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...

Platelet activation via Fc␥ receptor IIA (Fc␥RIIA) is a critical event in immunemediated thrombocytopenia and thrombosis syndromes (ITT). We recently identified signaling by the guanine nucleotide exchange factor CalDAG-GEFI and the adenosine diphosphate receptor P2Y12 as independent pathways leading to Rap1 small GTPase activation and platelet aggregation. Here, we evaluated the contribution of CalDAG-GEFI and P2Y12 signaling to platelet activation in ITT. Mice transgenic for the human Fc␥RIIA (hFcR) and deficient in CalDAG-GEFI ؊/؊ (hFcR/CDGI ؊/؊ ) were generated. Compared with controls, aggregation of hFcR/ CDGI ؊/؊ platelets or P2Y12 inhibitortreated hFcR platelets required more than 5-fold and approximately 2-fold higher concentrations of a Fc␥RIIA stimulating antibody against CD9, respectively. Aggregation and Rap1 activation were abolished in P2Y12 inhibitor-treated hFcR/ CDGI ؊/؊ platelets. For in vivo studies, a novel model for antibody-induced thrombocytopenia and thrombosis was established. Fc␥RIIA-dependent platelet thrombosis was induced by infusion of Alexa750-labeled antibodies to glycoprotein IX (CD42a), and pulmonary thrombi were detected by near-infrared imaging technology. Anti-GPIX antibodies dosedependently caused thrombocytopenia and pulmonary thrombosis in hFcRtransgenic but not wild-type mice. CalDAG-GEFI-deficient but not clopidogreltreated hFcR-transgenic mice were completely protected from ITT. In summary, we established a novel mouse model for ITT, which was used to identify CalDAG-GEFI as a potential new target in the treatment of ITT. (Blood. 2011;118(4): 1113-1120) IntroductionPlatelets are essential components of the hemostatic response to vascular injury. However, platelets also play a role in pathologic conditions, such as atherothrombosis, and in immune-mediated thrombocytopenia and thrombosis (ITT). Several ITT syndromes, including heparin-induced thrombocytopenia and thrombosis (HIT), 1-3 bacterial sepsis-associated thrombocytopenia and disseminated intravascular coagulation, 4,5 and the thrombotic manifestations of antiphospholipid syndromes 6 are characterized by immunemediated platelet activation through the platelet Fc␥ receptor, Fc␥RIIA. In addition, thrombotic complications have been observed with the expanded use of therapeutic IgG antibodies, such as bevacizumab. [7][8][9] One of the barriers to successful treatment of these thrombotic syndromes is that therapeutic targeting of platelet activation pathways to prevent thrombosis is either not effective or comes with an inherent risk of bleeding complications.In humans, Fc␥RIIA is expressed on platelets, neutrophils, monocytes, and macrophages and activates these cells following the binding of the Fc region of IgG-coated cells or IgG-containing immune complexes. 10 Mice lack the genetic equivalent of human Fc␥RIIA and, indeed, do not express a platelet Fc receptor. Consequently, most of the studies on the role of Fc␥RIIA in platelet activation were entirely dependent on the use of inhibitors, and they did not provide in...

Key Points TULA-2 negatively regulates platelet FcγRIIA signaling by dephosphorylating Syk. miR-148a targets TULA-2 and inhibition of miR-148a decreases FcγRIIA-mediated platelet activation and thrombosis in vivo.

Early onset torsion dystonia is an autosomal dominant movement disorder of variable caused by a glutamic acid, i.e. ΔE, deletion in DYT1, encoding the protein torsinA. Genetic and structural data implicate basal ganglia dysfunction in dystonia. TorsinA, however, is diffusely expressed, and therefore the primary source of dysfunction may be obscured in pan-neuronal transgenic mouse models. We utilized the tyrosine hydroxylase (TH) promoter to direct transgene expression specifically to dopaminergic neurons of the midbrain to identify cell-autonomous abnormalities. Expression of both the human wild type (hTorsinA) and mutant (ΔE-hTorsinA) protein resulted in alterations of dopamine release as detected by microdialysis and fast cycle voltammetry. Motor abnormalities detected in these mice mimicked those noted in transgenic mice with pan-neuronal transgene expression. The locomotor response to cocaine in both TH-hTorsinA and TH-ΔE-hTorsinA, in the face of abnormal extracellular DA levels relative to non-transgenic mice, suggests compensatory, post-synaptic alterations in striatal DA transmission. This is the first cell-subtype-specific DYT1 transgenic mouse that can serve to differentiate between primary and secondary changes in dystonia, thereby helping to target disease therapies.

To explore the potential for monoclonal antibodies as a treatment for immune thrombocytopenia (ITP) and to further explore their mechanisms of action, we tested 8 monoclonal CD44 antibodies in murine ITP and found 4 antibodies that could successfully ameliorate ITP; 2 of these antibodies function at a full 3-log fold lower dosage compared with IVIg. Further characterization of the 2 most successful antibodies (5035-41.1D and KM114) demonstrated that, similar to IVIg: (1) the presence of the inhibitory IgG receptor FcγRIIB was required for their ameliorative function, (2) complement-deficient mice responded to anti-CD44 treatment, and (3) human transgenic FcγRIIA-expressing mice also responded to the CD44 therapeutic modality. Dissimilar to IVIg, the Fc portion of the CD44 antibody was not required. These data demonstrate that CD44 antibodies can function therapeutically in murine ITP and that they could potentially provide a very-low-dose recombinant therapy for the amelioration of human ITP.