The Platelet-Derived Microparticles Related to the Clinical Phenotype Heterogeneity of Hemophlia a

Keywords: Hemophlia A, Phenotype; Heterogeneity; Flowcytometry ; Mircoparticle

Background: The clinical phenotype for hemophlia A are typically recognized sub-clinical, mild, moderate and severe according to the residue FVIII level of the plasma. For recent years, there are evidences indicate that the residue FVIII level is not the only factor relate to the clinical phenotype of patients with severe hemophlia A. Platelets are an important role in hemostasis and platelet-derived microparticles (PMPs) are known to mediate a prothrombotic state in patients, we hypothesized that PMPs contributes to the clinical phenotype heterogeneity of hemophilia A patients.

Methods: Thirty-one patients with severe hemophlia A (FVIII:C<1%, age 18-33 yrs, median 23.5 yrs) treated on demand were enrolled in this study, including 10 patients with mild type(FVIII:C<1%, but annualized bleed rates<6) and 21 patients with severe type (FVIII:C<1% and annualized bleed rates≥24). Seventeen healthy male donors were selected as normal controls. We also compared the baseline PMPs counts from eleven patients on demand treatment to that when they had three months prophylactic treatment. Venous blood was anticoagulated with sodium citrate (1:9). Plasma was isolated (1550g for 20 min at RT) and then cell free supernatant was ultracentrifuged (18,000g for 20 min at 20°C). The supernatant plasma samples were measured for baseline PMPs counts, with phycoerythrin (PE)-conjugated monoclonal antibodies CD41, CD42b, CD61, CD62P, and CD63 specific for PMPs derived from platelets respectively. Analyzed by flow cytometry, Forward scatter was set in scale using fluorescent microspheres of 1.0μm and standard fluorescent microbeads (0-1.0μm) in diameter were used to set the microparticle gate. Statistics were performed using independent-samples T test

Results: There was a trend toward higher the mean level of PMPs counts in mild type patients compared to severe type (mean ± SD: 2.06±1.33 vs 0.95±0.64, p = 0.02), the PMPs counts of normal individuals was significantly higher than severe type(mean ± SD: 1.06±1.20 v s0.95±0.64, p = 0.02), significantly lower than mild type (mean ± SD: 1.06±1.20 vs 2.06±1.33, p = 0.02). The PMPs counts decreased after 3 months low dose (10 iu/kg, 2-3/w) prophylacticlly treatment (mean ± SD: 6.50±0.51 vs 0.45±0.50, p = 0.00).

Conclusion/Discussion: Platelet-derived Microparticles (PMPs) are generated in response to platelet activation, our observations demonstrate that the PMPs counts differs in patients of severe hemophlia A, the mean lever of PMPs in mild clinical phenotype patients was higher than the severe type, prophylacticlly treated patients has a lower PMPs lever, PMPs is likely to play a role in clincal phenotype heterogeneity of sever hemophilia A.