Von Willebrand Factor Fragments Research Articles

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.

What a polyclonal antibody sees in von Willebrand factor

Introduction Von Willebrand factor (VWF) plays a critical role in hemostasis by carrying factor VIII (FVIII) and binding to specific ligands on the surface of blood platelets and within the blood vessel wall. Materials and methods We constructed a gene-specific phage display library containing small, random VWF fragments. Using this library, we mapped the repertoire of immune epitopes recognized by a commonly used commercial rabbit antihuman VWF polyclonal antibody. Results A total of eight discrete epitopes within the VWF protein were identified, including two dominant epitopes that account for 74% of immuno selected VWF fragments. Comparison with previously mapped epitopes for mouse monoclonal antibodies reveals four overlapping regions that may identify common antigenic determinants. The distribution of these epitopes was not readily predicted from primary amino acid sequence divergence among these mammalian species or standard algorithms for the prediction of antigenicity, hydrophobicity, or surface probability. Conclusion Taken together with previous monoclonal antibody epitope mapping studies, our results suggest that a limited number of exposed domains on the surface of the human VWF protein may be the primary determinants of immunogenicity.

The Middle and Distal Carboxyl Terminal Domains of ADAMTS13 Are Critical for Docking ADAMTS13 to von Willebrand Factor.

ADAMTS13 cleaves von Willebrand factor (VWF) at the Tyr1605-Met1606 bond of the central A2 domain. However, the domains of ADAMTS13 required for recognition and cleavage of VWF remain controversial. On the one hand, we and others have shown that the proximal carboxyl terminal domains (i.e. Cys-rich and spacer domains) of ADAMTS13 appeared to be sufficient for recognition of VWF under the static and denatured conditions (Zheng et al, JBC, 2003; Soejima et al, Blood, 2003, Ai et al, JBC, 2005). On the other hand, the distal carboxyl terminus (i.e. the first CUB domain) of ADAMTS13 has been proposed to dock ADAMTS13 to VWF under flow (Tao et al, Blood, 2005). To determine the interaction between ADAMTS13 and native VWF in real time, we employed Biacore technology based on surface plasmon resonance. To avoid conformational change or activation of VWF during the ligand immobilization, we chose to flow purified VWF or A2 fragment (VWF73) over the carboxymethylated dextran (CM5) surface to which was covalently attached via amine coupling either purified full-length recombinant ADAMTS13 (FL-A13) (~8,200 RU), the variant truncated after the 8th thrombospondin type 1 repeat (delCUB) (~7,200 RU), or that truncated after the spacer domain (MDTCS) (~4,200 RU). The control surface was prepared similarly by activation and deactivation processes without protein being coupled. The VWF at various concentrations in 20 mM HEPES buffer, pH 7.5, 150 mM NaCl, 0.02% Tween 20 and 0.05% BSA was flown at 20 μl/min over the surfaces as prepared above. The sensorgrams were recorded simutaneouly in all four microfluid cells and the kinetic parameters were determined. We showed that VWF bound immobilized FL-A13 in a dose and time-dependent manner with an equilibrium dissociation constant (Kd) of 50.0 ± 21.8 nM (n=6). Removal of the CUB domains (construct delCUB) reduced its affinity for VWF by ~5.5 fold (Kd=274 ± 158 nM (n=6). Further removal of the TSP1 2~8 repeats (construct MDTCS) almost completely abolished its binding to VWF (n=4) (Kd not determined). The affinity between ADAMTS13 (or variants) and VWF was not significantly altered by addition of 10 mM EDTA. Pre-denaturization of VWF with 1.5 M guanidine-HCl at 37 °C for 1 h, however, modestly enhanced VWF binding to immobilized delCUB (Kd=185 ± 96 nM, n=4) and MDTCS (Kd not determined), but reduced VWF binding to FL-A13 by ~2 fold, perhaps due to the mild denaturization of FL-A13 by 0.15 M guanidine-HCl in the flowing buffer. Interestingly, VWF73 bound FL-A13, delCUB and MDTCS with the Kd (s) of 2.6 ± 0.5 nM, 4.9 ± 1.0 nM and 3.0 ± 0.5 nM, respectively, which was at least 20~55 fold higher than native VWF did, suggesting that the protein structure surrounding the cleavage site at the central A2 domain of VWF negatively regulates ADAMTS13-VWF interaction. Moreover, the Michael-Menton constant Km (s) for cleavage of the denatured VWF (or VWF73) by FL-A13, delCUB and MDTCS were 260 ± 117 nM, 276 ± 118 nM and 534 ± 657 nM (or 1.8 ± 1.5 μM, 0.3 ± 0.1 μM and 0.6 ± 0.2 μM), respectively. These data demonstrate that the interaction between ADAMTS13 and native VWF is a high affinity one and that the middle and distal carboxyl terminal domains of ADAMTS13 may be critical for initial docking of ADAMTS13 to the exosites of native VWF in solution.

Impact of mutations in the von Willebrand factor A2 domain on ADAMTS13-dependent proteolysis

AbstractClassical von Willebrand disease (VWD) type 2A, the most common qualitative defect of VWD, is caused by loss of high-molecular-weight multimers (HMWMs) of von Willebrand factor (VWF). Underlying mutations cluster in the A2 domain of VWF around its cleavage site for ADAMTS13. We investigated the impact of mutations commonly found in patients with VWD type 2A on ADAMTS13-dependent proteolysis of VWF. We used recombinant human ADAMTS13 (rhuADAMTS13) to digest recombinant full-length VWF and a VWF fragment spanning the VWF A1 through A3 domains, harboring 13 different VWD type 2A mutations (C1272S, G1505E, G1505R, S1506L, M1528V, R1569del, R1597W, V1607D, G1609R, I1628T, G1629E, G1631D, and E1638K). With the exception of G1505E and I1628T, all mutations in the VWF A2 domain increased specific proteolysis of VWF independent of the expression level. Proteolytic susceptibility of mutant VWF in vitro closely correlated with the in vivo phenotype in patients. The results imply that increased VWF susceptibility for ADAMTS13 is a constitutive property of classical VWD type 2A, thus explaining the pronounced proteolytic fragments and loss of HMWM seen in multimer analysis in patients.

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.

ナノ接合・分離プロセス 中赤外レーザーを用いたｖｏｎ Ｗｉｌｌｅｂｒａｎｄ Ｆａｃｔｏｒ（ｖＷＦ）―生体高分子―の分解

ナノ接合・分離プロセス 中赤外レーザーを用いたｖｏｎ Ｗｉｌｌｅｂｒａｎｄ Ｆａｃｔｏｒ（ｖＷＦ）―生体高分子―の分解

Impaired dimerization of von Willebrand factor subunit due to mutation A2801D in the CK domain results in a recessive type 2A subtype IID von Willebrand disease

The CK domain of von Willebrand factor (VWF) is involved in the dimerization of the protein. We identified the homozygous substitution A2801D of the CK domain in two siblings. Patients had low levels of VWF in plasma, abnormal ristocetin-induced binding to platelets and abnormal multimeric pattern with a lack of high molecular weight (HMW) forms and the presence of intervening bands between normal multimers. Accordingly, they were classified in type 2A, subtype IID, von Willebrand disease (VWD). Both asymptomatic parents carried the mutation at the heterozygous state. Their plasmaVWF exhibited the full range of multimers found in normal plasma. When analyzed by high resolution gel electrophoresis, very faint bands corresponding to the position of intervening bands of the propositus can be observed. The mutated recombinant (r)VWF-D2801, the hybrid rVWF-A/D2801 and the mutated C-terminal VWF fragment rSPII-D2801 were expressed in COS-7 cells. rVWF-D2801 showed an abnormal multimeric distribution similar to that of the propositus'VWF with intervening bands and a lack of HMW species. rVWF-A/D2801 exhibited the full range of multimers and the aberrant sized forms observed both in propositus'VWF and in rVWF-D2801. rSPII-WT assembled correctly into a dimer of 220 kDa. rSPII-D2801 appeared as a mixture of monomeric and dimeric forms which may be related to the abnormal multimeric pattern of the propositus and both mutated rVWF. We concluded that mutation A2801D disturbs the folding of the CK domain, which may result in a mixture of monomers and dimers of VWF. Multimers containing either an odd or even number of mature subunits are produced, and the presence of monomers appears to limit the degree of multimerization. In the heterozygousVWF, the presence of normal dimers improves the multimerization process. In conclusion, the mutation A2801D appears to be responsible for a recessive type 2A, subtype IID, VWD.

Development and Performance of the GTI ADAMTS-13 Activity Assay.

Thrombotic Thrombocytopenic Purpura (TTP) is a disease characterized by the sudden onset of a classical pentad of symptoms: thrombocytopenia, fever, renal insufficiency, neurologic deficit, and microangiopathic hemolytic anemia. It was recently discovered that ADAMTS-13 is the protease responsible for cleaving von Willebrand Factor; deficiency of ADAMTS-13 activity has been demonstrated in the plasma of TTP patients. The lack of ADAMTS-13 activity results in the accumulation of multimers of vWF in the plasma and ultimately intravascular platelet aggregation resulting in the clinical symptoms associated with TTP. Currently there are no commercially available diagnostic assays for TTP. Several laboratories have developed “in house” assays that measure the activity of the ADAMTS-13 protease. However, these assays are cumbersome, complex, difficult to perform, and there is a long turn around time, as they require long incubations. The results are difficult to interpret and are not easily quantitated. In addition, there is no standardization of results from laboratory to laboratory, mainly because the assays use various technologies. Determining the extent to which the ADAMTS-13 enzyme activity is decreased is believed to be important in distinguishing a patient with TTP from those presenting with similar clinical symptoms. Until ADAMTS-13 assays are more quantitative, there is not a good method for studying patients with severe decreases in ADAMTS-13 levels versus those who have some intermediate but less than normal level of enzyme activity. Furthermore, the lack of standardized results makes the assays less useful in monitoring the effectiveness of treatment of the TTP patients. GTI has developed a rapid, quantitative assay for ADAMTS-13 activity that is based on the cleavage of a substrate which consists of a peptide fragment of von Willebrand Factor and subsequent fluorescent detection. An initial study using plasma (obtained from the Blood Center of Wisconsin) from 5 TTP patients demonstrated good correlation between the GTI assay and the collagen binding assay, thus demonstrating feasibility of the assay. A more extensive evaluation using 40 normal samples and 55 samples from suspected TTP patients was initiated, the results of which will be reported. Assay sensitivity/limit of detection (based on dilution studies) was 3% normal ADAMTS-13 activity. Assay precision using 3 samples (mean values of 96%, 55%, and 9% normal ADAMTS-13 activity) run in duplicate over 8 days was evaluated. Assay imprecision was less than 10% cv for all samples tested.

Characterization of a myeloma patient with a life-threatening hemorrhagic diathesis: presence of a lambda dimer protein inhibiting shear-induced platelet aggregation by binding to the A1 domain of vonWillebrand factor

We have identified a patient with IgD lambda-type multiple myeloma who was characterized by a severe bleeding tendency, especially after puncture of arterial vessels. Both the bleeding time (>25 min) and activated partial thromboplastin time (APTT) were prolonged. To clarify the underlying pathogenesis, we purified the APTT-prolonging activity from the patient's serum. The purified protein was a highly negatively-charged homodimer of the lambda light chain. The lambda dimer protein (M-protein) inhibited ristocetinand high shear-induced platelet aggregation, dependent on platelet glycoprotein Ibalpha (GPIbalpha), but not epinephrine-, collagen-, ADP-, thrombin-, or botrocetin-induced platelet aggregation. The lambda dimer protein inhibited the binding of platelets to immobilized or ristocetin-treated von Willebrand factor (VWF). Furthermore, a 39/34 kD fragment of VWF encompassing the A1 domain specifically bound to the immobilized lambda dimer protein in the presence of ristocetin, suggesting that the lambda dimer protein directly binds to the A1 domain of VWF. To help elucidate the binding site within the A1 domain, binding of ristocetin-treated VWF to the immobilized lambda dimer protein was assayed in the presence of various anti-A1 domain monoclonal antibodies. Based on these data, we conclude that the lambda dimer protein binds to the region of the A1 domain composed of helices alpha3 and alpha4 and thus interferes with VWF-GPIbalpha interaction. The existence of a protein that inhibits high shear-induced platelet aggregation in acquired von Willebrand disease (VWD) has only rarely been reported. The results suggest that the hemostatic function in arteries with high shear force is profoundly disrupted if the binding of GPIbalpha to VWF is abrogated, supporting the relevance of shear-induced VWF interaction with GPIbalpha in the initiation of the hemostatic process.

High Shear Stress and Immobilization of Von Willebrand Factor Promotes the VWF-Cleaving Protease Activity as Assessed by the Cone and Plate(let) Analyzer.

The metalloprotease ADAMTS-13 cleaves von Willebrand factor (VWF) that is released from endothelial cells as a large multimeric protein. However, the specific physiological conditions required for the function of this von Willebrand factor-cleaving protease (VWF-CP) are not yet established. In this study we determined the optimal conditions for the VWF-CP activity using the Cone and Plate(let) Analyzer (CPA). Proteolysis of a soluble recombinant VWF by a recombinant VWF-CP in the absence or the presence of BaCl2 (for induction of VWF-CP) was performed under static and flow (2050 s−1) conditions. The resulted fragments of the soluble VWF were immobilized on a polystyrene surface and non-adhering sites were blocked with 1% BSA. In parallel, polystyrene surface immobilized VWF was similarly treated by VWF-CP. The immobilized cleaved VWF fragments by the above protocols served as a substrate for citrated whole blood platelet adhesion under flow (2050 s−1). Reduction of platelet adhesion (surface coverage, SC) in BaCl2 treated compared to untrerated sample reflects the degree of VWF-CP activity.Platelet adhesion (SC, %) to VWF proteolytic products generated under the indicated conditions:Static w/o BaCl2Static + BaCl2Flow w/o BaCl2Flow + BaCl2Soluble VWF15.0 ± 3.411.1 ± 2.713.6 ± 2.28.0 ± 2.1Immobil. VWF19.1 ± 3.012.0 ± 4.16.5 ± 1.11.0 ± 0.6Maximal VWF-CP activity as reflected by maximal reduction (85%) of platelet adhesion was observed when immobilized VWF was treated by VWF-CP under flow. Minimal effect of VWF-CP activity was observed when soluble VWF was treated under static condition (26% reduction). Intermediate effect was observed with soluble VWF under flow (41%) and with immobilized VWF under static condition (37%). It should be noted that in the case of immobilized VWF application of flow alone (without BaCl2) was sufficient to induce a remarkable decrease of platelet adhesion (66%). In conclusion, both immobilization of VWF and high shear stress are important conditions for VWF-CP function, suggesting that stretching of immobilized VWF under flow exposes the VWF-cleavage site and thereby may serve as a control mechanism of platelet adhesion at the site of thrombus formation.

A VWF Fragment Spanning the A1, A2, and A3 Domains Supports Platelet Adhesion Better Than the Isolated A1 Domain and Is Considerably More Resistant to Cleavage by Adamts-13 Than the Isolated A2 Domain.

von Willebrand factor (VWF) is a large multimeric protein whose monomers are of the domain structure D'-D3-A1-A2-A3-D4-B1-B2-B3C1-C2. The three tandem A domains mediate several important functions of the protein including supporting platelet adhesion, through an interaction of A1 with the platelet GP Ib-IX-V complex, and binding collagen, through A3 and A1. Further, the A2 domain contains the cleavage site for the plasma metalloprotease ADAMTS-13, which processes the newly synthesized and extremely adhesive ultra-large forms of VWF (ULVWF) to the forms normally found in plasma. Our goal was to compare the functions of the individual A domains as isolated domains to their function in the context of the A1-A2-A3 fragment. All recombinant domains were produced in bacteria. The A1-A2-A3 fragment contained an N-terminal thioredoxin-tag and a C-terminal 6Xhis tag and was purified in 2 steps using nickel and heparin columns. The recombinant protein was correctly folded, as indicated by the fact that it was recognized by a number of conformation-specific antibodies. Using ELISA, we examined the interaction of the fragment with both glycocalicin (the soluble extracellular domain of GP Iba) and collagen. At equivalent concentrations, A1-A2-A3 bound collagen better than did the isolated A1 and A3 domains, but the binding to glycocalicin was approximately 7.5 times lower than the A1 domain. We then tested the ability of the fragment to support platelet adhesion under flow, by coating the fragment onto a coverslip that made up the bottom of a parallel-plate flow chamber. In contrast to the findings with immobilized glycocalicin, immobilized A1-A2-A3 supported the attachment, rolling, and firm adhesion of the platelets to the surface, whereas A1 did not support firm adhesion. Finally, we tested cleavage of the fragment by plasma ADAMTS-13, using both ELISA and western blotting to detect cleavage. The rate of cleavage of the A1-A2-A3 fragment was much slower than that of the recombinant A2 domain, needing a longer incubation time to achieve a similar percentage of cleavage in a 15 fold higher concentration of plasma. These results show clearly that within the triplicate A domains of VWF, adjacent A domains influence each other's functions, possibly by binding each other or by conformational modulation. The fact that the fragment is able to support firm platelet adhesion in isolation suggests that it may contain a second, previously unidentified binding site for platelet receptors.

An enzyme immunoassay of ADAMTS13 distinguishes patients with thrombotic thrombocytopenic purpura from normal individuals and carriers of ADAMTS13 mutations.

Recent studies demonstrate that assay of ADAMTS13, a circulating zinc metalloprotease that cleaves von Willebrand factor (VWF) at the Y1605-M1606 bond, is an important tool in the diagnosis of thrombotic thrombocytopenic purpura (TTP). In order to develop a method that could be adapted for general use, we describe an enzyme immunoassay (EIA)-based method for measuring the activity of ADAMTS13 in patient plasma samples. A monomeric peptide consisting of the amino acid residues D1596-R1668 of VWF was produced that spanned the ADAMTS13 cleavage site and was franked by glutathione s-transferase (GST) and a 6His sequences at the amino and carboxyl termini respectively. When probed with either anti-GST or anti-6His, the VWF fragment appeared as a 38.1-kDa band. After incubation with normal plasma, the VWF fragment was replaced by a 30.4-kDa band, which was recognized by anti-GST but not by anti-6His, consistent with the expected cleavage at the Y1605-M1606 bond. EDTA or plasma samples from patients with TTP inhibited this cleavage. After incubation with normal plasma, the VWF fusion protein immobilized onto anti-GST coated microtiter plate wells lost its anti-6His binding activity in a time- and plasma concentration-dependent manner. By using this EIA, the ADAMTS13 activity level was less than 0.12 U/mL in patients with acquired or hereditary TTP, distinguishing these patients from normal individuals or carriers of one copy of mutant ADAMTS13 allele. These results suggest the EIA method based on the VWF fusion protein is a simple but promising alternative for measuring ADAMTS13 activity.

Assays of ADAMTS-13 activity

Various assays for determination of ADAMTS-13 activity in plasma have been developed, all comprising two steps. The first step consists of proteolyzing a substrate by ADAMTS-13. Substrates were either exogenous von Willebrand factor (VWF) (purified from human plasma concentrates or recombinant VWF [rVWF]), purified VWF fragments, or endogenous VWF (from the tested plasma sample). All assays required a step in which substrate unfolding is performed using either urea or guanidine. Test plasmas were used at various dilutions and ADAMTS-13 was activated in most cases by divalent cations. The second step consists of quantifying the digestion products or the residual VWF remaining after proteolysis. Cleavage of VWF was thus estimated using electrophoresis (generation of proteolytic fragments or decrease of the size of multimers), functional methods (decrease of the collagen binding activity or of ristocetin cofactor activity), or immunological methods (enzyme-linked immunosorbent assay [ELISA] or immunoradiometric assay (IRMA) using selected monoclonal antibodies to VWF). Since 1998, all of these assays have been used to demonstrate the relevance of a deficient ADAMTS-13 activity in thrombotic thrombocytopenic purpura (TTP). However, improvements are required, as these methods remain cumbersome, time-consuming, and too remote from physiology to be routinely helpful for rapid laboratory diagnosis.

Deficiency of ADAMTS-13 in thrombotic and thrombocytopenic purpura

In his article refuting the association between thrombotic thrombocytopenic purpura (TTP) and ADAMTS-13 deficiency, Remuzzi correctly emphasizes that neurological lesions are not uncommon in patients with the infectious form of hemolytic uremic syndrome (HUS) [1]. This is contrary to a recent report in which he and his colleagues used the absence of neurological abnormalities as a main criterion for distinguishing HUS from TTP [2]. Also, Remuzzi states that since the proteolytic fragments of von Willebrand factor (VWF) resulting from cleavage by ADAMTS-13 account for a higher percentage of the VWF protein, cleavage of VWF by ADAMTS-13 is increased in patients with either TTP or HUS.

Acquired von Willebrand syndromes: clinical features, aetiology, pathophysiology, classification and management

Acquired von Willebrand syndrome (AVWS) associated with hypothyroidism is of type 1, results from a decreased synthesis of factor VIII and von Willebrand factor (VWF), responds to desmopressin with normal half-life times for factor VIII and VWF parameters, and disappears after treatment with l-thyroxine. AVWS type I or III, which occurs in a minority of patients with Wilms' tumour in the complete absence of an inhibitor against VWF and no absorption of factor VIII or VWF onto nephroblastoma cells, responds to chemotherapy and/or tumour resection. Hyaluronic acid produced by nephroblastoma cells may be the causative factor in atypical AVWS in Wilms' tumour. AVWS associated with thrombocythaemia of various myeloproliferative disorders is characterized by normal factor VIII and von Willebrand factor antigen (VWF:Ag) levels and a selective deficiency of functional ristocetin co-factor activity (VWF:RCo) and collagen-binding activity (VWF:CBA). AVWS type II in thrombocythaemia is caused by a platelet-dependent proteolysis of large VWF multimers, given the inverse relationship between platelet count and large VWF multimers in plasma and specific increases in the number of proteolytic VWF fragments in plasma.The laboratory findings of AVWS associated with systemic lupus erythematosus or IgG benign monoclonal gammopathy are characterized by a prolonged bleeding time and activated partial thromboplastin time, decreased or absent ristocetin-induced platelet activity, low to very low levels of factor VIII coagulant activity (mean 15%), VWF:Ag (mean<10·7%) and VWF:RCo (mean<6·2%), and a type II multimeric pattern of VWF. Neutralizing and non-neutralizing anti-VWF autoantibodies, usually IgG, have been detected in patient plasma either free or tightly bound to the intermediate and high molecular weight VWF factor VIII particles. The bound auto antibody–antigen complex is rapidly cleared from the circulation, resulting in low levels of factor VIII, VWF parameters as documented by a poor response to desmopressin and VWF factor VIII concentrate. High-dose intravenous immunoglobulin transiently corrects the factor VIII coagulant and VWF levels, lasting for a few weeks in AVWS type II associated with systemic lupus erythematosus or IgG benign monoclonal gammopathy. Prednisolone is effective in AVWS associated with autoimmune disorder. Prednisolone and chemotherapy will not affect AVWS associated with IgG benign monoclonal gammopathy because the monoclonal IgG protein remains to act as an anti-VWF autoantibody.An absorption of VWF to malignant cells has been documented in a few patients with various lymphoproliferative disorders or adrenal carcinoma and suggested to result in a depletion of VWF. The clinical picture of AVWS associated with early-stage IgG multiple myeloma, chronic lymphocytic leukaemia or non-Hodgkin's lymphoma without a paraprotein or no detectable underlying disorder is similar to that of AVWS type II in IgG benign monoclonal gammopathy but poorly documented with regard to the underlying immune mechanism of AVWS. The mechanical destruction of large VWF multimers may be of relevance in conditions in which the shear rate of flowing blood is increased, as may occur in cases of aortic stenosis, other heart valve defects or stenosed vessels. Drug-induced AVWS has been described in association with the use of pesticides valproic acid, ciprofloxacin, griseofulvin, tetracycline, thrombolytic agents and hydroxyethyl starch.

Testing of Platelet Deposition on Polystyrene Surface Under Flow Conditions by the Cone and Plate(let) Analyzer: Role of Platelet Activation, Fibrinogen and von Willebrand Factor

Recently, we described a method of testing platelet deposition on extracellular matrix under flow conditions. The method was used for assessment of platelet function in various platelet disorders, for monitoring of replacement and anti-platelet therapy. In the present study, we investigated platelet deposition on a polystyrene surface compared with that on extracellular matrix, under defined shear rates, using the original Cone and Plate(let) Analyzer. A correlation of adhesion rate (surface coverage) and aggregate formation (average size) of platelets from normal citrated blood between polystyrene and extracellular matrix was observed. Blocking of von Willebrand factor binding to glycoprotein Ib by a recombinant von Willebrand factor fragment substantially decreased platelet adhesion to both surfaces. Blocking of GPIIb-IIIa by Arg–Gly–Asp–Ser peptide prevented platelet adhesion to the polystyrene while an extensive adhesion of single platelets to extracellular matrix was observed. Furthermore, platelet adhesion to polystyrene but not to extracellular matrix was completely inhibited by platelet inactivation with prostaglandin E 1. Platelets from patients with severe von Willebrand disease yielded very low adhesion to both polystyrene and extracellular matrix. The addition of von Willebrand factor to the blood of these patients or pre-coating of polystyrene surface with von Willebrand factor restored the ability of platelets to adhere and aggregate on the surface. Platelets from patients with Glanzmann's thrombasthenia and afibrinogenemia adhered to extracellular matrix (with defective aggregate formation), while they failed to adhere to the polystyrene. Fibrinogen added to afibrinogenemia blood or pre-coating of the polystyrene with fibrinogen restored the ability of platelets to adhere and aggregate on the surface. In conclusion, the polystyrene surface, like extracellular matrix, can be used to assess platelet function disorders taking in account that platelet deposition on polystyrene under flow is absolutely dependent on platelet activation and on the presence of fibrinogen, von Willebrand factor, and their receptors.

Role of A and B blood group antigens in the expression of adhesive activity of von Willebrand factor.

ABO (H) blood group antigens are covalently linked to the oligosaccharide side-chains of von Willebrand factor (VWF). In this study, we investigated the role of the A and B antigens in the expression of VWF adhesive activity. VWF of type A, B or O was purified from fresh frozen plasma. Presence of A or B antigen on the VWF was confirmed by enzyme-linked immunosorbent assay (ELISA) and by immunoblotting with monoclonal anti-A or anti-B. The A or B antigen was also detected in the 48/52-kDa fragment of the respective VWF after trypsin digestion. Removal of A antigen with alpha-N-acetylgalactosaminidase or B antigen with alpha-galactosidase did not affect its multimer size or antigenic level, but decreased the ristocetin cofactor (RCoF) activity of the respective VWF by 33-39% (P < 0.01-0.002). Removal of A or B antigen from VWF did not affect the binding of the VWF to immobilized type III collagen. A and B antigens were not detected in platelet VWF. These results indicate that AB structures play a role in platelet aggregating activity of VWF.

Interaction of von Willebrand Factor Domain A1 with Platelet Glycoprotein Ibα-(1–289): SLOW INTRINSIC BINDING KINETICS MEDIATE RAPID PLATELET ADHESION

We investigated the crucial hemostatic interaction between von Willebrand factor (VWF) and platelet glycoprotein (GP) Ibalpha. Recombinant VWF A1 domain (residues Glu(497)-Pro(705) of VWF) bound stoichiometrically to a GPIbalpha-calmodulin fusion protein (residues His(1)-Val(289) of GPIbalpha; GPIbalpha-CaM) immobilized on W-7-agarose with a K(d) of 3.3 microM. The variant VWF A1(R545A) bound to GPIbalpha-CaM 20-fold more tightly, mainly because the association rate constant k(on) increased from 1,100 to 8,800 M(-1) s(-1). The GPIbalpha mutations G233V and M239V cause platelet-type pseudo-von Willebrand disease, and VWF A1 bound to GPIbalpha(G233V)-CaM and GPIbalpha(M239V)-CaM with a K(d) of 1.0 and 0.63 microM, respectively. The increased affinity of VWF A1 for GPIbalpha(M239V)-CaM was explained by an increase in k(on) to 4,500 M(-1) s(-1). GPIbalpha-CaM bound with similar affinity to recombinant VWF A1, to multimeric plasma VWF, and to a fragment of dispase-digested plasma VWF (residues Leu(480)/Val(481)-Gly(718)). VWF A1 and A1(R545A) bound to platelets with affinities and rate constants similar to those for binding to GPIbalpha-CaM, and botrocetin had the expected positively cooperative effect on the binding of VWF A1 to GPIbalpha-CaM. Therefore, allosteric regulation by botrocetin of VWF A1 binding to GPIbalpha, and the increased binding affinity caused by mutations in VWF or GPIbalpha, are reproduced by isolated structural domains. The substantial increase in k(on) caused by mutations in either A1 or GPIbalpha suggests that productive interaction requires rate-limiting conformational changes in both binding sites. The exceptionally slow k(on) and k(off) provide important new constraints on models for rapid platelet tethering at high wall shear rates.

S-Nitrosoderivative of a Recombinant Fragment of von Willebrand Factor (S-Nitroso-AR545C) Inhibits Thrombus Formation in Guinea Pig Carotid Artery Thrombosis Model

Antiplatelet drugs are the mainstays of therapy for acute and chronic cardiovascular diseases. S-nitroso-AR545C - an S-nitrosoderivative of a recombinant von Willebrand factor fragment AR545C spanning Ala 444 to Asp 730 and containing an Arg 545 Cys mutation, was previously found to inhibit ristocetin- and ADP-induced platelet aggregation and the interaction of platelets with extracellular matrix (ECM). In the current study we tested the antithrombotic properties of S-nitroso-AR545C on guinea pig platelets and in a platelet-rich thrombosis model in the guinea pig. Preincubation of guinea pig platelets with 0.1 microM of S-nitroso-AR545C decreased ristocetin-induced agglutination by 40% (p = 0.009) and completely abolished ADP-induced aggregation (p <0.0001). At concentration of 1.0 microM, S-nitroso-AR545C completely inhibited platelet adhesion (represented by surface coverage - SC) and decreased aggregate formation (represented by average aggregate size - AS) by more than 50%. Treatment of guinea pigs with 1.0 mg/kg S-nitroso-AR545C resulted in a significantly delayed time to arterial occlusion (31.7+/-6.0 min vs. 13.9+/-3.2 min, p <0.02). Similarly, total patency time was longer in the group injected with S-nitroso-AR545C compared to the control group. However, the difference was not statistically significant (33.8+/-6.3 min vs. 20.2+/-3.3 min, p = 0.07). No change in platelet count, hematocrit and bleeding time was observed 60 min after injection compared to baseline. In contrast, a significant decrease in SC (p <0.0001) and AS (p <0.01) were observed 60 min after the injection of S-nitroso-AR545C, whereas no change in these parameters was observed in the control group. These observations indicate that S-nitroso-AR545C exhibits significant antiadhesive and antiaggregating effects in-vitro and inhibits clot formation in-vivo suggesting that this compound may have potential therapeutic advantages.

Unique Antiplatelet Effects of a Novel S-Nitrosoderivative of a Recombinant Fragment of von Willebrand Factor, AR545C: In Vitro and Ex Vivo Inhibition of Platelet Function

AbstractThe recombinant fragment of von Willebrand factor (vWF) spanning Ala444 to Asp730 and containing an Arg545Cys mutation (denoted AR545C) has antithrombotic properties that are principally a consequence of its ability to inhibit platelet adhesion to subendothelial matrix. Endothelial-derived nitric oxide (NO) can also inhibit platelet function, both as a consequence of inhibiting adhesion as well as activation and aggregation. Nitric oxide can react with thiol functional groups in the presence of oxygen to form S-nitrosothiols, which are naturally occurring NO derivatives that prolong the biological actions of NO. Because AR545C has a single free cysteine (Cys545), we attempted to synthesize the S-nitroso-derivative of AR545C and to characterize its antiplatelet effects. We successfully synthesized S-nitroso-AR545C and found that it contained 0.96 mol S-NO per mole peptide. S-nitroso-AR545C was approximately 5-fold more potent at inhibiting platelet agglutination than was the unmodified peptide (IC50 = 0.02 ± 0.006 μmol/L v 0.1 ± 0.03 μmol/L, P = .001). In addition and by contrast, S-nitroso-AR545C was a powerful inhibitor of adenosine diphosphate–induced platelet aggregation (IC50 = 0.018 ± 0.002 μmol/L), while AR545C had no effect on aggregation. These effects were confirmed in studies of adhesion to and aggregation on extracellular matrix under conditions of shear stress in a cone-plate viscometer, where 1.5 μmol/L S-nitroso-AR545C inhibited platelet adhesion by 83% and essentially completely inhibited aggregate formation, while the same concentration of AR545C inhibited platelet adhesion by 74% and had significantly lesser effect on aggregate formation on matrix (P ≤ .004 for each parameter by ANOVA). In an ex vivo rabbit model, we also found that S-nitroso-AR545C had a more marked and more durable inhibitory effect on botrocetin-induced platelet aggregation than did AR545C, and these differences were also reflected in the extent and duration of effect on the prolongation of the bleeding time in these animals. These data show that S-nitroso-AR545C has significant and unique antiplatelet effects, inhibiting both adhesion and aggregation, by blocking platelet GPIb receptor through the AR545C moiety and elevating platelet cyclic 3′,5′-guanosine monophosphate through the -SNO moiety. These observations suggest that this NO-modified fragment of vWF may have potential therapeutic benefits as a unique antithrombotic agent.