Interaction Of Von Willebrand Factor Research Articles

Redundant Mechanism of Platelet Adhesion to Laminin and Collagen under Flow: INVOLVEMENT OF VON WILLEBRAND FACTOR AND GLYCOPROTEIN Ib-IX-V

Although the role of collagen in thrombosis has been extensively investigated, the contribution of other extracellular matrices is still unclear. We have recently reported that laminin stimulates platelet spreading through integrin alpha(6)beta(1)-dependent activation of the collagen receptor glycoprotein (GP) VI under static condition. Under physiological high and low shear conditions, platelets adhered to laminin, and this was strongly inhibited by an antibody that blocks association between GPIb-IX-V and von Willebrand factor (VWF). Moreover, platelets of type III von Willebrand disease or Bernard-Soulier syndrome adhered to laminin at a low shear condition but not at a high shear condition. The specific binding of laminin to VWF was confirmed by surface plasmin resonance spectroscopy (BIAcore). These findings suggest that laminin supports platelet adhesion depending on the interaction of VWF and GPIb-IX-V under pathophysiological high shear flow. This mechanism is similar to that of collagen. We propose that integrins, GPVI, GPIb-IX-V, and VWF represent a general paradigm for the interaction between platelets and subendothelial matrices.

Microvascular Injury, Thrombosis, Inflammation, and Apoptosis in the Pathogenesis of Heatstroke

Severe heatstroke is a leading cause of morbidity and mortality during heat waves. The pathogenesis of tissue injury, organ failure, and death in heatstroke is not well understood. We investigated the pathways of heatstroke-induced tissue injury and cell death in anesthetized baboons (Papio hamadyras) subjected to environmental heat stress until core temperature attained 42.5 degrees C (moderate heatstroke; n = 3) or onset of severe heatstroke (n = 4) signaled by a fall in systolic blood pressure to < 90 mm Hg and rise in core temperature to 43.1+/-0.1 degrees C. Three sham-heated animals served as controls. Light and electron microscopy revealed widespread hemorrhage and thrombosis, transmural migration of leukocytes, and microvascular endothelium injury in severe heatstroke. Immunohistology and ultrastructural analysis demonstrated increased staining of endothelial von Willebrand factor (vWF), tissue factor (TF), and endothelial leukocyte-platelet interaction. Extensive apoptosis was noted in spleen, gut, and lung, and in hematopoeitic cells populating these organs. Double-labeling studies colocalized active caspase-3 and TF with apoptotic cells. Findings in sham-heated animals were unremarkable. These data suggested that microvascular injury, thrombosis, inflammation, and apoptosis may play an important role in the pathogenesis of heatstroke injury.

Identification of amino acid residues responsible for von Willebrand factor binding to sulfatide by charged-to-alanine-scanning mutagenesis

von Willebrand factor (VWF) performs its hemostatic functions through binding to various proteins. The A1 domain of VWF contains binding sites of not only physiologically important ligands, but also exogenous modulators that induce VWF-platelet aggregation. Sulfatides, 3-sulfated galactosyl ceramides, that are expressed on oligodendrocytes, renal tubular cells, certain tumor cells and platelets, have been suggested to interact with VWF under some pathological conditions. The binding of VWF to sulfatide requires the A1 domain, but its binding sites have not been precisely identified. Here, we report that alanine mutations at Arg1392, Arg1395, Arg1399 and Lys1423 led to decreased VWF–sulfatide binding. These sites have been reported to be the binding sites for platelet membrane glycoprotein (GP) Ib and/or snake venom botrocetin, and, interestingly, are identical to the monoclonal antibody (mAb) NMC4 epitope previously reported to inhibit the VWF-GPIb interaction. We observed that NMC4 also inhibited VWF interaction with sulfatides in a dose-dependent manner. Thus, we conclude that VWF binding sites of sulfatide overlap those of platelet GPIb and botrocetin.

Role of erythrocytes in platelet activation during exposure to pathophysiological shear stress

Interaction of von Willebrand factor (VWF) with circulating blood platelets is the initial trigger for thrombosis in an arterial stenosis. Exposure of blood to a high shear stress promotes such interaction. These events have previously been studied in vitro under conditions where the platelets adhere to a VWF‐coated surface. Our approach assesses platelet activation in the absence of adhesion. Whole blood is perfused through polyetheretherketone (PEEK) tubing that includes an artificial stenosis, comprising a short (4 cm) section of narrow‐bore (130 μm) tubing. The pressure differential across the stenosis was measured with a pressure transducer. Platelet activation was evaluated from secretion of [14C]serotonin, and erythrocyte lysis from release of hemoglobin. The pressure drop across the stenosis matched the predictions for laminar flow without turbulence. Platelet activation rose progressively with increasing perfusion rate (2–12 ml/min). We examined the role of erythrocytes in platelet activation by adjusting the hematocrit of the perfused blood. Platelet activation was minimal (mean increment of 1.9% in serotonin secretion at 12 ml/min flow) when the hematocrit was low (30%), but substantial (increment of 15.5%) with a high hematocrit (60%). In contrast, changes in the platelet count did not affect the extent of platelet activation. [Supported by the National Science Foundation.]

A New Binding Assay of von Willebrand Factor and Glycoprotein Ib Using Solid-Phase Biotinylated Platelets

To obtain compounds that inhibit the interaction of von Willebrand factor (vWF) and glycoprotein (GP) Ib, a novel binding assay was established. The binding of fixed platelets to vWF-R497 mutant was quantified by a solid phase assay. In this assay, fixed platelets bound to the vWF-R497 mutant, carrying the deletion of Glu497-Tyr508 and the missense mutation of Arg545 to Ala, without binding modulators such as ristocetin. The Kd value of the binding was 2.8 nM, which was consistent with the result from liquid binding assay. The binding was inhibited by aurin tricarboxylic acid (ATA) and an anti GPIb antibody, AK2. Using this binding assay, we screened our library compounds and obtained D74-3736. This compound also inhibited ristocetin-induced platelet aggregation in the human platelet-rich plasma.

Diagnosis and treatment of von Willebrand disease: new perspectives and nuances

In 1926, Erik von Willebrand, a Finnish internist andacademic, evaluated and described a 5-year-old girlwith extreme bleeding and bruising due to what hadbeen designated the A˚landic haemorrhagic diseaseby inhabitants of this archipelago island in the Gulfof Bothnia. Hjordis, the propositus, was the ninth of12 children born to a pedigree in which four femalesiblings had already died before the age of 4 withuncontrolled haemorrhage and in which 23 of 66family members, predominantly females, had expe-rienced significant bleeding and bruising complica-tions [1]. In fact, Hjordis herself eventually died atthe age of 13 during her fourth menstrual cycle.Professor von Willebrand mistakenly concluded thatthis bleeding diathesis was an unusual form ofhaemophilia and decided to label the new diseaseas pseudo-haemophilia to differentiate it from thesex-linked recessive haemophilia A. Little did vonWillebrand realize that this disease, eventually tobecomeeponymous[vonWillebranddisease(VWD)],would become the most commonly diagnosed con-genital bleeding disorder, with a prevalence rangingbetween 1 per 10 000 individuals to 1.3% [2]. Type1 VWD is the most common subtype, representingup to 75–80% of all cases while the subtype 3 VWDoccurs in approximately 1 per million population inthe United States and Europe [2]. Determination ofthe exact prevalence of VWD is hindered somewhatby the heterogeneity of clinical and diagnosticlaboratory features. Even Professor von Willebrandappreciated the challenges of diagnosing VWD as hecollaborated with Professor Rudolf Ju¨gens at theBerlin University to examine patients blood sampleswith a newly invented kapilla¨rtrombometer appa-ratus [3]. Although they were technically mistakenwhen they attributed the bleeding manifestations ofVWD to a platelet defect, their observations wereremarkably prescient since it was not until early1970s that the existence of a specific von Willebrandfactor (VWF) glycoprotein separate from factor VIIIwas finally appreciated and demonstrated to supportplatelet adhesion to the subendothelial matrix ofdamaged blood vessels.Over the last 81 years since von Willebranddescribed the bleeding disorder, there have beennumerous attempts and approaches to refine thediagnosis of the disease so that appropriate andefficacious treatment can be delivered. The clinicalphenotype of VWD includes, in part, information onwhether bleeding is spontaneous or related to surgi-cal or physical trauma; family history and inheri-tance pattern; menstrual history in women; age ofonset of bleeding (to distinguish between acquired vs.inherited coagulation disorders); and sites of bleed-ing(mucocutaneousvs.visceral,intra-articular,intra-muscular, or soft tissue locations). Subsequentlaboratory testing is necessary to exclude or confirmthe diagnosis and to further classify the subtype ofVWD. This article will focus on selected vagariesassociated with the ability to utilize clinical pheno-typic information gathered through the history andphysical examination to predict the presence ofVWD. The clinical diagnosis of VWD must be based,however, on more than physician suspicion andintuition. Thus, the need for confirmatory testing inthe laboratory. The frailties of conventional labora-tory testing for diagnosing VWD and the use of anin vitro surrogate of the bleeding time will also be

A Small Fragment of ADAMTS13 Enhances Cleavage of von Willebrand Factor by the Intact Enzyme.

ADAMTS13 regulates hemostasis by cleaving von Willebrand Factor (VWF) to smaller less thrombogenic fragments. Absence of ADAMTS13 activity leads to thrombotic thrombocytopenic purpura, an often fatal disease that is characterized by microangiopathic hemolytic anemia, thrombocytopenia and microvascular thrombosis especially in renal and central nervous system vasculature. ADAMTS13 like other ADAMTS family members has a characteristic domain structure consisting of a metalloprotease domain, a disintegrin domain, a thrombospondin type 1 repeat (TSR), a cysteine-rich domain, a spacer domain, 7 additional TSRs and 2 carboxyl terminal CUB domains. Regions in the spacer and C-terminal thrombospondin type 1 repeats (TSR) and CUB domains have been shown previously to be important for the interaction of ADAMTS13 and VWF. To further characterize the interaction of ADAMTS13 and VWF, we generated a construct consisting of the spacer domain and the second TSR of ADAMTS13. This construct is called ST2 for Spacer and Tsr2. Although many constructs of ADAMTS13 fail to be secreted because of poor folding, ST2 is folded since it is secreted from human embryonic kidney 293 cells. Previous work has shown that the spacer domain alone can bind VWF under static conditions, therefore ST2 was included in VWF cleaving assays. Unexpectedly, ST2 was found to enhance the cleavage of VWF by ADAMTS13 by approximately 10-fold in assays using either guanidine or urea-denatured VWF. To understand how ST2 enhanced the cleavage of VWF by ADAMTS13, interactions of ST2 with ADAMTS13 and VWF were studied. ST2 was found to specifically co-immunoprecipitate with VWF but not with ADAMTS13. A Kd calculated from these co-immunoprecipitation experiments was 8.7 nM and the stoichiometry of binding was approximately 1 mole/mole. One explanation for the enhanced cleavage of VWF by ADAMTS13 in the presence of ST2 is that ST2 may induce a conformational change in VWF that makes the cleavage site more accessible. To test this hypothesis, VWF in the presence and absence of ST2 was subjected to trypsin digestion at different concentrations of trypsin. Differential cleavage of VWF was seen in the presence and absence of ST2 at all concentrations of trypsin tested. This is consistent with induction of a conformational change in VWF by ST2. These studies provide additional information on the structure-function relationship of these two key proteins in the control of hemostasis.

Functional Assay of VWF Measured by Its Interaction with Platelet GPIb without the Use of Ristocetin.

The clinical laboratory assessment of von Willebrand factor (VWF) includes measurement of the amount of VWF protein (VWF:Ag) and the functional interaction of VWF with platelets. The universally accepted measure of VWF function utilizes the ability of VWF to bind to platelets (or platelet GPIb) in the presence of ristocetin resulting in VWF-dependent agglutination of platelets measured quantitatively as ristocetin cofactor activity (VWF:RCo) by platelet aggregometry or turbidometry. An international reference standard has been assigned a biologic activity in international units by the WHO and the SSC of the ISTH. VWF:RCo has a recognized wide variability and reproducibility using different assays or when performed in different clinical laboratories. Alternative assays with improved reproducibility have recently been suggested that use recombinant GPIb in an ELISA assay of VWF binding, but this binding still requires ristocetin. Patients have been reported that have reduced interaction with ristocetin such that the VWF:RCo is markedly reduced (<0.12 IU/dL) yet patients have no bleeding symptoms even with major surgical challenge. Furthermore, recent studies done on normals entered into the ZPMCB-VWD have demonstrated a systematic problem with the measurement of VWF:RCo in African Americans in whom there VWF:RCo is significantly reduced compared with their VWF:Ag. Since this difference does not appear to have a clinical phenotype, a functional assay of VWF in the absence of ristocetin might be desirable. Such an assay should be sensitive to the measurement of the more-functional large VWF multimers, correlate with VWF:Ag in patients with reduced VWF function, and remain unaffected by mutations that affect VWF binding of ristocetin but do not have a bleeding phenotype. Since, platelet-type VWD variants of GPIb bind normal plasma VWF in the absence of added ristocetin and preferentially bind large VWF multimers, we explored an assay using combinations of the three GPIb mutations, G233V, D235Y, M239V, either individually or together to measure VWF function in the absence of ristocetin. Initially cell-based assays using mutant GPIB expressed in HEK293T cells were used in flow cytometry assays in which test VWF binding to mutant GPIb was measured using a labeled anti-VWF monoclonal antibody. Results were expressed in VWF:IbCo units. A normal curve was developed using serial dilutions of reference plasma previously standardized against both the ISTH and WHO VWF standards based on the VWF:Ag international standard. Patient samples included 41 normal, 16 type 2M, 5 type 2B, and 5 type 2A VWD patient plasmas. Samples included those with apparent type 2M VWD but without clinical symptoms and African American samples with a reduced VWF:RCo/VWF:Ag (RCo/Ag) ratio. Of the 16 type 2M VWD samples, 7 had markedly reduced VWF:IbCo (consistent with the VWF:RCo assay) and 9 had normal VWF:IbCo. African Americans with the SNPs associated with reduced RCo/Ag ratios had VWF:IbCo assays that correlated with their VWF:Ag in contrast to the abnormal RCo/Ag ratios identified by standard assays. Type 2A patients exhibited reduced VWF:IbCo assays and multimer size seemed to correlate with VWF:IbCo activity. Thus, measurement of VWF function using the VWF:IbCo assay may more directly correlate with VWF function and avoid some of the pitfalls and functional variability of VWF:RCo assays.

Evidence from limited proteolysis of a ristocetin-induced conformational change in human von Willebrand factor that promotes its binding to platelet glycoprotein Ib-IX-V

von Willebrand factor (VWF) does not normally interact with platelets in the bloodstream. Binding to exposed vascular subendothelium, however, enables VWF to interact with the platelet glycoprotein Ib-IX-V complex (GP Ib-IX-V). This change in function may reflect a change in its conformation. Ristocetin also promotes interaction of VWF with GP Ib-IX-V; it thus provides a model for changes in VWF conformation and function that may occur in vivo. The fluid-phase conformation of VWF was evaluated from its susceptibility to proteolytic digestion. Ristocetin markedly altered the pattern of VWF digestion by trypsin, increasing the prevalence of two major proteolytic fragments (109 and 160 kDa), and decreasing that of four fragments (130, 145, 181 and 199 kDa). Vancomycin, a structurally related antibiotic, did not affect the digestion pattern. However, it partially reversed the ristocetin-induced change in digestion. Changes in prevalence of five of the tryptic fragments of VWF with ristocetin and vancomycin correlated closely with changes in VWF binding to GP Ib-IX-V. Heparin also partially inhibited the ristocetin-induced changes in tryptic digestion of VWF. These observations suggest that ristocetin may modulate VWF conformation in such a way as to expose its GP Ib-binding domain and enable it to interact with the platelet. Such modulation also exposes a cryptic site (or sites) for proteolytic cleavage by trypsin.

Von Willebrand Factor, von Willebrand Factor-Cleaving Protease, and Shear Stress

von Willebrand factor (VWF) is a multimeric plasma glycoprotein (GP) involved in platelet adhesion at the site of vascular damage, which acts as a bridge between the injured subendothelium and the platelet receptors. The multimeric structure of VWF allows it to support multiple interactions with platelets and endothelial components under high shear stress. Rapid flow conditions induce a conformational transition of the VWF molecule, thus allowing its functional binding domains to be exposed. A specific VWF-cleaving protease (ADAMTS-13) physiologically down regulates the multimeric size of newly released and circulating VWF in order to prevent unwanted platelet thrombus formation. The occurrence of microvascular platelet aggregation in thrombotic thrombocytopenic purpura, which is caused by an ADAMTS-13 deficiency, well-demonstrates the important role of the protease in regulating the adhesive activity of VWF. Better knowledge of VWF function would contribute to the development of novel anti-thrombotic strategies based on the selective inhibition of the VWF interaction with platelet receptors and endothelial components in areas of the circulation characterised by elevated fluid dynamic forces.

Characterization of the interaction between von Willebrand factor and osteoprotegerin.

Osteoprotegerin (OPG), a member of the tumor necrosis-factor receptor superfamily, plays an important role in bone remodeling and is also involved in vascular diseases. OPG is physically associated with von Willebrand factor (VWF), a glycoprotein involved in primary hemostasis, within the Weibel-Palade bodies (WPBs) of endothelial cells and in plasma. The present study aimed to elucidate the molecular mechanisms underlying the interaction between OPG and VWF. In a solid-phase binding assay, VWF was able to bind specifically to OPG in a calcium-dependent manner. This interaction displayed strong pH dependence with optimal binding occurring at pH 6.5 and was severely impaired by chloride-ion concentrations above 40 mm. Using a series of purified VWF derivatives the functional site that supports VWF interaction with OPG was localized on its Al domain. Fluorescence microscopy on human umbilical vein endothelial cells showed co-localization of VWF and OPG in WPBs. When secretion was induced, OPG remained associated with VWF in extracellular patches of release under biochemical conditions found in blood plasma. Our observations demonstrate the existence of an interactive site for OPG within the VWF A1-domain. This study established that the optimal biochemical parameters allowing a complex formation between VWF and OPG are those thought to prevail in the trans-Golgi network. These conditions would allow VWF to act as a cargo targeting OPG to WPBs. Finally, blood environments appear suitable to preserve the complex, which may participate in vascular injury, arterial calcification and inflammation.

A mechanism to safeguard platelet adhesion under high‐shear flow: von Willebrand factor‐glycoprotein Ib and integrin α2β1‐collagen interactions make complementary, collagen‐type‐specific contributions to adhesion: reply to a rebuttal

A mechanism to safeguard platelet adhesion under high‐shear flow: von Willebrand factor‐glycoprotein Ib and integrin α2β1‐collagen interactions make complementary, collagen‐type‐specific contributions to adhesion: reply to a rebuttal

Cloning of the cDNA for murine von Willebrand factor and identification of orthologous genes reveals the extent of conservation among diverse species

Interaction of von Willebrand factor (VWF) with circulating platelets promotes hemostasis when a blood vessel is injured. The A1 domain of VWF is responsible for the initial interaction with platelets and is well conserved among species. Knowledge of the cDNA and genomic DNA sequences for human VWF allowed us to predict the cDNA sequence for murine VWF in silico and amplify its entire coding region by RT–PCR. The murine VWF cDNA has an open reading frame of 8,442 bp, encoding a protein of 2,813 amino acid residues with 83% identity to human pre-pro-VWF. The same strategy was used to predict in silico the cDNA sequence for the ortholog of VWF in a further six species. Many of these predictions diverged substantially from the putative Reference Sequences derived by ab initio methods. Our predicted sequences indicated that the VWF gene has a conserved structure of 52 exons in all seven mammalian species examined, as well as in the chicken. There is a minor structural variation in the pufferfish Takifugu rubripes insofar as the VWF gene in this species has 53 exons. Comparison of the translated amino acid sequences also revealed a high degree of conservation. In particular, the cysteine residues are conserved precisely throughout both the pro-peptide and the mature VWF sequence in all species, with a minor exception in the pufferfish VWF ortholog where two adjacent cysteine residues are omitted. The marked conservation of cysteine residues emphasizes the importance of the intricate pattern of disulfide bonds in governing the structure of pro-VWF and regulating the function of the mature VWF protein. It should also be emphasized that many of the conserved features of the VWF gene and protein were obscured when the comparison among species was based on the putative Reference Sequences instead of our predicted cDNA sequences.

Diagnosis of Inherited von Willebrand Disease: A Clinical Perspective

von Willebrand disease (VWD) is the most frequent inherited disorder of hemostasis and is due to quantitative (types 1 and 3) or qualitative (type 2) defects of von Willebrand factor (VWF). Due to the large heterogeneity of VWF defects and to the external variables influencing VWF levels in the circulation, VWD diagnosis can be difficult, especially in relatively mild forms. Three criteria should be always satisfied for a correct VWD diagnosis: (1) a positive bleeding history since childhood; (2) reduced levels of VWF activity in plasma; and (3) autosomal dominant or recessive inheritance patterns within the family (in most cases). According to clinical prospective studies, a bleeding history in VWD patients should be derived from a detailed questionnaire, with calculated bleeding scores. The ristocetin cofactor activity is the most useful test for VWD screening in the general population because it reproduces in vitro the first VWF interactions with its platelet receptor; however other assays are required to identify and classify VWD types. The current classification (types 1, 2A, 2B, 2M, 2N, and 3) is important to understand the basic mechanisms of VWF defects, to determine the risk of bleeding, and to select the best therapeutic approach. Molecular screening can be important to confirm phenotypic diagnosis for tracking VWF defects within families. Compared with hemophilia, most VWD patients show relatively mild bleeding symptoms. Therefore, prenatal diagnosis is required only for women already known to be carriers of VWD type 3. No spontaneous bleedings usually occur at birth in severe type 3 VWD. Neonatal diagnosis of VWD should always be compared with other affected members within the same family. Given that young children with VWD type 3 might carry deletions of VWF gene that predispose to the alloantibodies to VWF, every new child with VWD type 3 should be investigated intensively for VWF gene deletions before starting extensive therapy with exogenous VWF concentrates.

Shielding of the A1 Domain by the D′D3 Domains of von Willebrand Factor Modulates Its Interaction with Platelet Glycoprotein Ib-IX-V

Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ibalpha and needs immobilization and/or high shear stress to enable binding of its A1 domain to the receptor. The previously described anti-VWF monoclonal antibody 1C1E7 enhances VWF/GPIbalpha binding and recognizes an epitope in the amino acids 764-1035 region in the N-terminal D'D3 domains. In this study we demonstrated that the D'D3 region negatively modulates the VWF/GPIb-IX-V interaction; (i) deletion of the D'D3 region in VWF augmented binding to GPIbalpha, suggesting an inhibitory role for this region, (ii) the isolated D'D3 region inhibited the GPIbalpha interaction of a VWF deletion mutant lacking this region, indicating that intramolecular interactions limit the accessibility of the A1 domain, (iii) using a panel of anti-VWF monoclonal antibodies, we next showed that the D'D3 region is in close proximity with the A1 domain in soluble VWF but not when VWF was immobilized; (iv) destroying the epitope of 1C1E7 resulted in a mutant VWF with an increased affinity for GPIbalpha. Our results support a model of domain translocation in VWF that allows interaction with GPIbalpha. The suggested shielding interaction of the A1 domain by the D'D3 region then becomes disrupted by VWF immobilization.

Paratope Determination of the Antithrombotic Antibody 82D6A3 Based on the Crystal Structure of Its Complex with the von Willebrand Factor A3-Domain

The antithrombotic monoclonal antibody 82D6A3 is directed against amino acids Arg-963, Pro-981, Asp-1009, Arg-1016, Ser-1020, Met-1022, and His-1023 of the von Willebrand factor A3-domain (Vanhoorelbeke, K., Depraetere, H., Romijn, R. A., Huizinga, E., De Maeyer, M., and Deckmyn, H. (2003) J. Biol. Chem. 278, 37815-37821). By this, it potently inhibits the interaction of von Willebrand factor to collagens, which is a prerequisite for blood platelet adhesion to the injured vessel wall at sites of high shear. To fully understand the mode of action of 82D6A3 at the molecular level, we resolved its crystal structure in complex with the A3-domain and fine mapped its paratope by construction and characterization of 13 mutants. The paratope predominantly consists of two short sequences in the heavy chain CDR1 (Asn-31 and Tyr-32) and CDR3 (Asp-99, Pro-101, Tyr-102 and Tyr-103), forming one patch on the surface of the antibody. Trp-50 of the heavy and His-49 of the light chain, both situated adjacent to the patch, play ancillary roles in antigen binding. The crystal structure furthermore confirms the epitope location, which largely overlaps with the collagen binding site deduced from mutagenesis of the A3-domain (Romijn, R. A., Westein, E., Bouma, B., Schiphorst, M. E., Sixma, J. J., Lenting, P. J., and Huizinga, E. G. (2003) J. Biol. Chem. 278, 15035-15039). We herewith further consolidate the location of the collagen binding site and reveal that the potent action of the antibody is due to direct competition for the same interaction site. This information allows the design of a paratope-mimicking peptide with antithrombotic properties.

Identification of the von Willebrand Factor Binding Site in Collagen Using Triple Helical Peptides.

The interaction of the plasma protein von Willebrand factor (VWF) with subendothelial collagen initiates adhesion of blood platelets to the damaged vessel wall or ruptured atherosclerotic plaque. A detailed molecular description of the VWF-collagen interaction may facilitate development of a novel class of antithrombotic drugs that inhibits this vital step in platelet thrombus formation. We have previously used site-directed mutagenesis to map the collagen-binding site in the VWF A3 domain. Here, we report the identification of a 9-aminoacid sequence in collagen type III which mediates VWF binding. We have synthesized a set of 57 peptides, each containing 27 amino acids of native collagen sequence flanked at each end by five GPP (standard amino acid nomenclature) triplets which support the triple helical structure that is essential for ligand recognition by collagen. The sequence of each peptide overlaps by 9 amino acids with that of each adjacent peptide. A single peptide from this set (#23) was shown to bind VWF in a solid-phase binding assay. The affinity of peptide #23 for VWF was comparable to that of native collagen type III. The peptide #23-VWF interaction was abolished by a monoclonal antibody directed against the collagen-binding site on the VWF A3 domain. Furthermore, recombinant VWF variants that were previously shown to lack collagen-binding capacity (delta A3, His1023Ala) were not able to bind to the peptide. Using surface plasmon resonance, we showed that the peptide bound strongly to the isolated VWF A3 domain (Kd 300 nM). Immobilized peptide #23 also supported platelet adhesion from whole blood under flow conditions and washed platelet adhesion under static conditions. Platelet adhesion to peptide #23 could be abrogated by a monoclonal antibody directed against the VWF A3 domain, which inhibits the interaction of VWF with full-length collagen. We subsequently synthesized a set of truncated and alanine-modified triple helical peptides based on the sequence of #23, which were all tested for VWF and platelet binding from whole blood under flow conditions. Modified peptides either strongly interacted with both VWF and platelets, or lacked both VWF and platelet binding. Based on these experiments, we identified the sequence RGQOGVMGF (O is hydroxyproline) as the minimal VWF binding sequence in collagen type III. Mutation of either Q or M to alanine (A) did not affect VWF binding, whereas replacement of R, O, V, and F by A completely abolished VWF binding. Glycine residues were not replaced, as they are essential for triple helix formation. A model of the VWF A3 domain with this nonapeptide collagen sequence was constructed to give detailed insight into the VWF-collagen interaction. In conclusion, we have identified a 9 amino acid sequence in collagen type III that is entirely responsible for high affinity binding to VWF. The detailed molecular description of the VWF-collagen interaction described here may facilitate development of agents disrupting this interaction, which may have potential as antithrombotic drugs.

Role of Chloride Ions in Modulation of the Interaction between von Willebrand Factor and ADAMTS-13

The degradation of von Willebrand factor (VWF) depends on the activity of a zinc protease (referred to as ADAMTS-13), which cleaves VWF at the Tyr(1605)-Met(1606) peptide bond. Little information is available on the physiological mechanisms involved in regulation of AD-AMTS-13 activity. In this study, the role of ions on the ADAMTS-13/VWF interaction was investigated. In the presence of 1.5 m urea, the protease cleaved multimeric VWF in the absence of NaCl at pH 8.00 and 37 degrees C, with an apparent k(cat)/K(m) congruent with 3.4 x 10(4) M(-1) s(-1), but this value decreased by approximately 10-fold in the presence of 0.15 M NaCl. Using several monovalent salts, the inhibitory effect was attributed mostly to anions, whose potency was inversely related to the corresponding Jones-Dole viscosity B coefficients (ClO(4)(-) > Cl(-) > F(-)). The specific inhibitory effect of anions was due to their binding to VWF, which caused a conformational change responsible for quenching the intrinsic fluorescence of the protein and reducing tyrosine exposition to bulk solvent. Ristocetin binding to VWF could reduce the apparent affinity and reverse the inhibitory effect of chloride. We hypothesize that, after secretion into the extracellular compartment, VWF is bound by chloride ions abundantly present in this milieu, becoming unavailable to proteolysis by AD-AMTS-13. Shear forces, which facilitate GpIbalpha binding (this effect being artificially obtained by ristocetin), can reverse the inhibitory effect of chloride, whose concentration gradient across the cell membrane may represent a simple but efficient strategy to regulate the enzymatic activity of ADAMTS-13.

Binding of ADAMTS13 to von Willebrand Factor

ADAMTS13, a metalloprotease, cleaves von Willebrand factor (VWF) in plasma to generate smaller, less thrombogenic fragments. The interaction of von Willebrand factor with specific ADAMTS13 domains was characterized with a binding assay employing von Willebrand factor immobilized on a plastic surface. ADAMTS13 binding was saturable and reversible. Equilibrium binding occurred within 2 h and the half-time for dissociation was approximately 4 h. Binding to von Willebrand factor was similar with either recombinant ADAMTS13 or normal plasma ADAMTS13; plasma from a patient who lacked ADAMTS13 activity showed no binding. The stoichiometry of binding was one ADAMTS13 per two von Willebrand factor monomers, and the K(d) was 14 nm. The ADAMTS13 metalloprotease and disintegrin domains did not bind VWF detectably. ADAMTS13 truncated after the first thrombospondin type 1 repeat bound VWF with a K(d) of 206 nm, whereas ADAMTS13 truncated after the spacer domain had a K(d) of 23 nm, which is comparable with that of full-length ADAMTS13. Truncation after the eighth thrombospondin type 1 repeat reduced the binding affinity by approximately 3-fold and truncation after the seventh thrombospondin type 1 repeat in addition to the CUB domains increased the affinity for von Willebrand factor by approximately 2-fold. Therefore, the spacer domain is required for ADAMTS13 binding to von Willebrand factor. The first thrombospondin repeat also affects binding, and the C-terminal thrombospondin type 1 and CUB domains of ADAMTS13 may modulate this interaction.

Structure and function of snake venom toxins interacting with human von Willebrand factor

Hemostatic plug formation is a complex event mediated by platelets, subendothelial matrices and von Willebrand factor (VWF) at the vascular injury. Snake venom proteins have an excellent potency to regulate the interaction between VWF and platelet membrane receptors in vitro. Two protein families, C-type lectin-like proteins and Zn 2+-metalloproteinases, have been found to affect platelet–VWF interaction. Botrocetin and bitiscetin from viper venom are disulfide-linked heterodimers with C-type lectin-like motif, and modulate VWF to elicit platelet glycoprotein Ib (GPIb)-binding activity via the A1 domain of VWF leading to the platelet agglutination. The crystal structures of botrocetin and bitiscetin together with complex from the VWF A1 domain indicate the following: (1) a central concave domain formed by two subunits of botrocetin or bitiscetin provides the binding site for VWF, (2) these modulators directly bind to the A1 domain of VWF in close proximity to the GPIb binding site, (3) both modulators induce no significant conformational change on the GPIb-binding site of the A1 domain but could provide a supplemental platform fitting for GPIb. These results suggest that the modulating mechanisms of these venoms are different from those performed by either antibiotic ristocetin in vitro or extremely high shear stress in vivo. Other modulator toxins include kaouthiagin and jararhagin, chimeric proteins composed of metalloproteinase, disintegrin-like and Cys-rich domains. These toxins cleave VWF and reduce its platelet agglutinating or collagen-binding activity. Kaouthiagin from cobra venom specifically cleaves between Pro708 and Asp709 in the C-terminal VWF A1 domain resulting in the decrease of the multimer structure of VWF. Recently a plasma proteinase, which specifically cleaves VWF into a smaller multimer, has been elucidated to be a reprolysin-like metalloproteinase with thrombospondin motif family (ADAMTS). This endogenous metalloproteinase (ADAMTS-13) specifically cleaves between Tyr842 and Met843 in the A2 domain of VWF regulating its physiological hemostatic activity. These VWF-binding snake venom proteins are suitable probes for basic research on platelet plug formation mediated by VWF, for subsidiary diagnostic use for von Willebrand disease or platelet disorder, and might be potently applicable to the regulation of VWF in thrombosis and hemostasis. Structural information of these venom proteins together with recombinant technology might strongly promote the construction of a new antihemostatic drug in the near future.