"Thrombin" receptor-directed ligand accounts for activation by thrombin of platelet phospholipase C and accumulation of 3-phosphorylated phosphoinositides.
Using three experimental approaches, we have addressed the questions of whether the presence of saturably bound thrombin plays a role in potentiating the activation of platelet phospholipase C (PLC) and/or accumulation of the 3-phosphorylated phosphoinositides (3-PPI), i.e. phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, and whether the generation of tethered ligand (Vu, T-K.H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. (1991) Cell 64, 1057-1068) by thrombin can account fully for thrombin's proteolytic effects in activating platelets, as gauged by the above parameters. We have 1) measured PLC activation or 3-PPI after we have exposed platelets to thrombin for various periods and either blocked thrombin's proteolytic activity without interrupting its binding or blocked both binding and proteolytic activity of thrombin; 2) attempted to potentiate 3-PPI accumulation, using combinations of protein kinase C stimulation, Ca2+ elevation, and saturating but proteolytically inactive thrombins; and 3) compared the activation of platelets by thrombin with activation by the "thrombin" receptor-directed peptide, SFLLRNPNDKYEPF (SFLL; a portion of the tethered ligand created by thrombin's proteolytic activity), and examined the effect of thrombin on this latter activation. We conclude that the initial and sustained effects of thrombin in stimulating PLC and the accumulation of 3-PPI are completely attributable to thrombin's proteolytic activity. Further, thrombin's effects in promoting these responses can be accounted for by the actions of SFLL peptide, and by implication, formation of tethered ligand.
Huang
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RS
Department of Biochemistry, University of Vermont College of Medicine, Burlington 05405-0068.
Sorisky
A
A
Church
W R
WR
Simons
E R
ER
Rittenhouse
S E
SE
D000595:Amino Acid Sequence; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D003619:Dansyl Compounds / Q000494:pharmacology; D004789:Enzyme Activation; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism*; D010766:Phosphorylation; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism*
D003619:Dansyl Compounds; D006629:Hirudins; D008024:Ligands; D010446:Peptide Fragments; D010716:Phosphatidylinositols; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; C072032:thrombin receptor peptide (42-55); C019162:dansylarginine N-(3-ethyl-1,5-pentanediyl)amide; D001120:Arginine; D010738:Type C Phospholipases; D013917:Thrombin