Differential Involvement of Tyrosine and Serine/Threonine Kinases in Platelet Integrin α _IIb β ₃ Exposure

Abstract

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 ...

Protein kinase C- and calcium-regulated pathways independently synergize with Gi pathways in agonist-induced fibrinogen receptor activation

“…Watson et al [31] treated platelets with staurosporine (1 µM) for relatively shorter time periods and observed that thrombin-induced secretion is abolished with some effect on platelet aggregation. Likewise, bisindolylmaleimide I was shown to block thrombin-and platelet-activating-factorinduced, but not ADP-induced, fibrinogen binding [29]. These data are similar to our observations in that Ro 31-8220 inhibited thrombin-and SFLLRN-induced platelet aggregation to comparable levels, and that ADP-induced fibrinogen receptor activation is unaffected by Ro 31-8220.…”

“…In turn, aggregation results when platelets are cross-linked by fibrinogen molecules bound to activated αIIbβ3 receptors on the platelet surface. Previous studies have shown that phorbol esters, activating PKC and calcium ionophores, causing increases in intracellular calcium, can cause platelet aggregation [2,26,[29][30][31]. However, these agents are not physiological agonists, and the physiological relevance of these pathways is not clear.…”

“…Previously, several investigators have used different PKC inhibitors to investigate the role of PKC in platelet fibrinogen receptor activation [26,[29][30][31]41,42]. However, these results have some inconsistencies.…”

“…Non-physiological platelet-activating agents such as calcium ionophores and phorbol esters have been shown to cause platelet fibrinogen receptor activation through calcium and PKC pathways, respectively [2,26,[29][30][31]. However, the relevance of these pathways in agonist-induced fibrinogen receptor activation has not been studied in detail.…”

Low-Density Lipoprotein Enhances Platelet Secretion Via Integrin-α IIb β 3 –Mediated Signaling

“…46 On the other hand, ADP-induced ligand binding seems not to depend on PKC. 47,48 Normally, signal generation by platelet agonists is extremely rapid, raising cytosolic Ca 2ϩ and activating PKC within seconds, which results in immediate exposure of ␣ IIb ␤ 3 and induction of secretion responses. In the current study, a relatively long incubation with LDL was required before an effect on the platelets could be observed.…”