Recombinant fragment of yon Willebrand factor AR545c inhibits platelet binding to thrombin and platelet adhesion to thrombin-treated endothelial cells

Rima Dardik, David Varon, Regina Eskaraev, Ilya Tamarin, Aida Inbal

Research output: Contribution to journalArticlepeer-review

Abstract

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 μg/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 μM) 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 μM 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).

Original languageEnglish
Pages (from-to)512-518
Number of pages7
JournalBritish Journal of Haematology
Volume109
Issue number3
DOIs
StatePublished - 2000
Externally publishedYes

Keywords

  • AR545C
  • Endothelial cells
  • Platelet adhesion
  • Thrombin receptor

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