Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

Abstract

Integrin ␣2␤1-mediated adhesion of human platelets to monomeric type I collagen or to the GFOGER peptide caused a time-dependent activation of PI3K and Akt phosphorylation. This process was abrogated by pharmacologic inhibition of PI3K␤, but not of PI3K␥ or PI3K␣. Moreover, Akt phosphorylation was undetectable in murine platelets expressing a kinase-dead mutant of PI3K␤ (PI3K␤ KD ), but occurred normally in PI3K␥ KD platelets. Integrin ␣2␤1 failed to stimulate PI3K␤ in platelets from phospholipase C␥2 (PLC␥2)-knockout mice, and we found that intracellular Ca 2؉ linked PLC␥2 to PI3K␤ activation. Integrin ␣2␤1 also caused a time-dependent stimulation of the focal kinase Pyk2 downstream of PLC␥2 and intracellular Ca 2؉ . Whereas activation of Pyk2 occurred normally in PI3K␤ KD platelets, stimulation of PI3K␤ was strongly reduced in Pyk2-knockout mice. Neither Pyk2 nor PI3K␤ was required for ␣2␤1-mediated adhesion and spreading. However, activation of Rap1b and inside-out stimulation of integrin ␣IIb␤3 were reduced after inhibition of PI3K␤ and were significantly impaired in Pyk2-deficient platelets. Finally, both PI3K␤ and Pyk2 significantly contributed to thrombus formation under flow. These results demonstrate that Pyk2 regulates PI3K␤ downstream of integrin ␣2␤1, and document a novel role for Pyk2 and PI3K␤ in integrin ␣2␤1 promoted inside-out activation of integrin ␣IIb␤3 and thrombus formation. (Blood. 2012;119(3):847-856) IntroductionClass I PI3Ks are key signaling enzymes that phosphorylate the inositol ring of membrane phospholipids and generate different 3-phosphoinositides, important intracellular messengers that regulate several cellular processes through the downstream activation of the protein Ser/Thr kinase Akt. 1 Circulating blood platelets express all members of the class I PI3K family, which includes the PI3K␣, PI3K␤, PI3K␦, and PI3K␥ isoforms. PI3K activity is essential for platelet aggregation and thrombus formation, 1,2 and therefore these enzymes are potential novel targets for antithrombotic agents. For this reason, it is essential to recognize the precise contribution of every PI3K isoform in platelet activation induced by different extracellular agonists.Pharmacologic and genetic evidence indicates that PI3K␤ plays a predominant role in the regulation of platelet function. [3][4][5][6][7] Selective inactivation of PI3K␤ completely prevents platelet aggregation induced by the collagen receptor glycoprotein VI (GPVI) and reduces occlusive thrombus formation. 4,5 PI3K␤ is also implicated in the platelet response to agonists that stimulate G-protein coupled receptors (GPCRs) such as ADP or thromboxane A 2 (TxA 2 ). 3,4,8,9 Whereas PI3K␦ has been demonstrated to play a minor role in platelet activation, 10 PI3K␣ was recently proposed to be as important as PI3K␤ in GPVI signaling. 7 Similarly, several reports have documented that, in addition to PI3K␤, PI3K␥ is also implicated in GPCR-mediated platelet activation. 4,9,11,12 These observations are indicative of a still poorly appreciated interplay...

Impaired thrombin-induced platelet activation and thrombus formation in mice lacking the Ca2+-dependent tyrosine kinase Pyk2

“…47 In a recent study, we have also found that ex vivo thrombus formation under flow conditions on a collagen matrix was strongly reduced in the absence of Pyk2. 14 Our findings also indicate that Pyk2 is relevant, but not essential, for hemostasis because the bleeding time was mildly increased in Pyk2-KO mice. However, we observed a high variability in the time required to stop bleeding in the absence of Pyk2, and a clear bleeding tendency was observed in only approximately one-third of the analyzed mice.…”

“…[9][10][11][12][13] Phosphorylation of Pyk2 downstream of integrin ␣IIb␤3 and ␣2␤1 engagement has also been reported. 10,14 In thrombin-and VWF-activated platelets, Pyk2 interacts with the actin-based cytoskeleton. 9,11 Other studies have documented its association with PI3K, Cas, Shc, and Hic-5.…”

Altered Toll-Like Receptor 9 Signaling in Mycobacterium avium subsp. paratuberculosis-Infected Bovine Monocytes Reveals Potential Therapeutic Targets

“…Activation of Pyk2-dependent signaling events appears to represent a bifurcation of TLR induced signaling as inhibitors of Src block the Pyk2-mediated responses without influencing IRAK-1-mediated signal transduction (51). Furthermore, Pyk2 has recently been shown to be critical for PI3KB integrin-mediated activation of platelet adhesion (57). However, there is likely cross talk between canonical TLR signaling and Pyk2-mediated signaling because Pyk2-deficient macrophages undergo reduced LPSinduced activation of NF-B and IL-1 expression (53).…”

Platelet CD40L Modulates Thrombus Growth Via Phosphatidylinositol 3-Kinase β, and Not Via CD40 and IκB Kinase α

“…This role of PI3K-β is in good agreement with the literature, demonstrating that this kinase is a major regulator of Akt phosphorylation and downstream integrin activation in response to various platelet agonists. 31,[41][42][43] Other authors have suggested that CD40L in platelets signals via p38 mitogen-activated protein kinase, 19 or via tyrosine phosphorylation of the integrin β 3 chain. 22 These pathways may coexist and help to support the establishment of platelet-platelet interactions.…”