Inhibitors of coagulation factor Xa (fXa) have emerged as a new class of antithrombotics but lack effective antidotes for patients experiencing serious bleeding. We designed and expressed a modified form of fXa as an antidote for fXa inhibitors. This recombinant protein (r-Antidote, PRT064445) is catalytically inactive and lacks the membrane-binding γ-carboxyglutamic acid domain of native fXa but retains the ability of native fXa to bind direct fXa inhibitors as well as low molecular weight heparin-activated antithrombin III (ATIII). r-Antidote dose-dependently reversed the inhibition of fXa by direct fXa inhibitors and corrected the prolongation of ex vivo clotting times by such inhibitors. In rabbits treated with the direct fXa inhibitor rivaroxaban, r-Antidote restored hemostasis in a liver laceration model. The effect of r-Antidote was mediated by reducing plasma anti-fXa activity and the non-protein bound fraction of the fXa inhibitor in plasma. In rats, r-Antidote administration dose-dependently and completely corrected increases in blood loss resulting from ATIII-dependent anticoagulation by enoxaparin or fondaparinux. r-Antidote has the potential to be used as a universal antidote for a broad range of fXa inhibitors.

Integrins not only bind adhesive ligands, they also act as signalling receptors. Both functions allow the integrin alphaIIbbeta3 to mediate platelet aggregation. Platelet agonists activate alphaIIbbeta3 (inside-out signalling) to allow the binding of soluble fibrinogen. Subsequent platelet aggregation leads to outside-in alphaIIbbeta3 signalling, which results in calcium mobilization, tyrosine phosphorylation of numerous proteins including beta3 itself, increased cytoskeletal reorganisation and further activation of alphaIIbbeta3. Thus, outside-in signals enhance aggregation, although the mechanisms and functional consequences of specific signalling events remain unclear. Here we describe a mouse that expresses an alphaIIbbeta3 in which the tyrosines in the integrin cytoplasmic tyrosine motif have been mutated to phenylalanines. These mice are selectively impaired in outside-in alphaIIbbeta3 signalling, with defective aggregation and clot-retraction responses in vitro, and an in vivo bleeding defect which is characterized by a pronounced tendency to rebleed. These data provide evidence for an important role of outside-in signalling in platelet physiology. Furthermore, they identify the integrin cytoplasmic tyrosine motif as a key mediator of beta-integrin signals and a potential target for new therapeutic agents.

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Glycoprotein (GP) V is a major substrate cleaved by the protease thrombin during thrombin-induced platelet activation. Previous analysis of platelets from GP V-null mice suggested a role for GP V as a negative modulator of platelet activation by thrombin. We now report the mechanism by which thrombin activates GP V ؊͞؊ platelets. We show that proteolytically inactive forms of thrombin induce robust stimulatory responses in GP V null mouse platelets, via the platelet GP Ib-IX-V complex. Because proteolytically inactive thrombin can activate wild-type mouse and human platelets after treatment with thrombin to cleave GP V, this mechanism is involved in thrombin-induced platelet aggregation. Platelet activation through GP Ib-IX depends on ADP secretion, and specific inhibitors demonstrate that the recently cloned P2Y 12 ADP receptor (Gi-coupled ADP receptor) is involved in this pathway, and that the P2Y1 receptor (G q-coupled ADP receptor) may play a less significant role. Thrombosis was generated in GP V null mice only in response to catalytically inactive thrombin, whereas thrombosis occurred in both genotypes (wild type and GP V null) in response to active thrombin. These data support a thrombin receptor function for the platelet membrane GP Ib-IX-V complex, and describe a novel thrombin signaling mechanism involving an initiating proteolytic event followed by stimulation of the GP Ib-IX via thrombin acting as a ligand, resulting in platelet activation.G lycoprotein (GP) Ib-IX-V is a major complex on the platelet surface, second only to ␣⌱⌱b␤3. This complex consists of several subunits: GP Ib␣, GP Ib␤, GP IX, and GP V in the ratio of 2:2:2:1. Absence of GP Ib-IX-V results in a severe bleeding disorder known as Bernard Soulier syndrome characterized by giant platelets and impaired von Willebrand factor (vWf) binding (1). GP Ib␣ is a receptor for vWf, and the GP Ib-IX-V complex is critical for platelet adhesion under arterial shear conditions (2). A role for GP Ib-IX-V in platelet activation has been proposed on the basis of observations that the signaling molecule 14-3-3 (3, 4) is associated with the complex, and that phosphorylation of pp72 syk occurs upon vWf binding to GP Ib␣ (5). In fact, Zaffran et al. (6) recently showed that in heterologous Chinese hamster ovary (CHO) cells expressing both ␣⌱⌱b␤3 and GP Ib-IX, inside-out activation of ␣⌱⌱b␤3 could occur upon vWf adhesion.The GP Ib␣ subunit also has a thrombin binding site on the extracellular domain that overlaps the vWf binding domain (7). Additionally, the complex has a platelet-specific thrombin substrate, GP V, that is cleaved very early during thrombin-induced platelet aggregation (8). Platelets from Bernard Soulier syndrome patients show an impaired response to thrombin (9), and antibodies that block thrombin binding to GP Ib␣ also partially inhibit platelet responses to thrombin (9). More recently, thrombin binding to GP Ib␣ has been shown to enhance platelet procoagulant activity (10). However, the physiological significance of this interaction ha...

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...

The critical role for ADP in arterial thrombogenesis was established by the clinical success of P2Y12 antagonists, currently used at doses that block 40–50% of the P2Y12 on platelets. This study was designed to determine the role of P2Y12 in platelet thrombosis and how its complete absence affects the thrombotic process. P2Y12-null mice were generated by a gene-targeting strategy. Using an in vivo mesenteric artery injury model and real-time continuous analysis of the thrombotic process, we observed that the time for appearance of first thrombus was delayed and that only small, unstable thrombi formed in P2Y12–/– mice without reaching occlusive size, in the absence of aspirin. Platelet adhesion to vWF was impaired in P2Y12–/– platelets. While adhesion to fibrinogen and collagen appeared normal, the platelets in thrombi from P2Y12–/– mice on collagen were less dense and less activated than their WT counterparts. P2Y12–/– platelet activation was also reduced in response to ADP or a PAR-4–activating peptide. Thus, P2Y12 is involved in several key steps of thrombosis: platelet adhesion/activation, thrombus growth, and stability. The data suggest that more aggressive strategies of P2Y12 antagonism will be antithrombotic without the requirement of aspirin cotherapy and may provide benefits even to the aspirin-nonresponder population

A role for glycoprotein (GP)V in platelet function has been proposed on the basis of observations that GP V is the major thrombin substrate on intact platelets cleaved during thrombin-induced platelet aggregation, and that GP V promotes GP Ib-IX surface expression in heterologous cells. We tested the hypotheses that GP V is involved in thrombin-induced platelet activation, in GP Ib-IX expression, and in other platelet responses by generating GP V null mice. Contrary to expectations, GP V ؊͞؊ platelets were normal in size and expressed normal amounts of GP Ib-IX that was functional in von Willebrand factor binding, explaining why defects in GP V have not been observed in Bernard-Soulier syndrome, a bleeding disorder caused by a lack of functional GP Ib-IX-V. Moreover, in vitro analysis demonstrated that GP V ؊͞؊ platelets were hyperresponsive to thrombin, resulting in increased fibrinogen binding and an increased aggregation response. Consistent with these findings, GP V ؊͞؊ mice had a shorter bleeding time. These data support a role for GP V as a negative modulator of platelet activation. Furthermore, they suggest a new mechanism by which thrombin enhances platelet responsiveness independent of activation of the classical G-protein-coupled thrombin receptors. P latelet thrombosis and hemostasis are complex reactions that depend on adhesive interactions mediated by specific receptors. A major platelet complex is glycoprotein (GP) Ib-IX-V. The initial adhesion of platelets is primarily mediated by binding of platelet membrane GP Ib-IX-V to von Willebrand factor (vWf) found on damaged vessel walls (1). After adhesion, binding of other agonists such as thrombin, ADP, and collagen induce signaling events that ultimately activate the receptor function of ␣⌱⌱b␤3 for soluble fibrinogen, leading to platelet aggregation (2). Although platelet aggregates are required for normal hemostasis, they can in addition cause arterial thrombosis in atherosclerotic arteries, e.g., acute myocardial infarction and stroke, inducing ischemic complications of cardiovascular disease (3, 4).The importance of the GP Ib-IX-V complex in normal platelet function is underscored by the study of Bernard-Soulier syndrome (BSS), an inherited bleeding disorder characterized by large platelets that are defective in adhesion to damaged vessel walls (1). This genetic disorder is caused by a lack of functional GP Ib-IX-V and has been linked to defects in either GP Ib or GP IX (5). The activities mapped to the GP Ib subunit of the GP Ib-IX-V complex include vWf (6) and thrombin binding (7, 8) on the extracellular domain and actin-binding protein (9-11) and 14-3-3 (12-15) binding on the cytoplasmic domain.Several studies indicate functional activities for the GP V subunit of the GP Ib-IX-V complex. In one example, GP V has been shown to be cleaved by thrombin from the platelet surface during thrombin-mediated platelet stimulation (16), but the role of GP V cleavage in this thrombin-induced platelet response is unresolved (17). In another example, the signa...

Based on genetic studies that establish the role of spleen tyrosine kinase (Syk) in immune function, inhibitors of this kinase are being investigated as therapeutic agents for inflammatory diseases. Because genetic studies eliminate both adapter functions and kinase activity of Syk, it is difficult to delineate the effect of kinase inhibition alone as would be the goal with small-molecule kinase inhibitors. We tested the hypothesis that specific pharmacological inhibition of Syk activity retains the immunomodulatory potential of Syk genetic deficiency. We report here on the discovery of (4-(3-(2H-1,2,3-triazol-2-yl)phenylamino)-2-((1R,2S)-2-aminocyclohexylamino) pyrimidine-5-carboxamide acetate (P505-15), a highly specific and potent inhibitor of purified Syk (IC 50 1-2 nM). In human whole blood, P505-15 potently inhibited B cell antigen receptor-mediated B cell signaling and activation (IC 50 0.27 and 0.28 M, respectively) and Fc receptor 1-mediated basophil degranulation (IC 50 0.15 M). Similar levels of ex vivo inhibition were measured after dosing in mice (Syk signaling IC 50 0.32 M). Syk-independent signaling and activation were unaffected at much higher concentrations, demonstrating the specificity of kinase inhibition in cellular systems. Oral administration of P505-15 produced dose-dependent anti-inflammatory activity in two rodent models of rheumatoid arthritis. Statistically significant efficacy was observed at concentrations that specifically suppressed Syk activity by ϳ67%. Thus specific Syk inhibition can mimic Syk genetic deficiency to modulate immune function, providing a therapeutic strategy in P505-15 for the treatment of human diseases.