| Background and objectsAntiphospholipid syndrome(APS) is a clinical syndrome caused by antiphospholipid(aPL)antibodies.At present,the diagnosis of APS still follows the 2006 Sydney classification criteria in which only two events of thrombosis and morbid pregnancy was included.Thrombocytopenia is one of the most common and important clinical manifestations of APS.Recently,more and more scholars support to add thrombocytopenia into the updated APS classification criteria.However,many questions still exist in APS-related thrombocytopenia,including the clinical characteristics of these patients,the correlation between thrombocytopenia and the clinical manifestations and aPL antibodies,and the risk of symptoms recurrence.Research on the pathogenesis of APS-related thrombocytopenia is also very limited.Most studies focus on the increase of platelet activation and clearance induced by aPL antibodies and the increase of platelet destruction mediated by platelet-specific antibodies.Studies have shown that anti-platelet antibodies can inhibit megakaryocyte maturation and platelet release ex vivo.It has not been reported whether immune-mediated thrombocytopenia in APS is related to decreased megakaryocyte production,maturation disorders or reduced platelet release.Our study was designed to answer those questions,explore the mechanism of aPL antibodies in APS related thrombocytopenia,and provide guidance for the diagnosis and management of APS patients with thrombocytopenia.Materials and methodsIn part one,the inpatients with primary APS(PAPS)in Peking Union Medical College Hospital from 2009 to 2019 were analyzed retrospectively.Using the collected clinical and laboratory data the clinical characteristics and the recurrence risk in the PAPS patients with thrombocytopenia were compared with those in the PAPS patients with normal platelet counts.Univariate and multivariate logistic regression analysis were used to screen the risk factors for thrombocytopenia.In part two,we established our ex vivo culture system for expansion and differentiation of megakaryocytes from cord blood CD34+cells and determined the optimal time of cell harvest according to the status on differentiation and maturation of megakaryocytes.The effect of serum from PAPS patients with or without thrombocytopenia on expansion,differentiation and maturation of megakaryocytes and release of platelet were evaluated.During the evaluation,serum from primary immune thrombocytopenia(PITP)and healthy volunteers were used as control.In part three,the apoptosis and the Caspase3 activation of megakaryocytes were analyzed.RNA-Seq high-throughput sequencing was performed to compare the difference of the gene expression.The serum aPL IgG antibody titer and TPO level of PAPS patients were further clarified by ELISA.ResultsIn part one,127 patients with PAPS were enrolled,of which 36(28.3%)had thrombocytopenia,with a median age of 38 years and 63.9%of women.In the thrombocytopenia group,the average platelet count was 58.9±27×109/L and the prevalence of thrombosis and morbid pregnancy was not significantly different from that in the normal platelet group.The frequency of autoimmune hemolytic anemia(19.4%),reticular leukoplakia(16.7%),chronic kidney disease(25%).aPL triple positive(61.1%)and hypocomplementemia and the adjusted Global APS Score(aGAPSS)were significantly increased in the thrombocytopenia group.In multivariate logistic regression analysis,hypocomplementemia(OR value 5.032,95%CI 3.118-22.095)is an independent risk factor for thrombocytopenia.In part two,umbilical cord blood CD34+ cells were cultured in serum-free medium with rhTPO of 50ng/mL.Day 14 was confirmed as the time point of optimal expansion and differentiation of megakaryocytes.A total of 10 PAPS patients with thrombocytopenia(group A1),10 PAPS patients with normal platelets(group A2),4 patients with PITP(group B)and 8 healthy controls(group C)were included in our study.In comparison to group C,the total number of megakaryocytes were increased in group A1 and A2.However,the proportions of mature megakaryocytes,polyploid.proplatelet-bearing megakaryocytes and production of platelet were significantly reduced in group A1 and A2.Compared with group B,no significant difference of total megakaryocytes production and mature megakaryocytes proportion was found in group A1 and A2.Whereas group A2 had lower proportion of polyploid megakaryocytes.higher proportion of proplatelet-bearing megakaryocytes and platelet production than group B.Group A2 had a higher proportion of aPL triple positive and a significantly lower proportion of proplatelet-bearing megakaryocytes and platelets production than those in group A1.In part three,the levels of aCL and anti-(32GPI IgG antibodies in groups A1 and A2 were significantly higher than those in group C,whereas there was no correlation between antibody titers and the degree of megakaryocyte maturation inhibition.In comparison to group C,apoptosis was significantly reduced in group A1 and had inhibition tendency in group A2.No significant difference was found between group B and group A.The result of RNA-Seq showed that the expression of related substrate genes in the end stage of apoptosis was significantly decreased in group A1.The TPO level of PAPS patients with thrombocytopenia was 132.4±75.8 pg/mL.Although it was higher than healthy subjects(43.0±19.5pg/mL),the degree of increase was still relatively insufficient.ConclusionThe PAPS patients with thrombocytopenia had significantly higher aGAPSS scores,and their serum TPO levels were relatively insufficient.Hypocomplementemia may be the independent risk factor of thrombocytopenia in PAPS patients.We found for the first time that the serum of PAPS patients can inhibit ex vivo megakaryocyte maturation and platelet production regardless of clinical thrombocytopenia.The inhibitory effect of PAPS serum on thrombopoiesis might be induced by the inhibition of apoptosis and has no dose-dependent with the titer of aCL and anti-β2GPI IgG antibodies. |
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