遗传性凝血因子V缺乏症分子病理机制的研究和2A型vWD的临床研究
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摘要
遗传性凝血相关蛋白异常导致的遗传性出血性疾病包括各种凝血因子活性缺乏或功能缺陷。对血管性血友病（von Willebrand Disease, vWD）、血友病A及血友病B等较常见的遗传性出血性疾病，已进行了深入的分子生物学研究，目前发现的基因异常已达1000多种，发达国家已进入基因治疗的研究。而对一些少见的凝血因子缺陷，如FV、FⅪ、凝血酶原及纤维蛋白原等缺乏所致的遗传性出血性疾病，由于疾病资源的限制，世界范围内仅有为数不多的报道，我国则处于起步阶段。
凝血因子V（factorV，FV）是分子量约333KD的单链糖蛋白，生理情况下几乎以无活性的状态存在于血浆中。在凝血酶的作用下，血浆FV转变为具有凝血活性的活化FV（activated FV，FVa）。血浆中的FVa作为辅因子，与FⅩa、钙离子及磷脂（细胞膜）共同构成凝血酶原复合物，参与凝血酶原的活化。由于FVa的参与，使得FⅩa催化凝血酶原活化的能力增加了104-5倍。FVa又可通过活化的蛋白C（activated protein C , APC）裂解而被灭活，从而通过APC途径直接或间接参与凝血过程。总之，FV在凝血过程中处于促凝血途径和抗凝血途径的交叉点，对维持凝血平衡起着至关重要的作用。
FV分子由2196个氨基酸残基组成，包括3个同源的A区，1个B区及2个同源的C区，排列顺序为A1-A2-B-A3-C1-C2。其分子包括重链区和轻链区，重链区位于氨基端，由A1和A2区组成，分子量约为105KD；轻链区位于羧基端，由A3、C1和C2区组成,分子量约为74 KD；重链和轻链由B区相连。FV基因位于染色体1q23，全长约80 Kb，包含25个外显子和24个内含子。其cDNA全长约7Kb，mRNA编码28个氨基酸的前导肽和2196个氨基酸的成熟蛋白质。其中第1-12外显子编码信号肽和A1-A2区，第13外显子编码完整的B区，第14-25外显子编码A3-C1-C2区。根据产生机制的不同，FV缺陷可导致遗传性FV缺乏症，引起出血；或导致APCR，引起血栓。
遗传性凝血因子V缺乏症(hereditary deficiency of factor V)是一种罕见的常染色体隐性遗传性出血性疾病，发病率约为百万分之一。该病是由于血浆中缺乏正常活性的凝血因子V所致。其杂合子通常无症状，纯合子或复合杂合子临床表现为程度不等的出血，但大多较血友病轻微，并具有以下实验室检查特点：凝血酶原时间（prothrombin time, PT）及活化的部分凝血活酶时间（activated partial thromboplastin time, APTT）均延长，且可被正常吸附血浆所纠正。确诊常需进行凝血因子活性和抗原测定。此外，应同时注意除外FV与其它凝血因子的联合缺乏。
由于遗传性FV缺乏症发病率低和FV基因的复杂性，目前对其分子病理机制的研究很少。迄今，全世界有关遗传性FV缺乏症个例报道约200余例，报道了约20余种遗传性FV缺乏症的基因突变类型，基因变异的种类包括碱基替换、碱基插入和碱基缺失等，导致无义突变、移码突变和错义突变。已报道的突变均集中在外显子编码区，几乎都累及单一的外显子，且基因突变好发于编码FV B区的第13号外显子，这可能与外显子13较大（占FV cDNA全长的约40%）有关。
vWD（von Willebrand disease）是最常见的遗传性出血性疾病之一，是由于血浆中vWF（von Willebrand factor）质或量的缺陷所致，且这种缺陷是由于vWF基因突变所导致。根据多中心研究的结果估计，有症状的vWD患者达113/106或更高，而具有vWD危险的人数高达1480-3580/106。vWF是一种多聚血浆糖蛋白。血浆中vWF以多种形式参与体内止血过程。一方面，vWF在初期止血中起着重要作用：内皮细胞损伤后, vWF与内皮下胶原结合,并与血小板膜糖蛋白GpⅨ和GpⅡb/Ⅲa结合,促使血小板活化、粘附、聚集。另一方面，血浆中vWF与FⅧ以非共价键的形式结合，起着稳定FⅧ，延长其半衰期的作用。
vWD是一种高度异质性的疾病。随着对vWF分子基础的不断研究，1994年国际止血与血栓委员会根据vWD的临床表现、病理特点和分子病理机制，提出了新的分型标准，将vWD分为3型。1型vWD是指vWF量的部分缺乏。血浆vWF功能正常，多聚物分布正常或接近正常。2型vWD是指vWF质的异常，也常伴有量的异常，占vWD的20-30%。又分为2A、2B、2M和2N四种
亚型。3型vWD是指vWF的原发性完全缺乏，可表现初期止血和凝血的联合缺陷。FⅧ水平常小于正常的10%，少数可小于1%，可发生关节和软组织的自发性出血。
2A 型vWD是最常见的vWF质异常的类型，其特点是血浆中大、中分子量的多聚物消失，而导致vWF依赖的血小板粘附功能缺陷。临床表现为轻到中度的出血，APTT可正常或轻度延长，程度不等的vWF抗原和活性的减低，反映vWF与血小板相互作用的瑞斯托霉素诱导的血小板聚集试验（ristocetin-induced platelet aggregation,RIPA）明显降低。
本课题在对具有先天性出血倾向的门诊患者进行筛查的过程中，发现了一例重型凝血因子V缺乏症患者。随后，采用比浊法、凝血因子活性测定、BA-ELISA法、Western blot、PCR及其相关技术等生物化学、免疫学及分子生物学等多种方法，对先证者及其家系进行了研究。依据临床特征，先证者临床表型属重型：出生后较早出现较严重的出血症状，包括颅内血肿、消化道出血，以及近两年发生的关节内出血和多次腹腔出血。PT、APTT
Functional defect or quantitative deficiency of coagulation factors are involved in congenital bleeding diseases caused by the abnormality of coagulation related proteins. Molecular biology research was performed on a few diseases such as von Willebrand disease, hemophilia A and B, which are more common in population, and more than 1000 gene defects were founded. Gene therapy is being carried on in some of these diseases. Because of the limitation of disease resource, there are a few reports in the world on some rare bleeding diseases caused by coagulation factors deficiency including factor V, factor Ⅺ, fibrinogen, thrombinogen, and et al. But the researches on these diseases are beginning in our country.
Human coagulation factor V (FV) is a single-chain glycoprotein in plasma with a molecular weight of about 330kd. It is produced by megakaryocytes and circulates in blood as an inactive procoagulant cofactor. FVa forms an essential part of the prothrombinase complex that catalyzes the conversion of prothrombin to thrombin by factor Xa in the presence of calcium and a phospholipid membrane. Activated protein C (APC) inactivates FVa through claevage of FVa and FV is required as a cofactor in the APC-mediated inactivation of factor Ⅷa (FⅧa). Thus, coagulation factor V (FV) plays an important role in maintaining the hemostatic balance in both the formation of thrombin in procoagulant pathway as well as in the protein C anticoagulant pathway.
Factor V consists of 3 homologous A-type domains and 2 homologous C-type domains connected by a B domain in the following order: A1-A2-B-A3-C1-C2. Removal of the large B domain from inactive FV by
thrombin cleavage yields the activated factor V (FVa), which is made up of a heavy chain of approximately 105kd(A1-A2 domains), and a light chain of approximately 74kd(A3-C1-C2 domains). The gene coding for coagulation FV has been mapped to chromosome 1q23 and spans more than 80kb. It consists of 24 introns and 25 exons, and the messenger RNA (mRNA) encodes a 28-amino-acid leader peptide and a 2196-amino-acid mature protein. Roughly, the heavy chain is encoded by exons 1 to 12 and the light chain by exons 14 to 25. The entire B domain is encoded by a single exon, exon13.
Inheritary deficiency of factor V (parahemophillia) is a rare autosomal recessive hereditary bleeding disorder with the estimated morbility approximately is one per one million. The phenotypic expression of FV deficiency is variable. Heterozygotes are usually asymptomatic, whereas homozygous or compound heterozygous patients show mild, moderate, or severe bleeding symptoms during their entire life. Easy bruising, epitaxis, hematomas and, in females, menorrhagia are the usual clinical manifestations. The diagnosis of factor V deficiency can be easily made on the basis of the following criteria: a prolonged prothrombin time (PT) and a partial thromboplastin time (PTT) corrected by the addition of adsorbed normal plasma. No other clotting factor deficiency yields such a pattern. A factor V coagulation assay in needed to establish the precise nature of the defect. Both the activity and immunological assay are important. This combination has shown to be of critical importance in the diagnosis of combined defects.
Because of low prevalence of FV deficiency with variable phenotypic expression and complex of FV gene, little is known about the molecular basis underlying this disease. Up to date, more than 200 factor V deficiency cases have been reported in the literature, but the molecular basis for factor V deficiency has been established in only about 20 cases. The mutations in patients with factor V deficiency were identified including point mutation, frameshift mutation, deletion and insertion mutation. Most of them are cluster in 13 exon of
FV gene, which represents more then 40% of the whole cDNA.
von Willebrand disease (vWD), which is caused by the qualitative and quantitative defects of von Willebrand factor(vWF), is one of the most common inherited human bleeding disorders. Approximately 113
凝血因子V（factorV，FV）是分子量约333KD的单链糖蛋白，生理情况下几乎以无活性的状态存在于血浆中。在凝血酶的作用下，血浆FV转变为具有凝血活性的活化FV（activated FV，FVa）。血浆中的FVa作为辅因子，与FⅩa、钙离子及磷脂（细胞膜）共同构成凝血酶原复合物，参与凝血酶原的活化。由于FVa的参与，使得FⅩa催化凝血酶原活化的能力增加了104-5倍。FVa又可通过活化的蛋白C（activated protein C , APC）裂解而被灭活，从而通过APC途径直接或间接参与凝血过程。总之，FV在凝血过程中处于促凝血途径和抗凝血途径的交叉点，对维持凝血平衡起着至关重要的作用。
FV分子由2196个氨基酸残基组成，包括3个同源的A区，1个B区及2个同源的C区，排列顺序为A1-A2-B-A3-C1-C2。其分子包括重链区和轻链区，重链区位于氨基端，由A1和A2区组成，分子量约为105KD；轻链区位于羧基端，由A3、C1和C2区组成,分子量约为74 KD；重链和轻链由B区相连。FV基因位于染色体1q23，全长约80 Kb，包含25个外显子和24个内含子。其cDNA全长约7Kb，mRNA编码28个氨基酸的前导肽和2196个氨基酸的成熟蛋白质。其中第1-12外显子编码信号肽和A1-A2区，第13外显子编码完整的B区，第14-25外显子编码A3-C1-C2区。根据产生机制的不同，FV缺陷可导致遗传性FV缺乏症，引起出血；或导致APCR，引起血栓。
遗传性凝血因子V缺乏症(hereditary deficiency of factor V)是一种罕见的常染色体隐性遗传性出血性疾病，发病率约为百万分之一。该病是由于血浆中缺乏正常活性的凝血因子V所致。其杂合子通常无症状，纯合子或复合杂合子临床表现为程度不等的出血，但大多较血友病轻微，并具有以下实验室检查特点：凝血酶原时间（prothrombin time, PT）及活化的部分凝血活酶时间（activated partial thromboplastin time, APTT）均延长，且可被正常吸附血浆所纠正。确诊常需进行凝血因子活性和抗原测定。此外，应同时注意除外FV与其它凝血因子的联合缺乏。
由于遗传性FV缺乏症发病率低和FV基因的复杂性，目前对其分子病理机制的研究很少。迄今，全世界有关遗传性FV缺乏症个例报道约200余例，报道了约20余种遗传性FV缺乏症的基因突变类型，基因变异的种类包括碱基替换、碱基插入和碱基缺失等，导致无义突变、移码突变和错义突变。已报道的突变均集中在外显子编码区，几乎都累及单一的外显子，且基因突变好发于编码FV B区的第13号外显子，这可能与外显子13较大（占FV cDNA全长的约40%）有关。
vWD（von Willebrand disease）是最常见的遗传性出血性疾病之一，是由于血浆中vWF（von Willebrand factor）质或量的缺陷所致，且这种缺陷是由于vWF基因突变所导致。根据多中心研究的结果估计，有症状的vWD患者达113/106或更高，而具有vWD危险的人数高达1480-3580/106。vWF是一种多聚血浆糖蛋白。血浆中vWF以多种形式参与体内止血过程。一方面，vWF在初期止血中起着重要作用：内皮细胞损伤后, vWF与内皮下胶原结合,并与血小板膜糖蛋白GpⅨ和GpⅡb/Ⅲa结合,促使血小板活化、粘附、聚集。另一方面，血浆中vWF与FⅧ以非共价键的形式结合，起着稳定FⅧ，延长其半衰期的作用。
vWD是一种高度异质性的疾病。随着对vWF分子基础的不断研究，1994年国际止血与血栓委员会根据vWD的临床表现、病理特点和分子病理机制，提出了新的分型标准，将vWD分为3型。1型vWD是指vWF量的部分缺乏。血浆vWF功能正常，多聚物分布正常或接近正常。2型vWD是指vWF质的异常，也常伴有量的异常，占vWD的20-30%。又分为2A、2B、2M和2N四种
亚型。3型vWD是指vWF的原发性完全缺乏，可表现初期止血和凝血的联合缺陷。FⅧ水平常小于正常的10%，少数可小于1%，可发生关节和软组织的自发性出血。
2A 型vWD是最常见的vWF质异常的类型，其特点是血浆中大、中分子量的多聚物消失，而导致vWF依赖的血小板粘附功能缺陷。临床表现为轻到中度的出血，APTT可正常或轻度延长，程度不等的vWF抗原和活性的减低，反映vWF与血小板相互作用的瑞斯托霉素诱导的血小板聚集试验（ristocetin-induced platelet aggregation,RIPA）明显降低。
本课题在对具有先天性出血倾向的门诊患者进行筛查的过程中，发现了一例重型凝血因子V缺乏症患者。随后，采用比浊法、凝血因子活性测定、BA-ELISA法、Western blot、PCR及其相关技术等生物化学、免疫学及分子生物学等多种方法，对先证者及其家系进行了研究。依据临床特征，先证者临床表型属重型：出生后较早出现较严重的出血症状，包括颅内血肿、消化道出血，以及近两年发生的关节内出血和多次腹腔出血。PT、APTT
Functional defect or quantitative deficiency of coagulation factors are involved in congenital bleeding diseases caused by the abnormality of coagulation related proteins. Molecular biology research was performed on a few diseases such as von Willebrand disease, hemophilia A and B, which are more common in population, and more than 1000 gene defects were founded. Gene therapy is being carried on in some of these diseases. Because of the limitation of disease resource, there are a few reports in the world on some rare bleeding diseases caused by coagulation factors deficiency including factor V, factor Ⅺ, fibrinogen, thrombinogen, and et al. But the researches on these diseases are beginning in our country.
Human coagulation factor V (FV) is a single-chain glycoprotein in plasma with a molecular weight of about 330kd. It is produced by megakaryocytes and circulates in blood as an inactive procoagulant cofactor. FVa forms an essential part of the prothrombinase complex that catalyzes the conversion of prothrombin to thrombin by factor Xa in the presence of calcium and a phospholipid membrane. Activated protein C (APC) inactivates FVa through claevage of FVa and FV is required as a cofactor in the APC-mediated inactivation of factor Ⅷa (FⅧa). Thus, coagulation factor V (FV) plays an important role in maintaining the hemostatic balance in both the formation of thrombin in procoagulant pathway as well as in the protein C anticoagulant pathway.
Factor V consists of 3 homologous A-type domains and 2 homologous C-type domains connected by a B domain in the following order: A1-A2-B-A3-C1-C2. Removal of the large B domain from inactive FV by
thrombin cleavage yields the activated factor V (FVa), which is made up of a heavy chain of approximately 105kd(A1-A2 domains), and a light chain of approximately 74kd(A3-C1-C2 domains). The gene coding for coagulation FV has been mapped to chromosome 1q23 and spans more than 80kb. It consists of 24 introns and 25 exons, and the messenger RNA (mRNA) encodes a 28-amino-acid leader peptide and a 2196-amino-acid mature protein. Roughly, the heavy chain is encoded by exons 1 to 12 and the light chain by exons 14 to 25. The entire B domain is encoded by a single exon, exon13.
Inheritary deficiency of factor V (parahemophillia) is a rare autosomal recessive hereditary bleeding disorder with the estimated morbility approximately is one per one million. The phenotypic expression of FV deficiency is variable. Heterozygotes are usually asymptomatic, whereas homozygous or compound heterozygous patients show mild, moderate, or severe bleeding symptoms during their entire life. Easy bruising, epitaxis, hematomas and, in females, menorrhagia are the usual clinical manifestations. The diagnosis of factor V deficiency can be easily made on the basis of the following criteria: a prolonged prothrombin time (PT) and a partial thromboplastin time (PTT) corrected by the addition of adsorbed normal plasma. No other clotting factor deficiency yields such a pattern. A factor V coagulation assay in needed to establish the precise nature of the defect. Both the activity and immunological assay are important. This combination has shown to be of critical importance in the diagnosis of combined defects.
Because of low prevalence of FV deficiency with variable phenotypic expression and complex of FV gene, little is known about the molecular basis underlying this disease. Up to date, more than 200 factor V deficiency cases have been reported in the literature, but the molecular basis for factor V deficiency has been established in only about 20 cases. The mutations in patients with factor V deficiency were identified including point mutation, frameshift mutation, deletion and insertion mutation. Most of them are cluster in 13 exon of
FV gene, which represents more then 40% of the whole cDNA.
von Willebrand disease (vWD), which is caused by the qualitative and quantitative defects of von Willebrand factor(vWF), is one of the most common inherited human bleeding disorders. Approximately 113
引文
Girolami A, Simioni P, Scarano L, et al. Hemorrhagic and thromboitic disorders due to factor V deficiencies and abnormalities: an updated classification. Blood Rew. 1998, 12: 45-51.
谢飞，程烽，朱晓辉等.一例遗传性凝血因子V缺乏症发病机制研究.中华血液学杂志，2001，22：453-456.
Jenny RJ, Pittman DD, Toole JJ,et al. Complete cDNA and derived amino
acid sequence of human factor V. Biochemistry, 1987,84:4846-4850.
Cripe L.D, Moore K.D, Kane W.H. Structure of the gene for human factor V. Biochemistry. 1992,31:3777-3785.
Lak M, Sharifian R, Peyvandi F, et al.Symptoms of inherited factor V deficiency in 35 Iranian patients. Br J Haemotol, 1998 , 103:1067-1069.
von Wijk R, Nieuwenhuis K, Berg M, et al. Five novel mutations in the gene for human blood coagulation factor V associated with type I factor V deficiency. Blood, 2001,98:358-367.
VilloutreIX BO and Dahlback B. Structure investigation of the A domains of human blood coagulation factor V by molecular modeling. Protein Science. 1998,7:1317-1325.
Murray JM, Rand MD, Egan JO, et al. Factor VNew Brunswick: Ala221-to-Val substitution results in reduced cofactor activity. Blood. 1995,86:1820-1827.
Guasch JF, Cannegieter S, Reitsma PH, et al. Severe coagulation factor V deficiency caused by a 4bp deletion in the factor V gene. Br J Haematol. 1998,101:32-39.
Yang TL, Cui J, Taylor J.M, et al. Rescue of fatal neonatal heamorrhage in factor V deficient mice by low level transgene expression. Thromb Haemost. 2000,83:70-77.
Montefusco MC, Duga S, Asselta R, et al .A novel two base pair deletion in the factor V gene associated with severe factor V deficiency. Br J Haematol.2000, 111:1240-1246.
Zehnder JL, Hiraki D.D, Jones C.D, et al. Familial coagulation factor V deficiency caused by a novel 4 base pair insertion in the factor V gene: factor V Stanford. Thromb Haemost. 1999,82:1097-1099.
Schrijver I, Houissa-Kastally R, Jones CD,et al. Novel factor V C2-domain mutation (R2074H) in two families with factor V deficiency and bleeding.
Thromb Haemost. 2002,87:294-299.
Ajzner E, Balogh I, Szabo T, et al. Severe coagulation factor V deficiency caused by 2 novel frameshift mutations: 2952delT in exon 13 and 5493insGin exon 16 of factor 5 gene. Blood, 2002,99:702-705.
Schrijver I, Koerper MA, Jones CD, et al. Homozygoous factor V splice site mutation associated with severe factor V deficiency. Blood, 2002,99:3063-3065.
谢飞，程烽，朱晓辉等.一例遗传性凝血因子V缺乏症发病机制研究.中华血液学杂志，2001，22：453-456.
Jenny RJ, Pittman DD, Toole JJ,et al. Complete cDNA and derived amino
acid sequence of human factor V. Biochemistry, 1987,84:4846-4850.
Cripe L.D, Moore K.D, Kane W.H. Structure of the gene for human factor V. Biochemistry. 1992,31:3777-3785.
Lak M, Sharifian R, Peyvandi F, et al.Symptoms of inherited factor V deficiency in 35 Iranian patients. Br J Haemotol, 1998 , 103:1067-1069.
von Wijk R, Nieuwenhuis K, Berg M, et al. Five novel mutations in the gene for human blood coagulation factor V associated with type I factor V deficiency. Blood, 2001,98:358-367.
VilloutreIX BO and Dahlback B. Structure investigation of the A domains of human blood coagulation factor V by molecular modeling. Protein Science. 1998,7:1317-1325.
Murray JM, Rand MD, Egan JO, et al. Factor VNew Brunswick: Ala221-to-Val substitution results in reduced cofactor activity. Blood. 1995,86:1820-1827.
Guasch JF, Cannegieter S, Reitsma PH, et al. Severe coagulation factor V deficiency caused by a 4bp deletion in the factor V gene. Br J Haematol. 1998,101:32-39.
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