In contrast to native low density lipoprotein (LDL), mildly oxidized LDL (mox-LDL) induced platelet shape change and stimulated during shape change the tyrosine phosphorylation of specific proteins including Syk; the translocation of Src, Fyn, and Syk to the cytoskeleton; and the increase of cytosolic Ca 2؉ due to mainly Ca 2؉ entry. The stimulation of these early signal pathways by mox-LDL was inhibited by desensitization of the lysophosphatidic acid (LPA) receptor and specific LPA receptor antagonists, was independent of the ␣ IIb ␤ 3 -integrin, and was mimicked by LPA. Stimulation of tyrosine phosphorylation and Syk activation were independent of the increase of cytosolic Ca 2؉ and were suppressed by genistein and two specific inhibitors of the Src family tyrosine kinases, PP1 and PD173956. In contrast to PP1 and PD 173956, genistein prevented shape change by mox-LDL. The results indicate that mox-LDL, through activation of the LPA receptor, stimulates two separate early signal pathways, (a) Src family and Syk tyrosine kinases, and (b) Ca 2؉ entry. The activation of these early signaling pathways by mox-LDL probably plays a role in platelet responses subsequent to shape change. The inhibition of mox-LDL-induced platelet activation by LPA receptor antagonists or dietary isoflavonoids such as genistein could have implications in the prevention and therapy of cardiovascular diseases.Oxidative modification of LDL 1 plays an important role in the pathogenesis of atherosclerosis. Soft, lipid-rich plaques containing LDL and oxidized LDL (1, 2) are vulnerable and upon rupture may expose thrombogenic LDL particles that activate circulating platelets, causing them to aggregate and to form an intravascular plug that eventually leads to stroke and myocardial infarction (3,4). Indeed, previous studies indicate that oxidatively modified LDL stimulates platelets, mildly oxidized LDL (mox-LDL) being more active than heavily oxidized LDL (ox-LDL) (5, 6).mox-LDL or minimally modified LDL (mm-LDL) could also be present in the circulation. A subfraction of LDL that consists of electronegative, small dense, slightly oxidized LDL particles has been found in peripheral blood (7). An interaction of platelets with circulating mm-LDL may explain the enhanced platelet aggregation often observed in patients with cardiovascular disease. Indeed, a higher prevalence of small dense circulating LDL particles in coronary heart disease and in conditions commonly associated with atherogenesis has been described (8). The interaction of circulating mm-LDL with platelets may favor intravascular thrombus formation at hemodynamically critical sites of stenotic coronary and carotid arteries.Whereas ox-LDL has often toxic effects on cells (9, 10), mm-LDL and mox-LDL seem to alter the function of cells of the vessel wall and platelets through stimulation of specific signal transduction pathways (1,6,11). The elucidation of signal transduction mechanisms of mox-LDL and mm-LDL may provide a basis for new preventive and therapeutic strategies in atheroscl...

In platelets, ␣ IIb ␤ 3 exists in a form that cannot bind adhesive proteins in the plasma; although it can interact with immobilized fibrinogen it cannot interact with immobilized von Willebrand factor in the vessel wall. Soluble agonists such as thrombin convert ␣ IIb ␤ 3 to a form that recognizes soluble and immobilized ligands. Attempts to reconstitute ␣ IIb ␤ 3 activation in a non-hematopoietic, nucleated cell system have been unsuccessful. In the present study, we have developed a transfected Chinese hamster ovary cell model in which ␣ IIb ␤ 3 activation is induced by signaling across glycoprotein (GP) Ib-IX by its ligand, von Willebrand factor. GPIb-IX activates not only the transfected ␣ IIb ␤ 3 but also endogenous ␣ v ␤ 3 . Activation of the pathways leading to integrin activation occurred even in cells transfected with GPIb-IX lacking the domain on GPIb␣ that binds 14-3-3 or that which binds actin-binding protein. These studies demonstrate that signals induced by interaction of GPIb-IX with von Willebrand factor lead to ␣ IIb ␤ 3 activation and suggest that the signaling pathways by which GPIb-IX induces ␣ IIb ␤ 3 activation are different to those used by thrombin. Elucidation of these differences may provide insights into therapeutic ways in which to inhibit integrin activation in selective clinical settings.

Signaling across integrins is regulated by interaction of these receptors with cytoskeletal proteins and signaling molecules. To identify molecules interacting with the cytoplasmic domain of the ␤ 3 -integrin subunit (glycoprotein IIIa), a placental cDNA library was screened in the yeast two-hybrid system. Two identical clones coding for a 96-amino acid sequence were identified. This sequence was 100% identical to a sequence in skelemin, a protein identified previously in skeletal muscle. Skelemin is a member of a superfamily of cytoskeletal proteins that contain fibronectin-type III-like motifs and immunoglobulin C2-like motifs and that regulate the organization of myosin filaments in muscle. The amino acid residues in the isolated clones encompassed C2 motifs 4 and 5 of skelemin. A recombinant skelemin protein consisting of C2 motifs 3-7 interacted with ␤ 1 -and ␤ 3 -integrin cytoplasmic domains expressed as glu- Members of the integrin family of adhesion receptors consist of ␣-and ␤-subunits and are involved in a variety of cellular functions such as spreading, migration, cell division, and differentiation (1-6). Integrins are often in an inactive state and can be rapidly induced to bind ligand through intracellular signaling (7, 8), a process known as "inside-out" signaling. Ligand binding, in turn, induces "outside-in" signaling that results in numerous intracellular changes such as clustering of integrins, association of clustered integrins with cytoskeletal proteins (9) , recruitment of signaling molecules to integrincytoskeletal protein complexes (9 -11), increased phosphorylation of intracellular proteins (12-16), activation of calpain (17), reorganization of the cytoskeleton (9, 10, 18 -20), and altered gene expression (21). The cytoplasmic tails of the ␤-subunits are responsible for mediating the interaction of integrins with the cytoskeleton (22) and play an important role in mediating both inside-out and outside-in signaling events (22)(23)(24)(25)(26)(27)(28)(29).To elucidate the mechanisms involved in transmission of signals across integrins, it will be important to identify the intracellular proteins with which the cytoplasmic tails interact. Immunofluorescence studies in cultured cells have identified numerous cytoskeletal proteins and signaling molecules that colocalize with integrins in focal adhesions (11). Some of these molecules, such as talin, ␣-actinin, and focal adhesion kinase, have been shown to bind directly to the cytoplasmic domain of ␤ 1 -or ␤ 3 -integrin in in vitro assays (30 -34). Additional integrin-binding proteins have been identified using integrin cytoplasmic domains in a yeast two-hybrid screen. For example, integrin-linked kinase (35) and integrin cytoplasmic domainassociated protein (36) have been identified using the ␤ 1 -integrin cytoplasmic domain; endonexin was identified using the ␤ 3 -integrin cytoplasmic domain (37); and the SEC7 homologue cytohesin was identified using the ␤ 2 -integrin cytoplasmic domain (38). Yet other proteins such as CIB (calcium-and integrin...

Activation of human platelets by the peptide YFLLRNP has been shown to induce shape change but not secretion, Ca2+ mobilization, or pleckstrin phosphorylation (Rasmussen, U.B., Gachet, C., Schlesinger, Y., Hanau, D., Ohlmann, P., Van Obberghen-Schilling, E., Pouyssegur, J., Cazenave, J.P., and Pavirani, A. (1993) J. Biol. Chem. 268, 14322-14328). YFLLRNP was added to washed human platelets that had been pretreated with EGTA at 37 degrees C or preincubated with the fibrinogen receptor antagonist RGDS to preclude the activation of the integrin alpha IIb beta 3 (fibrinogen receptor). YFLLRNP induced shape change and stimulated the tyrosine phosphorylation of proteins of 62, 68, and 130 kDa within 7 s. Tyrosine phosphorylation of these proteins reached maximum levels (2-3-fold) 15-30 s after addition of YFLLRNP and decreased subsequently. The chelation of intracellular Ca2+ by BAPTA-AM decreased basal tyrosine protein phosphorylation but did not inhibit the increase of tyrosine phosphorylation of P62, P68, and P130 or the shape change induced by YFLLRNP. Preincubation of platelets with the tyrosine kinase inhibitors genistein or tyrphostin A23 completely inhibited platelet shape change and protein tyrosine phosphorylation induced by YFLLRNP. The inactive structural analogs daidzein and tyrphostin A1 were barely inhibitory. P62, P68, and P130, which exhibited increased tyrosine phosphorylation upon stimulation with YFLLRNP, were found in the cytoskeleton. P130 was not identical to vinculin or the focal adhesion kinase pp125FAK. The results indicate that stimulation of G-protein-coupled thrombin receptors rapidly induces protein tyrosine kinase activation through a Ca(2+)- and integrin-independent mechanism. Protein tyrosine kinase activation and tyrosine phosphorylation of novel protein substrates seem to play an essential role in the induction of platelet shape change.

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