THROMBIN-INDUCED GPIB-IX CENTRALIZATION ON THE PLATELET SURFACE REQUIRES ACTIN ASSEMBLY AND MYOSIN-II ACTIVATION

In resting platelets, the CPIb-IX complex, the receptor for the von Wi llebrand factor (vWF), is linked to underlying actin filaments by acti n-binding protein (ABP-280). Thrombin stimulation of human platelets l eads to a decrease in the surface expression of the GPIb-IX complex, w hich is redistributed from the platelet surface into the open canalicu lar system (OCS). Because the centralization of GPIb-IX is inhibited b y cytochalasin, it is believed to be linked to actin cytoskeletal rear rangements that take place during platelet activation. We have further characterized the mechanism of GPIb-IX centralization in platelets in suspension. Following thrombin stimulation, GPIb-IX shifts from the m embrane skeleton of the resting cell to the cytoskeleton of the activa ted cell in a reaction sensitive to cytochalasin B. The cytoskeletal a ssociation of GPIb-IX involves ABP-280, as it correlates with the inco rporation of ABP-280 into the activated cytoskeleton and because no di ssociation of the ABP-280/GPIb-IX complexes is detected after thrombin activation. However, the incorporation of GPIb-IX into the cytoskelet on is complete within 1 minute, whereas GPIb-IX centralization require s 5 to 10 minutes for completion. The movement of GPIb-IX to the cytos keleton of activated platelets is therefore necessary, but not suffici ent for GPIb-IX centralization. Blockage of cytosolic calcium increase s induced by thrombin by loading with the cell permeant calcium chelat or Ouin-2 AM inhibited GPIb-IX centralization by 70%, but did not prev ent its association with the activated cytoskeleton. Quin-2 loading di d, however, decrease the incorporation of myosin II into the activated cytoskeleton. The role of myosin II was further probed using the myos in light chain kinase (MLCK) inhibitor wortmannin. Wortmannin prevents myosin II association to the activated cytoskeleton and inhibits GPIb -IX centralization by 50%, without affecting actin assembly or the ass ociation of GPIb-IX to the cytoskeleton. Only micromolar concentration s of wortmannin, high enough to inhibit MLCK, prevent GPIb-IX centrali zation. These results indicate that thrombin-induced GPIb-IX centraliz ation requires a minimum of two steps, one associating GPIb-IX to the activated cytoskeleton and the second requiring myosin II activation. The involvement of myosin II implies that GPIb-IX/ABP-280 complexes, l inked to actin filaments, are pulled into the cell center, and that pl atelets may exert contractile tension on vWF bound to its receptor. (C ) 1996 by The American Society of Hematology.