Chromogenic assay for functional determination of TFP

Due to the manifold functions of tissue factor (TF), also tissue factor pathway inhibitor (TFPI), its endogenous antagonist, is of interest. For the determination of TFPI, either its antigen concentration or its activity can be measured. METHOD: A chromogenic assay for the functional determination of TFPI is presented and modified for its application in the routine laboratory. Its suitability for daily use was evaluated by testing more than 7000 plasma samples from a heparin study in healthy volunteers. In addition, our data confirm that the method records the activity of both free- and lipoprotein-associated TFPI. CONCLUSION: The chromogenic assay proved to be rapid and easy to perform and features a high reproducibility. Due to excellent correlation of the results with the total TFPI antigen concentrations, the method represents a more rapid and economic alternative to the determination of total TFPI antigen by ELISA in plasma samples after heparin application.     

Disposition of tissue factor pathway inhibitor during cardiopulmonary bypass

BACKGROUND: The tissue factor (TF) factor (F) VIIa complex activates coagulation FIX and FX to initiate coagulation, and also cleaves protease activated receptors (PARs) to initiate inflammatory processes in vascular cells. Tissue factor pathway inhibitor (TFPI) is the only specific inhibitor of the TF-FVIIa complex, regulating both its procoagulant and pro-inflammatory properties. Upon heparin infusion during cardiopulmonary bypass (CPB), a heparin releasable pool of endothelial associated TFPI circulates in plasma. Following protamine neutralization of heparin, the plasma TFPI level decreases, but does not return completely to baseline, suggesting that during CPB a fraction of the plasma TFPI becomes heparin-independent. We have investigated the structural and functional properties of plasma TFPI during CPB to further characterize how TFPI is altered during this procedure. METHODS: We enrolled 17 patients undergoing first-time cardiac surgery involving CPB. Plasma samples were obtained at baseline, 5 min and 1 h after start of CPB (receiving heparin), 10 min after protamine administration (off CPB) and 24 h following surgery. Samples were analyzed for full-length and free (non-lipoprotein bound) TFPI antigen by enzyme-linked immunosorbent assay (ELISA) and for TFPI anticoagulant activity using an amidolytic assay. Western blot analysis was used to identify TFPI species of varying molecular weights in three additional patients. Dunnett's test for post hoc comparisons was used for statistical analysis. RESULTS: The ELISA and Western blot data indicated that an increase in full-length TFPI accounted for most of the heparin releasable TFPI. Following heparin neutralization with protamine, the full-length TFPI antigen returned to baseline levels while the free TFPI antigen and the total plasma TFPI activity remained elevated. This was associated with the appearance of a new 38 kDa form of plasma TFPI identified by Western blot analysis. The 38 kDa form of TFPI did not react with an antibody directed against the C-terminal region of TFPI indicating it has undergone proteolysis within this region. All TFPI measurements returned to baseline 24 h following CPB. CONCLUSIONS: During CPB the full-length form of TFPI is the predominant form in plasma because of its prompt release from the endothelial surface following heparin administration. Upon heparin neutralization with protamine, full-length TFPI redistributes back to the endothelial surface. However, a new 38 kDa TFPI fragment is generated during CPB and remains circulating in plasma, indicating that TFPI undergoes proteolytic degradation during CPB. This degradation may result in a decrease in endothelium-associated TFPI immediately post-CPB, and may contribute to the procoagulant and proinflammatory state that often complicates CPB.     

组织因子途径在急性心肌梗死患者发作期间的比值变化及其意义

本研究旨在观察急性心肌梗死（AMI）患者发作期间组织因子途径（TFP）的变化及其意义。采用手工法检测血浆复钙时间,酶联免疫吸附法（ELISA）检测血浆中的组织因子（TF）、组织因子途径抑制物（TFPI）、凝血因子Ⅶ抗原（FVU：Ag）、激活的FⅦ（FⅦa）及D-二聚体（D—dimer）,采用发色底物法检测TF活性（TFact）;采用一期凝固法测定凝血因子Ⅶ促凝活性（FⅦ：C）。观察对象包括59名AMI患者和84名健康中老年人。结果表明,①AMI患者血浆复钙时间比正常对照者明显缩短（p〈0．01）;②与对照组相比,AMI患者血浆中TF活性无显著增高（p〉0．05）;TF与TFPI的抗原均显著增加（p〈0．01）,但TF增高程度较TFPI更为显著,故TF／TFPI比值升高;总TFPI（t-TFPI）和全长TFPI（fl-TFPI）含量明显增加（p〈0．01）,截短TFPI（tr-TFPI）含量明显降低（p〈0．01）,TF／t—TFPI比值高于正常组,TF／fl—TFPI比值低于正常组（p〈0．05）,但在AMI组TF／tr-TFPJ与fl—TFPL／t—TFPI比值明显高于对照组（p〈0．01）,而tr—TFPUt—TFPI与tr—TFPL／fl-TFPI比值降低（p〈0．01）;③与对照组比较,血浆FⅦ：C、FⅦa在AMI组是增高的（p〈0．05）,FⅦ：Ag无显著性变化,FⅦa／FⅦ：Ag比值明显升高（p〈0．01）,但FⅦa／FⅦ：C及FⅦ：C／FⅦ：Ag比值并无显著性差异（p〉0．05）;④与正常对照组相比,AMI组D—dimer明显增高（P〈0．01）。结论：AMI发作期间体内TFP被启动,提示AMI患者血液呈现高凝状态,为评估AMI及其危险事件的发生,以多因子同时测定为好,而且用相对比值表示高凝状态与危险因子比单测更为可靠。     

Automated amidolytic method for determining heparin, a heparinoid, and a low-Mr heparin fragment, based on their anti-Xa activity

Using the chromogenic substrate S-2222, we have optimized and automated an amidolytic assay for heparin. The assay is based on the detection of anti-Xa activity generated by heparin in plasma. The method is reproducible (intra- and interassay CVs of 2.4 and 3.3%, respectively) and reliable in antithrombin III-deficient plasma. Results of this assay, obtained for plasma samples from patients and volunteers treated with heparin, correlate well (r = 0.899) with those of the test for activated partial thromboplastin time. Upon administration of a low-Mr heparinoid (Org 10172) and heparin fragment ( Kabi 2165), however, the activated partial thromboplastin time failed to detect anticoagulant activity, whereas the chromogenic heparin assay revealed anti-Xa activity. This automated amidolytic assay for heparin is therefore suitable not only for monitoring standard therapy with heparin but also for measuring the activity of recently developed heparin fractions.     

Regulation of TFPI function by protein S

Summary.  Protein S is an anticoagulant cofactor of full-length tissue factor pathway inhibitor (TFPI) that facilitates optimal factor Xa-inhibition and efficient down-regulation of thrombin generation in plasma. Protein S and TFPI are constitutively active in plasma and therefore provide an effective anticoagulant barrier against unwanted procoagulant activity in the circulation. In this review, we describe the current status on how TFPI-activity depends on protein S, and show that TFPI and protein S are major regulators of thrombin generation both in the absence and presence of activated protein C (APC). As there is covariation of plasma TFPI and protein S levels both in health and in disease, these findings suggest that the risk of venous thrombosis associated with protein S deficiency states might be in part explained by the accompanying low plasma TFPI levels.     

Poor anticoagulant response to tissue factor pathway inhibitor in patients with venous thrombosis

Summary.  Tissue factor pathway inhibitor (TFPI) is of major importance in regulating the coagulation triggering effects of tissue factor. An association between TFPI deficiency and thrombosis has still not been clearly demonstrated. We evaluated the anticoagulant activity of exogenous TFPI added either to the plasma of patients with venous thrombosis ( n  = 118) or to the plasma of healthy controls similar in terms of mean age and sex ratio ( n  = 107). A poor anticoagulant response to TFPI, defined as TFPI resistance, was observed in 4.7% of controls and in 11.0% of patients. TFPI resistance was associated with an almost threefold increase in the risk of thrombosis and could therefore represent a novel hemostatic risk factor for venous thrombosis.     

急性脑梗死发作期间组织因子途径改变的观察

目的 :观察急性脑梗死 (ACI)与组织因子途径变化以及与该途径有关的其他凝血因子的关系。方法 :71例经 CT确诊为 ACI患者和 5 0名正常人被纳入研究范围。血浆中组织因子 (TF)和组织因子途径抑制物(TFPI)活性测定采用发色底物法 ;抗原测定用酶联免疫吸附法 (ELISA) ;血浆中 F 促凝活性 (F ∶C)和F 促凝活性 (F ∶C)测定用一期凝固法 ;凝血酶原 (F )的活性测定采用 Ecarin法 ;纤维蛋白原 (Fbg)的活性测定采用凝血酶法 ;抗凝血酶 (AT )的测定用肝素辅因子法。结果 :与正常对照组比较 ,ACI患者血浆中TF活性显著增加 (P<0 .0 5 ) ,TF抗原含量显著增加 (P<0 .0 5 ) ,TFPI的活性降低 (P<0 .0 5 ) ,TFPI抗原含量明显降低 (P<0 .0 5 ) ;血浆 F ∶ C显著增加 (P<0 .0 1) ,血浆 F ∶ C明显下降 (P<0 .0 5 ) ,F 活性显著增加(P<0 .0 1) ,Fbg的活性显著增加 (P<0 .0 1) ;AT 活性显著降低 (P<0 .0 1)。结论 :ACI的发生与组织因子途径的启动有关 ,在 ACI早期患者血液呈高凝状态     

Increased tissue factor pathway inhibitor activity is associated with myocardial infarction in young women: results from the RATIO study

Summary. Background: The tissue factor pathway inhibitor (TFPI)/protein S anticoagulant system is a potent inhibitor of blood coagulation. TFPI and protein S are major determinants of thrombin generation (TG) tests determined at low tissue factor (TF) and at high TF concentrations in the presence of activated protein C (APC). Both TFPI and protein S protect against venous thrombosis, but the importance of the TFPI/protein S system in arterial thrombosis remains unclear. Objectives: To investigate the influence of the TFPI/protein S anticoagulant system on the risk of myocardial infarction (MI) in young women. Methods: The RATIO study is a case–control study in women under 50 years of age, including 205 patients and 638 controls. TFPI and protein S were quantified using ELISA. The TFPI/protein S activity (nTFPIr) and the APC sensitivity ratio (nAPCsr) were determined using TG tests. Odds ratios (ORs) adjusted for putative confounders and corresponding 95% confidence intervals (95% CI) were determined. Results: Women with MI had higher TFPI levels than controls (135.9 ± 40% vs. 124.2 ± 41%), resulting in increased TFPI/protein S activities and increased APC sensitivity. Furthermore, an increased TFPI activity was associated with MI [nTFPIr: adjusted OR Q1 vs. Q4 = 2.1 (95%CI 1.1–4.1)]. Additionally, an increased APC sensitivity was associated with MI [nAPCsr: adjusted OR Q1 vs. Q4 = 1.7 (95% CI 0.9–3.2)] Conclusion: Women with MI had increased TFPI levels compared with controls. Consequently, the TFPI/protein S activity and APC sensitivity are increased in women with MI. Whether this increase in TFPI activity acts as a compensating mechanism for an increased procoagulant state or is a marker of endothelial damage remains to be investigated.     

Thrombin generation‐based assays to measure the activity of the TFPI–protein S pathway in plasma from normal and protein S‐deficient individuals

Summary. Background: Protein S acts as a cofactor for full‐length tissue factor pathway inhibitor (TFPI) in the downregulation of thrombin formation. Objective: To develop a functional test to measure the activity of the TFPI–protein S system in plasma. Methods/Patients: Using calibrated automated thrombography, we quantified the activity of the TFPI–protein S system in plasma by measuring thrombin generation in the absence and presence of neutralizing antibodies against protein S or TFPI. Moreover, we designed an enzyme‐linked immunosorbent assay (ELISA) to determine the level of full‐length TFPI in plasma. The performance of these assays was examined in plasma from 85 normal individuals and from 35 members of protein S‐deficient families. Results: The ratio of thrombin peaks determined in the absence and presence of anti‐protein S antibodies (protein S ratio = 0.5 in normal plasma) is a measure of the TFPI cofactor activity of protein S, whereas the ratio of thrombin peaks determined in the absence and presence of anti‐TFPI antibodies (TFPI ratio = 0.25 in normal plasma) is a measure of the overall activity of the TFPI–protein S system. Protein S and TFPI ratios were elevated in protein S‐deficient individuals, indicating an impairment of the TFPI–protein S system. Both ratios correlated well with full‐length TFPI levels, which were significantly lower in protein S‐deficient patients than in normal family members. Conclusions: Functional assays for the TFPI–protein S system and an ELISA for full‐length TFPI were developed. These assays show that the activity of the TFPI–protein S anticoagulant pathway is impaired in individuals with congenital protein S deficiency.     

抗磷脂综合征合并静脉血栓和蛋白C活性异常1例研究报告

目的：探讨抗磷脂综合征（antiphospholipid syndrome，APS）患者静脉血栓形成的原因。方法：对1例APS患者用发色底物法测定蛋白C活性（PC：A）、蛋白S活性（PS：A）和抗凝血酶活性；用ELISA方法测定PC、血浆纤溶酶原、血浆组织型纤溶酶原激活物、血浆纤溶酶原激活抑制物-1、α2-抗纤溶酶抗原和抗心磷脂抗体（ACA）。活化蛋白C抵抗（APC—R）检测结果以受检者血浆加入APC后的APTT与未加APC血浆的APTT的比值表示，比值〈2．0时为APC-R阳性。狼疮抗凝物质（LA）检测使用以dRVVT为基础的商品化试剂盒。采用PCR扩增和直接测序，检测PC的基因及FVLeiden和凝血酶原G20210A的突变。结果：本例患者LA和APC—R阳性，PC：A降低，PC抗原量增加，其他结果正常．PC基因所有外显子测字结果正常，FVLeiden突变和凝血酶原G20210A突变未检出。结论：LA可能通过抑制PC途径导致患者发生血栓，联合检测ACA、APC-R、抗凝蛋白抗原及活性有利于血栓性疾病的病因学诊断。     

Factor XII assay with the chromogenic substrate chromozym PK

A chromogenic assay for the determination of factor XII using the chromogenic substrate Chromozym PK was evaluated. The assay was linear in the range 10 U/l to more than 200 U/l. Using the assay, the normal range of factor XII (50 healthy volunteers at random) was 136 U/l +/- 27 U/l. Kallikrein-inhibiting concentrations of aprotinin did not influence factor XII. Comparison of the chromogenic with the clotting assay resulted in a correlation coefficient of r = 0.831 (p-value less than 0.001). In patients with deep vein thrombosis, factor XII level was found to be reduced to about 60% of normal activity.     

High titers of autoantibodies to tissue factor pathway inhibitor are associated with the antiphospholipid syndrome

Summary.  As the activity of the tissue factor pathway inhibitor (TFPI) may be impaired in patients with antiphospholipid antibodies (aPL), 162 aPL patients were evaluated for autoantibodies to recombinant TFPI (anti-TFPI) using an optimized ELISA. Anti-TFPI (>18 U mL⁻¹ for IgG and/or > 15 U mL⁻¹ for IgM) were detected in 54 patients with aPL (33.3%) and in three out of 79 normal controls (3.8%, P  < 0.0001). Among aPL patients, the prevalence of positive anti-TFPI was 38.3 and 28.4% in those with or without diagnosis of definite antiphospholipid syndrome (APS). Patients with definite APS had a significantly greater frequency of high titer (>50 U mL⁻¹) anti-TFPI than aPL patients from the no definite APS group (18.5% vs. 6.2%, OR 3.7, P = 0.017). Most aPL recognized full-length TFPI, but not a truncated form of TFPI lacking the C-terminus of the molecule. Isolated IgGs from subjects with anti-TFPI impaired the dose-dependent inhibitory effect of TFPI on factor Xa activity in the presence, but not in the absence of phospholipid vesicles. Thus, aPL with high titer anti-TFPI limit TFPI action and are associated with the APS.     

Biochemical characterization of plasma-derived tissue factor pathway inhibitor: post-translational modification of free, full-length form with particular reference to the sugar chain

Summary.  Background:  Tissue factor pathway inhibitor (TFPI) is a physiological protease inhibitor that inhibits the initial reactions of the extrinsic blood coagulation pathway. Most TFPI in human plasma is associated with lipoproteins; however, the most functionally active form is thought to be the free, full-length form (f-pTFPI). Cell culture derived TFPI and recombinant TFPI (rTFPI) exhibit variations in their respective anticoagulant activity, which may be caused by post-translational modifications, such as the frequent differences in sugar chain structures among recombinant proteins. Sugar chain structures in rTFPI expressed in Chinese hamster ovary (CHO) cells have been reported previously, but those of plasma TFPI have not been. Objectives:  To purify f-pTFPI and analyze the sugar chain structures. Results and conclusion:  f-pTFPI was purified to homogeneity from blood plasma using a combination of anion-exchange, heparin affinity, immunoaffinity, and reversed-phase chromatographies, resulting in a yield of 76%. f-pTFPI showed a partially phosphorylated glycoprotein comprising a total of 276 amino acids by peptide mapping. The sugar chain structures were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion of the pyridylamino sugar chains and the following results were obtained. (Sialyl) Galβ1-3GalNAc was linked to Thr¹⁷⁵, partially to Thr¹⁴ and Ser¹⁷⁴; sialyl complex-type sugar chains to Asn¹¹⁷ and Asn¹⁶⁷, whereas Asn²²⁸ was not glycosylated. Neuraminidase-resistant acidic sugar chains including sulfated sugar chains were not observed significantly. The protease inhibitory activities of f-pTFPI towards activated factor (F) X and tissue factor-activated FVII complex were identical to those of full-length rTFPI expressed in CHO cells.     

Development and characterization of an automated assay of effective heparin activity in plasma

We describe a fully automated assay for determining effective heparin activity in plasma, based on heparin-catalyzed inhibition of Factor Xa (EC 3.4.21.6) by antithrombin III (AT III). Residual Factor Xa is determined kinetically by the Du Pont aca discrete clinical analyzer with a chromogenic substrate and is inversely related to heparin activity. Because the test plasma is the sole source of AT III, the assay result is dependent on AT III activity and reflects effective rather than total heparin activity. The assay range is 20-1200 USP units/L, and the assay shows equivalent sensitivity to standard and low-molecular-mass heparins. Within-run reproducibility (CV) is 1.6% at 390 units/L. There was no interference from common blood components or drugs. Results agreed well with those by the Coatest heparin kit (Kabi) adapted to the Cobas-Bio analyzer (r = 0.85, n = 122).     

Optimum conditions for the stabilization and measurement of tissue plasminogen activator activity in human plasma

Current studies show a more than 50-fold variation in the estimated level of tissue-type plasminogen activator (t-PA) activity in normal resting blood samples that result from major differences in the methods used to sample, preserve, and assay t-PA activity in blood. In this study we developed optimized methods for stabilizing and measuring t-PA activity in plasma by using a coupled plasminogen-chromogenic substrate (amidolytic) assay. To maximize the recovery of t-PA activity, blood should be acidified within 60 seconds after being drawn by adding 2 parts whole blood to 1 part 0.5 mol/L sodium acetate, pH 4.2. This method prevents hemolysis and eliminates 70% of the alpha 2-plasmin inhibitor. Optimum conditions for measuring t-PA activity are pH 8.0 to 8.3 (37 degrees C), ionic strength 0.02 to 0.04, 0.5 mumol/L plasminogen, 80 micrograms/ml CNBr-cleaved fibrinogen, and a chromogenic substrate concentration of 0.65 mmol/L D-valyl-leucyl-lysyl-p-nitroanilide, 0.25 mmol/L D-valyl-phenylalanyl-lysyl-p-nitroanilide, or 0.2 mmol/L D-norleucyl-hexahydrotyrosyl-lysyl-p-nitroanilide. The final assay is linear with respect to added one-chain t-PA, two-chain t-PA, and acidified plasma. There was no difference in t-PA activity measured with ethylenediaminetetraacetic acid anticoagulant versus that measured with citrate anticoagulant after correction for dilution effects (average resting t-PA activity in plasma from 20 healthy individuals = 1.59 IU/ml). We conclude that assay conditions can have major effects on the measurement of t-PA activity in plasma and that suboptimal conditions may result in a significant underestimation of t-PA activity.     

Centrifugal analysis for plasma kallikrein activity, with use of the chromogenic substrate S-2302

The amidolytic activity of activated kallikrein in plasma can be measured by use of the chromogenic substrate, S-2302 (H-D-Pro-Phe-Arg-pNA). Plasma prekallikrein was activated to kallikrein by exposure to 50 mg/L dextran sulfate in acetone/water (35/65 by vol) at 0 degrees C for 15 min. The acetone slows anti-kallikrein activity and increases the kallikrein activity by 30%. The 37 degrees C reaction mixture contained 0.54 mmol of S-2302 substrate per liter of Tris buffer (pH 7.5 at 37 degrees C). We monitored the change in absorbance at 405 nm for 60 s. The specificity of the substrate for kallikrein was demonstrated by using plasma deficient in prekallikrein (Fletcher trait) diluted with pooled normal human plasma. We recommend collecting blood specimens with sodium citrate as the anticoagulant and with use of a double-syringe technique and all-plastic containers. Plasma kallikrein activity with Chromozym-PK (Bz-Pro-Phe-Arg-pNA) as substrate (y-axis) compared with S-2302 as substrate (x-axis) gave the relation: y = 0.28x + 0.82 (r = 0.94). Day-to-day analytical variation was 2.4% for a pooled plasma with a mean value of 85.9 mukat/L. The mean (and 2 SD) for 50 healthy adults was 86.4 (32.4) mukat/L.     

Kinetic measurement of total amylase and isoamylase activities with a centrifugal analyzer

We evaluated a new kinetic assay for alpha-amylase (Phadebas IsoAmylase Test), using modified starch as the substrate and a CentrifiChem 400 centrifugal analyzer system. We determined isoamylase activities by using a selective inhibitor. Results were compared with those obtained with the chromogenic Phadebas dyed-starch technique. The method for total amylase appeared to be rapid and precise (CV = 4%) and results correlated well with the chromogenic method. Samples with activities up to 3000 arb. units/L can be analyzed without dilution. Glucose and pyruvate interfere with the assay, but hemoglobin and bilirubin do not. Pancreatic (P) and salivary (S) isoamylase activity can be determined with acceptable precision (CVP = 8%, CVS = 10%) by an automated routine procedure with commercially available reagents.     

冠心病患者血浆TF、TFPI水平的变化及其临床意义

目的　观察冠心病患者血浆组织因子 (TF)、组织因子途径抑制物 (TFPI)水平的变化及其临床意义。方法　 79例临床确诊的冠心病患者 ,其中急性心肌梗死 (AMI组 ) 32例、不稳定型心绞痛 (UAP组 ) 2 7例、稳定型心绞痛 (SAP组 ) 2 0例 ,15例健康对照组作为对照组。采用发色底物法测定血浆TF、TFPI活性。结果　与对照组、SAP组比较 ,AMI组血浆TF、TFPI显著增高 ,三组差异具有显著性意义 (P<0 0 5 ) ;AMI组血浆TF活性与UAP组比较差异无统计学意义 (P >0 0 5 ) ,但TFPI活性较UAP组明显升高 ,两者差异具有显著性意义 (P <0 0 5 )。UAP组血浆TF活性较健康对照组及SAP组明显升高 ,三组间差异具有显著性意义 (P <0 0 5 ) ,UAP组血浆TFPI活性较健康对照组及SAP组差异无统计学意义 (P >0 0 5 ) ;SAP组血浆TF、TFPI活性较健康对照组差异无统计学意义 (P >0 0 5 )。结论　 (1)AMI、UAP患者存在异常激活的高凝状态 ,TF触发的外源性凝血途径在冠脉内血栓形成上发挥重要作用。 (2 )AMI、UAP患者体内TF、TFPI系统存在严重失衡     

Comparison of cell-surface TFPIα and β

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is mainly produced by endothelial cells and alternative mRNA splicing generates two forms, TFPIalpha and TFPIbeta. A portion of expressed TFPI remains associated with the cell surface through both direct (TFPIbeta) and indirect (TFPIalpha) glycosylphosphatidyl-inositol (GPT)-mediated anchorage. OBJECTIVE: Compare the structure and properties of TFPIalpha and TFPIbeta. METHODS: TFPIalpha and TFPIbeta, with protein molecular masses of 36 and 28 kDa, respectively, migrate similarly (46 kDa) on SDS-PAGE. Experiments using specific glycosidases were carried out to determine the different glycosylation pattern of the two forms. ECV304 cells, a cell line with some endothelial properties, were stimulated with IL-lbeta, LPS, and TNFalpha for up to 24 hrs and mRNA levels and protein synthesis were determined. Stable clones of ECV304 cells that express reduced levels of TFPIalpha, TFPIbeta or both were produced using a plasmid-based small-interfering RNA technique. Surface TFPI activity was determined by a two-stage chromogenic assay based on the ability of each form to inhibit FXa activation by FVIIa on cells with comparable amount of tissue factor (TF). RESULTS AND CONCLUSIONS: The deglycosylation studies show that the difference in molecular masses is due to a greater degree of sialylation in O-linked carbohydrate in TFPIbeta. The mRNA and protein levels of neither form of TFPI were affected by stimulation of cells with inflammatory stimuli. Although TFPIalpha comprises 80% of the surface-TFPI, TFPIbeta was responsible for the bulk of the cellular FVIIa/TF inhibitory activity, suggesting a potential alternative role for cell surface TFPIalpha.     

1例遗传性蛋白S缺陷症患者的基因分析

目的研究一个遗传性蛋白S(PS)缺陷症家系的表型诊断及基因特征。方法PS活性(PS:A)用发色底物法测定,PS抗原(PS:Ag)用ELISA方法测定。用PCR扩增PS基因各个外显子及侧翼序列,用直接测序法检测突变点。结果先证者的PS:Ag和PS:A分别为8·3mg/L和29%,均低于正常。基因检测发现在14号外显子Gln522(CAG)→Stop(TAG)。结论本家系PS基因在14号外显子Gln522(CAG)→Stop(TAG),为国内首次报道的一个新的基因突变。