Phosphoinositide 3-Kinase p110β Regulates Integrin αIIbβ3 Avidity and the Cellular Transmission of Contractile Forces

Abstract

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin ␣ IIb ␤ 3 , necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin ␣ IIb ␤ 3 activation (affinity modulation) primarily occurs downstream of G i -coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin ␣ IIb ␤ 3 bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110␤ isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin ␣ IIb ␤ 3 activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin ␣ IIb ␤ 3 association with the contractile cytoskeleton. Analysis of integrin ␣ IIb ␤ 3 adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin ␣ IIb ␤ 3 bonds. These studies demonstrate an important role for PI3K p110␤ in regulating the avidity of high affinity integrin ␣ IIb ␤ 3 receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110␤ inhibitors.Platelet adhesion and aggregation at sites of vascular injury are essential for the formation of the primary hemostatic plug and for the development of arterial thrombi (1, 2). Platelet thrombus growth is critically dependent on the adhesive function of the platelet integrin, ␣ IIb ␤ 3 . This receptor engages multiple adhesive ligands, including von Willebrand factor, fibrinogen, and fibronectin, and is indispensable for the formation of stable platelet-platelet adhesion contacts during thrombus development. Qualitative or quantitative defects in integrin ␣ IIb ␤ 3 are associated with a profound defect in platelet aggregation, leading to a severe bleeding disorder. Inhibitors of integrin ␣ IIb ␤ 3 have been developed therapeutically, and these agents are effective at preventing arterial thrombus development in patients with the acute coronary syndromes (3).The ligand binding function of integrin ␣ IIb ␤ 3 is subject to tight regulation by intracellular signaling processes that modulate the conformation of the receptor complex (affinity regulation) (4, 5). Once engaged by adhesive ligand, integrins transduce signals into the cell (outside-in signaling) necessary for cytoskeletal remodeling and clustering of integrin receptors. These latter ch...

Platelet PI3Kβ and GSK3 regulate thrombus stability at a high shear rate

“…Although the molecular mechanisms involved in modulating a IIb b 3 affinity have not been completely defined as yet, the literature points to several signaling pathways controlling a IIb b 3 activation in a PI3Kb-dependent way. 11 Among them, Rap1b and the Ser/Thr kinase Akt have been implicated in a IIb b 3 inside-out and/or outside-in signaling in a PI3Kb-dependent manner. 6,19,[31][32][33][34][35][36][37] Using PDK1-deficient mice, it was proposed that the PDK1/Akt axis modulates platelet aggregation and clot retraction.…”

“…These platelets exhibit a delay in fibrin clot retraction and a decrease in the interaction of platelets with fibrinogen under flow, suggesting that PI3Kb is an important actor downstream of a IIb b 3 . 6,10,11 Because PI3Kb inhibition prevents occlusive thrombus formation in mice, rats, and dogs with a limited increase in bleeding risk and acceptable safety in humans, this kinase has been proposed as a potential antithrombotic drug target. 12,13 However, the role of platelet PI3Kb in integrated in vivo or ex vivo models of thrombosis on high shear-rate condition remains incompletely documented.…”

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

“…PI3K␤ has also been proposed to be involved in integrin ␣IIb␤3-mediated outside-in signaling, a process essential for platelet spreading, stable thrombus formation, and clot retraction. [3][4][5]13 In contrast, very little is known about the role and regulation of PI3K downstream of the other major platelet integrin, integrin ␣2␤1.…”

“…PI3K␤ has also been proposed to be involved in integrin ␣IIb␤3-mediated outside-in signaling, a process essential for platelet spreading, stable thrombus formation, and clot retraction. [3][4][5]13 In contrast, very little is known about the role and regulation of PI3K downstream of the other major platelet integrin, integrin ␣2␤1.Together with GPVI, integrin ␣2␤1 is a platelet receptor for collagen, 14 but it can also interact with other ligands, including decorin and tenascin. 15,16 Although some controversies persist, the role of integrin ␣2␤1 in adhesion to collagen, platelet activation, and thrombus formation is well documented.…”

Src family kinases: at the forefront of platelet activation

“…30,33 Downstream Src-dependent phosphorylation events include Tyr773 and Tyr785 in human b3 (mouse Tyr747 and Tyr759) that provide docking sites for myosin light chain and the adaptor Shc, 34 adaptor and cytoskeletal proteins (paxillin, vinculin, and actinin) that provide docking sites for assembly of signaling complexes, [35][36][37] tyrosine kinases (FAK, Pyk, and Syk), 38 small guanosine triphosphatase (Rap1B), guanine nucleotide exchange factors (Vav1 and Vav3), 39,40 and the lipid kinase phosphoinositide 3 kinase (PI3K) that propagate the signal. 38,41 Src-dependent activation of phospholipase Cg2 (PLCg2) results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI4,5P 2 ) to the second messengers diacylglycerol (DAG) and inositol triphosphate (IP 3 ) that activate the serine/threonine protein kinase C (PKC) family and facilitate Ca 21 mobilization, respectively. 42 Tyrosine phosphorylation of the ITAM-containing FcgRIIA receptor by SFKs provides a high-affinity docking site For personal use only.…”