Activation of endothelial nitric oxide synthase is dependent on its interaction with globular actin in human umbilical vein endothelial cells.

Endothelial nitric oxide synthase (eNOS) has been reported to associate with globular actin, and this association increases eNOS activity. Adenosine, histamine, salbutamol and thrombin cause activation of eNOS through widely different mechanisms. Whether these eNOS agonists can regulate eNOS activity through affecting its association with actin is unknown. As previously reported, we confirmed in cultured human umbilical vein endothelial cells (HUVEC) that histamine and thrombin increased intracellular Ca(2+) whereas adenosine and salbutamol did not, and that these four agonists caused different effects on actin filament structure. Nevertheless, despite their divergent effects on intracellular Ca(2+) and on actin filament structure, we found by immunoprecipitation that adenosine, histamine, salbutamol and thrombin all caused an increase in association between eNOS and globular actin. This increase of association was inhibited by pre-treatment with phalloidin, an actin filament stabilizer. All of these agonists also increased phosphorylation of eNOS on serine residue 1177, eNOS activity, and cyclic guanosine-3', 5'-monophosphate, and these increases were all attenuated by phalloidin. Agonist-induced phosphorylation of eNOS on serine 1177 was attenuated by Akt inhibition, whereas association of eNOS with actin was not. We also found, in HEK-293 cells transfected with the eNOS mutants eNOS-S1177A or eNOS-S1177D, that the association between eNOS and globular actin was decreased as compared to cells transfected with wild-type eNOS. We conclude that association of globular actin with eNOS plays an essential and necessary role in agonist-induced eNOS activation, through enabling its phosphorylation by Akt at serine residue 1177.

Impaired platelet-dependent thrombin generation associated with thrombocytopenia is improved by prothrombin complex concentrates in vitro.

BACKGROUND: Impaired thrombin generation (TG) in patients with acquired coagulopathy, is due to low coagulation factors and thrombocytopenia. The latter is typically treated with platelet transfusions and the former with plasma and occasionally with prothrombin complex concentrates (PCCs). We hypothesized that manipulating the concentrations of coagulation factors might result in restoration of platelet-dependent TG over and above that of simple replacement therapy. OBJECTIVE: To investigate the influence of PCCs on impaired TG secondary to thrombocytopenia. METHODS: TG was evaluated by thrombin generation assay using a thrombocytopenia model in which normal plasma samples with varying platelet counts (20-300 × 109/L) were spiked with PCCs (25%-150% increase in plasma PCC levels). RESULTS: PCCs and platelets significantly increased TG in a dose-dependent manner in vitro. Two-way repeated measures of analysis of variance showed variance in peak height, area under the curve, time to peak, and velocity. This variance explained, respectively, by levels of PCC was 47, 59, 25 and 53%; by platelet count was 45, 28, 44, and 14%; by the combination was 80, 67, 70, and 62% variance; and a combination with additional interaction was 91, 84, 76, and 68%. TG at a platelet count 40 × 109/L with an approximate 25% increase in PCC concentration was similar to TG at 150 × 109/L. Similarly, patient samples spiked ex vivo with PCCs also showed highly significant improvements in TG. CONCLUSIONS: Impaired TG of thrombocytopenia is improved by PCCs, supporting the need for additional studies in complex coagulopathies characterized by mild to moderate thrombocytopenia and abnormal coagulation.

Effects of multiple-dose ponesimod, a selective S1P1 receptor modulator, on lymphocyte subsets in healthy humans.

This study investigated the effects of ponesimod, a selective S1P1 receptor modulator, on T lymphocyte subsets in 16 healthy subjects. Lymphocyte subset proportions and absolute numbers were determined at baseline and on Day 10, after once-daily administration of ponesimod (10 mg, 20 mg, and 40 mg each consecutively for 3 days) or placebo (ratio 3:1). The overall change from baseline in lymphocyte count was -1,292±340×106 cells/L and 275±486×106 cells/L in ponesimod- and placebo-treated subjects, respectively. This included a decrease in both T and B lymphocytes following ponesimod treatment. A decrease in naïve CD4+ T cells (CD45RA+CCR7+) from baseline was observed only after ponesimod treatment (-113±98×106 cells/L, placebo: 0±18×106 cells/L). The number of T-cytotoxic (CD3+CD8+) and T-helper (CD3+CD4+) cells was significantly altered following ponesimod treatment compared with placebo. Furthermore, ponesimod treatment resulted in marked decreases in CD4+ T-central memory (CD45RA-CCR7+) cells (-437±164×106 cells/L) and CD4+ T-effector memory (CD45RA-CCR7-) cells (-131±57×106 cells/L). In addition, ponesimod treatment led to a decrease of -228±90×106 cells/L of gut-homing T cells (CLA-integrin ß7+). In contrast, when compared with placebo, CD8+ T-effector memory and natural killer (NK) cells were not significantly reduced following multiple-dose administration of ponesimod. In summary, ponesimod treatment led to a marked reduction in overall T and B cells. Further investigations revealed that the number of CD4+ cells was dramatically reduced, whereas CD8+ and NK cells were less affected, allowing the body to preserve critical viral-clearing functions.

Monocyte-platelet interaction induces a pro-inflammatory phenotype in circulating monocytes.

BACKGROUND: Activated platelets exert a pro-inflammatory action that can be largely ascribed to their ability to interact with leukocytes and modulate their activity. We hypothesized that platelet activation and consequent formation of monocyte-platelet aggregates (MPA) induces a pro-inflammatory phenotype in circulating monocytes. METHODOLOGY/PRINCIPAL FINDINGS: CD62P(+) platelets and MPA were measured, and monocytes characterized, by whole blood flow cytometry in healthy subjects, before and two days after receiving influenza immunization. Three monocytic subsets were identified: CD14(+)CD16(-), CD14(high)CD16(+)and CD14(low)CD16(+). The increase in high sensitivity C-reactive protein post-immunization was accompanied by increased platelet activation and MPA formation (25.02±12.57 vs 41.48±16.81; p = 0.01), along with enhancement of circulating CD14(high)CD16(+) cells (4.7±3.6 vs 10.4±4.8; p = 0.003), their percentage being linearly related to levels of CD62P(+)-platelets (r(2) = 0.4347; p = 0.0008). In separate in vitro experiments, co-incubation of CD14(+)CD16(-) cells, isolated from healthy donor subjects, with autologous platelets gave rise to up-regulation of CD16 on monocytes as compared with those maintained in medium alone (% change in CD14(+)CD16(+) cells following 48 h co-incubation of monocytes with platelets was +106±51% vs monocytes in medium alone; p<0.001). This effect correlated directly with degree of MPA formation (r(2) = 0.7731; p<0.0001) and was associated with increased monocyte adhesion to endothelial cells. P-selectin glycoprotein ligand-1 (PSGL-1) blocking antibody, which abrogates MPA formation, abolished these effects, as did the cyclooxygenase (COX)-2 selective inhibitor NS-398, aspirin and the EP1/EP2-selective antagonist AH6809. CONCLUSIONS/SIGNIFICANCE: These data suggest that MPA formation, as occurs in the blood under pro-inflammatory conditions, expands the pool of circulating CD14(high)CD16(+) monocytes in a COX-2 dependent manner, and these monocytes exhibit increased adhesion to endothelium. Our findings delineate a novel mechanism underlying the pro-inflammatory effect of platelet activation.