Abstract
Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin alpha(IIb)beta(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 alpha(IIb)beta(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 alpha(IIb)beta(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 beta 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 alpha(IIb)beta(3) activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin alpha(IIb)beta(3) association with the contractile cytoskeleton. Analysis of integrin alpha(IIb)beta(3) adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin alpha(IIb)beta(3) bonds. These studies demonstrate an important role for PI3K p110 beta in regulating the avidity of high affinity integrin alpha(IIb)beta(3) receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110 beta inhibitors.
Highlights
Maintaining integrin ␣IIb3 receptors in their high affinity state is critical for sustained platelet aggregation and thrombus development and requires the ADP purinergic receptor, P2Y12, the major Gi-coupled signaling receptor linked to the activation of PI3K3 and RapIb [12,13,14,15,16]
We have investigated the potential role of PI3K signaling processes in regulating the avidity of integrin ␣IIb3 receptors after they have been converted to a high affinity state by high dose thrombin
Our studies demonstrate that fully activated integrin ␣IIb3 receptors are capable of supporting irreversible platelet aggregation independent of PI3K signaling; these receptors have a deficit in their ability to mediate stable platelet interactions with a fibrin matrix, leading to reduced fibrin clot retraction
Summary
Inhibition of PI3K Selectively Inhibits Clot Retraction in Thrombin-stimulated Platelets—PI3Ks have a well defined role in promoting sustained integrin ␣IIb3 activation downstream of Gi-coupled receptors, necessary for stable platelet aggregation and spreading [12,13,14, 16, 29, 40]. Thrombin (0.025– 0.05 unit/ml); above 0.1 units/ml, PI3K inhibitors produced a similar defect in clot neither LY294002 nor wortmannin had a significant inhibitory retraction in platelets pretreated with ADP/TxA2 antagonists effect on the rate, extent, or stability of thrombin-stimulated compared with untreated controls (Fig. 2B, ii), confirming that platelet aggregation.
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