The thrombin receptors PAR1 and PAR4 and their relative role in platelet activation

Sammanfattning: Many blood cell mechanisms in the human body are working all the time to maintain haemostasis in the blood vessels. Once a wound arises platelets are alerted via different substances to cover the wound and prevent loss of blood. Most of the times these mechanisms do stop the blood, and further heal the wound. During other circumstances the platelet-covering continues to form a thrombus, preventing the blood to flow and instead causes myocardial infarction or stroke. There are several risk factors triggering development of circulatory diseases such as obesity, lack of exercise, smoking, infection and stress.This thesis describes the interaction between the two platelet thrombin receptors PAR1 and PAR4, together with the interaction of the oral pathogen Porphyromonas gingivalis (with thrombin-like gingipains), and the cross talk with the stress hormone epinephrine and its α2A adrenergic receptor. Until now PAR1 is thought to be the most important thrombin receptor due to its high affinity for thrombin. From a phylogenetical and patophysiological point of view there must be a reason why platelets express two different thrombin receptors. Today PAR4 is considered less important, but this thesis implies that PAR4 plays an important role in platelet signaling and haemostasis.The results show that bacteria pre-stimulated platelets, followed by epinephrine gives a strong and full aggregation and calcium mobilization, in both aspirinated and non-aspirinated human platelets. The amount of bacteria does not itself, or epinephrine alone give aggregation or calcium mobilization. This mechanism is dependent on both Rgp type gingipain released from P. gingivalis, and PARs in an interaction with the α2A adrenergic receptor.Further, results reveal that PAR4 interacts and cross talks with the platelet α2A-adrenergic receptor in aspirinated platelets. Neither of the two platelet purinergic P2Y-receptors (P2Y12 and P2Y1) contribute to this action, but the purinergic P2X1 does. In aggregation studies a low dose of PAR4 activating peptide (AP), but not PAR1-AP, followed by epinephrine results in a strong aggregation and in a calcium mobilization. ATP secretion measurements did reveal that ATP was released during epinephrine stimulation, which indicate that ATP and P2X1 have a key role in this event. By blocking P2X1 both aggregation and calcium mobilization were abolished, but not by blocking P2Y12 and P2Y1. Inhibition of PI3-kinase, both epinephrine-induced calcium mobilization and aggregation were significant reduced. In non-aspirinated platelets PAR1 synergizes with the α2A adrenergic receptor and P2X1.In conclusion, this thesis suggests that PAR4 plays an intriguing and important role in platelets with inactived cyclooxygenase 1.  The results described in this thesis contribute to an increased knowledge of the platelet thrombin receptors.