| Objective: Paroxysmal nocturnal hemoglobinuria(PNH) is an acquired clonal disorder of hematopoietic stem cell gene mutation.The pathological mechanism of PNH is that the deficiency of anchor protein induced by PIG-A gene mutation resulted in the lake of functional protein. The primary clinical manifestations are intravascular hemolysis, marrow failure and thrombophilia. Historically, thrombosis was considered to be a major contributor of fatality. Although complement-mediated hemolysis, consumption of nitric oxide(NO), impairment of the fibrinolytic system and platelet activation are all related to hemolysis, the mechanism of thrombosis in PNH is not clearly. Recently, study showed serine protease3(PR3), “indirect anchor protein, ” was an important regulatory factor of anticoagulant, which was expressed abnormal in patients with PNH. We detected PR3 in patients with PNH on the level of gene and functional proteins to explore the role of PR3 in PNH thrombosis.Methods: We enrolled 27 patients with PNH and 25 healthy people as the control group over the same period. The expressions of NB1 and PR3 on the neutrophil's cell membranes in PNH patients and normal controls were detected by flow cytometry. Plasma PR3, NB1 and PAR-1 in patients and control group were detected by ELISA. The level of m RNA of PR3 and NB1 were detected by RT-PCR.PR3 in the neutrophil's cytoplasm were detected by western blotting. Clinical data including D-dimer were retrospectively recorded from patients and normal controls, and the correlations between D-dimer and PR3,between D-dimer and PAR-1 were analyzed respectively.Results: 1.The expressions of NB1 and PR3 on the neutrophil's cell membranes in patients with PNH are decreased.The expression of PR3 on the PNH neutrophil's cell membranes(CD59-PR3+/CD59-) in PNH group [(70.40±29.86)%] was lower than that on the CD59+ neutrophil's cell membranes(CD59+PR3+/CD59+)in control group[(93.28±10.53)%](P=0.01).The expression of PR3 on the CD59+ neutrophil's cell membranes(CD59+PR3+/CD59+)in PNH group[(85.68±22.21)%] was not different from that on the CD59+ neutrophil's cell membranes(CD59+PR3+/CD59+)[(93.28±10.53)%](P=0.131)in control group. In PNH patients, the expression of PR3 on the PNH neutrophil's cell membranes(CD59-PR3+/CD59-) [(70.40±29.86)%] was lower than that on the CD59+ neutrophil's cell membranes(CD59+PR3+/CD59+) [(85.68±22.21)%](P=0.046). The expression of NB1 on the PNH neutrophil's cell membranes(CD59-NB1+/CD59-)in PNH group[(20.61±26.07)%] was lower than that on the CD59+ neutrophil's cell membranes(CD59+NB1+/CD59+) [(67.72±19.65)%](P=0.001) in control group. The expression of NB1 on the CD59+ neutrophil's cell membranes(CD59+NB1+/CD59+)in PNH group [(72.25±25.62)%] was not different from that on the CD59+ neutrophil's cell membranes(CD59+NB1+/CD59+)[(67.72±19.65)%](P=0.46) in control group. In PNH patients, the expression of NB1 on the PNH neutrophil's cell membranes(CD59-NB1+/CD59-)[(20.61±26.07)%] was lower than that on the CD59+ neutrophil's cell membranes(CD59+NB1+/CD59+) [(70.15±27.28)%](P=0.001).2. The level of plasma PR3 in PNH group(2262.72±802.80)pg/ml was lower than that(3292.92±651.68)pg/ml in control group(P=0.001), but the level of plasma NB1 in PNH group was not different from that in control group(P=0.32)3. There were no difference on the levels of m RNA of PR3 and NB1 between PNH group and control group(P=0.629,P=0.836).4. There were no difference on the level PR3 in the neutrophil's cytoplasm between PNH group and control group.5. The level of plasma PAR-1 in PNH group(1.38±0.96)ng/ml was higher than that(0.47±0.29)ng/ml in control group(P<0.01).6. The level of D-Dimer of 5 patients with thrombosis was higher than 22 patients withnot thrombosis.7. There is negative correlation between PR3 level and plasma PAR-1 level(r = 0.456, P = 0.043). There is negative correlation between PR3 level and D-Dimer level(r = 0.503, P = 0.503).There is positive correlation between PAR-1 level and D-Dimer level(r = 0.65, P = 0.65) in patients with PNH.Conclusion: 1. In PNH patients, the expression of indirect anchor protein PR3 on the neutrophil's cell membranes and level of plasma PR3 are both decreased.2. Low level of PR3 and high level of PAR-1 in the plasma of PNH patients are closely related to thrombosis. |
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