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 endothelial cells (ECs). We evaluated the effect of a recombinant von Willebrand 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 conditions (350/s) resulted in minimal platelet adhesion. The adhesion was enhanced two- to threefold after either platelet activation by thrombin receptor agonist peptide (TRAP) or EC pretreatment with thrombin. The enhancing effect of thrombin was abolished by addition of either hirudin (10 u/ml) or PGE1 (1 microg/ml). Preincubation of resting platelets with increasing concentrations of AR545C under static or flow conditions resulted in a dose-dependent inhibition of thrombin-induced enhanced adhesion to ECs. AR545C (0.3 microM) completely abolished the effect of thrombin, reducing platelet adhesion to the control level observed with non-treated ECs. In contrast, the same concentration of AR545C had no effect on the adhesion of TRAP-activated platelets to ECs. AR545C also inhibited thrombin-induced platelet aggregation and binding in a dose-dependent manner. In addition, 0.3 microM of AR545C reduced thrombin-induced serotonin release by 57%, whereas monoclonal antibody AN51, which inhibits ristocetin-induced platelet aggregation, had no effect on either thrombin-induced platelet aggregation or binding or on serotonin release. Similarly, AR545C had no effect on TRAP-induced serotonin release. These findings suggest that (i) AR545C inhibits platelet activation mediated by thrombin and this inhibition occurs through blocking the high-affinity thrombin binding sites on the GPIb/IX complex and (ii) AR545C has no effect on the moderate affinity thrombin receptor (seven transmembrane domain thrombin receptor; STDR).
Dardik
R
R
Institute of Thrombosis and Haemostasis and National Haemophilia Centre, Sheba Medical Centre, Tel-Hashomer, Sackler Faculty of Medicine, Tel Aviv University, Israel.
Varon
D
D
Eskaraev
R
R
Tamarin
I
I
Inbal
A
A
D001792:Blood Platelets / Q000378:metabolism; D002478:Cells, Cultured; D046508:Culture Techniques; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular; D006801:Humans; D010446:Peptide Fragments / Q000494:pharmacology; D015539:Platelet Activation; D010973:Platelet Adhesiveness / Q000187:drug effects*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D018179:Receptors, Thrombin / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology*; D014841:von Willebrand Factor / Q000378:metabolism / Q000494:pharmacology*
D010446:Peptide Fragments; D019038:Platelet Glycoprotein GPIb-IX Complex; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D013917:Thrombin