Previous studies have demonstrated that activation of platelets by collagen results in a dramatic increase in tyrosine phosphorylation of several cellular proteins, including pp125FAK, through the interaction of collagen with integrin alpha 2 beta 1 (GP Ia-IIa). In this study, we report that p72syk is a potential candidate for the protein-tyrosine phosphorylation event following collagen stimulation in porcine platelets. Washed platelets were stimulated with collagen and the activation of p72syk was assessed in an immunoprecipitation kinase assay. The activity of p72syk increased within 1 min, reached a maximum at 5 min after stimulation by collagen, and the phosphorylation at tyrosine residues of p72syk in platelets also occurred in the same time course as the activation of p72syk. Prior treatment of platelets with cytochalasin D to inhibit actin polymerization, or with aspirin and apyrase to inhibit the secondary reaction, or EGTA and the acetoxymethyl ester of 5,5'-dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid to chelate both extracellular and intracellular Ca2+, did not affect the activation of p72syk induced by collagen. Furthermore, herbimycin A, a potent protein-tyrosine-kinase inhibitor, was capable of reducing collagen-evoked p72syk activation, Ca2+ mobilization and platelet aggregation. These results suggest that upon stimulation by collagen p72syk is physically activated by a process that is independent of the effects of Ca2+, ADP, and actin polymerization, and may participate in the regulation of Ca2+ mobilization mediated by collagen in platelets.

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Previous studies have demonstrated that activation of platelets by collagen results in a dramatic increase in tyrosine phosphorylation of several cellular proteins, including ppl 25FAK, through the interaction of collagen with integrin (GP Ia-IIa). In this study, we report that p7Tyk is a potential candidate for the protein-tyrosine phosphorylation event following collagen stimulation in porcine platelets. Washed platelets were stimulated with collagen and the activation of p7Tyk was assessed in an immunoprecipitation kinase assay. The activity of p72"yk increased within 1 min, reached a maximum at 5 min after stimulation by collagen, and the phosphorylation at tyrosine residues of p7TYk in platelets also occurred in the same time course as the activation of ~7 2 "~. Prior treatment of platelets with cytochalasin D to inhibit actin polymerization, or with aspirin and apyrase to inhibit the secondary reaction, or EGTA and the acetoxymethyl ester of 5,5'-dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid to chelate both extracellular and intracellular Ca", did not affect the activation of p7Tyk induced by collagen. Furthermore, herbimycin A, a potent proteintyrosine-kinase inhibitor, was capable of reducing collagen-evoked p7Tyk activation, Ca2+ mobilization and platelet aggregation. These results suggest that upon stimulation by collagen p7Tyk is physically activated by a process that is independent of the effects of Ca", ADP, and actin polymerization, and may participate in the regulation of Caz+ mobilization mediated by collagen in platelets.Following injury to the blood-vessel wall, thrombogenic components in the vessel wall and perivascular space, in particular the collagenous elements, are brought into contact with the circulating blood. Platelets are the circulating blood cells that mainly participate in hemostatic reactions and are activated by diverse substances of which collagen is one of the strongest agonists, especially in the vascular subendothelium [l -31. Activation of platelets by collagen causes many events including shape change, aggregation and secretion that are considered to be regulated through coordination of intracellular signaling pathways, including Ca2+ mobilization and protein-kinase-C activation. Previous studies have demonstrated that activation of platelets by collagen results in a dramatic increase in tyrosine phosphorylation of several cellular proteins including ~~1 2 5~~~ through the interaction of collagen with integrin a& (GP Ia-IIa). This protein-tyrosine phosphorylation is independent of both GPIIb-IIIa and ADP and is accompanied by cytoskeletal reorganization and changes in cell shape [4-71. However, the key enzymes that Correspondence to H. Yamamura, Department of Biochemistry, Fukui Medical School, Matsuoka, Fukui, Japan 910-11Abbreviations. SH2 and SH3, src homology region-2 and src homology region-3 ; AM, acetoxymethyl; BAFTA, 53'-dimethyl- Enzymes. Protein-tyrosine kinase (EC 2.7.1.112); protein kinase C (EC 2.7.1.37).control the level of protein-tyrosine...

References1. Weinreb RN. Compliance with medical treatments of glaucoma. J Glaucoma 1992;1:134-136. 2. Camras CB, United States Latanoprost Study Group. Comparison of latanoprost and timolol in patients with ocular hypertension and glaucoma: a six-month, masked, multicenter trial in the United States. Ophthalmology 1996;103:138-147. 3. Hedner J, Events B, Möller CS. Latanoprost and respiratory function in asthmatic patients: randomized, double-masked, placebocontrolled crossover evaluation. Arch Ophthalmol 1999;117: 1305-1309. 4. O'Connor DJ, Martone JF, Mead A. Additive intraocular pressure lowering effect of various medications with latanoprost. Am J Ophthalmol 2002;133:836-837. 5. Alm A, Widengard I, Kjellen D, et al. Latanoprost administered once daily maintained reduction of intraocular pressure in glaucoma patients treated concomitantly with timolol. Br

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