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Resistance to thromboembolism in PI3K-deficient mice ¹

EMILIO HIRSCH^*2, ORNELLA BOSCO, PHILIPPE TROPEL^*, MURIEL LAFFARGUE, RONAN CALVEZ, FIORELLA ALTRUDA^*, MATTHIAS WYMANN^3, and GIUSEPPE MONTRUCCHIO^3,

^* Dipartimento di Genetica, Biologia e Biochimica, Università di Torino, 10126 Torino, Italy;
Dipartimento di Fisiopatologia Clinica, Università di Torino, 10126 Torino, Italy; and
Institute of Biochemistry, CH-1700 Fribourg, Switzerland

²Correspondence: Dipartimento di Genetica, Biologia e Biochimica, Università di Torino, Via Santena 5 bis, 10126 Torino, Italy. E-mail: emilio.hirsch{at}unito.it

SPECIFIC AIMS

PI3K has been suggested as a key downstream effector of G-protein-coupled receptors (GPCR) but its specific role in thrombocyte function is still obscure. We investigated the role of PI3K in platelet activation using mice where PI3K expression was genetically ablated. We analyzed platelet aggregation after stimulation with GPCR agonists like ADP and thrombin. We evaluated PKB and fibrinogen receptor activation. Finally, to assess the in vivo consequences of the absence of PI3K in platelets, we analyzed bleeding time and challenged mice in a model of ADP-dependent thromboembolic vascular occlusion.

PRINCIPAL FINDINGS

1. PI3K-null platelets show impaired aggregation after stimulation with the GPCR agonist ADP
Acting via GPCRs, ADP is an important signal in the formation of thrombus and involves activation of phosphoinositide 3-kinases (PI3K). Western blot analysis of murine platelets indicates that they express PI3K, ß, and , but not . Platelet aggregation was analyzed in whole blood, platelet-rich plasma, and washed platelets of mice that lack the G-protein-activated PI3K isoform. Physiological platelet agonists that act dependently and independently of GPCRs were tested. In all preparations, aggregation was impaired after stimulation with ADP (Fig. 1A -D ). When used on PI3K-null platelets, collagen, an agonist able to signal through tyrosine kinase-coupled receptors, evoked a normal response (Fig. 1E ). Thrombin, an agonist that relays signals through a GPCR, displayed an unexpected normal activation in platelets lacking PI3K (Fig. 1F ). These results indicated that PI3K is specifically required for ADP-induced maximal aggregation of platelets.

View larger version (27K): [in this window] [in a new window]   Figure 1. PI3K deletion affects platelet aggregation. White and black bars correspond to mean ± SE of wild-type and PI3K-null samples, respectively. A) Representative traces of ADP-induced aggregation in wild-type (+/+) and PI3K-null (-/-) whole blood. B) Quantification of the maximal aggregation response after ADP (0.5 µM) and PMA (100 nM) stimulation of whole blood. C) Aggregation of platelet-rich plasma after increasing doses of ADP stimulation. D) Quantification of the maximal aggregation response after ADP and PMA stimulation of platelet-rich plasma (PRP) or washed platelets (WP). E) Representative traces of collagen and F) thrombin-induced aggregation in PRP and WP, respectively. Arrowheads indicate the addition of the indicated agonists. *P < 0.05, 2-tailed Student’s t test.

2. PKB phosphorylation and fibrinogen binding is decreased in ADP-stimulated PI3K-null platelets
To understand the biochemical mechanism leading to impaired aggregation in ADP-stimulated PI3K-null platelets, we investigated a defect in activation of possible downstream targets of PI3K. Thus, activation of the serine/threonine kinase PKB/Akt, which becomes phosphorylated in a PI3K-dependent manner, was tested. ADP stimulation induced PKB phosphorylation in wild-type platelets but not in PI3K-null thrombocytes. Analysis of PKB activation in wild-type platelets in the presence of specific inhibitors of each of the two ADP receptors revealed that PI3K is uniquely activated by the G_i-coupled P2Y₁₂ receptor. Consistent with the aggregation results, the absence of PI3K did not affect thrombin-induced PKB phosphorylation.

To further clarify the mechanism underlying PI3K function in ADP-stimulated platelets, we investigated the ADP-dependent activation of the fibrinogen receptor, the _IIbß₃ integrin, in PI3K-null thrombocytes. Binding of fluorescently labeled fibrinogen was analyzed by FACS after stimulation of platelets of both genotypes with ADP or collagen. Collagen, which did not evoke abnormal responses in aggregation studies, induced similar levels of fibrinogen binding. In contrast, ADP stimulation of PI3K-null platelets caused 30% less fibrinogen binding than in wild-type controls. Western blot analysis revealed that this difference is not due to altered fibrinogen receptor expression. These data thus demonstrate that PI3K is needed for ADP-induced PKB activation and for a complete switch of the fibrinogen receptor into the high-affinity state.

3. Reduced thrombosis in PI3K-null mice with normal bleeding time
The in vivo effect on the hemostatic process of the aggregation defect of ADP-stimulated PI3K-null platelets was first assessed by bleeding time assays. In agreement with the fact that PI3K-deficient mice have never suffered from spontaneous hemorrhage, bleeding time measured after cutting the tail tip showed no significant differences. The time required for the hemorrhage to cease was equal in wild-type and mutant mice (319±37 vs. 300±37 s; mean±SE, n=37 each, P=0.8, 2-tailed Student’s t test).

To evaluate the in vivo consequences of a lack of PI3K in thrombotic processes, a model was examined where ADP-induced thrombosis causes a series of platelet activating events culminating in lung thromboembolism and death. These studies reveal that after intravenous (i.v.) ADP administration, PI3K-null mice accumulate less platelets in the lungs (Fig. 2A ). Furthermore, the lack of PI3K was sufficient to protect from ADP-induced, platelet-dependent thromboembolic vascular occlusion and subsequent death (Fig. 2B ).

View larger version (14K): [in this window] [in a new window]   Figure 2. The lack of PI3K reduces acute platelet-dependent thromboembolism. A) Mortality after thrombotic challenge of wild-type (black bars) and PI3K-deficient (white bars) platelets. Results are expressed as the percentage of the total number of animals tested. *P = 0.02, Fischer’s exact test. B) Accumulation of 111In-labeled platelets in lungs of wild-type (white bars) and PI3K-null (black bars) mice after i.v. administration of either saline or 300 mg/kg ADP (n=4). *P = 0.0001, vs. saline and wild-type + ADP group as determined by ANOVA with Schiff’s test for pairwise comparisons.

CONCLUSIONS

Platelet activation and aggregation are crucial steps for the control of hemostasis and the development of thrombotic diseases. An understanding of signal transduction pathways that lead to thrombocyte activation can provide fundamental knowledge to treat pathologies like stroke and myocardial infarction. We show that murine platelets express at least three different members of the PI3K family of signaling enzymes. We demonstrate that thrombocytes express high amounts of PI3K, a PI3K isoform that can be activated by GPCRs. To gain insight into the role of this specific isoform, we analyzed platelet function in mice that lack PI3K. PI3K-null platelets showed impaired response to selective GPCR agonist like ADP. In vivo, PI3K-null mice were protected from thromboembolism but displayed normal bleeding time.

That PI3K is specifically activated by GPCR agonists has been supported by experimental evidence. Unexpectedly, PI3K-null platelets show decreased aggregation after stimulation by ADP but not by another GPCR agonist like thrombin (Fig. 3 ). Thrombin binds a G_q-associated receptor, and we found that PI3K is activated by the G_i-but not by the G_q-coupled ADP receptor. Thrombin has complex multiple signaling functions and it is conceivable that, for example, its protease activity relays other intracellular signals that might overcome the lack of PI3K. Consistent with this view, thrombin has been reported to trigger the activation of Src-like tyrosine kinases that subsequently induce PtdIns (3,4,5)P₃ production via phosphotyrosine dependent p85-associated PI3K or ß (Fig. 3) .

View larger version (27K): [in this window] [in a new window]   Figure 3. The role of PI3K in ADP-mediated aggregation. ADP is released by activated platelets and damaged tissues and enforces platelet aggregation. Here we show that PtdIns (3,4,5)P3 produced by a functional PI3K is necessary to fully activate the fibrinogen receptor (IIbß3) and induce full aggregation. PI3K is activated by the Gi-coupled P2Y12 ADP receptor and forms a complex with Gß subunits and its accessory protein p101. A role for protein kinase B (PKB) was proposed based on the previously described in vitro phosphorylation of the fibrinogen. When platelets are activated with thrombin, Src-like protein tyrosine kinases contribute to class IA PI3K activation (p110 and p110ß isoforms), thus explaining why this response is not affected in PI3K-null platelets. For references, see the full text online.

The specific molecular mechanism by which PI3K exerts its function in ADP-mediated platelet aggregation is obscure. We show, however, that PI3K plays a significant role in the ADP-mediated activation of the serine/threonine kinase Akt/PKB. We also demonstrate that PI3K is necessary for full ADP-induced activation of the _IIbß₃ integrin fibrinogen receptor, a key player in the aggregation process. Recent evidence suggests that a possible downstream target of PKB is the cytoplasmic tail of the ß₃ subunit of the fibrinogen receptor. PKB-dependent phosphorylation occurs at a site known to be important for modulation of receptor activity. Hence, we can speculate that in platelets lacking PI3K, the effect on _IIbß₃ might be related to the impaired activation of PKB (Fig. 3) . Nevertheless, we cannot rule out the involvement of other, not yet identified, mechanisms by which PI3K relays the aggregation signals.

The lack of PI3K has limited consequences on in vivo hemostasis. The finding that bleeding time after cutting the tail tip is normal in PI3K-null mice agrees with the observation that these animals show no spontaneous bleeding tendency. In the tail bleeding assay, time for hemorrhage to cease is thought to depend on the coagulation cascade and thrombin generation. The fact that the thrombin response in PI3K-null platelets is normal therefore might explain the unaltered bleeding time.

Thromboembolism is a major cause of a variety of pathological processes such as atherosclerosis, occlusion of vascular grafts, or acute restenosis after angioplasty. Inhibitors of the P2Y₁₂ ADP receptor, like the thienopyridines ticlopidine and clopidogrel, can be used as antithrombotic therapy. Treatment of patients with these drugs (particularly ticlopidine) can, however, induce serious adverse effects. The finding that PI3K-null mice appear healthy in standard conditions but are protected against acute thromboembolism indicates PI3K as a target for antithrombotic drugs that will eventually lack major side effects.

Although platelets express several PI3K isoforms, our results define a relevant role of PI3K for the full response to only selective GPCR agonists such as ADP. The lack of PI3K does not interfere with thrombin-induced platelet aggregation or bleeding time. We previously suggested that PI3K could be an interesting target for new anti-inflammatory drugs in humans. The fact that PI3K-null mice do not show alteration of hemostasis suggests that future use of specific PI3K inhibitors as therapeutic agents will not increase the risk of hemorrhage. In addition, our findings indicate that treatment with such compounds might help to prevent thrombosis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0810fje ; to cite this article, use FASEB J. (July 9, 2001) 10.1096/fj.00-0810fje

³ These authors contributed equally to this work.

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