Abstract：
BACKGROUND: ; Factor (F)V is pivotal in both procoagulant and anticoagulant mechanisms. The present report describes a novel F5 mutation in a FV-deficient patient (FV:C 6 IU/dL, FV:Ag 32 IU/dL), complicated with recurrent deep vein thrombosis. The patient demonstrated activated protein C resistance (APCR) with compound heterozygous mutations consisting of FV-Y1961C (FVKanazawa) and FV-1982_1983del. AIM;: To clarify thrombotic mechanisms associated with this FV abnormality.
RESULTS: Levels of FV-1982_1983del were below the detection sensitivity in our expression experiments using HEK293T cells, and analyses were targeted, therefore on the FV-Y1961C mutation. APTT-based clotting assays demonstrated that FV-Y1961C exhibited APCR, and that the reduced APC susceptibility in FVa-Y1961C resulted in a marked depression of APC-catalyzed inactivation with delayed cleavage at Arg506 and little cleavage at Arg306 with or without protein (P)S. The APC cofactor activity of FV-Y1961C in APC-catalyzed FVIIIa inactivation promoted by Arg336 cleavage in FVIII was impaired. The binding affinity of FVa-Y1961C to phospholipid membranes was reduced in reactions involving APC/PS-catalyzed inactivation and in prothrombinase activity. Furthermore, the addition of FVa-Y1961C to plasma failed to inhibit tissue factor (TF)-induced procoagulant function. These characteristics were similar to those of FV-W1920R (FVNara) and FV-A2086D (FVBesançon).
CONCLUSIONS: ; We identified a compound heterozygous. FV-Y1961C mutation in the C1 domain representing a novel FV mutation (FVKanazawa) resulting in not only APCR due to impaired FVa susceptibility and FV cofactor activity for APC function, but impaired inhibition of TF-induced procoagulant function. These defects in anticoagulant function associated with FV in FV-Y1961C contributed to a prothrombotic state.
RESULTS: Levels of FV-1982_1983del were below the detection sensitivity in our expression experiments using HEK293T cells, and analyses were targeted, therefore on the FV-Y1961C mutation. APTT-based clotting assays demonstrated that FV-Y1961C exhibited APCR, and that the reduced APC susceptibility in FVa-Y1961C resulted in a marked depression of APC-catalyzed inactivation with delayed cleavage at Arg506 and little cleavage at Arg306 with or without protein (P)S. The APC cofactor activity of FV-Y1961C in APC-catalyzed FVIIIa inactivation promoted by Arg336 cleavage in FVIII was impaired. The binding affinity of FVa-Y1961C to phospholipid membranes was reduced in reactions involving APC/PS-catalyzed inactivation and in prothrombinase activity. Furthermore, the addition of FVa-Y1961C to plasma failed to inhibit tissue factor (TF)-induced procoagulant function. These characteristics were similar to those of FV-W1920R (FVNara) and FV-A2086D (FVBesançon).
CONCLUSIONS: ; We identified a compound heterozygous. FV-Y1961C mutation in the C1 domain representing a novel FV mutation (FVKanazawa) resulting in not only APCR due to impaired FVa susceptibility and FV cofactor activity for APC function, but impaired inhibition of TF-induced procoagulant function. These defects in anticoagulant function associated with FV in FV-Y1961C contributed to a prothrombotic state.
摘要：
背景：;因子（F）V在促凝血和抗凝血机制中都至关重要。本报告描述了FV缺陷患者的新F5突变（FV：C6IU/dL，FV：Ag32IU/dL），并发复发性深静脉血栓。患者表现出活化的蛋白C抗性（APCR），具有由FV-Y1961C（FVKanazawa）和FV-1982_1983del组成的复合杂合突变。目的：阐明与这种FV异常相关的血栓形成机制。
结果：在我们使用HEK293T细胞的表达实验中，FV-1982_1983del的水平低于检测灵敏度，分析是有针对性的，因此FV-Y1961C突变。基于APTT的凝血试验表明FV-Y1961C表现出APCR，并且FVa-Y1961C中APC敏感性的降低导致APC催化失活的显着抑制，在Arg506处延迟裂解，在Arg306处几乎没有裂解，有或没有蛋白质（P）S。FV-Y1961C在由FVIII中的Arg336裂解促进的APC催化的FVIIIa失活中的APC辅因子活性受损。在涉及APC/PS催化的失活和凝血酶原酶活性的反应中，FVa-Y1961C与磷脂膜的结合亲和力降低。此外，血浆中添加FVa-Y1961C未能抑制组织因子（TF）诱导的促凝血功能。这些特征类似于FV-W1920R(FVNara)和FV-A2086D(FVBesançon)。
结论:;我们鉴定了复合杂合。C1结构域中的FV-Y1961C突变代表新的FV突变（FVKanazawa），不仅由于FVa易感性和FV辅因子对APC功能的活性受损而导致APCR，但TF诱导的促凝血功能抑制受损。这些与FV-Y1961C中FV相关的抗凝功能缺陷导致了血栓前状态。
结果：在我们使用HEK293T细胞的表达实验中，FV-1982_1983del的水平低于检测灵敏度，分析是有针对性的，因此FV-Y1961C突变。基于APTT的凝血试验表明FV-Y1961C表现出APCR，并且FVa-Y1961C中APC敏感性的降低导致APC催化失活的显着抑制，在Arg506处延迟裂解，在Arg306处几乎没有裂解，有或没有蛋白质（P）S。FV-Y1961C在由FVIII中的Arg336裂解促进的APC催化的FVIIIa失活中的APC辅因子活性受损。在涉及APC/PS催化的失活和凝血酶原酶活性的反应中，FVa-Y1961C与磷脂膜的结合亲和力降低。此外，血浆中添加FVa-Y1961C未能抑制组织因子（TF）诱导的促凝血功能。这些特征类似于FV-W1920R(FVNara)和FV-A2086D(FVBesançon)。
结论:;我们鉴定了复合杂合。C1结构域中的FV-Y1961C突变代表新的FV突变（FVKanazawa），不仅由于FVa易感性和FV辅因子对APC功能的活性受损而导致APCR，但TF诱导的促凝血功能抑制受损。这些与FV-Y1961C中FV相关的抗凝功能缺陷导致了血栓前状态。
