Asialo von Willebrand factor interactions with platelets. Interdependence of glycoproteins Ib and IIb/IIIa for binding and aggregation.

Abstract

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...

Furin gene (fur) regulation in differentiating human megakaryoblastic Dami cells: involvement of the proximal GATA recognition motif in the P1 promoter and impact on the maturation of furin substrates

“…48,63-66 A body of literature has firmly established that VWF and ␣ IIb are essential for thrombus formation at the vascular injury site, whereas platelet ␣-granule-derived TGF-␤ and PDGF are major players of endothelial cell proliferation and vessel repair. [67][68][69][70] In addition, TGF-␤1 has been shown to enhance platelet aggregation through a nontranscriptional effect on the fibrinogen receptor. 71 Furin has also been identified as the convertase responsible for the maturation of Notch-1, a cell-surface receptor involved in multiple developmental processes.…”

Von Willebrand's disease with spontaneous platelet aggregation induced by an abnormal plasma von Willebrand factor.

“…EDTA added in vitro inhibits the platelet aggregation of the PRP of platelet-type vWd seen after adding cryoprecipitate or plasma and the post-DDAVP platelet aggregation in type Ilb vWd patients (2,5,23). Calcium ions are also necessary for the platelet aggregation that occurs after addition of AS-vWf to platelets (see below) (7)(8)(9)(10)25).…”

“…Asialo vWf (AS vWf) has been shown to aggregate normal platelets in the absence of ristocetin (7)(8)(9)(10)25). We and others have recently shown that AS-vWf binds to both the GPIb and the GPIlb/IlIa complexes (10, 1 1).…”

“…EDTA was used in two of the original anticoagulants in which the blood was collected at 1 and 5 mM final concentration (see above). Dibutryl cyclic AMP (db-cAMP) 4.5-20 mM (Sigma Chemical Co.); apyrase, 20-200 ,g/ml (Sigma Chemical Co.); and prostaglandin 12 (PG12), 4- (10,15,16). The monoclonal antibodies were added to the PRP 3 min before adding the stirring bar.…”

Identification of the thrombin receptor on human platelets by chemical crosslinking.

“…Platelets Solum) were tested. The vWf was purified as previously described (13). The monoclonal antibody 6D1 is directed against the site on GPIb which vWf binds in the presence of ristocetin (14).…”