A B ST R A CT The binding of '251-von Willebrand factor to platelets stimulated by thrombin, ADP, and a combination of ADP + epinephrine (EPI) is specific, saturable, and reversible. Active platelet metabolism and divalent cations are required for binding induced by these stimuli, but not by ristocetin, suggesting the existence of different mechanisms involved in the vWF-platelet interaction. A monoclonal antibody directed against an epitope of membrane glycoprotein (GP) lb had no effect on the binding of '251-vWF to normal platelets stimulated by thrombin or a combination of ADP + EPI, but completely blocked ristocetin-induced binding. Binding induced by thrombin to GPIb-blocked platelets was specific. Moreover, thrombin-induced binding of 1251-vWF was increased, rather than decreased, in two patients with the Bernard-Soulier syndrome whose platelets lacked GPIb. Conversely, monoclonal antibodies directed against the GPIIb/IIIa complex had no effect on ristocetininduced binding of (vWF)' is a large multimeric glycoprotein that circulates in blood complexed with the Factor VIII procoagulant activity protein (1). It plays an essential role in platelet function as shown by the prolonged bleeding time in von Willebrand disease (1). Specific binding sites for vWF are induced on the platelet membrane by the antibiotic ristocetin (2), a nonphysiologic agent, as well as by thrombin (3, 4) and ADP (5). Platelet membrane glycoprotein (GP) lb is considered to function as the surface receptor for vWF (6). In the Bernard-Soulier syndrome, a congenital bleeding disorder, platelets lack GPIb (7) and the ristocetin-induced binding of vWF is decreased (8). On the other hand, in Glanzmann thrombasthenia, another congenital platelet abnormality, there is a marked decrease of the membrane GP complex Ilb/Illa but normal content of GPIb (9). The ristocetin-induced binding of vWF to thrombasthenic platelets has been reported to be normal (8). In contrast, we have recently shown that binding of vWF to thrombasthenic platelets stimulated by thrombin is severely deficient (4). Therefore, we postulated that different sites are involved in ' Abbreviations used Marguerie et al. (12). The final platelet suspensions were all in modified calcium-free Tyrode buffer, containing 137 mM NaCI, 2 mM MgCI2, 0.42 mM NaH2PO4, 11.9 mM NaHCO3, 2.9 mM KCI, 5.5 mM glucose, 10 mM Hepes, pH 7.35, and 20 mg/ml bovine serum albumin (Fraction V, CalbiochemBehring Corp., American Hoechst Corp., San Diego, CA). When experiments of serotonin release were performed, PRP was labeled before washing by incubating 20 ml with 0. (3.3,M). However, ADG-washed platelets usually gave reversible aggregation to ADP and were unresponsive to epinephrine (EPI). On the contrary, gel-filtered platelets always gave irreversible aggregation to ADP, and responded to 20 MM EPI in the presence of fibrinogen (3.3 ,M) with a typical two-wave aggregation. Contamination of plasma vWF in all washed platelet preparations was below 5 X 10' U/dl (1 dl of a normal plasma pool contains ...

Abstract. Mitogenesis, cellular aggregation, and motility follow upon the interaction of fibrinogen with certain defined cell surface receptors. In addition to circulating platelets and vascular endothelium, monocytes express what appears to be a receptor for fibrinogen.

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).

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.