Thrombotest mixing experiments in congenital coagulation disorders of the prothrombin complex and in coumarin treated patients. An additional evidence against the presence of an inhibitor in the latter.

Thrombotest clotting times of mixtures of coumarin plasmas and normal plasma yielded a patterm similar to that observed in mixtures of plasma with congenital coagulation disorders and normal plasma. The presence of 10 or 20% of test plasma in the mixture failed to affect the clotting times which resulted in normal limits. The only exception to this rule was the hemophilia BM plasma. In this case even the presence of 10-20% of patient plasma in the mixture caused a prolongation of the clotting time. This indicates that no inhibitor is present in coumarin plasmas and in the plasma of congenital coagulation disorders of the prothrombin complex save for hemophilia BM plasma which does contain an inhibitor.     

人凝血酶原复合物生产过程中凝血因子活化分析

目的通过比较以组分Ⅲ沉淀和血浆为原料制备人凝血酶原复合物(Prothrombin complex concentrates,PCC)过程中凝血因子活化情况,为选择最适PCC制备原料提供数据支持。方法分别对以组分Ⅲ沉淀和血浆为原料制备PCC过程中中间品的活化的凝血因子活性和人凝血酶活性两个项目进行检定,分析凝血因子的活化情况。观察以组分Ⅲ沉淀为原料制备PCC过程中添加肝素能否抑制PCC中凝血因子的活化。结果以组分Ⅲ沉淀为原料制备的PCC中间品活化的凝血因子活性和人凝血酶活性两个项目均不合格。以组分Ⅲ沉淀为原料制备PCC生产过程中添加肝素后,PCC中间品的活化的凝血因子活性和人凝血酶活性均不合格。以血浆为原料制备的PCC中间品活化的凝血因子活性和人凝血酶活性两个项目均合格。结论组分Ⅲ沉淀为原料制备PCC会增加凝血因子活化的风险,新鲜冰冻血浆可作为制备PCC的原料。     

人凝血酶原复合物在制备过程中凝血因子活性的检测和分析

目的对人凝血酶原复合物在制备工艺中凝血因子效价进行检测和分析。方法对PCC制备过程中的血浆、S/D病毒灭活、冻干工艺、干热病毒灭活前和后分别取样,检测分析凝血因子Ⅱ、Ⅶ、Ⅸ和Ⅹ效价,分别分析S/D病毒灭活、冻干工艺、干热病毒灭活前和后凝血因子效价的变化情况。分析PCC干热病毒灭活后的样品中四种凝血因子的效价比例。结果三批血浆中凝血因子Ⅱ、Ⅶ、Ⅸ和Ⅹ的活性在0.8~1.2 IU/m L之间。S/D病毒灭活前后PCC冻干工艺前后PCC中四种凝血因子活性无明显变化,而干热病毒灭活后PCC中四种凝血因子活性均有明显下降。三批PCC中四种凝血因子的比例基本一致,但是凝血因子Ⅶ效价较低,凝血因子Ⅱ效价偏高。结论通过PCC制备工艺中凝血因子效价的检测和分析可实现良好的质量控制。     

凝血酶原复合物中影响激活凝血因子测定因素的探讨

含肝素的凝血酶原复合物（ＰＣＣ）用适量的硫酸鱼精蛋白中和后,作１：１０和１：１００稀稀测定激活凝血因子。肝素影响激活凝血因子测定,不同活性单位的ＰＣＣ对某些厂家生产的ＰＣＣ激活凝血因子测定无影响,但对有厂家生产的ＰＣＣ激活凝血因子测定有影响。对国内５家血制品生产单位生产的１５批ＰＣＣ进行激活疑血因子测定,凝血酶活性通过者激活凝血因子也能通过。     

凝血酶原复合物中肝素含量的测定

凝血酶原复合物按标示量加蒸馏水溶解成１０ＰＥ／ｍｌ。通过加入适量的硫酸鱼精蛋白中和凝血酶原复合物样品中肝素。取中和后的样品加入缺血小板血浆、脑磷脂和氯化钙,记录凝固时间,挑选凝固时间最短的样品管计算肝素含量。该方法误差２０％。以５家血制品生产单位生产的２４批ＰＣＣ肝素含量的测定,按ＰＣＣ效价标示量小于等于１３０％计算,不合格６批占２５％。     

Identification of distinct sequences in human blood coagulation factor Xa and prothrombin essential for substrate and cofactor recognition in the prothrombinase complex

To identify amino acid sequences in factor Xa (fXa) and prothrombin (fII) that may be involved in prothrombinase complex (fXa.factor Va.fII.phospholipids) assembly, synthetic peptides based on fXa and fII sequences were prepared and screened for their ability to inhibit fXa-induced clotting of normal plasma. One fII peptide (PT557-571 homologous to chymotrypsin (CHT) residues 225-239) and two fXa peptides (X404-418, CHT231-244, and X415-429, CHT241-252C) potently inhibited plasma clotting and prothrombinase activity with 50% inhibition between 41 and 115 microM peptide. Inhibition of prothrombinase by PT557-571 and X415-429 was fVa-independent, whereas the inhibition by X404-418 was fVa-dependent. X404-418 inhibited the binding of fVa to fluorescein-labeled, inhibited fXai in the presence of phosphatidylcholine/phosphatidylserine vesicles, whereas X415-429 inhibited binding of fII to phospholipid-bound fluorescein-labeled, inhibited fXai. PT557-571 altered the fluorescence emission of fluorescein-labeled fXai, showing that PT557-571 binds to fXai. These data suggest that residues 404-418 in fXa provide fVa binding sites, whereas residues 557-571 in fII and 415-429 in fXa mediate interactions between fXa and fII in the prothrombinase complex.     

Liver function disorders and blood coagulation disorders in the course of EPH gestosis]

Three cases of EPH-gestosis complicated with liver function and blood coagulation disorders (HELLP syndrome) are presented. The most frequent diagnostic errors and subsequent risk for both mother and fetus have also been discussed. Basing on the available literature, the safest management of pregnancy and delivery is critically assessed.     

Comparison of prothrombin complex concentrate and vitamin K1 in oral anticoagulant reversal.

A randomised clinical trial was undertaken to compare the value of a factor II, IX, and X concentrate (Prothromplex) with intravenous vitamin K1 (2-5 mg) in reversing an overdose of oral anticoagulants. Rapid partial correction of the prothrombin time, partial thromboplastin time, and the clotting factor assays were observed with the concentrate, but these changes were not always sustained. In contrast vitamin K1 did not show any great effect at two hours but at 24 hours there was always over-correction despite the conservative dosage, prothrombin times being shorter than the therapeutic range. The prothrombin complex concentrate provides a quicker, more controlled but less sustained method of reversing the coumarin defect than vitamin K1. But there remains a significant risk of hepatitis even with a preparation for which strenuous efforts have been made to minimise this risk by screening for hepatitis B virus. The risk should be carefully considered before such concentrates are infused in non-urgent conditions.     

Update and perspectives on congenital disorders of glycosylation.

Defects in nine genes of the N-linked glycosylation pathway cause congenital disorders of glycosylation (CDGs) and serious medical consequences. Although glycobiology is seldom featured in a general medical education, an increasing number of physicians are becoming acquainted with the field because it directly impacts patient diagnosis and care. Medical practice and attitudes will change in the postgenomic era, and glycobiology has an opportunity to be a cornerstone of part of that new perspective. This review of recent developments in the CDG field describes the biochemical and molecular basis of these disorders, describes successful experimental approaches, and points out a few perspectives on current problems. The broad, multisystemic presentations of these patients emphasize that glycobiology is very much a general medical science, cutting across many traditional medical specialties. The glycobiology community is well poised to provide novel perspectives for the dedicated clinicians treating both well-known and emerging human diseases.     

Effects of prothrombin on the individual activated protein C-mediated cleavages of coagulation factor Va

The factor Va (FVa) inactivation by activated protein C (APC), mediated by cleavages at Arg306 and Arg506 in FVa, is inhibited by both factor Xa (FXa) and prothrombin. Although FXa is known to specifically inhibit the Arg506 cleavage, the effect of prothrombin has not been confined to one cleavage site. We used recombinant FV variants, FV:R506Q/R679Q and FV:R306Q/R679Q, to investigate the effect of prothrombin on the individual cleavage sites. The APC-mediated FVa inhibition was monitored by a prothrombinase-based FVa assay, and apparent first order rate constants were calculated for each of the cleavage sites both in the presence and absence of prothrombin. Prothrombin impaired cleavages at both Arg306 and Arg506 and the inhibition correlated with a delayed appearance of proteolytic products on Western blots. Almost complete inhibition was obtained at around 3 microm prothrombin, whereas half-maximal inhibition was obtained at 0.7 microm prothrombin. After cleavage of prothrombin by thrombin, the inhibitory activity was lost. The inhibitory effect of prothrombin on APC-mediated inhibition of FVa was seen both in the presence and absence of protein S, but in particular for the Arg306 sites, it was more pronounced in the presence of protein S. Thus, prothrombin inhibition of APC inactivation of FVa appears to be due to both impaired APC function and decreased APC cofactor function of protein S. In conclusion, FVa, being part of the prothrombinase complex, is protected from APC by both FXa and prothrombin. Release of products of prothrombin activation from the prothrombinase complex would alleviate the protection, allowing APC-mediated inactivation of FVa.