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

Abstract

We have investigated the role of the thrombin/GPIbalpha 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 GPIbalpha 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 GPIbalpha also inhibited platelet adhesion on fibrin, showing the importance of the thrombin/GPIbalpha interaction.We then looked at the involvement of alphaIIbbeta3 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 GPIbalpha induced an intracellular signaling leading to the interaction of the platelet integrin alphaIIbbeta3 with the fibrin-dodecapeptide sequence.

Platelet-Mediated NET Release Amplifies Coagulopathy and Drives Lung Pathology During Severe Influenza Infection

“…Moreover, CD18 has been implicated in the binding of platelets to neutrophils ( 20 ), and this binding subsequently triggers neutrophil activation and migration ( 39 ). The integrin CD41/CD61 (GPIIb/IIIa), which is found solely on platelets, is responsible for the formation of fibrinogen bridges among platelets, facilitating platelet cohesion, aggregation, and subsequent thrombus growth ( 40 , 41 ).…”

Thrombin-induced reactive oxygen species generation in platelets: A novel role for protease-activated receptor 4 and GPIbα

“…Thrombin, a potent platelet activator, can signal through GPIbα and protease activated receptor (PAR)1 and PAR4 on platelets [9] . PAR1 and PAR4 are known as the major thrombin receptors on platelets [10] ; however, thrombin additionally binds GPIbα initiating the phosphorylation of signalling molecules required for integrin α IIb β 3 activation [11] , [12] . This signalling is inhibited by treatment of platelets with the purified snake venom-derived metalloproteinases, mocarhagin and Naja kaouthia (Nk) protease [12] , [13] , which cleave GPIbα between amino acids Glu282-Asp283 and Tyr276-Asp277, respectively, to remove the GPIbα ectodomain including a sulphated tyrosine sequence that binds thrombin [14] , [15] .…”

“…PAR1 and PAR4 are known as the major thrombin receptors on platelets [10] ; however, thrombin additionally binds GPIbα initiating the phosphorylation of signalling molecules required for integrin α IIb β 3 activation [11] , [12] . This signalling is inhibited by treatment of platelets with the purified snake venom-derived metalloproteinases, mocarhagin and Naja kaouthia (Nk) protease [12] , [13] , which cleave GPIbα between amino acids Glu282-Asp283 and Tyr276-Asp277, respectively, to remove the GPIbα ectodomain including a sulphated tyrosine sequence that binds thrombin [14] , [15] . In human platelets both PAR1 and PAR4 initiate platelet activation through G-protein signalling, and PAR1 contains a thrombin-binding sequence, which is absent in PAR4, allowing thrombin to bind more readily (20–70-fold faster rate of activation than PAR4) and at lower concentrations [16] , [17] .…”