Formation of PI 3-kinase products in platelets by thrombin, but not collagen, is dependent on synergistic autocrine stimulation, particularly through secreted ADP.
Platelet activation by thrombin or collagen results in secretion and synthesis of several platelet agonists that enhance the responses to the primary agonists (autocrine stimulation). To disclose the effects of thrombin and collagen on the phosphorylation of 3-phosphoinositides per se we incubated platelets with five inhibitors of platelet autocrine stimulation (IAS) that act extracellularly. We found that IAS almost totally blocked thrombin-induced production of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. In contrast, collagen induced massive production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) in the presence of IAS. When testing the effect of each inhibitor individually we found the strongest inhibition of thrombin-induced PtdIns(3,4)P(2) production with the ADP scavenger system CP/CPK. Furthermore, we found a strong synergistic effect between exogenously added ADP and thrombin on production of PtdIns(3,4)P(2). In contrast to the results from 3-phosphorylated phosphoinositides, CP/CPK had little effect on thrombin-induced protein tyrosine phosphorylation. Our results show the importance of autocrine stimulation in thrombin-induced accumulation of 3-phosphorylated phosphoinositides and raise the question as to whether thrombin by itself is capable of inducing PI 3-K activation. In marked contrast to thrombin, collagen per se appears to be able to trigger increased production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3).
Selheim
F
F
Department of Biochemistry and Molecular Biology, University of Bergen, Bergen, Norway. Frode.Selheim@pki.uib.no
Idsøe
R
R
Fukami
M H
MH
Holmsen
H
H
Vassbotn
F S
FS
D000244:Adenosine Diphosphate / Q000097:blood*; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D019086:Bridged Bicyclo Compounds, Heterocyclic; D003094:Collagen / Q000494:pharmacology*; D003402:Creatine Kinase / Q000494:pharmacology; D003533:Cyproheptadine / Q000494:pharmacology; D004224:Diterpenes; D004357:Drug Synergism; D005231:Fatty Acids, Unsaturated; D046934:Ginkgolides; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D066298:In Vitro Techniques; D007783:Lactones / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000097:blood*; D018129:Phosphatidylinositol Phosphates / Q000097:blood*; D010716:Phosphatidylinositols / Q000097:blood / Q000302:isolation & purification; D010725:Phosphocreatine / Q000494:pharmacology; D015539:Platelet Activation; D010974:Platelet Aggregation; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology*
D019086:Bridged Bicyclo Compounds, Heterocyclic; D004224:Diterpenes; D005231:Fatty Acids, Unsaturated; D046934:Ginkgolides; D006834:Hydrazines; D007783:Lactones; D009842:Oligopeptides; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010975:Platelet Aggregation Inhibitors; D010725:Phosphocreatine; D003533:Cyproheptadine; D000244:Adenosine Diphosphate; C045749:SQ 29548; D003094:Collagen; C046617:arginyl-glycyl-aspartyl-serine; C045856:ginkgolide B; D019869:Phosphatidylinositol 3-Kinases; D003402:Creatine Kinase; D013917:Thrombin