Regulation of the phosphoinositide hydrolysis pathway in thrombin-stimulated platelets by a pertussis toxin-sensitive guanine nucleotide-binding protein. Evaluation of its contribution to platelet activation and comparisons with the adenylate cyclase inhibitory protein, G(i)

L. F. Brass, M. Laposata, H. S. Banga, S. E. Rittenhouse

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139 Scopus citations

Abstract

In platelets activated by thrombin, the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C produces inositol 1,4,5-triphosphate (IP3) and diacylglycerol, metabolites which are known to cause Ca2+ release from the platelet dense tubular system and granule secretion. Previous studies suggest that phospholipase C activation is coupled to platelet thrombin receptors by a guanine nucleotide-binding protein or G protein. The present studies examine the contribution of this protein to thrombin-induced platelet activation and compare its properties with those of G(i), the G protein which mediates inhibition of adenylate cyclase by thrombin. In platelets permeabilized with saponin, nonhydrolyzable GTP analogs reproduced the effects of thrombin by causing diacylglycerol formation, Ca2+ release from the dense tubular system and serotonin secretion. In intact platelets, fluoride, which by-passes the thrombin receptor and directly activates G proteins, caused phosphoinositide hydrolysis and secretion. Fluoride also caused an increase in the platelet cytosolic free Ca2+ concentration that appeared to be due to a combination of Ca2+ release from the dense tubular system and increased Ca2+ influx across the platelet plasma membrane. Guanosine 5'-O-(2-thiodiphosphate) (GDPβS), which inhibits G protein function, inhibited the ability of thrombin to cause IP3 and diacylglycerol formation, granule secretion, and Ca2+ release from the dense tubular system in saponin-treated platelets. Increasing the thrombin concentration overcame the effects of GDPβS on secretion without restoring diacylglycerol formation. The effects of GDPβS on platelet responses to thrombin which had been subjected to partial proteolysis (γ-thrombin) were similar to those obtained with native α-thrombin despite the fact that γ-thrombin is a less potent inhibitor of adenylate cyclase than is α-thrombin. Thrombin-induced diacylglycerol formation and 45Ca release were also inhibited when the saponin-treated platelets were preincubated with pertussis toxin, an event that was associated with the ADP-ribosylation of a protein with M(r) = 41.7 kDa. At each concentration tested, the inhibition of thrombin-induced diacylglycerol formation by pertussis toxin paralleled the inhibition of thrombin's ability to suppress PGI2-stimulated cAMP formation. These data suggest 1) that a G protein couples thrombin receptors to the phosphoinositide hydrolysis pathway in platelets and plays a central role in Ca2+ entry into the cytosol as well as secretion, 2) that this G protein is affected by pertussis toxin in a manner similar to G(i), and 3) that a pathway exists for thrombin-induced secretion that is independent of phosphoinositide hydrolysis.

Original languageEnglish (US)
Pages (from-to)16838-16847
Number of pages10
JournalJournal of Biological Chemistry
Volume261
Issue number36
StatePublished - 1986

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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