Roelse J C, De Laaf R T, Timmermans S M, Peters M, Van Mourik J A, Voorberg J
Department of Blood Coagulation, CLB, Amsterdam, The Netherlands.
Br J Haematol. 2000 Feb;108(2):241-6. doi: 10.1046/j.1365-2141.2000.01834.x.
Patients with cross-reacting material (CRM)-reduced haemophilia A exhibit reduced levels of factor VIII antigen. In this study, we determined the molecular basis of the genetic defect in the factor VIII gene induced by either the Arg593-->Cys or the Asn618-->Ser missense mutation, identified in two CRM-reduced haemophilia A patients. We introduced either the Arg593-->Cys or the Asn618-->Ser mutation into a B-domain-deleted factor VIII cDNA and expressed the modified cDNAs in C127 cells. Reduced levels of factor VIII activity and factor VIII antigen in conditioned medium of transfected cells indicated that the secretion of both factor VIII variants was impaired. The ratio of factor VIII antigen present in cell extract to that in conditioned medium was 1.9 and 2.4 times higher for rFVIII-R593C and rFVIII-N618S, respectively, than for rFVIII. Metabolic labelling and immunoprecipitation of transfected cells revealed that rFVIII-R593C and rFVIII-N618S persisted somewhat longer inside the cell than factor rFVIII. Intracellular accumulation and subsequent degradation of factor VIII-R593C and factor VIII-N618S may explain the reduced levels of both factor VIII activity and antigen in plasma of mild haemophilia A patients with corresponding genetic defects.
交叉反应物质（CRM）降低的A型血友病患者的凝血因子VIII抗原水平降低。在本研究中，我们确定了在两名CRM降低的A型血友病患者中鉴定出的由Arg593→Cys或Asn618→Ser错义突变诱导的凝血因子VIII基因遗传缺陷的分子基础。我们将Arg593→Cys或Asn618→Ser突变引入缺失B结构域的凝血因子VIII cDNA中，并在C127细胞中表达修饰后的cDNA。转染细胞条件培养基中凝血因子VIII活性和凝血因子VIII抗原水平降低表明两种凝血因子VIII变体的分泌均受损。rFVIII-R593C和rFVIII-N618S的细胞提取物中存在的凝血因子VIII抗原与条件培养基中的凝血因子VIII抗原的比率分别比rFVIII高1.9倍和2.4倍。对转染细胞进行代谢标记和免疫沉淀显示，rFVIII-R593C和rFVIII-N618S在细胞内的持续时间比凝血因子rFVIII稍长。凝血因子VIII-R593C和凝血因子VIII-N618S在细胞内的积累及随后的降解可能解释了具有相应遗传缺陷的轻度A型血友病患者血浆中凝血因子VIII活性和抗原水平降低的原因。
