PRT-060318, a novel Syk inhibitor, prevents heparin-induced thrombocytopenia and thrombosis in a transgenic mouse model

Abstract

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...

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

“…However previous work has shown that the tyrosine kinase inhibitor, Genistein which has been shown to inhibit agonist-evoked Syk activation in human platelets [34], has no effect on thrombin-or ADP-evoked Ca 2+ release [35,36]. In addition, a recent report using a specific Syk inhibitor, PRT-060318, found no effect on TRAP-induced Ca 2+ signals at concentrations found to inhibit over 90% of the convulxin-induced Ca 2+ response [37]. These results therefore suggest that any effect of Gö6976 observed on platelet Ca 2+ signalling is limited to its effect on conventional PKC isoforms.…”

Anti–miR-148a regulates platelet FcγRIIA signaling and decreases thrombosis in vivo in mice

“…The observation that a Syk inhibitor is able to prevent HIT in our FcgRIIA/PF4 transgenic mouse model demonstrated the central role of Syk in the FcgRIIA pathway and HIT. 6 The signaling is further transmitted by phosphorylation of phospholipase Cg2 (PLCg2), phosphatidylinositide 3-kinases (PI3Ks), and the linker for activation of T cells (LAT), followed by calcium mobilization and protein kinase C activation. These signals ultimately lead to platelet activation and thrombus formation.…”

“…7 Recently, FcgRIIA was also identified as a key regulator in platelet integrin outside-in signaling. 6,8,9 There is considerable interindividual variation in platelet activation via FcgRIIA among healthy donors and patients. The genetic mechanisms behind this phenotypic variation are incompletely understood.…”

Małopłytkowość indukowana protaminą — nowy problem czy inny typ małopłytkowości poheparynowej?

“…Mimo to grupa doustnych antykoagulantów stanowi niewystarczającą ochronę przeciwzakrzepową i nadal brakuje twardych dowodów na ich skuteczność w terapii HIT [30,[35][36][37]. Najnowsze metody leczenia HIT znajdują się w fazie badań przedklinicznych i należą do nich między innymi: antagoniści PF4, niepatogenne, monoklonalne przeciwciała HIT abs zakłócające powstanie kompleksów PF4-heparyna oraz blokery FcγRIIa uniemożliwiające wiązanie kompleksów HIT z receptorami i aktywację płytek krwi [38,39].…”