Involvement of Protein‐Tyrosine Kinase p72^syk in Collagen‐Induced Signal Transduction in Platelets

Abstract

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...

ELMO1 deficiency enhances platelet function

“…All materials were obtained from ThermoFisher Scientific unless noted. PI (3,4,5)P3 PIP Beads were obtained from Echelon (Salt Lake City, UT). 2-MeSADP, acetylsalicylic acid, apyrase (type V), and anti-ELMO1 antibody were obtained from Sigma (St. Louis, MO).…”

“…The PIP 3 pull-down assay was performed as per the manufacturer's instructions. Briefly, cleared lysates were incubated with 100 mL PI (3,4,5)P3 PIP beads or control beads overnight at 4°C. Beads were washed 3 times with 1x cold NP-40 buffer, eluted with 2x SDS-sample buffer (Boston BioProducts), and boiled at 95°C for 10 minutes.…”

“…1 The interaction of collagen to the GPVI/FcRg complex leads to phosphorylation of the immune tyrosine-based activation motif by the constitutively associated Src-family tyrosine kinases Fyn and Lyn, 2,3 initiating the activation of spleen tyrosine kinase (Syk). [4][5][6] The activation of Syk leads to downstream signaling events, including the activation of class I phosphoinositide 3-kinases (PI3Ks). 7 In platelets, the activation of class I PI3K is not restricted to the GPVI pathway but is also activated downstream of P2Y12 8 and aIIbb3 receptors.…”

“…8 The inhibition of class I PI3K with pharmacological and genetic approaches leads to diminished platelet functional responses. [8][9][10] PI3K phosphorylates the 3-hydroxyl group of the inositol ring of phosphatidylinositol, leading to the generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) from phosphatidylinositol 4,5-bisphosphate in the plasma membrane. 8 PIP 3 formation in turn leads to the recruitment of pleckstrin homology (PH) domain-containing proteins to the plasma membrane 11 such as Bruton tyrosine kinase (Btk) [12][13][14] and Ras GTPaseactivating protein 3 (RASA3).…”

Platelets undergo phosphorylation of Syk at Y525/526 and Y352 in response to pathophysiological shear stress

“…Syk is a 72-kDa nonreceptor tyrosine kinase, the activity of which is correlated to its autophosphorylation at specific tyrosine residues (53). Inside-out signaling phenomena involving Syk include, but are not limited to, collagen-induced activation, through glycoprotein VI (GPVI) and the FcR ␥-chain (GPVI-FcR␥ complex), and thrombin-induced activation (15,49,65). Syk also participates in outside-in signaling following ligand occupancy of activated GPIIb-IIIa, where it is downstream of Src kinase (16,31,44).…”