Recombinant fragment of von Willebrand factor AR545C inhibits platelet binding to thrombin and platelet adhesion to thrombin-treated endothelial cells

Activated, but not resting, platelets are capable of adhering to intact end othelial cells (ECs). We evaluated the effect of a recombinant von Willebra nd factor (VWF) fragment AR545C, which inhibits glycoprotein Ib (GPIb)/VWF binding, on platelet adhesion to human ECs under static or flow conditions. Incubation of resting platelets with intact endothelium under flow conditi ons (350/s) resulted in minimal platelet adhesion. The adhesion was enhance d two- to threefold after either platelet activation by thrombin receptor a gonist peptide (TRAP) or EC pretreatment with thrombin. The enhancing effec t of thrombin was abolished by addition of either hirudin (10 u/ml) or PGE( 1) (1 mu g/ml). Preincubation of resting platelets with increasing concentr ations of AR545C under static or flow conditions resulted in a dose-depende nt inhibition of thrombin-induced enhanced adhesion to ECs. AR545C (0.3 mu M) completely abolished the effect of thrombin, reducing platelet adhesion to the control level observed with non-treated ECs. In contrast, the same c oncentration of AR545C had no effect on the adhesion of TRAP-activated plat elets to ECs. AR545C also inhibited thrombin-induced platelet aggregation a nd binding in a dose-dependent manner In addition, 0.3 mu M Of AR545C reduc ed thrombin-induced serotonin release by 57%, whereas monoclonal antibody A N51, which inhibits ristocetin-induced platelet aggregation, had no effect on either thrombin-induced platelet aggregation or binding or on serotonin release. Similarly, AN545C had no effect on TRAP-induced serotonin release, These findings suggest that (i) AR545C inhibits platelet activation mediat ed by thrombin and this inhibition occurs through blocking the high-affinit y thrombin binding sites on the GPIb/IX complex and (ii) AR545C has no effe ct on the moderate affinity thrombin receptor (seven transmembrane domain t hrombin receptor: STDR).