The biogenesis of trimeric G proteins was investigated by measurement of the expression of alpha-subunits in the megakaryoblastic cell lines MEG-01, DAMI, and CHRF-288-11, representing stages of increasing maturation, and compared with platelets. Megakaryoblasts and platelets contained approximately equal amounts of Gi alpha-1/2, Gi alpha-3, Gq alpha, and G12 alpha protein. Maturation was accompanied by (1) downregulation of mRNA for Gs alpha and disappearance of iloprost-induced Ca2+ mobilization, (2) upregulation of the long form of Gs alpha protein (Gs alpha-L) and an increase in iloprost-induced cAMP formation, and (3) upregulation of G16 alpha mRNA and G16 alpha protein and appearance of thromboxane A2-induced signaling (Ca2+ mobilization and stimulation of prostaglandin I2-induced cAMP formation). Gz alpha protein was absent in the megakaryoblasts despite weak expression of Gz alpha mRNA in DAMI and relatively high levels of Gz alpha mRNA and Gz alpha protein in platelets. These findings reveal major changes in G protein-mediated signal transduction during megakaryocytopoiesis and indicate that G16 alpha couples the thromboxane receptor to phospholipase C beta.

Abstract-The relative contributions of protein tyrosine kinases (PTKs) and protein kinase C isoenzymes (PKCs), a family of serine/threonine kinases, in integrin ␣ IIb ␤ 3 (glycoprotein IIb/IIIa) exposure are the subject of much controversy. In the present study we measured the effect of the PTK inhibitor herbimycin A and the PKC inhibitor bisindolylmaleimide I on 125 I-fibrinogen binding to ␣ IIb ␤ 3 and on aggregation/secretion induced by different agonists. Dose-response studies showed complete inhibition of ␣ IIb ␤ 3 exposure by 30 mol/L (ADP stimulation) and 35 to 40 mol/L (␣-thrombin stimulation) herbimycin A. In contrast, inhibition of exposure by bisindolylmaleimide I varied from none (for ADP and epinephrine), to 30% (for platelet-activating factor), and to Ϸ80% (for ␣-thrombin). Studies with a submaximal dose of herbimycin A (Ϸ50% inhibition of the ADP-response) and a maximal dose of bisindolylmaleimide I showed that optical aggregation had a similar sensitivity to the inhibitors as ␣ IIb ␤ 3 exposure with minimal interference by secreted ADP. Thus, the relative contributions of tyrosine and serine/threonine kinases in ␣ IIb ␤ 3 exposure and aggregation differ among the different agonists, with an exclusive role for PTKs in ADP-and epinephrine-induced responses and a role for both PTKs and PKCs in responses induced by platelet-activating factor and ␣-thrombin. (Arterioscler Thromb Vasc Biol. 1998;18:404-414.)Key Words: integrin ␣ IIb ␤ 3 Ⅲ fibrinogen binding Ⅲ aggregation Ⅲ human platelets Ⅲ protein phosphorylation P latelet aggregation is mediated by the coupling of fibrinogen to integrin ␣ IIb ␤ 3 (ie, glycoprotein IIb/IIIa) via binding sites that are exposed when these cells are activated. Our understanding of the intracellular mechanisms that control the exposure of ligand-binding sites on platelet integrin ␣ IIb ␤ 3 is far from complete. A major stimulating pathway in platelets involves tyrosine kinases, which may signal to the processes mediating platelet-platelet contact and formation of focal adhesions.1 A second stimulating route involves hydrolysis of polyphosphoinositides and formation of diacylglycerol, inositol 1,4,5-tris-phosphate, and phosphatidic acid. Diacylglycerol activates PKC, whereas inositol 1,4,5-tris-phosphate mobilizes Ca 2ϩ , thereby inducing rearrangements of the plasma membrane and cytoskeleton that facilitate anchorage of ␣ IIb ␤ 3 and possibly exposure of the binding sites. 1,2There is uncertainty regarding the role of protein kinases in inside-out signaling to ␣ IIb ␤ 3 . Platelet activation is accompanied by tyrosine phosphorylation of 60-, 64-, 75-, and 130-kDa proteins, a step that precedes ligand binding to ␣ IIb ␤ 3 and subsequent outside-in signaling through this integrin. 3,4 One or more of these tyrosine phosphorylations may therefore function in the intracellular control of ␣ IIb ␤ 3 , which accords with the effect of certain PTK inhibitors. For instance, genistein, erbstatin, and tyrphostins inhibit aggregation and secretion induced by ␣-thrombin, collagen, and ...

Abstract-␣-Thrombin stimulation of human platelets initiates inside-out signaling to integrin ␣ IIb ␤ 3 (glycoprotein IIb/IIIa), resulting in the exposure of ligand binding sites. In the present study, the regulation of ␣ IIb ␤ 3 via protein kinases was investigated in platelets permeabilized with streptolysin O by introducing peptides that interfere with these enzymes and with possible regulatory domains in the cytosolic tail of the ␤ 3 subunit. Compared with intact platelets, the permeabilized platelets preserved Ͼ80% of the aggregation, secretion, and ␣ IIb ␤ 3 ligand binding capacity. The peptide YIYGSFK, a substrate for Src kinases, inhibited ␣-thrombin-induced ligand binding to ␣ IIb ␤ 3 , but a reversed peptide with Y3 F substitutions (KFSGFIF) had no effect. Ligand binding to ␣ IIb ␤ 3 was also inhibited by the peptide RKRCLRRL, which binds irreversibly to the catalytic domain of protein kinase C. Peptides corresponding to parts of the protein C inhibitor and ␤ 2 -glycoprotein I were used as negative controls and failed to interfere with ligand binding. Possible target domains for protein kinases are present in the cytoplasmic tail of the ␤ 3 subunit. The LLITIHDR peptide, matching the membrane-proximal domain of ␤ 3 (residues 717 to 724), had no effect, but NNPLYKEA (residues 743 to 750), EATSTFTN (residues 749 to 756), and TNITYRGT (residues 755 to 762), which mimicked overlapping domains of the carboxy-terminal part of ␤ 3 , reduced ␣-thrombin-induced ligand binding by 60Ϯ4%, 97Ϯ1%, and 97Ϯ2% (nϭ3) at 500 mol/L peptide, respectively. These observations indicate that Src kinases and protein kinase C take part in inside-out signaling to integrin ␣ IIb ␤ 3 and identify target domains in ␤ 3 that contribute to the regulation of this integrin. Key Words: integrin ␣ IIb ␤ 3 Ⅲ glycoprotein IIb/IIIa Ⅲ protein kinase C Ⅲ protein tyrosine kinase Ⅲ platelets I ntegrins are heterodimeric cell surface receptors consisting of noncovalently associated ␣ and ␤ subunits. 1 Each subunit contains a large extracellular domain, a transmembrane domain, and a relatively short cytoplasmic tail (typically Ͻ70 residues for each subunit). Integrins function in a variety of biological processes, such as the differentiation, growth, and migration of cells, and in inflammation and wound healing. Many integrins are subject to intracellular modulation of their activation state for ligands, a process known as inside-out signaling. [2][3][4] An increase in binding affinity and subsequent ligand binding generates signals into the cell, a process known as outside-in signaling. [5][6][7][8] The regulation of platelet integrin ␣ IIb ␤ 3 is a good illustration of the significance of the rapid affinity regulation of integrins. Unstimulated platelets express ␣ IIb ␤ 3 in a conformation inaccessible to ligands, thereby preventing plateletplatelet interaction. On activation, inside-out signaling converts the integrin to a functional receptor for fibrinogen, fibronectin, von Willebrand factor, and vitronectin. 9 Fibrinogen binding to ␣ IIb ␤ 3 ...

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