Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII.
Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII.
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DOI：
10.1182/blood.v81.11.2925.2925
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发表时间：
1993-06
期刊：
影响因子：
20.3
通讯作者：
Debra D. Pittman;E. Alderman;Kathleen N. Tomkinson;Jack H. Wang;Alan R. Giles;Randal J. Kaufman
中科院分区：
文献类型：
--
作者：
Debra D. Pittman;E. Alderman;Kathleen N. Tomkinson;Jack H. Wang;Alan R. Giles;Randal J. Kaufman
凝血因子VIII(FVIII)是凝血的内在途径中的一种辅因子，缺乏凝血因子可导致出血性疾病。凝血因子VIII含有A1-A2-B-A3-C1-C2的结构域，其中B结构域是体外促凝血活性所必需的。在这份报告中，我们比较了B结构域缺失的FVIII(残基760到1639，命名为LA-VIII)和野生型重组FVIII的性质。在转基因的中国仓鼠卵巢(CHO)细胞中，LA-的表达水平是野生型FVIII的10-20倍。纯化的LA-VIII的比活性与野生型重组FVIII没有区别，两者的凝血酶活化系数相似。野生型重组FVIII和LA-VIII也显示出相似的凝血酶激活和重链断裂的时间过程。然而，与野生型重组衍生的FVIII相比，LA-VIII的轻链被凝血酶切割的速度快了5倍。加入纯化的von Willebrand因子(VWF)并不改变凝血酶切割或激活野生型重组FVIII或LA-VIII的动力学。在一种新的小鼠新生儿耐受诱导模型中，比较了LA-VIII和野生型FVIII的免疫原性。结果没有检测到野生型FVIII和LA-之间的任何免疫学差异，表明LA-不包含野生型FVIII所没有的重要新表位。LA-VIII在FVIII基因缺陷犬体内耐受性良好，能纠正皮肤出血时间，与野生型重组因子VIII相似。在体内，LA-VIII与犬vWF结合，半衰期与野生型重组FVIII相似。这些研究支持B结构域缺失的FVIII可能在治疗人类血友病A方面有效。
Coagulation factor VIII (FVIII) is a cofactor in the intrinsic pathway of blood coagulation for which deficiency results in the bleeding disorder hemophilia A. FVIII contains a domain structure of A1-A2-B-A3-C1-C2 of which the B domain is dispensable for procoagulant activity in vitro. In this report, we compare the properties of B-domain-deleted FVIII (residues 760 through 1639, designated LA-VIII) to wildtype recombinant FVIII. In transfected Chinese hamster ovary (CHO) cells, LA-VIII was expressed at a 10- to 20-fold greater level compared with wildtype FVIII. The specific activity of purified LA-VIII was indistinguishable from wild-type recombinant FVIII and both exhibited similar thrombin activation coefficients. Wildtype recombinant-derived FVIII and LA-VIII also displayed similar timecourses of thrombin activation and heavy chain cleavage. However, compared with wildtype recombinant-derived FVIII, the light chain of LA-VIII was cleaved fivefold more rapidly by thrombin. Addition of purified von Willebrand factor (vWF) did not alter the kinetics of thrombin cleavage or activation of either wildtype recombinant-derived FVIII or LA-VIII. The immunogenicity of LA-VIII was compared with wildtype FVIII in a novel model of neonatal tolerance induction in mice. The results did not detect any immunologic differences between wildtype FVIII and LA-VIII, suggesting that LA-VIII does not contain significant new epitopes that are absent in wildtype FVIII. LA-VIII was tolerated well on infusion into FVIII-deficient dogs and was able to correct the cuticle bleeding time similar to wildtype recombinant factor VIII. In vivo, LA-VIII was bound to canine vWF and exhibited a half-life similar to wildtype recombinant FVIII. These studies support that B-domain-deleted FVIII may be efficacious in treatment of hemophilia A in humans.