Differences between platelet phosphoinositide metabolism stimulated by thrombin or SFLLRN are not accounted for by interaction of thrombin with glycoprotein Ib.
The formation of inositol phosphates was compared in aspirin-treated, washed human platelets suspended in Tyrode's-albumin solution containing 2 mM calcium and stimulated with SFLLRN (thrombin receptor-activating peptide) or thrombin. SFLLRN (20 microM) and thrombin (1 U/ml) resulted in maximal irreversible aggregation and 80-90% secretion of dense granule contents. SFLLRN (50-100 microM) caused larger increases at 10 sec than 20 microM SFLLRN in the formation of inositol trisphosphate (IP3, measured as [3H]inositol label). These increases were not significantly less than those caused by thrombin (1 unit/ml). However, whereas the labeling of IP3 increased from 10-60 sec with thrombin, with SFLLRN it was much less at 60 sec than that at 10 sec. The decrease was not due to degradation of SFLLRN by ectopeptidases, since it was not prevented by amastatin, an inhibitor of ectopeptidases. Degradation of glycoprotein Ib (GPIb) with an O-sialoglycoprotein endopeptidase did not affect the thrombin-stimulated labeling of inositol phosphates, indicating that binding to GPIb is not involved in the sustained thrombin-induced formation of inositol phosphates. The finding that the thrombin-stimulated formation of IP3 was not dependent on Ca2+ in the medium (EGTA added) indicates that the transient SFLLRN-induced formation of IP3 is not due to failure to cause Ca2+ influx. The finding that formation of IP3 was transient in SFLLRN-stimulated platelets, whereas platelet aggregation and secretion were maximal, indicates that the sustained activation of phospholipase C caused by thrombin may have roles related to later processes in which platelets participate.
Vickers
J D
JD
Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
Packham
M A
MA
Kinlough-Rathbone
R L
RL
D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000378:metabolism*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology*
D010446:Peptide Fragments; D010716:Phosphatidylinositols; D019038:Platelet Glycoprotein GPIb-IX Complex; D018179:Receptors, Thrombin; C082835:thrombin receptor peptide (42-47); D013917:Thrombin; D002118:Calcium