Thrombin per se does not induce tyrosine protein phosphorylation in human platelets as judged by western blotting, while collagen does: the significance of synergistic, autocrine stimulation.
Thrombin elicits responses in platelets such as shape change, aggregation, arachidonate liberation and secretion of the contents of three storage granules, processes that coincide with serine/threonine and tyrosine phosphorylation of numerous proteins, hydrolysis of polyphosphoinositides and mobilisation of Ca2+ within the cell. However, the significance of these parallel signal transduction processes has not been clearly elucidated in the light of the prevalent autocrine stimulation in platelets: a great amplification of the thrombin signal through secreted ADP, by production of thromboxane A2 from the liberated arachidonic acid, by the close cell contact produced by aggregation caused by exposure of integrin receptors that become ligated by fibrinogen and other platelet-produced factors. In the present communication five pathways of autocrine stimulation have been prevented by appropriate inhibitors. Under these conditions thrombin stimulated platelet secretion with little tyrosine phosphorylation, except for a 125-130 kDa protein that was tyrosine-phosphorylated in response to one of the inhibitors, the peptide Arg-Gly-Asp-Ser (RGDS) used to block aggregation. In sharp contrast, collagen elicits massive tyrosine phosphorylation and platelet secretion in the absence of autocrine stimulation. When the thrombin-induced tyrosine phosphorylations was corrected for RGDS-induced phosphorylation, the presence of inhibitors of autocrine stimulation reduced the thrombin-induced phosphorylation by 97%. Our results strongly suggests that tyrosine phosphorylation is not part of the signal transduction pathway initiated by thrombin per se, but it represents an integral part of signal transduction initiated by collagen.
Ryningen
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Department of Biochemistry and Molecular Biology, University of Bergen, Norway. Anita.Ryningen@pki.uib.no
Holmsen
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D019898:Autocrine Communication; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D015153:Blotting, Western; D003094:Collagen / Q000378:metabolism*; D004357:Drug Synergism; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism; D018179:Receptors, Thrombin / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D018179:Receptors, Thrombin; D014443:Tyrosine; D005340:Fibrinogen; D003094:Collagen; C046617:arginyl-glycyl-aspartyl-serine; D013917:Thrombin