THE PLATELET SURFACE EXPRESSION OF GLYCOPROTEIN-V IS REGULATED BY 2 INDEPENDENT MECHANISMS - PROTEOLYSIS AND A REVERSIBLE CYTOSKELETAL-MEDIATED REDISTRIBUTION TO THE SURFACE-CONNECTED CANALICULAR SYSTEM

In this study, we show that the platelet surface expression of glycopr otein (GP) V is regulated by two independent mechanisms. While confirm ing that both thrombin and neutrophil elastase proteolyse GPV, we show that neutrophil cathepsin G, thrombin receptor activating peptide (TR AP), and a combination of ADP and epinephrine can each result in a dec rease in the platelet surface expression of GPV by a nonproteolytic me chanism: a cytoskeletal-mediated redistribution of platelet surface GP V to the surface-connected canalicular system (SCCS). Four independent lines of evidence documented the nonproteolytic nature of this decrea se in the platelet surface expression of GPV. First, flow cytometric s tudies showed that cathepsin G, TRAP, and ADP/epinephrine decreased th e platelet surface expression of GPV without changing the total platel et content of GPV. Second, immunoelectron microscopy directly demonstr ated translocation of GPV from the platelet surface to the SCCS. Third , the cathepsin G-, TRAP-, and ADP/epinephrine-induced decreases in pl atelet surface GPV were fully reversible. Fourth, cytochalasin B, an i nhibitor of actin polymerization, completely inhibited the cathepsin G -, TRAP-, and ADP/epinephrine-induced decreases in platelet surface GP V. The cytoskeletal-mediated redistribution of GPV occurred in a whole blood milieu and at physiologic temperatures (37 degrees C) and extra cellular calcium concentrations (2 mmol/L). This study also defines th e diverse effects on GPV, GPIb, and GPIX of multiple important platele t agonists. Cathepsin G proteolysed platelet surface GPlb alpha, but r edistributed platelet surface GPIX and GPV to the SCCS. Thrombin prote olysed platelet surface GPV, but redistributed platelet surface GPIb a nd GPIX to the SCCS. Both TRAP and ADP/epinephrine redistributed plate let surface GPIb, GPIX, and GPV to the SCCS. Elastase proteolysed plat elet surface GPIb alpha and GPV, but, unlike the other agonists tested , neither proteolysed nor redistributed platelet surface GPIX. The exp eriments with TRAP showed that activation of the seven-transmembrane d omain thrombin receptor can result in translocation of GPIb, GPIX, and GPV to the SCCS independently of the GPIb-mediated pathway of thrombi n-induced platelet activation. This study also provides two additional lines of support for the recent report that GPV is noncovalently comp lexed with GPIb and GPIX in the platelet surface membrane. First, alth ough only the GPIb alpha subunit of this putative complex is known to be directly linked to the platelet cytoskeleton via actin-binding prot ein, cytochalasin B inhibited the ADP/epinephrine-, cathepsin G-, and TRAP-induced decrease in platelet surface GPV. Second, triple labeling flow cytometric experiments showed that, on each individual platelet, the ADP/epinephrine-induced decrease and subsequent return of the pla telet surface expression of GPV occurred simultaneously with the decre ase and subsequent return of the platelet surface expression of GPIb. In summary, the platelet surface expression of GPV is regulated by two independent mechanisms: proteolysis and a reversible, cytoskeletal-me diated redistribution to the SCCS. (C) 1996 by The American Society of Hematology.