Scott M. Taylor

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

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 heparin-platelet factor 4 complexes lead to FcγRIIA-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.

Thrombopoietin following transfusion of platelets in preterm neonates

Thrombocytopenia is common in the neonatal intensive care unit. Transfusion of platelets is often required. The purpose of our study was to determine changes in thrombopoietin (Tpo) following transfusion of platelets in preterm neonates. Preterm neonates undergoing platelet transfusion were randomized to receive a transfusion volume of either 10 or 15 ml/kg. Blood was obtained for Tpo measurement pre-transfusion, one and 24 hours post-transfusion. Platelet Factor 4 (PF4) was also measured to quantify platelet activation. Statistical analysis was performed using repeated measures ANOVA, and Mann-Whitney U test as appropriate. Ten infants were enrolled in each group. Gestational age, birth weight, etiology of thrombocytopenia, and timing of transfusion did not differ between the 10 and 15 ml/kg groups. There were no differences between the groups in platelet count prior to and/or following transfusion. Both transfusion volumes were equally well tolerated. Tpo and PF4 did not differ between groups at any of the study time points. When both groups were analysed together, Tpo dropped 43% (95% confidence 37–49%, p = 0.01) 1-hour post compared to pre-transfusion. In conclusion the observed decrease in Tpo following platelet transfusion suggests that Tpo kinetics in neonates is similar to adults following transfusion. PF4 was not affected by transfusion. There was not an increase in platelet count following transfusion volume of 15 ml/kg compared to 10 ml/kg.

Immune-mediated Thrombocytopenia is More Severe in Human Fc|[gamma]|RIIa Transgenic Mice than in Wild-type Mice |[bull]| 782

Human FcγRIIa is the platelet receptor for IgG. FcγRIIa is also expressed in man along with the FcγRI/γ and FcγRIIa/γ complexes on spleen macrophages. Mice lack an equivalent of FcγRIIa although it is likely to be a key receptor in immune-mediated thrombocytopenias. We established transgenic mouse lines that express human platelet FcγRIIa at levels equivalent to human platelets in order to determine if immune-mediated thrombocytopenia would be more severe in these transgenic mice which more fully recapitulate the human Fc receptor endowment. We showed that the human receptor is functional on mouse platelets using platelet aggregation stimulated by antibody in vitro. To determine whether this receptor was functional in vivo, we injected mice from this transgenic line with 70 μg of 4A5 antibody, a previously characterized rat anti-mouse platelet antibody found to cause moderate thrombocytopenia in wild type mice. We compared the transgenic mice to wild type mice. We followed the time course of platelet counts in mice injected with saline or isotype control (n=6 each) versus wild type mice (n=6) and FcγRIIa transgenic mice (n=9) receiving a single intraperitoneal dose of antibody on day 0. Intravenous injection gave comparable nadir platelet counts, with more rapid kinetics as expected. The average platelet count in wild type mice decreased to a nadir of 0.60 × 106 / μL on day 6 from a pre-treatment count of 1.13 × 106/ μL following injection of the antibody, in keeping with literature results on this antibody. In FcγRIIa transgenic mice there was a greater response to 4A5. The average count reached a nadir on day 6 after injection of 0.12 × 106 / μL (n = 9; statistically significantly different from wild-type, p < 0.05), while the pre-treatment and recovery counts of the transgenic mice were similar to the controls, with platelet counts of 0.96 × 106 / μL. In FcR γ-chain knockout mice which express no FcγRI or RIII, no 4A5-induced thrombocytopenia was seen (n = 6). Human FcγRIIa transgenic mice have more severe antibody-mediated thrombocytopenia in vivo, suggesting that the human FcγRIIa receptor is functional in vivo in these transgenic mice and contributes to the increased clearance of platelets. Our human FcγRIIa transgenic mice will be a valuable model of the pathophysiology and therapy of human immune-mediated thrombocytopenia disorders, including neonatal alloimmune thrombocytopenia.