Thrombin binding to GPIbα induces integrin αIIbβ3 dependent platelet adhesion to fibrin in ex vivo flowing whole blood

Summary

We have investigated the role of the thrombin/GPIbα interaction in the adhesion of platelets to fibrin in a whole blood ex vivo perfusion model at a shear rate of 280 s-1. Blood was perfused through parallel-plate chambers containing coverslips coated with cells expressing tissue factor, leading to the generation of thrombin and thus, deposition of fibrin onto the exposed cells. Adhesion of platelets to fibrin and thrombus growth were analyzed. Interestingly, when GPIbα was removed from the platelet surface by action of mocarhagin, platelet adhesion on fibrin was inhibited. Furthermore, a monoclonal antibody, VM16d, directed against the thrombin binding site on GPIbα also inhibited platelet adhesion on fibrin, showing the importance of the thrombin/GPIbα interaction. We then looked at the involvement of αIIbβ3 and showed that platelet adhesion and thrombus growth on fibrin were inhibited by the dodecapeptide, whereas lamifiban only inhibited the growth of the platelet thrombus. These results indicated that binding of thrombin to GPIbα induced an intracellular signaling leading to the interaction of the platelet integrin αIIbβ3 with the fibrindodecapeptide sequence.

Keywords

Platelet adhesion - GPIb-IX-V - thrombin - αIIbβ3 - fibrin

• References

• 1 Hantgan RR, Hindriks G, Taylor RG. et al. Glycoprotein Ib, von Willebrand factor, and glycoprotein IIb:IIIa are all involved in platelet adhesion to fibrin in flowing whole blood. Blood 1990; 76: 345-53.
• 2 Herrick S, Blanc-Brude O, Gray A, Laurent G. Fibrinogen. Int J Biochem Cell Biol 1999; 31: 741-746.
• 3 Farrell DH, Thiagarajan P, Chung DW, Davie EW. Role of fibrinogen alpha and gamma chain sites in platelet aggregation. Proc Natl Acad Sci U S A 1992; 89: 10729-32.
• 4 Dubois C, Steiner B, Kieffer N, Meyer SCReigner. Thrombin binding to GPIbalpha induces platelet aggregation and fibrin clot retraction supported by resting alphaIIbbeta3 interaction with polymerized fibrin. Thromb Haemost 2003; 89: 853-65.
• 5 Kirchhofer D, Tschopp TB, Baumgartner HR. Active site-blocked factors VIIa and IXa differentially inhibit fibrin formation in a human ex vivo thrombosis model. Arterioscler Thromb Vasc Biol 1995; 15: 1098-106.
• 6 Kirchhofer D, Tschopp TB, Steiner B. et al. Role of collagen-adherent platelets in mediating fibrin formation in flowing whole blood. Blood 1995; 86: 3815-22.
• 7 Bodnar RJ, Xi X, Li Z, Berndt MC, Du X. Regulation of Glycoprotein Ib-IX-von Willebrand Factor Interaction by cAMPdependent Protein Kinase-mediated Phosphorylation at Ser 166 of Glycoprotein Ibbeta. J Biol Chem 2002; 277: 47080-7.
• 8 Hantgan RR, Endenburg SC, Cavero I. et al. Inhibition of platelet adhesion to fibrin(ogen) in flowing whole blood by Arg-Gly-Asp and fibrinogen gamma-chain carboxy terminal peptides. Thromb Haemost 1992; 68: 694-700.
• 9 Hantgan RR, Stahle MC, Jerome WG. et al. Tirofiban blocks platelet adhesion to fibrin with minimal perturbation of GpIIb/IIIa structure. Thromb Haemost 2002; 87: 910-7.
• 10 Hantgan RR, Endenburg SC, Sixma JJ, de Groot PG. Evidence that fibrin alpha-chain RGDX sequences are not required for platelet adhesion in flowing whole blood. Blood 1995; 86: 1001-9.