Rho Associated Coiled-Coil Kinase-1 Regulates Collagen-Induced Phosphatidylserine Exposure in Platelets (original) (raw)
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FEBS Letters, 1999
In this report we have studied the role of phosphatidylinositol 3'-kinase (PI3-K) and tyrosine phosphatase activation on platelet activation by Convulxin (Cvx). Wortmannin, a specific PI3-K inhibitor, and phenylarsine oxide (PAO), a sulfhydryl reagent that inhibits tyrosine phosphatase (PTPase), block Cvx-induced platelet aggregation, granule secretion, inositol phosphate production, and increase in [Ca2+]i. However, PAO does not inhibit Cvx-induced tyrosine phosphorylation of platelet proteins, including Syk and PLCgamma2, but blocked collagen-induced platelet aggregation as well as tyrosine phosphorylation of PLCgamma2. In contrast, Cvx-induced PLCgamma2 tyrosyl phosphorylation was partially inhibited by wortmannin. We conclude that (i) although Cvx and collagen activate platelets by a similar mechanism, different regulatory processes are specific to each agonist; (ii) mechanisms other than tyrosine phosphorylation regulate PLCgamma2 activity; and (iii) besides protein tyrosine kinases, PI3-K (and PTPase) positively modulate platelet activation by both Cvx and collagen, and this enzyme is required for effective transmission of GPVI-Fc receptor gamma chain signal to result in full activation and tyrosine phosphorylation of PLCgamma2 in Cvx-stimulated platelets.
Journal of Biological Chemistry, 2003
The ATP-gated P2X 1 ion channel is the only P2X subtype expressed in human platelets. Via transmission electron microscopy, we found that P2X 1 mediates fast, reversible platelet shape change, secretory granule centralization, and pseudopodia formation. In washed human platelets, the stable P2X 1 agonist ␣,-methylene ATP (␣,-meATP) causes rapid, transient (2-5 s), and dosedependent myosin light chain (MLC) phosphorylation, requiring extracellular Ca 2؉. Phosphorylation was inhibited by the calmodulin (CaM) inhibitor W-7, but not by the Rho kinase inhibitor HA-1077, i.e. it is exclusively regulated by Ca 2؉ /CaM-dependent MLC kinase. Correspondingly, the P2X 1-induced platelet shape change was inhibited by W-7 and by the MLC kinase inhibitor ML-7 but not by HA-1077. W-7, ML-7, the protein kinase C inhibitor GF109203-X, and the Src family kinase inhibitor PP1 inhibited the collagen and convulxin-induced early platelet degranulation, shape change, and subsequent aggregation, indicating a role for Ca 2؉ /CaM and MLC kinase in these glycoprotein VI-related platelet responses. The secreted ATP-mediated P2X 1-dependent ERK2 activation induced by low collagen concentrations contributes to MLC kinase activation since P2X 1 desensitization or blockade of ERK2 phosphorylation by U0126 strongly attenuated MLC phosphorylation, degranulation, and aggregation. We therefore conclude that at low doses of collagen, glycoprotein VI activation leads to early protein kinase C-and MLC kinase-dependent degranulation. Rapidly released ATP triggers P2X 1-mediated Ca 2؉ influx, activating ERK2, in turn amplifying platelet secretion by reinforcing the early MLC kinase phosphorylation. Hence, the P2X 1-ERK2-MLC axis contributes to collagen-induced platelet activation by enhancing platelet degranulation. * This work was supported by grants from the bilateral scientific and technological cooperation between Flanders and Hungary (BIL00/12) and from the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (FWO) project G.0227.03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Both authors contributed equally to this work. ¶ The recipient of a doctoral KULeuven scholarship. ʈ Holds a postdoctoral research mandate of the FWO.
Collagen-induced platelet activation mainly involves the protein kinase C pathway
The Biochemical journal, 1990
This study analyses early biochemical events in collagen-induced platelet activation. An early metabolic event occurring during the lag phase was the activation of PtdIns(4,5)P2-specific phospholipase C. Phosphatidic acid (PtdOH) formation, phosphorylation of P43 and P20, thromboxane B2 (TXB2) synthesis and platelet secretion began after the lag phase, and were similarly time-dependent, except for TXB2 synthesis, which was delayed. Collagen induced extensive P43 phosphorylation, whereas P20 phosphorylation was weak and always lower than with thrombin. The dose-response curves of P43 phosphorylation and granule secretion were similar, and both reached a peak at 7.5 micrograms of collagen/ml, a dose which induced half-maximal PtdOH and TXB2 formation. Sphingosine, assumed to inhibit protein kinase C, inhibited P43 phosphorylation and secretion in parallel. However, sphingosine was not specific for protein kinase C, since a 15 microM concentration, which did not inhibit P43 phosphoryla...
Cytosolic calcium as a second messenger for collagen-induced platelet responses
Biochemical Journal, 1992
We showed previously that direct platelet activation by collagen involves an increase in the platelet cytosolic free Ca2+ concentration ([Ca2+]i) but that this increase is not required for the adhesion of platelets to collagen. We now report that collagen-induced arachidonic acid liberation, myosin phosphorylation and 5-hydroxytryptamine secretion are dependent on increases in [Ca2+]i, as they were markedly inhibited in platelets loaded with the acetoxymethyl ester of the Ca2+ chelator BAPTA but not in cells loaded with the acetoxymethyl ester of the non-chelating diazo-3. BAPTA also partially inhibited the rate of collagen-induced phosphatidic acid (PtdA) formation but had little effect on increases in phosphorylation of pleckstrin (47 kDa protein; P47). From these results we infer that collagen-induced increases in [Ca2+]i are required for dense granule secretion and arachidonic acid liberation, but are not necessary for stimulation of the protein kinase C pathway.
A role for Bruton's tyrosine kinase (Btk) in platelet activation by collagen
Current Biology, 1998
Bruton's tyrosine kinase (Btk) is essential for normal B-cell receptor signalling. The lack of expression of functional Btk in humans leads to the B-cell deficiency X-linked agammaglobulinaemia (XLA). We report here that Btk is also important for signalling via the collagen receptor glycoprotein VI (GPVI) in platelets. GPVI is coupled to the Fc receptor g chain (FcRg). The FcRgchain contains a consensus sequence known as the immune-receptor tyrosine-based activation motif (ITAM). Tyrosine phosphorylation of the ITAM upon GPVI stimulation is the initial step in the regulation of phospholipase C g2 (PLCg2) isoforms via the tyrosine kinase p72 Syk (Syk) in platelets. Here we show that collagen and a collagen-related peptide (CRP), which binds to GPVI but does not bind to the integrin a 2 b 1 , induced Btk tyrosine phosphorylation in platelets. Aggregation, dense granule secretion and calcium mobilisation were significantly diminished but not completely abolished in platelets from XLA patients in response to collagen and CRP. These effects were associated with a reduction in tyrosine phosphorylation of PLCg2. In contrast, aggregation and secretion stimulated by thrombin in Btk-deficient platelets were not significantly altered. Our results demonstrate that Btk is important for collagen signalling via GPVI, but is not essential for thrombin-mediated platelet activation.
2007
Sustained stimulation of platelets with protease-activated receptor agonists in presence of extracellular calcium was associated with tyrosine dephosphorylation of specific proteins of relative mobilities 35, 67, and 75 kDa. From phosphatase assays and inhibitor studies SHP1, a Src homology 2 (SH2) domain-containing tyrosine phosphatase expressed abundantly in hemopoietic cells, was found to be upregulated in platelets between 25 and 30 min following thrombin stimulation. Concomitantly, SHP1 was tyrosine phosphorylated by, and coprecipitated with, Src tyrosine kinase. SHP1 activation, association with Src and dephosphorylation of specific proteins were dependent on extracellular calcium and maintenance of a higher cytosolic calcium plateau. There was progressive impairment of platelet functions like aggregability and clot retraction, associated with downregulation of fibrinogen-binding affinity of integrin α IIb β 3 , in the platelets exposed to thrombin for 45 min. This could reflect the late physiological changes in platelets when the cells are consistently exposed to stimulatory signals under thrombogenic environment in vivo.
Involvement of Protein-Tyrosine Kinase p72syk in Collagen-Induced Signal Transduction in Platelets
European Journal of Biochemistry, 2008
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
Biochemical Journal, 1999
The collagen receptor glycoprotein VI (GPVI) induces platelet activation through a similar pathway to that used by immune receptors. In the present study we have investigated the role of phosphatidylinositol 3-kinase (PI 3-kinase) in GPVI signalling. Our results show that collagen-related peptide oCRP : [GCP* (GPP*) "! GCP*G] n ; P* l hydroxyprolineq, which is selective to GPVI, induces formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P $ ] and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P # ] in platelets. The increase in the two 3-phosphorylated lipids is inhibited completely by wortmannin and by LY294002, two structurally unrelated inhibitors of PI 3-kinase. The formation of inositol phosphates and phosphatidic acid (PA), two markers of phospholipase C (PLC) activation, by CRP are inhibited by between 50 and 85 % in the presence of wortmannin and LY294002. This is associated with inhibition of elevation of intracellular Ca# + ([Ca# + ] i ) and aggregation. Wortmannin and