Michael P. Reilly

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.

Cholesterol Enrichment of Human Monocyte/Macrophages Induces Surface Exposure of Phosphatidylserine and the Release of Biologically-Active Tissue Factor–Positive Microvesicles

Objective—Biologically significant amounts of two procoagulant molecules, phosphatidylserine (PS) and tissue factor (TF), are transported by monocyte/macrophage-derived microvesicles (MVs). Because cellular cholesterol accumulation is an important feature of atherosclerotic vascular disease, we now examined effects of cholesterol enrichment on MV release from human monocytes and macrophages. Methods and Results—Cholesterol enrichment of human THP-1 monocytes, alone or in combination with lipopolysaccharide (LPS), tripled their total MV generation, as quantified by flow cytometry based on particle size and PS exposure. The subset of these MVs that were also TF-positive was likewise increased by cellular cholesterol enrichment, and these TF-positive MVs exhibited a striking 10-fold increase in procoagulant activity. Moreover, cholesterol enrichment of primary human monocyte-derived macrophages also increased their total as well as TF-positive MV release, and these TF-positive MVs exhibited a similar 10-fold increase in procoagulant activity. To explore the mechanisms of enhanced MV release, we found that cholesterol enrichment of monocytes caused PS exposure on the cell surface by as early as 2 hours and genomic DNA fragmentation in a minority of cells by 20 hours. Addition of a caspase inhibitor at the beginning of these incubations blunted both cholesterol-induced apoptosis and MV release. Conclusions—Cholesterol enrichment of human monocyte/macrophages induces the generation of highly biologically active, PS-positive MVs, at least in part through induction of apoptosis. Cholesterol-induced monocyte/macrophage MVs, both TF-positive and TF-negative, may be novel contributors to atherothrombosis.

CalDAG-GEFI deficiency protects mice in a novel model of FcγRIIA-mediated thrombosis and thrombocytopenia

Platelet activation via Fcγ receptor IIA (FcγRIIA) is a critical event in immune-mediated 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 FcγRIIA (hFcR) and deficient in CalDAG-GEFI(-/-) (hFcR/CDGI(-/-)) were generated. Compared with controls, aggregation of hFcR/CDGI(-/-) platelets or P2Y12 inhibitor-treated hFcR platelets required more than 5-fold and approximately 2-fold higher concentrations of a FcγRIIA 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. FcγRIIA-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 dose-dependently caused thrombocytopenia and pulmonary thrombosis in hFcR-transgenic but not wild-type mice. CalDAG-GEFI-deficient but not clopidogrel-treated 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.

Dominant Expression of the Inhibitory FcγRIIB Prevents Antigen Presentation by Murine Plasmacytoid Dendritic Cells

Plasmacytoid dendritic cells (pDCs) are key regulators of the innate immune response, yet their direct role as APCs in the adaptive immune response is unclear. We found that unlike conventional DCs, immune complex (IC) exposed murine pDCs neither up-regulated costimulatory molecules nor activated Ag-specific CD4+ and CD8+ T cells. The inability of murine pDCs to promote T cell activation was due to inefficient proteolytic processing of internalized ICs. This defect in the IC processing capacity of pDCs results from a lack of activating FcγR expression (FcγRI, III, IV) and the dominant expression of the inhibitory receptor FcγRIIB. Consistent with this idea, transgenic expression of the activating human FcγRIIA gene, not present in the mouse genome, recapitulated the human situation and rescued IC antigenic presentation capacity by murine pDCs. The selective expression of FcγRIIB by murine pDCs was not strain dependent and was maintained even following stimulation with TLR ligands and inflammatory cytokines. The unexpected difference between the mouse and human in the expression of activating/inhibitory FcγRs has implications for the role of pDCs in Ab-modulated autoimmunity and anti-viral immunity.

Contribution of the P2Y12 receptor-mediated pathway to platelet hyperreactivity in hypercholesterolemia.

Summary.  Background: In hypercholesterolemia, platelets demonstrate increased reactivity and promote the development of cardiovascular disease. Objective: This study was carried out to investigate the contribution of the ADP receptor P2Y12‐mediated pathway to platelet hyperreactivity due to hypercholesterolemia. Methods: Low‐density lipoprotein receptor‐deficient mice and C57Bl/6 wild‐type mice were fed on normal chow and high‐fat (Western or Paigen) diets for 8 weeks to generate differently elevated cholesterol levels. P2Y12 receptor‐induced functional responses via Gi signaling were studied ex vivo when washed murine platelets were activated by 2MeSADP and PAR4 agonist AYPGKF in the presence and absence of indomethacin. Platelet aggregation and secretion, αIIbβ3 receptor activation and the phosphorylation of extracellular signal‐regulated protein kinase (ERK) and Akt were analyzed. Results: Plasma cholesterol levels ranged from 69 ± 10 to 1011 ± 185 mg dL−1 depending on diet in mice with different genotypes. Agonist‐dependent aggregation, dense and α‐granule secretion and JON/A binding were gradually and significantly (P < 0.05) augmented at low agonist concentration in correlation with the increasing plasma cholesterol levels, even if elevated thromboxane generation was blocked. These functional responses were induced via increased levels of Gi‐mediated ERK and Akt phosphorylation in hypercholesterolemic mice vs. normocholesterolemic animals. In addition, blocking of the P2Y12 receptor by AR‐C69931MX (Cangrelor) resulted in strongly reduced platelet aggregation in mice with elevated cholesterol levels compared with normocholesterolemic controls. Conclusions: These data revealed that the P2Y12 receptor pathway was substantially involved in platelet hyperreactivity associated with mild and severe hypercholesterolemia.

Insights from mouse models of heparin-induced thrombocytopenia and thrombosis.

Heparin-induced thrombocytopenia/thrombosis (HIT/T) is the most frequent cause of drug-induced antibody-mediated thrombocytopenia, a common cause of life-and limb-threatening platelet activation and thrombosis. Although antibodies to heparin–platelet factor 4 (PF4) complexes are found in essentially all patients with HIT/T, it is unclear how antibody formation is initiated, why only a small subset of these antibodies cause disease, or the mechanism by which they initiate thrombosis. The recent development of a transgenic mouse model of HIT/T showed, for the first time in vivo, that heparin, PF4, antibodies to the heparin–PF4 complex, and Fc&ggr;RIIA are necessary and sufficient to recapitulate the severe thrombocytopenia and thrombosis seen in patients. This model can be expanded to systematically study individual factors important to HIT/T development in vivo, to determine their contribution to the spectrum of human disease. Several recent papers have examined the role of other contributors, such as monocyte tissue factor, endothelial cell activation, and leukocyte-platelet aggregates in the pathogenesis of the disease. Mouse models will provide a means to test new diagnostic and therapeutic approaches.

Amelioration of murine immune thrombocytopenia by CD44 antibodies: a potential therapy for ITP?

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.

The effects of prenatal PCBs on adult social behavior in rats

Endocrine disrupting chemical (EDC) exposures during critical periods of development may influence neuronal development and the manifestation of sexually dimorphic sociability and social novelty behaviors in adulthood. In this study, we assessed the effects of gestational exposure to PCBs on the social behavior of males and females later in adulthood. A weakly estrogenic PCB mixture, Aroclor 1221 (A1221, 0.5 or 1mg/kg) was administered to pregnant Sprague-Dawley rat dams. Both a positive control (estradiol benzoate; EB, 50μg/kg) and negative control (dimethylsulfoxide; DMSO in sesame oil vehicle) were similarly administered to separate sets of dams. The sexes responded differently in two tasks essential to sociality. Using a three-chamber apparatus that contained a caged, same-sex, gonadectomized stimulus animal and an empty stimulus cage, we found that both sexes showed a strong preference for affiliating with a stimulus animal (vs. an empty cage), an effect that was much more pronounced in the males. In the second task, a novel and a familiar stimulus animal were caged at opposite ends of the same apparatus. Females displayed a higher degree of novelty preference than the males. During both tests, females had significantly higher social approach behaviors while male engaged in significantly more interactive behaviors with the conspecific. Of particular interest, males born of dams that received prenatal A1221 (0.5mg/kg) exhibited an overall decrease in nose-to-nose investigations. These behavioral data suggest that the males are more sensitive to A1221 treatment than are females. In addition to behavioral analysis, serum corticosterone was measured. Females born of dams treated with A1221 (0.5mg/kg) had significantly higher concentrations of corticosterone than the DMSO female group; males were unaffected. Females also had significantly higher corticosterone concentrations than did males. Overall, our results suggest that the effects of gestational exposure to PCBs on adult social behavior are relatively limited within this particular paradigm.

Anxiety-like behaviors in adulthood are altered in male but not female rats exposed to low dosages of polychlorinated biphenyls in utero.

&NA; Exposure to polychlorinated biphenyls (PCBs), a class of endocrine‐disrupting chemicals, can result in altered reproductive behavior in adulthood, especially when exposure occurs during critical periods of brain sexual differentiation in the fetus. Whether PCBs alter other sexually dimorphic behaviors such as those involved in anxiety is poorly understood. To address this, pregnant rat dams were injected twice, on gestational days 16 and 18, with the weakly estrogenic PCB mixture Aroclor 1221 (A1221) at one of two low dosages (0.5 mg/kg or 1.0 mg/kg, hereafter 1.0 and 0.5), estradiol benzoate (EB; 50 &mgr;g/kg) as a positive estrogenic control, or the vehicle (3% DMSO in sesame oil). We also conducted a comprehensive assessment of developmental milestones of the F1 male and female offspring. There were no effects of treatment on sex ratio at birth and age at eye opening. Puberty, assessed by vaginal opening in females and preputial separation in males, was not affected in females but was advanced in males treated with A1221 (1.0). Males and females treated with A1221 (both dosages) were heavier in early adulthood relative to controls. The earliest manifestation of this effect developed in males prior to puberty and in females slightly later, during puberty. Anxiety‐like behaviors were tested using the light:dark box and elevated plus maze tests in adulthood. In females, anxiety behaviors were unaffected by treatment. Males treated with A1221 (1.0) showed reduced indices of anxiety and increased activity in the light:dark box but not the elevated plus maze. EB failed to replicate the phenotype produced by A1221 for any of the developmental and behavioral endpoints. Collectively, these results indicate that PCBs increase body weight in both sexes, but their effects on anxiety‐like behaviors are specific to males. Furthermore, differences between the results of A1221 and EB suggest that the PCBs are likely acting through mechanisms distinct from their estrogenic activity. HighlightsAnxiety‐like behavior is decreased and exploration increased by low dose PCB exposure in males.Behaviors in the elevated plus and light:dark box are differentially affected by PCBs.Body weight is increased in males and females exposed to PCBs during late gestation.The effects of transient low dose PCB exposure are dose‐ and sex‐specific.

Prepubertal exposure to arsenic(III) suppresses circulating insulin-like growth factor-1 (IGF-1) delaying sexual maturation in female rats

Arsenic (As) is a prevalent environmental toxin readily accessible for human consumption and has been identified as an endocrine disruptor. However, it is not known what impact As has on female sexual maturation. Therefore, in the present study, we investigated the effects of prepubertal exposure on mammary gland development and pubertal onset in female rats. Results showed that prepubertal exposure to 10 mg/kg of arsenite (As(III)) delayed vaginal opening (VO) and prepubertal mammary gland maturation. We determined that As accumulates in the liver, disrupts hepatocyte function and suppresses serum levels of the puberty related hormone insulin-like growth factor 1 (IGF-1) in prepubertal animals. Overall, this is the first study to show that prepubertal exposure to As(III) acts peripherally to suppress circulating levels of IGF-1 resulting in delayed sexual maturation. Furthermore, this study identifies a critical window of increased susceptibility to As(III) that may have a lasting impact on female reproductive function.