Monocyte-Derived Microparticles Improve Hemostasis in Factor VIIIDeficient Mice: Exploring the Role of PSGL-1/P-Selectin.

Abstract

Introduction: Microparticles derived from leukocytes contribute to fibrin formation at thrombi in vivo and factor VIII-deficient (FVIII) mice treated with an agent that elevates their microparticles have decreased bleeding. A novel therapy for hemophilia patients with inhibitors is needed. We evaluated whether microparticles generated in vitro could improve hemostasis in FVIII mice.Methods: Mouse CD11b positive monocytes, isolated by MACS, or cultured monocytic cells (WEHI274.1) were treated with the calcium ionophore A23187. The resulting microparticles (isolated by differential centrifugation, and defined as CD18 positive events less than 1 μm diameter) or PIPES buffer were infused intravenously into FVIII-deficient mice (B6.129S4-F8tm1Kaz) or control wild type B6.129 mice prior to evaluation. The amount of platelets in laser-generated thrombi in cremaster muscle arterioles was evaluated using high-speed intravital fluorescence microscopy. The amount of hemoglobin shed from a 2 mm tail tip amputation measured blood loss.Results: Infusion of MPs at doses above 1000/g resulted in the death of wild type mice; FVIII-deficient mice tolerated MPs at doses up to 4000/g. Blood loss after tail clip in FVIII-deficient mice was 6-fold higher than blood loss from wild type mice. Blood loss after tail clip in FVIII-deficient mice was reduced to normal after the infusion of MPs at concentrations as low as 400/g. MPs, at 400/g, from CD11b positive cells isolated from wild type, FVIII-deficient mice or PSGL-1-deficient mice all similarly reduced blood loss after tail clip in FVIII-deficient mice. The biological half life of MP effect on tail-bleeding was 3 hours.Platelet accumulation in cremaster arteriolar thrombi was impaired in FVIII-deficient mice. Infusion of MPs at a concentration of 1000/g normalized platelet accumulation, but infusion of MPs at a lower concentration (400/g) did not.Conclusion: Abnormal hemostasis in FVIII-deficient mice can be temporarily reversed by the infusion of in vitro generated monocyte-derived MPs, including MPs derived from monocytes from FVIII-deficient or PSGL-1-deficient mice. The dose whereby MPs normalize FVIII-deficient mice is different between the hemostasis and thrombosis models. To explore whether P-selectin at injuries is required for the effect of MPs, we have generated by cross-breeding FVIII/P-selectin double deficient mice. These mice are born at expected mendelian frequency. Two of 20 male FVIII/P-selectin double deficient mice had spontaneous bleeding at 8 weeks of age, one in the thigh and one from the ear. FVIII/P-selectin double deficient mice also have prolonged tail bleeding times, which will serve as a model for testing the P-selectin targeting of microparticles.