The association of pp125FAK, pp60Src, CDC42Hs and Rap1B with the cytoskeleton of aggregated platelets is a reversible process regulated by calcium (original) (raw)
Related papers
The EMBO Journal, 1996
Thrombin-stimulated aggregation of human platelets promotes an increase in the phosphatidylinositol 4-phosphate (Ptdlns 4-P) 5-kinase (PIPkin) activity in the cytoskeleton. This phenomenon is associated with translocation of PIPkin isoform C to the cytoskeleton and with an increase in the amount of phosphatidylinositol bisphosphate (PtdInsP2) bound to the cytoskeletal pellet. All three of these effects are prevented if the platelets are not stirred or if RGD-containing peptides are present, demonstrating that they require integrin activation. All three are also abolished by pretreatment with okadaic acid, which also prevents the aggregation-dependent translocation of pp6OC-src to the cytoskeleton. The results point to the existence of a cytoskeletally associated PtdInsP2 pool under the control of integrin-mediated signals that act via PIPkin C and suggest that a common, okadaic acid-sensitive mechanism may underlie the aggregation-dependent translocation of certain signalling molecules to the platelet cytoskeleton.
Biochemical Journal, 2000
We have recently reported that store-mediated Ca# + entry in platelets is likely to be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, a model termed ' secretion-like coupling '. In this model the actin cytoskeleton plays a key regulatory role. Since tyrosine kinases have been shown to be important for Ca# + entry in platelets and other cells, we have now investigated the possible involvement of tyrosine kinases in the secretion-like-coupling model. Treatment of platelets with thrombin or thapsigargin induced actin polymerization by a calcium-independent pathway. Methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor, prevented thrombin-or thapsigargin-induced actin polymerization. The effects of tyrosine kinases in store-mediated Ca# + entry
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2004
Signaling pathways elicited by protease-activated receptor-1 (PAR-1) agonists, thrombin receptor-activating peptide (TRAP) and thrombin, are markedly different. Here we show that TRAP-induced disaggregation of platelets is a function of extracellular calcium. Chelation of calcium with EGTA after the onset of aggregation precluded subsequent destabilization of the aggregates in TRAP-stimulated platelets, whereas disaggregation was not observed in the platelets stimulated with thrombin. TRAP-induced disaggregation was independent of the activity of the calcium-dependent thiol protease, calpain. Inhibition of phosphoinositide 3-kinase activity provoked further destabilization of the platelet aggregates in the presence of calcium; however, EGTA attenuated this effect. Activation of protein kinase C (PKC) by phorbol ester prevented disaggregation of the TRAP-stimulated platelets independent of the extracellular calcium. Two proteins of relative mobilities 67 and 75 kD were found to be significantly dephosphorylated on tyrosine in calcium-pretreated platelets as compared to the EGTA-treated platelets following continued stimulation with either TRAP or thrombin for 15 min. Inhibition of phosphoinositide 3kinase by two pharmacologically independent inhibitors also caused dephosphorylation of p67, which was completely abrogated by chelation of extracellular calcium. Platelet activation by phorbol ester was not associated with disaggregation, although dephosphorylation of p67 was induced under this condition. SHP-1, an abundant tyrosine phosphatase in platelets, co-migrated with the p67 protein and co-localized to the actin-based cytoskeleton of aggregated platelets; however, its identity with p67 was ruled out from immunoprecipitation studies.
Blood, 2002
We found that the interaction of platelets with immobilized von Willebrand factor (VWF) under flow induces distinct elevations of cytosolic Ca ؉؉ concentration ([Ca ؉؉ ] i ) that are associated with sequential stages of integrin ␣ IIb  3 activation. Fluid-dynamic conditions that are compatible with the existence of tensile stress on the bonds between glycoprotein Ib␣ (GPIb␣) and the VWF A1 domain led to Ca ؉؉ release from intracellular stores (type ␣/ peaks), which preceded stationary platelet adhesion. Raised levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate, as well as membrane-permeable calcium chelators, inhibited these [Ca ؉؉ ] i oscilla-tions and prevented stable adhesion without affecting the dynamic characteristics of the typical platelet translocation on VWF mediated by GPIb␣. Once adhesion was established through the integrin ␣ IIb  3 , new [Ca ؉؉ ] i oscillations (type ␥) of greater amplitude and duration, and involving a transmembrane ion flux, developed in association with the recruitment of additional platelets into aggregates. Degradation of released adenosine diphosphate (ADP) to AMP or inhibition of phosphatidylinositol 3-kinase (PI3-K) prevented this response without affecting stationary adhesion and blocked aggregation. These findings indicate that an initial signal induced by stressed GPIb␣-VWF bonds leads to ␣ IIb  3 activation sufficient to support localized platelet adhesion. Then, additional signals from ADP receptors and possibly ligand-occupied ␣ IIb  3 , with the contribution of a pathway involving PI3-K, amplify platelet activation to the level required for aggregation. Our conclusions modify those proposed by others regarding the mechanisms that regulate signaling between GPIb␣ and ␣ IIb  3 and lead to platelet adhesion and aggregation on immobilized VWF. (Blood.
Blood, 2002
We found that the interaction of platelets with immobilized von Willebrand factor (VWF) under flow induces distinct elevations of cytosolic Ca++ concentration ([Ca++]i) that are associated with sequential stages of integrin αIIbβ3 activation. Fluid-dynamic conditions that are compatible with the existence of tensile stress on the bonds between glycoprotein Ibα (GPIbα) and the VWF A1 domain led to Ca++ release from intracellular stores (type α/β peaks), which preceded stationary platelet adhesion. Raised levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate, as well as membrane-permeable calcium chelators, inhibited these [Ca++]ioscillations and prevented stable adhesion without affecting the dynamic characteristics of the typical platelet translocation on VWF mediated by GPIbα. Once adhesion was established through the integrin αIIbβ3, new [Ca++]i oscillations (type γ) of greater amplitude and duration, and involving a transmembrane ion flux, developed i...
Archives of Biochemistry and Biophysics, 2007
Thrombin induces platelet activation through a variety of intracellular mechanisms, including Ca 2+ mobilization. The protein of the exocytotic machinery SNAP-25, but not VAMPs, is required for store-operated Ca 2+ entry, the main mechanism for Ca 2+ influx in platelets. Hence, we have investigated the role of the SNAP-25 and VAMPs in thrombin-induced platelet aggregation. Platelet stimulation with thrombin or selective activation of thrombin receptors PAR-1, PAR-4 or GPIb-IX-V results in platelet aggregation that, except for GPIb-IX-V receptor, requires Ca 2+ entry for full activation. Depletion of the intracellular Ca 2+ stores using pharmacological tools was unable to induce aggregation except when cytosolic Ca 2+ concentration reached a critical level (around 1.5 lM). Electrotransjection of cells with anti-SNAP-25 antibody reduced thrombin-evoked platelet aggregation, while electrotransjection of anti-VAMP-1, -2 and -3 antibody had no effect. These findings support a role for SNAP-25 but not VAMP-1, -2 and -3 in platelet aggregation, which is likely mediated by the regulation of Ca 2+ mobilization in human platelets.
Journal of Biological Chemistry, 1997
A key regulatory event controlling platelet activation is mediated through the phosphorylation of several cellular proteins by protein-tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK) is a novel cytoplasmic tyrosine kinase and a member of the focal adhesion kinase (FAK) gene family. FAK phosphorylation in platelets is integrin-dependent, occurs in a late stage of platelet activation, and is dependent on platelet aggregation. In this study, we have investigated the involvement of RAFTK phosphorylation during different stages of platelet activation. Treatment of platelets with thrombin induced, in as early as 10 s, a rapid tyrosine phosphorylation of RAFTK in a time-and concentrationdependent manner. Treatment of platelets with thrombin in the absence of stirring or pretreatment of platelets with RGDS peptide prevented platelet aggregation, but not RAFTK phosphorylation. Furthermore, phosphorylation of RAFTK did not require integrin engagement since platelets treated with the 7E3 inhibitory antibodies that block fibrinogen binding to glycoprotein IIb-IIIa did not inhibit RAFTK phosphorylation. Similarly, platelets treated with LIBS6 antibodies, which specifically activate glycoprotein IIb-IIIa, did not induce RAFTK phosphorylation. Stimulation of platelets by several agonists such as collagen, ADP, epinephrine, and calcium ionophore A23187 induced RAFTK phosphorylation. Tyrosine phosphorylation of RAFTK in platelets is regulated by calcium and is mediated through the protein kinase C pathway. Phosphorylation of RAFTK is dependent upon the formation of actin cytoskeleton as disruption of actin polymerization by cytochalasin D significantly inhibited this phosphorylation. The RAFTK protein appears to be proteolytically cleaved by calpain in an aggregation dependent manner upon thrombin stimulation. These results demonstrate that RAFTK is tyrosine-phosphorylated during an early phase of platelet activation by an integrin-independent mechanism and is not dependent on platelet aggregation, suggesting different mechanisms of regulation for FAK and RAFTK phosphorylation during platelet activation.
Integrin-independent tyrosine phosphorylation of p125fak in human platelets stimulated by collagen
Journal of Biological …, 2001
Collagen fibers or a glycoprotein VI-specific collagenrelated peptide (CRP-XL) stimulated tyrosine phosphorylation of the focal adhesion kinase, p125 fak (FAK), in human platelets. An integrin ␣ 2  1-specific triple-helical peptide ligand, containing the sequence GFOGER (single-letter nomenclature, O ؍ Hyp) was without effect. Antibodies to the ␣ 2 and  1 integrin subunits did not inhibit platelet FAK tyrosine phosphorylation caused by either collagen fibers or CRP-XL. Tyrosine phosphorylation of FAK caused by CRP-XL or thrombin, but not that caused by collagen fibers, was partially inhibited by GR144053F, an antagonist of integrin ␣ IIb  3. The intracellular Ca 2؉ chelator, BAPTA, and the protein kinase C inhibitor, Ro31-8220, were each highly effective inhibitors of the FAK tyrosine phosphorylation caused by collagen or CRP-XL. These data suggest that, in human platelets, 1) occupation or clustering of the integrin ␣ 2  1 is neither sufficient nor necessary for activation of FAK, 2) the fibrinogen receptor ␣ IIb  3 is not required for activation of FAK by collagen fibers, and 3) both intracellular Ca 2؉ and protein kinase C activity are essential intermediaries of FAK activation.
Journal of Biological Chemistry, 1991
The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), serves as the receptor for fibrinogen. This study examined what effect GPIIb-IIIa receptor occupancy had on the cytoskeleton of resting and activated platelets. Triton X-100-insoluble residues (cytoskeletons) were isolated from resting washed platelets incubated with either 500)IM RGDS or 500)IM RGES and examined for protein content. RGDS did not increase the amount of GPIIb-IIIa associated with the cytoskeletal residues which sedimented at either 15,800 X g or 100,000 X g. To determine the effect of receptor occupancy on the formation of the activated platelet cytoskeleton, stirred and nonstirred RGDS-treated platelets in plasma were activated with ADP. Triton X-100insoluble residues were isolated and examined for both protein content and retention of GPIIb-IIIa. Further, morphological studies were performed on the RGDS-ADP-stimulated platelets. The results of this study suggest that 1) RGDS peptide receptor occupancy does not lead to GPIIb-IIIa linkage to the cytoskeleton, 2) ADPstimulated platelet shape change, polymerization of actin, and association of myosin with the cytoskeleton are unaffected by RGDS peptide receptor occupancy, 3) RGDS inhibits an aggregation-dependent incorporation of ABP, a-actinin, talin, and GPIIb-IIIa into the Triton-insoluble residue. Platelets contain a number of adhesive protein receptors belonging to the integrin superfamily (1,2). These include the glycoprotein complexes Ia-IIa (VLA-2), IC-IIa (VLA-5), VLA-6, a,-GPIIIa, and GPIIb-IIIa (3-9). As members of the integrin superfamily, these receptors are proposed to bind extracellular ligands and to interact with the cytoskeleton. Interestingly, GPIa-IIa has been shown to be linked to the resting platelet cytoskeleton via actin-binding protein while GPIIb-IIIa has been suggested to become associated with the cytoskeleton following platelet aggregation (10, 11). Therefore, it is of considerable interest to determine what regulates the interactions of these receptors with the cytoskeleton. On the activated platelet, GPIIb-IIIa serves as the receptor for fibrinogen (12-16). GPIIb-IIIa can also bind fibronectin, von Willebrand factor, and vitronectin (17-24). The binding