Mechanisms Of Actions Of Protease-activated Receptors 1 And 4 In Platelet Activation

Platelets play a major role in haemostasis and thrombosis. Platelet activation in vivo results from injury of the vessel wall, which is followed by spreading, secretion of granules, aggregation and procoagulant activity. This latter response leads to the generation of thrombin that is involved in the activation of platelets arriving in the blood flow at the site. Then, thrombin activates platelets at least in part through protease-activated receptors (PARs) which belong to the family of G protein-coupled receptor. Human platelets express PAR1 and PAR4, and activation of either is sufficient to trigger platelet secretion and aggregation. Thrombin activates human platelets by cleavage of the amino-terminal exodomain of PAR1 and PAR4, thereby-exposing a new amino terminus, which serves as a tethered peptide ligand. Binding of the tethered peptide ligand to the body of the receptor induces transinembrane signaling, leading to platelet secretion and aggregation. Another receptor for thrombin on platelets is the glycoprotein complex Ib-iX-V. Glycoprotein (GP) Ib is a sialoglycoprotein, which is implicated in platelet adhesion to the subendothelium expressing von willebrand factor (vWf). The critical roles played by GP Ib is emphasised by the Bernard-souliers syndrome where either a impaired expression of a component of the GP Ib-IX-V complex or a unfunctional complex is associated with a bleeding disorder. Platelet GP Ib moves into the open canalicular system (OCS) upon thrombin activation, and then returned to the platelet surface along time.showing a reversible translocation of GP Ib.In order to compare the participation of PAR-1 and PAR-4 during platelet activation, to determine whether they can induce GPIb redistribution, and then to investigate the role of the cytoskeleton in GP Ib centralisation, we use two peptides corresponding to PAR1-AP (SFLLRNPNDKYEPF) and PAR4-AP(AYPGKF) for stimulation. Using these peptides to stimulate platelets, we follow the redistribution of GP Ib and the link of GP Ibu, with actin and myosin. To study by which mechanism PARs induce the redistribution of GP Ib and the role of actin and myosin. a series of inhibitors were used in our experiments, among them Cytochalasin D (inhibitor of actin polymerization). BAPTA/AM (calcium chelator). Wortmannin (inhibitor of PB-kinasc and myosin light chain kinase). Apyrase VII (low ATP/ADPase). Ro-31-2220 (inhibitor of protein kinase C) and Y27632 (inhibitor of Rho kinase).Stimulation of either PAR1 or PAR4 leads to platelet aggregation with different efficiency. PAR-l-AP being the more potent, the following sequence was found: PAR 1 (SFLLRNPNDKY)>PAR4 (AYPGKF)>PAR4 (GYPGKF)> PAR4 (G YPGQV). In the following study, we used the more potent peptide for PAR1 and PAR4. Concomitant with a shrinkage of platelets, both flow cytoinctry and immtinofluorescence microscopy showed a transient redistribution of GP Ib in response to either PAR1-AP or PAR4-AP. detected as a decrease of approximately 50% of platelet surface GPIb. All over the time course experiment a different kinetic of redistribution of GP Ib was detected for the two peptides (p<0.05). Although an initial reduction of GP Ibalpha is apparent at 1 minute for both peptides. a maximum decrease is detected at 2 minutes upon PAR1-AP and 5 minutes upon PAR4-AP incubation, followed by a return of GP Iba to the surface, but slower for PAR4-AP. Similar results were obtained by various concentrations of PAR1-AP or PAR4-AP. but in a condition of enough concentration to induce platelet shape change. GP Ibainternalisation and shape change were blocked by Cytochalasin D or BAPTA/AM, although the former had a more dramatic effect. For P-selectin expression, there is a significant increase at 1 minute and maintained at a maximum state at 2 minutes.Following PAR stimulation, GP Iba became incorporated into the insoluble cytoskeleton. The fraction of GP Iba detected in the cytoskeleton reached a maximum at 1 minute in response to PAR1-AP, whereas it peaked at 5 minutes upon PAR4-AP activation, to...