Abstract/Summary

Background. The Glycoprotein VI (GPVI) receptor and the protease-activated receptor 1 (PAR1) are key platelet receptors and are activated by collagen and fibrin, or via thrombin, respectively. GPVI and PAR1 play a role in platelet activation and thrombus formation, two crucial processes for thrombosis and haemostasis. Furthermore, they are currently interesting targets for novel antiplatelet therapy. There is, therefore, a need to increase the understanding of the working mechanisms and the potential ligands for GPVI and PAR1. Aims. The goal of this thesis was to increase the understanding of acute and persistent effects of platelet activation mediated through GPVI and PAR1. Results. The role of GPVI and Syk in thrombus formation on collagens and collagen�like peptides was studied. Using the Syk inhibitor PRT060318, we showed that Syk supports platelet activation induced by collagens and collagen-like peptides, regardless of the presence of the GPVI binding motif GPO. More pronounced platelet activating effects of collagens were observed after immobilization, compared to when collagens were soluble. Since GPVI is also known to be the receptor for the coagulation-generated product fibrin, the role of GPVI in other coagulation proteins was studied. Through the use of GPVI and Syk inhibitors, it was evident that GPVI and Syk were involved in platelet activation induced by FXIIIa. Moreover, we found that APC induced platelet spreading through PAR1, which had previously only been shown for endothelial cells. In line with platelet activation induced by collagens, effects induced by FXIIIa and APC were more pronounced when the proteins were immobilized on a surface compared to in solution. Short- and long-term effects through the GPVI and the GPCR receptors PAR1 and P2Y1/12 were compared. Platelet activation through GPVI was more persistent, while responses evoked by PAR1 or P2Y1/12 stimulation were rather transient. Interestingly, pre-activated platelets, which started to return to a resting state (integrin inactivation, morphological change), but still expressed P-selectin on their surface, could be activated again upon restimulation. Conclusions. Taken together, the data presented in this thesis increase our understanding of how platelet functions are regulated through GPVI and PAR1. FXIIIa and APC were identified as novel ligands for GPVI and PAR1, respectively. Further, stimulation through GPVI was persistent, while stimulation through GPCRs (PAR1 or P2Y1/12) was transient. In addition, when platelets reverse, they can be reactivated. Since platelet function is highly regulated through GPVI and PAR1, the increased knowledge of the working mechanisms of these receptors, as well as on short- and long-term effects after activation, will contribute to an enhanced understanding of pre-activated circulating platelets in vivo in patients who are continuously exposed to activating components and will contribute to the improvement and development of more effective antiplatelet therapy.