Biomedical subjects

G Tans

Publications and source records attributed to G Tans.

At least 19 recordsLinked to original sources

PubMed2000

Mutations in the R2 FV gene affect the ratio between the two FV isoforms in plasma.

Molecular genetics and biochemical studies were performed in homozygotes for the R2 allele (4070G) in the factor V gene, most of them affected by coronary artery disease. Novel polymorphisms (G642T, 156Ser; T1328C, 385Met/Thr), among which a functional candidate (A6755G, 2194Asp/Gly) located in the C2 domain of FV, were identified in the R2 gene. In chromatographic studies R2 FV appeared qualitatively identical to normal FV. However, a relative increase of the more thrombogenic and more glycosylated FV isoform (FV1) was observed in plasma of 2194Gly homozygotes (mean FV1/FV2 ratio 0.71, 95% CI 0.66-0.77) as compared to R2-free controls (0.37, 95% CI 0.34-0.40). We conclude that carriership of the R2 FV gene is associated with an imbalance between the two functionally different FV isoforms, and propose that genetically determined differential glycosylation of FV could represent a novel mechanism of thrombotic disease.

Aged↗

PubMed2000

Effects on coagulation of levonorgestrel- and desogestrel-containing low dose oral contraceptives: a cross-over study.

Combined oral contraceptives (OC) are known to increase the risk of venous thromboembolism. The aim of this randomized, cycle-controlled, cross-over study in 28 healthy volunteers was to assess potential differences between the effects of an OC containing 150 microg levonorgestrel (as representative of the so-called second generation OC) and an OC containing 150 microg desogestrel (as representative of the third generation OC) in combination with 30 microg ethinylestradiol on several coagulation factors and markers of thrombin formation. All participants used each OC for two cycles, and were switched to the other OC after a washout period of two menstrual cycles. The plasma concentrations of factors II, VII, X, and fibrinogen significantly increased during use of both the levonorgestrel- and desogestrel-containing OC's. The plasma concentrations of factor VIII increased, and of factor V decreased, changes which only reached statistical significance during the use of the desogestrel-containing OC. During exposure to the desogestrel-containing OC, as compared with the levonorgestrel-containing OC, both factor VII and factor II showed a greater increase (FVII: 32% and 12% respectively; p <0.0001; FII: 16% and 12% respectively; p = 0.048), whereas factor V showed a greater decrease (-11% and -3% respectively; p = 0.010). Only one of the markers for ongoing coagulation (prothrombin fragment 1+2) showed a significant increase during OC use, whereas concentrations of thrombin-antithrombin complexes and soluble fibrin remained unchanged. For these markers, there was no difference between the tested OC's. We conclude that there are differences between the effects of levonorgestrel and desogestrel-containing OC's on some coagulation factors. Whether these changes provide a biological explanation for the reported differences in venous thromboembolic risk is as yet unclear. The real challenge now becomes to define a pattern of changes in the various systems which, if affected simultaneously, may tip the hemostatic balance towards a prethrombotic state and may lead to overt clinical venous thromboembolism.

Adolescent↗

PubMed2000

Increased fibrinolytic activity during use of oral contraceptives is counteracted by an enhanced factor XI-independent down regulation of fibrinolysis: a randomized cross-over study of two low-dose oral contraceptives.

The effect of oral contraceptives (OC) on fibrinolytic parameters was investigated in a cycle-controlled cross-over study in which 28 non-OC using women were randomly prescribed either a representative of the so-called second (30 microg ethinylestradiol, 150 microg levonorgestrel) or third generation OC (30 microg ethinylestradiol, 150 microg desogestrel) and who switched OC after a two month wash out period. During the use of OC, the levels of tissue-type plasminogen activator (tPA) activity, plasminogen, plasmin-alpha2-antiplasmin complexes and D-dimer significantly increased (by 30 to 80%), while the levels of plasminogen activator inhibitor- (PAI-1) antigen, PAI-1 activity and tPA antigen significantly decreased (25 to 50%), suggesting an increase in endogenous fibrinolytic activity. These OC-induced changes were not different between the two contraceptive pills. TAFI (thrombin-activatable fibrinolysis inhibitor) levels increased on levonorgestrel, and even further increased on desogestrel. A clot lysis assay that probes both fibrinolytic activity and the efficacy of the coagulation system to generate thrombin necessary to down regulate fibrinolysis via TAFI showed no change of the clot lysis time during OC use. This finding suggests that the OC-induced increase in endogenous fibrinolytic activity is counteracted by an increased capacity of the coagulation system to down regulate fibrinolysis via TAFI. Indeed we observed that during OC use there was a significant increase of F1+2 generation during clot formation. When these assays were performed in the presence of an antibody against factor XI, we observed that the clot lysis time was significantly increased during OC use and that the increase in F1+2 generation during OC therapy was due to a factor XI-independent process, which was significantly higher on desogestrel than on levonorgestrel. These data indicate that the OC-induced inhibition of endogenous fibrinolysis takes place in a factor XI-independent way and is more pronounced on desogestrel than on levonorgestrel-containing OC.

Adolescent↗

PubMed2000

A randomized cross-over study on the effects of levonorgestrel- and desogestrel-containing oral contraceptives on the anticoagulant pathways.

The use of oral contraceptives (OC) causes disturbances of the procoagulant, anticoagulant and fibrinolytic pathways of blood coagulation which may contribute to the increased risk of venous thrombosis associated with OC therapy. Here we report the results of a cycle-controlled randomized cross-over study, in which we determined the effects of so-called second and third generation OC's on a number of anticoagulant parameters. In this study, 28 non-OC using women were randomly prescribed either a second generation (150 microg levonorgestrel/30 microg ethinylestradiol) or a third generation OC (150 microg desogestrel/30 microg ethinylestradiol) and who switched to the other OC after a two month wash out period. The anticoagulant parameters determined were: antithrombin (AT), alpha2-macroglobulin (alpha2-M), alpha1-antitrypsin, protein C inhibitor (PCI), protein C, total and free protein S and activated protein C sensitivity ratios (APC-sr) measured with two functional APC resistance tests which quantify the effect of APC on either the activated partial thromboplastin time (aPTT) or on the endogenous thrombin potential (ETP). During the use of desogestrel-containing OC the plasma levels of alpha2-M, alpha1-antitrypsin, PCI and protein C significantly increased, whereas AT and protein S significantly decreased. Similar trends were observed with levonorgestrel-containing OC, although on this kind of OC the changes in AT, PCI and protein S (which was even slightly increased) did not reach significance. Compared with levonorgestrel, desogestrel-containing OC caused a significant decrease of total (p <0.005) as well as free protein S (p <0.0001) and more pronounced APC resistance in both the aPTT (p = 0.02) and ETP-based (p <0.0001) APC resistance tests. These observations indicate that the activity of the anticoagulant pathways in plasma from users of desogestrel-containing OC is more extensively impaired than in plasma from users of levonorgestrel-containing OC.

Activated Protein C Resistance↗

PubMed1999

C-terminal residues 621-635 of protein S are essential for binding to factor Va.

Protein S is anticoagulant in the absence of activated protein C because of direct interactions with coagulation Factors Xa and Va. Synthetic peptides corresponding to amino acid sequences of protein S were tested for their ability to inhibit prothrombinase activity. The peptide containing the C-terminal sequence of protein S, residues 621-635 (PSP14), reversibly inhibited prothrombinase activity in the presence but not in the absence of Factor Va (K(i) approximately 2 microM). PSP14 inhibition of prothrombinase was independent of phospholipids but could be competitively overcome by increasing Factor Xa concentrations, suggesting that the C-terminal region of protein S may compete for a Factor Xa binding site on Factor Va. Studies using peptides with amino acid substitutions suggested that lysines 630, 631, and 633 were critical residues. PSP14 inhibited Factor Va activity in Factor Xa-one-stage clotting assays. PSP14 inhibited protein S binding to immobilized Factor Va. When preincubated with protein S, antibodies raised against PSP14 inhibited binding of protein S to Factor Va and blocked inhibition of prothrombinase activity by protein S. These results show that the C-terminal region of protein S containing residues 621-635 is essential for binding of protein S to Factor Va and that this interaction contributes to anticoagulant action.

Amino Acid Sequence↗

PubMed1999

Low-dose oral contraceptives and acquired resistance to activated protein C: a randomised cross-over study.

BACKGROUND: We have reported previously that, compared with use of second-generation oral contraceptives, the use of third-generation oral contraceptives is associated with increased resistance to the anticoagulant action of activated protein C (APC). Owing to the cross-sectional design of that study, these observations may have been subject to unknown bias or uncontrolled effects of the menstrual cycle. We aimed to overcome these sources of bias by doing a cycle-controlled randomised cross-over trial. METHODS: The response to APC in plasma was assessed in 33 women who received two consecutive cycles of a second-generation oral contraceptive (150 microg levonorgestrel and 30 microg ethinyloestradiol) or a third-generation oral contraceptive (150 microg desogestrel and 30 microg ethinyloestradiol), and who switched preparations after two pill-free cycles. Normalised APC sensitivity ratios were calculated by measurement of the effect of APC on thrombin generation in the plasma of these women and in pooled plasma from 90 controls. FINDINGS: Of the 33 women, five were excluded because not all required plasma samples were available. In the remaining 28 women, the normalised APC sensitivity ratio increased during treatment with both preparations. Compared with levonorgestrel, desogestrel-containing oral-contraceptive treatment caused a highly significant (p<0.0001) additional increase in normalised APC sensitivity ratio (0.51 [95% CI 0.37-0.66]). Normalised APC sensitivity ratios during oral-contraceptive treatment correlated with the values before oral-contraceptive use. INTERPRETATION: Oral-contraceptive treatment diminishes the efficacy with which APC down-regulates in-vitro thrombin formation. This phenomenon, designated as acquired APC resistance, is more pronounced in women using desogestrel-containing oral contraceptives than in women using levonorgestrel-containing preparations. Whether acquired APC resistance induced by oral contraceptives explains the increased risk of venous thromboembolism in oral-contraceptive users remains to be established.

Activated Protein C Resistance↗

PubMed1999

Effects of oral contraceptives on hemostasis and thrombosis.

OBJECTIVE: The object of the study was to determine the effects of oral contraceptives on blood coagulation, in particular on the protein C pathway. STUDY DESIGN: Plasma samples from healthy men, from healthy female users and nonusers of oral contraceptives, and from heterozygous and homozygous male and female carriers of the factor V Leiden mutation (some of whom used oral contraceptives) were tested for their sensitivity to activated protein C by means of a new activated protein C resistance test developed in our laboratory. This assay is based on measurement of the effect of activated protein C on the endogenous thrombin potential, the time integral of thrombin generation initiated in plasma through the extrinsic coagulation pathway. RESULTS: The normalized activated protein C sensitivity ratio ([ETP+APC/ETP-APC]plasma/[ETP+APC/ETP-APC]normal plasma, where ETP is endogenous thrombin potential, +APC is with activated protein C, and -APC is without activated protein C) of men was lower than that of healthy female nonusers of oral contraceptives. The normalized activated protein C sensitivity ratio of the users of oral contraceptives was significantly higher than that of nonusers of oral contraceptives. The normalized activated protein C sensitivity ratio of women who were using oral contraceptives with third-generation progestogens was higher than that of users of oral contraceptives with second-generation progestogens. Furthermore, the normalized activated protein C sensitivity ratio of 80% of the users of third-generation preparations fell within the 5th to 95th percentile of the normalized activated protein C sensitivity ratio of female carriers of factor V Leiden, a mutation that is associated with hereditary resistance to activated protein C and with an increased risk of venous thromboembolism. CONCLUSION: Acquired activated protein C resistance may explain the increased risk of venous thromboembolism among users of oral contraceptives reported in epidemiologic studies and the higher risk of venous thromboembolism among users of oral contraceptives with third- versus second-generation progestogens.

Contraceptives, Oral↗

PubMed1999

Acquired APC resistance and oral contraceptives: differences between two functional tests.

Resistance to activated protein C (APC) is often associated with a mutation in factor V (factor VLeiden). Individuals without factor VLeiden who exhibit a response in functional APC-resistance tests similar to that of carriers of factor VLeiden are considered to be acquired APC resistant. This phenomenon is particularly observed in women using oral contraceptives (OC). In the present study we compared the response to APC in plasma from normal individuals, carriers of factor VLeiden and women who use OC using functional tests that either quantify the effect of APC on the endogenous thrombin potential (ETP) or on the activated partial thromboplastin time (aPTT). Both tests discriminated equally well between individuals with and without factor VLeiden who were not using OC. In contrast to the aPTT-based test, the ETP-based assay yielded significant differences in sensitivity to APC between non-OC users and OC users and between users of second and third generation OC. Since there was no correlation between APC-sensitivity determined with both assays in non-carriers of factor VLeiden and in women who use OC and a poor correlation in carriers of factor VLeiden, we propose that other plasma components differentially modulate the response to APC in the aPTT- and ETP-based APC-resistance tests and that OC change the level of plasma protein(s) that modulate the effect of APC on thrombin formation initiated via the extrinsic coagulation pathway.

Activated Protein C Resistance↗

PubMed1998

A kinetic assay to determine prothrombin binding to membranes.

Activation of prothrombin by multisquamase, the prothrombin activator from the venom of Echis multisquamatus (Central Asian sand viper), is inhibited by membranes containing negatively charged anionic phospholipids. This inhibition appears to be due to the fact that the venom activator cannot activate membrane-bound prothrombin. Initial steady state rates of prothrombin activation by multisquamase in the presence of phospholipids appeared to depend on the fraction unbound prothrombin only and this phenomenon was used to quantitate binding of prothrombin to membranes of varying phospholipid composition. In this method, the initial rate of prothrombin activation by multisquamase is measured in the absence (total prothrombin) and in the presence of a procoagulant surface (rate depending only on free prothrombin) and from the difference in activation rates the amount of membrane-bound prothrombin is calculated. The validity of the method was established by determination of the binding parameters for prothrombin binding to 100 microM phospholipid vesicles composed of 20 mole% phosphatidylserine and 80 mole% phosphatidylcholine. The binding parameters obtained were Kd=0.84 microM and n=0.021 micromoles prothrombin bound per micromole phospholipid which is in agreement with literature. Due to the nature of the measurement the method is especially suitable to quantitate binding of prothrombin at concentrations as low as 5 nM prothrombin.

Animals↗

PubMed1998

Autocatalytic peptide bond cleavages in prothrombin and meizothrombin.

During factor Xa-catalyzed prothrombin activation, several other reaction products accumulate as a result of proteolysis of prothrombin and its activation products by thrombin and meizothrombin. Gel electrophoretic analysis and N-terminal sequencing of reaction products showed that in the absence of Ca2+ ions thrombin cleaved the following peptide bonds: Arg51-Thr52/Arg54-Asp55 in the fragment 1 (F1) domain (k = 0.4 x 10(4) M-1 s-1), Arg155-Ser156 in prothrombin (k = 2 x 10(4) M-1 s-1), and Arg284-Thr285 in prethrombin 1 (k = 0.02 x 10(4) M-1 s-1). In the presence of 2.5 mM CaCl2, cleavage in fragment 1 (Arg51-Thr52/Arg54-Asp55) was not detectable, whereas cleavage at Arg155-Ser156 (i.e., removal of F1) was inhibited 25-fold. Cleavage at Arg284-Thr285 (formation of prethrombin 2 des-1-13) was not affected by the presence of Ca2+ ions. Meizothrombin rapidly converted itself into meizothrombin des-F1. The half-life (t1/2 = approximately 30 s) of this reaction was independent of the meizothrombin concentration (0.1-1 microM meizothrombin), which is indicative for intramolecular autocatalysis (k = 0.02 s-1 in the presence of 2.5 mM Ca2+ ions). Since the rapid removal of fragment 1 precludes investigations of the cleavage at Arg284-Thr285 in intact meizothrombin, we analyzed the cleavage of this peptide bond in R155A-meizothrombin, a recombinant product that is resistant to autocatalytic removal of the fragment 1 domain. In the absence of phospholipids, R155A-meizothrombin converted itself into thrombin des-1-13 by a combination of intramolecular (k = 0.8 x 10(-4) s-1) and intermolecular autocatalysis (k = 0.2 x 10(3) M-1 s-1). Intramolecular autocatalytic conversion of R155A-meizothrombin into thrombin was not affected by the presence of phospholipids (k = 0.8 x 10(-4) s-1), whereas intermolecular autocatalysis was accelerated 25-fold (k = 5.6 x 10(3) M-1 s-1) by phospholipid vesicles. Since factor Xa/Va-catalyzed conversion of meizothrombin into thrombin occurs with k = 5.5 x 10(8) M-1 s-1, we conclude that in reaction systems containing purified proteins autocatalysis of meizothrombin hardly contributes to thrombin formation during factor Xa-catalyzed prothrombin activation.

Animals↗

PubMed1998

Molecular biology and pathophysiology of APC resistance: current insights and clinical implications.

APC resistance is often associated with the occurrence of a single point mutation in factor V (factor VLeiden) at a predominant cleavage site for the natural anticoagulant, activated protein C (APC). In this article we will discuss the effects of this mutation (Arg506-->Gln) on the down-regulation of factor Va cofactor activity and on thrombin formation by APC in model systems and in plasma. Our studies on the effects of APC on thrombin formation in plasma resulted in the development of a new method for the screening of APC resistance that is based on measurement of the effect of APC on the endogenous thrombin potential (the time integral of thrombin generated in clotting plasma). It appeared that sensitivities for APC determined via this method were considerably affected by the use of oral contraceptives (OC) and that women who use OC become acquired APC resistant. The fact that acquired APC resistance in women who use third-generation OC was more pronounced than in users of second-generation OC may explain the further increased risk for venous thrombosis associated with the use of third-generation OC.

Activated Protein C Resistance↗

PubMed1997

Purification and characterization of multisquamase, the prothrombin activator present in Echis multisquamatus venom.

The venom of Echis multisquamatus (Central Asian sand viper) contains a single prothrombin activator, designated multisquamase, which is structurally and functionally different from ecarin, the prothrombin activator from the venom of Echis carinatus (saw-scaled viper). Multisquamase is comprised of a 58000 Mr and a 23000 Mr subunit that consists of two disulfide-linked chains of 12000 Mr and 10000 Mr, respectively. In contrast to ecarin, which activates prothrombin and prethrombin 1 at comparable rates, and whose activity is hardly affected by Ca2+ or by changes in ionic strength, multisquamase hardly activates prethrombin 1; prothrombin activation requires Ca2+ and is strongly inhibited at high ionic strength. The most favourable kinetic parameters are observed at 1 mM Ca2+ and at low ionic strength (Km=0.085 microM and kcat=0.68 s(-1) at I approximately 0.04). An increase in ionic strength considerably reduces the rate of prothrombin activation, due to an increase of the Km (Km=0.8 microM and kcat=1.03 s(-1) at I approximately 0.2). Studies in plasmas from patients on oral anticoagulant therapy show that E. Multisquamatus venom only activates carboxylated prothrombin, whereas E. carinatus activates both prothrombin and descarboxyprothrombin. Thus, multisquamase-dependent prothrombin activation appears to require post-translational modification of the gla-domain. This venom prothrombin activator may, therefore, become a useful tool to quantitate prothrombin and descarboxyprothrombin in cases where vitamin K-dependent carboxylation of prothrombin is impaired.

Enzymes↗

PubMed1997

The suicide substrate reaction between plasminogen activator inhibitor 1 and thrombin is regulated by the cofactors vitronectin and heparin.

The interaction of thrombin with plasminogen activator inhibitor 1 (PAI-1) is shown to result in the simultaneous formation of both cleaved PAI-1 and a sodium dodecyl sulfate-stable thrombin-PAI-1 complex. The kinetics of this reaction can be described by a "suicide substrate" mechanism that includes a branched reaction pathway, which terminates in either the stable inhibitor-enzyme complex or the cleaved inhibitor plus free enzyme. Because of the branched pathway, approximately three moles of PAI-1 are needed to completely inhibit one mole of thrombin. Heparin and vitronectin enhance the rate of inhibition from 9.8 x 10(2) L mol(-1) s(-1) to 6.2 x 10(4) L mol(-1) s(-1) and 2.1 x 10(5) L mol(-1) s(-1), respectively, under optimal conditions. In addition to enhancing the rate of inhibition, both cofactors increase the apparent stoichiometry of the PAI-1-thrombin interaction, with cofactor concentration dependencies similar to the inhibition reaction. Thus, at 37 degrees C approximately six cleavage reactions occur per inhibition reaction. Therefore, thrombin will efficiently inactivate PAI-1 in the presence of either vitronectin or heparin, unless a sufficient excess of the inhibitor is present. These results show that physiological cofactors are able to switch a protease-serpin inhibition reaction to a substrate reaction, depending on the local concentrations of each of the components.

Animals↗

PubMed1997

Human factor Va1 and factor Va2: properties in the procoagulant and anticoagulant pathways.

Human plasma factor V is heterogeneous and yields two forms of activated factor V that bind with low (factor Va1) and high affinity (factor Va2) to phospholipids. The properties of factor Va1 and factor Va2 in the anticoagulant and procoagulant pathways were evaluated by comparing their sensitivity for inactivation by APC and their ability to act as cofactor in prothrombin activation. At low phospholipid concentrations and on membranes containing low amounts of phosphatidylserine (PS), factor Va1 was inactivated by APC at 15-fold lower rates than factor Va2, both in the absence and in the presence of protein S. At high phospholipid concentrations and on membranes with more than 15 mol % PS, factor Va1 and factor Va2 were inactivated with equal efficiency. Differences between cofactor activities of factor Va1 and factor Va2 in prothrombin activation were only observed on membranes with less than 7.5 mol % PS. Due to the different phospholipid requirements of APC-catalyzed factor Va inactivation and of expression of factor Va cofactor activity in prothrombin activation, the thrombin-forming capacity of factor V1 was 7-fold higher than that of factor V2 in a reaction system containing factor Xa, prothrombin, APC, protein S, vesicles with a phospholipid composition resembling that of activated platelets, and traces of thrombin to initiate prothrombin activation. This shows that in the process of generation, expression, and down-regulation of factor Va cofactor activity on physiological membranes, the overall procoagulant activity of factor V1 can considerably exceed that of factor V2.

Blood Coagulation↗

PubMed1997

Factor V.

Factor V is a single chain glycoprotein that plays an essential role in the regulation of blood coagulation. After initiation of coagulation, factor V is converted into factor Va through limited proteolysis. Factor Va acts as protein cofactor in the prothrombin-activating complex, which is comprised of the serine protease factor Xa, Ca2+ ions and a procoagulant membrane surface. Factor Va accelerates factor Xa-catalysed conversion of prothrombin into thrombin more than 10(4)-fold. The cofactor activity of factor Va in prothrombin activation is down-regulated by activated protein C (APC). The physiological importance of this regulatory pathway is demonstrated by the occurrence of hereditary thrombophilia in individuals with a genetic defect that makes factor Va less sensitive to proteolytic inactivation by APC (APC resistance).

Blood Coagulation↗

PubMed1997

Oral contraceptives and venous thrombosis: different sensitivities to activated protein C in women using second- and third-generation oral contraceptives.

Epidemiological studies have shown that women who use third-generation oral contraceptives (OC) containing desogestrel, gestodene or norgestimate have a higher risk of venous thrombosis than women who use second-generation OC containing levonorgestrel. It is also known that a mutation in factor V (factor V(Leiden)), which results in resistance to activated protein C (APC) and which is the most common cause of hereditary thrombophilia, potentiates the prothrombotic effect of OC. Effects of APC on thrombin generation in the plasma of women using OC were compared to the response to APC in non-OC users and in individuals that were heterozygous or homozygous for factor V(Leiden). The response towards APC was evaluated on basis of the ratio (APC-sr) of the time integrals of thrombin formation determined in the presence and absence of APC. Compared with women not using OC, women who used OC exhibited a significantly decreased sensitivity to APC (P<0.001), independent of the kind of OC used. Women who used third-generation monophasic OC were significantly less sensitive to APC than women using second-generation OC (P<0.001) and had APC-sr that did not significantly differ from heterozygous female carriers of factor V(Leiden) who did not use OC. Women who were heterozygous for factor V(Leiden) and used OC had APC-sr in the range of homozygous carriers of factor V(Leiden). Two women who started OC therapy had significantly elevated APC-sr within 3 d. Acquired APC resistance may explain the epidemiological observation of increased risk for venous thrombosis in OC users, especially in women using third-generation OC.

Adolescent↗

PubMed1997

The clinical application of a new specific functional assay to detect the factor V(Leiden) mutation associated with activated protein C resistance.

Resistance to activated protein C (APC) is the most common defect found in patients who have venous thromboembolism. The molecular basis of APC resistance is a single-point mutation (arginine506-glutamine) in the gene that encodes for coagulation factor V. This mutation results in a factor V molecule (factor V(Leiden)) that is less effectively downregulated by APC than is normal factor V. The gold standard for the detection of this defect is DNA analysis. Several functional tests, which are based on activated partial thromboplastin time clotting assays, are also commercially available for the detection of APC resistance. These tests, however, have not been satisfactory. Compared with the results of DNA analysis, the results of these tests are frequently discordant. Further, in some patients (eg, those who have lupus anticoagulant or have been receiving heparin), these tests cannot be performed at all. DNA analysis is therefore required in most patients to distinguish congenital APC resistance (factor V(Leiden)) from other causes of abnormal response in functional APC-resistance tests. The purpose of this study was to investigate the clinical use of a new chromogenic APC-resistance assay that is based on direct measurement of the effect of APC on factor Va cofactor activity in highly diluted, thrombin-activated plasma specimens. All individuals who provided plasma samples for the study underwent DNA analysis to detect the presence of the factor V mutation. In all patient subgroups, including patients who had lupus anticoagulant and those who were receiving unfractionated heparin or coumarin derivatives, the chromogenic test showed excellent discrimination between normal individuals and those who were heterozygous or homozygous for the factor V(Leiden) mutation. No discordant results with DNA analysis were found in 150 cases. The new test easily can be incorporated in any laboratory that has an automated coagulation apparatus with an option for chromogenic measurements. All reagents are commercially available at low cost, and the test is easy to perform and is not time-consuming. This new, sensitive, and specific test allows large-scale screening for the factor V(Leiden) mutation without the need for DNA analysis.

Blood Coagulation Disorders↗

PubMed1997

Effect of activated protein C on thrombin generation and on the thrombin potential in plasma of normal and APC-resistant individuals.

In this paper we describe the effect of activated protein C (APC) on thrombin generation initiated in platelet-poor plasma via the extrinsic or the intrinsic pathway. Thrombin was determined with a specific chromogenic substrate and quantitated by calculating the time integral of the thrombin generation curve, i.e. the endogenous thrombin potential (ETP). Addition of APC to normal plasma after both extrinsic and intrinsic initiation of coagulation resulted in a dose-dependent inhibition of thrombin generation as reflected by the decrease in ETP. Data obtained in intrinsically triggered plasma of normal individuals were subject to large variation. Therefore, the effect of APC on thrombin generation in APC-resistant plasmas was only studied in extrinsically stimulated reaction systems. APC had much less effect on the ETP of plasma from individuals that were heterozygous or homozygous for the mutation Arg506-->Gln506 in factor V (APC resistance). There appears to be a linear relationship between the ETP and the amount of alpha 2-macro-globulin-thrombin complex (alpha 2 M-IIa) that accumulates in plasma during thrombin formation. Since the alpha 2M-IIa complex possesses amidolytic activity, we measured the effect of APC on thrombin generation via the so-called normalized APC sensitivity ratio (APC-sr). The latter was defined as the ratio of the end levels of amidolytic activity of the alpha 2M-IIa complex determined in the presence and absence of 50 nM APC (alpha 2M-IIa + APC/(alpha 2M-IIa - APC) divided by the ratio of a normal plasma pool. Significant differences (P < 0.001) were observed between APC-sr of plasmas from normal individuals (APC-sr: 0.5-1.9, n = 25) and of plasmas from individuals that were heterozygous (APC-sr: 2.1-6.7, n = 17) or homozygous APC resistant (APC-sr: 3.9-5.9, n = 5). There was no overlap between APC-sr of normal plasmas and plasma from individuals, bearing the factor. V mutation. Abnormal APC-sr in certain plasmas (pregnancy, use of oral contraceptives, anticoagulant therapy, protein S deficiency or lupus anticoagulant) were corrected by performing the assay on a plasma sample that was diluted 10-fold in factor V-deficient plasma. Our data show that measurement of the effect of APC on the ETP yields valuable information about the (pro)thrombotic status of plasma (e.g. APC resistance, pregnancy, use of oral contraceptives).

Drug Resistance↗