G12/13 Signaling Pathways Substitute for Integrin αIIbβ3-Signaling for Thromboxane Generation in Platelets

We have previously shown that ADP-induced TXA(2) generation requires signaling from αIIbβ3 integrin in platelets. Here we observed that, unlike ADP, protease-activated receptor (PAR)-mediated TXA(2) generation occurs independently of αIIbβ3. PAR agonists, but not ADP, activate G(12/13) signaling pathways. Hence, we evaluated the role of these pathways in TXA(2) generation.Inhibition of ADP-induced thromboxane generation by fibrinogen receptor antagonist SC57101 was rescued by co-stimulation of G(12/13) pathways with YFLLRNP. This observation suggested an existence of a common signaling effector downstream of integrins and G(12/13) pathways. Hence, we evaluated role of three potential tyrosine kinases; c-Src, Syk and FAK (Focal Adhesion Kinase) that are known to be activated by integrins. c-Src and Syk kinase did not play a role in ADP-induced functional responses in platelets. Selective activation of G(12/13) pathways resulted in the activation of FAK, in the absence of integrin signaling. Interestingly, αIIbβ3-mediated FAK activation occurred in a Src family kinase (SFK)-independent manner whereas G(12/13) pathway caused FAK activation in a SFK and RhoA-dependent manner. A FAK selective inhibitor TAE-226, blocked TXA(2) generation. However, in comparison to WT mice, Pf4-Cre/Fak-Floxed mice did not show any difference in platelet TXA(2) generation.Therefore, we conclude that differential activation of FAK occurs downstream of Integrins and G(12/13) pathways. However, the common effector molecule, possibly a tyrosine kinase downstream of integrins and G(12/13) pathways contributing to TXA(2) generation in platelets remains elusive

Central role of the P2Y(12) receptor in platelet activation

Platelet activation occurs in response to vessel injury and is important for the arrest of bleeding. Platelet activation during disease states leads to vascular occlusion and ischemic damage. The P2Y(12) receptor, activated by ADP, plays a central role in platelet activation and is the target of P2Y(12) receptor antagonists that have proven therapeutic value

Src family kinase–mediated and Erk-mediated thromboxane A2 generation are essential for VWF/GPIb-induced fibrinogen receptor activation in human platelets

The binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein Ib-IX (GPIb-IX) results in platelet activation. In this study, we sought to clarify previous conflicting reports and to elucidate the mechanism of activation and the precise role of extracellular signal-regulated kinase (Erk) in VWF-induced platelet activation. Erk2 is activated in platelets on stimulation with VWF/ristocetin in a time-dependent manner. VWF-induced Erk2 phosphorylation and thromboxane A2 (TXA2) release were completely blocked by PP2, an Src family kinase inhibitor, suggesting that Erk is downstream of Src family kinases. U73122, a phospholipase C inhibitor, also abolished TXA2 generation and Erk phosphorylation. Although VWF fostered the agglutination of platelets regardless of any additional treatment, the inhibition of mitogen-activated protein kinase kinase (MEK) with U0126 abolished VWF-induced platelet aggregation and thromboxane production in non–aspirin-treated washed platelets. However, in platelets treated with aspirin, VWF failed to cause any aggregation. Thus, we conclude that VWF stimulation of platelets results in phospholipase A2 activation through Erk stimulation and that Src family kinases and phospholipase C play essential roles in this event. We further conclude that VWF-induced platelet aggregation does not directly depend on Erk activation but has an absolute requirement for Src/Erk-mediated TXA2 generation

Protein kinase C- and calcium-regulated pathways independently synergize with Gi pathways in agonist-induced fibrinogen receptor activation.

Platelet fibrinogen receptor activation is a critical step in platelet plug formation. The fibrinogen receptor (integrin alphaIIbbeta3) is activated by agonist-mediated G(q) stimulation and resultant phospholipase C activation. We investigated the role of downstream signalling events from phospholipase C, namely the activation of protein kinase C (PKC) and rise in intracellular calcium, in agonist-induced fibrinogen receptor activation using Ro 31-8220 (a PKC inhibitor) or dimethyl BAPTA [5,5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N', N'-tetra-acetic acid], a high-affinity calcium chelator. All the experiments were performed with human platelets treated with aspirin, to avoid positive feedback from thromboxane A2. In the presence of Ro 31-8220, platelet aggregation caused by U46619 was completely inhibited while no effect or partial inhibition was seen with ADP and the thrombin-receptor-activating peptide SFLLRN, respectively. In the presence of intracellular dimethyl BAPTA, ADP- and U46619-induced aggregation and anti-alphaIIbbeta3 antibody PAC-1 binding were completely abolished. However, similar to the effects of Ro 31-8220, dimethyl BAPTA only partially inhibited SFLLRN-induced aggregation, and was accompanied by diminished dense-granule secretion. When either PKC activation or intracellular calcium release was abrogated, aggregation and fibrinogen receptor activation with U46619 or SFLLRN was partially restored by additional selective activation of the G(i) signalling pathway. In contrast, when both PKC activity and intracellular calcium increase were simultaneously inhibited, the complete inhibition of aggregation that occurred in response to either U46619 or SFLLRN could not be restored with concomitant G(i) signalling. We conclude that, while the PKC- and calcium-regulated signalling pathways are capable of inducing activating fibrinogen receptor independently and that each can synergize with G(i) signalling to cause irreversible fibrinogen receptor activation, both pathways act synergistically to effect irreversible fibrinogen receptor activation

Comparative analysis of various platelet glycoprotein IIb/IIIa antagonists on shear-induced platelet activation and adhesion. J Pharmacol Exp Ther 303:1114–1120

ABSTRACT Platelet accretion into arterial thrombus in stenotic arterial vessels involves shear-induced platelet activation and adhesion. The Cone and Plate(let) Analyzer (CPA) is designed to simulate such conditions in vitro under a rotating high shear rate in whole blood. In the present study, we evaluated various experimental conditions (including aspirin, temperature, and calcium concentration) and investigated the effects of small molecules along with peptide glycoprotein IIb/IIIa antagonists on platelet adhesion using the CPA system. Concentration-dependent effect of glycoprotein IIb/IIIa antagonists on shear-induced platelet adhesion showed marked differences in potencies: IC 50 ϭ 34, 35, 91, 438, and 606 nM for DPC802 (a specific glycoprotein IIb/IIIa antagonist), roxifiban, sibrafiban, lotrafiban, and orbofiban (free acid forms), respectively, and IC 50 values of 43, 430, and 5781 nM for abciximab, tirofiban, and eptifibatide, respectively. Parallel study was also conducted to evaluate the effect of glycoprotein IIb/IIIa inhibitors using optical aggregometry. The potency of fibans in blocking shear-induced platelet adhesion correlated well with their binding affinity to the resting and activated glycoprotein IIb/IIIa receptors, as well as their "off-rates". Nevertheless, none of these fibans was able to effectively block shear-induced platelet adhesion at targeted clinical dosing regimens except for abciximab. These data suggest that glycoprotein IIb/IIIa antagonists that show similar efficacy in the inhibition of platelet aggregation in a static in vitro assay may differ substantially in a shear-based system of platelet adhesion. The clinical significance of this phenomenon awaits further investigation

Comparative Analysis of Various Platelet Glycoprotein IIb/IIIa Antagonists on Shear-Induced Platelet Activation and Adhesion

Platelet accretion into arterial thrombus in stenotic arterial ves-sels involves shear-induced platelet activation and adhesion. The Cone and Plate(let) Analyzer (CPA) is designed to simulate such conditions in vitro under a rotating high shear rate in whole blood. In the present study, we evaluated various experimental conditions (including aspirin, temperature, and calcium con-centration) and investigated the effects of small molecules along with peptide glycoprotein IIb/IIIa antagonists on platelet adhesion using the CPA system. Concentration-dependent ef-fect of glycoprotein IIb/IIIa antagonists on shear-induced plate-let adhesion showed marked differences in potencies: IC50 34, 35, 91, 438, and 606 nM for DPC802 (a specific glycopro-tein IIb/IIIa antagonist), roxifiban, sibrafiban, lotrafiban, and or-bofiban (free acid forms), respectively, and IC50 values of 43

Role of c-Src and Syk downstream of integrin αIIbβ3 in thromboxane generation in platelets.

<p>Aspirin–treated, washed platelets were stimulated with 2MeSADP (100 nM) at various time points (A) or with varying concentrations of 2MeSADP (B) for one minute under stirring conditions at 37°C. The lysates were then subjected to western blotting analysis and probed with anti-phospho-(Y416) and total c-Src antibodies as lane loading control. Washed murine (WT or c-Src KO) platelets, without aspirin-treatment, were stimulated with 2MeSADP (100 nM) for 3.5 minutes and TXB₂ levels were analyzed (C) as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016586#pone-0016586-g001" target="_blank">Figure 1</a>. The data are represented as the % Fold increase over the control. Aspirin-treated washed platelets were stimulated with 2MeSADP (100 nM) for (30–120 seconds) or convulxin (100 ng/ml) for 30 seconds under stirring conditions at 37°C (D). The lysates were then subjected to western blotting analysis and probed with anti- phospho- Syk(Y 525/526) and total Syk antibodies as lane loading control. The data are representative of at least 3 separate experiments.</p

Model depicting the regulation of TXA₂ generation by G_12/13 pathways and integrins through FAK.

<p>Integrin clustering leads to FAK activation (1). Fibrinogen receptor antagonist SC57101 prevents integrin clustering hence inhibits FAK activation (2). FAK can be activated in an integrin-independent manner by G_12/13 pathways (3). FAK is activated downstream of Rho kinase and SFKs upon stimulation of G_12/13 pathways (4). FAK can be inhibited by TAE-226 (5). Common effector molecule downstream of integrins and G_12/13 pathways contributing to thromboxane generation are unknown (6).</p

Evaluation of FAK as a common signaling effector molecule regulating thromboxane generation downstream of integrins and G_12/13 pathways.

<p>Non-aspirin-treated, washed human platelets were pre-treated with varying concentrations of TAE-226 for 5 minutes at 37°C (A) and murine platelets from WT and Pf4-Cre/Fak-Floxed mice (B) were stimulated with 2MeSADP (100 nM) for 3.5 minutes and TXB₂ levels were analyzed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016586#pone-0016586-g001" target="_blank">Figure 1</a>. Aggregation tracings were measured from WT and Pf4-Cre/Fak-Floxed mice and representative tracings are shown (C). The values are representative of 3 independent experiments mean ± S.E.M (n = 3). The data were analyzed by ANOVA and student t-test, * P≤0.05 was considered significant.</p