习惯性流产妇女中凝血因子V基因三种多态性和APC-R、LA的检测

目的研究习惯性流产妇女与活化蛋白C抵抗(APC-R)、狼疮抗凝物质(LA)和凝血因子V(FV)基因多态性的关系.方法测定习惯性流产妇女(未妊娠期)和正常对照妇女的APC-R、LA,并用限制性内切酶片段多态性的方法检测FV G1691→A、G1091→C、A1090→G 3种基因多态性的发生情况.结果习惯性流产妇女的APC-R和3种FV基因多态性测定未见阳性,LA阳性率达15.6%.结论中国妇女习惯性流产与APC-R和3种FV基因多态性可能关系不大,LA的存在可能与之有关.     

习惯性流产妇女中凝血因子V基因三种多态性和APC—R、LA的检测

目的　研究习惯性流产妇女与活化蛋白C抵抗 (APC R)、狼疮抗凝物质 (LA)和凝血因子Ⅴ (FⅤ )基因多态性的关系。方法　测定习惯性流产妇女 (未妊娠期 )和正常对照妇女的APC R、LA ,并用限制性内切酶片段多态性的方法检测FⅤG16 91→A、G10 91→C、A10 90→G 3种基因多态性的发生情况。结果　习惯性流产妇女的APC R和 3种FⅤ基因多态性测定未见阳性 ,LA阳性率达 15 .6 %。结论　中国妇女习惯性流产与APC R和 3种FⅤ基因多态性可能关系不大 ,LA的存在可能与之有关     

静脉血栓栓塞症患者活化蛋白C抗性及凝血因子V活性和基因多态性研究

本研究旨在观察静脉血栓栓塞患者凝血因子V(Factor V,F V)活性改变,检测活化蛋白C抗性(activated protein C resistance,APCR)和凝血因子V基因F V Leiden、F V Cambridge、F V Hong Kong、F V Asp79His和F V I359T多态性.对95例静脉血栓栓塞患者(其中下肢深静脉血栓79例,肺栓塞4例,下肢深静脉血栓合并肺栓塞12例)和95例正常对照者.应用限制性内切酶片段长度多态性测定FV Leiden、F V Cambridge和F V Hong Kong多态性,用MassARRAYTM技术检测F V Asp79His、F V I359T多态性.以一期法和校正的APTT试验分别对其中的65例静脉血栓栓塞患者和60例正常对照者行凝血因子V活性水平和活化蛋白C抵抗检测.结果表明静脉血栓栓塞患者血浆凝血因子V活性水平(108.03%±28.29%)高于对照组(95.17%±29.75%),差异有显著性统计学意义(P=0.008);静脉血栓栓塞组活化蛋白C抵抗阳性13例(20.0%),对照组3例(5.0%),二组比较,有统计学意义(P=0.012).二组均未发现FV Leiden、F V Cambridge、F V Hong Kong、FV Asp79His和FV I359T多态性.结论凝血因子V活性升高和活化蛋白C抵抗是静脉血栓栓塞的危险因素,但APCR与F V Leiden、F V Cambridge、F V HongKong、F V Asp79His和F V I359T多态性无关.     

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基因点突变造成造成Ｆ－Ⅴ分子第５０６信精氨酸被谷氨酰胺替代是造成ＡＰＲＣ的分子机制。     

正常妊娠妇女血中活化的蛋白C抵抗、狼疮样抗凝物质与血栓前状态分子标志物测定

目的研究活化的蛋白 Ｃ抵抗（Ａｃｔｉｖａｔｅｄ ｐｒｏｔｅｉｎ Ｃ ｒｅｓｉｓｔａｎｃｅ，ＡＰＧＲ）在正常妊娠中的发生情况，探讨狼疮抗凝物质（Ｌｕ－ｐｕｓ－ｌｉｋｅ ａｎｔｉｃｏａｇｕｌａｎｔ，ＬＡ）对妊娠性 ＡＰＣ－Ｒ的影响及二者与凝血酶生成、继发性纤溶的关系。方法采用 ＡＰＴＴ－ＡＰＣ法检测ＡＰＣ－Ｒ、ｄＲＶＶＴ法测定ＬＡ水平，并用 ＥＬＩＳＡ法测定了凝血酶原片段Ｆ１＋２和Ｄ－二聚体（Ｄ－ｄｉｍｅｒ，Ｄ－Ｄ）的含量。结果检测３０例正常妇女对照（ＮＣ）和５０例正常妊娠妇女，ＮＣ组ＡＰＣ－Ｒ比率为２．８８±０．３７，ＮＰ组为２．０４±０．３１（ＡＰＣ－Ｒ阳性率为４２％）；ＮＣ组 ＬＡ阳性率为 ０， ＮＰ组为 ３６．７％； ＮＣ组 Ｆ１＋２为（０．７３４ ± ０．４２） ｎｍｏｌ／Ｌ， ＮＰ组为（ １．０５ ± ０．６９） ｎｍｏｌ／Ｌ； ＮＣ组Ｄ－Ｄ为（０．４８±０．０５）ｍｇ／Ｌ，ＮＰ组为（０．６３±０．１１）ｍｇ/Ｌ；ＮＰ组的ＡＰＣ比率、Ｆ１＋２和Ｄ－Ｄ的测定结果均较ＮＣ组有显著性差异。结论妊娠可能发生与ＬＡ升高有关的ＡＰＣ－Ｒ，并导致了凝血酶激活物生成增加以及凝血酶、纤溶酶的激活和继发性纤溶的发生。     

凝血因子基因多态性与血栓形成

1993年以前 ,由于遗传性抗凝血蛋白缺陷引起的静脉血栓栓塞 ,占总静脉血栓栓塞的 5 %～ 15 % ,大多与抗凝血酶(ＡＴ) ,蛋白Ｃ(ＰＣ)和蛋白Ｓ(ＰＳ)缺陷有关。 90年代中后期 ,新的与血栓形成有关的遗传性危险因子不断被发现 ,一方面 ,它强化了危险因素与血栓形成之间可能的病理生理联系 ;另一方面 ,它可被用来作为预测血栓形成或监测高凝状态的生物学指标。1　因子Ⅴ (ＦⅤ )基因多态性与血栓形成　　 1993年 ,Ｄａｈｌｂ ｃｋ等[1] 发现在家族性易栓症 (Ｔｈｒｏｍ ｂｏｐｈｉｌｉａ)患者血浆中存在一种对活化的蛋白Ｃ(ＡＰＣ)抗凝活性耐受…     

凝血因子V缺陷：血栓形成与出血转换的分子基础

第Ｖ因子（ＦＶ）是分子量为３３０ＫＤ的单链糖蛋白,与第Ⅷ因子（ＦⅧ）及铜蓝蛋白之间在结构上有许多相似之处,ＦＶ参与凝血过程中的凝血酶的生成,促进凝血,同时,活化的蛋白Ｃ（ＡＰＣ）通过灭活ＦＶ来抑制凝血,ＦＶ陷根据产生机制不同可导致对ＡＰＣ的抵抗（ＡＰＣＲＥ）引起血栓病或遗传性ＦＶ缺乏症,导致出血,目前已证实部分ＡＰＣＲ和遗传性ＥＦＶ缺乏症均存在分子缺陷,本文主要对ＦＶ的结构,功能,活化蛋白Ｃ抵抗及     

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。     

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。     

妊娠妇女活化蛋白C抵抗与狼疮抗凝物和抗心磷脂抗体的关系

目的 探讨妊娠期妇女血浆中活化蛋白C抵抗(APCR)现象发生的原因及其与凝血因子V(FV)基因多态性、狼疮抗凝物(LA)、抗心磷脂抗体IgG(ACA IgG)的关系.方法 对50例健康非孕妇女、50例健康妊娠妇女、30例妊娠高血压综合征(下称妊高征)患者及20例习惯性流产患者应用多聚酶链反应-限制性内切酶长度多态性分析法对FV基因多态性进行分析,同时用APTT-APC法、简化的单管稀释蝰蛇毒时间测定法(DVVT)以及酶联免疫吸附双抗体夹心法检测APCR敏感性比值、LA水平及ACA IgG含量.结果 健康非孕妇女正常化APCR敏感指数的比值(n-APCR-SR)为0.95±0.16,以n-APCR-SR＜0.63为APCR阳性标准,则对照组、妊娠组、妊高征组及习惯性流产组APCR阳性率分别为4.0%、44.0%、40.0%、35.0%.各组研究对象均未检测到FV基因突变.发现LA及ACA Ig G阳性率与APCR阳性率在妊娠各组有较好的一致性.结论 本研究发现APCR是妊娠期高凝状态的一个危险因素,但并非由FV基因点突变引起;LA和ACA Ig G可能是获得性APCR的一个重要原因.     

Search for mutations in the genes for coagulation factors V and VIII with a possible predisposition to activated protein C resistance

A total of 74 non-pregnant women with a previous episode of thrombosis were investigated for activated protein C (APC) resistance in the aPTT-based and factor IXa-X-based assays and for the presence of mutations in all APC-cleavage sites in the heavy chains of factor V and factor VIII. DNA fragments were amplified with the polymerase chain reaction (PCR) and those encoding for the Arg-306 and Arg-506 (factor V) and for Arg-740 (factor VIII) cleavage sites were subjected to restriction enzyme analysis. DNA fragments of 29 selected patients corresponding to the Arg-306 and Arg-679 cleavage sites in factor V, and to the Arg-336 and Arg-562 cleavage sites in factor VIII were sequenced. APC resistance was found in 40 cases, using the aPTT-based assay and in 35, using the factor IXa-X-based assay (23 patients were APC resistant in both assays), whereas 22 individuals had a normal response to APC. Forty-three patients carried Arg-506 to Gln mutation in factor V. No other polymorphism or mutation was found in the genes for factors V or VIII in the vicinity of the APC cleavage sites. It was concluded that the difference in response to APC in the two assays may not be explained by the presence of mutations in the APC cleavage sites of factor V and factor VIII in this group of patients. The data do not exclude the presence of mutations elsewhere in the factor V or factor VIII genes.     

Detection of a common mutation in factor V gene responsible for resistance to activated protein C causing predisposition to thrombosis

Hereditary predisposition to thrombosis due to activated protein C resistance (APCR) has been attributed to a missense mutation in the factor V gene at nucleotide 1691 (G to A), causing replacement of arginine at codon 506 with glutamine. Using an RFLP-PCR assay to detect this mutation, we measured a prevalence of 3.3% in healthy Caucasians and 1.25% in healthy African-Americans. In addition, we evaluated a total of 90 consecutive specimens submitted to the coagulation laboratory at the Medical College of Virginia for the presence of this mutation. We compared our results for 78 of these specimens with the values measured by a modified partial thromboplastin assay, the COATEST. Twelve of the 90 samples could not be tested using the COATEST because the patients were undergoing anticoagulant therapy. One of the latter 12 specimens was positive by the RFLP-PCR test. Using the genetic test as the definitive assay and the cutoff value established for distinguishing between normal and abnormal results by the COATEST, the COATEST had a sensitivity of 50% and specificity of 93% for the detection of factor V mutation. Analysis of the 90 samples stratified by ethnic groups revealed a frequency of mutation of 13.3% for Caucasians and 6.88% for African-Americans, although with the present sample size, the difference was not statistically significant. Although the COATEST is technically simpler to perform than the genetic test for diagnosing the presence of the factor V mutation, its use for this purpose is limited due to low sensitivity. Thus where this disorder is clinically suspected, submission of the specimen directly for genetic testing by RFLP-PCR or equivalent assay should be considered. J. Clin. Lab. Anal. 11:328–335, 1997. © 1997 Wiley-Liss, Inc.     

The discovery of activated protein C resistance

Summary.  Venous thrombosis is a multifactorial disease, with the pathogenesis involving genetic and environmental risk factors. The most common genetic risk factor known to date is a single point mutation in the gene of coagulation factor V (FV), which results in the replacement of Arg506 with Gln (FV Leiden). Arg506 is one of several cleavage sites in FV for anticoagulant activated protein C (APC) and the mutation results in the loss of the cleavage site. Via a complicated series of reactions, this results in impaired APC-mediated degradation of both FVa and FVIIIa. The associated hypercoagulable condition, which causes a lifelong increased risk of thrombosis, is known as APC resistance. APC resistance was discovered in my laboratory in the late 1980s and we published the first report almost exactly 10 years ago. This started an avalanche of research activities and several thousand articles have since been published on this topic. Analyses for APC resistance and FV Leiden have made their way into clinical medicine and are now performed routinely all over the world. I have been asked to write a personal historical annotation about the discovery of APC resistance, the early research activities and the rapid progress in this field.     

血管内皮生长因子基因多态性与复发性自然流产的相关性研究

目的：探讨血管内皮生长因子基因多态性（VEGF-1154 G/A）与中国南方妇女复发性自然流产（RSA）的相关性。方法：采用等位基因特异性扩增-聚合酶链反应法,检测271例RSA患者（RSA组）和250名健康妇女VEGF-1154 G/A多态位点基因型频率分布情况。结果：RSA组和对照组VEGF-1154 G/A的3种基因型频率（GA、GG、AA）分别为39.5%、55.0%、5.5%和30.0%、67.2%、2.8%,两组3种基因型频率分布不相同,比较差异有统计学意义（P〈0.05）;RSA组和对照组等位基因频率（G、A）分别为74.7%、25.3%和82.2%、17.8%,携带A等位基因发生RSA的风险大于携带G等位基因（OR=1.562,95%CI1.157～2.109,P〈0.01）。结论：VEGF-1154G/A多态性与中国南方妇女RSA的发病风险相关,携带A等位基因的孕妇发生RSA的危险性较高。     

Does activated protein C‐resistant factor V contribute to thrombin generation in hemophilic plasma?

In this study we assessed the role of factor V (FV) inactivation in hemophilic plasma with particular reference to the activated protein C (APC)-resistant variants FV-R506Q (FV Leiden) and FV-R306T (FV Cambridge). Purified recombinant full-length FV carrying these single substitutions and FV-R306T/R506Q were used in thrombin generation experiments. Plasma was first immunodepleted of FV, and subsequently of factors VIII, IX, or combinations thereof. Thrombin generation was initiated by low concentrations of recombinant tissue factor. Recombinant soluble thrombomodulin (TM) was used to trigger the APC system. Surprisingly, TM concentrations that reduced thrombin generation in normal plasma by no more than 50% virtually abolished thrombin formation in plasma deficient in the factor VIII/IX complex. This was already apparent at TM levels as low as 0.1 nmol L(-1). By varying the concentrations of purified (activated) protein C to plasma that was additionally depleted of protein C, we confirmed that impaired thrombin generation indeed was the result of the action of APC. In contrast, this did not occur when FV-depleted plasma had been reconstituted with FV-R306T/R506Q. Addition of FV-R306T or FV-R506Q partially reduced prothrombin activation, demonstrating the involvement of both APC cleavage sites. FV inactivation also occurred on the surface of human microvascular endothelial cells. Apparently, these cells express sufficient TM to down-regulate thrombin production via the APC pathway. We further conclude that in hemophilic plasma this pathway can induce a secondary defect because of premature FV inactivation. It therefore seems conceivable that APC-resistant FV has the potential of alleviating hemophilic bleeding.     

The differential association of conjugated equine estrogen and esterified estrogen with activated protein C resistance in postmenopausal women

OBJECTIVES: Clinical trials have demonstrated that oral conjugated equine estrogen (CEE) therapy with or without medroxyprogesterone (MPA) increases venous thrombotic risk but this safety issue has not been investigated for other oral estrogens. Based on observational study findings that esterified estrogen (EE) was not associated with venous thrombotic risk whereas CEE was, we hypothesized that CEE users would be more resistant to activated protein C (APC), a prothrombotic phenotype, than EE users. METHODS: We conducted an observational, cross-sectional study of postmenopausal women 30-89 years old who were controls in a case-control study of venous thrombosis. Use of CEE, EE, and MPA at the time of phlebotomy was determined using computerized pharmacy records. APC resistance was measured in plasma by the endogenous thrombin potential normalized APC sensitivity ratio. Adjusted mean APC resistance values were compared across estrogen type and CEE:EE ratios are presented. RESULTS: There were 119 CEE and 92 EE users at the time of phlebotomy. Compared with EE users, CEE users had APC resistance measures that were 52% higher (1.52; 95% confidence intervals: 1.07-2.17) in adjusted analyses. Restricting to modal dose users (0.625 mg) and stratifying by MPA use did not materially change associations. CONCLUSIONS: CEE use was associated with higher levels of APC resistance when compared with EE use in postmenopausal women. These findings might provide an explanation for the higher risk of venous thromboembolism previously observed with CEE compared with EE use and, if replicated, may have safety implications for women when choosing an estrogen for symptom relief.