Amidolytic Assay Research Articles

Amidolytic assay of factor XI in human plasma-significance of kallikrein for the activity measured

Factor XI (FXI) deficiency is associated with an abnormal bleeding state. The extent of bleeding does not correlate well with the plasma concentration of FXI, and it has been suggested that also unknown factors interfere with the bleeding tendency. In a recent paper ( Thromb. Res. 74, 477–485, 1994) we found that FXIa activated in human plasma was present in association with part of factor XIIa (FXIIa) and part of kallikrein, influencing their functional activities. Should the activity level of FXIa also be altered by the other contact factors this might provide one approach to the problem of the failure of assays of FXIa to correlate with bleeding tendency. In the present study we have developed an assay procedure for FXIa based on its amidolytic (S-2366) activity, and allowing at the same time a quantification of the amount of FXIa associated to kallikrein. The total amidase activity obtained was separated into two main fractions by use of soybean trypsin inhibtor (STI), corn inhibitor (CI) and lima bean trypsin inhibitor (LTI). One fraction contained free FXIa which could be specifically blocked by LTI. An inhibitor resistant fraction was found to contain FXIa inactive in association with kallikrein. The content of FXIa could be assessed in experiments with mixtures of normal plasma and plasma deficient in prekallikrein, and was taken into account in the calculations. This fraction increased during storage of plasma at −70°C. To obtain stable and comparable assay conditions the method was based on plasma stored for at least four weeks. The specificity of the method was verified by parallel radial immunodiffusion tests. The results imply that the activity level of FXIa is dependent on kallikrein present. If the experimental results has relevance to the situation under physiological conditions, they indicate one possible cause of the failure of assays of FXI to correlate with bleeding tendency.

Studies on the thrombogenic effects of recombinant tissue factor. In vivo versus ex vivo findings.

An exposure of blood to tissue factor (TF) activates the coagulation system by the extrinsic pathway and may cause clot formation. Recombinant TF (r-TF) has been produced and subsequently reconstituted into phospholipid vesicles. The aim of these studies was to elucidate the in vitro procoagulant effects and the in vivo thrombogenicity of r-TF using a rabbit jugular vein stasis thrombosis model. The in vitro studies exhibited a clear concentration-dependent decrease in the clotting time when rabbit brain thromboplastin was replaced by r-TF in the prothrombin time assay. The in vivo studies revealed a dose-dependent thrombogenicity between 1.6 ng/kg and 50 ng/kg. Electron microscope scanning of the surface of representative clots revealed fibrin-rich structures of heterogeneous density. In comparison, thrombi obtained when FEIBA was utilized as the thrombogenic agent were more homogeneous. The injection of r-TF caused a slight transient drop in blood pressure with little or no effects on the pulse rate, complete blood count (CBC) profile, clotting and amidolytic assays when compared to sham control animals. In contrast, the whole blood clotting parameters (activated clotting time and thrombelastograph) were prolonged dose-dependently after r-TF injection. The antithrombotic activity of heparin was assessed in this model and compared to the antithrombotic activity when FEIBA is used as the thrombogenic agent. The apparent ED50 of heparin was found to be 4 times higher in the r-TF system. In control studies, no thrombogenic effects were observed by the phospholipid vesicles alone nor by r-TF not embedded in phospholipid vesicles. These data demonstrate that lipidated r-TF is a potent thrombogenic challenge that activates the hemostatic system by the extrinsic pathway.

Pharmacologic profile of a low-molecular-weight heparin depolymerized by gamma-irradiation.

Low molecular weight heparins (LMWHs) are considered to be the agent of choice for the prophylaxis of DVT in medical and surgical patients. Conventionally, these agents have been produced by fractionation of or by chemical or enzymatic depolymerization of native heparin. The fractionated heparin retains many of its biological properties such as AT III affinity and sulfate content gamma-irradiation (60Co) has been used to depolymerize GAGs (De Ambrosi et al. In: biomedical and Biotechnological Advances in Industrial Polysaccharides, pp. 45-53). This procedures has now been used for the preparation of LMWH derivatives of varying molecular weight. The current studies examine the biochemical and pharmacologic profile of one such gamma-irradiated depolymerized heparin. In standard clotting and amidolytic antiprotease assays (PT, APTT, AXa, Alla), gamma-irradiated depolymerized heparin produced equal or stronger activity when compared to a LMWH produced by nitrous acid depolymerization and retained the ability to active AT III and HCHII. Initial results indicate that LMWHs produced by gamma-irradiation exhibit comparable antithrombotic actions to those produced by chemical depolymerization when measured in animal models of thrombosis. gamma-Irradiation may be a useful method for the production of LMWHs.

Enzymatic Removal of Sialic Acid from Human Factor IX and Factor X Has No Effect on Their Coagulant Activity

Factor IX and factor X have sialic acid in O-linked and N-linked oligosaccharides on their activation peptides, and a terminal sialic acid is found on a recently described O-linked tetrasaccharide at Ser-61 in the light chain of human factor IXa. In studies presented here, the potential role of sialic acid residues in mediating activity of human coagulation factors IX and X was tested after enzymatic removal of sialic acid residues. In contrast to previous reports, treatment of factor IX or factor IXa with recombinant sialidase did not decrease the rate of factor IX activation or proteolytic properties of human factor IXa. The activation rates of factor IX and desialated factor IX were indistinguishable when treated with factor XIa, with factor VIIa/tissue factor complex, and with the factor X activating enzyme from Russell's viper venom. Desialated human factor IXa showed full activity in the non-activated partial thromboplastin time assay and retained full "tenase" activity in a coupled amidolytic assay. Similar experiments with human factor X showed no detectable loss of clotting activity in the prothrombin time assay after desialation. Additionally, desialated human factor X was cleaved by the factor X activating enzyme from Russell's viper venom and intrinsic tenase at the same rate as untreated factor X when analyzed by SDS-polyacrylamide gel electrophoresis. These studies have shown that factor IX and factor X clotting activity are not dependent on sialic acid content. Further studies are needed to determine whether desialated factor IX binds to endothelial cells, and whether factors IX and X are more rapidly cleared from circulation or have altered susceptibility to proteolysis after enzymatic removal of sialic acid.

The Activation of Type 1 and Type 2 Plasminogen by Type I and Type II Tissue Plasminogen Activator

Tissue plasminogen activator (tPA) was fractionated using lysine-Sepharose affinity chromatography. Type I, type II, and a minor peak with high affinity for lysine (designated type D) tPA were recovered. In an indirect amidolytic assay involving native human Glu-plasminogen and fibrin, type II tPA showed a 2-fold higher activity than type I. To explore the combinatorial effect of the variable glycosylation status of both tPA and plasminogen, kinetic constants for fibrin-dependent plasminogen activation were determined for combinations of type I, II, and D tPA with type 1 and 2 plasminogen. Within a 4-fold range, the fastest rate was achieved from the combination of type D (type II + D) tPA and type 2 plasminogen. N-Glycosylation of plasminogen increased the Km value for activation by all tPA variants; N-glycosylation of type I tPA at Asn184 decreased the kcat (turnover) values for the fibrin-dependent activation of plasminogen over type II tPA, while type D tPA showed the highest turnover rate. In the presence of fibrinogen fragments, N-glycosylation of plasminogen at site 289 modulates the kinetics of association of enzyme and substrate, while N-glycosylation at site 184 on tPA modulates the turnover rate of the enzyme.

Urinary Levels of Kallikrein and Kallistatin in Pregnancy-Induced Hypertension

Objective: We evaluated if urinary excretion of kallikrein and kallistatin (a glandular kallikrein inhibitor) is altered early during gestation in patients who will ultimately develop pregnancy-induced hypertension.Methods: The protocol was designed as a nested, longitudinal case-control study. Overnight urine collections were obtained at 12-20, 24-26, and 30-32 weeks of gestation in a consecutive series of 350 primigravidae. Urinary kallikrein (amidolytic assay) and kallistatin (enzyme-linked immunosorbent assay) levels were measured in all patients (n = 24) who were found to be affected by pregnancy-induced hypertension. In addition, urinary kallikrein and kallistatin levels were measured in 24 women used as controls. They were chosen from the group of 326 normotensive subjects by matching them with the hypertensive patients for age and gestational time at the occasion of urine collections.Results: Prevalence of pregnancy-induced hypertension with or without proteinuria was 2.3% and 4.6%, respectively. ...

The Strong Positive Correlation between Factor VII Clotting Activity Using Bovine Thromboplastin and the Activated Factor VII Level

We compared factor VII clotting activity (FVIIc) assays using different thromboplastins to determine which is the most sensitive for activated FVII (FVIIa) or for FVII antigen (FVIIag). FVIIc levels were measured using thromboplastins derived from bovine brain (FVIIc Bov), human placenta (FVIIc Hum), and rabbit brain (FVIIc Rab). FVIIa levels were measured by fluorogenic assays using human soluble tissue factor (rsTF) or bovine rsTF. We also measured FVII activity by an amidolytic assay (FVIIc:am Hum) using human thromboplastin and a chromogenic substrate for thrombin. FVIIag levels were determined by ELISA. In the FVIIa assay, the reaction time obtained from using bovine rsTF was shorter than that with human rsTF, suggesting that the interaction of plasma FVIIa with bovine rsTF was stronger than with human rsTF. The plasma FVIIa levels measured using human rsTF and bovine rsTF were almost the same (r = 0.947, p < 0.0001). Among the three FVIIc assays, FVIIc Bov had the strongest positive correlation with the plasma FVIIa level (r = 0.886, p < 0.0001), but had no correlation with FVIIag. An increase of 1 ng/ml in the plasma FVIIa level yielded a 27.9% increase of FVIIc Bov. Plasma FVIIc Hum and FVIIc:am Hum showed moderate correlations with both FVIIa (r = 0.520, p < 0.02 and r = 0.569, p < 0.01, respectively) and FVIIag (r = 0.438, p < 0.05 and r = 0.468, p < 0.05, respectively). FVIIc Rab had the lowest correlation with FVIIa (r = 0.367, p < 0.1), but had a moderate correlation with FVIIag (r = 0.436, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-induced release of plasminogen activator from human blood platelets

Incubation of washed platelets in Tyrode buffer, pH 7.5, with insulin (200 microU/ml) and CaCl2 (1.2 mM) at 37 degrees C for 3 h resulted in a threefold increase of plasminogen activator activity in the supernatant over the basal level as determined by both the amidolytic assay and the proteolysis of alpha-casein through the formation of plasmin from plasminogen. This plasminogen activator showed no plasmin-like activity and was inhibited by anti-tissue plasminogen activator antibody as well as by type 1 plasminogen activator inhibitor. The substrate specificity and the inhibition of the enzymic activity by various inhibitors indicated that the platelet plasminogen activator (pPA) was related to tissue-type plasminogen activator of relative molecular weight 56,000. Fibrinolytic activity of pPA and its insulin-dependent release were demonstrated by the shortening of euglobulin lysis time and by the clot lysis time of platelet-rich plasma from normal and type I diabetes mellitus patients. Treatment of platelet membranes with insulin also increased the release of pPA. Increased levels of adenosine 3',5'-cyclic monophosphate (cAMP) in platelets by incubation with various agents completely inhibited the insulin-induced release of the activator. On the other hand, inhibition of platelet aggregation by aspirin had no effect on the release of pPA, indicating that the effect of cAMP was not due to the inhibition of platelet aggregation by the nucleotide.

The International and “NIH” Units for Thrombin - How Do They Compare?

The current International Standard (IS) for alpha-thrombin was established in 1991. It was related in unitage with the 1st IS for Thrombin established in 1975 and contains 100 IU of thrombin activity. The National Institute of Health (NIH) standard (Lot J) is in common use for the calibration of commercial thrombin reagents and this study reports on a comparison of the thrombin units defined by these 2 standards. This study has indicated that one "NIH" unit is equivalent to 1.1 to 1.3 IU of thrombin, depending on the influence of PEG in the assay. A unitage ratio figure of 1.15 was recommended following analysis of data obtained in the presence and absence of PEG in the comparative assays. This was confirmed by amidolytic assay data.

Surface plasmon resonance studies of the interaction between factor VII and tissue factor. Demonstration of defective tissue factor binding in a variant FVII molecule (FVII-R79Q).

The blood coagulation cascade is initiated when vessel injury allows factor VII (FVII) to form a complex with tissue factor (TF). Complete deficiency of FVII causes a lethal bleeding diathesis, but individuals with moderately reduced FVII levels are often asymptomatic. Some of these individuals have circulating partially functional FVII, as a result of point missense mutations in critical parts of the molecule. One such mutation has been reported at position 79 in the first epidermal growth factor-like (EGF) domain of FVII, where an arginine residue has been replaced by glutamine. There is controversy as to whether or not this mutation reduces the affinity of the FVII/TF interaction compared to wild-type FVII. To address this problem, we have expressed recombinant FVII-R79Q and subjected it to detailed biochemical analysis. One-stage FVII:C assays show the variant FVII to have reduced activity with respect to the wild type. Rates of autoactivation and activation by FXa to the two-chain molecule were identical for wild-type and variant FVII. The Vmax for FX activation was lower for the mutant as measured using an amidolytic assay for FX activity. In contrast, the Km for FX was lower for the variant than the wild-type molecule. Peptidyl substrate hydrolysis was virtually identical for both variant and normal FVIIa in the presence and absence of TF. The variant has reduced affinity for TF as measured by surface plasmon resonance. FVII-R79Q has an association rate constant (kassoc) one-fifth of that of normal FVII, but a similar kdiss, resulting in a decrease in the affinity of the enzyme for its cofactor.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticoagulant and antiprotease activities of aprosulate sodium, a new synthetic polyanion, in human plasma and purified systems.

Aprosulate sodium, a new synthetic polyanion, was evaluated for anticoagulant/antiprotease activities in vitro. Aprosulate prolonged activated partial thromboplastin time (aPTT), Heptest and thrombin time (TT) with the sensitivity order of aPTT > Heptest > TT. Prothrombin time (PT) was hardly affected by the agent. Since these results indicated that inhibition of the intrinsic coagulation cascade was important for the anticoagulant activity of aprosulate, amidolytic assays were subsequently performed to characterize antiprotease activities of the agent in the common and intrinsic pathways. Aprosulate inhibited amidolytic activity of thrombin in the presence of heparin cofactor II, but was devoid of direct and antithrombin-mediated anti-factor Xa, and direct anti-factor XIIa activities. However, aprosulate was found to be a potent inhibitor of amidolytic activity of factor Xase (IXa/VIIIa). These data suggest that the direct anti-factor Xase activity of aprosulate primarily contributes to its anticoagulant activity in the intrinsic coagulation cascade.

Elastin-derived peptide binding to a hydrophobic domain on neutrophil elastase.

To understand the contributions of binding of elastin to domains removed from the active site of neutrophil elastase, we isolated an elastin-derived peptide (EDP) fraction, which we have previously shown was tightly linked to neutrophil elastase after prolonged digestion of elastin but which can be released from the enzyme with hydroxylamine. Elastin from human aorta was incubated with human neutrophil elastase under conditions favoring proteolysis. Low molecular weight species, including free EDP, were separated from the protein fraction by a small centrifuged gel filtration column. The high molecular weight protein fraction was subjected directly to 0.5 M hydroxylamine. The reaction mixture was then fractionated on a phosphocellulose column using an ionic gradient. A fraction was collected that exhibited fluorescence with a peak at approximately 410 nm when excited at 320 nm, indicating the presence of desmosine and (or) isodesmosine. A second peak with amidolytic activity towards methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroaniline (MeOSucAAPVpNa), but no fluorescence at 410 nm was also detected at the same elution volume where free elastase appeared. After removal of low molecular weight digestion products but prior to treatment with hydroxylamine, the putative elastase-EDP complex possessed no amidolytic activity towards MeOSucAAPVpNa. When the liberated EDP was added to elastase in an amidolytic assay, the EDP behaved as only a partial noncompetitive inhibitor (Vmax/Vmax approximately 90%), but bound with high affinity to neutrophil elastase (Ki congruent to 29 nM), as detected by its ability to quench elastase endogenous fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of the APC-resistance phenomenon

We developed an amidolytic assay for the determination of APC-resistance by inhibition of factor Xa (F Xa) generation, expecting it to be a result of factor VIIIa (F VIIIa) inactivation. Thirty-nine samples were tested with the proposed assay and compared to an APTT-based assay performed using the APC-resistance kit (Chromogenix, Mölndal, Sweden). In both assays APC was added at the last stage of the reaction together with CaCl 2. The response to APC was calculated as a ratio of absorbances (or of clotting times) of a sample tested with or without APC and as the amount of F VIII activity generated in the presence of APC. An APC-ratio less than 2.0 and residual F VIII activity over 55% indicated APC-resistance. The results demonstrated that APC-response was heterogenic and dependent on the assay used for the determination. Only 1 3 of the tested samples were resistant in both APTT-based and in amidolytic assays. A number of samples developed an adequate decrease in F VIII activity, but the APC-resistance was still registered by the APTT-based assay. Other samples had a sufficient APC-response in APTT-based assay, in spite of diminished inactivation of F VIIIa. The obtained data presented the response to APC as a complex process with a chain of reactions. It indicated that poor degradation of coagulation factors may not be sufficient for its development.

A kallikrein-like enzyme in the aorta of normotensive and hypertensive rats.

We evaluated whether vascular kallikrein is altered in rats with genetic or experimental hypertension. Group 1 was infused intraperitoneally with angiotensin II (Ang II) or vehicle for 4 weeks; group 2 was injected subcutaneously with deoxycorticosterone (75 mumol/kg once a week) or vehicle for 4 weeks; group 3 consisted of uninephrectomized rats on high sodium intake given deoxycorticosterone or vehicle; and group 4 consisted of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. Active and total kallikrein activity was measured in abdominal aortic homogenates using an amidolytic assay. Ang II increased systolic blood pressure at a dose of 400 nmol/kg per day (146 +/- 6 versus 123 +/- 3 mm Hg in controls, P < .01) but not at 80 nmol/kg per day. Deoxycorticosterone did not increase blood pressure except in uninephrectomized rats on high salt (173 +/- 6 versus 135 +/- 4 mm Hg in controls, P < .01). Blood pressure averaged 194 +/- 2 mm Hg in SHR and 123 +/- 3 mm Hg in WKY rats. Vascular kallikrein was similar in rats given Ang II or vehicle. In deoxycorticosterone-treated rats total kallikrein was higher than in controls (9.2 +/- 0.8 versus 3.5 +/- 0.1 pkat/mg protein, P < .05), whereas active kallikrein did not differ (0.09 +/- 0.04 versus 0.09 +/- 0.03 pkat/mg protein, P = NS). A similar pattern was observed in uninephrectomized deoxycorticosterone-treated rats (active, 0.09 +/- 0.03 versus 0.10 +/- 0.04, P = NS; total, 7.4 +/- 0.7 versus 4.1 +/- 0.2 pkat/mg protein, P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Binding and activation of plasminogen on the surface of osteosarcoma cells.

Plasmin (Pm) is a broad action serine protease implicated in numerous physiological functions. In bone, Pm may play a role in growth, resorption, metastasis, and the activation of growth factors. The various components of the Pm system are known to bind and function on the cell surface of various cell types, but no pertinent data are available describing membrane-bound Pm or its zymogen, plasminogen (Pg), in either normal or neoplastic bone cells. We report here that Pg binds to the surface of the human osteosarcoma cell line MG-63 and is activated to Pm by endogenous urokinase plasminogen activator (uPA). These conclusions are based on experiments utilizing radiolabeled compounds and a cell surface proteolytic assay measuring amidolytic activity of Pm. 125I-Pg binding to cells was time dependent, saturable, reversible, and specific. Binding was characterized by a relatively low affinity (Kd approximately 0.9 microM) and a high capacity (approximately 7.5 x 10(6) sites/cell). The binding of 125I-Pg was associated with lysine binding sites of the plasminogen molecule. Activation of 125I-Pg to 125I-Pm occurred on the cell surface and was dependent upon cell bound uPA, as determined by inhibitory antibodies. Binding of Pg to MG-63 monolayers represented approximately 80% bound specifically to the cell surface and the remainder to the surrounding extra-cellular matrix. Either co-incubation with uPA or pre-incubation with Pm resulted in increased 125I-Pg binding to osteosarcoma cells. Cell surface Pm proteolytic activity was confirmed by an amidolytic chromogenic assay. Both Pm and Pg bound to cells with Pg being activated by endogenous uPA. Plasmin activated on the cell surface was partially protected from inhibition by alpha 2-antiPm (requiring Pm lysine binding site interaction) but inhibited by aprotinin, (interacting directly with the Pm catalytic site). Resistance of cell bound Pm to alpha 2-antiPm inhibition suggests that cell surface proteolysis can occur in the presence of a soluble Pm inhibitor known to exist in the extracellular space. Based on these results, we speculate that the various bone physiological processes implicating Pm may occur at or near the bone cell surface.

Factor VIIa levels are decreased in patients with hemophilia B

American Journal of HematologyVolume 45, Issue 2 p. 196-197 Letters and CorrespondenceFree Access Factor VIIa levels are decreased in patients with hemophilia B Juzo Matsuda, Juzo Matsuda Department of Medicine, Teikyo University School of Medicine, Tokyo, JapanSearch for more papers by this authorNoriko Saitoh, Noriko Saitoh Department of Medicine, Teikyo University School of Medicine, Tokyo, JapanSearch for more papers by this authorKengo Gohchi, Kengo Gohchi Department of Medicine, Teikyo University School of Medicine, Tokyo, JapanSearch for more papers by this authorMiyo Tsukamoto, Miyo Tsukamoto Department of Medicine, Teikyo University School of Medicine, Tokyo, JapanSearch for more papers by this authorMoritaka Gotoh, Moritaka Gotoh Department of Medicine, Teikyo University School of Medicine, Tokyo, JapanSearch for more papers by this authorHideji Hanabusa, Hideji Hanabusa Department of Pediatrics, Ogikubo Hospital, Tokyo, JapanSearch for more papers by this authorMinoru Inagaki, Minoru Inagaki Department of Pediatrics, Ogikubo Hospital, Tokyo, JapanSearch for more papers by this author Juzo Matsuda, Juzo Matsuda Department of Medicine, Teikyo University School of Medicine, Tokyo, JapanSearch for more papers by this authorNoriko Saitoh, Noriko Saitoh Department of Medicine, Teikyo University School of Medicine, Tokyo, JapanSearch for more papers by this authorKengo Gohchi, Kengo Gohchi Department of Medicine, Teikyo University School of Medicine, Tokyo, JapanSearch for more papers by this authorMiyo Tsukamoto, Miyo Tsukamoto Department of Medicine, Teikyo University School of Medicine, Tokyo, JapanSearch for more papers by this authorMoritaka Gotoh, Moritaka Gotoh Department of Medicine, Teikyo University School of Medicine, Tokyo, JapanSearch for more papers by this authorHideji Hanabusa, Hideji Hanabusa Department of Pediatrics, Ogikubo Hospital, Tokyo, JapanSearch for more papers by this authorMinoru Inagaki, Minoru Inagaki Department of Pediatrics, Ogikubo Hospital, Tokyo, JapanSearch for more papers by this author First published: February 1994 https://doi.org/10.1002/ajh.2830450229AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References 1 Nagasaki T, Foster DC, Derkner KL, Kisiel W: Initiation of the extrinsic pathway of blood coagulation: Evidence for the tissue factor dependent autoactivation of human blood coagulation factor VII. Biochemistry 30: 10819, 1991. 2 Wildgoose P, Nemerson Y, Lyng L, Nielsen FE, Glazer S, Hendner U: Measurement of basal levels of factor VIIa in hemophilia A and B patients. Blood 80: 25, 1992. 3 Seligsohn U, Osterud B, Rapaport SI: Coupled amidolytic assay for factor VII: Its use with a clotting assay to determine the activity state of factor VII. Blood 52: 978, 1978. 4 Miller BC, Hultin ME, Jesty J: Altered factor VII activity in hemophilia. Blood 65: 845, 1985. 5 Kario K, Matsuo T, Kario T, Kobayashi H: Sex-related differences in the relationship between plasma factor VII levels and serum lipids, lipoproteins. and apolipoproteins. Thromb Res 64: 769, 1991. Volume45, Issue2February 1994Pages 196-197 ReferencesRelatedInformation

Influence of Six Mutations of the Protein C Gene on the Gla Domain Conformation and Calcium Affinity

The protein C Gla domain was studied in six families presenting a type II hereditary deficiency characterized by low activity in a coagulation assay and normal activity in an amidolytic assay. Five of these mutations, previously described by our group, affected Arg-5, Arg-1, Arg 229 and Ser 252. We report here the first natural Glu 7 to Asp mutation in a sixth family. We evaluated the binding of the mutated protein C to H11, a monoclonal antibody (mAb) known to recognize the sequence Phe4 to Arg9 of the Gla domain; the presence of calcium ions suppresses the recognition of this epitope by H11. Mutation of Arg229 to Gln and Ser252 to Asn did not modify the inhibition of protein C binding, whereas the Arg-1 to His mutation resulted in a loss of inhibition in the presence of CaCl2. This suggests that the protein C of this patient shows impaired carboxylation. The protein C from patients bearing the mutations Arg-5 to Trp, Arg-1 to Cys and Glu 7 to Asp bound poorly to H11 mAb, even in the absence of calcium ions. The calcium affinity of the Gla domain was studied by pseudo-affinity chromatography, in which protein C was successively eluted from a Mono Q column by CaCl2 10 mM and NaCl 0.6 M. Protein C from the patient bearing the Arg-5 to Asp mutation had a normal elution profile, suggesting that a modification of the propeptide cleavage site impairs the conformation of the Gla domain but not carboxylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of clivarin® a new low molecular weight heparin in healthy volunteers

The antithrombotic activity and pharmacokinetics of Clivarin®, a low molecular weight heparin was randomly studied in 10 healthy male volunteers. Doses of 20, 40, 60 and 80 anti F Xa U/kg BW were injected intravenously and subcutaneously in crossover fashion. The heparin concentrations were measured by inhibition of clotting assays (anti Ila and anti Xa activities using amidolytic assays and (dilute) thrombin time. The pharmacokinetic profile of Clivarin® is characterized by a linear relationship between dose and absorption, relatively low clearance and a long elimination half-life, and a high anti Xa/anti Ila ratio of 5.3.

Activation and Inactivation of Human Protein C by Plasmin

Transient procoagulant states resulting in failure of recanalization or rethrombosis of the reperfused artery during thrombolytic therapy might be due to an inhibitory effect of plasmin on the anticoagulant properties of protein C. We therefore studied the effect of plasmin on protein C (PC) and activated protein C (APC) using purified human proteins. Incubation of 70 nM purified human PC with 40-400 nM human plasmin resulted in rapid activation and subsequent inactivation of PC as measured by amidolytic and anticoagulant assays. The rates of activation and inactivation were dependent on the concentration of plasmin. Lower concentrations of plasmin resulted in higher peaks of generated APC and more sustained activity, while at higher concentrations, both activation and inactivation were more rapid. Anticoagulant activity appeared more sensitive to inactivation by plasmin than amidolytic activity; e. g., while amidolytic activity reached a maximum of 13.8 nM in 6 min and declined to approximately 6 nM after 30 min, anticoagulant activity reached its maximum of only 1.4 nM within 30 s and completely disappeared within 90 s. Plasmin rapidly destroyed both the anticoagulant and amidolytic activity of purified APC, with second order rate constants of 2.8 x 10(5) M-1 s-1 and 1.2 x 10(4) M-1 s-1, respectively, for 70 nM APC. The rates of activation and subsequent inactivation were slowed by the presence of CaCl2. The second order rate constant of inactivation of APC amidolytic activity decreased to 6.6 x 10(3) M-1 s-1 in the presence of 5 mM CaCl2.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of fibrinolysis by ionizing radiation.

Radiation-induced damage to the central nervous system is believed to be targeted to glial or endothelial cells or both, although the pathophysiology of this process is still poorly understood. A series of experiments were, therefore, conducted, including irradiation to primary rat astrocytes (in vitro) and rat spinal cords (in vivo). The levels of plasminogen activators (uPA and tPA) and their inhibitors (PNI and PAI-1) were determined by fibrin zymography, ELISA, amidolytic activity assay, complex formation, and Western blot analysis. Fibrin zymography revealed the presence of M(r) 48,000 (uPA) and M(r) 68,000 (tPA) lytic bands that were increased in irradiated samples. Three- to four-fold higher levels of tPA and 8- to 10-fold higher levels of uPA were detected in irradiated samples. Western blot analysis confirmed the presence of a 51-kDa band (PAI-1) in irradiated samples. PAI-1 is undetectable in nonirradiated spinal cord. Serum-free medium and cell and spinal cord extracts of nonirradiated samples showed a 43-kDa band (PNI), the intensity of which is decreased in irradiated samples. Four- to five-fold decreased levels of PNI were detected in irradiated serum-free media and cell extracts, but no levels of PNI were detected in irradiated spinal cord extracts. This study provides additional information regarding the proposed roles of plasminogen activators and their inhibitors in the development of CNS damage after irradiation.