A functional assay of protein C in human plasma

A functional assay for protein C in plasma is described in which barium eluates of plasma are incubated with bovine thrombin and rabbit thrombomodulin to activate protein C. The activated protein C solution is added to an activated partial thromboplastin time (APTT) system containing normal plasma and an APTT reagent (Dade ActinR). The prolongation of coagulation time after recalcification in this system is taken as a measure of the anticoagulant activity of protein C. When expressed as per cent of the value in pooled normal plasma, the results obtained by this method in 34 normal controls and in 3 untreated patients with protein C deficiency were very similar to those obtained by radioimmunoassay of protein C. In 2 patients with protein C deficiency and 23 patients without, all on dicoumarol or warfarin treatment, the anticoagulant activity of protein C was less than its antigen concentration. The day to day analytical coefficient of variation (SD/mean) was 12% at the 100% level (n = 12), and 10% at the 25% level (n = 12).     

Fast functional protein C assay using Protac, a novel protein C activator

A simple and rapid clotting method for the quantitative determination of protein C (PC) in plasma consists of the conversion of PC into activated PC (APC) by means of Protac, an activator protein isolated from Agkistrodon contortrix contortrix venom, of the subsequent degradation of factors V and VIII in PC immuno-depleted plasma by the generated APC and of the measurement of the prolongation of the activated partial thromboplastin time (APTT) which is proportional to the amount of PC in the sample. In 33 normal individuals a mean PC level of 97.1% of a normal pooled plasma was found. Comparison with an enzyme-immunoassay for PC in 33 patients with liver disease revealed a good correlation (r = 0.986). Patients under warfarin therapy (n = 34) had a mean PC level of 19.8%; a comparison with the immunological assay (mean value = 55.3%) in the same population suggested that the assay did not co-estimate acarboxy forms of PC. The assay proved to be insensitive to heparin concentration lower than 1 U/ml. Due to its simplicity, it should be suitable for diagnostic routine and monitoring of patients with abnormal PC level, even if under anticoagulation.     

A simplified PTT-based protein C activity assay using the thrombin-thrombomodulin complex

A simplified assay for protein C activity in plasma is described which uses the ability of rabbit lung thrombomodulin to inhibit the procoagulant activity of thrombin while stimulating protein C activation. Barium eluates of plasma are activated for one hour at 37 degrees C by a mixture of human thrombin and rabbit lung thrombomodulin at concentrations which neutralize each other's effect on the kaolin-cephalin activated partial thromboplastin time (PTT). Protein C anticoagulant activity in the activated eluates is then measured directly in the PTT. The method is independent of protein S levels in the test samples, and is suitable for warfarinized and heparinized plasma. Protein C levels obtained with this method correlate closely with functional levels of vitamin K-dependent procoagulants as measured by the prothrombin and proconvertin time (P&P) in normal subjects and in patients receiving warfarin, indicating specificity for gamma-carboxylated protein C. The method has the potential to detect molecular variants defective in any of the interactions required for generation of anticoagulant activity in vivo.     

Plasma protein S activity measured using Protac, a snake venom derived activator of protein C

Functional activity of protein S, a cofactor of activated protein C-dependent inhibition of blood coagulation, in human plasma was measured by using Protac, a snake venom derived activator of protein C. This assay appeared to be specific for protein S, because 1) the activated partial thromboplastin time of protein S-depleted plasma depended on the purified protein S added in the presence of Protac; and 2) the level of protein C in plasma sample (0 to 10 micrograms/ml) had no influence on the clotting time. The cofactor activity of protein S in the plasma of normal men (n = 16) and women (n = 14) was 99.4 +/- 23.8% and 98.6 +/- 24.5% respectively. The protein S activity in the plasma of pregnant women at pre- and post-partum (n = 14), and that in the plasma of patients under warfarin therapy (n = 20) were 46.2 +/- 18.9%, 45.8 +/- 19.6% and 24.0 +/- 15.7%, respectively. In these plasmas, the levels of protein S activity were lower than those of total protein S antigen, but were similar to those of free protein S antigen. In 16 patients out of two families with congenital protein S deficiency, the protein S activity, the free antigen and the total antigen were 9.4 +/- 6.9%, 13.3 +/- 4.6% and 57.4 +/- 20.7%, respectively. There was no significant relationship between the level of protein S activity and that of a complemental C4b-binding protein antigen in any of these patients.     

Protein C inhibitor and other components of the protein C pathway in patients with acute deep vein thrombosis during heparin treatment

The protein C inhibitor (PCI) concentration and other parameters of the protein C pathway were studied in 19 patients with symptomatic acute deep vein thrombosis before and during the first 5 days of heparin treatment. The mean levels of PCI antigen and activity decreased rapidly and significantly during the first day of heparin therapy from 83 and 84% to 60 and 59% of the pooled normal human plasma (p less than 0.01), respectively, and to 56 and 54% after 5 days of treatment (p less than 0.01). In contrast, antithrombin III decreased progressively 25% during 5 days of heparin treatment. Protein C antigen and activity and total protein S remained unchanged during heparin treatment. Free protein S was decreased before heparin treatment (83%, p less than 0.05) and increased to normal values after 5 days of treatment. C4b-binding protein was significantly increased before and during heparin treatment (p less than 0.01). Activated protein C (APC) complexed to its two major plasma inhibitors, PCI and alpha 1-antitrypsin (alpha 1AT) were measured by specific ELISA's. Before treatment, 18 of the 19 patients studied had increased levels of APC:alpha 1AT complexes with a mean value of 27 +/- 22 ng/ml (range, 6-86 ng/ml) compared to normal values (8 +/- 2 ng/ml) and 12 of the patients also had detectable APC:PCI complex levels with a mean value of 11 +/- 17 ng/ml (range, 5-68 ng/ml). Both APC:inhibitor complexes decreased significantly during heparin treatment.     

Determination of plasma protein C inhibitor and of two activated protein C-inhibitor complexes in normals and in patients with intravascular coagulation and thrombotic disease

We developed an ELISA to quantitate complexes of activated protein C (APC) with a major plasma APC inhibitor, alpha 1-antitrypsin (alpha 1AT) in human plasma based on the sandwich principle using two different antibodies directed towards protein C and alpha 1AT, respectively. This ELISA test was specific for APC:alpha 1AT complexes and sensitive to greater than or equal to 150 pg complex. Fifty-one of 56 healthy donors had APC:alpha 1AT complex levels above the detection limit (3 ng/ml) ranging from 4 to 14 ng/ml (mean value +/- SD: 7.6 +/- 2.5 ng/ml). Patients (n = 10) with disseminated intravascular coagulation (DIC) had detectable levels of APC:alpha 1AT complex ranging from 21 to 125 ng/ml (median: 69 ng/ml). Complexes of APC with plasma protein C inhibitor (PCI) were also measured using an ELISA sandwich assay. None of the 30 healthy donors had detectable levels (greater than or equal to 5 ng/ml) of APC:PCI complex, and plasma samples from 9 of 10 DIC patients had detectable concentrations of APC:PCI complex ranging from 10 to 63 ng/ml (median: 22 ng/ml). APC:alpha 1AT complex was detected in 25 of 26 patients with deep venous thrombosis (DVT), with levels ranging from 5 to 136 ng/ml (median: 23 ng/ml), whereas APC:PCI was detected in only 6 DVT patients, with levels between 11 and 105 ng/ml. PCI antigen levels in 70 normals ranged from 56 to 175% (mean +/- SD: 99.1% +/- 24.2%). PCI antigen levels were decreased in DIC patients, in patients with cerebral arterial thrombosis, and in DVT patients undergoing heparin therapy, but not in patients with myocardial infarction. PCI antigen levels were decreased much further in DVT patients receiving heparin compared to those not receiving heparin, showing that heparin therapy is associated with a decrease in PCI levels. The detection in normal subjects and in thrombotic patients of circulating APC:inhibitor complexes supports the view that the protein C pathway is activated during DIC and DVT. Moreover, it emphasizes that both PCI and alpha 1AT are physiologic inhibitors of APC. Thus, measurement of APC complexes may provide sensitive parameters for specific detection of activation of the clotting and protein C pathways.     

Heparin assays and bleeding complications in treatment of deep venous thrombosis with particular reference to retroperitoneal bleeding

Bleeding complications occurred in 30 (11%) out of 280 patients who received continuous heparin infusion for deep venous thrombosis (DVT). 22 (8%) had minor while 8 patients (3%) had major bleeding complications (1 intrathoracic [fatal], 2 gastrointestinal and 5 retroperitoneal). Heparin activity, in daily drawn blood samples, was determined by four assays (chromogenic substrate [CS] assay, activated partial thromboplastin time [APTT], thrombin time with citrated plasma [CiTT] and thrombin time with recalcified plasma [CaTT]). The differences in median heparin activity between patients with minor bleeding and patients with no bleeding did not reach significance for any of the tests. In patients with major bleeding, the differences were significant with the CS (p = .011) and the CaTT (p = .030) assays. Patients with retroperitoneal bleeding had significantly increased median activity judged by all four assays: CS (p = .002), CaTT (p = .003), APTT (p = .010), CiTT (p = .029). The difference was most pronounced after four days of heparin treatment, but there was a considerable overlap with patients without bleeding.     

Characterisation and some properties of the protein C activator from Agkistrodon Contortrix Contortrix venom

The protein C activator (PCA) detectable in the venom of Agkistrodon Contortrix Contortrix (ACCV, Southern Copperhead) by specific immunochromometric assay and anticoagulant activity has been isolated and partially characterized. Chromatography of the crude venom on SP-Sephadex followed by Con A Sepharose and finally on hydroxylapatite was necessary to achieve an electrophoretically - pure product. The isolated PCA is a single chain glycoprotein with strong positive charge and an apparent molecular weight of 36,000. It had an immediate-inhibiting effect on the activated partial thromboplastin time (APTT) of normal plasma with no noticeable effect on the prothrombin time. Its prolonging effect on the APTT was dependent on protein C and it appeared to interfere with the contact mechanism rather than with factors V and VIII. It had enzymatic activity on some tripeptide chromogenic substrates sensitive to thrombin and kallikrein. When mixed with normal plasma it generated activity on substrates sensitive to activated protein C and should be useful for studies of protein C.     

A novel functional assay of protein C in human plasma and its comparison with amidolytic and anticoagulant assays.

A simple and fast method for the quantitative determination of protein C activity in plasma is here described. The first step consists in the conversion of protein C in the test sample into activated protein C by means of an activator isolated from Southern Copperhead venom. Subsequently, the degradation of factor Va, in presence of protein C-deficient plasma, is measured by the prolongation of the prothrombin time which is proportional to the amount of protein C in the sample. The dose-response curve showed a linear relationship from 6 to 150% protein C activity and the inter- and intra-assay reproducibility was 3.5% and 5.6% respectively. In normal subjects, a mean of protein C level of 98 +/- 15% of normal pooled plasma was found. Comparison with the anticoagulant assay in samples of patients with oral anticoagulant, liver cirrhosis, disseminated intravascular coagulation and severe preeclampsia revealed an excellent correlation (r = 0.94, p less than 0.001). Also, a similar correlation (r = 0.93, p less than 0.001) existed between amidolytic assay and the method here proposed for all the samples studied without including the oral anticoagulant group. These results allowed us to infer that this method evaluates the ability of protein C to interact with protein S, phospholipids, calcium ions and factor Va.     

Effect of protein C and activated protein C on coagulation and fibrinolysis in normal human subjects

Although protein C (PC) and activated protein C (APC) have been postulated to be useful for treating patients with thrombosis, their critical effect remains to be studied in human subjects. To examine whether purified PC or APC are useful for treating patients with thrombosis without showing any adverse effect, we studied effects on coagulation and fibrinolysis in normal human subjects. When highly purified human PC was administered intravenously to healthy subjects, plasma levels of immunoreactive PC decreased with a half-life of 10.9 h. Intravenously administered APC decreased with a half-life of 23 min as measured by prolongation of activated partial thromboplastin time (APTT). However, 1.7 h was obtained for the plasma half-life of APC when it was measured immunologically. These findings suggested that a significant fraction of the administered APC was rapidly inhibited by plasma inhibitor. Upon administration of APC, APTT was prolonged and plasma levels of clotting factor VIII (F-VIII) decreased transiently as measured by clotting assay. However, when determined by a chromogenic assay method in which 120-fold diluted plasma samples were used, plasma levels of F-VIII remained unchanged. Plasma levels of F-V did not decrease after APC administration. These findings suggested that prolongation of APTT and apparent decrease in plasma F-VIII clotting activity might be due to the in vitro-effect of APC present in plasma samples used. Diurnal fluctuation of plasminogen activator inhibitor in normal subject was not affected by administration of APC. Thus, PC or APC seems to function selectively at the site of thrombin-formation without lowering plasma levels of coagulation factors.     

Monoclonal antibodies against the heparin-dependent protein C inhibitor suitable for inhibitor purification and assay of inhibitor complexes

Two different monoclonal antibodies against the heparin-dependent inhibitor of human activated protein C were produced, using cleaved modified inhibitor for immunization and partially purified inhibitor for screening of the hybridomas. One of the antibodies recognized free and complexed forms of the inhibitor in immunoblotting experiments. The other antibody was used to develop an assay for APC-PCI inhibitor complexes. Using the assay the formation of complexes was studied in plasma, both in the presence and absence of heparin. The rate of complex formation was similar to that reported previously for the loss of activated protein C amidolytic activity in plasma. The same antibody was also immobilized on Sepharose and used to purify the inhibitor from fresh human plasma. The purified material appeared as two narrowly spaced bands with Mr about 57,000 in SDS-PAGE. The average yield from 1 liter of fresh plasma was 1 mg of inhibitor. The purified inhibitor formed SDS stable complexes with activated protein C and urokinase that could be identified in immunoblots using specific antibodies.     

Congenital protein C deficiency and myocardial infarction:concomitant factor VII hyperactivity may play a role in the onset of arterial thrombosis.

A 29-year-old man with congenital protein C deficiency and acute myocardial infarction is reported. Four hours after the onset of chest pain, he was treated intravenously with tissue-type plasminogen activator. Subsequent coronary angiography revealed only slight stenosis of the left anterior descending coronary artery without any atherosclerosis. The propositus, his brother, and his mother, showed low levels of both protein C activity and antigen, while plasma thrombomodulin levels were normal. His grandfather had died from acute myocardial infarction at 38 years of age. We investigated several other risk factors for arterial thrombosis, including factor VII, fibrinogen, heparin cofactor II, lipoprotein (a), and anticardiolipin antibodies. No other haemostatic abnormalities apart from factor VII hyperactivity were detected in this family. To study the effects of protein C and factor VII on procoagulant activity, prothrombin time was measured after the addition of activated protein C and factor VII to protein C-deficient plasma. The prothrombin time ratio decreased along with an increase in the factor VII level. It also decreased with a decrease in the activated protein C level. These findings indicated that the procoagulant activity of factor VII was enhanced by low protein C levels, suggesting that concomitant factor VII hyperactivity may cause acute myocardial infarction in patients with protein C deficiency.     

EVALUATION OF A NEW SCREENING ASSAY PROC® GLOBAL FOR IDENTIFICATION OF DEFECTS IN THE PROTEIN C/PROTEIN S ANTICOAGULANT PATHWAY

In the present study a new assay, ProC® Global, globally estimating the activity of the main plasma components of anticoagulant protein C/protein S pathway, was evaluated with respect to test characteristics and its sensitivity in the detection of deficiency states of protein C and protein S and of increased aPCR. In the ProC® Global assay procedure protein C is activated in patient's plasma by an activator reagent (venom from agkistrodon contortrix). The extent of the prolongation of a sample's aPTT, caused by the activation of protein C, is taken as a measure for its anticoagulant capacity. Ninetyeight patients with one of the above mentioned defects were investigated. Decreased plasma protein C activity and increased aPCR were detected with a sensitivity of 1.0, while only 11 of 14 patients with decreased levels of free protein S antigen showed abnormal results in the ProC® Global assay (sensitivity=0.79). The test can be used in heparinized samples up to 1.0 anti Xa U/ml heparin (UFH and LMWH). When samples from patients on oral anticoagulant treatment are prediluted with factor V deficient plasma the test is sensitive for increased aPCR. © 1997 Elsevier Science Ltd     

Prediction of recurrent venous thromboembolism by measuring ProC Global

In patients with venous thromboembolism (VTE) a laboratory assay that globally measures the overall thrombophilic tendency is not available. We hypothesized that determination of ProC((R)) Global, a plasma assay which tests the global function of the protein C pathway, could be used to stratify patients according to their risk of recurrent VTE. We prospectively followed 774 patients with first spontaneousVTE for a mean time of 52 months. ProC Global normalized ratio (NR) was measured in plasma by use of a commercially available assay based on activated partial thromboplastin time. Ninety-eight of the 774 patients had recurrentVTE. Patients with ProC Global NR > or = 0.75 had a relative risk of recurrence of 0.59 (95% CI 0.40-0.87) as compared with those with lower ratio. After four years, cumulative probability of recurrence was 8.6% in patients with ProC Global NR > or = 0.75 and 17.4% in patients with a lower ratio (p = 0.006). Patients with a high ProC Global NR have a low risk of recurrent VTE. ProC Global NR can be used to stratify patients with a first unprovoked VTE according to their risk of recurrence.     

Evaluation of an amidolytic heparin assay method: Increased sensitivity by adding purified antithrombin III

A recently described heparin assay using a chromogenic substrate for activated factor X (Xa) has been improved using purified antithrombin III (At-III), leading to increased sensitivity with maintained high accuracy even in pathological plasma samples. Minimum detectable heparin concentration in normal plasma samples was about 0.010 U/ml for the At-III modification and about 0.025 U/ml for the original pooled plasma modification. Blood should be kept on ice from it is drawn and centrifuged at 4 °C to obtain less than 15 000 thrombocytes per μl plasma. Storage of plasma at 0–4 °C for 24 hours, prolonged storage at -20 °C and freezing and thawing did not cause significant loss of heparin activity. When found heparin concentration (heparin added to a final concentration of 0.05 U/ml) was correlated to the amount of platelet factor 4 (PF4) in 11 pathological plasma samples, a correlation coefficient of -0.69 (p <0.02) was found, suggesting that PF4 accounts for a considerable part of the “error” of the amidolytic assay in the low heparin concentration range.The At-III concentration of the test plasma had a moderate influence on the results of the assay in the low heparin concentration range, but this error may be corrected by a simple formula, if the At-III concentration of the test plasma is known.     

The dilute phospholipid APTT: a sensitive assay for verification of lupus anticoagulants

A simple sensitive method for verification of lupus anticoagulants utilizing dilution of phospholipid in the activated partial thromboplastin time (APTT) system is described. Patient plasma, mixed with an equal volume of normal plasma, is activated with micronized silica. To this mixture are added different dilutions of Thrombofax and then calcium chloride. Clotting times are plotted linearly against the logarithm of the phospholipid dilutions and slopes are calculated by regression analysis. In this assay the mean negative slope of 19 plasmas that contained anti-phospholipid activity was five times greater than those of normal plasma or those obtained from patients having single or multiple coagulation factor deficiencies such as those induced by warfarin. The assay can be modified to test heparinized plasmas. Thus, it is a sensitive means by which to verify the presence of lupus anticoagulants in patients who have congenital or acquired factor deficiencies or who are receiving anticoagulant therapy.     

Anticoagulant effects of a low molecular weight heparinoid (Org 10172) in human volunteers and haemodialysis patients

Org 10172, a low MW heparinoid derived from animal intestinal mucosal tissue, has a mean molecular weight of 6500 D and a specific activity of 8.0 anti-Xa U/mg. Its elimination half-life after i.v. administration is 18 hours. Six human volunteers received repeated single i.v. injections of 800 and 3200 anti-Xa units of Org 10172, 5000 IU heparin or placebo. Bleeding time, platelet count and plasma beta thromboglobulin were not affected by Org 10172 or heparin. Heparin stimulated ADP-induced platelet aggregation (0.2 uM; p less than 0.05) and inhibited thrombin induced aggregation (0.3 U/ml; p less than 0.05), while the heparinoid lacked these effects. Heparin increased plasma platelet factor 4, whereas Org 10172 had no effect. In contrast to heparin Org 10172 had only a minor effect on the activated partial thromboplastin time and thrombin time, while both compounds induced anti-Xa activity in plasma. In a crossover study in six haemodialysis patients, both heparin and Org 10172 (34.4 and 22.4 anti-Xa units/kg/body weight) successfully prevented clotting of the extracorporeal circuit. Microscopical analysis of the artificial kidney membranes showed that the 34.4 unit Org 10172 dosage was as effective as heparin in preventing fibrin deposition. The haemostatic and coagulation effects were as expected from those observed in the volunteers except that there was a slower elimination of the plasma anti-Xa response. In addition heparin and Org 10172 (34.4 anti-Xa units/kg) inhibited the Xa-induced platelet aggregation (0.5 U/ml; p less than 0.01 and p less than 0.001 respectively).     

Preparation and partial characterization of human plasma depleted of antithrombin-III by heparin-sepharose affinity chromatography

A simple and rapid method for the complete depletion of functional antithrombin-III from human plasma is described. The procedure involves affinity chromatography of pooled normal plasma on heparin-Sepharose equilibrated with 0.5M NaCl. Only antithrombin-III was removed by this procedure. The prothrombin time, activated partial thromboplastin time, and thrombin clotting time of the antithrombin-III depleted plasma were unchanged relative to the starting plasma. Plasma depleted of antithrombin-III contained no functional antithrombin-III, but contained 8 ug/ml of immunologically detectable antithrombin-III. Removal of this residual antithrombin-III by immunodepletion resulted in plasma which had an identical response to heparin as the plasma subjected only to heparin-Sepharose chromatography.     

In vitro comparison of the effect of heparin,enoxaparin and fondaparinux on tests of coagulation

Low-molecular weight heparin (LMWH) is increasingly used in place of unfractionated heparin (UFH) in patients with unstable angina, and phase II clinical trials using fondaparinux for this indication are underway. Because unstable angina patients often require urgent percutaneous coronary interventions (PCI) or aortocoronary bypass surgery, a point-of-care test is needed to monitor the anticoagulant effect of these agents. The activated clotting time (ACT) and activated partial thromboplastin time (aPTT) are the tests most often used to monitor heparin. The purpose of this in vitro study was to determine whether the ACT or the aPTT can be used to monitor the anticoagulant effect of low-molecular weight heparin and fondaparinux.The ACT and aPTT were measured after heparin, enoxaparin or fondaparinux was added to the blood of healthy volunteers, in doses with equivalent inhibitory activity against activated factor X (factor Xa). To mimic the clinical scenario where an unstable angina patient, who has already received enoxaparin, is urgently taken for PCI or bypass surgery, the ACT was determined after heparin was added to blood containing clinically relevant doses of enoxaparin.We determined that enoxaparin produced significantly less prolongation of both the ACT and the aPTT than heparin, whereas fondaparinux had no effect on either of these tests. Addition of enoxaparin to heparin-containing plasma did not prolong the ACT beyond that produced by heparin alone. The ACT and aPTT therefore cannot be used to monitor low-molecular weight heparin or fondaparinux, highlighting the need for a point-of-care anti-factor Xa assay.     

A second plasma inhibitor of activated protein C: alpha 1-antitrypsin

Inactivation of activated protein C (APC) in normal human plasma was studied in the absence and presence of heparin. In the absence of heparin APC inactivation followed pseudo-first order kinetics. In the presence of heparin the neutralization of APC was found to be biphasic. Up to 500 nM APC could be readily inactivated in normal plasma, indicating that the concentration of the APC inhibitor must be higher than previously assumed. Plasma deficient in the protein C inhibitor (PCI-I, as described by Suzuki and coworkers) and deficient in beta 2-glycoprotein I still possessed APC neutralizing capacity, presumably through the formation of complexes of APC with another plasma protein as was demonstrated by immunoblotting with anti-protein C antibodies. Together these data made us to conclude that a second inhibitor of APC (PCI-II) must be present in normal human plasma. This second inhibitor should be heparin independent, have a relatively high plasma concentration and form complexes with APC. Subsequently, we purified this PCI-II by isolating APC-PCI-II complexes from plasma deficient of vitamin K dependent proteins, PCI-I and beta 2-glycoprotein-I, to which purified human APC had been added. Purified PCI-II has a molecular weight of 50,000 daltons and aminoacid analysis revealed that PCI-II is identical with alpha 1-antitrypsin (alpha 1-AT). The second order rate constant for the reaction between purified alpha 1-AT and APC was found to be 269 M-1 min-1 in the absence of calcium and 602 M-1 min-1 in the presence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)