The platelet surface expression of glycoprotein V is regulated by two 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 glycoprotein (GP) V is regulated by two independent mechanisms. While confirming that both thrombin and neutrophil elastase proteolyse GPV, we show that neutrophil cathepsin G, thrombin receptor activating peptide (TRAP), and a combination of ADP and epinephrine can each result in a decrease in the platelet surface expression of GPV by a nonproteolytic mechanism: a cytoskeletal-mediated redistribution of platelet surface GPV to the surface-connected canalicular system (SCCS). Four independent lines of evidence documented the nonproteolytic nature of this decrease in the platelet surface expression of GPV. First, flow cytometric studies showed that cathepsin G, TRAP, and ADP/epinephrine decreased the platelet surface expression of GPV without changing the total platelet content of GPV. Second, immunoelectron microscopy directly demonstrated translocation of GPV from the platelet surface to the SCCS. Third, the cathepsin G-, TRAP-, and ADP/epinephrine-induced decreases in platelet surface GPV were fully reversible. Fourth, cytochalasin B, an inhibitor of actin polymerization, completely inhibited the cathepsin G-, TRAP-, and ADP/epinephrine-induced decreases in platelet surface GPV. The cytoskeletal-mediated redistribution of GPV occurred in a whole blood milieu and at physiologic temperatures (37 degrees C) and extracellular calcium concentrations (2 mmol/L). This study also defines the diverse effects on GPV, GPIb, and GPIX of multiple important platelet agonists. Cathepsin G proteolysed platelet surface GPIb alpha, but redistributed platelet surface GPIX and GPV to the SCCS. Thrombin proteolysed platelet surface GPV, but redistributed platelet surface GPIb and GPIX to the SCCS. Both TRAP and ADP/epinephrine redistributed platelet surface GPIb, GPIX, and GPV to the SCCS. Elastase proteolysed platelet surface GPIb alpha and GPV, but, unlike the other agonists tested, neither proteolysed nor redistributed platelet surface GPIX. The experiments with TRAP showed that activation of the seven-transmembrane domain thrombin receptor can result in translocation of GPIb, GPIX, and GPV to the SCCS independently of the GPIb-mediated pathway of thrombin-induced platelet activation. This study also provides two additional lines of support for the recent report that GPV is noncovalently complexed with GPIb and GPIX in the platelet surface membrane. First, although only the GPIb alpha subunit of this putative complex is known to be directly linked to the platelet cytoskeleton via actin-binding protein, 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 platelet surface expression of GPV occurred simultaneously with the decrease 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-mediated redistribution to the SCCS.
Michelson
A D
AD
Department of Pediatrics, University of Massachusetts Medical School, Worcester 01655, USA.
Benoit
S E
SE
Furman
M I
MI
Barnard
M R
MR
Nurden
P
P
Nurden
A T
AT
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000328:Adult; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000378:metabolism* / Q000648:ultrastructure; D002118:Calcium / Q000502:physiology; D056649:Cathepsin G; D002403:Cathepsins / Q000494:pharmacology; D002451:Cell Compartmentation; D015550:Cell Degranulation; D003599:Cytoskeleton / Q000502:physiology; D004837:Epinephrine / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D019272:Leukocyte Elastase / Q000494:pharmacology; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D012697:Serine Endopeptidases; D013917:Thrombin / Q000494:pharmacology
D000199:Actins; D010446:Peptide Fragments; D019038:Platelet Glycoprotein GPIb-IX Complex; C072032:thrombin receptor peptide (42-55); D000244:Adenosine Diphosphate; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D013917:Thrombin; D002118:Calcium; D004837:Epinephrine