Platelets play a critical role in maintaining vascular integrity during inflammation, but little is known about the underlying molecular mechanisms. Here we report that platelet immunoreceptor tyrosine activation motif (ITAM) signaling, but not GPCR signaling, is critical for the prevention of inflammation-induced hemorrhage. To generate mice with partial or complete defects in these signaling pathways, we developed a protocol for adoptive transfer of genetically and/or chemically inhibited platelets into thrombocytopenic (TP) mice. Unexpectedly, platelets with impaired GPCR signaling, a crucial component of platelet plug formation and hemostasis, were indistinguishable from WT platelets in their ability to prevent hemorrhage at sites of inflammation. In contrast, inhibition of GPVI or genetic deletion of Clec2, the only ITAM receptors expressed on mouse platelets, significantly reduced the ability of platelets to prevent inflammation-induced hemorrhage. Moreover, transfusion of platelets without ITAM receptor function or platelets lacking the adapter protein SLP-76 into TP mice had no significant effect on vascular integrity during inflammation. These results indicate that the control of vascular integrity is a major function of immune-type receptors in platelets, highlighting a potential clinical complication of novel antithrombotic agents directed toward the ITAM signaling pathway.

Background-The membrane-bound chemokine fractalkine (CX 3 CL1) is expressed on various cell types such as activated endothelial cells and has been implicated in the inflammatory process of atherosclerosis. The aim of the present study was to dissect the role of fractalkine in leukocyte recruitment to inflamed endothelium under arterial shear forces. Methods and Results-With the use of immunofluorescence and laminar flow assays, the present study shows that human umbilical vein endothelial cells stimulated with tumor necrosis factor-␣ and interferon-␥ abundantly express CX 3 CL1 and promote substantial leukocyte accumulation under arterial flow conditions. In the presence of high shear, firm adhesion of leukocytes to inflamed endothelial cells is reduced by Ϸ40% by a function-blocking anti-fractalkine antibody or by an antibody directed against the fractalkine receptor (CX 3 CR1). With the use of intravital video-fluorescence microscopy we demonstrate that inhibition of fractalkine signaling attenuates leukocyte adhesion to the atherosclerotic carotid artery of apolipoprotein E-deficient mice, which suggests that the CX 3 CL1-CX 3 CR1 axis is critically involved in leukocyte adhesion to inflamed endothelial cells under high shear forces both in vitro and in vivo. Surprisingly, platelets were strictly required for fractalkine-induced leukocyte adhesion at high shear rates. Correspondingly, specific inhibition of platelet adhesion to inflamed endothelial cells also significantly reduced leukocyte accumulation. We show that both soluble and membrane-bound fractalkine induces platelet degranulation and subsequent surface expression of P-selectin, which thereby promotes direct platelet-leukocyte interaction.

Heparin-induced thrombocytopenia (HIT)is a major cause of morbidity and mortality resulting from the associated thrombosis. Extensive studies using our transgenic mouse model of HIT have shown that antibodies reactive with heparinplatelet factor 4 complexes lead to Fc␥RIIA-mediated platelet activation in vitro as well as thrombocytopenia and thrombosis in vivo. We tested PRT-060318 (PRT318), a novel selective inhibitor of the tyrosine kinase Syk, as an approach to HIT treatment. PRT318 completely inhibited HIT immune complex-induced aggregation of both human and transgenic HIT mouse platelets. Transgenic HIT model mice were treated with KKO, a mouse monoclonal HIT-like antibody, and heparin. The experimental group received orally dosed PRT318, whereas the control group received vehicle. Nadir platelet counts of PRT318-treated mice were significantly higher than those of control mice. When examined with a novel thrombosis visualization technique, mice treated with PRT318 had significantly reduced thrombosis. The Syk inhibitor PRT318 thus prevented both HIT immune complex-induced thrombocytopenia and thrombosis in vivo, demonstrating its activity in HIT. (Blood. 2011;117(7): 2241-2246) IntroductionHeparin-induced thrombocytopenia (HIT), characterized by antibodies to macromolecular complexes formed by heparin and platelet factor 4 (PF4), is the most frequent drug-induced immune thrombocytopenia. Patients with HIT are at an increased risk for thrombosis, a major cause of morbidity and mortality in treated patients. Despite this potential side effect, heparins (unfractionated or low molecular weight) remain the drug of choice in clinical situations where high-intensity therapy is needed along with the ability to rapidly modulate the anticoagulant level. 1 The incidence of HIT has therefore not decreased, notwithstanding the introduction of new anticoagulants, primarily because no drug has replaced heparin for the immediate therapy of acute deep vein thrombosis, arterial thrombosis, or extracorporeal circuits during surgery. In addition, indications for its use in the aging population continue to increase.Multiple factors influence the incidence and severity of HIT. The pathogenesis of the disease is well understood, 2-5 although additional progress is being made. Extensive studies in vitro 4,6,7 and in vivo using our transgenic mouse model of HIT 8 show that antibodies reactive with heparin-PF4 complexes lead to Fc receptormediated platelet activation. This activation leads to platelet aggregation, a procoagulant surface, and release of prothrombotic microparticles. In addition, monocytes and other leukocytes bearing Fc␥ receptors can become activated by the HIT immune complex (IC), generating tissue factor and resulting in other prothrombotic and proadhesive changes. [9][10][11] Blocking Fc␥RIIA signaling is an attractive target for therapeutic intervention because Fc␥RIIA-mediated platelet activation (and possibly concurrent monocyte activation) is central to the disease.Fc␥RIIA, like other activating receptors, i...

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...