Functions of Thrombin Receptors in the Reversible Distribution of Platelet Glycoprotein Ib within Activated Platelets.

Platelets are critical in normal haemostasis and arterial thrombosis. As a potent activator, thrombin activates human platelets by cleavage of the protease-activated receptors PARs, exposing new amino terminus which serve as tethered peptide ligands, binding to the receptors induce transmembrane signaling. Thrombin also binds to platelet glycoprotein Ib, whose redistribution within platelet was showed to be reversible upon to thrombin or to PAR1 and PAR4 peptides. In an attempt to understand the reversible expression of platelet GPIb and cytoskeleton reorganization during platelet activation, then determine functions of thrombin receptors in GPIb redistribution. we used peptide SFLLRNPNDKYEPF (PAR1-AP, trap) and AYPGKF (PAR4-AP) for stimulating platelet at different time points (0~60minute), detected the platelet surface GPIbα and P-selectin with flowcytometry, and compared the alteration of GPIbα, actin and myosin in cytoskeleton by western-blot, then analyzed the membrane cytoskeleton followed by GPIbα immunoprecipitation.

As expected, a reversible interalisation of GPIbα was obtained by PAR1 or PAR4 activation, and a transient change of actin, myosin and GPIbα /myosin GPIbα /actin association were also found in this course. These transient internalisations were apparently blocked by Cytochalasin D (inhibitor of actin polymerisation) or BAPTA/AM (calcium chelator). At the same time, ApyraseVII had weak effect on GPIbα interalisation, although the return of GPIbα to platelet surface was accelerated by this low ATP/ADPase, which quickened GPIbα dissappearance in cytoskeleton and the dissociation of GPIb/myosin or GPIb/actin during PAR1-AP activation.100nM and 10μM wortmannin were used to inhibit phosphatidylinositol 3-kinase (PI3-K) and/or myosin light chain kinase (MLCK) in our experiments. In response to both PARs activation, GPIbα interalisation was partly inhibited by 100nM wortmannin but little change for 10μM wortmannin, and a delayed restoration of surface GPIbα was observed in the presence of 10μM wortmannin upon PAR1-AP activation.. Apart from the above reagents, Ro-31-2220 (inhibitor of protein kinase C) induced a decreased GPIbα centralisation in response to PAR1 activation, and blocked the pool of GPIbα inside platelet in the latter course of PAR4 activation (p<0.05 at 10,30 min). Globally, Our study confirmed that thrombin receptors are important for platelet signal transmission, Stimulation of either receptor is sufficient to trigger platelet activation and induce GP Ib redistribution, which is correlated with cytoskeleton reorganisation, depending on the actin polymerisation and calcium mobilisation and implicate both myosin and actin. Our results also suggested critical roles of ADP, PI3-K or protein kinase C (PKC) in the GPIb redistribution.