von Willebrand factor and factor VIII are independently required to form stable occlusive thrombi in injured veins

von Willebrand factor (VWF) protects factor VIII (FVIII) from proteolysis and mediates the initial contact of platelets with the injured vessel wall, thus playing an important role in hemostasis and thrombosis. VWF is crucial for the formation of occlusive thrombi at arterial shear rates. However, with only a few conflicting studies published, the role of VWF in venous thrombosis is still unclear. Using gene-targeted mice, we show that in ferric chloride-injured veins platelet adhesion to subendothelium is decreased and thrombus growth is impaired in VWF(-/-) mice when compared with wild type (WT). We also observed increased embolization in the VWF(-/-) mice, which was due to lower FVIII levels in these mice as recombinant factor VIII (r-FVIII) restored thrombus stability. Despite normalization of blood clotting time and thrombus stability after r-FVIII infusion, the VWF(-/-) venules did not occlude. Transgenic platelets lacking the VWF receptor GPIbalpha extracellular domain showed decreased adhesion to injured veins. But, after a delay, all the injured venules occluded in these transgenic mice. Thus, VWF likely uses other adhesion receptors besides GPIbalpha in thrombus growth under venous shear conditions. Our studies document crucial roles for VWF and FVIII in experimental thrombosis under venous flow conditions in vivo.     

von Willebrand factor is present on the surface of platelets stimulated in plasma by ADP

von Willebrand factor (vWf) can bind to glycoprotein (GP) IIb/IIIa on activated platelets. The significance of this interaction is unclear, however, because it has not been possible to detect vWf binding to GPIIb/IIIa on platelets stimulated in plasma. We have developed an indirect, flow cytometry assay that uses fluorescein-labeled antibodies to detect vWf and fibrinogen on platelets. Using this assay, we found vWf on the surface of platelets stimulated in plasma by ADP. The number of platelets that bound vWf increased in proportion to ADP concentration and incubation time. Washed platelets in a protein-free buffer activated by 1 mumol/L calcium ionophore A23187 or 10 mumol/L ADP also bound vWf, suggesting that we were detecting surface binding of alpha-granule-derived vWf. Monoclonal antibodies against the vWf binding site on GPIb (6D1) and the vWf and fibrinogen binding sites on GPIIb/IIIa (LJP5 and LJ-CP8, respectively) were used to characterize the mechanism of vWf binding to stimulated platelets. Ristocetin- induced binding of vWf was inhibited by 6D1, and ADP-induced binding of fibrinogen was inhibited by LJ-CP8. None of these antibodies inhibited ADP-induced vWf binding. Aspirin and prostaglandin E1 also inhibited ADP-induced binding of vWf in platelet-rich plasma. During platelet activation in plasma, vWf derived from alpha-granules becomes bound to the platelet surface possibly being transferred already associated with a binding site.     

Platelets roll on stimulated endothelium in vivo: an interaction mediated by endothelial P-selectin.

P-selectin, found in storage granules of platelets and endothelial cells, can be rapidly expressed upon stimulation. Mice lacking this membrane receptor exhibit a severe impairment of leukocyte rolling. We observed that, in addition to leukocytes, platelets were rolling in mesenteric venules of wild-type mice. To investigate the role of P-selectin in this process, resting or activated platelets from wild-type or P-selectin-deficient mice were fluorescently labeled and transfused into recipients of either genotype. Platelet-endothelial interactions were monitored by intravital microscopy. We observed rolling of either wild-type or P-selectin-deficient resting platelets on wild-type endothelium. Endothelial stimulation with the calcium ionophore A23187 increased the number of platelets rolling 4-fold. Activated P-selectin-deficient platelets behaved similarly, whereas activated wild-type platelets bound to leukocytes and were seen rolling together. Platelets of either genotype, resting or activated, interacted minimally with mutant endothelium even after A23187 treatment. The velocity of platelet rolling was 6- to 9-fold greater than that of leukocytes. Our results demonstrate that (i) platelets roll on endothelium in vivo, (ii) this interaction requires endothelial but not platelet P-selectin, and (iii) platelet rolling appears to be independent of platelet activation, indicating constitutive expression of a P-selectin ligand(s) on platelets. We have therefore observed an interesting parallel between platelets and leukocytes in that both of these blood cell types roll on stimulated vessel wall and that this process is dependent on the expression of endothelial P-selectin.     

Canine von Willebrand factor expresses a multimeric composition similar to human von Willebrand factor

Canine von Willebrand factor (vWf) was compared to human vWf. Antisera raised against human vWf or canine vWf cross-reacted with both heterologous proteins and reactions of partial identity were seen using crossed immunoelectrophoresis. Similar patterns of multimerization were obtained for vWf from both canine and human sources using the enzyme-linked immunoelectrotransfer blot method. However, the canine protein displayed an altered electrophoretic mobility. The molecular weight of the vWf monomer was estimated by SDS-PAGE and found to be indistinguishable from that of human vWf monomer. Canine vWf is decreased in animals with clinically evident hypothyroidism and in heterozygous "carriers" of von Willebrand's disease (vWd) that display no clinical symptoms of vWd. Results expand the concept that von Willebrand's disease in dogs may be a useful model for study of vWd in humans, and that immunochemical methods established for studies of human vWf appear appropriate for studies of canine vWf.     

Acquired von Willebrand disease in multiple myeloma secondary to absorption of von Willebrand factor by plasma cells

A case of acquired von Willebrand disease (AvWD) associated with an IgA lambda multiple myeloma is reported. No form of inhibitor could be detected. SDS-agarose gel electrophoresis patterns of von Willebrand factor (vWF) both in plasma and platelet lysates were normal but a decrease in all-sized multimers with a type IA pattern was seen. After 1-deamino-8-D arginine vasopressin (DDAVP) infusion, vWF multimers larger than those seen in the resting state appeared in patient plasma, which were progressively cleared. Indirect immunofluorescence studies with a monoclonal antibody to vWF showed that vWF was selectively absorbed into myelomatous cells. This is the first case of AvWD associated with multiple myeloma resulting from the selective absorption of vWF into abnormal plasma cells. This feature established a new pathophysiological mechanism of AvWD in multiple myeloma and probably in other lymphoproliferative diseases.     

Induced antibodies to von Willebrand factor

Plasma from 15 patients with haemophilia A and 4 patients with von Willebrand's disease (VWD) has been investigated for the presence of antibodies to von Willebrand factor (VWF) using ELISA and immunoblotting systems. All plasma samples were also analyzed for anticoagulant activity against factor VIII coagulant activity (FVIII:C) and ristocetin co-factor activity of VWF (RCof). The patients were on regular prophylaxis or treated 'on demand' with different commercial FVIII-VWF complex concentrates. Neutralizing activity against FVIII:C was only found in haemophiliacs (in 3 out of 15), whereas anti-RC of was only found in VWD patients (in 2 out of 4). In ELISA, antibodies to VWF were detected in 3 of the VWD patients. However, using the immunoblotting system, antibodies to the multimeric structure of VWF were found not only in VWD patients (in 3 out of 4); remarkably 9 out of 15 haemophiliacs also reacted with VWF, including some that did not show FVIII:C inhibitors. The pattern of VWF multimers obtained with the antibodies showed differences among the patients. These findings are discussed together with the mechanisms by which such antibodies could have developed in haemophilia A patients who are not deficient in this particular protein (VWF).     

Differential regulation by cytokines of constitutive and stimulated secretion of von Willebrand factor from endothelial cells

We examined the effect of cytokines on basal and agonist-stimulated release of von Willebrand factor (vWf) by human endothelial cells. Treatment of endothelial cells for up to 48 hours with human recombinant or purified interleukin 1 (IL-1) or human recombinant tumor necrosis factor-alpha (TNF-alpha) did not significantly affect constitutive secretion of vWf or intracellular levels of vWf, although basal prostacyclin (PGI2) production was markedly enhanced. In contrast, both IL-1 and TNF-alpha modulated vWf release in response to thrombin or phorbol ester. Pretreatment of endothelial cells for 2 hours with either cytokine enhanced by up to threefold the stimulatory effect of a subsequent 60-minute exposure to thrombin. Addition of cycloheximide (5 micrograms/mL) during the preincubation abolished this enhancement. Moreover, if the cytokine pretreatment time was extended to 24 hours, agonist-stimulated vWf release was significantly suppressed. Cytokine treatment for 2 or 24 hours had no detectable effect on levels of vWf messenger RNA. The effects of cytokines were not the result of contamination with bacterial lipopolysaccharide and were not attributable to endothelial cell injury. These results show that cytokines have little or no direct effect on vWf release from endothelial cells but can significantly modulate its acute release in response to other stimuli in a complex time- and dose-dependent manner.     

Microparticles adhere to collagen type I, fibrinogen, von Willebrand factor and surface immobilised platelets at physiological shear rates

Microparticles (MPs), shed during the storage of platelets, support blood coagulation and could be helpful in restoring the haemostatic system in thrombocytopenic patients. The mechanisms by which MPs support haemostasis under flow conditions were investigated. Fluorescent-labelled MPs were perfused at shear rates of 100 and 1000/s over surfaces coated with collagen, fibrinogen, von Willebrand factor (VWF) or surface-adherent platelets. Adhesion was monitored in real-time by fluorescence microscopy. In addition, thrombin-antithrombin (TAT) complex formation was measured in flowing thrombocytopenic blood. MPs attained the capacity to firmly adhere to collagen, VWF, fibrinogen and surface-adherent platelets at high and low shear rate. Antibodies against glycoprotein Ibalpha and alpha(IIb)beta(3) were used to demonstrate the specificities of these interactions. The addition of MPs to thrombocytopenic blood did not affect platelet adhesion. TAT complex formation was increased in the presence of MPs in capillaries coated with fibrinogen, but not on collagen fibres. We confirmed that MPs adhere to a damaged vascular bed in vivo after infusion in denuded arteries in a mouse model. MPs have platelet-like adhering properties and accelerate thrombin generation. These properties strongly support the notion that MPs can be beneficial in maintaining normal haemostasis when platelet function is impaired or reduced, as in thrombocytopenic patients.     

Life-threatening reaction to factor VIII concentrate in a patient with severe von Willebrand disease and alloantibodies to von Willebrand factor

A 16-yr-old girl with severe von Willebrand disease complicated by the development of precipitating alloantibodies to von Willebrand factor (anti-VWF) had a life-threatening anaphylactoid reaction immediately after the infusion of a commercial plasma concentrate of factor VIII/von Willebrand factor. An early post-infusion activation of the complement system was demonstrated by the appearance of C3 split products and by the drop of serum CH50 activity, occurring in parallel with a post-infusion drop in the anti-VWF antibody levels. Immune complexes remained unchanged in the early post-infusion period and rose to a moderate extent only after 24 h. We conclude that biologically active products of the complement system contributed to the onset of this life-threatening reaction which occurred after concentrate infusion.     

Increased factor VIII/von Willebrand factor antigen and von Willebrand factor activity in renal failure.

Factor VIII/von Willebrand factor antigen and von Willebrand factor activity (ristocetin assay) were studied in 12 patients in renal failure. A dramatic increase in both activities was observed (antigen 315 +/- 30 per cent in patients verus 104 +/- 9 per cent in control subjects; activity 402 +/- 48 per cent in patients versus 111 +/- 5 per cent in control subjects; p less than 0.001 for both). Since von Willebrand factor is thought to play at least a facilitative role in the development of arteriosclerosis, these increased activities may contribute to the premature arteriosclerosis reported in patients with chronic renal failure undergoing dialysis.     

Changes in von Willebrand factor level and von Willebrand activity with age in type 1 von Willebrand disease

In a normal population, VWF plasma levels (VWF:Ag) and VWF activity (VWF:RCo) increase by approximately 0.17 and 0.15 IU mL^?1 per decade, but the influence of age is unknown in patients with type 1 von Willebrand disease (VWD). In a retrospective cohort study, the medical records of 31 type 1 VWD patients over the age of 30, who had been followed for ≥5 years, were reviewed for baseline clinical data and previously performed VWF:Ag, VWF:RCo and factor VIII levels (FVIII:C). VWF multimer analysis was normal in 28/31 cases performed. Mean age at diagnosis was 33 (range 16–60 years), and duration of follow‐up ranged from 5 to 26 years (mean 11 years). Patients had 2–10 time points of VWD testing (mean of 5.2). The mean VWF:Ag, VWF:RCo and FVIII:C at time of diagnosis were 0.44 IU mL^?1 0.34 IU mL^?1 and 0.75 IU mL^?1. At last follow‐up, the mean VWF:Ag, VWF:RCo and FVIII:C were significantly increased to 0.71 IU L^?1, 0.56 IU mL^?1 and 0.90 IU mL^?1 (P ≤ 0.001, <0.001, and 0.0081 respectively). Here 18/31 patients had VWF:Ag, VWF:RCo and FVIII: C levels that increased into the normal range. The rate of change in VWF:Ag, VWF:RCo and FVIII was 0.30 IU mL^?1 (0.21–0.39, CI 95%, P < 0.0001), 0.20 IU mL^?1 per decade (0.13–0.27, CI 95%, P = 0.0001) and 0.20 IU mL^?1 (0.11–0.29, CI 95%, P = 0.0011). Patients with type 1 VWD experience age‐related increases to VWF:Ag and VWF:RCo which can result in normalization of VWF levels. Further studies are required to determine if the bleeding phenotype resolves with the increases in VWF:Ag and VWF:RCo levels.     

Atherosclerosis and von Willebrand factor

S ummary. The prevalence of von Willebrand's disease (VWD) in Western European countries and Israel was assessed. Possible patients were identified initially by questionnaire and those with plasma levels of von Willebrand factor antigen (WFAg; factor VIII related antigen) less than 1 u/dl (<1%) were confirmed by immunoradiometric assay performed at four reference centres. The prevalences of severe VWD in Scandinavian countries were similar to each other and about 10 times greater than those in other Western European countries. The prevalence in Israel was intermediate. The Registry will form the basis for future non-invasive epidemiological studies of atherosclerosis in these individuals.     

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.     

Optimizing therapy with factor VIII/von Willebrand factor concentrates in von Willebrand disease

In von Willebrand disease, the main goals of treatment are to correct the dual defect of haemostasis caused by a reduced or abnormal von Willebrand factor (vWF), i.e. the prolonged bleeding time (BT) and the deficiency of factor VIII coagulant activity (FVIII:C). The synthetic vasopressin analogue, desmopressin (DDAVP), has reduced the need for transfusions in most of the mild forms of von Willebrand disease but DDAVP is ineffective in type 3 and in other severe cases of types 1 and 2 von Willebrand disease. For many years cryoprecipitate has been the mainstay of replacement therapy but, after the introduction of virucidal methods, concentrates containing FVIII/vWF have been considered much safer than cryoprecipitate and proposed in von Willebrand disease management. FVIII/vWF concentrates have been produced and tested by many authors but there is only one report describing four virus-inactivated FVIII/vWF concentrates evaluated in a cross-over randomized trial. According to these in vitro and pharmacokinetic data, the following information can be derived: (a) no FVIII/vWF concentrate had an intact multimeric structure similar to that of normal plasma or of cryoprecipitate; (b) all FVIII/vWF concentrates were equally effective in attaining normal and sustained levels of FVIII:C postinfusion, although peak levels were more delayed in the concentrate devoid of FVIII:C; (c) no FVIII/vWF concentrate consistently normalized the BT in a sustained fashion. On the other hand, clinical haemostasis can be achieved in the management of bleeding episodes and of surgery for most of von Willebrand disease cases regardless of whether the BT is corrected; in the few rare cases with mucosal bleeding not controlled by FVIII/vWF concentrates, infusion of DDAVP or platelet concentrates can be administered in addition.     

Hybridoma antibodies to human von Willebrand factor

Twenty-seven stable subclones of seven independent cellular hybrids producing murine monoclonal antibodies to human von Willebrand factor (vWF) have been established. The specificity of the hybridoma antibodies for vWF has been substantiated by a variety of methods including binding to highly purified vWF, absence of binding to plasma or cryoprecipitate from severe von Willebrand's disease, binding to different size multimers in normal plasma, and binding to low molecular weight multimers in type IIA von Willebrand's disease plasma. Monoclonality of the hybridoma derived antibodies has been sought by repeated cloning at limiting dilutions. All seven specificities of hybridoma antibodies cross-react to a variable degree with porcine vWF and all but one with bovine vWF, indicating that they bind to structural loci that are relatively though not identically conserved between species. Hybridoma antibodies bind native as well as denatured vWF, suggesting that the epitopes may be determined to a considerable extent by primary structure rather than entirely by tertiary or quaternary conformation. The hybridoma antibodies define precise epitopes in the vWF molecule, extend the horizon of direct analysis of vWF to the intramolecular level, and may be useful in assigning functional loci to parts of the molecule.