Blood platelets are key players in haemostasis, the system that maintains an intact circulation. Circulating platelets are constantly on guard for vessel wall injury and will momentarily adhere to exposed subendothelial proteins, mainly collagens, at site of the injury. In parallel with platelet adhesion, tissue damage will also lead to the exposure of tissue factor, which will initiate thrombin generation. Platelet adhesion to collagen and the formation of thrombin will work in concert to induce the formation of a lose platelet plug as a result of platelet activation. This process is amplified by platelet agonists, mainly ADP, released from platelets themselves upon activation. The lose platelet plug is then stabilised by the formation of an armouring network of fibrin as a result of a burst in thrombin generation, dependent on the assembly of coagulation factors on the surface of activated platelets.
This thesis presents studies on thrombin- and ADP-induced platelet activation, and drug intervention in vitro, mainly using tlow cytometry as a tool for platelet function evaluation in diluted whole blood. Thrombin-induced platelet activation is composed of an initial response to low, and a secondary response to high concentrations of the agonist. Low concentrations will result in the activation of the high affinity thrombin receptor [protease activated receptor 1 (PAR1)], which in turn is sufficient to induce ADP release. Higher concentrations are needed to activate the low affinity PAR4. The platelet response to thrombin is thus the combined effect of signalling from PAR1, PAR4 and the ADP receptors, P2Y1 and P2Y12.
Reversible and irreversible or tight binding thrombin inhibitors differentially inhibited thrombin-induced platelet activation. Reversible inhibitors inhibited the thrombin response via a potent indirect inhibition of PAR4, and a partial inhibition of PAR1. Irreversible or tight binding inhibitors were potent indirect inhibitors of both PAR4 and PAR1. This can be explained by the different aftinity of thrombin for the two receptors and the different affinity of the inhibitors for thrombin. It was possible to completely block the ADP component in the thrombin response by inhibition of P2Y12, which resulted in a 15-80% relative inhibition depending on the thrombin concentration, whereas P2Y1 inhibition was ineffective. This was true not only in man but also in dog, mouse, rat and guinea pig. Blockade of P2Y12 gave in all species an effect equal to total removal of ADP. The relative inhibitory effect was most pronounced at low thrombin concentrations just enough to cleave PAR1 and thus release all ADP. The difference in effect between PSY1 and PSY12 inhibition is likely explained by the need for both a Gαi and Gαq signal in order to obtain potent platelet activation and that the only strong Gαq signal after thrombin stimulation is via PSY12. PSY1 on the other hand couples to Gαq, which is also activated by thrombin it-self via both PARI and PAR4. PSY1 dependent Gαq-signalling is therefore more or less redundant when thrombin is used as agonist. Finally, by a concomitant inhibition of a Gαq and a Gαisignalling true synergistic inhibition of both the thrombin and ADP response could be achieved.
We conclude that reversible thrombin inhibitors are potent platelet inhibitors by indirect inhibition of primarily PAR4 and to a less extent PAR1. Thrombin-induced platelet activation can also be potently inhibited by PSY12 antagonists, especially at low thrombin concentrations, just enough to cleave PAR1 and release all ADP. Synergistic inhibition of thrombin- and ADP induced platelet activation can be achieved by a concomitant inhibition of thrombin/P2Y12 and PSY1/P2Y12, respectively.
Göteborg: Intellecta Docusys , 2005. , 76 p.
2005-03-17, Elsa Brändströmsalen, Universitetssjukhuset, Linköping, 13:00 (Swedish)