脑血栓形成患者APCR现象观察及ATⅢ、PC水平的变化

1993年,Dahlback等1]在家族性血栓症患者中发现活化蛋白C抵抗现象(activated protein C resistance,APCR);此后,在欧美国家静脉血栓性患者中发现凝血因子Ⅴ(FV)基因第1691位的核苷酸G→A点突变(即FV Leiden),是发生APCR的主要机制.国内静脉血栓形成患者也存在APCR现象[2],但FV Leiden突变仅在国外的中国人群中发现2例[3].对动脉血栓形成与APCR和FVLeiden关系的研究,国外的报道不一[4],国内现有文献认为有待更多的研究.为此,本文就正常人和脑血栓形成患者是否存在APCR现象及FV Leiden突变再作初步探讨.     

中国脑血栓患者活化蛋白C抵抗的检测

目的 由凝血因子V Arg506→Gln突变（FV Leiden突变）引起的活化蛋白C抵抗（APCR）是深静脉血栓形成的主要危险因素，然而它与缺血性脑卒中的相关性尚不明确。本研究为探讨APCR和FV Leiden突变在中国人群中脑血栓形成中的作用。方法应用以活化部分凝血酶时间为基础的APCR测定方法对61例急性脑血栓的住院病人和39例正常健康人进行检测。APC敏感性比率（APC-SR）=（APTT＋ARC）／（ATIT-APC），并计算出正常化APC-SR。用多聚酶链反应-限制性内切酶长度多态性（PCR-RFLP）分析检测FV Leiden突变。结果61例脑血栓患者中有2例（3．3％）存在APCR现象，但对照组中未发现APCR。在正常健康人和脑血栓患者中均未发现有FV Leiden突变。结论在中国人群中，低发病率的非FV Leiden突变的APCR可能参与了脑血栓的形成。     

凝血因子V基因编码区单核苷酸多态性与静脉血栓栓塞症的相关性分析

目的研究凝血因子Ⅴ（FⅤ）基因编码区单核苷酸多态性与静脉血栓栓塞症的关系。方法检测静脉血栓患者及正常对照者血浆中的FⅤ的活性（FⅤ：C）（一期法）及FⅤ抗原（FⅤ：Ag）（ELISA法），用Premier5软件设计5对与静脉血栓症相关的基因多态性（Asp79His、Arg306The、Arg306Gly、Arg506Gln、Ile359The和His1299Arg）引物，进行PCR扩增、产物纯化、限制性片段长度多态性聚合酶链反应（PCR-RFLP）检测。对所发现的多态性用直接测序法证实，并对含多态性的标本用直接测序法进行FⅤ基因全部外显子编码区的筛查。结果静脉血栓组（VTE组）FⅤ：C（106．9±28．0）％，FⅤ：Ag（110．4±33．3）％；对照组FⅤ：C（102．4±30．9）％，FⅤ：Ag（102．1±24．1）％。VTE组FⅤ：Ag明显高于对照组（P〈0．05），FⅤ：C两者之间差异无明显统计学意义。VTE组和对照组均无Asp79His、Arg306The、Arg306Gly、Arg506Gln和Ile359The多态性，5例静脉血栓栓塞症患者及3名正常人含有His1299Arg多态性。5例静脉血栓栓塞症患者同时伴有Met1736Val多态性，其中3例尚含Asp2194Gly多态性。结论FⅤ含量增高与静脉血栓栓塞症相关，静脉血栓栓塞症与Asp79His、Arg306The、Arg306Gly、Arg506Gln和Ile359The多态性基本无关；His1299Arg在VTE组中的分布频率高于对照组，但差异无统计学意义。Hisl299Arg与Metl736Ⅴal和As02194Gly多态性在人群中有连锁不平衡特性。     

血栓及血栓前状态与凝血因子V基因多态性和APCR及HHcy的相关性研究

目的:探讨血栓患者与高同型半胱氨酸血症(HHcy)、活化蛋白C抗性(APCR)及凝血因子v基因多态性的关系。方法:300例健康查体者为正常对照;纳入研究的223例血栓患者中,经计算机断层显像(CT)的脑梗塞(CI)80例、心肌梗塞(MI)82例和静脉血栓栓塞(VTE)61例;另纳入研究的270例血栓前状态患者中,妊高症(PTH)76例、慢性阻塞性肺疾病(COPD)62例、糖尿病(DM)60例和癌症(CA)72例。以循环酶法和APTT凝固法分别测定病例组和正常对照血浆中HHcy及APCR,并用限制性内切酶片段多态性(RFLP)测定FV G1691-A、G1091-C、A1090-G等3种基因多态性的发生情况。结果:静脉血栓患者APCR阳性率最高(62.29%),正常对照组APCR阳性很低(7.33%),而其HHcy阳性分别为68.42%及10.00%。发现3例FV基因杂合突变静脉血栓患者为APCR阳性。结论:静脉血栓患者HHcy阳性率明显高于正常对照,HHcy是引起静脉血栓患者血栓形成的重要原因之一,静脉血栓患者存在APCR,而APCR阳性可能与凝血因子V基因多态性有关。     

抗磷脂蛋白抗体、活化蛋白C抵抗与深静脉血栓形成的关系研究

目的探讨抗磷脂蛋白抗体（APA）、活化蛋白C抵抗（APCR）与深静脉血栓形成（DVT）之间的关系及其临床意义。方法选取100例DVT患者和100例正常对照者,分别采用ELISA法检测ACA（IgG,IgM,IgA）,APTT法检测LA,APTT＋／-APC法检测APCR。PCR-限制性片断长度多态性方法（PCR—RFLP）检测FV Leiden变异。结果DVT组患者APA、APCR的总阳性率分别为34％、4％,与对照组相比差异显著;APA阳性组APCR阳性率（8．82％）明显高于APA阴性组（1．51％）,4例APCR阳性患者均未检测到FV Leiden突变。4例APCR阳性患者中有3例APA阳性。并能被血小板磷脂纠正。结论抗磷脂抗体、活化蛋白C抵抗与深静脉血栓形成密切相关,抗磷脂蛋白抗体及其产生获得性APCR是高凝状态和深静脉血栓形成的重要原因。     

活化蛋白C抵抗试验及其临床意义

1993年Dahlback等发现静脉血栓形成与活化蛋白C抵抗（APCR）现象密切相关，其发生率在欧美人群中为3％～5％。已知凝血因子V（FV）基因点突变（第1691位减基G→A，169iG6A）造成FV分子第506位精氨酸被谷氨酰胺替代（FV：Q506）是造成APCR的分子机制。用活化部分凝血活酶时间（APTT）法可筛检并确定APCR现象；用分子生物学方法可明确APCR是否与FV基因突变有关。有高凝状态、血栓形成史的人群应作APCR筛检试验，以降低或预防血栓的发生。     

深静脉血栓患者抗凝蛋白水平检测及临床意义

抗凝蛋白缺陷是与深静脉血栓形成（deep venous thrombosis，DVT）密切相关的易栓病因，这些遗传因素在中国人群血栓病患者中的分布与西方人群存在较大差异。由凝血因子V Leiden突变导致的血浆活化蛋白C抵抗（activated protein c resistance，APCR）已被证实是西方欧美人群DVT发病最为相关的危险因素。然而在我国，APCR的发病率并不像国外那样高，而且FV Leiden突变更为少见。中国人群DVT的主要发病原因还不清楚。为此，我们检测了100例DVT患者的抗凝血酶（antithrombin，AT）、蛋白C（protein C，PC）、蛋白S（protein S，PS）活性水平及APCR，旨在探讨抗凝蛋白缺陷在中国人群深静脉血栓形成中的作用，为临床提供经济、方便、有效的早期实验室筛查方法。     

四个遗传性凝血因子V缺陷症家系临床表型和基因型变化的研究

目的 研究4个遗传性凝血因子V(FV)缺陷症家系的临床表型和基因突变.方法 测定家系成员活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)及FV促凝活性(FV:C)和FV抗原(FV:Ag)含量进行表型诊断;用PCR法扩增先证者F5基因的25个外显子及其侧翼序列,PCR产物纯化后直接测序,检测其基因突变.结果 4例先证者APTT、PT明显延长,血浆FV:C和FV:Ag含量均显著降低.基因分析发现,先证者1的F5基因存在G16088C(Asp68His)杂合错义突变及4种位于同一条染色体上的杂合多态性T35788C(Met385Thr)、A47295G(Hisl299Arg)、A58668G(Metl736Val)和A74083G(Asp2194Gly);先证者2的F5基因存在CA6253T(Arg952Cys)和CA6724T (Glnl 109stop)两种纯合突变;先证者3的F5基因存在C67793G(Pr02006Ala)纯合错义突变;先证者4的F5基因存在C74022T(Arg2174Cys)纯合错义突变.结论 Asp68His、Arg952Cys、Glnl109stop、Pro2006Ala和Arg2174Cys这5种突变,及Met385Thr、Hisl299Arg、Metl736Val和Asp2194Gly这4种多态性是导致相应先证者I型遗传性FV缺陷症的原因.其中,Glnl109stop、Pro2006Ala和Arg2174Cys是国际七首次报道的新突变.     

对检测FVLeiden组织因子依赖性FV试验的评价:证明其作为扰活化蛋白C常规检测方法的高度敏感性和特异性

目前用于检测抗活化蛋白C(APCR)的APTT方法有许多缺陷:重复性差以及受血浆处理、试剂标准化和应用抗凝剂或有狼疮抗凝物质等因素影响,因此限制了其常规应用。本文评价了一种新的检测APCR的一期组织因子依赖性因子V(FV)试验,并以双盲方式与检测FV Leiden分子缺陷的聚合酶链反应(PCR)法相比较。 研究对象:82例血栓病患者和35例无血栓病史     

动脉血栓性疾病凝血因子Ⅴ基因的研究

1993年 ,Dahlback等[1] 发现静脉血栓患者抗活化蛋白C(activatedproteinCresistance,APCR)现象。在欧洲 ,已证实凝血因子Ⅴ基因Leiden (FⅤL)G16 91A突变是遗传性静脉血栓的主要机制之一[2 ,3 ] 。我们研究了我国汉族人脑血栓(CT)和急性心肌梗死组 (AMI)患者APCR和凝血因子Ⅴ(FⅤ )基因突变。现将结果报道如下。对象和方法1　研究对象1.1　FⅤ基因检测 :健康人组 :共 10 5名 (上海地区 6 0名 ,苏州医学院DNA检测用标本 4 5份 ) ,经体检无器质性病变 ,无用药史。CT组 :共 10 2例 (上海地区 5 7例 ,苏州医学院DNA检测用标本 4 5…     

Genetic modulation of the FVLeiden/normal FV ratio and risk of venous thrombosis in factor V Leiden heterozygotes

Summary. Background and Objectives: The factor (F) V Leiden mutation causes activated protein C (APC) resistance by decreasing the susceptibility of FVa to APC‐mediated inactivation and by impairing the APC‐cofactor activity of FV in FVIIIa inactivation. However, APC resistance and the risk of venous thromboembolism (VTE) vary widely among FV Leiden heterozygotes. Common F5 genetic variation probably contributes to this variability. Patients/methods: APC resistance was determined in 250 FV Leiden heterozygotes and 133 normal relatives using the prothrombinase‐based assay, which specifically measures the susceptibility of plasma FVa to APC. The effects of 12 F5 single‐nucleotide polymorphisms (SNPs) on the normalized APC sensitivity ratio (nAPCsr) and on FV levels were determined by multiple regression analysis. Results: In FV Leiden heterozygotes, VTE risk increased with increasing nAPCsr, reaching an odds ratio (OR) of 9.9 (95% confidence interval [CI] 1.2–80.5) in the highest nAPCsr quartile. The minor alleles of several F5 SNPs, including 327 A/G (Q51Q), 409 G/C (D79H), 2663 A/G (K830R, T2 haplotype), 6533 T/C (M2120T) and 6755 A/G (D2194G, R2 haplotype), increased the nAPCsr in FV Leiden heterozygotes, but not in their normal relatives. Most of these effects could be attributed to a shift in the FV_Leiden/normal FV ratio. Four FV Leiden heterozygotes with extremely high nAPCsr turned out to be pseudo‐homozygotes, i.e. they carried a deleterious mutation on the non‐Leiden allele. Conclusions: In FV Leiden heterozygotes, the prothrombinase‐based nAPCsr is a marker of VTE risk and is modulated by common F5 SNPs that affect the FV_Leiden/normal FV ratio in plasma.     

血栓栓塞症患者抗活化的蛋白C的研究

目的 探讨抗活化的蛋白Ｃ（ＡＰＣＲ）现象在中国人血栓栓塞症发病中的作用。方法 用ＡＰＴＴ法检测ＡＰＣＲ敏感比率（ＡＰＣＲ－ＳＲ）来研究４０例深静脉血栓症（ＤＶＴ）患者、５２例脑血栓形成患者和１００例正常人的ＡＰＣＲ发生率;用多聚酶链反应－限制性内切酶长度多态性（ＰＣＲ－ＲＦＬＰ）来检测ＦＶ Ｌｅｉｄｅｎ突变;用免疫火箭电泳测血浆总蛋白Ｓ和游离蛋白Ｓ。结果 正常人的正常化ＡＰＣＲ－ＳＲ（ｎ－ＡＰＣＲ     

The factor V Glu1608Lys mutation is recurrent in familial thrombophilia

BACKGROUND: Co-inheritance of heterozygous factor V deficiency with FV Leiden enhances the activated protein C resistance (APCR) associated with this mutation, resulting in pseudo-homozygous APCR. The role of FV deficiency in modulating thrombotic risk in this rare condition is poorly understood. METHODS AND RESULTS: We have identified in thrombophilic patients with FV deficiency a novel FV gene mutation (c. 4996G>A), predicting the Glu1608Lys substitution in the A3 domain. The heterozygous mutation was detected in three unrelated patients, two carriers of the FV Leiden mutation, and one of the FVHR2 haplotype. The Glu1608Lys change was also present in two subjects with mild FV deficiency, and absent in 200 controls. The FV1608Lys carriers showed reduced mean FV activity (42% +/- 12%) and antigen (53% +/- 18%) levels and, in Western blot analysis, reduced amounts of intact platelet FV. The restriction fragment length polymorphism (RFLP) study identified two haplotypes underlying the mutation, which suggests that it is recurrent. In heterozygous subjects the amount of FV1608Lys mRNA in white blood cells was similar to that produced by the counterpart alleles (FVWt or FVHR2). Recombinant FV1608Lys (rFV1608Lys), detected by Western blot in the conditioned medium, was indistinguishable from rFVWt and FV antigen and activity were found to be respectively 44% +/- 20% and 13% +/- 4% of rFVWt. CONCLUSIONS: Our data indicate that FVGlu1608Lys predicts a CRM (plasma)/CRMred (cell culture) FV deficiency, and may contribute to thrombophilia in carriers of FV Leiden and FVHR2 haplotype via a pseudo-homozygosity mechanism. Our findings help to define the molecular bases of FV deficiency and thrombophilia.     

ABO blood group, other risk factors and incidence of venous thromboembolism: the Longitudinal Investigation of Thromboembolism Etiology (LITE)

Summary.  Background:  Numerous case–control studies have reported higher prevalence of non-O blood type among venous thromboembolism (VTE) patients than controls, but potential mechanisms or effect modifiers for the association are not fully established. Patients/methods:  Using a nested case–control design combining the Atherosclerosis Risk in Communities and the Cardiovascular Health Study cohort, ABO blood type and other VTE risk factors were measured on pre-event blood samples of 492 participants who subsequently developed VTE and 1008 participants who remained free of VTE. Results:  A total of 64.4% of cases and 52.5% of controls had non-O blood type. Among controls, mean values of factor VIIIc (FVIIIc) and von Willebrand factor among the non-O blood type group were higher than among the O group. Compared with O blood type, the age-adjusted odds ratio (OR) of VTE for non-O blood type was 1.64 (95% CI, 1.32–2.05) and was similar for the two parent studies and race groups. Further adjustment for sex, race, body mass index, diabetes mellitus and FVIIIc reduced the OR: 1.31 (95% CI, 1.02–1.68). Factor V Leiden (FV Leiden) appeared to modify the non-O blood type association with VTE in a supra-additive fashion, with an age-, sex- and race-adjusted OR of 6.77 (95% CI, 3.65–12.6) for having both risk factors. Conclusions:  Non-O blood type was independently associated with risk of VTE, and added to the risk associated with FV Leiden.     

Prothrombin A19911G polymorphism and the risk of venous thromboembolism

BACKGROUND: The A > G polymorphism at position 19911 of the prothrombin gene is associated with increased plasma prothrombin levels but its role as a risk factor for venous thromboembolism (VTE) is not established. OBJECTIVE: To investigate the role of prothrombin 19911 A > G polymorphism in the risk of VTE in patients with heterozygous prothrombin 20210GA or factor (F) V Leiden and in those without thrombophilia. PATIENTS AND METHODS: Case-control study of 793 patients with prothrombin 20210 GA (n = 167) or FV Leiden (n = 198), and without thrombophilia (n = 428), and of 795 healthy individuals with the corresponding coagulation profile, investigated for the presence of prothrombin 19911 A > G. Plasma prothrombin levels were measured in 342 individuals. RESULTS: Prothrombin 19911 A > G did not increase the risk of VTE in carriers of prothrombin 20210 GA [odds ratio (OR) 1.2, 95% CI (95% CI) 0.8-1.8] but significantly increased the risk in carriers of FV Leiden (OR 2.1, 95% CI 1.3-3.4) and in patients without thrombophilia (OR 1.5, 95% CI 1.0-2.2). Higher plasma prothrombin levels in carriers of prothrombin 19911 A > G polymorphism than in non-carriers were found among individuals without thrombophilia (P =0.05) and with FV Leiden (P = 0.07), but not in carriers of prothrombin 20210 GA (P = 0.2). CONCLUSIONS: Prothrombin 19911 A > G polymorphism was independently associated with a 1.5-fold increased risk of VTE and increased 2-fold the risk of VTE associated with FV Leiden, both increases statistically significant. No effect was observed in carriers of prothrombin 20210 GA, perhaps because this polymorphism has a stronger influence on plasma prothrombin levels than the prothrombin 19911 polymorphism.     

Low-density lipoprotein receptor-related protein 1 polymorphism 663 C > T affects clotting factor VIII activity and increases the risk of venous thromboembolism

BACKGROUND: Clotting factor (F) VIII is an independent risk factor for primary and recurrent venous thromboembolism (VTE). The causes for high plasma FVIII levels are not fully understood, but an involvement of genetic factors has been demonstrated. A multifunctional endocytic receptor, low-density lipoprotein receptor-related protein 1 (LRP1), mediates cellular uptake and subsequent degradation of FVIII and may contribute to variations in FVIII levels. OBJECTIVE: We assessed the association of a genetic variation of LRP1 (663C > T) with basal FVIII levels and the risk of venous thrombosis in a group of high-risk patients and in healthy controls. PATIENTS AND METHODS: One hundred and fifty-two patients with a history of recurrent VTE (median age 56 years, 47% women) were compared with 198 age- and sex-matched controls (median age 53 years, 50% women). The LRP1 663C > T genotype was analyzed by mutagenic separated polymerase chain reaction assay and heterozygosity was confirmed by sequence analysis. RESULTS: LRP1 663C > T genotype distribution differed significantly between patients (663CC n = 138, 663CT n = 14) and controls (663CC n = 190, 663CT n = 8; P = 0.048). In multivariable linear regression analysis including LRP1 663C > T, ABO blood group, von Willebrand factor antigen, C-reactive protein and age, LRP1 663CT was independently associated with FVIII activity (P = 0.02). LRP1 663CT was also associated with increased odds for VTE following adjustment for blood group O, FV Leiden and the prothrombin variation 20210G > A in multivariate analysis (odds ratio 3.3, 95% CI 1.3-8.5). CONCLUSIONS: According to our data the LRP1 663C > T polymorphism influences plasma FVIII levels independently of blood group, C-reactive protein and von Willebrand factor and is significantly associated with the risk of VTE.     

Vitamin K epoxide reductase genetic polymorphism is associated with venous thromboembolism: results from the EDITH Study

BACKGROUND: The vitamin K epoxide reductase complex subunit 1 (VKORC1) recycles endogenous vitamin K, a cofactor for vitamin K-dependent coagulation factor synthesis. Common polymorphisms in VKORC1, the gene coding for VKORC1, have been found to affect the dose response to vitamin K antagonists, and to confer an increased risk of vascular diseases in a Chinese population. The aim of this study was to evaluate the association between the VKORC1 1173C > T polymorphism and venous thromboembolism (VTE). METHODS: We report the results of a case-control study designed to evaluate interactions between acquired and inherited risk factors of VTE. We studied 439 cases hospitalized with a first venous thromboembolic event that was not related to a major acquired risk factor for VTE, and 439 matched controls. The VKORC1 1173C > T polymorphism was selected for genotyping as the tagging single-nucleotide polymorphism for previously identified VKORC1 haplotypes. RESULTS: The relationship between VTE and the VKORCI 1173C > T polymorphism was consistent with a recessive model. The frequency of the VKORCI TT genotype was lower in cases than in controls. The odds ratio (OR) (95% CI) was 0.62 (0.41-0.94) for the TT genotype as compared to CT/CC genotypes. Adjustment on cardiovascular diseases, body mass index, factor V (FV) and prothrombin gene mutations did not alter the results. CONCLUSIONS: In this case-control study, the frequency of the VKORCI TT genotype was lower in patients with VTE than in matched controls. The clinical consequence of these results remains to be determined, but gives new perspectives for exploration of the role of VKORCI polymorphism in the pathogenesis of VTE.     

Expression of the normal factor V allele modulates the APC resistance phenotype in heterozygous carriers of the factor V Leiden mutation

BACKGROUND: Functional defects of the protein C pathway, detectable in plasma as activated protein C (APC) resistance, are a prevalent risk factor for venous thrombosis. The factor V (FV) Leiden mutation causes APC resistance by interfering with the APC-mediated inactivation of both FVa and FVIIIa. Co-inheritance of FV Leiden and quantitative FV deficiency on different alleles, a rare condition known as pseudo-homozygous APC resistance, is associated with pronounced APC resistance and 50% reduced FV levels, because of non-expression of the non-Leiden FV allele. OBJECTIVES: The role of normal FV in modulating the APC resistance phenotype in carriers of FV Leiden was investigated in patients with pseudo-homozygous APC resistance and in model systems. PATIENTS/METHODS: Four functional plasma assays probing both components of APC resistance (susceptibility of FVa to APC and cofactor activity of FV in FVIIIa inactivation) were employed to compare seven clinically and genetically characterized FV Leiden pseudo-homozygotes to 30 relatives with different FV genotypes (including 12 FV Leiden heterozygotes and seven carriers of FV deficiency) and to 32 unrelated FV Leiden homozygotes. RESULTS AND CONCLUSIONS: All assays consistently indicated that FV Leiden pseudo-homozygotes are significantly more APC-resistant than heterozygotes and indistinguishable from homozygotes. Thrombin generation measurements in FV-deficient plasma reconstituted with purified normal FV and FV Leiden confirmed these observations and showed that the expression of the normal FV allele is an important modulator of APC resistance in FV Leiden heterozygotes. These findings provide an explanation for the higher thrombotic risk of FV Leiden pseudo-homozygotes when compared with heterozygotes.     

Thrombophilic polymorphisms – factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T mutations – and preterm birth

AIM OF THE STUDY: To evaluate the influence of three common thrombophilic polymorphisms, factor V Leiden (FV), prothrombin G20210A (PT), and methylenetetrahydrofolate reductase (MTHFR) C677T mutations, on preterm birth of unknown cause. PATIENTS AND METHODS: A single-centre case-control study of women with preterm infants < or =35 weeks of gestation, in whom obvious maternal, uterine, and fetal causes responsible for preterm birth were excluded (n = 35). The controls were 54 women with term infants hospitalised in the same ward. RESULTS: There were no significant differences between the groups of mothers in history of fetal loss, venous or familial thrombosis, or previous preterm birth. FV was found in 8.6% of the cases, PT in 5.7%, and MTHFR mutation (homozygous) in 4.8% compared with 5.4% (p=0.292, OR 1.594, CI95% 0.303-8.384), 7.4% (p=0.379, OR 0.758, CI95% 0.131-4.374), and 4.5% (p = 0.485, OR 1.050, CI95% 0.090-12.276), respectively, in the controls. Differences in the three thrombophilic polymorphisms in the two groups of infants were also not significant. CONCLUSION: We could not demonstrate a distinct association between these thrombophilic polymorphisms and preterm birth.