Biomedical subjects

J Rosing

Publications and source records attributed to J Rosing.

At least 37 records · Page 2Linked to original sources

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 Ca2+-mobilizing potency of alpha-thrombin and thrombin-receptor-activating peptide on human platelets -- concentration and time effects of thrombin-induced Ca2+ signaling.

In single platelets and in suspensions of platelets, alpha-thrombin evokes dose-dependent, transient increases in cytosolic Ca2+ concentration, [Ca2+]i, which are more prolonged than the [Ca2+]i transients evoked by other platelet agonists such as the thrombin-receptor-activating hexapeptide SFLLRN, thromboxane A2 analog U46619, and ADP. As a quantity taking into account both the magnitude and length of the Ca2+ response, we defined the Ca2+-mobilizing potency (CMP) of an agonist as the integrated rise in [Ca2+]i during the time of the Ca2+ signal. It was observed that: (a) the CMP increased with the agonist concentration in a saturating way, its maximal value being about four-times higher with alpha-thrombin than with SFLLRN; (b) the high CMP of alpha-thrombin was for only a small part due to endogenous production of ADP or thromboxane, and was mainly a consequence of prolonged influx of external Ca2+; (c) the CMP declined when alpha-thrombin was inactivated during the course of the Ca2+ signal; (d) CMP values increased with the agonist concentration upon sequential addition of increasing amounts of alpha-thrombin or SFLLRN; (e) when alpha-thrombin was gradually added to the platelets or formed by an in situ reconstituted prothrombinase system (with factor Xa, factor Va, and prothrombin), integrated Ca2+ responses were a function of the product of the alpha-thrombin concentration and the time of its presence. However, in these cases, the final CMP values were independent of the rate of alpha-thrombin addition or formation. We conclude that alpha-thrombin-induced Ca2+ signals in platelets rely largely upon Ca2+ influx, are not, or only slightly, subjected to homologous desensitization, and reflect the enzymatic capacity of alpha-thrombin to cleave protease-activated receptors. Thus, the high and prolonged Ca2+ signal induced by alpha-thrombin is due to continuous receptor cleavage without desensitizing effects of previously cleaved receptors.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

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↗

PubMed1997

Coagulation factor V: an old star shines again.

Blood coagulation factor V plays an important role in the regulation of thrombin formation. Activation of factor V by traces of activated coagulation factors (thrombin, factor Xa or meizothrombin) yields factor Va, the non-enzymatic cofactor of the prothrombinase complex. Since factor Va accelerates prothrombin activation under physiological conditions more than 10(4)-fold it is not surprising that down-regulation of factor Va cofactor activity by the protein C pathway is a very effective way for maintaining the hemostatic balance. In this paper we have reviewed the present status of structural knowledge of factor V and Va, the molecular changes in factor V that occur during factor V activation, the function of factor Va in prothrombin activation and the molecular mechanism of inactivation of factor Va by APC. Although considerable insight in the structure-function relationship of factor V and Va has been achieved, the study of mutated factor V molecules obtained by recombinant DNA technology will undoubtedly resolve remaining questions. The latter is illustrated by the fact that the discovery of factor VaLeiden has significantly contributed to our present knowledge on the regulation of the cofactor activity of factor Va via the protein C pathway. It appears that modulation of the activity of APC by protein S and factor Xa will strongly affect the in vivo activity of this pathway. Factor V not only plays an important role in the regulation of the activity of the prothrombinase complex but also acts as cofactor in APC-mediated inactivation of factor VIIIa. This gives rise to a rather intricate mechanism of regulation of thrombin formation by APC that thus far has been mainly studied in model systems containing purified proteins. Thus, extensive studies in plasma will be required in order to get more insight in the in vivo regulation of thrombin formation via the protein C pathway.

Arginine↗

PubMed1997

Regulation of thrombin formation by activated protein C: effect of the factor V Leiden mutation.

Activated Protein C (APC) resistance, one of the most common genetic risk factors for venous thrombosis, is caused by a single base mutation (G1691-->A) in the factor V (FV) gene resulting in the replacement of Arg506 by Gln at a predominant cleavage site for APC. Great progress in understanding the mechanism of downregulation of FVa activity via the protein C pathway has been achieved by studying APC-mediated inactivation of FVa purified from homozygous APC-resistant individuals. This review briefly summarizes the role of FVa in prothrombin activation and the structure-function relationship of FV and FVa. Subsequently, APC-dependent inactivation of FVa and FVa Leiden and its modulation by protein S and factor Xa in model systems containing purified proteins is discussed. FV also has a function in increasing the inactivation of FVIII/VIIIa by APC. This cofactor activity appears diminished in FV Leiden. Thus, an intricate mechanism of regulation of thrombin formation via the protein C pathway is starting to emerge. Extensive studies in plasma milieu will be needed to gain more insight into the relation between the presence of FV Leiden and impaired downregulation of thrombin formation in APC-resistant individuals.

Factor V↗

PubMed1996

Protein C activation on endothelial cells by prothrombin activation products generated in situ: meizothrombin is a better protein C activator than alpha-thrombin.

The conversion of protein C into activated protein C (APC) by the thrombin-thrombomodulin complex on the surface of endothelial cells initiates an essential negative feedback reaction on blood coagulation. APC, together with its non-enzymic cofactor protein S, inactivates factors Va and VIIIa, the non-enzymic protein cofactors of the prothrombinase and intrinsic tenase complex, by proteolytic degradation. In this study we report that prothrombin activation products, generated by the prothrombinase complex on the surface of quiescent endothelial cells, are able to activate protein C. Subsequent inactivation of factor Va by the APC that was formed decreased the rate of prothrombin activation, thus demonstrating in vitro the negative feedback loop on coagulation factor activation. The anticoagulant feedback reaction of APC on the prothrombinase complex was stimulated 3-4-fold by the addition of protein S but not by thrombin-cleaved protein S or by protein S complexed with C4b-binding protein. Stimulation of endothelial cells with 50 pM tumour necrosis factor (TNF) or 500 pM interleukin 1 (IL-1) resulted in a 70% decrease in activation of protein C by exogenously added alpha-thrombin, which seemed to be due to down-regulation of thrombomodulin activity on the surface of endothelial cells. However, when prothrombin activation products generated in situ were allowed to activate protein C, stimulation of endothelial cells with TNF and IL-1 resulted in only a 25% decrease in activation of protein C. Stimulation with TNF or IL-1 did not affect the ability of endothelial cells to support prothrombinase activity. We investigated whether the differences in extent of protein C activation by exogenously added alpha-thrombin and by prothrombin activation products generated in situ were due to meizothrombin formed during prothrombin activation. Previous reports from our groups revealed that meizothrombin is generated as a transient intermediate during prothrombin activation on phospholipid vesicles and endothelial cells. Here we show that meizothrombin is at least a 6-fold better activator of protein C on the surface of endothelial cells than is alpha-thrombin. These results demonstrate that meizothrombin, formed during the initial phase of prothrombin activation, efficiently down-regulates both its own formation and that of thrombin.

Cells, Cultured↗

PubMed1996

Factor V enhances the cofactor function of protein S in the APC-mediated inactivation of factor VIII: influence of the factor VR506Q mutation.

Factor V and protein S are cofactors of activated protein C (APC) which accelerate APC-mediated factor VIII inactivation. The effects of factor V and protein S were quantitated in a reaction system in which plasma factor VIII was inactivated by APC and the loss of factor VIII activity was monitored in a factor X-activating system in which a chromogenic substrate was used to probe factor Xa formation. Factor V increased the rate of APC-mediated factor VIII inactivation in a dose-dependent manner in representative plasma samples with protein S or factor V deficiency, abnormal factor V (heterozygous or homozygous for factor VR506Q), or a combination of heterozygous protein S deficiency and heterozygous factor VR506Q. This effect was much less pronounced in the plasma samples with a decreased protein S level, but the impaired response in these plasmas was corrected by addition of protein S, indicating that both factor V and protein S are required for optimal inactivation of factor VIII by APC. The effects of factor V and protein S were also studied in a reaction system with purified proteins. APC-catalysed factor VIII inactivation was enhanced 3.7-fold in the presence of 1.1 nM factor V and 1.5-fold in the presence of 2.4 nM protein S. When both 1.1 nM factor V and 2.4 nM protein were present the rate enhancement was 11-fold. Factor V is a more potent cofactor than protein S, as can be concluded from the fact that 0.04 nM factor V gave the same stimulation as 2.4 nM protein S. Protein S lost its cofactor function after complexation with C4b binding protein, which indicates that it is free protein S that acts as a cofactor. To investigate the effect of the R506Q mutation in factor V on APC-mediated factor VIII inactivation, factor V was purified from the plasma of patients homozygous for factor VR506Q. In the absence of protein S, factor VR506Q did not enhance factor VIII inactivation by APC, but in the presence of 2.4 nM protein S a slight enhancement was observed. The APC cofactor activity of factor V was lost when factor V was activated with thrombin or with the factor V activator from Russell's viper venom. These data indicate that optimal inactivation of factor VIII by APC requires the presence of an intact factor V molecule and free protein S.

Arginine↗

PubMed1996

A prothrombinase-based assay for detection of resistance to activated protein C.

In this paper we present a new method for the detection of resistance to activated protein C (APC) that is based on direct measurement of the effect of APC on the cofactor activity of plasma factor Va. The factor V present in a diluted plasma sample was activated with thrombin and its sensitivity towards APC was subsequently determined by incubation with phospholipids and APC. The loss of factor Va cofactor activity was quantified in a prothrombinase system containing purified prothrombin, factor Xa and phospholipid vesicles and using a chromogenic assay for quantitation of thrombin formation. The reaction conditions were optimized in order to distinguish normal, heterozygous and homozygous APC-resistant plasmas. Maximal differences in the response of these plasmas towards APC were observed when factor Va was inactivated by APC in the absence of protein S and when the cofactor activity of factor Va was determined at a low factor Xa concentration (0.3 nM). Addition of 0.2 nM APC and 20 microM phospholipid vesicles to a 1000-fold diluted sample of thrombin-activated normal plasma resulted in loss of more than 85% of the cofactor activity factor Va within 6 min. Under the same conditions, APC inactivated approximately 60% and approximately 20% of the factor Va present in plasma samples from APC-resistant individuals that were heterozygous or homozygous for the mutation Arg506-->Gln in factor V, respectively. Discrimination between the plasma samples from normal and heterozygous and homozygous APC-resistant individuals was facilitated by introduction of the so-called APC-sensitivity ratio (APC-sr). The APC-sr was defined as the ratio of the factor Va cofactor activities determined in thrombin-activated plasma samples after 6 min incubation with or without 0.2 nM APC and was multiplied by 100 to obtain integers (APC-sr = ¿factor Va+APC square root of factor Va-APC¿ x 100). Clear differences were observed between the APC-sr of plasmas from normal healthy volunteers (APC-sr: 8-20, n = 33) and from individuals that were heterozygous (APC-sr: 35-50, n = 17) or homozygous APC resistant (APC-sr: 82-88, n = 7). There was no mutual overlap between the APC-sr of normal plasmas and plasmas from heterozygous or homozygous APC resistant individuals (p < 0.0001). In all cases our test gave the same result at the DNA-based assay. Since the test is performed on a highly diluted plasma sample there is no interference by conditions that affect APC resistance tests that are based on clotting time determinations (e.g. coagulation factor deficiencies, oral anticoagulation, heparin treatment, the presence of lupus anticoagulants, pregnancy or the use of oral contraceptives). Furthermore, we show that part of the factor Va assay can be performed on an autoanalyzer which increases the number of plasma samples that can be handled simultaneously.

Biological Assay↗