Collagen-induced platelet activation mainly involves the protein kinase C pathway.
This study analyses early biochemical events in collagen-induced platelet activation. An early metabolic event occurring during the lag phase was the activation of PtdIns(4,5)P2-specific phospholipase C. Phosphatidic acid (PtdOH) formation, phosphorylation of P43 and P20, thromboxane B2 (TXB2) synthesis and platelet secretion began after the lag phase, and were similarly time-dependent, except for TXB2 synthesis, which was delayed. Collagen induced extensive P43 phosphorylation, whereas P20 phosphorylation was weak and always lower than with thrombin. The dose-response curves of P43 phosphorylation and granule secretion were similar, and both reached a peak at 7.5 micrograms of collagen/ml, a dose which induced half-maximal PtdOH and TXB2 formation. Sphingosine, assumed to inhibit protein kinase C, inhibited P43 phosphorylation and secretion in parallel. However, sphingosine was not specific for protein kinase C, since a 15 microM concentration, which did not inhibit P43 phosphorylation, blocked TXB2 synthesis by 50%. Sphingosine did not affect PtdOH formation at all, even at 100 microM, suggesting that collagen itself induced this PtdOH formation, independently of TXB2 generation. The absence of external Ca2+ allowed the cleavage of polyphosphoinositides and the accumulation of InsP3 to occur, but impaired P43 phosphorylation, PtdOH and TXB2 formation, and secretion; these were only restored by adding 0.11 microM-Ca2+. In conclusion, stimulation of platelet membrane receptors for collagen initiates a PtdInsP2-specific phospholipase C activation, which is independent of external Ca2+, and might be the immediate receptor-linked response. A Ca2+ influx is indispensable to the triggering of subsequent platelet responses. This stimulation predominantly involves the protein kinase C pathway associated with secretion, and appears not to be mediated by TXB2, at least during its initial stage.
Karniguian
A
A
INSERM U150-CNRS URA 334, Hôpital Saint-Louis, Paris, France.
Grelac
F
F
Levy-Toledano
S
S
Legrand
Y J
YJ
Rendu
F
F
D001792:Blood Platelets / Q000201:enzymology* / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D003094:Collagen / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation; D011493:Protein Kinase C / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism; D013110:Sphingosine / Q000494:pharmacology; D013929:Thromboxane B2 / Q000096:biosynthesis; D001620:beta-Thromboglobulin / Q000378:metabolism
D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D001620:beta-Thromboglobulin; D012701:Serotonin; D004533:Egtazic Acid; D013929:Thromboxane B2; D003094:Collagen; D011493:Protein Kinase C; D009219:Myosin-Light-Chain Kinase; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D013110:Sphingosine; D002118:Calcium