Light Chain of Factor VIII Is Sufficient for Accelerating Cleavage of von Willebrand Factor by ADAMTS13 Metalloprotease

We previously demonstrated that coagulation factor VIII (FVIII) accelerates proteolytic cleavage of von Willebrand factor (VWF) by A disintegrin and metalloprotease with thrombospondin type 1 repeats (ADAMTS13) under fluid shear stress. In this study, the structural elements of FVIII required for the rate-enhancing effect and the biological relevance of this cofactor activity are determined using a murine model. An isolated light chain of human FVIII (hFVIII-LC) increases proteolytic cleavage of VWF by ADAMTS13 under shear in a concentration-dependent manner. The maximal rate-enhancing effect of hFVIII-LC is ∼8-fold, which is comparable with human full-length FVIII and B-domain deleted FVIII (hFVIII-BDD). The heavy chain (hFVIII-HC) and the light chain lacking the acidic (a3) region (hFVIII-LCΔa3) have no effect in accelerating VWF proteolysis by ADAMTS13 under the same conditions. Although recombinant hFVIII-HC and hFVIII-LCΔa3 do not detectably bind immobilized VWF, recombinant hFVIII-LC binds VWF with high affinity (K_D, ∼15 nm). Moreover, ultra-large VWF multimers accumulate in the plasma of fVIII^−/− mice after hydrodynamic challenge but not in those reconstituted with either hFVIII-BDD or hFVIII-LC. These results suggest that the light chain of FVIII, which is not biologically active for clot formation, is sufficient for accelerating proteolytic cleavage of VWF by ADAMTS13 under fluid shear stress and (patho) physiological conditions. Our findings provide novel insight into the molecular mechanism of how FVIII regulates VWF homeostasis.     

Distinct Roles of Ser-764 and Lys-773 at the N Terminus of von Willebrand Factor in Complex Assembly with Coagulation Factor VIII

Complex formation between coagulation factor VIII (FVIII) and von Willebrand factor (VWF) is of critical importance to protect FVIII from rapid in vivo clearance and degradation. We have now employed a chemical footprinting approach to identify regions on VWF involved in FVIII binding. To this end, lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII, which does not bind VWF. Nano-LC-MS analysis showed that the lysine residues of almost all identified VWF peptides were not differentially modified upon incubation of VWF with FVIII or activated FVIII. However, Lys-773 of peptide Ser-766–Leu-774 was protected from chemical modification in the presence of FVIII. In addition, peptide Ser-764–Arg-782, which comprises the first 19 amino acid residues of mature VWF, showed a differential modification of both Lys-773 and the α-amino group of Ser-764. To verify the role of Lys-773 and the N-terminal Ser-764 in FVIII binding, we employed VWF variants in which either Lys-773 or Ser-764 was replaced with Ala. Surface plasmon resonance analysis and competition studies revealed that VWF(K773A) exhibited reduced binding to FVIII and the FVIII light chain, which harbors the VWF-binding site. In contrast, VWF(S764A) revealed more effective binding to FVIII and the FVIII light chain compared with WT VWF. The results of our study show that the N terminus of VWF is critical for the interaction with FVIII and that Ser-764 and Lys-773 have opposite roles in the binding mechanism.     

Factor VIII and Platelets Synergistically Accelerate Cleavage of von Willebrand Factor by ADAMTS13 under Fluid Shear Stress

Previous studies have demonstrated that factor VIII (FVIII) or platelets alone increase cleavage of von Willebrand factor (VWF) by ADAMTS13 under mechanically induced shear stresses. We show in this study that the combination of FVIII and platelets at the physiological concentrations is more effective than either one alone. In the absence of FVIII, lyophilized platelets increase the formation of cleavage product by 2–3-fold. However, in the presence of physiological concentration of FVIII (1 nm), the formation of VWF cleavage product increases dramatically as a function of increasing platelets with the maximal rate enhancement of ∼8-fold. Conversely, in the presence of a physiological concentration of lyophilized platelets (150 × 10³/μl), the half-maximal concentration of FVIII required to accelerate VWF proteolysis by ADAMTS13 reduces by ∼10-fold (to ∼0.3 nm) compared with that in the absence of platelets (∼3.0 nm). Further studies using the FVIII derivative that lacks an acidic region (a3), an antiplatelet glycoprotein 1bα IgG, and a purified recombinant VWF-A1 domain or glycoprotein 1bα-stripped platelets demonstrate that the synergistic rate-enhancing effect of FVIII and platelets depends on their specific binding interactions with VWF. Our findings suggest that FVIII and platelets are cofactors that regulate proteolysis of multimeric VWF by ADAMTS13 under physiological conditions.     

Structure and dynamics of the von Willebrand Factor C6 domain

Von Willebrand disease (VWD) is a bleeding disorder with different levels of severity. VWD-associated mutations are located in the von Willebrand factor (VWF) gene, coding for the large multidomain plasma protein VWF with essential roles in hemostasis and thrombosis. On the one hand, a variety of mutations in the C-domains of VWF are associated with increased bleeding upon vascular injury. On the other hand, VWF gain-of-function (GOF) mutations in the C4 domain have recently been identified, which induce an increased risk of myocardial infarction. Mechanistic insights into how these mutations affect the molecular behavior of VWF are scarce and holistic approaches are challenging due to the multidomain and multimeric character of this large protein. Here, we determine the structure and dynamics of the C6 domain and the single nucleotide polymorphism (SNP) variant G2705R in C6 by combining nuclear magnetic resonance spectroscopy, molecular dynamics simulations and aggregometry. Our findings indicate that this mutation mostly destabilizes VWF by leading to a more pronounced hinging between both subdomains of C6. Hemostatic parameters of variant G2705R are close to normal under static conditions, but the missense mutation results in a gain-of-function under flow conditions, due to decreased VWF stem stability. Together with the fact that two C4 variants also exhibit GOF characteristics, our data underline the importance of the VWF stem region in VWF’s hemostatic activity and the risk of mutation-associated prothrombotic properties in VWF C-domain variants due to altered stem dynamics.     

Evaluation of a new semi-automated Hydragel 11 von Willebrand factor multimers assay kit for routine use

BackgroundAccurate diagnosis and classification of von Willebrand disease (VWD) are essential for optimal management. The von Willebrand factor multimers analysis (VWF:MM) is an integral part of the diagnostic process in the phenotypic classification, especially in discrepant cases. The aim of this study was to evaluate the performance of a new Hydragel 11VWF multimer assay (H11VW).MethodsAnalytical performance characteristics such as repeatability (intra-assay variability, in gel between track variation), reproducibility (inter-assay variability, between gel variation), sensitivity, EQA performance and differences between two commercially available VWF:MM kits (H5VW and H11VW) were analysed in healthy volunteers'' plasmas using in-house prepared reference plasma.ResultsRepeatability and reproducibility results of H11VW demonstrated acceptable and equivalent performance with previously verified H5VW. Participation in EQA was successful. No statistically significant difference was detected between H5VW and H11VW kits for different fractions of multimers: LMWM p=0.807; IMWM p=0.183; HMWM p=0.774.ConclusionsH11VW demonstrated acceptable analytical performance characteristics. H11VW kit conveniently offers a more significant number of samples on a single gel. H5VW and H11VW kits can be used in daily practice interchangeably.     

Structural Basis of Regulation of von Willebrand Factor Binding to Glycoprotein Ib

Activation by elongational flow of von Willebrand factor (VWF) is critical for primary hemostasis. Mutations causing type 2B von Willebrand disease (VWD), platelet-type VWD (PT-VWD), and tensile force each increase affinity of the VWF A1 domain and platelet glycoprotein Ibα (GPIbα) for one another; however, the structural basis for these observations remains elusive. Directed evolution was used to discover a further gain-of-function mutation in A1 that shifts the long range disulfide bond by one residue. We solved multiple crystal structures of this mutant A1 and A1 containing two VWD mutations complexed with GPIbα containing two PT-VWD mutations. We observed a gained interaction between A1 and the central leucine-rich repeats (LRRs) of GPIbα, previously shown to be important at high shear stress, and verified its importance mutationally. These findings suggest that structural changes, including central GPIbα LRR-A1 contact, contribute to VWF affinity regulation. Among the mutant complexes, variation in contacts and poor complementarity between the GPIbα β-finger and the region of A1 harboring VWD mutations lead us to hypothesize that the structures are on a pathway to, but have not yet reached, a force-induced super high affinity state.     

双Intein介导3片段vWF的反式剪接

von Willebrand因子(vWF)基因突变导致血管性血友病(VWD),由于其基因过大在基因治疗研究中难以为多数病毒载体携带.利用双内含肽(intein)的蛋白质反式剪接功能研究断裂成3段的vWF基因分别表达后在蛋白水平的连接,旨在为vWF基因的3载体联合转移应用于VWD基因治疗研究提供依据.将vWF cDNA于满足剪接所需的保守性氨基酸Cys1099、Ser2004的密码子前断裂为3段(N、M和C),分别与splitSspDnaE intein的N端(En)、C端(Ec)和splitSspDnaB intein的N端(Bn)、C端(Bc)编码序列融合,构建到原核表达载体pET-28a(+)中的His-Tag的下游,得到3种表达载体pET-NEn、pET-EcMBn和pET-BcC.分别转化感受态大肠杆菌BL21(DE3)细胞,经IPTG诱导表达后,以SDS-PAGE分析融合蛋白的表达,并进一步用His-Tag的特异性抗体进行分析;亲和层析纯化分别表达的带His-Tag标签的3段蛋白,复性后体外混合进行剪接实验以观察3片段vWF的连接.结果显示,3段预期大小的融合intein的vWF蛋白均有表达,用His-Tag抗体进行的Western印迹得到进一步证实;3段纯化的蛋白混合后可见明显的剪接条带形成,与vWF的预期分子量大小一致,表明双intein通过蛋白质反式剪接可有效连接3个片段的vWF,为进一步应用蛋白质剪接技术的3重载体真核细胞转vWF基因奠定了基础.     

Phylogenetic and functional analysis of histidine residues essential for pH-dependent multimerization of von Willebrand factor

von Willebrand factor (VWF) is a multimeric plasma protein that mediates platelet adhesion to sites of vascular injury. The hemostatic function of VWF depends upon the formation of disulfide-linked multimers, which requires the VWF propeptide (D1D2 domains) and adjacent D′D3 domains. VWF multimer assembly occurs in the trans-Golgi at pH ∼6.2 but not at pH 7.4, which suggests that protonation of one or more His residues (pK_a ∼6.0) mediates the pH dependence of multimerization. Alignment of 30 vertebrate VWF sequences identified 13 highly conserved His residues in the D1D2D′D3 domains, and His-to-Ala mutagenesis identified His³⁹⁵ and His⁴⁶⁰ in the D2 domain as critical for VWF multimerization. Replacement of His³⁹⁵ with Lys or Arg prevented multimer assembly, suggesting that reversible protonation of this His residue is essential. In contrast, replacement of His⁴⁶⁰ with Lys or Arg preserved normal multimer assembly, whereas Leu, Met, and Gln did not, indicating that the function of His⁴⁶⁰ depends primarily upon the presence of a positive charge. These results suggest that pH sensing by evolutionarily conserved His residues facilitates the assembly and packaging of VWF multimers upon arrival in the trans-Golgi.     

Interaction of Shiga Toxin with the A-domains and Multimers of von Willebrand Factor

Shiga toxin (Stx) produced by enterohemorrhagic Escherichia coli causes diarrhea-associated hemolytic-uremic syndrome (DHUS), a severe renal thrombotic microangiopathy. We investigated the interaction between Stx and von Willebrand Factor (VWF), a multimeric plasma glycoprotein that mediates platelet adhesion, activation, and aggregation. Stx bound to ultra-large VWF (ULVWF) secreted from and anchored to stimulated human umbilical vein endothelial cells, as well as to immobilized VWF-rich human umbilical vein endothelial cell supernatant. This Stx binding was localized to the A1 and A2 domain of VWF monomeric subunits and reduced the rate of ADAMTS-13-mediated cleavage of the Tyr¹⁶⁰⁵-Met¹⁶⁰⁶ peptide bond in the A2 domain. Stx-VWF interaction and the associated delay in ADAMTS-13-mediated cleavage of VWF may contribute to the pathophysiology of DHUS.     

Stability of von Willebrand Factor secretion in different human endothelial hybrid cell lines

Endothelial cells form a highly differentiated tissue on the inner surface of blood vessels. One of the typical characteristics is the expression of von Willebrand Factor, a protein that participates in blood coagulation. Thein vitro cultivation of endothelial cells is limited by the fact that primary cells become senescent after 40 generation doublings. We have immortalized human endothelial cells by somatic cell hybridization. Primary cells were fused to different tumor cell lines of murine and human origin. The degree of differentiation of the resulting hybrids was analyzed by characterizing the expression of von Willebrand Factor. This protein was identified intracellularly and in the culture supernatant. During long-term cultivation the hybrid cells showed a tendency to lose this differentiated property even after several subcloning steps. However by fusing them with primary endothelial cells a second time, cell lines expressing von Willebrand Factor for more than 180 population doublings were generated.     

Intracellular storage and regulated secretion of von Willebrand factor in quantitative von Willebrand disease

Several missense mutations in the von Willebrand Factor (VWF) gene of von Willebrand disease (VWD) patients have been shown to cause impaired constitutive secretion and intracellular retention of VWF. However, the effects of those mutations on the intracellular storage in Weibel-Palade bodies (WPBs) of endothelial cells and regulated secretion of VWF remain unknown. We demonstrate, by expression of quantitative VWF mutants in HEK293 cells, that four missense mutations in the D3 and CK-domain of VWF diminished the storage in pseudo-WPBs, and led to retention of VWF within the endoplasmic reticulum (ER). Immunofluorescence and electron microscopy data showed that the pseudo-WPBs formed by missense mutant C1060Y are indistinguishable from those formed by normal VWF. C1149R, C2739Y, and C2754W formed relatively few pseudo-WPBs, which were often short and sometimes round rather than cigar-shaped. The regulated secretion of VWF was impaired slightly for C1060Y but severely for C1149R, C2739Y, and C2754W. Upon co-transfection with wild-type VWF, both intracellular storage and regulated secretion of all mutants were (partly) corrected. In conclusion, defects in the intracellular storage and regulated secretion of VWF following ER retention may be a common mechanism underlying VWD with a quantitative deficiency of VWF.     

Rearranging Exosites in Noncatalytic Domains Can Redirect the Substrate Specificity of ADAMTS Proteases

ADAMTS proteases typically employ some combination of ancillary C-terminal disintegrin-like, thrombospondin-1, cysteine-rich, and spacer domains to bind substrates and facilitate proteolysis by an N-terminal metalloprotease domain. We constructed chimeric proteases and substrates to examine the role of C-terminal domains of ADAMTS13 and ADAMTS5 in the recognition of their physiological cleavage sites in von Willebrand factor (VWF) and aggrecan, respectively. ADAMTS5 cleaves Glu(373)-Ala(374) and Glu(1480)-Gly(1481) bonds in bovine aggrecan but does not cleave VWF. Conversely, ADAMTS13 cleaves the Tyr(1605)-Met(1606) bond of VWF, which is exposed by fluid shear stress but cannot cleave aggrecan. Replacing the thrombospondin-1/cysteine-rich/spacer domains of ADAMTS5 with those of ADAMTS13 conferred the ability to cleave the Glu(1615)-Ile(1616) bond of VWF domain A2 in peptide substrates or VWF multimers that had been sheared; native (unsheared) VWF multimers were resistant. Thus, by recombining exosites, we engineered ADAMTS5 to cleave a new bond in VWF, preserving physiological regulation by fluid shear stress. The results demonstrate that noncatalytic thrombospondin-1/cysteine-rich/spacer domains are principal modifiers of substrate recognition and cleavage by both ADAMTS5 and ADAMTS13. Noncatalytic domains may perform similar functions in other ADAMTS family members.     

Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction

In vivo, clotting Factor VIII (FVIII) circulates in plasma bound to von Willebrand factor (vWF), and the vWF:FVIII complex prevents binding of FVIII to phosphatidylserine (PS). Activation of FVIII by thrombin releases FVIII from vWF, and subsequently FVIII binds to PS exposed on activated platelets and forms the tenase complex together with clotting Factor IX. In vitro, during serum free production of recombinant FVIII (rFVIII), production cells also expose PS, and since vWF is not present to hinder interaction of secreted rFVIII with PS, rFVIII is partly associated with the cell membrane of the production cells. Recently, we showed that as much as 90% of secreted rFVIII is bound to transiently transfected production cells during serum free conditions. In this study, we investigated the effect of including vWF in the serum free medium, and demonstrate that addition of vWF results in release of active membrane bound rFVIII to the culture medium. Moreover, the attachment of rFVIII to cell membranes of un-transfected HEK293 cells was studied in the presence of compounds that competes for interactions between rFVIII and PS. Competitive assays between iodinated rFVIII (¹²⁵I-rFVIII) and annexin V or ortho-phospho-l-serine (OPLS) demonstrated that annexin V and OPLS were able to reduce the membrane bound fraction of rFVIII by 70% and 30%, respectively. Finally, adding OPLS to CHO cells stably expressing FVIII increased the yield by 50%. Using this new knowledge, the recovery of rFVIII could be increased considerably during serum free production of this therapeutic protein.     

Structure determination of the major asparagine-linked sugar chain of human factor VIII--von Willebrand factor

N-glycosidically-linked glycans released by hydrazinolysis of human factor VIII/von Willebrand factor (FVIII/vWf) were separated by high-voltage electrophoresis. Five fractions were obtained, one of them representing 60% of the total amount of the N-glycosidically-linked glycans of FVIII/vWf. On the basis of the carbohydrate composition, methylation analysis and 500 MHz 1H-NMR spectroscopy, we describe the primary structure of this major glycan which is of the monosialylated and monofucosylated biantennary N-acetyllactosaminic type.     

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.     

Mutation G1629E Increases von Willebrand Factor Cleavage via a Cooperative Destabilization Mechanism

The large multimeric glycoprotein von Willebrand Factor (VWF) plays a pivotal adhesive role during primary hemostasis. VWF is cleaved by the protease ADAMTS13 as a down-regulatory mechanism to prevent excessive VWF-mediated platelet aggregation. For each VWF monomer, the ADAMTS13 cleavage site is located deeply buried inside the VWF A2 domain. External forces in vivo or denaturants in vitro trigger the unfolding of this domain, thereby leaving the cleavage site solvent-exposed and ready for cleavage. Mutations in the VWF A2 domain, facilitating the cleavage process, cause a distinct form of von Willebrand disease (VWD), VWD type 2A. In particular, the VWD type 2A Gly1629Glu mutation drastically accelerates the proteolytic cleavage activity, even in the absence of forces or denaturants. However, the effect of this mutation has not yet been quantified, in terms of kinetics or thermodynamics, nor has the underlying molecular mechanism been revealed. In this study, we addressed these questions by using fluorescence correlation spectroscopy, molecular dynamics simulations, and free energy calculations. The measured enzyme kinetics revealed a 20-fold increase in the cleavage rate for the Gly1629Glu mutant compared with the wild-type VWF. Cleavage was found cooperative with a cooperativity coefficient n = 2.3, suggesting that the mutant VWF gives access to multiple cleavage sites of the VWF multimer at the same time. According to our simulations and free energy calculations, the Gly1629Glu mutation causes structural perturbation in the A2 domain and thereby destabilizes the domain by ～10 kJ/mol, promoting its unfolding. Taken together, the enhanced proteolytic activity of Gly1629Glu can be readily explained by an increased availability of the ADAMTS13 cleavage site through A2-domain-fold thermodynamic destabilization. Our study puts forward the Gly1629Glu mutant as a very efficient enzyme substrate for ADAMTS13 activity assays.     

内含肽介导三段vWF基因真核细胞转移的翻译后连接及其功能性多聚体形成

vWF(von Willebrand factor)是一种超大分子质量的血浆多聚体糖蛋白,在血栓形成和生理凝血过程中发挥重要作用,其质和/或量的缺陷导致血管性血友病(VWD),由于VWD为单基因病,且vWF为分泌性蛋白,基于基因转移的基因治疗无需特异的靶器官,因此VWD特别适合于基因治疗,但vWF基因过大(8.4 kb),难以为多数病毒载体特别是优点较多的腺相关病毒(AAV)载体承载.运用内含肽(intein)的蛋白质反式剪接功能,研究了三重载体真核细胞共转断裂3段的vWF基因,以期通过转基因翻译后的蛋白质剪接作用形成完整的功能性vWF蛋白.将vWF的cDNA于满足剪接所需的保守性氨基酸Cys1099、Ser2004的密码子前断裂为3段,分别与2种不同的内含肽即Ssp DnaE内含肽和Ssp DnaB内含肽编码序列融合,构建到真核表达载体pcDNA3.1(+),得到3个分别融合内含肽的vWF片段基因真核表达载体,共转染培养的293细胞,通过瞬时表达,电泳观察培养上清中的vWF多聚体形态,分析vWF蛋白量和凝血Ⅷ因子(FⅧ)结合力;通过共转FⅧ基因,分析了培养上清中的FⅧ蛋白量及生物活性.结果显示,通过内含肽的蛋白质反式剪接作用,共转内含肽融合的三片段vWF基因细胞上清,表现与正常人血浆和转vWF基因阳性对照细胞相似的vWF多聚体模式和FⅧ结合力,而且可明显提高转FⅧ基因后表达的FⅧ蛋白的分泌量和活性,提示剪接vWF蛋白的FⅧ载体功能的恢复.结果表明,内含肽可作为一种有效的技术手段进行三重载体共转断裂的vWF基因,为进一步基于内含肽的三重AAV转断裂vWF基因应用于VWD基因治疗研究、克服AAV的容量限制提供了依据.     

Influence of Von Willebrand Disease (VWD) and pregnancy on the expression of angiogenic factors in the porcine female reproductive tract

Von Willebrand Disease (VWD) is a heritable disorder caused by defects of the Von Willebrand Factor (VWF), leading to deficiencies in coagulation and also angiogenesis. Women affected by VWD frequently show bleeding concerning the reproductive tract and may present with increased rates of miscarriages. We used a porcine model representing VWD type 1 and type 3 as well as the wildtype. Samples were obtained from the reproductive tract of non-pregnant sows and sows pregnant at time of placentation. Relative expression of the genes CALR, CCN2, CXCL8, ECE1, EDN1, F8, IGFBP7, and LGALS3 was analyzed. CCN2 and FVIII proteins were additionally analyzed using immunohistochemistry. In uterus and ovary significant upregulation of CCN2 was seen in non-pregnant pigs affected by VWD. This might be caused by the higher VEGFA-levels in these pigs and could have an influence angiogenesis. During pregnancy, CCN2 expression increased in wildtype pig uteri but hardly changed in those of pregnant pigs affected by VWD, presumably because the expression level in the latter pigs already was significantly increased before pregnancy. F8 expression was significantly reduced in uterus and ovary of VWD-affected pigs. VWF is known to protect FVIII from decomposition and a lack of VWF leads to lower levels of FVIII. Our results suggest that a reduced F8 expression primarily might contribute to those reduced FVIII levels in VWD-affected pigs. Additional significant results involving the pregnant pigs were detected for CALR, EDN1, and LGALS3. These genes are promising candidates for more detailed future studies.     

Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate.     

The N-terminal Flanking Region of the A1 Domain Regulates the Force-dependent Binding of von Willebrand Factor to Platelet Glycoprotein Ibα

Binding of platelet glycoprotein Ibα (GPIbα) to von Willebrand factor (VWF) initiates platelet adhesion to disrupted vascular surface under arterial blood flow. Flow exerts forces on the platelet that are transmitted to VWF-GPIbα bonds, which regulate their dissociation. Mutations in VWF and/or GPIbα may alter the mechanical regulation of platelet adhesion to cause hemostatic defects as found in patients with von Willebrand disease (VWD). Using a biomembrane force probe, we observed biphasic force-decelerated (catch) and force-accelerated (slip) dissociation of GPIbα from VWF. The VWF A1 domain that contains the N-terminal flanking sequence Gln¹²³⁸–Glu¹²⁶⁰ (1238-A1) formed triphasic slip-catch-slip bonds with GPIbα. By comparison, using a short form of A1 that deletes this sequence (1261-A1) abolished the catch bond, destabilizing its binding to GPIbα at high forces. Importantly, shear-dependent platelet rolling velocities on these VWF ligands in a flow chamber system mirrored the force-dependent single-bond lifetimes. Adding the Gln¹²³⁸–Glu¹²⁶⁰ peptide, which interacted with GPIbα and 1261-A1 but not 1238-A1, to whole blood decreased platelet attachment under shear stress. Soluble Gln¹²³⁸–Glu¹²⁶⁰ reduced the lifetimes of GPIbα bonds with VWF and 1238-A1 but rescued the catch bond of GPIbα with 1261-A1. A type 2B VWD 1238-A1 mutation eliminated the catch bond by prolonging lifetimes at low forces, a type 2M VWD 1238-A1 mutation shifted the respective slip-catch and catch-slip transition points to higher forces, whereas a platelet type VWD GPIbα mutation enhanced the bond lifetime in the entire force regime. These data reveal the structural determinants of VWF activation by hemodynamic force of the circulation.