65	3.80	+/-LHStaple

Table S2. Proteins in the Protein Data Bank that contain disulphide bonds linking 5 adjacent cysteine residues in the polypeptide chain. The search was performed using the Disulphide Bond Analysis tool (38). The solvent accessibility of the disulphide bond, the secondary structure in which it is found, the distance between the α-carbon atoms of the cysteine residues and the configuration of the disulphide bond is indicated.

Category	Name	PDB identifiers	Disulphide¹	Cys1+Cys2 solvent accessibility (~A²)	Secondary structure	a-carbon atom distance (A)	Configuration
Enzymes
Transferase	transglutaminase 2	2Q3Z	370-371	20	within βloop	2.9	-RHStaple
Glycosylases	arabinanase	1WD3, 1GYH, 1WL7, 1UV4, 3CU9, IGYD, IGYE, 3D5Y, 3D5Z, 3D60, 3D61, 1WD4, 2D43, 2D44	176-177 (1WD3)	33	within βloop	2.9	-LHStaple
fucosidase	2WSP, 2ZWY, 2ZX5, 2ZX6, 2ZX7, 2ZX8, 2ZX9, 2ZXA, 2ZXB, 2ZXD, 1HL8, 1HL9,	364-365 (2WSP)	60	within βloop	3.7	+/-LHStaple

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		1ODU
amylase	1MXD, 1MW0	153-154 (1MXD)	146	within βloop	3.7	+/-LHStaple
galactosidase	1TG7, 1XC6	205-206 (1TG7)	137	within βloop	3.7	+/-LHStaple
glucosaminidase	2VZS, 2VZT, 2VZU, 2VZV, 2X05, 2X09	419-420 (2VZS)	20	within strandhelix link	3.7	+/-LHStaple
pullulanase	2FHF, 2FGZ, 2FH6, 2FH8, 2FHB, 2FHC	643-644 (2FHF)	118	within βloop	3.7	+/-LHStaple
Lipidases	palmitoyl esterase	3GRO, 1EH5, 1EI9, 1EXW	45-46 (3GRO)	65	within βloop	3.7	+/-LHStaple
ceraminidase	2ZXC	503-504	162	within βloop	3.8	+/-LHStaple
Peptidases	carboxypeptidase	3PRT, 1OBR, 3QNV	155-156 (3PRT)	117	within helix-helix link	3.7	+/-LHStaple
trypsin-like	1YM0	150-151	98	within βloop	2.9	+/-LHStaple
peptidase	2WYR	231-232	45	within strandhelix link	2.9	-RHStaple
Oxidases & Reductases	thioredoxin reductase	2J3N	497-498	86	undefined	3.8	+LHStaple
alcohol dehydrogenase	1G72, 1FLG, 1KB0, 1YIQ, 1KV9, 1H4I, 1H4J, 1LRW, 1W6S, 2AD6, 2AD7, 2AD8, 2D0V, 4AAH	103-104 (1G72)	73	within strandhelix link	3.7	+/-LHStaple
copper amine oxidase	3ALA, 1PU4, 1US1, 2C10, 2C11, 2Y73, 2Y74, 1TU5, 2PNC	198-199 (3ALA)	6	end of ahelix	3.8	+/-LHStaple
mercuric reductase	1ZK7	464-465	169	undefined	3.7	+RHStaple

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Lyases	alginate lyase	1QAZ, 4E23, 4E25, 1HV6, 3EVH, 3EVL, 3EW4, 4E1Y	188-189 (1QAZ)	58	end of ahelix	3.7	+/-LHStaple
methyl-isocitrate lyase	3EOO	238-239	20	end of ahelix	3.8	+/-LHStaple
carbonic anhydrase	2FGY	283-284	130	end of ahelix	3.8	+/-LHStaple
Hydrolase	amidohydrolase	3E2V	116-117	68	end of ahelix	3.7	+/-LHStaple
Esterase	carboxylesterase	1QLW, 2WKW	71-72 (1QLW)	12	within strandhelix link	3.8	+/-LHStaple
RNA polymerase	RNA polymerase	3GTK 2NVZ 3GTQ	45-46 (3GTK)	0	end of ahelix	3.8	+/-LHStaple
Receptors
von Willebrand factor	3ZQK 3GXB	1669-1670 (3ZQK)	20	end of ahelix	3.7	+/-LHStaple
acetylcholine receptor	3SQ9, 3SQ6 2QC1	186-187 (3SQ9)	112	within βloop	3.8	+/-RHStaple
acetylcholine binding protein	2Y7Y, 2BR7, 2BR8, 2BYN, 2BYP, 2BYQ, 2C9T, 2PGZ, 2PH9, 2UZ6, 2W8E, 2W8G, 2WN9, 2WNC, 2WNJ, 2WNL, 2WZY, 2X00, 2XNT, 2XNU, 2XNV, 2XYS, 2XYT, 2XZ5, 2XZ6, 2Y54, 2Y56, 2Y57, 2Y58, 3C79, 3C84, 3GUA, 3PEO, 3PMZ,	188-189 (2Y7Y)	140	within βloop	3.8	+/-LHStaple

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	3SH1, 3SIO, 3T4M, 4DBM, 1UX2, 119B, 3U8J, 3U8M, 3U8K, 3U8L, 1UV6, 1UW6, 2BJ0, 2BYR, 2BYS, 2ZJU, 2ZJV, 1YI5
glutamate binding protein	1US4	89-90	66	end of ahelix	3.7	+/-LHStaple
Toll-like receptor 4	3T6Q, 2Z63, 2Z64, 2Z66, 3FXI, 3B2D, 3T6Q, 3RG1	387-388 (3T6Q)	2	within βloop	3.0	-RPIStaple
variable lymphocyte receptor	3E6J	129-130	56	within strandhelix link	3.7	+/-LHStaple
transferrin binding protein	3HOL, 3V8U, 3PQU, 3PQS, 3VE2, 3VE1	351-352 (3HOL)	4	within βloop	3.0	-RPIStaple
fucolectin	3CQO, 1K12	74-75 (3CQO)	75	end of ahelix	3.8	+/-LHStaple
Other
bone morphogenetic protein 1	3EDH	64-65	167	within βloop	2.9	-RPIStaple
ABC transporter	3RPW	100-101	54	end of ahelix	3.7	+/-LHStaple
heterochromatin-associated protein	2H4R	406-407	26	undefined	3.7	+/-LHStaple
polypyrimidine tract-binding protein	3ZZY, 3ZZZ	250-251 (3ZZY)	7	within βloop	3.7	+/-LHStaple
profilin-2	2VK3	15-16	18	within strandhelix link	3.7	+/-LHStaple
pumilio homolog 2	3Q0Q, 3Q0R, 3Q0S	982-983 (3Q0Q)	45	end of ahelix	3.8	+/-LHStaple
Leptospira protein	3BWS	346-347	106	within βloop	3.7	+/-LHStaple
antibiotic peptide	3SR3	301-302	0	within βloop	2.8	-RPIStaple

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When more than one structure, the numbering and other measures are for the PDB in bracket.

Example 9 Oxidation of VWF by oxidised DTT.

Purified human plasma VWF (10 pg/mL) was incubated with 0.5 mM oxidised DTT (4,55 dihydroxy-1,2-dithiane; Sigma) in argon-flushed phosphate-buffered saline containing

0.1 mM EDTA for 18 h at room temperature. Microcentrifuge tubes were flushed with argon prior to sealing to prevent ambient air oxidation during the incubation period.

References and Notes

1. J. E. Sadler, Biochemistry and genetics of von Willebrand factor. Annu. Rev. 0 Biochem. 67, 395-424 (1998).

2. M. Furlan, R. Robles, B. Lammle, Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis. Blood 87, 4223-4234 (1996).

3. G. G. Levy, W. C. Nichols, E. C. Lian, T. Foroud, J. N. McClintick, B. M. McGee, 5 A. Y. Yang, D. R. Siemieniak, K. R. Stark, R. Gruppo, R. Sarode, S. B. Shurin, V.

Chandrasekaran, S. P. Stabler, H. Sabio, E. E. Bouhassira, J. D. Upshaw, Jr., D. Ginsburg, Η. M. Tsai, Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 413, 488-494 (2001).

4. J. L. Moake, C. K. Rudy, J. H. Troll, M. J. Weinstein, N. M. Colannino, J. 0 Azocar, R. H. Seder, S. L. Hong, D. Deykin, Unusually large plasma factor VIILvon

Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura. N. Engl. J. Med. 307, 1432-1435 (1982).

5. Q. Zhang, Y. F. Zhou, C. Z. Zhang, X. Zhang, C. Lu, T. A. Springer, Structural specializations of A2, a force-sensing domain in the ultralarge vascular protein von

Willebrand factor. Proc. Natl. Acad. Sci. U. S. A. 106, 9226-9231 (2009).

6. S. W. Schneider, S. Nuschele, A. Wixforth, C. Gorzelanny, A. Alexander-Katz, R. R. Netz, M. F. Schneider, Shear-induced unfolding triggers adhesion of von Willebrand factor fibers. Proc. Natl. Acad. Sci. U. S. A. 104, 7899-7903 (2007).

7. C. Baldauf, R. Schneppenheim, W. Stacklies, T. Obser, A. Pieconka, S. 30 Schneppenheim, U. Budde, J. Zhou, F. Grater, Shear-induced unfolding activates von

Willebrand factor A2 domain for proteolysis. J Thromb Haemost 7, 2096-2105 (2009).

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8. T. Marti, S. J. Rosselet, K. Titani, K. A. Walsh, Identification of disulfide-bridged substructures within human von Willebrand factor. Biochemistry 26, 8099-8109 (1987).

9. B. Schmidt, L. Ho, P. J. Hogg, Allosteric disulfide bonds. Biochemistry 45, 74297433 (2006).

10. B. Schmidt, P. J. Hogg, Search for allosteric disulfide bonds in NMR structures.

BMC Struct Biol 7, 49 (2007).

11. A. J. Jakobi, A. Mashaghi, S. J. Tans, E. G. Huizinga, Calcium modulates force sensing by the von Willebrand factor A2 domain. Nature communications 2, 385 (2011).

12. D. Butera, K. M. Cook, J. Chiu, J. W. Wong, P. J. Hogg, Control of blood proteins by functional disulfide bonds. Blood 123, 2000-2007 (2014).

13. K. M. Cook, P. J. Hogg, Post-translational control of protein function by disulfide bond cleavage. Antioxidants & redox signaling 18, 1987-2015 (2013).

14. P. J. Hogg, Targeting allosteric disulphide bonds in cancer. Nature reviews. Cancer 13, 425-431 (2013).

15. C. Pasquarello, J. C. Sanchez, D. F. Hochstrasser, G. L. Corthals, N-tbutyliodoacetamide and iodoacetanilide: two new cysteine alkylating reagents for relative quantitation of proteins. Rapid Commun. Mass Spectrom. 18, 117-127 (2004).

16. Μ. H. Kroll, J. D. Heliums, L. V. McIntire, A. I. Schafer, J. L. Moake, Platelets and shear stress. Blood 88, 1525-1541 (1996).

^0 17. M. Al-Tamimi, C. W. Tan, J. Qiao, G. J. Pennings, A. Javadzadegan, A. S.

Yong, J. F. Arthur, A. K. Davis, J. Jing, F. T. Mu, J. R. Hamilton, S. P. Jackson, A. Ludwig, M. C. Berndt, C. M. Ward, L. Kritharides, R. K. Andrews, E. E. Gardiner, Pathologic shear triggers shedding of vascular receptors: a novel mechanism for downregulation of platelet glycoprotein VI in stenosed coronary vessels. Blood 119, 431125 4320(2012).

18. L. Ju, J. F. Dong, M. A. Cruz, C. Zhu, The N-terminal flanking region of the A1 domain regulates the force-dependent binding of von Willebrand factor to platelet glycoprotein Ibalpha. J. Biol. Chem. 288, 32289-32301 (2013).

19. L. Ju, J. Lou, Y. Chen, Z. Li, C. Zhu, Force-Induced Unfolding of Leucine-Rich

Repeats of Glycoprotein Ibalpha Strengthens Ligand Interaction. Biophys. J. 109, 17811784 (2015).

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2016204172 20 Jun 2016

20. L. Ju, J. Qian, C. Zhu, Transport regulation of two-dimensional receptor-ligand association. Biophys. J. 108, 1773-1784 (2015).

21. B. M. Luken, L. Y. Winn, J. Emsley, D. A. Lane, J. T. Crawley, The importance of vicinal cysteines, C1669 and C1670, for von Willebrand factor A2 domain function.

Blood 115, 4910-4913 (2010).

22. C. Seifert, F. Grater, Force distribution reveals signal transduction in E. coli Hsp90. Biophys. J. 103, 2195-2202 (2012).

23. A. L. Meyer, D. Malehsa, U. Budde, C. Bara, A. Haverich, M. Strueber, Acquired von Willebrand syndrome in patients with a centrifugal or axial continuous flow left ventricular assist device. JACC. Heart failure 2, 141-145 (2014).

24. D. A. Murphy, L. E. Hockings, R. K. Andrews, C. Aubron, E. E. Gardiner, V. A. Pellegrino, A. K. Davis, Extracorporeal membrane oxygenation-hemostatic complications. Transfus. Med. Rev. 29, 90-101 (2015).

25. G. Krause, J. Lundstrom, J. L. Barea, C. Pueyo de la Cuesta, A. Holmgren, 5 Mimicking the active site of protein disulfide-isomerase by substitution of proline 34 in

Escherichia coli thioredoxin. J. Biol. Chem. 266, 9494-9500 (1991).

26. C. H. Lillig, A. Holmgren, Thioredoxin and related molecules-from biology to health and disease. Antioxidants & redox signaling 9, 25-47 (2007).

27. Y. Yamada, H. Nakamura, T. Adachi, S. Sannohe, H. Oyamada, H. Kayaba, J. 0 Yodoi, J. Chihara, Elevated serum levels of thioredoxin in patients with acute exacerbation of asthma. Immunol. Lett. 86, 199-205 (2003).

28. A. Burke-Gaffney, Μ. E. Callister, H. Nakamura, Thioredoxin: friend or foe in human disease? Trends Pharmacol. Sci. 26, 398-404 (2005).

29. T. Jikimoto, Y. Nishikubo, M. Koshiba, S. Kanagawa, S. Morinobu, A. Morinobu, 25 R. Saura, K. Mizuno, S. Kondo, S. Toyokuni, H. Nakamura, J. Yodoi, S. Kumagai,

Thioredoxin as a biomarker for oxidative stress in patients with rheumatoid arthritis. Mol. Immunol. 38, 765-772 (2002).

30. H. Lemarechal, Y. Allanore, C. Chenevier-Gobeaux, O. G. Ekindjian, A. Kahan, D. Borderie, High redox thioredoxin but low thioredoxin reductase activities in the serum of patients with rheumatoid arthritis. Clin. Chim. Acta 367, 156-161 (2006).

31. I. B. Baldus, F. Grater, Mechanical force can fine-tune redox potentials of

1001489620

2016204172 20 Jun 2016 disulfide bonds. Biophys. J. 102, 622-629 (2012).

32. A. P. Wiita, R. Perez-Jimenez, K. A. Walther, F. Grater, B. J. Berne, A. Holmgren, J. M. Sanchez-Ruiz, J. M. Fernandez, Probing the chemistry of thioredoxin catalysis with force. Nature 450, 124-127 (2007).

33. P. A. Fernandes, M. J. Ramos, Theoretical insights into the mechanism for thiol/disulfide exchange. Chemistry (Weinheim an der Bergstrasse, Germany) 10, 257266 (2004).

34. A. P. Wiita, S. R. Ainavarapu, Η. H. Huang, J. M. Fernandez, Force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques.

Proc. Natl. Acad. Sci. U. S. A. 103, 7222-7227 (2006).

35. J. Chen, X. Fu, Y. Wang, M. Ling, B. McMullen, J. Kulman, D. W. Chung, J. A. Lopez, Oxidative modification of von Willebrand factor by neutrophil oxidants inhibits its cleavage by ADAMTS13. Blood 115, 706-712 (2010).

36. D. Lillicrap, von Willebrand disease: advances in pathogenetic understanding, diagnosis, and therapy. Hematology I the Education Program of the American Society of

Hematology. American Society of Hematology. Education Program 2013, 254-260 (2013).

37. P. M. Mannucci, C. Kempton, C. Millar, E. Romond, A. Shapiro, I. Birschmann, Μ. V. Ragni, J. C. Gill, T. T. Yee, R. Klamroth, W. Y. Wong, M. Chapman, W. Engl, P. L.

Turecek, T. M. Suiter, B. M. Ewenstein, V. W. F. A. H. S. G. r, Pharmacokinetics and safety of a novel recombinant human von Willebrand factor manufactured with a plasma-free method: a prospective clinical trial. Blood 122, 648-657 (2013).

38. J. W. Wong, P. J. Hogg, Analysis of disulfide bonds in protein structures. J Thromb Haemost 8, 2345 (2010).

39. R. R. Montgomery, T. S. Zimmerman, von Willebrand's disease antigen II. A new plasma and platelet antigen deficient in severe von Willebrand's disease. J. Clin. Invest. 61, 1498-1507 (1978).

40. Y. Jin, T. Manabe, Analysis of PEG-fractionated high-molecular-mass proteins in human plasma by non-denaturing micro 2-DE and MALDI-MS PMF. Electrophoresis

30,3613-3621(2009).

41. T. A. McKinnon, A. C. Chion, A. J. Millington, D. A. Lane, M. A. Laffan, N-linked

1001489620

2016204172 20 Jun 2016 glycosylation of VWF modulates its interaction with ADAMTS13. Blood 111,3042-3049 (2008).

42. T. Ganderton, J. W. Wong, C. Schroeder, P. J. Hogg, Lateral self-association of VWF involves the Cys2431-Cys2453 disulfide/dithiol in the C2 domain. Blood 118,

5312-5318(2011).

43. L. Xie, C. N. Chesterman, P. J. Hogg, Control of von Willebrand factor multimer size by thrombospondin-1. J. Exp. Med. 193, 1341-1349 (2001).

44. J. Chiu, J. W. Wong, P. J. Hogg, Redox regulation of methionine aminopeptidase 2 activity. J. Biol. Chem. 289, 15035-15043 (2014).

45. K. M. Cook, Η. P. McNeil, P. J. Hogg, Allosteric control of betall-tryptase by a redox active disulfide bond. J. Biol. Chem. 288, 34920-34929 (2013).

46. W. S. Nesbitt, F. J. Tovar-Lopez, E. Westein, I. S. Harper, S. P. Jackson, A multimode-TIRFM and microfluidic technique to examine platelet adhesion dynamics. Methods Mol. Biol. 1046, 39-58 (2013).

47. Y. I. Choi, J. S. Duke-Cohan, W. Chen, B. Liu, J. Rossy, T. Tabarin, L. Ju, J.

Gui, K. Gaus, C. Zhu, E. L. Reinherz, Dynamic control of betal integrin adhesion by the plexinDI -sema3E axis. Proc. Natl. Acad. Sci. U. S. A. 111,379-384 (2014).

48. V. F. Fiore, L. Ju, Y. Chen, C. Zhu, T. H. Barker, Dynamic catch of a Thy-1alpha5beta1+syndecan-4 trimolecular complex. Nature communications 5, 4886 (2014).

49. L. Ju, Y. Chen, F. Zhou, H. Lu, M. A. Cruz, C. Zhu, Von Willebrand factor-A1 domain binds platelet glycoprotein Ibalpha in multiple states with distinctive forcedependent dissociation kinetics. Thromb. Res. 136, 606-612 (2015).

50. Y. Chen, B. Liu, L. Ju, J. Hong, Q. Ji, W. Chen, C. Zhu, Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and

Binding-induced Intracellular Signaling on a Single Cell. Journal of visualized experiments : JoVE, e52975 (2015).

51. W. Pos, J. T. Crawley, R. Fijnheer, J. Voorberg, D. A. Lane, B. M. Luken, An autoantibody epitope comprising residues R660, Y661, and Y665 in the ADAMTS13 spacer domain identifies a binding site for the A2 domain of VWF. Blood 115, 164030 1649(2010).

52. S. Zanardelli, J. T. Crawley, C. K. Chion, J. K. Lam, R. J. Preston, D. A. Lane,

1001489620

2016204172 20 Jun 2016

ADAMTS13 substrate recognition of von Willebrand factor A2 domain. J. Biol. Chem. 281, 1555-1563 (2006).

53. Y. Han, J. Xiao, E. Falls, X. L. Zheng, A shear-based assay for assessing plasma ADAMTS13 activity and inhibitors in patients with thrombotic thrombocytopenic purpura. Transfusion (Paris). 51, 1580-1591 (2011).

54. V. Hornak, R. Abel, A. Okur, B. Strockbine, A. Roitberg, C. Simmerling, Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins 65, 712-725 (2006).

55. R. B. Best, G. Hummer, Optimized molecular dynamics force fields applied to the helix-coil transition of polypeptides. The journal of physical chemistry. B 113, 90049015 (2009).

56. K. Lindorff-Larsen, S. Piana, K. Palmo, P. Maragakis, J. L. Klepeis, R. O. Dror, D. E. Shaw, Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins 78, 1950-1958 (2010).

57. D. Van Der Spoel, E. Lindahl, B. Hess, G. Groenhof, A. E. Mark, H. J.

Berendsen, GROMACS: fast, flexible, and free. J Comput Chem 26, 1701 -1718 (2005).

58. B. Hess, C. Kutzner, D. van der Spoel, E. Lindahl, GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput 4, 435-447 (2008).

59. W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W. Impey, M. L. Klein,

Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 79, 926-935 (1983).

60. I. S. Joung, T. E. Cheatham, Determination of alkali and halide monovalent ion parameters for use in explicitly solvated biomolecular simulations. Journal of Physical

Chemistry B 112, 9020-9041 (2008).

61. H. J. C. Berendsen, J. P. M. Postma, W. F. Vangunsteren, A. Dinola, J. R. Haak, Molecular-Dynamics with Coupling to an External Bath. J. Chem. Phys. 81,36843690 (1984).

62. G. Bussi, D. Donadio, M. Parrinello, Canonical sampling through velocity rescaling. J. Chem. Phys. 126, 014101 (2007).

63. M. Parrinello, A. Rahman, Polymorphic Transitions in Single-Crystals - a New

1001489620

2016204172 20 Jun 2016

Molecular-Dynamics Method. J Appl Phys 52, 7182-7190 (1981).

64. T. Darden, D. York, L. Pedersen, Particle Mesh Ewald - an N.Log(N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 98, 10089-10092 (1993).

65. U. Essmann, L. Perera, M. L. Berkowitz, T. Darden, H. Lee, L. G. Pedersen, A 5 Smooth Particle Mesh Ewald Method. J. Chem. Phys. 103, 8577-8593 (1995).

66. B. Hess, H. Bekker, H. J. C. Berendsen, J. G. E. M. Fraaije, LINCS: A linear constraint solver for molecular simulations. J Comput Chem 18, 1463-1472 (1997).

67. K. A. Feenstra, B. Hess, H. J. C. Berendsen, Improving efficiency of large timescale molecular dynamics simulations of hydrogen-rich systems. J Comput Chem 20,

786-798(1999).

68. S. Miyamoto, P. A. «oilman, Settle - an Analytical Version of the Shake and Rattle Algorithm for Rigid Water Models. J Comput Chem 13, 952-962 (1992).

69. A. Amadei, A. B. Linssen, H. J. Berendsen, Essential dynamics of proteins. Proteins 17, 412-425 (1993).

70. L. L. C. Schrodinger, The PyMOL Molecular Graphics System, Version-1,3rl.

(DeLano Scientific, San Carlos, CA, USA, 2002).

71. E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng, T. E. Ferrin, UCSF chimera - A visualization system for exploratory research and analysis. J Comput Chem 25, 1605-1612 (2004).

^0 72. B. I. Costescu, F. Grater, Time-resolved force distribution analysis. BMC

Biophys 6, 5 (2013).

73. D. M. Rothwarf, H. A. Scheraga, Equilibrium and kinetic constants for the thioldisulfide interchange reaction between glutathione and dithiothreitol. Proc. Natl. Acad. Sci. U. S. A. 89, 7944-7948 (1992).

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Claims

1. A recombinant or synthetic peptide having an amino acid sequence of a von Willebrand factor (VWF),

- the amino acid sequence including a VWF A2 region in the form of an amino acid sequence for formation of a VWF domain A2,

- the VWF A2 region including adjacent selenocysteine residues,

- the adjacent selenocysteine residues being positioned in the VWF A2 region to enable the formation of a covalent bond between the selenium atoms of the selenocysteine residues.

2. The peptide of claim 1 wherein the formation of a covalent bond between the selenium atoms of the adjacent selenocysteine residues confers on the peptide a reduced susceptibility to cleavage of the peptide by ADAMTS13.

3. The peptide of any one of the preceding claims wherein the formation of a covalent bond between the selenium atoms of the adjacent selenocysteine residues confers on the peptide an increased affinity for binding of the peptide with glycoprotein lb.

4. The peptide of any one of the preceding claims wherein the peptide has an amino acid sequence at least 90%, preferably 95% identical, preferably identity to the sequence shown in SEQ ID No: 2, provided that the peptide has selenocysteine at position 172 and 173.

5. The peptide of any one of the preceding claims wherein the peptide has an amino acid sequence at least 90%, preferably 95% homologous, preferably identity to the sequence shown in SEQ ID No: 1, provided that the peptide has selenocysteine at position 1669 and 1670.

6. A composition including a peptide of any one of the preceding claims.

7. The composition of claim 6 including a recombinant form of VWF in which adjacent cysteine residues in domain A2 of the VWF are linked by a covalent bond between the sulfur atoms of the adjacent cysteine residues, and/or including a recombinant form of VWF in which adjacent cysteine residues in domain A2 of the VWF are not linked by a covalent bond between the sulfur

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8. The composition of claim 6 including a blood or plasma -derived form of VWF in which adjacent cysteine residues in domain A2 of the VWF are linked by a covalent bond between the sulfur atoms of the adjacent cysteine residues

5 and/or including a blood or plasma -derived form of VWF in which adjacent cysteine residues in domain A2 of the VWF are not linked by a covalent bond between the sulfur atoms of the adjacent cysteine residues.

9. The composition of claim 6 further including cells and/or platelets.

10. The composition of claim 6 further including blood or plasma -derived

0 proteins.

11. A method for increasing the relative abundance of oxidised VWF in a composition including reduced VWF, the method including:

- providing a composition including reduced VWF;

- contacting the composition with an oxidising agent for selectively oxidising

5 adjacent cysteine residues in domain A2 of reduced VWF, thereby forming a covalent bond between the sulfur atoms of the adjacent cysteine residues;

0 thereby increasing the relative abundance of oxidised VWF in the composition.

12. The method of claim 11 wherein the oxidising agent is 4,5-dihydroxy-1,2dithiane (oxoDTT).

13. The method of claim 11 or 12 wherein the relative abundance of oxidised VWF in the composition is increased by at least 10%, preferably 50%,

25 preferably 100% or more.

14. The method of claim 11 or 12 wherein at least 75% of VWF in the composition prior to contact with the oxidising agent is reduced VWF.

15. A composition produced by the method of claim 11.

16. A method for increasing the relative abundance of oxidised VWF in a

30 composition including reduced VWF, the method including:

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- providing a composition including reduced VWF;

- adding a composition of claim 15 or a peptide of claim 1 to the composition;

wherein the addition of the composition of claim 15 or peptide of claim 1 increases the relative abundance of oxidised VWF in the composition.

5 17. A method for determining the potential of a VWF composition to induce the formation of a thrombus in an individual, the method including:

- providing a VWF composition;

- measuring the relative abundance of oxidised VWF in the composition, the oxidised VWF being a form of VWF in which adjacent cysteine residues in the A2

0 domain of VWF are linked by a covalent bond between sulfur atoms of the adjacent cysteine resides

- determining that the VWF composition has a high likelihood for inducing formation of a thrombus in the individual where the amount of oxidised VWF in the composition is measured to be greater than about 25% of the total amount of

5 VWF in the composition.

18. A method for treating an individual for von Willebrand disease (VWD), the method including the following steps:

- providing an individual having VWD;

- administering a peptide or composition of any one of the preceding claims to ^0 the individual, thereby treating the individual for VWD.

19. A recombinant or synthetic peptide having an amino acid sequence of a von Willebrand factor (VWF),

25 - the VWF A2 region being devoid of adjacent cysteine residues or adjacent selenocysteine residues, a residue of the region thereby being incapable of forming a covalent bond with another residue in the region.

20. The peptide of claim 19 wherein the absence of formation of a covalent bond between residues of the region confers on the peptide an increased

30 susceptibility to cleavage of the peptide by ADAMTS13.

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21 .The peptide of claim 19 or 20 wherein the absence of formation of a covalent bond between residues of the region confers on the peptide an decreased affinity for binding of the peptide with glycoprotein lb.

22. The peptide of claim 19 Or 20 wherein the peptide has an amino acid

5 sequence at least 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 2, provided that the peptide has a residue other than cysteine or selenocysteine at position 172.

23. The peptide of claim 22 wherein the peptide has glycine, alanine or serine at position 172.

0 24. The peptide of claim 22 wherein the peptide may have a cysteine or selenocysteine at position 173.

25. .The peptide of claim 19 wherein the peptide has an amino acid sequence at least 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 1, provided that the peptide has a residue other than cysteine

5 or selenocysteine at position 173.

26. The peptide of claim 25 wherein the peptide has glycine, alanine or serine at position 173.

27. The peptide of claim 25 wherein the peptide may have cysteine or selenocysteine at position 172.

0 28. The peptide of claim 19 wherein the peptide has an amino acid sequence at least 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 1, provided that the peptide has an amino acid other than cysteine and selenocysteine at position 1669 or at position 1670.

29. A composition including a peptide of claim 19.

25 30. The composition of claim 29 including a recombinant form of VWF in which adjacent cysteine residues in domain A2 of the VWF are linked by a covalent bond between the sulfur atoms of the adjacent cysteine residues, and/or including a recombinant form of VWF in which adjacent cysteine residues in domain A2 of the VWF are not linked by a covalent bond between the sulfur

30 atoms of the adjacent cysteine residues.

31. The composition of claim 29 including a blood or plasma -derived form of

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VWF in which adjacent cysteine residues in domain A2 of the VWF are linked by a covalent bond between the sulfur atoms of the adjacent cysteine residues and/or a blood or plasma -derived form of VWF in which adjacent cysteine residues in domain A2 of the VWF are not linked by a covalent bond 5 between the sulfur atoms of the adjacent cysteine residues.

32. The composition of claim 29 further including cells and/or platelets.

33. The composition of claim 29 further including blood or plasma derived proteins.

34. A method for increasing the relative abundance of a reduced VWF in a

0 composition including oxidised VWF, the method including:

- providing a composition including oxidised VWF;

- contacting the composition with an reducing agent in conditions enabling selective reduction of adjacent cysteine residues in domain A2 of oxidised VWF, thereby breaking a covalent bond between the sulfur atoms of the adjacent

5 cysteine residues in oxidised VWF;

thereby increasing the relative abundance of reduced VWF in the composition.

0 35. The method of claim 34 wherein the reducing agent is selected from the group consisting of dithiothreitol (DTT), 2 mercapto-ethanol and an oxidoreductase.

36. The method of claim 34 wherein the relative abundance of reduced VWF in the composition is increased by at least 10%, preferably 50%, preferably

25 100% or more.

37. The method of claim 34 wherein at least 25% of VWF in the composition prior to contact with the reducing agent is oxidised VWF.

38. A composition produced by the method of claim 34.

39. A method for increasing the relative abundance of reduced VWF in a

30 composition including oxidised VWF, the method including:

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- providing a composition including oxidised VWF;

- adding a composition of claim 38 or a peptide of claim 19 to the composition;

wherein the addition of the composition of claim 38 or peptide of claim 19 increases the relative abundance of reduced VWF in the composition.

5 40. A method for treating for or preventing an individual from developing AVWS, the method including the following steps:

- providing an individual having, or at risk of developing AVWS;

- administering a peptide or composition of any one of claims 19 to 38 to the individual, thereby treating the individual for AVWS, or preventing the individual

0 from developing AVWS.

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Figure 1

Cys1669-Cys1670

1/20

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Figure 2

SH ! 1¾

T and healthy donors

SH :PA

-/,12

35 40' 45 ,50' ,55 ft0' <65: 7Q. 75;

fraction number

35' 40 45 SCI 55 Η11 l< 5 7:0 ?5 fraction number

2/20

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Figure 3

A wt^:WVF reduced (mutant) B WA

I SO dyn/cm²

D E F

3/20

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-_Ί VWF cleavage ^J products

VWF, pg/mL

4/20

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Figure 5

5/20

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Figure 6 A _ 1.0 o 0.8 <: M t3 heart failure LVAD ECMO :- .1.0 ca 5 ΐ 0.8.

Ϊ5

0.5 >

:T3

S-0.2

CD ΰ,.ο * ril ........

β ^: +cxidised Tnc A -«educed Tra no . 32 cfj-'n/arr · shear stress shear stress heart failure LW ECMO

6/20

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Figure 7

SEQ ID No: 1

10 20 30 40 50 MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM 60 70 80 90 100 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG 110 120 130 140 150 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL 160 170 180 190 200 SDRYFNKTCG LCGNFNIFAE DDEMTQEGTL TSDPYDFANS WALSSGEQWC 210 220 230 240 250 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 260 270 280 290 300 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 310 320 330 340 350 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 360 370 380 390 400 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 410 420 430 440 450 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 460 470 480 490 500 LHNSLVKLKH GAGVAMDGQD VQLPLLKGDL RIQHTVTASV RLSYGEDLQM 510 520 530 540 550 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 560 570 580 590 600 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS

7/20

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610 620 630 640 650 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 660 670 680 690 700 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 710 720 730 740 750 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGEMHCTM SGVPGSLLPD 760 770 780 790 800 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 810 820 830 840 850 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 860 870 880 890 900 CQDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS 910 920 930 940 950 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE 960 970 980 990 1000 THFEWESGR YIILLLGKAL SWWDRHLSI SWLKQTYQE KVCGLCGNFD 1010 1020 1030 1040 1050 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1060 1070 1080 1090 1100 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCACF 1110 1120 1130 1140 1150 CDTIAAYAHV CAQHGKWTW RTATLCPQSC EERNLRENGY ECEWRYNSCA 1160 1170 1180 1190 1200 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1210 1220 1230 1240 1250 VAGRRFASGK KVTLNPSDPE HCQICHCDW NLTCEACQEP GGLWPPTDA 1260 1270 1280 1290 1300

8/20

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PVSPTTLYVE DISEPPLHDF YCSRLLDLVF LLDGSSRLSE AEFEVLKAFV 1310 1320 1330 1340 1350 VDMMERLRIS QKWVRVAWE YHDGSHAYIG LKDRKRPSEL RRIASQVKYA 1360 1370 1380 1390 1400 GSQVASTSEV LKYTLFQIFS KIDRPEASRI TLLLMASQEP QRMSRNFVRY 1410 1420 1430 1440 1450 VQGLKKKKVI VIPVGIGPHA NLKQIRLIEK QAPENKAFVL SSVDELEQQR 1460 1470 1480 1490 1500 DEIVSYLCDL APEAPPPTLP PDMAQVTVGP GLLGVSTLGP KRNSMVLDVA 1510 1520 1530 1540 1550 FVLEGSDKIG EADFNRSKEF MEEVIQRMDV GQDSIHVTVL QYSYMVTVEY 1560 1570 1580 1590 1600 PFSEAQSKGD ILQRVREIRY QGGNRTNTGL ALRYLSDHSF LVSQGDREQA 1610 1620 1630 1640 1650 PNLVYMVTGN PASDEIKRLP GDIQWPIGV GPNANVQELE RIGWPNAPIL 1660 1670 1680 1690 1700 IQDFETLPRE APDLVLQRCC SGEGLQIPTL SPAPDCSQPL DVILLLDGSS 1710 1720 1730 1740 1750 SFPASYFDEM KSFAKAFISK ANIGPRLTQV SVLQYGSITT IDVPWNWPE 1760 1770 1780 1790 1800 KAHLLSLVDV MQREGGPSQI GDALGFAVRY LTSEMHGARP GASKAWILV 1810 1820 1830 1840 1850 TDVSVDSVDA AADAARSNRV TVFPIGIGDR YDAAQLRILA GPAGDSNWK 1860 1870 1880 1890 1900 LQRIEDLPTM VTLGNSFLHK LCSGFVRICM DEDGNEKRPG DVWTLPDQCH 1910 1920 1930 1940 1950 TVTCQPDGQT LLKSHRVNCD RGLRPSCPNS QSPVKVEETC GCRWTCPCVC

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1960 1970 1980 1990 2000 TGSSTRHIVT FDGQNFKLTG SCSYVLFQNK EQDLEVILHN GACSPGARQG 2010 2020 2030 2040 2050 CMKSIEVKHS ALSVELHSDM EVTVNGRLVS VPYVGGNMEV NVYGAIMHEV 2060 2070 2080 2090 2100 RFNHLGHIFT FTPQNNEFQL QLSPKTFASK TYGLCGICDE NGANDEMLRD 2110 2120 2130 2140 2150 GTVTTDWKTL VQEWTVQRPG QTCQPILEEQ CLVPDSSHCQ VLLLPLFAEC 2160 2170 2180 2190 2200 HKVLAPATFY AICQQDSCHQ EQVCEVIASY AHLCRTNGVC VDWRTPDFCA 2210 2220 2230 2240 2250 MSCPPSLVYN HCEHGCPRHC DGNVSSCGDH PSEGCFCPPD KVMLEGSCVP 2260 2270 2280 2290 2300 EEACTQCIGE DGVQHQFLEA WVPDHQPCQI CTCLSGRKVN CTTQPCPTAK 2310 2320 2330 2340 2350 APTCGLCEVA RLRQNADQCC PEYECVCDPV SCDLPPVPHC ERGLQPTLTN 2360 2370 2380 2390 2400 PGECRPNFTC ACRKEECKRV SPPSCPPHRL PTLRKTQCCD EYECACNCVN 2410 2420 2430 2440 2450 STVSCPLGYL ASTATNDCGC TTTTCLPDKV CVHRSTIYPV GQFWEEGCDV 2460 2470 2480 2490 2500 CTCTDMEDAV MGLRVAQCSQ KPCEDSCRSG FTYVLHEGEC CGRCLPSACE 2510 2520 2530 2540 2550 WTGSPRGDS QSSWKSVGSQ WASPENPCLI NECVRVKEEV FIQQRNVSCP 2560 2570 2580 2590 2600 QLEVPVCPSG FQLSCKTSAC CPSCRCERME ACMLNGTVIG PGKTVMIDVC 2610 2620 2630 2640 2650

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TTCRCMVQVG VISGFKLECR KTTCNPCPLG YKEENNTGEC CGRCLPTACT 2660 2670 2680 2690 2700 IQLRGGQIMT LKRDETLQDG CDTHFCKVNE RGEYFWEKRV TGCPPFDEHK 2710 2720 2730 2740 2750 CLAEGGKIMK IPGTCCDTCE EPECNDITAR LQYVKVGSCK SEVEVDIHYC 2760 2770 2780 2790 2800 QGKCASKAMY SIDINDVQDQ CSCCSPTRTE PMQVALHCTN GSWYHEVLN

2810

AMECKCSPRK CSK

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Figure 8

SEQ ID No: 2

DVA

13 23 33 43 53 FVLEGSDKIG EADFNRSKEF MEEVIQRMDV GQDSIHVTVL QYSYMVTVEY 63 73 83 93 103 PFSEAQSKGD ILQRVREIRY QGGNRTNTGL ALRYLSDHSF LVSQGDREQA 113 123 133 143 153 PNLVYMVTGN PASDEIKRLP GDIQWPIGV GPNANVQELE RIGWPNAPIL 163 173 183 193 203

IQDFETLPRE APDLVLQRCC

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Figure 9

SEQ ID No:3

10 20 30 40 50 MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM 60 70 80 90 100 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG 110 120 130 140 150 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL 160 170 180 190 200 SDRYFNKTCG LCGNFNIFAE DDEMTQEGTL TSDPYDFANS WALSSGEQWC 210 220 230 240 250 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 260 270 280 290 300 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 310 320 330 340 350 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 360 370 380 390 400 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 410 420 430 440 450 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 460 470 480 490 500 LHNSLVKLKH GAGVAMDGQD VQLPLLKGDL RIQHTVTASV RLSYGEDLQM 510 520 530 540 550 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 560 570 580 590 600

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NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 610 620 630 640 650 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 660 670 680 690 700 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 710 720 730 740 750 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGEMHCTM SGVPGSLLPD 760 770 780 790 800 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 810 820 830 840 850 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 860 870 880 890 900 CQDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS 910 920 930 940 950 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE 960 970 980 990 1000 THFEWESGR YIILLLGKAL SWWDRHLSI SWLKQTYQE KVCGLCGNFD 1010 1020 1030 1040 1050 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1060 1070 1080 1090 1100 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCACF 1110 1120 1130 1140 1150 CDTIAAYAHV CAQHGKWTW RTATLCPQSC EERNLRENGY ECEWRYNSCA 1160 1170 1180 1190 1200 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1210 1220 1230 1240 1250 VAGRRFASGK KVTLNPSDPE HCQICHCDW NLTCEACQEP GGLWPPTDA

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1260 1270 1280 1290 1300 PVSPTTLYVE DISEPPLHDF YCSRLLDLVF LLDGSSRLSE AEFEVLKAFV 1310 1320 1330 1340 1350 VDMMERLRIS QKWVRVAWE YHDGSHAYIG LKDRKRPSEL RRIASQVKYA 1360 1370 1380 1390 1400 GSQVASTSEV LKYTLFQIFS KIDRPEASRI TLLLMASQEP QRMSRNFVRY 1410 1420 1430 1440 1450 VQGLKKKKVI VIPVGIGPHA NLKQIRLIEK QAPENKAFVL SSVDELEQQR 1460 1470 1480 1490 1500 DEIVSYLCDL APEAPPPTLP PDMAQVTVGP GLLGVSTLGP KRNSMVLDVA 1510 1520 1530 1540 1550 FVLEGSDKIG EADFNRSKEF MEEVIQRMDV GQDSIHVTVL QYSYMVTVEY 1560 1570 1580 1590 1600 PFSEAQSKGD ILQRVREIRY QGGNRTNTGL ALRYLSDHSF LVSQGDREQA 1610 1620 1630 1640 1650 PNLVYMVTGN PASDEIKRLP GDIQWPIGV GPNANVQELE RIGWPNAPIL 1660 1670 1680 1690 1700 IQDFETLPRE APDLVLQRGG SGEGLQIPTL SPAPDCSQPL DVILLLDGSS 1710 1720 1730 1740 1750 SFPASYFDEM KSFAKAFISK ANIGPRLTQV SVLQYGSITT IDVPWNWPE 1760 1770 1780 1790 1800 KAHLLSLVDV MQREGGPSQI GDALGFAVRY LTSEMHGARP GASKAWILV 1810 1820 1830 1840 1850 TDVSVDSVDA AADAARSNRV TVFPIGIGDR YDAAQLRILA GPAGDSNWK 1860 1870 1880 1890 1900 LQRIEDLPTM VTLGNSFLHK LCSGFVRICM DEDGNEKRPG DVWTLPDQCH 1910 1920 1930 1940 1950

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TVTCQPDGQT LLKSHRVNCD RGLRPSCPNS QSPVKVEETC GCRWTCPCVC 1960 1970 1980 1990 2000 TGSSTRHIVT FDGQNFKLTG SCSYVLFQNK EQDLEVILHN GACSPGARQG 2010 2020 2030 2040 2050 CMKSIEVKHS ALSVELHSDM EVTVNGRLVS VPYVGGNMEV NVYGAIMHEV 2060 2070 2080 2090 2100 RFNHLGHIFT FTPQNNEFQL QLSPKTFASK TYGLCGICDE NGANDEMLRD 2110 2120 2130 2140 2150 GTVTTDWKTL VQEWTVQRPG QTCQPILEEQ CLVPDSSHCQ VLLLPLFAEC 2160 2170 2180 2190 2200 HKVLAPATFY AICQQDSCHQ EQVCEVIASY AHLCRTNGVC VDWRTPDFCA 2210 2220 2230 2240 2250 MSCPPSLVYN HCEHGCPRHC DGNVSSCGDH PSEGCFCPPD KVMLEGSCVP 2260 2270 2280 2290 2300 EEACTQCIGE DGVQHQFLEA WVPDHQPCQI CTCLSGRKVN CTTQPCPTAK 2310 2320 2330 2340 2350 APTCGLCEVA RLRQNADQCC PEYECVCDPV SCDLPPVPHC ERGLQPTLTN 2360 2370 2380 2390 2400 PGECRPNFTC ACRKEECKRV SPPSCPPHRL PTLRKTQCCD EYECACNCVN 2410 2420 2430 2440 2450 STVSCPLGYL ASTATNDCGC TTTTCLPDKV CVHRSTIYPV GQFWEEGCDV 2460 2470 2480 2490 2500 CTCTDMEDAV MGLRVAQCSQ KPCEDSCRSG FTYVLHEGEC CGRCLPSACE 2510 2520 2530 2540 2550 WTGSPRGDS QSSWKSVGSQ WASPENPCLI NECVRVKEEV FIQQRNVSCP 2560 2570 2580 2590 2600 QLEVPVCPSG FQLSCKTSAC CPSCRCERME ACMLNGTVIG PGKTVMIDVC

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2610 2620 2630 2640 2650 TTCRCMVQVG VISGFKLECR KTTCNPCPLG YKEENNTGEC CGRCLPTACT 2660 2670 2680 2690 2700 IQLRGGQIMT LKRDETLQDG CDTHFCKVNE RGEYFWEKRV TGCPPFDEHK 2710 2720 2730 2740 2750 CLAEGGKIMK IPGTCCDTCE EPECNDITAR LQYVKVGSCK SEVEVDIHYC 2760 2770 2780 2790 2800 QGKCASKAMY SIDINDVQDQ CSCCSPTRTE PMQVALHCTN GSWYHEVLN

2810

AMECKCSPRK CSK

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Figure S1

11536644937ws VWF Iff cleaved WVF

N N N N NN

L'CRC' C i' lit

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A B

Figure S2 electrophoresis

Figure S3

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Figure S4

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