The kinetics of αIIbβ3 activation determines the size and stability of thrombi in mice: implications for antiplatelet therapy

Abstract

Two major pathways contribute to Rasproximate-1-mediated integrin activation in stimulated platelets. Calcium and diacyglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI, Ras-GRP2) mediates the rapid but reversible activation of integrin ␣IIb␤3, while the adenosine diphosphate receptor P2Y12, the target for antiplatelet drugs like clopidogrel, facilitates delayed but sustained integrin activation. To establish CalDAG-GEFI as a target for antiplatelet therapy, we compared how each pathway contributes to thrombosis and hemostasis in mice. Ex vivo, thrombus formation at arterial or venous shear rates was markedly reduced in CalDAG-GEFI ؊/؊ blood, even in the presence of exogenous adenosine diphosphate and thromboxane A 2 . In vivo, thrombosis was virtually abolished in arterioles and arteries of CalDAG-GEFI ؊/؊ mice, while small, hemostatically active thrombi formed in venules. Specific deletion of the C1-like domain of CalDAG-GEFI in circulating platelets also led to protection from thrombus formation at arterial flow conditions, while it only marginally increased blood loss in mice. In comparison, thrombi in the micro-and macrovasculature of clopidogrel-treated wild-type mice grew rapidly and frequently embolized but were hemostatically inactive. Together, these data suggest that inhibition of the catalytic or the C1 regulatory domain in CalDAG-GEFI will provide strong protection from atherothrombotic complications while maintaining a better safety profile than P2Y12 inhibitors like clopidogrel. (Blood. 2011; 117(3):1005-1013) IntroductionArterial thrombosis in the coronary or cerebrovascular circulation is the principal pathological process underlying acute coronary syndrome and ischemic stroke, which together represent the leading cause of morbidity and mortality in industrialized countries. 1 Platelet activation is a central event in the pathogenesis of arterial thrombosis. Currently, the most powerful antiplatelet agents used in the clinic are inhibitors of cyclooxygenase-1 (acetylsalicylic acid, aspirin), the platelet adenosine diphosphate (ADP) receptor P2Y12 (eg, clopiodgrel or Plavix), and integrin ␣IIb␤3 (eg, abciximab or Reopro). 2,3 These agents have all been shown to improve clinical outcomes in large-scale randomized controlled trials. However, all therapies have limitations that include uncertainty about optimal dosing, questions about resistance, and issues regarding the lack of reversibility in situations where bleeding risks are high.␣IIb␤3, the platelet fibrinogen receptor, is the best-studied member of the integrin family. 4,5 Like most integrins, especially those regulating adhesion and trafficking of blood cells, it is expressed in a low-affinity state on resting platelets. Engagement of agonist receptors on the platelet surface triggers intracellular signaling events, which lead to inside-out activation of ␣IIb␤3. Deficiency in ␣IIb␤3 completely inhibits the ability of platelets to aggregate and adhere to sites of injury. 6,7 Consequently, inhibitors to integrin ␣IIb␤3 show...

RASA3 is a critical inhibitor of RAP1-dependent platelet activation

“…No significant difference in the expression levels of major adhesion receptors ( Figure 4A) or RAP1 protein ( Figure 4B) Figure 2A). Consistent with the critical role of CalDAG-GEFI in platelet signaling (16)(17)(18)24), both Caldaggef1 -/-and Caldaggef1 -/-Rasa3 hlb/hlb platelets exhibited impaired αIIbβ3 activation in response to cellular stimulation ( Figure 4C). Agonist-induced activation of αIIbβ3, however, was significantly increased in Caldaggef1 -/-Rasa3 hlb/hlb platelets when compared with that in Caldaggef1 -/-controls.…”

“…Furthermore, αIIbβ3 activation in Caldaggef1 -/-platelets was modulated by the presence of an exogenous P2Y12 agonist (ADP) or P2Y12 inhibitor (2-methylthio-AMP triethylammonium salt hydrate [2-MeSAMP]) ( Figure 5D), while no such modulation was observed in Caldaggef1 -/-Rasa3 hlb/hlb cells ( Figure 5D). Consistent with the documented role for Ca 2+ /CalDAG-GEFI in the rapid activation of RAP1 (17,24),…”

“…Second, we and others identified two independent yet synergistic pathways critical to RAP1 activation in mouse and human platelets (11,21,23,27). Fast but reversible RAP1 activation is dependent on calcium and CalDAG-GEFI, while sustained RAP1 activation requires signaling by P2Y12 and PI3K (11,13,17,24). Significantly, the importance of a CalDAG-GEFI-dependent and -independent pathway to RAP1 activation was confirmed recently in patients with impaired CalDAG-GEFI function (21).…”

“…Upon cellular stimulation, CalDAG-GEFI is important for the rapid, calcium-dependent (Ca 2+ -dependent) activation of RAP1 and integrin αIIbβ3 (22)(23)(24)(25)(26). RAP1 activation in the absence of Ca 2+ /CalDAG-GEFI is comparatively slow but sustained (17) and requires signaling via PKC (23,27), P2Y12 (11,13,17), and PI3K (11,28).…”

“…They are regulated tightly by GEFs, which stimulate GTP loading, and GAPs, which catalyze GTP hydrolysis. Our recent work and that of others demonstrated that RAP1 is a central signaling node, regulating platelet adhesion and thrombosis (15)(16)(17), and that CalDAG-GEFI (also known as RASGRP2) is a critical RAP-GEF expressed in platelets (18)(19)(20)(21). Upon cellular stimulation, CalDAG-GEFI is important for the rapid, calcium-dependent (Ca 2+ -dependent) activation of RAP1 and integrin αIIbβ3 (22)(23)(24)(25)(26).…”