PI 3-kinase p110: a new target for antithrombotic therapy

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin _IIb3 (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110 isoform in regulating the formation and stability of integrin _IIb3 adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110 inhibitors have been developed which prevent formation of stable integrin _IIb3 adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110 as an important new target for antithrombotic therapy.

Platelet thrombus formation is crucially dependent on the adhesive and signaling functions of integrin _IIb3 (platelet GPIIb-IIIa), a major platelet integrin that mediates platelet−vessel wall and platelet-platelet adhesive interactions through engagement of multiple adhesive ligands, including von Willebrand factor (vWf), fibrinogen, fibronectin and soluble CD40L¹⁰. All existing antiplatelet agents target one or more key steps in the thrombotic mechanism, which ultimately downregulate the adhesive function of integrin _IIb3; however, a considerable limitation of current therapies is their deleterious effects on hemostasis, leading to bleeding complications¹¹. An ideal antithrombotic therapy would selectively target an event crucial for pathological thrombus formation without affecting hemostasis. One such strategy would be to attenuate the ability of platelets to form stable integrin _IIb3 adhesion contacts under high shear stress by targeting shear-sensitive signaling processes in platelets. To date, the identification of signaling elements crucial for shear activation of platelets, but not for hemostasis, have remained elusive.

An important signaling pathway promoting shear activation of platelets involves the PI3Ks^{12, 13}. These lipid kinases are divided into three distinct classes (Class I, II and III) based on their primary structure, mode of regulation and substrate specificity^{14, 15}. The Class I PI3Ks are responsible for agonist-induced production of the second messengers phosphoinositide (PI) (3,4,5)P₃ and PI(3,4)P₂, and participate in the regulation of a broad range of functional platelet responses, including activation of integrin _IIb3¹⁶. The Class I enzymes are further divided into , , and isoforms based on their distinct p110 catalytic subunits and modes of regulation. The Class Ia isoforms (p110, p110, p110) are classically regulated by tyrosine kinases, whereas the Class Ib isoform (p110) is typically activated by G-protein-coupled receptors¹⁷. Platelets contain all Class I PI3K isoforms, although the levels of p110 are much lower in platelets than in other hemopoietic cells^{18, 19, 20}. To date, the role of the individual PI3K isoforms in regulating platelet functional responses has been poorly defined and there is currently no information on the role of individual PI3K isoforms in regulating shear activation of platelets.

PI3K regulates integrin _IIb3 adhesive bond stability

Previous studies have defined a key role for PI3K in promoting the initial formation of integrin _IIb3 adhesive bonds under high shear^{12, 21} and there is evidence that PI3Ks operate downstream of each of the major platelet receptors involved in shear-induced platelet activation, notably glycoprotein (GP) Ib^{22, 23}, integrin _IIb3²¹ and the ADP purinergic receptor P2Y₁₂^{13, 24}. The specific PI3K isoforms involved in this process and the importance of these enzymes in maintaining the stability of formed integrin _IIb3 adhesion bonds have not been established. To investigate this, platelets were perfused through fibrinogen-coated microcapillary tubes at low shear to enable formation of integrin _IIb3 adhesion bonds, then exposed to sudden accelerations in blood flow, thereby increasing tensile stress on formed bonds. Of platelets adherent to fibrinogen, 63.8 6.0% were able to resist the detaching effects of rapid increases in shear, whereas 27.4 6.3% of platelets pretreated with the pharmacological PI3K inhibitor LY294002 were able to maintain firm adhesion (Fig. 1a). Previous studies have established an important role for integrin _IIb3−dependent calcium flux, in combination with released ADP, in promoting shear-resistant platelet adhesion²⁵. Consistent with an important role for PI3Ks in this process, LY294002 completely inhibited calcium flux in fibrinogen-adherent platelets (Fig. 1b), either in the presence or absence of ADP receptor antagonists (data not shown). To investigate the identity of the PI3K isoforms involved in this process, flow-based adhesion assays were performed on mouse platelets deficient in the Type IA PI3K p110 or Type 1B PI3K p110 isoform. Similar to human platelets, maintenance of mouse integrin _IIb3 adhesive bonds on a fibrinogen matrix is dependent on the cooperative signaling function of integrin _IIb3 and released ADP; however, in the mouse system, the contribution of integrin _IIb3−dependent signaling processes to sustained adhesion is most clearly manifested in the presence of ADP receptor antagonists²⁵. This species difference probably reflects differences in the binding kinetics of mouse versus human integrin _IIb3 to human fibrinogen. In the presence of ADP receptor antagonists, 71.4 3.2% of wild-type mouse platelets maintained stable adhesion contacts with fibrinogen after exposure to rapid increases in shear (Fig. 1c). In contrast, 39.8 5% of wild-type platelets treated with LY294002 and ADP receptor antagonists were able to maintain stable adhesive interactions under the same experimental conditions. As shown in Fig. 1, analysis of PI3K p110−deficient and PI3K p110−deficient platelets showed no significant difference in shear-resistant platelet adhesion relative to wild-type matched controls (Fig. 1c,e). Similarly, deficiency of PI3K p110 and PI3K p110 had no significant effect on integrin _IIb3−dependent cytosolic calcium flux, in the presence or absence of ADP receptor antagonists (Fig. 1d,f), excluding an important role for these isoforms in maintaining integrin _IIb3 adhesive contacts under high shear.

Figure 1. PI3K promotes cytosolic calcium flux and stable platelet adhesion on immobilized fibrinogen following exposure to rapid increases in shear. Calcium dye−loaded human platelets (a,b) or those isolated from PI3K p110−containing (+), p110−deficient (-) (c,d) or PI3K p110−deficient (-) (d,e) mice were pretreated with vehicle alone (Me2SO), the non-isoform-selective PI3K inhibitor LY294002 (LY, 25 M), and/or ADP inhibitors (ØADP: apyrase (1.5 U/ml), MRS-2179 (100 M), AR-C69931MX (10 M)), as indicated. The results represent the percentage of platelets (a,c,e) becoming displaced from the point of initial contact, and (b,d) undergoing a sustained oscillatory calcium response following an increase in shear from 150 s-1 to 1,800 s-1 for human and 600 s-1 to 10,000 s-1 for mouse platelets (arrow) (mean s.e.m., n = 3). Full Figure and legend (36K)

Development of isoform-selective PI3K p110 inhibitors

Establishing the roles of the PI3K p110 and p110 isoforms in signaling pathways is complicated by the lack of available mouse models and specific isoform-selective inhibitors. To overcome this problem we developed isoform−selective PI3K inhibitors based on detailed structure and function analysis of LY294002 (Fig. 2a). Replacing the chromone nucleus with the pyrido[1,2-a]pyrimidin-4-one nucleus and elaboration of the pendant aryl ring yielded compounds with increased potency and selectivity against the Type I PI3K p110 isoform (Fig. 2b and Supplementary Fig. 1 online). One of the most potent members of this group (TGX-221) has an IC₅₀ of 5 nM against the purified kinase (phosphatidylinositol as substrate in the presence of 50 M ATP; IC₅₀ 10 nM with PI(4,5)P₂ as substrate) and shows 1,000-fold selectivity over PI3K p110 (Fig. 2c). TGX-221 had minimal inhibitory effect on Type II PI3K C2 and PI4K and showed >1,000-fold selectivity for PI3K p110 over a broad range of protein kinases (Supplementary Table 1 online), and similar to LY294002, had minimal effect on global protein phosphorylation in thrombin-stimulated platelets (data not shown). Similar to LY294002, the inhibitory effects of TGX-221 were influenced by the concentration of ATP. At physiologically relevant cytosolic ATP levels (typically 1,000 M) the IC₅₀ against PI3K p110 increased from 5 nM to 50 nM, indicating that TGX-221 was probably acting as a competitive ATP antagonist. Analysis of the effects of TGX-221 on PI3K lipid product generation in vivo showed that TGX-221 inhibited shear-induced PI(3,4)P₂ production in a dose-dependent manner (Fig. 2d and Supplementary Fig. 1 online), without altering the cellular levels of PI(3)P and the conventional phosphoinositides, PI(4)P and PI(4,5)P₂ (Fig. 2d). Increases in PI(3,4,5)P3 levels were not detected under these experimental conditions, presumably as a result of the rapid dephosphorylation of PI(3,4,5)P3 to PI(3,4)P₂ by SHIP-1 (ref. 26). The IC50 for inhibiting PI(3,4)P₂ production in platelets (50 nM) was 50−100-fold lower than that observed with LY294002 (2.5−5 M), which correlated well with the relative IC₅₀s for these compounds against PI3K p110 at physiological ATP concentrations. These studies show that TGX-221 is a potent cell-permeable inhibitor of PI3K p110 and furthermore, show an important role for PI3K p110 in promoting shear-dependent PI(3,4)P₂ generation.

Figure 2. Development of a new PI3K p110 isoform−selective inhibitor. (a) Chemical structure of LY294002. Key features for PI3K activity and selectivity include the chromone nucleus (i), the 2-morpholinyl substitution (ii) and the pendant aryl ring (iii)42. These features were systematically modified in structure-function studies. (b) Chemical structures and PI3K p110 inhibitory potency of 2-morpholinyl-pyrido-[1,2-a] pyrimidinones. Successively more potent inhibitors of the PI3K p110 isoform were identified, such as TGX-126 and TGX-221 (Supplementary Fig. 1 online). (c) Relative dose-response inhibition of PI3K p110, p110 and p110 (expressed as percent activity relative to Me2SO control) by TGX-221 (p110 and , n = 5; p110, n = 10, mean s.e.m.). (d) Effect of TGX-221 (221, 0.1 M) on PI(4)P, PI(3)P, PI(4,5)P2 and PI(3,4)P2 levels in shear-activated platelets. Histograms represent the level of each lipid expressed as a percent of total lipid (all groups n = 3, mean s.e.m., ***P < 0.0001; ns, P > 0.1, Student's t-test). Full Figure and legend (70K)

PI3K p110 regulates integrin _IIb3 bond stability

Analysis of the effects of TGX-221 on shear-resistant adhesion of human platelets on a fibrinogen matrix showed a defect in integrin _IIb3-dependent calcium flux and firm platelet adhesion similar in magnitude to that observed with LY294002-treated platelets (Fig. 3a,b). Similarly, PI3K p110−deficient and PI3K p110−deficient mouse platelets were equally sensitive to the inhibitory effects of TGX-221 and LY294002, defining a major role for PI3K p110 in promoting integrin _IIb3−dependent calcium flux (Fig. 3c−f). To investigate the importance of PI3K p110 in initiating the formation of integrin _IIb3 adhesive bonds under high shear, flow-based adhesion studies were performed on an immobilized human vWf matrix. TGX-221 abolished stable platelet adhesion and thrombus formation on a vWf substrate at elevated shear rates (1,800 s^-1; Fig. 3g,h). This reduction in firm platelet adhesion was associated with inhibition of cytosolic calcium flux and integrin _IIb3 activation (data not shown). Similarly, shear-induced aggregation of platelets in suspension (5,000 s^-1) was inhibited by TGX-221 by >90% (data not shown). Recent studies have defined an important role for rapid increases in shear (temporal shear gradients) in promoting integrin _IIb3−dependent calcium flux, necessary for efficient platelet activation on a vWf matrix²⁷. As shown in Figure 4, TGX-221 completely eliminated cytosolic calcium flux (Fig. 4a,c) and integrin _IIb3 activation (Fig. 4f) induced by temporal shear gradients (0−10,000 s^-1), confirming a major role for PI3K p110 in integrin _IIb3 mechanotransduction. In control studies, a PI3K p110 inhibitor (D-010 (ref. 28); Supplementary Fig. 2 online) had no inhibitory effects on platelet activation induced by high shear stress (data not shown) or by temporal shear gradients (Supplementary Fig. 2 online). Similar findings were found with PI3K p110−deficient platelets (Supplementary Fig. 2 online). Furthermore, the effects of TGX-221 on calcium flux were selective, as it had no inhibitory effect on GPIb-dependent calcium signals (Fig. 4b) or on calcium signals induced by a range of physiological agonists (Fig. 4d,e and data not shown). These studies define a major role for PI3K p110 in promoting the formation and maintenance of integrin _IIb3 adhesive bonds under high shear through regulation of integrin _IIb3−dependent calcium flux.

Figure 3. PI3K p110 promotes cytosolic calcium flux and stable platelet adhesion in response to rapid increases in shear. Calcium dye−loaded human platelets (a,b), or those isolated from PI3K p110−deficient (-) (c,d) or PI3K p110−deficient (-) mice (e,f), were pretreated with vehicle alone (Me2SO) or 0.5 M TGX-221 (221). The results represent the percentage of platelets displaced (a,c,e) and the percentage of platelets exhibiting a sustained oscillatory calcium response (b,d,f) after the shear increase (arrow) (mean s.e.m., n = 3). (g) Differential interference contrast images of platelet thrombi formed on a vWf matrix in the presence (221) or absence (Me2SO) of TGX-221. (h) Quantification of the effect of TGX-221 on thrombus volume on a vWf matrix (mean s.e.m., n = 7). Full Figure and legend (198K)

Figure 4. Role of PI3K p110 in promoting platelet activation in response to fluid shear stress or soluble agonists. Representative single platelet recordings showing the effects of TGX-221 on (a) integrin IIb3−dependent calcium responses induced by temporal shear gradients or (b) GPIb-dependent [Ca2+]c responses occurring under steady-state shear conditions. (c) The proportion of platelets showing an integrin IIb3− or GPIb-dependent calcium response was quantified and expressed as mean s.e.m. (n = 6, mean s.e.m.). (d,e) Population-based analysis of [Ca2+]c in platelets activated with (d) TxA2 (U46619, 50 nM) or (e) thrombin receptor agonist peptide (TRAP, 0.4 M). (f) Effect of TGX-221 (221) on integrin IIb3 activation (assessed by PAC-1 binding, PAC-1) induced by temporal shear gradients. Full Figure and legend (170K)

PI3K p110 involvement in platelet aggregation and secretion

To investigate the importance of PI3K p110 in promoting platelet activation by physiological agonists, we performed platelet aggregation and granule release studies. TGX-221 had no inhibitory effects on platelet aggregation and alpha- and dense-granule release induced by thrombin, high-dose collagen or the TxA₂ analog U46619 (Fig. 5a−c, Supplementary Fig. 3 online and data not shown). Furthermore, TGX-221 had no inhibitory effect on the ability of these agonists to stimulate integrin _IIb3 activation (as assessed by PAC-1 binding; data not shown). But inhibition of PI3K 110 did inhibit sustained platelet aggregation induced by threshold concentrations of thrombin receptor agonist peptide (TRAP; Fig. 5c and Supplementary Fig. 3 online), collagen (Supplementary Fig. 3 online) and U46619 (data not shown), although alpha- and dense-granule release was only marginally affected by this inhibitor (Fig. 5a,b). These studies show that PI3K p110 has an important role in sustaining platelet aggregation in response to weak agonist stimulation.

Figure 5. Role of PI3K p110 in regulating platelet activation induced by physiological agonists. Effect of TGX-221 (221, 0.5 M) on thrombin (Thr, 0.01 or 1.0 U/ml)-, TxA2 (U46619, 0.01 or 1.0 M)- or collagen (Coll, 1.0 or 10 g/ml)-induced (a) P-selection expression and (b) serotonin release (n = 5, mean s.e.m., ***P < 0.005, **P < 0.05, paired Student t-test). Effect of TGX-221 (0.5 M) on the aggregation of washed platelets stimulated by (c) TRAP (5 or 10 M) or (d) ADP (5, 10 or 25 M). (e) Dose-dependent effects of TGX-221 (concentrations shown in M) on ADP (25 M)−induced platelet aggregation (n = 3, mean s.e.m.). (f) Effect of TGX-221 (221) or the P2Y1 antagonist A3P5PS (A3) (500 M) on calcium flux induced by ADP (25 M) (quantification of results in Supplementary Fig. 4 online). (g) Effect of TGX-221 on the P2Y12- or 2A-dependent potentiation of platelet aggregation induced by a threshold concentration of TRAP (T, 0.25 M); A, ADP; E, epinephrine. (n = 3, mean s.e.m.). (h) Effect of vehicle (Me2SO), TGX-221 (221) or the P2Y12 receptor agonist AR-C69931MX (ARC, 10 M), on ADP (25 M)-induced Rap1b activation (n = 3, mean s.e.m.). Full Figure and legend (88K)

Platelet aggregation induced by threshold concentrations of agonists is partially dependent on the granule release of ADP²⁹. TGX-221 inhibited ADP-induced platelet aggregation over a dose range consistent with the inhibition of PI3K p110. The inhibitory effects of TGX-221 were apparent over a broad range of ADP concentrations and were primarily manifested as an inability of ADP to sustain platelet aggregation (Fig. 5d,e). Inhibition of PI3K p110 had no effect on the ability of ADP to stimulate P2Y₁-dependent cytosolic calcium flux (Fig. 5f and Supplementary Fig. 4 online) or platelet shape change (Supplementary Fig. 4 online), suggesting a potentially important role for PI3K p110 in P2Y₁₂ signaling. Consistent with this, TGX-221 inhibited P2Y₁₂/G_i-dependent potentiation of platelet aggregation induced by threshold concentrations of other agonists (Fig. 5g). Similar findings were apparent when the G_i-linked _2A adrenergic receptor agonist epinephrine was used to potentiate platelet aggregation (Fig. 5g), suggesting an important role for PI3K p110 in platelet Gi signaling processes. Consistent with this, G_i-dependent Rap1b activation, a Ras family GTPase that potentiates integrin _IIb3 activation³⁰ was inhibited by >70% by TGX-221 (Fig. 5h). These studies define an important role for PI3K p110 in P2Y₁₂/G_i signaling processes linked to sustained platelet aggregation.

Antithrombotic effect of PI3K p110 inhibitors

To investigate the antithrombotic potential of TGX-221 in vivo, a modified 'Folts-type' thrombosis model was used in anesthetized rats and rabbits³¹. TGX-221 (2 mg/kg intravenous bolus) abolished occlusive thrombus formation in 100% of rats (n = 8) (Fig. 6a) and rabbits (n = 9) (Supplementary Fig. 5 online). The effects of TGX-221 in these models was selective, as a pharmacological PI3K p110 inhibitor (D-010) had no effect on arterial thrombotic occlusion (Fig. 6a). The onset of the antithrombotic effect of TGX-221 was rapid (occurring within 5 min of bolus injection; Supplementary Fig. 6 online) and dose dependent, with activity observed at doses as low as 0.5 mg/kg. In a separate rat carotid artery electrolytic injury model³², TGX-221 (2 mg/kg) prevented the development of occlusive thrombi (Fig. 6b, n = 6) and was more effective than aspirin at preserving carotid blood flow volume over the 60-min period after injury (Fig. 6b,c). TGX-221 had no effect on baseline arterial blood pressure, heart rate or blood flow in the uninjured carotid artery in both the Folts and electrolytic studies (data not shown). Analysis of a range of structural analogs of TGX-221 with varying inhibitory potencies against PI3K p110 showed a strong rank-order correlation between their inhibitory effects on the purified kinase with their ability to inhibit shear activation of platelets in vitro and occlusive thrombus formation in vivo (data not shown). Furthermore, analysis of the circulating concentrations of TGX-221 in rats confirmed that the antithrombotic activity of this compound occurred at serum levels relevant to inhibition of PI3K p110 in platelets (serum concentration of 2 and 0.2 M at 5 and 60 min after intravenous infusion of TGX-221, respectively). In further control studies, LY294002 (5−10 mg/kg) and wortmannin (1 mg/kg), both non-isoform-selective PI3K inhibitors, also prevented arterial thrombotic occlusion in the rat Folts-type and electrolytic preparations, although LY294002 was 10−20-fold less potent than TGX-221 (Fig. 6a and data not shown). PI3K p110 seemed to be the major PI3K isoform promoting occlusive thrombus formation as mice deficient in PI3K p110 showed normal arterial occlusion in several distinct arterial thrombosis models (Fig. 6a and Supplementary Fig. 5 online).

Figure 6. Antithrombotic activity of TGX-221. (a) The effect of PI3K inhibitors on CFRs in the Folts-type stenosis injury models of arterial thrombosis. The effects of an intravenous bolus of vehicle (propylene glycol 0.25 ml/kg; n = 10), LY294002 (LY, 5 mg/kg; n = 6), D-010 (5 mg/kg; n = 6) or TGX-221 (221, 2 mg/kg; n = 8) on the average number of CFRs in the rat, or PI3K p110−deficient (-) mice were examined. Gray bars represent the number of CFRs per 10 min before drug administration and black bars represent the number of CFRs per 10 min after drug administration (***P < 0.0001, paired Student t-test). (b) Effects of vehicle (propylene glycol 0.25 ml/kg; n = 10), aspirin (Asp, 200 mg/kg orally; n = 8) or TGX-221 (221, 2 mg/kg; n = 6) on carotid artery blood flow (ml/min per 100 g body weight) after electrolytic injury in rats (mean s.e.m., from repeated measures ANOVA). (c) Effect of treatments on carotid artery blood flow volume (area under the blood flow curve over the 60-min period after stimulation) after electrolytic injury in rats (**P < 0.01, one-way ANOVA, Dunnett post hoc test). (d) Tail bleeding time in rats, following the indicated drug treatments: baseline, before any drug (n = 16); TGX-221 (221, n = 4); aspirin (Asp; n = 6); clopidogrel (Clop; n = 6); TGX-221 + heparin (221+Hep; n = 4); aspirin + heparin (Asp+Hep; n = 10); clopidogrel + heparin (Clop+Hep; n = 10) (*P < 0.05, **P < 0.01, one-way ANOVA, Dunnett post hoc test). (e) Schematic model showing the central role of PI3K p110 in sustaining integrin IIb3 adhesive bonds necessary for shear-resistant platelet adhesion. Integrin IIb3 engagement of adhesive ligands (vWf-fibrinogen) induces outside-in signals linked to the mobilization of cytosolic calcium flux. These calcium signals positively feed back on the integrin itself, strengthening the affinity and avidity of the adhesive interaction21, 25. Released ADP potentiates integrin IIb3 activation through P2Y12-mediated activation of Rap1b, promoting shear-resistant platelet adhesion. PI3K p110 has a central role in this process by promoting integrin IIb3-dependent calcium flux (solid lines in 1) and Gi-dependent activation of Rap1b (dotted lines in 2). This molecular mechanism is also probably responsible for the sustained aggregation of platelets induced by ADP or threshold concentrations of other agonists. Full Figure and legend (132K)

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The studies presented here have defined a key role for the Type IA p110 isoform of PI3K in initiating and sustaining integrin _IIb3 adhesion contacts, particularly under conditions of high shear stress. Shear-induced platelet activation requires the cooperative adhesive and signaling function of GPIb and integrin _IIb3 in concert with amplification signals linked to the G_i-coupled purinergic receptor, P2Y₁₂^{33, 34}. We have shown that PI3K p110 is involved in G_i-dependent signaling processes linked to the activation of Rap1b, a Ras family GTPase that has a well-defined role in regulating integrin adhesive function³⁵. Thus, PI3K p110 has an important role in sustaining integrin _IIb3 activation and stable platelet aggregation by regulating both integrin _IIb3−dependent calcium flux and G_i-activation of Rap1b (Fig. 6e). These findings provide a mechanistic explanation as to why PI3K p110 inhibitors reduce platelet adhesion and aggregation under high shear and prevent arterial thrombotic occlusion in vivo.

By exploiting the unique structural features of PI3K^{36, 37} we have developed potent isoform-selective inhibitors against PI3K p110. A crucial determinant of LY294002 specificity is the 8-phenyl group, which occupies a relatively open pocket that interacts with the ribose moiety of ATP. The active site of PI3K is more open at this position than in protein kinases, explaining the specificity of LY294002 toward lipid kinases. We have shown here that elaboration of the pendant aryl ring yields compounds with increased potency and selectivity against PI3K p110, with TGX-221 showing 1,000-fold selectivity over the other two major Type I PI3Ks in platelets, p110 and p110. We have confirmed that p110 is minimally expressed in platelets^{18, 20} and studies using pharmacological p110 inhibitors and p110-deficient mouse platelets do not support a substantial role for this isoform in platelet function (data shown here and S.M. Schoenwaelder & S.P. Jackson, unpublished observations). Thus, utilizing TGX-221 at submicromolar concentrations, we have shown that the only functionally relevant platelet PI3K that is markedly inhibited by this compound is PI3K p110. Several lines of evidence suggest that the antithrombotic activity of TGX-221 observed in our animal models is probably the result of inhibition of PI3K p110. First, analysis of the circulating concentrations of TGX-221 in rats confirmed that the antithrombotic activity of this compound occurred at serum levels relevant to inhibition of PI3K p110 in platelets. Second, utilizing a series of structural analogs of TGX-221, we have shown a close rank-order correlation between the inhibition of PI3K p110 and the antithrombotic activity of these compounds in vivo. Third, direct comparison of the dose responses of LY294002 and TGX-221 showed that the latter compound was 10−20-fold more potent than the former at preventing arterial thrombotic occlusion in vivo. In contrast, the pharmacological inhibitor against PI3K p110, which has 20-fold higher potency against the isoform than LY294002, showed considerably less potency in in vivo models. This combined with the demonstration of a normal thrombotic response in PI3K p110−deficient mice provides strong experimental evidence that the major antithrombotic effect of LY294002 and TGX-221 is primarily through inhibition of PI3K p110.

A notable finding in this study is the demonstration that PI3K p110 inhibitors have no effect on bleeding time, even when administered at concentrations 40-fold higher than the minimal effective antithrombotic dose or when coadministered with an anticoagulant agent such as heparin. This is presumably a reflection of the fact that PI3K p110 is not essential for initial platelet aggregation and granule release induced by physiological agonists but is important for sustained integrin _IIb3 activation necessary for arterial thrombotic occlusion. These findings suggest that PI3K p110 represents a potentially important new target for antithrombotic therapy. Given the ubiquitous expression of PI3K p110, the challenge ahead will be to develop strategies to abolish PI3K p110 activation in platelets while minimizing potential systemic toxicities, particularly if long-term thromboprophylactic therapy is contemplated. Such strategies have previously been used successfully against other ubiquitous targets; the most notable involves irreversible acetylation of cyclo-oxygenases by aspirin.

Overall, our findings provide further evidence for functional specialization of different PI3K isoforms and show for the first time an important role for PI3K p110 in platelet function and thrombosis. Given the important roles of each of the Type I PI3K family members in various pathophysiological processes (PI3K p110 in oncogenesis, PI3K p110 in thrombosis, PI3K p110 in immune function and PI3K p110 in inflammation^{38, 39, 40}) isoform-selective PI3K inhibitors may ultimately have considerable therapeutic benefit in a wide range of human diseases.

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All materials were obtained from sources described elsewhere^{21, 26, 41}. The synthesis of TGX-221 and other compounds used in this study have been described⁴² or will be reported in detail elsewhere. PI3K p110−deficient mice were generated by D. Wu (University of Rochester, New York). PI3K p110−deficient mice were provided by M. Turner (Babraham Institute, UK). All protocols involving the use of animals were approved by the Alfred Medical Research Education Precinct Animal Ethics Committee, Alfred Medical Research Education Precinct, Victoria, Australia.

We measured surface expression of P-selectin using a P-selectin-specific monoclonal antibody (AK6, 1 g/ml), and analyzed it on a FACScalibur (Becton Dickinson).

Calcium mobilization in resting and ADP-stimulated washed platelets (200 10⁹/L) loaded with Fura-2 AM (5 M) was monitored over time using a fluorimeter (LS-50B; Supplementary Methods online). We performed measurements in the absence or presence of the indicated inhibitors (TGX-221, 0.5 M; A3P5PS, 300 M), with dual excitation at 340 and 380 nm and detected emission at 510 nm. Calcium measurements were converted to nM calcium using computer-assisted analysis (FL WinLAB).

All individual aggregation traces, calcium traces, and images were taken from one experiment representative of at least three independent experiments. All numerical results are presented as mean s.e.m. The significance between two data tests was tested using unpaired t-tests in most instances, unless otherwise indicated. Statistical significance was considered when P < 0.05.

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Competing interests statement:  The authors declare competing financial interests.