Functional analysis of a type IIB von Willebrand disease missense mutation: increased binding of large von Willebrand factor multimers to platelets.

Type IIB von Willebrand disease is an autosomal dominant bleeding disorder characterized by the selective loss of high molecular weight von Willebrand factor (vWF) multimers in plasma, presumably due to their abnormally increased reactivity with platelets. We and others have recently identified a panel of missense mutations clustered in the platelet glycoprotein Ib binding domain of vWF from patients with type IIB von Willebrand disease. We now report functional analysis of one of the most frequent type IIB missense mutations, Arg-543----Trp (vWF R543W). vWF from a human umbilical vein endothelial cell culture heterozygous for the vWF R543W mutation showed markedly increased binding of large vWF multimers to platelets in the presence of a low dose of ristocetin compared to vWF from a normal control culture. Recombinant vWF containing the vWF R543W mutation expressed in COS-7 cells also demonstrated increased binding of large vWF multimers. Mixed multimers obtained by cotransfection of mutant and wild-type cDNAs showed partial dominance of the vWF R543W mutation. Thus these data demonstrate that the vWF R543W mutation alone is sufficient to confer increased binding of large vWF multimers to platelets in a dominant fashion and that no other factors relating to vWF posttranslational processing or secretion in endothelial cells are required for this effect.     

Storage and secretion of von Willebrand factor by endothelial cells

Endothelial cells synthesize and store von Willebrand factor. We have studied the storage and secretion of von Willebrand factor in cultured human umbilical vein endothelial cells. In particular, we were interested in the nature of the storage compartment and the effects of perturbation on the storage and secretion processes. The storage compartment for von Willebrand factor was isolated from homogenates of endothelial cells. By an immunostaining technique the isolated vesicles stained for von Willebrand factor. The staining pattern was similar to that of Weibel-Palade bodies in intact endothelial cells. We concluded that the storage compartment containing von Willebrand factor is identical to the Weibel-Palade body. The von Willebrand factor of the isolated storage vesicles is predominantly constructed of polypeptide chains with a Mr of 220 kD. On the other hand, von Willebrand factor continuously secreted by endothelial cells is constructed of both a 220 kD and a larger precursor (apparent Mr of 275 kD) subunit. The storage vesicles contain von Willebrand factor that supports ristocetin-induced platelet aggregation. Thus, endothelial cells store fully processed, biologically active von Willebrand factor within Weibel-Palade bodies. Short-term (less than 1 h) treatment of endothelial cells with the perturbing phorbol ester 4 beta-phorbol-12-myristate-13-acetate (PMA) results in release of cellular stored von Willebrand factor. 24-48 h after exposure to PMA the endothelial cell distribution of von Willebrand factor is changed distinctly. While the contents of the von Willebrand factor storage sites in the cells are gradually restored within 48 h, enhanced amounts of von Willebrand factor are secreted into the medium. The number as well as the size of von Willebrand factor storage granules in the endothelial cells increase after exposure to phorbol ester, as determined by immunofluorescence microscopy. Phorbol ester treated cells release stored von Willebrand factor 48 h after they have been stimulated. PMA decreases the von Willebrand factor contents of the extracellular matrix; the deposition of von Willebrand factor in the subendothelium is blocked by PMA, whereas the degradation of matrix von Willebrand factor is not affected. Thus, perturbation of endothelial cells changes the cellular distribution of von Willebrand factor.     

Influence of mutations and size of multimers in type II von Willebrand disease upon the function of von Willebrand factor

We compared the properties of plasma von Willebrand factor (vWF) from normal individuals and from two patients with type IIA (Glu875Lys) and type IIB (duplication of Met 540) von Willebrand disease (vWD) with the corresponding fully multimerized recombinant proteins. We included cryosupernatant from normal human plasma and type IIA plasma (Cys509Arg). Functions of vWF were analyzed by binding assays to platelets in the presence of ristocetin or botrocetin. Parameters of binding (number of binding sites per vWF subunit, and dissociation constant Kd) were quantitatively estimated from the binding isotherms of 125I-botrocetin or glycocalicin to vWF, independently of the size of the multimers. We found that ristocetin- or botrocetin-induced binding to platelets was correlated in all cases with the size of vWF multimers. In the absence of inducer, only type IIB rvWF Met-Met540 spontaneously bound to platelets. No significant difference of binding of purified botrocetin to vWF was found between normal and patients' plasma, or between wild-type rvWF (rvWF-WT) and rvWF-Lys875. In contrast, affinity of botrocetin for type IIB rvWF Met-Met540 was decreased. Botrocetin-induced binding of glycocalicin to vWF from all plasma and cryosupernatant was similar. Compared with rvWF-WT, binding of glycocalicin to rvWF-Lys875 was normal. In contrast, the affinity for type IIB rvWF Met-Met540 was 10-fold greater. Thus, our data suggest that, in the patients tested, the abnormal IIA phenotype results from the lack of large-sized multimers and is independent of the point mutations. In contrast, the type IIB mutation is directly involved by providing a conformation to the vWF subunits that allows the high molecular weight multimers to spontaneously interact with platelet glycoprotein Ib.     

Leu 697-->Val mutation in mature von Willebrand factor is responsible for type IIB von Willebrand disease

Type IIB von Willebrand disease is characterized by the selective loss of high molecular weight von Willebrand factor (vWF) multimers from plasma and enhanced platelet agglutination of platelet-rich-plasma in the presence of low concentrations of ristocetin. We identified, in two related patients, a C-->G transversion resulting in the substitution of Valine for Leucine at position 697 of the mature subunit of vWF. We reproduced this mutation in vWF cDNA and expressed the recombinant protein in Cos-7 cells. The subunit composition and multimeric structure of mutated protein (rvWFLeu697Val) were similar to the wild- type recombinant (WTrvWF). Ristocetin-induced binding of rvWFLeu697Val to platelets was markedly increased in the presence of low doses of ristocetin and slightly increased with botrocetin as compared with that for WTrvWF, whereas collagen binding was not affected by the mutation. These data show that the Leu 697-->Val substitution is not a rare polymorphism but is responsible for the subtype IIB characteristic abnormalities identified in the two affected patients; however, it is not located in the area of vWF (amino acid 540 to amino acid 578) where most of the other type IIB mutations have already been reported.     

Interaction of purified type IIB von Willebrand factor with the platelet membrane glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and initiates aggregation.

Von Willebrand factor (vWF) was purified from the plasma of a patient with type IIB von Willebrand disease (vWF from such a patient, IIB vWF) who had a normal platelet count and showed no evidence of spontaneous platelet aggregation. Large multimers of IIB vWF were absent from purified preparations and from plasma. Ristocetin-induced platelet aggregation was enhanced by purified IIB vWF. The aggregation of washed normal platelets mixed with IIB vWF (0.4 microgram/ml) required lower amounts of ristocetin than the aggregation of normal platelets mixed with the same concentrations of normal vWF. Moreover, purified IIB vWF alone induced aggregation of platelet-rich plasma at concentrations as low as 10 micrograms of IIB vWF/ml in the absence of any other agonist. Aggregation was blocked by a monoclonal antibody against the platelet membrane glycoprotein, GPIb, as well as by an anti-GPIIb/IIIa antibody. Washed platelet suspensions were promptly aggregated by IIB vWF only when fibrinogen and CaCl2 were added to the mixture. Purified IIB vWF induces the binding of fibrinogen to platelets. Such binding was blocked by the anti-GPIb monoclonal antibody as well as by the anti-GPIIb/IIIa monoclonal antibody that inhibited aggregation. A second anti-GPIIb/IIIa antibody, which has the property of blocking vWF but not fibrinogen binding to platelets, blocked neither aggregation nor fibrinogen binding induced by IIB vWF. These studies demonstrate that platelet aggregation is triggered by the initial interaction of IIB vWF with GPIb which is followed by exposure of fibrinogen binding sites on GPIIb/IIIa. Fibrinogen binds to these sites and acts as a necessary cofactor for the aggregation response.     

von Willebrand's disease characterized by increased ristocetin sensitivity and the presence of all von Willebrand factor multimers in plasma

In eight members of one family, platelets in platelet-rich plasma aggregated at much lower ristocetin concentrations than normal. Ivy bleeding time was variously prolonged, and von Willebrand factor antigen (vWF:Ag), ristocetin cofactor activity, and factor VIII coagulant activity were decreased. Most of the affected members had had slight to rather severe bleeding symptoms. Platelet-type von Willebrand's disease (vWD) could be ruled out. All multimers of vWF:Ag were found in plasma as well as platelets. Administration of 1-desamino-8-D-arginine vasopressin (DDAVP) to the propositus did not cause thrombocytopenia, and platelet-poor plasma obtained immediately after did not aggregate normal platelets. The molecular defect in this family, inherited as an autosomal dominant, resembles the one in type IIB because of the response to ristocetin but differs from IIB because all vWF:Ag multimers are present in plasma and the response to DDAVP is atypical. We conclude that this family has a new subtype of vWD and propose that structural as well as functional criteria should be used for a proper classification of vWD.     

A probable double heterozygous type II von Willebrand's disease with increased ristocetin induced platelet aggregation.

We have identified a patient with von Willebrand's disease (vWD) resembling type IIB vWD, with increased ristocetin induced platelet aggregation (RIPA), the absence of the large multimers of von Willebrand factor (vWF) in plasma, and the presence of the large multimers in platelets in whom a family study indicated a probable double heterozygous inheritance pattern. The propositus was a 12-year-old boy with frequent epistaxis and bruising. Abnormal hemostatic findings included a prolonged bleeding time (BT), decreased levels of factor VIII coagulant activity (VIIIC), von Willebrand factor antigen (vWF:Ag), ristocetin cofactor (RCof), and an increased RIPA. In the presence of ristocetin, binding of the patient's plasma vWF to normal platelets was increased but binding of normal vWF to his platelets was normal. SDS-agarose gel (1.5%) electrophoresis revealed that plasma vWF lacked the large multimers, and 3.0% gel electrophoresis revealed that the multimers had a 5-band pattern similar to normal. The above findings were consistent with type IIB vWD, but 1-deamino[8-D-arginine]-vasopressin (DDAVP) infusion resulted in a shortened BT and the transient appearance of large multimers without a decrease in the platelet count. Family studies revealed that his mother has mild bleeding symptoms, decreased VIIIC, vWF:Ag, and RCof levels and normal to slightly reduced RIPA with a multimer pattern consistent with type I vWD. In contrast, the father, sister, and paternal grandfather were asymptomatic, with a slightly decreased VIIIC level but a normal BT and vWF:Ag and RCof levels. Their RIPA and vWF binding to normal platelets were increased, but unlike the propositus their plasma contained large multimers. We concluded that the propositus is a type IIB-like variant differing from previously reported IIB variants in two ways: 1) his response to DDAVP and 2) a possible double heterozygous mode of inheritance rather than the usual dominant route.     

A new von Willebrand variant (type I, New York): increased ristocetin-induced platelet aggregation and plasma von Willebrand factor containing the full range of multimers

We report three members of a family who had reduced levels of plasma von Willebrand factor (vWF) and increased ristocetin-induced platelet aggregation (RIPA) (aggregation of platelet-rich plasma with ristocetin at a concentration of 0.45 mg/mL), as previously reported in type IIB and pseudo-von Willebrand's disease (vWD). However, in contrast to the latter two disorders in which the larger vWF multimers are absent in plasma, the entire range of vWF multimers was observed in the patients' plasma after sodium dodecyl sulfate-agarose gel electrophoresis, and all vWF multimers (including the largest) were present in the same proportion as in normal plasma and type I vWD. Thus, despite increased RIPA, the levels and multimeric pattern of vWF in this family's plasma were indistinguishable from those in type I vWD in which RIPA is usually decreased. Addition of ristocetin to the patients' platelet- rich plasma resulted in the removal of vWF (and, more selectively, of the large multimers) at lower concentrations of ristocetin than normal, as in type IIB and pseudo-vWD. The defect in the patients was localized to their vWF, which had an enhanced capacity for aggregating washed normal platelets in the presence of low concentrations of ristocetin and for aggregating pseudo-vWD platelets (in the absence of ristocetin). Both glycoproteins (GP) Ib and IIb-IIIa were involved in the enhanced aggregation response. RIPA (at low ristocetin concentrations) in the patients' platelet-rich plasma was abolished by a monoclonal antibody (AP1) to GPIb and was markedly reduced by monoclonal antibodies (10E5 and LJP9) that block adenosine diphosphate and thrombin-induced binding of vWF and fibrinogen to GPIIb-IIIa but was unaffected by an antibody (LJP5) that only blocks vWF binding. Partial inhibition of the initial aggregation slope (and complete inhibition of second phase aggregation) was achieved with creatine phosphate/creatine phosphokinase. EDTA blocked second-phase aggregation but was without effect on the initial slope. The findings in this family combine some features of both type I vWD (normal pattern of vWF multimers in plasma) and type IIB vWD (increased RIPA) and further demonstrate the increasing complexity of the structure-function relationships in vWD.     

von Willebrand disease type B: a missense mutation selectively abolishes ristocetin-induced von Willebrand factor binding to platelet glycoprotein Ib.

von Willebrand factor (vWF) is a multimeric glycoprotein that mediates the adhesion of platelets to the subendothelium by binding to platelet glycoprotein Ib. For human vWF, this interaction can be induced in vitro by the antibiotic ristocetin or the snake venom protein botrocetin. A missense mutation, Gly-561-->Ser, was identified within the proposed glycoprotein Ib binding domain of vWF in the proband with von Willebrand disease type B, a unique variant characterized by no ristocetin-induced, but normal botrocetin-induced, binding to glycoprotein Ib. The corresponding mutant recombinant protein, rvWF(G561S), formed normal multimers and exhibited the same functional defect as the patient's plasma vWF, confirming that this mutation causes von Willebrand disease type B. These data show that botrocetin and ristocetin cofactor activities of vWF can be dissociated by a point mutation and confirm that these mediators promote vWF binding to platelets by different mechanisms. The normal botrocetin-induced binding and the defective ristocetin-induced binding of rvWF(G561S) suggest that the primary defect in von Willebrand disease type B may be a failure of normal allosteric regulation of the glycoprotein Ib binding function of vWF.     

Platelet--von Willebrand factor interactions in type IIB von Willebrand's disease

Type IIB von Willebrand's disease (vWD) is a distinct form of this disorder in which the largest multimers of the von Willebrand factor (vWF) are lacking in plasma but present in platelets. When the vasopressin analogue, 1-deamino-8-D-arginine vasopressin (DDAVP), is given to patients with type IIB vWD, an abnormal vWF is released to plasma. This vWF causes thrombocytopenia in vivo and platelet aggregation in vitro. Aggregation occurs in the plasma milieu and thus at physiological fibrinogen concentration. In this study we demonstrate that IIB post-DDAVP vWF aggregated only metabolically active platelets. The platelet aggregation was completely inhibited by EDTA and PGE1, and either inhibited or greatly weakened by ASA, demonstrating the role of divalent cations and thromboxane A2 formation. In spite of inhibiting platelet aggregation, EDTA, PGE1 and ASA did not prevent platelet binding of IIB post-DDAVP vWF. An antiserum against GP Ib made normal platelets less responsive to the IIB vWF although neither platelet aggregation nor vWF binding were completely prevented. The aggregation was fibrinogen-dependent and platelets from patients with Glanzmann's thrombasthenia were unresponsive. The studies provide evidence that IIB post-DDAVP vWF is bound to unstimulated platelets and that the interaction between vWF and platelets in type IIB vWD is different from ristocetin-induced as well as thrombin- and epinephrine-induced binding to platelets of normal vWF.     

Type I von Willebrand disease, subtype 'platelet low': decreased platelet adhesion can be explained by low synthesis of von Willebrand factor in endothelial cells

Summary Endotheial cells (EC) were isolated from the umbilical vein of a newborn girl with type I 'platelet low' von Willebrand disease (I vWD) and endothelial localization and release and the ability of subendothelial von Willebrand factor (vWF) to support platelet adhesion were compared with those of normal EC. vWF was detectable by immunofluorescence in Weibel-Palade bodies, but the number of Weibel-Palade bodies positive for vWF was lower than in control EC. Patient EC released into the medium significantly smaller amount of vWF, both constitutively and after their stimulation. The vWF content of the extracellular matrix of patient EC was 38% that of control EC matrix. Platelet adhesion studies were performed under flow conditions with umbilical arteries and EC matrices of cultured EC. Using normal citrated whole blood as perfusate, platelet adhesion was lower in the umbilical artery of the patient (9 ± 1% v 35 ± 4% for the control) and in her EC matrix (7 ± 1% v 21 ± 2% of control). When patient EC matrix was perfused with vWF-deficient reconstituted blood, adhesion was 17 ± 3% v 32 ± 3% for control EC matrix; preincubation of patient EC matrix with 1 U/ml vWF increased the adhesion to 30 ± 6%. These data establish that low contents of vWF in EC and subendothelium are important characteristics of type I vWD 'platelet low', and that such characteristics correlate with low platelet adhesion to the subendothelium.     

Platelet – von Willebrand factor interactions in Type IIB von Willebrand's disease

Type IIB von Willebrand's disease (vWD) is a distinct form of this disorder in which the largest multimers of the von Willebrand factor (vWF) are lacking in plasma but present in platelets. When the vasopressin analogue, l-deamino-8-D-arginine vasopressin (DDAVP), is given to patients with type IIB vWD, an abnormal vWF is released to plasma. This vWF causes thrombocytopenia in vivo and platelet aggregation in vitro. Aggregation occurs in the plasma milieu and thus at physiological fibrinogen concentration. In this study we demonstrate that IIB post-DDAVP vWF aggregated only metabolically active platelets. The platelet aggregation was completely inhibited by EDTA and PGE₁, and either inhibited or greatly weakened by ASA, demonstrating the role of divalent cations and thromboxane A₂ formation. In spite of inhibiting platelet aggregation, EDTA, PGE₁ and ASA did not prevent platelet binding of IIB post-DDAVP vWF. An antiserum against GP Ib made normal platelets less responsive to the IIB vWF although neither platelet aggregation nor vWF binding were completely prevented. The aggregation was fibrinogen-dependent and platelets from patients with Glanzmann's thrombasthenia were unresponsive. The studies provide evidence that IIB post-DDAVP vWF is bound to unstimulated platelets and that the interaction between vWF and platelets in type IIB vWD is different from ristocetin-induced as well as thrombin- and epinephrine-induced binding to platelets of normal vWF.     

Biosynthesis of human von Willebrand factor

Endothelium forms the inner lining of all blood vessels and, as a consequence, is in direct contact with the blood. Because of this and the synthesis and secretion of hemostatic components, the endothelium is able to modulate coagulation and fibrinolysis. An important hemostatic factor synthesized by endothelial cells is the von Willebrand factor (vWF). vWF is a large plasma glycoprotein which promotes the adhesion of platelets to the vessel wall after a vascular injury. vWF is initially synthesized as a pre-pro-polypeptide. During its transport to the outside of the cell, the single-chain polypeptides are assembled into multimers. The pro-polypeptide can be cleaved and also be secreted. Free pro-polypeptide is identified as von Willebrand antigen II, a plasma glycoprotein of unknown function. Plasma vWF consists of a heterogenous series of multimers, composed of an apparently single-type glycoprotein subunit, linked together by disulfide bonds. The hemostatic potency of vWF was shown to increase with increasing multimer size. Therefore, the multimeric assembly of vWF is a crucial aspect in vWF biosynthesis. Furthermore, vWF synthesized by endothelial cells can either be secreted constitutively or stored and released upon stimulation of the endothelial cell. In this review, data are presented which contribute to the understanding of the biosynthetic pathway and complex processing which vWF has to undergo before it is secreted by the endothelial cell. These data have allowed a prediction of the sequential events underlying vWF biosynthesis, processing, multimer assembly, and secretion.     

Heterogeneity of type I von Willebrand disease: evidence for a subgroup with an abnormal von Willebrand factor

Type I von Willebrand disease (vWD) is characterized by equally low plasma concentrations of von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor (RiCof) and by the presence of all vWF multimers in sodium dodecyl sulfate (SDS)-agarose gel electrophoresis. For 17 patients (13 kindreds) diagnosed with these criteria, we have studied the platelet contents of vWF:Ag and RiCof and the changes of these in plasma after DDAVP infusion. Platelet vWF:Ag and RiCof were normal in four kindreds (called "platelet normal" subgroup); following 1-deamino- 8-D-arginine vasopressin; plasma vWF:Ag, RiCof and the bleeding time (BT) became normal. In six kindreds, platelet vWF:Ag and RiCof were equally low (platelet low); after DDAVP, plasma vWF:Ag and RiCof remained low, and the BT was prolonged. In three additional kindreds, platelets contained normal concentrations of vWF:Ag, but RiCof was very low (platelet discordant); even though a complete set of multimers was found in plasma and platelets, there was a relatively small amount of large multimers. After DDAVP, plasma vWF:Ag became normal, but RiCof remained low and the BT was very prolonged. These findings demonstrated that there can be an abnormal vWF (RiCof less than vWF:Ag) even in type I vWD, coexisting with a complete set of vWF multimers (platelet discordant); that the abnormal vWF can be shown more clearly in platelets than in plasma or else in plasma after DDAVP infusion; and that DDAVP normalizes the BT only in those patients with normal platelet levels of both vWF:Ag and RiCof (platelet normal).     

von Willebrand factor released from Weibel-Palade bodies binds more avidly to extracellular matrix than that secreted constitutively

Large multimers of von Willebrand factor (vWf) are released from the Weibel-Palade bodies of cultured endothelial cells following treatment with a secretagogue (Sporn et al, Cell 46:185, 1986). These multimers were shown by immunofluorescent staining to bind more extensively to the extracellular matrix of human foreskin fibroblasts than constitutively secreted vWf, which is composed predominantly of dimeric molecules. Increased binding of A23187-released vWf was not due to another component present in the releasate, since releasate from which vWf was adsorbed, when added together with constitutively secreted vWf, did not promote binding. When iodinated plasma vWf was overlaid onto the fibroblasts, the large forms bound preferentially to the matrix. These results indicated that the enhanced binding of the vWf released from the Weibel-Palade bodies was likely due to its large multimeric size. It appears that multivalency is an important component of vWf interaction with the extracellular matrix, just as has been shown for vWf interaction with platelets. The pool of vWf contained within the Weibel-Palade bodies, therefore, is not only especially suited for platelet binding, but also for interaction with the extracellular matrix.     

Gamma-interferon modulates von Willebrand factor release by cultured human endothelial cells

Endothelial cells (EC) synthesize and secrete von Willebrand factor (vWF), a multimeric glycoprotein required for normal hemostasis. Within human endothelial cells, vWF multimers of extremely high molecular weight are stored in rod-shaped organelles known as Weibel-Palade bodies. Inflammatory mediators, such as interleukin-1, induce in vitro a variety of procoagulant responses by EC, including the secretion of stored vWF. We postulated that other inflammatory mediators might act to balance this procoagulant reaction, thereby assisting in the maintenance of blood fluidity during immune activation. Both gamma-interferon (gamma-IFN) and tumor necrosis factor (TNF) were found to act independently and cooperatively to depress the stimulated release of vWF from EC. Analysis of stored vWF in either gamma-IFN and/or TNF-treated EC demonstrated a loss of high molecular weight multimers while immunofluorescent studies documented a loss of visible Weibel-Palade bodies. This suggests that gamma-IFN and TNF interfere with normal vWF storage. gamma-IFN acted in a dose-, time-, and RNA-dependent fashion, and its inhibition of vWF release was reversible with time. No effect of gamma-IFN on EC was noted when anti-serum to gamma-IFN was added. Unlike gamma-IFN, alpha-interferon did not effect EC vWF. Therefore, gamma-IFN and TNF may be important in decreasing vWF release during inflammatory or immunologic episodes.     

Multimer size dependence of von Willebrand factor binding to crosslinked or noncrosslinked fibrin

von Willebrand factor (vWF) is synthesized in endothelial cells (EC) and may be either secreted constitutively or stored in Weibel-Palade bodies (WPB) for regulated release. Because fibrin stimulates rapid vWF release from EC, we examined the binding of EC synthesized vWF to fibrin. Culture medium containing constitutively secreted vWF was removed from metabolically labeled primary cultures of human umbilical vein EC, and vWF released from WPB was obtained after stimulation by A23187. vWF-deficient fibrinogen with or without factor XIII was added to releasate or media and clotted with thrombin to form crosslinked or noncrosslinked fibrin. vWF was immunopurified from releasate or media before and after clotting, and the amount and multimeric pattern of vWF bound was determined after sodium dodecyl sulfate agarose gel electrophoresis. High molecular weight multimers of vWF, whether secreted constitutively or released from WPB, bound preferentially to fibrin. Multimers of greater than 20 subunits represented 60% +/- 4% (SEM) of A23187 released vWF and 11% +/- 5% of media vWF, but binding to fibrin was similar, 96% +/- 1% and 94% +/- 2%, respectively. A progressively smaller proportion of vWF bound as multimer size decreased, and dimeric vWF binding was least, with 34% +/- 5% binding from A23187 releasate and 51% +/- 4% from media. The amount of vWF binding to crosslinked or noncrosslinked fibrin was similar, and preferential binding of high molecular weight multimers occurred with both. As measured by enzyme-linked immunosorbent assay, 45% +/- 2% of constitutively secreted vWF bound to crosslinked fibrin and 50% +/- 2% to noncrosslinked fibrin. The propolypeptide of vWF did not bind to fibrin. These findings indicate that binding of EC secreted vWF binding to fibrin depends on multimeric size but not on factor XIII crosslinking. This suggests that vWF released from EC in the presence of fibrin will bind locally, thereby facilitating platelet adhesion to the hemostatic plug or thrombus.     

Identification of a point mutation in type IIB von Willebrand disease illustrating the regulation of von Willebrand factor affinity for the platelet membrane glycoprotein Ib-IX receptor.

von Willebrand factor (vWF) supports platelet adhesion on thrombogenic surfaces by binding to platelet membrane glycoprotein (GP) Ib in the GP Ib-IX receptor complex. This interaction is physiologically regulated so that it does not occur between circulating vWF and platelets but, rather, only at a site of vascular injury. The abnormal vWF found in type IIB von Willebrand disease, however, has a characteristically increased affinity for GP Ib and binds to circulating platelets. We have analyzed the molecular basis of this abnormality by sequence analysis of a type IIB vWF cDNA and have identified a single amino acid change, Trp550 to Cys550, located in the GP Ib-binding domain of the molecule comprising residues 449-728. Bacterial expression of recombinant fragments corresponding to this vWF domain yielded molecules that, whether containing a normal Trp550 or a mutant Cys550 residue, bound directly to GP Ib in the absence of modulators and with similar affinity. In contrast, mammalian cell expression of the same segment of sequence yielded molecules that, when containing the normal Trp550, did not bind to GP Ib directly but, like native vWF, bound in the presence of ristocetin. However, molecules containing the point mutation (Cys550) behaved like type IIB vWF--namely, bound to GP Ib even without ristocetin modulation and, in the presence of ristocetin, had 10-fold higher affinity than molecules with normal sequence. These results identify a region of vWF that, although not thought to be directly involved in binding to GP Ib, may modulate the interaction through conformational changes.