We have investigated and characterized the abnormalities in four unrelated patients with von Willebrand's disease (vWd) who have (a) enhanced ristocetin-induced platelet aggregation (RIPA) at low ristocetin concentrations, (b) absence of the largest plasma von Willebrand factor (vWf) multimers, and (c) thrombocytopenia. The platelet-rich plasma of these patients aggregates spontaneously without the addition of any agonists. When isolated normal platelets are resuspended in patient plasma spontaneous aggregation occurs; however, the patients' plasmas did not induce platelet aggregation of normal washed formalinized platelets. When the patients' platelets are suspended in normal plasma, spontaneous aggregation is not observed. The spontaneous platelet aggregation (SPA) is associated with dense granule secretion as measured by ATP release and alpha granule release as measured by fl-thromboglobulin and platelet factor 4 release.The SPA is totally inhibited by 5 mM EDTA, prostaglandin I2, and dibutryl cyclic AMP, while it is only partially inhibited by 1 mM EDTA, acetylsalicylic acid, or apyrase. A monoclonal antibody directed against glycoprotein Ib (GPIb) and/or a monoclonal antibody against the glycoprotein IIb/IIIa (GPIIb/ IIIa) complex totally inhibits the SPA.The vWf was isolated from the plasma of one of these patients. The purified vWf induced platelet aggregation of normal platelets resuspended in either normal or severe vWd plasma, but the vWf did not induce platelet aggregation of normal platelets resuspended in afibrinognemic plasma. Sialic acid and galactose quantification ofthe patient's vWf revealed approximately a 50% reduction compared with normal vWf. These studies indicate that a form of vWd exists, which is characterized by SPA that is induced by the abnormal plasma vWf. The SPA is dependent on the presence of plasma fibrinogen, and the availability of the GPIb and the GPIIb/IIIa complex. In this variant form of vWd the abnormal vWf causes enhanced RIPA, SPA, and thrombocytopenia.

Asialo von Willebrand factor (AS-vWf) binds to and aggregates normal human platelets in the absence of ristocetin. Maximal specific binding of AS-vWf is 1-2 ug vWf protein/108 platelets.Despite the specificity of the binding, only 60% of the bound AS-vWf can be dissociated after equilibrium has been reached. We investigated the site of binding and the mechanism of aggregation of platelets by AS-vWf by (a) pre-incubating platelets with either of two monoclonal antibodies, one against glycoprotein lb (GPIb) to normal primary hemostasis, that is, the adhesion of platelets to subendothelial surfaces and the subsequent aggregation of platelets (1, 2). Human vWf in the presence of ristocetin binds to the glycoprotein lb (GPIb) of platelets that are either intact metabolically or have been fixed with formaldehyde. To date, no in vivo analogue of ristocetin has been found. DeMarco and Shapiro reported, however, that asialo vWf (AS-vWf) binds spontaneously and independently of ristocetin to the platelet surface and causes aggregation (3). They postulated that this represented a possible mechanism for the in vivo binding of vWf to human platelets and subsequent adhesion and aggregation.We have undertaken an investigation to determine the site or sites of AS-vWf binding to human platelets, the effects of other plasma proteins on the binding, and the mechanism(s) of platelet aggregation induced by AS-vWf. Our studies employed (a) monoclonal antibodies specific for GPIb and the glycoprotein lIb/Ila complex (GPIIb/IIIa), (b) plasmas containing variable concentrations of fibrinogen, and (c) the platelets from thrombasthenic patients. MethodsBlood was drawn from normal subjects and patients by use of a twosyringe technique. A 19-gauge needle was used and the initial 5-10 ml of blood was used for other laboratory tests. The blood from the second syringe was placed into polypropylene tubes containing 0.1 ml sodium citrate anticoagulant, final concentration 10.9 mM. Plateletrich plasma (PRP) was prepared from whole blood by removal of the plasma-platelet layer after centrifugation at 750 relative centrifugal force (RCF) for 3 min at 250C. In some experiments, the platelets were washed free of erythrocytes and leukocytes by the preparation of PRP and one wash of the erythrocytes with Hepes buffer, pH 7.35 (4). The PRP and wash were then placed on a discontinuous arabinogalactan gradient 20% (3 ml) and 10% (5 ml) and platelets were separated from plasma proteins by centrifugation at 2,000 RCF for 30 min. The platelets (>85% recovery) were then resuspended in autologous or homologous normal plasma or plasma from two patients with congenital afibrinogenemia. Plasma was obtained from two patients with congenital afibrinogenemia who had thrombin time > 300 s, and no detectable fibrinogen by the Clauss technique or tanned erythrocyte hemagglutination inhibition, i.e., a level < 0.5 isg/ml. Mixing studies were performed to ensure that neither afibrinogenemic plasma contained antifibrinogen antibodies. Each afibrinogenemic plasma...