Mechanics of transient platelet adhesion to von Willebrand factor under flow

A primary and critical step in platelet attachment to injured vascular endothelium is the formation of reversible tether bonds between the platelet glycoprotein receptor Ibalpha and the A1 domain of surface-bound von Willebrand factor (vWF). Due to the platelet's unique ellipsoidal shape, the force mechanics involved in its tether bond formation differs significantly from that of leukocytes and other spherical cells. We have investigated the mechanics of platelet tethering to surface-immobilized vWF-A1 under hydrodynamic shear flow. A computer algorithm was used to analyze digitized images recorded during flow-chamber experiments and track the microscale motions of platelets before, during, and after contact with the surface. An analytical two-dimensional model was developed to calculate the motion of a tethered platelet on a reactive surface in linear shear flow. Through comparison of the theoretical solution with experimental observations, we show that attachment of platelets occurs only in orientations that are predicted to result in compression along the length of the platelet and therefore on the bond being formed. These results suggest that hydrodynamic compressive forces may play an important role in initiating tether bond formation.     

Sequencing of the primary adhesion domain of bovine von Willebrand factor.

A cDNA library, constructed from bovine heart endothelial cell poly(A)+ RNA, was screened using a BstXI fragment of human von Willebrand and factor (vWF) cDNA as a probe. This probe codes for the major adhesion domain of vWF that includes the GPIb, collagen and heparin binding domains. Of the ten positive clones obtained, a clone that spanned the region of interest was sequenced by the dideoxynucleotide method yielding a sequence of 1550 bp. This region of the bovine cDNA codes for amino acids corresponding to #262 to #777 in human vWF and encompasses the entire pro adhesion domain. Both the nucleotide sequence and the deduced amino acid sequence are 82% homologous to those of human vWF. Cysteine residues #471, 474, 509 and 695, which form intrachain bonds in human vWF, are also present in the bovine vWF sequence.     

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.     

Targeting of liposomes carrying recombinant fragments of platelet membrane glycoprotein Ibalpha to immobilized von Willebrand factor under flow conditions

Liposomes with covalently bound recombinant fragments of platelet membrane glycoprotein Ibalpha that retain the von Willebrand factor (vWf)-binding function (rGPIbalpha-liposomes) were prepared. Their interactions with an immobilized vWf surface under flow conditions were evaluated with a recirculating flow chamber, mounted on an epifluorescence microscope, which allows real-time visualization of fluorescence-labeled liposomes interacting with the surface. The interaction of rGPIbalpha-liposomes with the vWf surface was directly related to shear rate. At high densities of rGPIbalpha and vWf, rGPIbalpha-liposomes establishing contact with the vWf surface exhibited continuous displacement with decreased velocity relative to the hydrodynamic flow, depending on receptor density and matrix concentration. At lower densities of rGPIbalpha and vWf, rGPIbalpha-liposomes stopped only transiently, in the millisecond range, on the surface. This is the first study to demonstrate that the targeting of rGPIbalpha-liposomes is specific to the vWf surface under flow conditions.     

Identification and functional analysis of a novel von Willebrand factor mutation in a family with type 2A von Willebrand disease

von Willebrand factor (VWF) is essential for normal hemostasis. VWF gene mutations cause the hemorrhagic von Willebrand disease (VWD). In this study, a 9-year-old boy was diagnosed as type 2A VWD, based on a history of abnormal bleeding, low plasma VWF antigen and activity, low plasma factor VIII activity, and lack of plasma high-molecular-weight (HMW) VWF multimers. Sequencing analysis detected a 6-bp deletion in exon 28 of his VWF gene, which created a mutant lacking D1529V1530 residues in VWF A2 domain. This mutation also existed in his family members with abnormal bleedings but not in >60 normal controls. In transfected HEK293 cells, recombinant VWF ΔD1529V1530 protein had markedly reduced levels in the conditioned medium (42±4% of wild-type (WT) VWF, p<0.01). The mutant VWF in the medium had less HMW multimers. In contrast, the intracellular levels of the mutant VWF in the transfected cells were significantly higher than that of WT (174±29%, p<0.05), indicating intracellular retention of the mutant VWF. In co-transfection experiments, the mutant reduced WT VWF secretion from the cells. By immunofluorescence staining, the retention of the mutant VWF was identified within the endoplasmic reticulum (ER). Together, we identified a unique VWF mutation responsible for the bleeding phenotype in a patient family with type 2A VWD. The mutation impaired VWF trafficking through the ER, thereby preventing VWF secretion from the cells. Our results illustrate the diversity of VWF gene mutations, which contributes to the wide spectrum of VWD.     

Purification of factor VIII and von Willebrand factor from human plasma by anion-exchange chromatography

Factor VIII (anti-hemophilia A factor) is isolated from human plasma. Purification is carried out by a combination of precipitation and chromatographic procedures. After precipitation, the first step in virus inactivation is achieved through the effect of a non-ionic detergent such as Tween 80, and a solvent, e.g. tri-n-butylphosphate (TnBP). By subsequent anion-exchange chromatography, a highly enriched product is isolated, consisting of a complex formed by factor VIII and von Willebrand factor (FVIII-vWF). This treatment also removes the virus-inactivating reagents to quantities in the low ppm range. The second step in virus inactivation is aimed specifically at the non-enveloped viruses and consists of pasteurization at temperatures higher than 60°C for 10 h. Through the addition of stabilizers, between 80% and 90% of the initial activity of FVIII is preserved during the modified pasteurisation. Along with the possibly denatured proteins the stabilizers, such as sugars, amino acids and bivalent cations, are subsequently removed by ion-exchange chromatography. The two-fold virus inactivation, by solvent/detergent treatment and subsequent pasteurisation, allows the destruction of both lipid-enveloped and non-enveloped viruses. During the procedure FVIII is stabilized through the high content of vWF. The complex consisting of FVIII and vWF can be dissociated by adding calcium ions. Subsequently both glycoproteins from this complex are separated from one another by further anion-exchange chromatography.     

Structural basis of von Willebrand factor activation by the snake toxin botrocetin

The A1 domain of von Willebrand factor (vWF) mediates platelet adhesion to sites of vascular injury by binding to the platelet receptor glycoprotein Ib (GpIb), an interaction that is regulated by hydrodynamic shear forces. The GpIb binding surface of A1 is distinct from a regulatory region, suggesting that ligand binding is controlled allosterically. Here we report the crystal structures of the "gain-of-function" mutant A1 domain (I546V) and its complex with the exogenous activator botrocetin. We show that botrocetin switches the mutant A1 back toward the wild-type conformation, suggesting that affinity is enhanced by augmenting the GpIb binding surface rather than through allosteric control. Functional studies of platelet adhesion under flow further suggest that the activation mechanism is distinct from that of the gain-of-function mutation.     

Interaction of the cysteine-rich domain of snake venom metalloproteinases with the A1 domain of von Willebrand factor promotes site-specific proteolysis of von Willebrand factor and inhibition of von Willebrand factor-mediated platelet aggregation

Snake venom metalloproteinases (SVMPs) have recently been shown to interact with proteins containing von Willebrand factor A (VWA) domains, including the extracellular matrix proteins collagen XII, collagen XIV, matrilins 1, 3 and 4, and von Willebrand factor (VWF) via their cysteine-rich domain. We extended those studies using surface plasmon resonance to investigate the interaction of SVMPs with VWF, and demonstrated that jararhagin, a PIII SVMP containing a metalloproteinase domain followed by disintegrin-like and cysteine-rich domains, catrocollastatin C, a disintegrin-like/cysteine-rich protein, and the recombinant cysteine-rich domain of atrolysin A (A/C) all interacted with immobilized VWF in a dose-dependent fashion. Binding of VWF in solution to immobilized A/C was inhibited by ristocetin and preincubation of platelets with A/C abolished ristocetin/VWF-induced platelet aggregation, indicating that the interaction of A/C with VWF is mediated by the VWA1 domain. Jararhagin cleaved VWF at sites adjacent to the VWA1 domain, whereas atrolysin C, a SVMP lacking the cysteine-rich domain, cleaved VWF at dispersed sites. A/C and catrocollastatin C completely inhibited the digestion of VWF by jararhagin, demonstrating that the specific interaction of jararhagin with VWF via the VWA1 domain is necessary for VWF proteolysis. In summary, we localized the binding site of PIII SVMPs in VWF to the A1 domain. This suggests additional mechanisms by which SVMPs may interfere with the adhesion of platelets at the site of envenoming. Thus, specific interaction of cysteine-rich domain-containing SVMPs with VWF may function to promote the hemorrhage caused by SVMP proteolysis of capillary basements and surrounding stromal extracellular matrix.     

von Willebrand factor is a cofactor for thrombin-catalyzed cleavage of the factor VIII light chain

The proteolytic activation of highly purified, heterodimeric porcine factor VIII and factor VIII-von Willebrand factor complex by thrombin was compared at I 0.17, pH 7.0, 22 degrees C. During the activation of factor VIII, heavy-chain cleavage is necessary to activate the procoagulant function, whereas light-chain cleavage is required to dissociate factor VIII from von Willebrand factor. The kinetics of activation of free factor VIII and factor VIII-von Willebrand factor complex were identical. The steady-state kinetics of thrombin-catalyzed heavy-chain cleavages and light-chain cleavage of factor VIII either free or in complex with von Willebrand factor were studied using sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis and scanning densitometry of fragments derived from 125I-labeled factor VIII. Association of factor VIII with von Willebrand factor resulted in an 8-fold increase in the catalytic efficiency (kcat/Km) of light-chain cleavage (from 7 x 10(6) to 54 x 10(6) M-1 s-1). The catalytic efficiencies of heavy-chain cleavage at position 372 (approximately 6 x 10(6) M-1 s-1) and position 740 (approximately 100 x 10(6) M-1 s-1) were not affected by von Willebrand factor. We conclude that von Willebrand factor promotes cleavage of the factor VIII light chain by thrombin which is followed by rapid dissociation of the complex, so that the rate-limiting step becomes heavy-chain cleavage at position 372. This accounts for the observation that von Willebrand factor has no effect on the kinetics of activation of factor VIII by thrombin.     

血管性血友病因子裂解蛋白酶的稳定表达及其活性检测

本研究旨在得到重组的血管性血友病因子裂解蛋白酶(ADAMTS13),进一步研究其在血栓止血中的作用。利用脂质体将编码ADAMTS13全长序列的重组质粒pSecTag-ADAMTS13转染Hela细胞,用潮霉素(Hygromycin-B)筛选得到阳性克隆细胞株,并扩大培养,收集上清。利用Ni-NTA琼脂糖柱、梯度咪唑淋洗法纯化蛋白,SDS-PAGE和Westernblotting鉴定纯化产品纯度和免疫学活性,采用GST-His双抗夹心法测定蛋白剪切活性。结果显示,成功获得一株能恒定分泌重组ADAMTS13蛋白的细胞株ADAMTS2-4,每1L培养上清可纯化得到5.8mg重组蛋白。Western blotting结果显示,ADAMTS13多抗能与重组蛋白在190kDa处显单一条带,并且蛋白具有6.4U/mL的剪切活性(每毫升正常人混合血浆中ADAMTS13活性为1U)。重组蛋白具有较好的免疫原活性和酶活性,为进一步研究ADAMTS13作用机理和运用奠定了良好的基础。     

Isolation of the factor VIII–von Willebrand factor complex directly from plasma by gel filtration

A high capacity gel filtration system was developed with the purpose of isolating factor VIII (FVIII) and von Willebrand factor (vWF) directly from plasma in significantly higher yields than obtained by cryoprecipitation, the technique most commonly used to recover FVIII–vWF from human plasma. After laboratory-scale gel filtration of plasma, a FVIII-containing fraction was collected containing about 90% of FVIII in the applied plasma and with almost tenfold higher purity than that obtained by cryoprecipitation. The gel filtration step has been scaled up for use as the initial step in the manufacturing process for a FVIII preparation (Nordiate).     

Nonsense mutations of the von Willebrand factor gene in patients with von Willebrand disease type III and type I.

von Willebrand disease (vWD) is the most common inherited bleeding disorder in humans. The disease is caused by qualitative and quantitative abnormalities of the von Willebrand factor (vWF). Genomic DNA from 25 patients with vWD type III, the most severe form of the disease, was studied using PCR followed by restriction-enzyme analysis and direct sequencing of the products. Nonsense mutations (CGA----TGA) were detected in exons 28, 32, and 45 by screening of all the 11 CGA arginine codons of the vWF gene. Two patients were found to be homozygous and five heterozygous for the mutation. Both parents and some of the relatives of the homozygous patients carry the mutation. These are the first reported examples of homozygous point mutations associated with the severe form of vWD. In the three heterozygous probands, one of the parents carried the mutation and had vWD type I. Family studies including parents and family members with or without vWD type I indicated that these three heterozygous patients are likely to be compound heterozygous. Twenty-one individuals from these seven families with vWD type I were found to be heterozygous for the mutation.     

Abnormal binding of factor VIII is linked with the substitution of glutamine for arginine 91 in von Willebrand factor in a variant form of von Willebrand disease

von Willebrand factor (vWf) is a multimeric plasma glycoprotein that functions in hemostasis as the initiator of platelet adhesion to damaged blood vessels and as the carrier of Factor VIII (FVIII). Montgomery et al. (Montgomery, R.R., Hathaway, W.E., Johnson, J., Jacobsen, L., and Muntean, W. (1982) Blood 60, 201-207) reported a variant of von Willebrand disease characterized by the abnormal interaction between FVIII and a defective vWf. To identify the molecular basis of this abnormal interaction, we isolated platelet RNA from members of one of the affected families and determined the nucleotide sequence of the FVIII-binding domain encoded by the vWf mRNA. A single G to A transition at nucleotide 2561 was linked with disease expression and results in the substitution of Gln for Arg91 in mature vWf. A restriction fragment containing this mutation was introduced into a full-length vWf expression vector, and both wild type and mutant vWf were expressed in COS-7 cells. In a solid-phase binding assay, expressed vWf was captured with anti-vWf monoclonal antibody AVW1 and then incubated with 6.25-400 milliunits of recombinant FVIII. After washing, vWf-bound FVIII activity was determined with a chromogenic assay. Mutant vWf showed reduced binding of FVIII compared with wild type, suggesting that the substitution of Gln for Arg91 is the likely basis for the abnormal vWf/FVIII interaction in this von Willebrand disease variant.     

Sulfation of Tyr1680 of human blood coagulation factor VIII is essential for the interaction of factor VIII with von Willebrand factor

The acidic region of the Factor VIII light chain was studied with regard to structural requirements for the formation of a functional von Willebrand factor (vWF)-binding site. Factor VIII mutants lacking the B domain, with additional deletions and an amino acid replacement within the sequence 1649-1689 were constructed using site-directed mutagenesis and expressed in Cos-1 cells. These mutants, which were recovered as single-chain molecules with similar specific activities, were compared in their binding to immobilized vWF. Deletion of amino acids 741-1648 or 741-1668 did not affect the binding of Factor VIII to vWF. However, a mutant with a deletion of residues 741-1689 was no longer capable of interacting with vWF. This indicates a role for residues within the sequence 1669-1689 in the formation of a vWF-binding site. When recombinant Factor VIII was expressed in the presence of chlorate, an inhibitor of protein sulfation, the resulting Factor VIII displayed strongly reduced binding to vWF. vWF binding was completely abolished when within the sequence 1669-1689 the tyrosine residue Tyr1680, which is part of a consensus tyrosine sulfation sequence, was replaced by phenylalanine. The Factor VIII sequence 1673-1689 was identified as a high affinity substrate for tyrosylprotein sulfotransferase (Km = 57 microM) in cell-free sulfation studies. It is concluded that sulfation of Tyr1680 is required for the interaction of Factor VIII with vWF. Two synthetic peptides that represent the sequence 1673-1689, but differ with respect to sulfation of Tyr1680 are shown to have vWF binding affinity that is considerably lower than the Factor VIII protein. Several models to accommodate our findings are discussed.     

The effect of thrombin on the complex between factor VIII and von Willebrand factor

Purified human factor FVIII (FVIII; 6000-8000 U/mg) was radiolabeled and bound to immobilized von Willebrand factor (vWF). The complex was incubated with human thrombin. Thrombin induced a release of 65% of the radioactivity initially bound. Released FVIII fragments and fragments remaining bound during incubation with thrombin were analyzed using gel electrophoresis. This led to the following observations. Released fragments largely consisted of Mr-70000 and Mr-50000 fragments; Mr-90000 and Mr-80000 fragments were only found in the fractions remaining bound to vWF and decreased with time. In contrast to these digestion products of FVIII, the Mr-42000 heavy-chain fragment remained bound to vWF, comprising the larger part of the radioactivity after a 2-h incubation. No thrombin-induced cleavages were observed in vWF. Furthermore, vWF-coated wells preincubated with thrombin were still able to bind 125I-FVIII. These results implicate a new concept for the activation of vWF-bound FVIII. Activation is a multistep process in which several cleavages are necessary to produce and release a coagulant-active FVIII molecule (FVIIIa), which is probably an Mr-50000/70000 heterodimer. Inactivation of FVIIIa is likely to be the result of a nonproteolytic dissociation due to loss of the joining divalent cation(s).     

Mechanism of inhibition by trinitrophenylation of the ristocetin cofactor activity of von Willebrand factor

In the presence of ristocetin, von Willebrand factor is capable of agglutinating washed platelets. Modification of only a small percentage of amino groups of von Willebrand factor with trinitrobenzenesulfonic acid markedly inhibits this platelet agglutinating activity. 90% of the platelet agglutinating activity is lost after modification of only 10% of the von Willebrand factor amino groups. Since only the higher molecular weight forms of the heterogeneous von Willebrand factor polymers possess this platelet agglutinating activity, it was important to demonstrate that trinitrophenylation did not alter the degree of von Willebrand factor polymerization. This was accomplished by agarose gel electrophoresis. Subsequent direct binding and competitive binding studies demonstrated that trinitrophenylation markedly impairs the ability of von Willebrand factor to bind to the platelet surface. Thus the loss of platelet agglutinating activity upon modification of only a small fraction of the amino groups of von Willebrand factor is attributable to impaired binding of the modified von Willebrand factor to the platelet surface.     

Isolation and characterization of a collagen binding domain in human von Willebrand factor

von Willebrand factor binds to fibrillar type I collagen in a rapid, temperature-independent, reversible, specific, and saturable manner. Evaluation of binding isotherms by Scatchard-type analysis demonstrated that 6-18 micrograms of von Willebrand factor bind per mg of collagen, with Ka between 2 and 8 X 10(8) M-1. Five distinct tryptic fragments, purified under denaturing and reducing conditions and representing over 75% of the molecular mass of the von Willebrand factor subunit, were tested for their capacity to inhibit the von Willebrand factor-collagen interaction. Complete inhibition was obtained with a 52/48-kDa fragment at a concentration of approximately 1 microM. The location of this fragment in the subunit was established to be between Val-449 and Lys-728. Fifteen monoclonal antibodies against the 52/48-kDa fragment inhibited von Willebrand factor binding to collagen. Six antibodies against other portions of the von Willebrand factor subunit had no inhibitory effect. The tryptic fragment was a competitive inhibitor of von Willebrand factor binding to collagen and, therefore, recognizes the same interaction site as the intact molecule. These studies precisely define a domain in the von Willebrand factor subunit that interacts with type I collagen.     

Regulation of von Willebrand factor binding to the platelet glycoprotein Ib-IX by a membrane skeleton-dependent inside-out signal

The platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX (GPIb-IX), mediates initial platelet adhesion and activation. We show here that the receptor function of GPIb-IX is regulated intracellularly via its link to the filamin-associated membrane skeleton. Deletion of the filamin binding site in GPIb(alpha) markedly enhances ristocetin- (or botrocetin)-induced vWF binding and allows GPIb-IX-expressing cells to adhere to immobilized vWF under both static and flow conditions. Cytochalasin D (CD) that depolymerizes actin also enhances vWF binding to wild type GPIb-IX. Thus, vWF binding to GPIb-IX is negatively regulated by the filamin-associated membrane skeleton. In contrast to native vWF, binding of the isolated recombinant vWF A1 domain to wild type and filamin binding-deficient mutants of GPIb-IX is comparable, suggesting that the membrane skeleton-associated GPIb-IX is in a state that prevents access to the A1 domain in macromolecular vWF. In platelets, there is a balance of membrane skeleton-associated and free forms of GPIb-IX. Treatment of platelets with CD increases the free form and enhances vWF binding. CD also reverses the inhibitory effects of prostaglandin E1 on vWF binding to GPIb-IX. Thus, GPIb-IX-dependent platelet adhesion is doubly controlled by vWF conformation and a membrane skeleton-dependent inside-out signal.