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, aggregat ion, arachidonate liberation and secretion of the contents of three st orage granules, processes that coincide with serine/threonine and tyro sine phosphorylation of numerous proteins, hydrolysis of polyphosphoin ositides and mobilisation of Ca2+ within the cell. However, the signif icance of these parallel signal transduction processes has not been cl early elucidated in the light of the prevalent autocrine stimulation i n platelets: a great amplification of the thrombin signal through secr eted ADP, by production of thromboxane A(2) from the liberated arachid onic 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 pat hways of autocrine stimulation have been prevented by appropriate inhi bitors. 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 sha rp contrast, collagen elicits massive tyrosine phosphorylation and pla telet secretion in the absence of autocrine stimulation. When the thro mbin-induced tyrosine phosphorylations was corrected for RODS-induced phosphorylation, the presence of inhibitors of autocrine stimulation r educed the thrombin-induced phosphorylation by 97%. Our results strong ly suggests that tyrosine phosphorylation is not part of the signal tr ansduction pathway initiated by thrombin per se, but it represents an integral part of signal transduction initiated by collagen. (C) 1998 A cademic Press.