Adhesive ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK

Tyrosine phosphorylation of multiple platelet proteins is stimulated by thrombin and other agonists that cause platelet aggregation and secretion. The phosphorylation of a subset of these proteins, including a protein tyrosine kinase, pp125FAK, is dependent on the platelet aggregation that follows fibrinogen binding to integrin alpha IIb beta 3. In this report, we examined whether fibrinogen binding, per se, triggers a process of tyrosine phosphorylation in the absence of exogenous agonists. Binding of soluble fibrinogen was induced with Fab fragments of an anti-beta 3 antibody (anti-LIBS6) that directly exposes the fibrinogen binding site in alpha IIb beta3. Proteins of 50-68 KD and 140 kD became phosphorylated on tyrosine residues in a fibrinogen-dependent manner. This response did not require prostaglandin synthesis, an increase in cytosolic free calcium, platelet aggregation or granule secretion, nor was it associated with tyrosine phosphorylation of pp125FAK. Tyrosine phosphorylation of the 50-68-kD and 140-kD proteins was also observed when (a) fibrinogen binding was stimulated by agonists such as epinephrine, ADP, or thrombin instead of by anti-LIBS6; (b) fragment X, a dimeric plasmin-derived fragment of fibrinogen was used instead of fibrinogen; or (c) alpha IIb beta 3 complexes were cross-linked by antibodies, even in the absence of fibrinogen. In contrast, no tyrosine phosphorylation was observed when the ligand consisted of monomeric cell recognition peptides derived from fibrinogen (RGDS or gamma 400-411). Fibrinogen-dependent tyrosine phosphorylation was inhibited by cytochalasin D. These studies demonstrate that fibrinogen binding to alpha IIb beta 3 initiates a process of tyrosine phosphorylation that precedes platelet aggregation and the phosphorylation of pp125FAK. This reaction may depend on the oligomerization of integrin receptors and on the state of actin polymerization, organizational processes that may juxtapose tyrosine kinases with their substrates.     

Regulation of the pp72syk protein tyrosine kinase by platelet integrin alpha IIb beta 3

pp72syk is essential for development and function of several hematopoietic cells, and it becomes activated through tandem SH2 interaction with ITAM motifs in immune response receptors. Since Syk is also activated through integrins, which do not contain ITAMs, a CHO cell model system was used to study Syk activation by the platelet integrin, alpha IIb beta 3. As in platelets, Syk underwent tyrosine phosphorylation and activation during CHO cell adhesion to alpha IIb beta 3 ligands, including fibrinogen. This involved Syk autophosphorylation and the tyrosine kinase activity of Src, and it exhibited two novel features. Firstly, unlike alpha IIb beta 3-mediated activation of pp125FAK, Syk activation could be triggered by the binding of soluble fibrinogen and abolished by truncation of the alpha IIb or beta 3 cytoplasmic tail, and it was resistant to inhibition by cytochalasin D. Secondly, it did not require phosphorylated ITAMs since it was unaffected by disruption of an ITAM-interaction motif in the SH2(C) domain of Syk or by simultaneous overexpression of the tandem SH2 domains. These studies demonstrate that Syk is a proximal component in alpha IIb beta 3 signaling and is regulated as a consequence of intimate functional relationships with the alpha IIb beta 3 cytoplasmic tails and with Src or a closely related kinase. Furthermore, there are fundamental differences in the activation of Syk by alpha IIb beta 3 and immune response receptors, suggesting a unique role for integrins in Syk function.     

Impaired platelet aggregation and sustained bleeding in mice lacking the fibrinogen motif bound by integrin alpha IIb beta 3

Blood loss at sites of vascular rupture is controlled by the adhesion and aggregation of platelets and the formation of an insoluble fibrin matrix. Fibrinogen is considered to be critical in these processes by both providing an abundant dimeric ligand for alpha IIb beta 3-mediated platelet aggregation, and serving as the fundamental building block of the fibrin polymer. To establish an in vivo model system to examine in detail the importance of alpha IIb beta 3-fibrinogen interactions in platelet function, hemostasis, response to injury and vasoocclusive disease, and to test the prevailing hypothesis that the C-terminal segment of the fibrinogen gamma chain is essential for alpha IIb beta 3 binding, we have used gene-targeting technology in mice to eliminate the last five residues (QAGDV) from the gamma chain. Mice homozygous for the modified gamma chain gene (gamma delta 5/gamma delta 5) displayed a generally normal hematological profile, including normal platelet count, plasma fibrinogen level, clotting time and fibrin crosslinking. However, both gamma delta 5-fibrinogen binding to alpha IIb beta 3 and platelet aggregation were highly defective. Remarkably, another alpha IIb beta 3-dependent process, clot retraction, was unaffected by the gamma delta 5 mutation. Despite the preservation of clotting function, gamma delta 5/gamma delta 5 mice were unable to control blood loss following a surgical challenge and occasionally developed fatal neonatal bleeding events.     

Studies on the role of platelet eicosanoid metabolism and integrin alpha IIb beta 3 in tumor-cell-induced platelet aggregation

Platelet eicosanoid metabolism resulting from tumor-cell-induced platelet aggregation (TCIPA) was examined in a homologous in vitro system. Rat Walker 256 carcinosarcoma cells induced the aggregation of rat platelets via a thrombin-dependent mechanism with concomitant production of eicosanoid metabolites (e.g., 12-HETE, TXA2). TCIPA was dependent on the concentration of tumor cells inducing aggregation, as well as cyclooxygenase and lipoxygenase products. Cyclooxygenase inhibitors, but not lipoxygenase inhibitors, blocked platelet aggregation induced in vitro by a low concentration of agonist. At a high agonist concentration, neither cyclooxygenase nor lipoxygenase inhibitors alone affected platelet aggregation; however, the combined inhibition of both the cyclooxygenase and lipoxygenase pathways resulted in subsequent inhibition of platelet aggregation regardless of agonist concentration. The extent of platelet TXA2 and 12-HETE biosynthesis was likewise dependent on and correlated with agonist concentration. The inhibitors used in this study did not significantly inhibit protein kinase C activity at the doses tested. Platelet surface glycoprotein alpha IIb beta 3 play an important role in platelet aggregation. The effect of platelet cyclooxygenase and lipoxygenase inhibition in regulating alpha IIb beta 3 surface expression was examined by flow cytometric analysis. Thrombin stimulation of washed rat platelets resulted in significantly increased surface expression of platelet alpha IIb beta 3 integrin complex. The enhanced surface expression was not inhibited by a cyclooxygenase inhibitor (aspirin), a thromboxane synthase inhibitor (CGS-14854) or a thromboxane receptor antagonist (SQ 29,548), nor was it stimulated by a thromboxane A2 mimic (pinane-thromboxane A2). However, alpha IIb beta 3 expression was blocked by lipoxygenase inhibition and stereospecifically increased by the platelet lipoxygenase metabolite 12(S)-HETE. These results suggest that both the platelet lipoxygenase and cyclooxygenase pathways are important for TCIPA but that different mechanisms of action are involved.     

Integrin alpha 8 beta 1 promotes attachment, cell spreading, and neurite outgrowth on fibronectin

The integrin alpha 8 subunit, isolated by low stringency hybridization, is a novel integrin subunit that associates with beta 1. To identify ligands, we have prepared a function-blocking antiserum to the extracellular domain of alpha 8, and we have established by transfection K562 cell lines that stably express alpha 8 beta 1 heterodimers on the cell surface. We demonstrate here by cell adhesion and neurite outgrowth assays that alpha 8 beta 1 is a fibronectin receptor. Studies on fibronectin fragments using RGD peptides as inhibitors show that alpha 8 beta 1 binds to the RGD site of fibronectin. In contrast to the endogenous alpha 5 beta 1 fibronectin receptor in K562 cells, alpha 8 beta 1 not only promotes cell attachment but also extensive cell spreading, suggesting functional differences between the two receptors. In chick embryo fibroblasts, alpha 8 beta 1 is localized to focal adhesions. We conclude that alpha 8 beta 1 is a receptor for fibronectin and can promote attachment, cell spreading, and neurite outgrowth on fibronectin.     

Lipid products of phosphoinositide 3-kinase and phosphatidylinositol 4',5'-bisphosphate are both required for ADP-dependent platelet spreading

We have shown previously that ADP released upon platelet adhesion mediated by alphaIIb beta3 integrin triggers accumulation of phosphatidylinositol 3',4'-bisphosphate (PtdIns-3,4-P2) (Gironcel, D. , Racaud-Sultan, C., Payrastre, B., Haricot, M., Borchert, G., Kieffer, N., Breton, M., and Chap, H. (1996) FEBS Lett. 389, 253-256). ADP has also been involved in platelet spreading. Therefore, in order to study a possible role of phosphoinositide 3-kinase in platelet morphological changes following adhesion, human platelets were pretreated with specific phosphoinositide 3-kinase inhibitors LY294002 and wortmannin. Under conditions where PtdIns-3, 4-P2 synthesis was totally inhibited (25 microM LY294002 or 100 nM wortmannin), platelets adhered to the fibrinogen matrix, extended pseudopodia, but did not spread. Moreover, addition of ADP to the medium did not reverse the inhibitory effects of phosphoinositide 3-kinase inhibitors on platelet spreading. Although synthetic dipalmitoyl PtdIns-3,4-P2 and dipalmitoyl phosphatidylinositol 3',4', 5'-trisphosphate restored only partially platelet spreading, phosphatidylinositol 4',5'-bisphosphate (PtdIns-4,5-P2) was able to trigger full spreading of wortmannin-treated adherent platelets. Following 32P labeling of intact platelets, the recovery of [32P]PtdIns-4,5-P2 in anti-talin immunoprecipitates from adherent platelets was found to be decreased upon treatment by wortmannin. These results suggest that the lipid products of phosphoinositide 3-kinase are required but not sufficient for ADP-induced spreading of adherent platelets and that PtdIns-4,5-P2 could be a downstream messenger of this signaling pathway.     

A novel regulatory epitope defined by a murine monoclonal antibody to the platelet GPIIb-IIIa complex (alpha IIb beta 3 integrin)

We characterized a murine monoclonal antibody, PT25-2 (IgG1), raised against washed human platelets. The antibody and its Fab fragments were both capable of inducing platelet aggregation in a fibrinogen-dependent manner and induced 125I-fibrinogen binding to unstimulated platelets (120,000 molecules/platelet at a 100 nM IgG concentration). The antibody immunoprecipitated the alpha IIb beta 3 complex from lysates of iodinated platelets but did not react with the respective subunits when complex formation was disrupted by treatment with 5 mM EDTA at 37 degrees C for 30 min. However, simply removing the extracellular divalent cation with EDTA had no effect on antibody binding indicating that the antibody's epitope depends upon a conformational structure maintained by alpha beta subunit association. Antibody binding to unstimulated, washed platelets yielded binding parameters (Kd = 40 nM, Bmax = 100,000 molecules/platelet), which were found to be virtually unchanged when binding was performed using thrombin or RGDS-peptide-stimulated platelets. Thus, the PT25-2 antibody defines a novel regulatory epitope expressed by the alpha IIb beta 3 integrin on unstimulated, quiescent platelets.     

T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin-mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1-mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR-CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1-dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.     

Difference of (Ca2+)i movements in platelets stimulated by thrombin and TRAP: the involvement of alpha(IIb)beta3-mediated TXA2 synthesis

This study investigated the difference of [Ca2+]i movement in platelets in response to thrombin and TRAP. The involvement of alpha(IIb)beta3 in this signaling was also studied. Stimulation of platelets with thrombin at 0.03 U/ml caused platelet aggregation and a two-peak increase in [Ca2+]i. The second peak of [Ca2+]i, but not the first peak was abolished by the inhibition of platelet aggregation with alpha(IIb)beta3 antagonists or by scavenging endogenous ADP with apyrase. A cyclooxygenase inhibitor, aspirin, and a TXA2 receptor antagonist, BM 13505, also abolished the second peak of [Ca2+]i but not the first peak, although these regents did not inhibit aggregation. Under the same assay conditions, measurement of TXB2 demonstrated that alpha(IIb)beta3 antagonists and aspirin almost completely inhibited the production of TXB2. In contrast to thrombin-stimulation, TRAP caused only a single peak of [Ca2+]i even in the presence of platelet aggregation, and a high level of [Ca2+]i increase was needed for the induction of platelet aggregation. The inhibition of aggregation with alpha(IIb)beta3 antagonists had no effect on [Ca2+]i change and TXB2 production induced by TRAP. Inhibition studies using anti-GPIb antibodies suggested that GPIb may be involved in the thrombin response, but not in the TRAP. Our findings suggest that low dose thrombin causes a different [Ca2+]i response and TXA2 producing signal from TRAP. Endogenous ADP release and fibrinogen binding to alpha(IIb)beta3 are responsible for the synthesis of TXA2 which results in the induction of the second peak of [Ca2+]i in low thrombin- but not TRAP-stimulated platelets.     

Immunological comparisons of integrin alpha IIb beta 3 (GPIIb-IIIa) expressed on platelets and human erythroleukemia cells: evidence for cell specific differences

Platelet glycoprotein IIb-IIIa (GPIIb-IIIa, alpha IIb beta 3) is expressed on the cell surface of the human erythroleukemia (HEL) cell line. Previous studies have demonstrated differences in GPIIb-IIIa ligand binding properties of HEL cells when compared to platelets. Although the mRNA sequences for GPIIb and GPIIIa are identical in platelets and HEL cells, cell specific differences in the conformation states of the GPIIb-IIIa complex may exist and may explain in part the contrasting functional properties. Two monoclonal antibodies (mAbs), an anti-GPIIb mAb C3 and an anti-GPIIIa mAb D3, were used to determine whether differences in GPIIb-IIIa conformational states could be measured. Initial studies in a purified system showed that the mAbs' binding to isolated GPIIb-IIIa conformers was increased to the active GPIIb-IIIa and to dissociated receptor subunits when compared to the inactive form. Furthermore, soluble active GPIIb-IIIa was a much better inhibitor of D3 binding to the immobilized receptor compared to soluble inactive GPIIb-IIIa. Extending these studies with intact cells, we detected at least two classes of binding sites for each mAb on each cell type. Differences in Bmax and in the relative affinities of the mAbs were identified and may represent subpopulations of GPIIb-IIIa conformations. Total HEL cell and platelet GPIIb-IIIa was determined in our binding assays using a radiolabeled GPIIb-IIIa complex specific mAb, 10E5. HEL cells express approximately five times more GPIIb-IIIa on a per cell basis. The percent of total GPIIb-IIIa that represented each class of mAb binding sites was determined. In summary, the relative differences in GPIIb-IIIa conformation found on platelets and HEL cells may be related to cell-specific ligand binding properties and activation states of the receptor.     

EGF stimulates tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin in rat pancreatic acini by a phospholipase C-independent process that depends on phosphatidylinositol 3-kinase, the small GTP-binding protein, p21rho, and the integrity of the actin cytoskeleton

Epidermal growth factor (EGF) is a potent mitogen in many cell types including pancreatic cells. Recent studies show that the effects of some growth factors on growth and cell migration are mediated by tyrosine phosphorylation of the cytosolic tyrosine kinase p125 focal adhesion kinase (p125FAK) and the cytoskeletal protein, paxillin. The aim of the present study was to determine whether EGF activates this pathway in rat pancreatic acini and causes tyrosine phosphorylation of each of these proteins, and to examine the intracellular pathways involved. Treatment of pancreatic acini with EGF induced a rapid, concentration-dependent increase in p125FAK and paxillin tyrosine phosphorylation. Depletion of the intracellular calcium pool or inhibition of PKC activation had no effect on the response to EGF. However, inhibition of the phosphatidylinositol 3-kinase (PI3-kinase) or inactivation of p21rho inhibited EGF-stimulated phosphorylation of p125FAK and paxillin by more than 70%. Finally, cytochalasin D, a selective disrupter of the actin filament network, completely inhibited EGF-stimulated tyrosine phosphorylation of both proteins. All these treatments did not modify EGF receptor autophosphorylation in response to EGF. These results identify p125FAK and paxillin as components of the intracellular pathways stimulated after EGF receptor occupation in rat pancreatic acini. Activation of this cascade requires activation of PI3-kinase and participation of p21rho, but not PKC activation and calcium mobilization.