Role of the N-terminal EGF module of coagulation factor IX in activation of factors IX and X

Absence or reduced activity of coagulation factor IX (FIX) causes the severe bleeding disorder haemophilia B. FIX contains a Gla module, two epidermal growth factor-like (EGF) modules, and a serine protease region. I characterized a monoclonal antibody and found that it recognizes an epitope around residues 72 and 80 in the C-terminal part of EGF1 in human FIX. The antibody exhibited 10-fold greater affinity for activated FIX (FIXa) than for the zymogen FIX, indicating the existence of intra-molecular communication between the serine protease region and EGF1. Binding of the antibody did not affect the amidolytic activity of FIXa, hence I could use the antibody during activation of FIX to show that the C-terminal part of EGF1 is of importance for the interaction with FXIa but not with FVIIa/TF. Considering activation of FX, it is a matter of debate whether EGF1 or FIXa interacts directly with FVIIIa. I activated FX in the presence and absence of the antibody and/or FVIIIa. The addition of antibody caused only a minor decrease in k cat,app , and the major increase in k cat,app caused by the addition of FVIIIa occurred even in the presence of the antibody. This implies that EGF1 of FIXa is not directly involved in interaction with FVIIIa in the Xase complex. A model of the FIXa-FVIIIa complex, based on my findings and results from the literature, was constructed and indicated that EGF1 of FIXa does not interact directly with FVIIIa.     

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.     

The P-selectin, tissue factor, coagulation triad

The primary importance of tissue factor (TF) in blood coagulation and thrombus propagation has been recognized for many years. Nevertheless, our view about the origin of TF activity, necessary for normal hemostasis and found in pathologic conditions, needs to be revised in the light of recent observations. Pioneering work by Yale Nemerson's group showed that circulating TF on microparticles (MPs), could promote thrombus growth. The origin and characteristics of this 'blood-borne' TF are targets of intense research as well as intense debate. Surprising observations now implicate the adhesion receptor P-selectin (P-sel), known for its role in inflammation, in these MPs' generation. P-sel, translocated from granules to the cell surfaces of activated platelets and endothelial cells, was recently found to play multiple roles in hemostasis. Expressed on endothelium, it can mediate platelet rolling. Signaling by P-sel through its receptor on leukocytes, P-selectin glycoprotein ligand 1 (PSGL-1), induces the generation of TF-positive, highly procoagulant MPs. In addition, P-sel on activated platelets helps to recruit these MPs specifically to thrombi. In this review, we discuss the roles of P-sel and TF-positive MPs and highlight strategies to modulate hemostasis by modulating the P-sel, TF, coagulation triad.     

Standardization of assays of factor VIII and factor IX

Summary The development of international standards over the last 15–20 years has led to improved interlaboratory agreement on assays of factor VIII and factor IX. In the most recent international collaborative study, the coefficient of variation for one-stage assays (26 laboratories) was 5.6%. However, in quality assurance surveys, carried out in the UK and USA, agreement between laboratories is much less good, with coefficients of variation ranging from 30% to over 50%. Improvements in agreement between clinical laboratories could be obtained by increasing the amount of testing on each sample, especially the number of dilutions, and reducing the number of reagent systems used. A large number of laboratories now use immunodepleted plasmas instead of congenitally deficient plasmas as substrates for one-stage assays. These plasmas may give satisfactory assays, but many of them have not been thoroughly evaluated in comparison with congenitally deficient plasma. In assessment of potency of very high purity (VHP) factor VIII concentrates, some immunodepleted plasmas were found to give lower potencies than hemophilic plasma. This is partly due to the fact that VHP concentrates contain little or no von Willebrand factor (vWF), and most immunodepleted plasmas are also deficient in vWF. In recent collaborative studies, assays of VHP factor VIII concentrate were much more variable, both within and between laboratories, than assays of intermediate purity concentrates. Standardization of these new products will require careful attention to methodological detail. Presented at the ‘2nd International Symposium on Standardization and Quality Control of Coagulation Tests: Implications for the Clinical Laboratory’, Rome, September 28–29, 1989.     

Specificity of coagulation factor signaling

Summary.  Coagulation serine proteases signal through protease-activated receptors (PARs). Thrombin-dependent PAR signaling on platelets is essential for the hemostatic response and vascular thrombosis, but regulation of inflammation by PAR signaling is now recognized as an important aspect of the pro- and anti-coagulant pathways. In tissue factor (TF)-dependent initiation of coagulation, factor (F) Xa is the PAR-1 or PAR-2-activating protease when associated with the transient TF–FVIIa–FXa complex. In the anticoagulant protein C (PC) pathway, the thrombin–thrombomodulin complex activates PC bound to the endothelial cell PC receptor (EPCR), which functions as a required coreceptor for activated PC-mediated signaling through endothelial cell PAR-1. Thus, the pro- and anti-inflammatory receptor cascades are mechanistically coupled to immediate cell signaling, which precedes systemic coagulant or anticoagulant effects. In contrast to the substrate-like recognition of PARs by thrombin, TF- or EPCR-targeted activation of PARs generates cell-type specificity, PAR selectivity and protease receptor cosignaling with the G-protein-coupled PAR response. Protease receptors are thus major determinants of the biological outcome of coagulation factor signaling on vascular cells.     

Immunoadsorption for coagulation factor inhibitors

Inhibitors to coagulation factors are among the most difficult problems in the management of coagulation disorders. Most presently available therapy does not assure hemostasis. An extracorporeal immunoadsorption system, which selectively binds IgG, was used to lower inhibitor levels in eight patients on 10 occasions. In this system, separated plasma is delivered to two staphylococcal protein A-Sepharose columns, which are coupled to an elution monitor. Columns are eluted sequentially and regenerated to maximize IgG removal. Successful removal of the inhibitor was accomplished in all six hemophiliacs on seven occasions, as well as in a patient with acquired von Willebrand disease. All patients whose inhibitors were lowered to less than 10 Bethesda units achieved measurable factor levels when factor concentrate replacement was given. Immunoadsorption facilitates efficient removal of inhibitors, which allows factor replacement therapy.     

人血浆凝血因子V双抗体夹心ELISA测定

目的 建立一种新的双抗体夹心酶联免疫吸附试验（ELISA），以测定人血浆凝血因子V（FV）抗原含量。方法 采用兔抗人FV抗体为包被抗体，FV单抗为检测抗体，首次对中国正常人（n=26)和一个遗传性FV缺乏家系的有关成员血浆中的FV抗原进行了检测。结果 所建方法线性范围广、特异性强、灵敏度高、重复性好，与FV活性测定有很好的相关性；正常人血浆FV抗原呈偏态分布；FV缺乏家系纯合子血浆FV抗原量只有正常人的2％，证实该家系为I型遗传性FV缺乏症。结论 该法是一种较好的FV蛋白定量测定法，可以对FV缺乏症进行辅助诊断和分型。     

Continuous infusion of coagulation factor products

OBJECTIVE: To review clinical information related to the use of continuous infusion factor products in patients with hemophilia. Specifically, case reports and open-label trials are summarized involving the use of factor VIII and recombinant factor VIIa for a variety of indications including surgical prophylaxis, acute bleeding, primary prevention, and management of inhibitors. In addition, issues surrounding the use of continuous infusion of factor products such as pharmacokinetic rationale, stability/sterility, and cost are reviewed. DATA SOURCES: Primary and review articles were identified through a MEDLINE search (1990-June 2001) and through secondary sources. STUDY SELECTION AND DATA EXTRACTION: All articles identified from the data sources were evaluated, and all information deemed relevant was included in this review. DATA SYNTHESIS: Data concerning the administration of factor products are primarily detailed in open-label trials and case reports. Comparisons between intermittent bolus injections and continuous infusion of factor products are limited and primarily compare continuous infusion regimens with historical controls. The rationale behind the continuous-infusion approach is linked to the pharmacokinetics of factor products administered via this route. Pharmacokinetic data reveal that, with continuous infusion of factor products, a reduction in clearance and a maintenance of factor serum concentrations are noted. CONCLUSIONS: Administration of factor products (factor VIII and recombinant factor VIIa) via continuous infusion has produced favorable hemostatic effects compared with intermittent bolus injections. The advantages of continuous infusion include maintenance of a constant factor concentration, thereby reducing risk of bleeding from excessively low trough concentrations, and a decrease in factor consumption related to a reduction in factor clearance with constant infusion. Manufacturers recommend using reconstituted factor products either immediately or within 1-3 hours after reconstitution; however, several studies have found the products to be stable and sterile for longer periods.     

Effects of hydroxyethyl starch on blood coagulation, particularly factor VIII

The effects of hydroxyethyl starch (HES) on hemostasis were investigated extensively. In order to simulate acute blood loss due to surgery or trauma, one unit (450 ml) of blood was drawn from normal healthy men. This was followed by a 1-liter infusion over 60 minutes of either 6 percent HES, 5 percent albumin, or 0.9 percent sodium chloride (NaCl) as replacement. Coagulation studies were performed before phlebotomy, before infusion and at 0, 4, 20, 27, and 92 hours following infusion. Following infusion of HES and albumin, plasma fibrinogen and antithrombin-III levels fell slightly due to plasma volume expansion and hemodilution. In subjects receiving HES, partial thromboplastin times (PTTs) were significantly (p less than .05) prolonged and factor VIII activities were significantly (p less than .05) decreased when compared to the albumin and NaCl groups. These findings could not be attributed solely to hemodilution. The effects of HES on PTT and factor VIII could not be correlated with plasma HES levels; neither could they be reproduced in vitro by mixing HES with normal plasma. Mean values of the following studies remained normal after infusion of all replacement fluids: prothrombin time, bleeding time, fibrin monomer, fibrin-fibrinogen degradation products, platelet adhesion, circulating platelet aggregates, and platelet count.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factor Va - factor Xa interactions: molecular sites involved in enzyme:cofactor assembly

The generation of thrombin by the prothrombinase complex is a key event in coagulation. In this complex, the activated form of coagulation factor V (FVa) serves as an essential cofactor to factor Xa (FXa) in the activation of prothrombin to thrombin. The enzyme FXa is virtually ineffective in the absence of its cofactor. The assembly of FXa with its cofactor FVa on negatively charged phospholipid membranes enhances its catalytic efficiency by several orders of magnitude. The non-activated procofactor factor V (FV) circulates in plasma with a domain organization of A1-A2-B-A3-C1-C2 expressing little procoagulant activity. Upon activation through limited proteolysis by either thrombin or FXa, the B-domain dissociates from FVa. After activation, the procoagulant activity of FVa is greatly enhanced. This report provides insight into the interaction of FV and FXa and the molecular events important in enzyme:cofactor assembly of the FXa:FVa complex. Furthermore, light is shed on the molecular events associated with the activation process, i.e. the release of the B-domain and exposure of binding sites for FXa. The assembly of FVa and FXa was studied using a set of recombinant FV mutants. The interaction between FVa and FXa on phospholipid was investigated with a functional prothrombin activation assay as well as in a novel direct binding assay in the absence of prothrombin. We found that all three thrombin cleavages in FV contribute to increasing the FXa affinity and that the B-domain in intact FV has an inhibitory effect on the FV-FXa interaction, which is important in prohibiting premature coagulation.     

Determination of coagulation factor VIII activity by a chromogenic substrate method on STA,an automated coagulation analyzer

This study was aimed at evaluating the performance of a chromogenic factor VIII assay on STA, an automated coagulation analyzer. Additionally, a correlation study was conducted with an aPTT-based one-stage factor VIII clotting assay. Throughout the study the performance of the chromogenic assay was tested in two ranges of factor VIII activity: a high range with activity between 20% and 150% and a low range with activity below 20%. Inter-assay coefficient of variation ranged from 1.9% to 8.9% and intra-assay coefficient of variation from 0.5% to 11.4%, depending on factor VIII concentration. Day-to-day reproducibility was tested over a 5-day period; between-day imprecision ranged from 7.1% to 9.4%. The chromogenic factor VIII assay showed a good correlation with the clotting assay in both ranges. The correlation coefficients were 0.924 and 0.792 for the high and low range, respectively. A statistically significant difference in mean values was observed in the high range (p<0.0001). Comparison between the chromogenic assay on STA versus on microplates showed a high correlation (0.991), which was highly significant (p<0.0001). In conclusion, the chromogenic assay for factor VIII on STA shows good analytical performance. It correlates well with the one-stage factor VIII clotting assay, although significant differences between individual samples occur. Probably these are partly related to differences in measurement principle and standardization. Altogether, this precise and rapid assay is suitable for determination of factor VIII by an automated procedure.     

Determination of coagulation factor VIII activity by a chromogenic substrate method on STA, an automated coagulation analyzer

This study was aimed at evaluating the performance of a chromogenic factor VIII assay on STA, an automated coagulation analyzer. Additionally, a correlation study was conducted with an aPTT-based one-stage factor VIII clotting assay. Throughout the study the performance of the chromogenic assay was tested in two ranges of factor VIII activity: a high range with activity between 20% and 150% and a low range with activity below 20%. Inter-assay coefficient of variation ranged from 1.9% to 8.9% and intra-assay coefficient of variation from 0.5% to 11.4%, depending on factor VIII concentration. Day-to-day reproducibility was tested over a 5-day period; between-day imprecision ranged from 7.1% to 9.4%. The chromogenic factor VIII assay showed a good correlation with the clotting assay in both ranges. The correlation coefficients were 0.924 and 0.792 for the high and low range, respectively. A statistically significant difference in mean values was observed in the high range (p < 0.0001). Comparison between the chromogenic assay on STA versus on microplates showed a high correlation (0.991), which was highly significant (p < 0.0001). In conclusion, the chromogenic assay for factor VIII on STA shows good analytical performance. It correlates well with the one-stage factor VIII clotting assay, although significant differences between individual samples occur. Probably these are partly related to differences in measurement principle and standardization. Altogether, this precise and rapid assay is suitable for determination of factor VIII by an automated procedure.     

A guide to murine coagulation factor structure, function, assays, and genetic alterations

Murine blood coagulation factors and function are quite similar to those of humans. Because of this similarity and the adaptability of mice to genetic manipulation, murine coagulation factors and inhibitors have been extensively studied. These studies have provided significant insights into human hemostasis. They have also provided useful experimental models for evaluation of the pathophysiology and treatment of thrombosis. This review contains recommendations for obtaining, processing and assaying mouse blood hemostatic components, and it summarizes the extensive literature on murine coagulation factor structure and function, assays and genetic alteration. It is intended to be a convenient reference source for investigators of hemostasis and thrombosis.