Granulocyte proteases do not process endothelial cell-derived unusually large von Willebrand factor multimers to plasma vWF in vivo

The unusually large von Willebrand factor (ULvWF) multimers present within endothelial cells and platelets are larger than the vWF multimers normally found in adult human plasma. Furthermore, ULvWF multimers are cleared rapidly from the circulation if they are released by intense endothelial cell stimulation. The mechanisms by which the ULvWF multimers are processed to large plasma vWF multimers are not known. It has been demonstrated that granulocyte proteases are capable of decreasing vWF multimer size in vitro, and that some patients with myeloproliferative syndromes have a relative absence of large plasma vWF multimers in sodium citrate-anticoagulated plasma samples. In order to assess the influence of granulocyte proteases on vWF multimer size, we evaluated the vWF multimeric patterns in 94 plasma samples from 60 patients with neutrophil counts that were either considerably elevated or extremely reduced. In 83 of 94 plasma samples, the vWF multimeric patterns were normal. No patients with very low neutrophil counts had ULvWF multimers present. These observations suggest that granulocyte proteases are not likely to be involved in vivo in the processing of ULvWF multimers from endothelial cells to the smaller vWF forms in circulation.     

Multimeric structure of platelet factor VIII/von Willebrand factor: the presence of larger multimers and their reassociation with thrombin- stimulated platelets

The multimeric structure of platelet factor VIII/von Willebrand factor (FVIII/vWF) in cell extracts and in collagen and thrombin releasates has been analyzed by SDS polyacrylamide gel electrophoresis followed by detection with 125I-anti-FVIII/vWF. Platelets contained larger multimers than those normally present in plasma. When secreted FVIII/vWF was analyzed, all platelets. In contrast, in thrombin releasates the larger multimers were lost in a manner dependent on divalent cations, time, and thrombin dose. This loss could not be accounted for by modification of FVIII/vWF by thrombin or platelet enzymes since no effect of thrombin on the multimeric structure of FVIII/vWF in the absence of platelets or in the presence of platelet lysates was observed. Large multimers of 125I-labeled purified FVIII/vWF underwent divalent cation-dependent association with platelets in the presence of thrombin, indicating that the loss of FVIII/vWF from thrombin releasates was due to reassociation with the platelet. These studies show a structural difference between platelet and plasma FVIII/vWF that suggests a specific role for platelet FVIII/vWF in hemostasis.     

Binding of von Willebrand factor to glycoproteins Ib and IIb/IIIa complex: affinity is related to multimeric size

We have separated von Willebrand factor (vWF) multimers of different size into several fractions which were characterized by SDS-agarose gel electrophoresis and by measuring the ratio between ristocetin cofactor activity (Ricof) and von Willebrand antigen (vWF:Ag) content. The pooled fractions contained vWF with multimeric structures and Ricof similar to those in plasma. The pool was labelled with 125I and used for inhibition binding studies with individual fractions to calculate the dissociation constants (Kd values expressed in mol/l) of the individual fractions for ristocetin-dependent binding to GP Ib and thrombin-induced binding to GP IIb/IIIa. Direct binding studies of the 125I-vWF pool gave mean Kd values of 2.02 +/- 0.05 x 10(-8) for GP Ib and 1.15 +/- 0.02 x 10(-8) for the GP IIb/IIIa complex. Inhibition binding studies gave Kd mean values one third to one tenth as high for larger multimers and 3-10 times higher for smaller multimers, for both GP Ib and IIb/IIIa complex. Similar results were observed when binding studies were carried out in the presence of platelets from a patient with afibrinogenaemia. These data on binding correlated very well with ristocetin- and thrombin-induced aggregation of afibrinogenaemic platelets, since equal concentrations of the higher molecular weight forms gave significantly higher aggregation rates. Based on these results, we conclude that the affinity of the vWF molecule for its two platelet receptors is greater for the largest multimers.     

Control of von Willebrand factor multimer size and implications for disease

Plasma von Willebrand factor (vWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury, however only the very large vWF multimers are haemostatically competent. Plasma vWF derives predominantly from the vascular endothelium and is of a smaller average multimer size to that found in the sub-endothelial matrix. The existence of plasma factors that control the size of vWF multimers and account for this difference has long been suspected. vWF cleaving protease (vWFCP), a metalloproteinase that regulates vWF multimer size by proteolytic cleavage, and thrombospondin-1 (TSP-1), a trimeric glycoprotein that uncouples multimers by reducing the disulfide-bonds which link individual subunits, are two such factors that have recently been identified. The large and ultra large vWF multimers play a central role in arterial thrombosis and agents that regulate their size hold promise as novel anti-thrombotic drugs.     

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.     

Elevated platelet count as a cause of abnormal von Willebrand factor multimer distribution in plasma

Twelve of 19 patients with myeloproliferative disorders showed a decrease of absence of the largest multimers of plasma von Willebrand factor (vWF) that correlated with elevated platelet counts but not with leukocyte counts. This suggested that platelets, rather than leukocytes, may be associated with the pathogenesis of the acquired vWF abnormality seen in these patients. To examine the hypothesis further, we studied 12 patients with reactive thrombocytosis after splenectomy. Increased platelet count (> 5 x 10(11)/L) after splenectomy was associated with vWF abnormalities indistinguishable from those detected in patients with myeloproliferative disorders. Accordingly, there was an inverse correlation between proportion of large vWF multimers and platelet, but not leukocyte, number: normalization of the platelet count was accompanied by restoration of a normal vWF multimeric pattern. These findings suggest that an increase in the number of platelets circulating in blood may favor the adsorption of larger vWF multimers onto the platelet membrane, resulting in their removal from the circulation and subsequent degradation.     

Membrane fluidity and platelet aggregation: dibucaine permits ristocetin-induced platelet aggregation with low-molecular-weight von Willebrand multimers.

The effects of brief incubation of platelets with dibucaine were studied. Membrane fluidity was increased after incubation with 1 mM dibucaine for 1 min as determined by fluorescence polarization. Platelet aggregation was increased when ristocetin was employed as modulator and normal plasma used as a source of von Willebrand factor (vWF). In contrast, aggregation was decreased with botrocetin. After cryoprecipitation the supernatant, which contained predominantly low-molecular-weight vWF multimers, was effective with ristocetin only after prior incubation of the platelets with dibucaine. This indicates that low-molecular-weight vWF multimers can also support ristocetin-induced aggregation when the membrane fluidity is increased. This idea was supported by the use of plasma from a patient with von Willebrand disease type IIa. Being a multimeric protein, von Willebrand factor contains multiple binding sites for glycoprotein Ib (GP Ib). GP Ib-dependent aggregation by agents that do not contain multiple binding sites, i.e. wheat germ agglutinin or polyclonal antibodies to GP Ib, was decreased after brief incubation with dibucaine. These observations are discussed in relation to the hypothesis that the increased membrane fluidity allows an increased number of GP Ib molecules to bind to each vWF multimer.     

Aurin tricarboxylic acid: a novel inhibitor of the association of von Willebrand factor and platelets

Shear stress activated platelets undergo aggregation in the presence of large or unusually large von Willebrand factor (vWF) multimers without the addition of ristocetin or any other exogenous chemical. This phenomenon may be analogous to the platelet aggregation that leads to thrombosis in the narrowed arteries and arterioles of patients with atherosclerosis or vasospasm. A triphenyl-methyl compound, aurin tricarboxylic acid (ATA), inhibits shear-induced, vWF-mediated platelet aggregation in platelet-rich plasma (PRP) in concentrations above 200 mumol/L and in buffer suspensions of washed platelets at a concentration of 0.1 mumol/L. In a concentration-dependent manner, ATA also inhibits ristocetin-induced, vWF-mediated platelet clumping in both fresh and formaldehyde-fixed platelet suspensions. This inhibition can be overcome by increasing the concentration of vWF, following the kinetics of first order competitive inhibition. ATA prevents the attachment to platelets of the largest vWF multimeric forms found in normal plasma and of the unusually large vWF multimers derived from endothelial cells. The rate of aggregation and degree of inhibition by ATA is not accounted for by the binding of ristocetin or calcium. Arachidonic acid- and adenosine diphosphate (ADP)-induced aggregation are not inhibited by ATA. Platelets incubated with ATA can be easily separated from the compound. However, ATA binds to large vWF multimeric forms and inhibits their ristocetin-induced interaction with platelet glycoprotein Ib. Because ATA also inhibits shear-induced, vWF-mediated platelet aggregation in vitro in the absence of ristocetin, it may be a useful prototype compound to impede the development of arterial thrombosis in vivo.     

ABO Blood Group Genotype and Plasma von Willebrand Factor in Normal Individuals

von Willebrand factor (vWF) is a multimeric plasma protein with ABO (H) blood group sugar chains. We investigated a total of 330 plasmas from normal individuals having various ABO genotypes, with special reference to vWF antigen and its platelet glycoprotein-Ib-related biological activities, termed ristocetin cofactor (RCof) and botrocetin cofactor (BCof). RCof reflects the biological activity of higher vWF multimers, while BCof reflects that of vWF of multimers of all sizes. Plasmas from normal individuals carrying one O gene (genotypes AO and BO) had slightly, but proportionally lower levels of vWF antigen, RCof, and BCof than those carrying no O gene (genotypes AA, AB, and BB). Normal plasmas from individuals carrying two O genes (genotype 00) showed much lower values for these parameters than the other plasmas, as previously reported. However, multimeric analysis of plasma vWF antigen revealed no differences among the different genotypes.     

Assays of von Willebrand factor-cleaving protease: a test for diagnosis of familial and acquired thrombotic thrombocytopenic purpura

Endothelial cells secrete von Willebrand factor (vWF) multimers that are larger than those found in the circulating plasma. These very large multimeric forms of vWF, capable of spontaneously binding to and agglutinating the blood platelets under conditions of high fluid shear rate, are degraded by a specific metalloprotease cleaving the peptide bond 842Tyr-843Met of the vWF subunit. The vWF-cleaving protease was found to be deficient in patients with familial thrombotic thrombocytopenic purpura (TTP). The acute events in these patients can be successfully treated and prophylactically prevented by repletion of the missing protease using fresh frozen plasma (FFP). In another, apparently more common, form of TTP, the protease deficiency is due to inhibiting circulating antibodies directed against the vWF-cleaving protease. Therapy of these patients should include immunosuppressive treatment in addition to plasma exchange and replacement with FFP. Normal activity of vWF-cleaving protease was established in patients with a clinically similar disorder: hemolytic-uremic syndrome (HUS). The level of vWF-cleaving protease activity is thus a laboratory parameter that provides important information for the differential diagnosis and treatment of patients with TTP/HUS. Several assays of vWF-cleaving protease have been described and are summarized here.     

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.     

Ultracentrifugal Analysis of Factor VIII and von Willebrand Factor in Therapeutic Preparations

Plasma and therapeutic preparations of factor VIII (1 recombinant factor VIII and two monoclonally purified plasma-derived factor VIII preparations, Kogenate, and AHF-M and Monoclate, respectively) were centrifuged in a sucrose density gradient, and the fractions were analyzed for factor VIII and von Willebrand factor (vWF). The residual vWF in the monoclonally purified factor VIII preparations sediments more slowly than the vWF of plasma. In the absence of added vWF, the factor VIII in all preparations sediments more slowly than plasma factor VIII. These same preparations of factor VIII added to hemophilic plasma as a source of vWF sediment differently. The addition of either recombinant factor VIII or AHF-M results in sedimentation of the factor VIII with the plasma vFW and in a position indistinguishable from factor VIII in plasma. In contrast, when Monoclate is added to hemophilic plasma in vitro, the factor VIII sediments more slowly than the vWF of the hemophilic plasma. However, 5 min after the infusion of Monoclate into a patient with hemophilia A, the factor VIII sediments with the plasma vWF. These results indicate that the addition of recombinant factor VIII and AHF-M results in random binding to all vWF multimers of plasma, while there is little exchange between the added factor VIII in Monoclate and the plasma vWF in vitro. In contrast, when the Monoclate is infused, there is rapid binding of factor VIII to the plasma vWF. This is presumably due to tight binding of the factor VIII to the vWF in Monoclate, whereas the factor VIII in Kogenate and AHF-M, containing no or very low amounts of vWF, are free to bind to plasma vWF. In vivo there are other conditions, as yet uninvestigated, which allow for the binding of factor VIII to vWF.     

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.     

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.     

Chromatographic Preparation of a Therapeutic Highly Purified von Willebrand Factor Concentrate from Human Cryoprecipitate

A therapeutic highly purified von Willebrand factor (vWF) concentrate has been prepared from cryoprecipitate by a three-step chromatographic procedure. After solvent/detergent treatment to inactivate viruses, the cryoprecipitate solution was chromatographed on DEAE-fractogel TSK 650 M to separate vWF from most cryoprecipitate proteins, including factor VIII (FVIII) and fibrinogen. A second DEAE-fractogel TSK 650 M was then performed to further purify vWF and to allow concentrating it to over 100 U ristocetin cofactor activity/ml. The last step on immobilized gelatin removed fibronectin and increased the purity of vWF. vWF was recovered with about 18 and 40% yield in antigen and collagen-binding (CB) activity, respectively, from cryoprecipitate. vWF was obtained in an essentially pure state corresponding to a purification factor of over 10,000-fold from plasma. Immunonephelometric and SDS-PAGE analyses of the concentrate did not reveal any detectable cryoprotein contaminants, especially fibrinogen, fibronectin, immunoglobulins and albumin. The content in intermediate- and high-molecular-weight multimers in the concentrate was similar or higher than that of plasma, as the ion-exchanger selectively favored the binding and concentration of the larger multimeric forms while reducing the amount of the smaller forms with abnormal structure and low activity. Other characteristics of the concentrate included a CB activity to antigen ratio of 1.69 and a high capacity (86%) to correct platelet adhesion in a perfusion system. Clinical use of this standardized vWF concentrate has been shown to be efficacious in the treatment of vWF patients.     

Defective von willebrand factor activity detected by the filterometer in three clinical conditions

When exposed to high levels of shear in a filterometer, platelets bind to von Willebrand factor (vWF) via receptors Ib and IIb/IIIa, forming aggregates that block the filteromer. In this study we used the filterometer to explore the mechanisms by which abnormal vWF-platelet interaction might occur. In the first phase of the study, the global vWF-platelet interaction in native blood was investigated. In the second phase, to eliminate the difference that platelets might contribute, samples of platelet-poor plasma from test individuals were added to normal control blood and the mixtures were investigated by the filterometer. The filterometer results were adjusted for the antigen concentrations to obtain vWF potency ratios. Sodium dodecyl sulphate (SDS) agarose gel electrophoresis and SDS-Polyacylamide gel electrophoresis (PAGE) were used to analyze multimeric size and proteolytic profiles of vWF. Pregnancy was associated with high platelet retention, high vWF antigen concentration, normal multimeric size distribution, but decreased vWF potency ratios. The plasma samples of pregnancy contained one 183-kDa fragment not detected in normal plasma. These results suggested that in pregnancy, platelets were highly active. However, presumably due to abnormal proteolytic cleavage, vWF potency was decreased. This decrease in vWF potency might minimize the risk of thrombosis in association with highly active platelets. Renal transplant patients had normal platelet retention but high vWF levels. The plasma vWF contained normal multimers. A decrease in vWF potency, presumably caused by toxic inhibitors in the plasma, was detected. Aortic valve stenosis patients had decreased platelet retention, normal or slightly increased vWF antigen concentration and a decrease in large multimers. As a result, the vWF potency was markedly decreased. However, the results obtained with the filterometer became normal when the studies were repeated 3 months postpartum, when renal function had improved after transplantation, and when the aortic valves were corrected by surgery. These results indicate that the filterometer is a useful tool for elucidating the mechanisms by which vWF-platelet interaction might be impaired in various clinical conditions.     

Antibody inhibitors to von Willebrand factor metalloproteinase and increased binding of von Willebrand factor to platelets in ticlopidine-associated thrombotic thrombocytopenic purpura

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) affects 1 in 1600 to 1 in 5000 patients who receive ticlopidine, but little is known about the pathogenesis of this complication. OBJECTIVE: To investigate whether von Willebrand factor (vWF), which has been associated with idiopathic TTP, is involved in the pathogenesis of ticlopidine-associated TTP. DESIGN: Case series. SETTING: Three tertiary care, university-affiliated medical centers. PATIENTS: Seven patients who developed TTP 2 to 7 weeks after initiation of ticlopidine therapy. Controls were 7 consecutive patients without thrombocytopenia who had been receiving ticlopidine for 3 to 5 weeks and 10 randomly selected hospitalized patients. MEASUREMENTS: Platelet-bound vWF in patients' EDTA-anticoagulated whole blood samples; vWF proteinase activity in patients' plasma samples; inhibitory activity of IgG isolated from patients' plasma samples against the proteinase from the controls' plasma samples; and vWF multimeric patterns in patients' EDTA-anticoagulated plasma samples. RESULTS: Binding of vWF to single platelets was increased in the three patients tested during the most thrombocytopenic phase of TTP episodes. Initial plasma samples from all seven patients lacked the largest vWF multimers and were severely deficient in vWF metalloproteinase. IgG molecules, isolated from plasma samples of five patients, inhibited metalloproteinase in plasma samples from the controls. In patients examined, these abnormalities resolved upon the remission that accompanied plasma exchange and discontinuation of ticlopidine therapy. CONCLUSION: In the patients who developed ticlopidine-associated TTP, autoantibodies to the vWF metalloproteinase were formed; this led to the same type of vWF abnormalities observed in patients with idiopathic acute TTP. The findings suggest that failure to process large and unusually large vWF multimers in vivo caused binding of vWF to platelets, systemic platelet thrombosis, and TTP.     

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.     

Defective von Willebrand factor activity detected by the filterometer in three clinical conditions

When exposed to high levels of shear in a filterometer, platelets bind to von Willebrand factor (vWF) via receptors Ib and IIb/IIIa, forming aggregates that block the filteromer. In this study we used the filterometer to explore the mechanisms by which abnormal vWF - platelet interaction might occur. In the first phase of the study, the global vWF - platelet interaction in native blood was investigated. In the second phase, to eliminate the difference that platelets might contribute, samples of platelet - poor plasma from test individuals were added to normal control blood and the mixtures were investigated by the filterometer. The filterometer results were adjusted for the antigen concentrations to obtain vWF potency ratios. Sodium dodecyl sulphate (SDS) agarose gel electrophoresis and SDS - Polyacylamide gel electrophoresis (PAGE) were used to analyze multimeric size and proteolytic profiles of vWF. Pregnancy was associated with high platelet retention, high vWF antigen concentration, normal multimeric size distribution, but decreased vWF potency ratios. The plasma samples of pregnancy contained one 183-kDa fragment not detected in normal plasma. These results suggested that in pregnancy, platelets were highly active. However, presumably due to abnormal proteolytic cleavage, vWF potency was decreased. This decrease in vWF potency might minimize the risk of thrombosis in association with highly active platelets. Renal transplant patients had normal platelet retention but high vWF levels. The plasma vWF contained normal multimers. A decrease in vWF potency, presumably caused by toxic inhibitors in the plasma, was detected. Aortic valve stenosis patients had decreased platelet retention, normal or slightly increased vWF antigen concentration and a decrease in large multimers. As a result, the vWF potency was markedly decreased. However, the results obtained with the filterometer became normal when the studies were repeated 3 months postpartum, when renal function had improved after transplantation, and when the aortic valves were corrected by surgery. These results indicate that the filterometer is a useful tool for elucidating the mechanisms by which vWF - platelet interaction might be impaired in various clinical conditions.     

Type IIB von Willebrand's disease associated with a complex thrombocytopenic thrombocytopathy

A familial bleeding disorder characterized by an association of Type IIB von Willebrand's disease (vWD) with a complex thrombocytopenic thrombocytopathy is described in two patients from the same generation. Findings typical of type IIB vWD included enhanced ristocetin-induced binding of patient von Willebrand factor (vWF) to platelets of patients and normal individuals in association with the absence of larger multimers from plasma. Abnormalities in platelet function included deficient platelet aggregation to ADP, collagen, epinephrine, and arachidonic acid; and defective release of 14C-serotonin, vWF, and platelet factor 4 (PF4) in response to thrombin, collagen, or ADP. Platelet factor 4 and platelet vWF were decreased when measured per mg of total platelet protein. In addition, the binding of normal vWF to patient platelets stimulated with thrombin was decreased. Platelet size was increased with a very heterogeneous distribution width. Electron microscopic evaluation showed giant platelets with dense and alpha bodies present. The platelet count was borderline or slightly decreased in the resting state and declined to frankly thrombocytopenic levels at the time of acute bleeding episodes; this state was associated with the presence of platelet aggregates in blood smears.