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.     

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.     

Evaluation of coagulometric assays in the assessment of protein C anticoagulant activity; variable sensitivity of commercial APTT reagents to the cofactor effect of protein S

In vivo expression of protein C activity is dependent on the availability of the activated protein C (APC) cofactor protein S. In the clinical laboratory, measurement of protein C anticoagulant activity is mostly performed in modified APTT assays. We have evaluated 13 commercial APTT reagents for their sensitivity to the cofactor effect of protein S by comparing APC-dependent clotting time prolongations in normal plasma and in protein S depleted plasma. In normal plasma, the sensitivities of the APTT reagents to the anticoagulant effect of APC were markedly different and correlated with the sensitivity of reagents to factor V and VIII. Reagents containing soy phosphatides appeared more sensitive than reagents containing phospholipid of animal origin. Analysis of dose-response curves obtained in normal plasma distinguished one group of reagents showing clotting time prolongations linearly related to the APC concentrations, a second group showing a log-linear relationship and a third group showing a log-log relationship. In protein S depleted plasma, sensitivity of APTT reagents to APC was in general proportional to that observed in normal plasma. However, for some reagents dose-response curves were qualitatively different in normal and in protein S depleted plasma. With all the APTT reagents, APC-dependent clotting time prolongations corresponding to 30-80% of APC anticoagulant activity observed in normal plasma, were observed in protein S depleted plasma. At variance, in a modified Xa one-stage clotting assay, negligible clotting time prolongations were observed in protein S depleted plasma, indicating that over 90% of the APC anticoagulant activity was protein S dependent in this assay system. Dilution of a relative insensitive APTT reagent effectively increased its sensitivity to the cofactor effect of protein S, suggesting that different phospholipid content and/or composition might be responsible for the different sensitivity of APTT reagents to protein S. These results question the validity of APTT based assays for the identification of qualitative protein C abnormalities with defective interaction with protein S.     

Hereditary protein C deficiency caused by the Ala267Thr mutation in the protein C gene is associated with symptomatic and asymptomatic venous thrombosis

Introduction

Protein C (PC) is a key anticoagulant that regulates hemostasis, and inherited deficiency of PC is an established risk factor for venous thrombosis (VT). The factor V Leiden mutation causing activated PC (APC) resistance is an additional risk factor for VT. Reduced PC levels in the circulation and/or APC resistance do not necessarily lead to thrombotic disease. In the present study we describe and characterize an ethnic Lebanese family in which individuals with reduced PC levels and APC resistance have various clinical symptoms.

Methods

PC activity and antigen levels and APC resistance in the family members were quantified with commercial kits. Sequencing of PC DNA and mRNA was performed with BigDye Terminator Cycle Sequencing kit on the ABI 3730 Genetic Analyzer.

Results

PC antigen and anticoagulant activity in the plasma of the proband and family members ranged from 9% to 69% and 3% to 63%, respectively, compared to levels measured in pooled normal plasma. Sequencing analysis of the PC gene of family members revealed that they were either homozygous or heterozygous for the Ala267Thr mutation. In addition, three of them exhibited APC resistance. None of the family members, except the proband, have had a history of VT despite that two of them have two genetic risk factors for thrombosis.     

Inhibitory effects of activated protein C and heparin on thrombotic arterial occlusion in rat mesenteric arteries

Effects of human activated protein C (APC) and heparin on thrombus formation were examined using small mesenteric arteries of rats and video-recording system attached to a microscope. To induce thrombosis we damaged the vessel wall over a short segment by compression and exposed the damaged media to the blood stream. Platelet-rich thrombus enlarged gradually at the damaged site, occluded the vascular lumen for a short period and then flowed away. Such thrombus formation was observed several times after a compression damage. An intravenous administration of APC significantly decreased the total occlusion time from 6.4 +/- 0.7 min at control to 2.2 +/- 0.4 min at 0.9 mg/kg given over 1 min (mean +/- SEM, n = 6, p less than 0.01), and from 6.5 +/- 1.0 min to 1.0 +/- 0.3 min at 3.0 mg/kg (n = 6, p less than 0.01). An intravenous heparin (300 and 1000 U/kg) also decreased the total occlusion time significantly from 6.2 +/- 0.8 to 2.2 +/- 0.8 min (n = 6, p less than 0.05) and from 5.4 +/- 0.8 to 0.8 +/- 0.7 min (n = 6, p less than 0.01), respectively. APC prolonged APTT from 11 +/- 1 sec (n = 5) at control to 50 +/- 5 sec (n = 5) at 0.9 mg/kg and to 87 +/- 8 sec (n = 5) at 3.0 mg/kg, while heparin prolonged APTT to more than 120 sec in all 5 rats at both doses. APTT prolongation by APC was significantly attenuated by inhibiting its residual activity in the plasma samples using monoclonal antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activated protein C resistance and the FV:R506Q mutation in a random population sample--associations with cardiovascular risk factors and coagulation variables.

Activated protein C (APC) resistance, defined as a low APC ratio, is associated with the factor V mutation R506Q (factor V Leiden). APC ratio may also be influenced by other clinical and coagulation variables, which we studied in 460 men and 495 women aged 25-74 years, from a random population sample (Glasgow MONICA Survey). APC ratio correlated positively with APTT; and inversely with factor VIIIc, factor IXc, antithrombin activity, prothrombin F1+2 fragment, and thrombin-antithrombin complexes; but not with other coagulation variables. APC ratio decreased with age, but APTT did not. APC ratio and APTT were significantly lower in women versus men, and were significantly lower in users of oral contraceptives or hormone replacement therapy. The FV:R506Q mutation (prevalence 2.5%) was associated with lower APC ratio and protein C and S activities and with higher factor VIIIc levels; but not with increases in F1+2 fragment or thrombin-antithrombin complexes. APC ratio correlated inversely with total cholesterol and diastolic blood pressure; and in women with triglycerides, systolic blood pressure, and body mass index. Obesity was associated with a significantly lower APC ratio. In contrast, smoking markers correlated positively with APC ratio in men. These associations of APC ratio may be relevant to the increased risks of venous thrombosis with age, female sex, oestrogen use, obesity and high factor VIIIc levels. The association of APC resistance with elevated plasma levels of coagulation markers suggests that this phenotype represents an in vivo hypercoagulable state.     

Sensitivity of the ProC global assay for protein C pathway abnormalities. clinical experience in 899 unselected patients with venous thromboembolism.

The ProC Global is a clotting assay designed to globally evaluate the functionality of the protein C (PC) pathway, which is found defective in up to 30% of the Caucasian patients with thrombophilia. It is based on the ability of endogenous activated protein C (APC), generated by activation of PC by a snake venom extract, to prolong an activated partial thromboplastin time (APTT). This retrospective study was carried out to evaluate the ability of this assay to distinguish patients with or without any PC pathway abnormalities in a cohort of 899 unselected patients with a history of thrombosis. The result of the ProC Global assay, expressed in PC activation time-normalized ratio (PCAT-NR), was significantly lower in patients in whom was previously demonstrated an abnormality of the PC pathway compared to those without. The cut-off level of PCAT-NR=0.75 was found to provide the best sensitivity-specificity ratio, since all the patients with the factor V (FV) Leiden mutation (n=71), APC resistance (n=3), PC deficiency (n=22), or combined defects (n=19) had a PCAT-NR below that value. The sensitivity of the ProC Global assay for a low protein S (PS) level (n=56) was only 66%, and was even weaker in the case of hereditary PS deficiency (46.6%, n=15). The assay did not perform well in samples from patients on oral anticoagulant treatment (OAT, n=64) or with liver failure (n=4), as the PCAT-NR was reduced in most cases, even in the absence of any abnormality. These results suggest that the ProC Global assay could be validly used as a first-step screening test for the FV Leiden-related APC resistance and PC deficiency in patients not on OAT. Given the moderate sensitivity of the assay for PS deficiency, this coagulation inhibitor must be determined in every case. However, the overall benefit of such a screening strategy is limited since more than 38% of the 659 patients without abnormality had a decreased PCAT-NR.     

Protein S levels modulate the activated protein C resistance phenotype induced by elevated prothrombin levels

Elevated plasma prothrombin levels, due to the prothrombin 20210 G/A mutation or to acquired causes, are a risk factor for venous thrombosis, partly because of prothrombin-mediated inhibition of the protein C anticoagulant pathway and consequent activated protein C (APC) resistance. We determined the effect of plasma prothrombin concentration on the APC resistance phenotype and evaluated the role of protein S levels as a modulating variable. The effect of prothrombin and protein S levels on APC resistance was investigated in reconstituted plasma systems and in a population of healthy individuals using both the aPTT-based and the thrombin generation-based APC resistance tests. In reconstituted plasma, APC resistance increased at increasing prothrombin concentration in both assays. Enhanced APC resistance was caused by the effect of prothrombin on the clotting time in the absence of APC in the aPTT-based test, and on thrombin formation in the presence of APC in the thrombin generation-based test. In plasma from healthy individuals prothrombin levels were highly correlated to protein S levels. Since prothrombin and protein S had opposite effects on the APC resistance phenotype, the prothrombin/protein S ratio was a better predictor of APC resistance than the levels of either protein alone. Prothrombin titrations in plasmas containing different amounts of protein S confirmed that protein S levels modulate the ability of prothrombin to induce APC resistance. These findings suggest that carriers of the prothrombin 20210 G/A mutation, who have a high prothrombin/protein S ratio, may experience a higher thrombosis risk than non-carriers with comparable prothrombin levels.     

Plasma cholesteryl ester transfer protein and blood coagulability

Dyslipoproteinemia involving low levels of high density lipoprotein (HDL) is linked to venous thrombosis in young male adults and to recurrence of venous thrombosis in patients who have experienced a previous unprovoked venous thrombosis episode. Plasma cholesteryl ester transfer protein (CETP) modulates HDL metabolism and some lipoproteins can affect blood coagulation reactions with either procoagulant or anticoagulant effects. Hence, we evaluated relationships between the mass of CETP and blood coagulability in plasma samples from 39 normal healthy adults. For clotting initiated by dilute tissue factor or factor XIa, clotting times significantly correlated with CETP antigen levels. Thus, coagulation initiated by either the extrinsic or intrinsic coagulation pathway is positively correlated with CETP plasma levels. When added to plasma, a recombinant CETP preparation dose-dependently shortened factor Xa-1-stage clotting times, showing that it augmented procoagulant activity in plasma. In reaction mixtures containing purified factors Xa and Va and prothrombin, the recombinant CETP preparation dose-dependently increased prothrombin activation, suggesting it specifically enhances prothrombinase activity. Thus, our data highlight a previously unknown positive relationship between CETP plasma levels and blood coagulability that might relate to risks for thrombotic events.     

Contribution of polymorphisms in the endothelial protein C receptor gene to soluble endothelial protein C receptor and circulating activated protein C levels, and thrombotic risk

Endothelial cell protein C receptor (EPCR) enhances the generation of activated protein C (APC) by the thrombin-thrombomodulin complex. A soluble form of EPCR (sEPCR), which is generated by metalloprotease activity, is present in plasma.The distribution of sEPCR levels in healthy populations is bimodal. Previously, we described two polymorphisms in exon 4 of the EPCR gene, 4600A/G that encodes the substitution of Ser219 by Gly in the transmembrane region of EPCR and 4678G/C in the 3'-UT region. The aim of this study was to investigate the relationship between these two polymorphisms and plasma sEPCR and APC levels and risk of venous thrombosis.We genotyped 401 healthy controls from the Spanish population and measured their plasma sEPCR and APC levels. Carriers of the 4600AG genotype had significantly higher sEPCR levels than those with the AA genotype, while the 4678CC genotype was associated, to a lesser extent, with elevated APC levels. To assess the effect of these polymorphisms on the risk of thrombosis, we genotyped 405 patients with venous thromboembolism. The frequency of the 4600AG genotype was very similar in patients and controls (p=0.975), whereas the 4678CC genotype was significantly more frequent in controls than in patients (p=0.008). In multivariate analysis, carriers of the 4678CC genotype had a decreased risk of thrombosis (OR=0.61, p=0.009).These data indicate that individuals carrying the 4600AG genotype have high sEPCR levels but do not have an increased risk of thrombosis, whereas individuals carrying the 4678CC genotype have higher APC levels and lower risk of venous thromboembolism.     

Brain-specific protein C activation during carotid artery occlusion in humans

BACKGROUND AND PURPOSE: Activation of plasma protein C (PC) zymogen by thrombin-thrombomodulin at the endothelial surface is an important endogenous antithrombotic mechanism. It is unknown whether activated protein C (APC) is generated in vivo in the cerebrovasculature, because there is only limited thrombomodulin expression in human brain vascular endothelium. Therefore, we tested the hypothesis that carotid occlusion produces brain-specific PC activation. METHODS: Blood samples were simultaneously collected from the ipsilateral internal jugular vein and radial artery before and during carotid cross-clamping and on "de-occlusion" in 8 awake patients undergoing routine carotid endarterectomy. Plasma PC zymogen and circulating APC levels were measured using enzyme immunocapture assay and expressed as percent of pooled plasma controls. RESULTS: Internal jugular vein APC levels increased 28% exclusively during carotid occlusion and then decreased 32% with de-occlusion (F=8.1, P<0.005). PC zymogen increased only 5.9% with occlusion (F=6.3, P<0.02), consistent with hemoconcentration. There were no changes in radial artery PC or APC levels. CONCLUSIONS: These findings demonstrate brain-specific protein C activation in humans during carotid occlusion and suggest a protective role for endogenous APC generation during cerebrovascular occlusion.     

Impairment of the protein C anticoagulant pathway in a patient with systemic lupus erythematosus, anticardiolipin antibodies and thrombosis

We have identified an inhibitor of the protein C anticoagulant pathway in the plasma of a patient with systemic lupus erythematosus and a history of recurrent deep vein thrombosis, fetal wastage, and seizures. The patient's plasma contained anticardiolipin antibodies as well as a weak lupus anticoagulant. Examination of this patient's plasma revealed normal levels of protein C and protein S antigen, normal levels of functional protein C, as well as essentially normal levels of every blood coagulation factor. In a modified prothrombin time assay, the activated protein C-mediated prolongation of the clotting time observed in normal plasma was not observed in this patient's plasma. Gel permeation chromatography of the patient's plasma revealed that the inhibitory material was a high molecular weight protein that coeluted with the IgM peak. The inhibitor did not appear to circulate as a complex with protein C, since the inhibitor could easily be separated from protein C during fractionation procedures, and did not interfere with the activation of protein C in plasma as assessed by a functional amidolytic assay. Our findings suggest that the recurrent thrombotic episodes observed in this patient may have occurred as a result of the patient's antiphospholipid antibody neutralizing specific phospholipids essential for the full expression of the anticoagulant activity of activated protein C.     

Comparison of three activated protein C resistance tests in the risk assessment of venous thrombosis in non-carriers of the factor V Leiden mutation

Activated protein C resistance (APCR), measured using the original assay described by Dahlb?ck, is a risk factor for venous thrombosis independent of the factor V Leiden (FVL) mutation. This assay is based on the activated partial thromboplastin time (APTT) after plasma exposure to activated protein C (APC). As this assay was sensitive to numerous interferences, new assays have been developed for FVL screening. The objectives of the study were to investigate the association of second generation assays for APCR with venous thrombosis in FVL non-carriers. One hundred ninety-seven subjects with a history of venous thrombosis and 211 controls were explored using 3 APCR assays, the original APTT-based assay (test A), an APTT-based assay with factor V depleted plasma pre-dilution (test B) and a direct factor X activation-based assay with the same pre-dilution (test C). We found that subjects with results in the lowest quartile of the APTT-based assays are at increased risk, compared to those in the highest quartile (test A Odds Ratio = 6.39; 95% CI 3.23-12.63; test B OR = 2.72; 95% CI 1.50-4.94). There was no significant risk increase associated with test C results. After adjusting for FVIII levels, the ORs of tests A and B were similar (test A OR = 3.22; 95% CI 1.47-7.08; test B OR = 3.10; 95% CI 1.54-6.21). In conclusion, APTT-based assays, but not direct factor X activation-based assays, effectively detect the risk for venous thrombosis independent of FVL. Pre-dilution in factor V depleted plasma is an effective way to directly assess the risk independent of FVIII levels.     

Preparation of succinylthrombin and its effects in vivo on the coagulation system

An innovative chemically modified thrombin, succinylthrombin, was prepared by the treatment of thrombin with succinic anhydride. Succinylthrombin showed a remarkable reduction of fibrinogen clotting activity with a reduction of the potency for protein C (PC) activation. However, the degree of the remaining potency for PC activation was greater than the degree of the remaining clotting activity. The potency for PC activation was enhanced by the addition of thrombomodulin and calcium ion. The degree of the enhancement was approximately 6-times greater than that of acetylthrombin, which we previously suggested as an effective anticoagulant. The infusion of succinylthrombin into rabbits induced a prolongation of the activated partial thromboplastin time (APTT). The fibrinogen level was not affected even by the infusion of succinylthrombin of a dose which cause the prolongation of the APTT to the same extent as that following an infusion of thrombin. The factor Xa clotting time was also prolonged by the succinylthrombin without any significant drop in the number of the circulating platelet.     

Activated protein C: alpha 1-antitrypsin (APC: alpha 1 AT) complex as a marker for in vitro diagnosis of prethrombotic states.

The purpose of the study was to test whether APC: alpha 1AT complex is a useful clinical marker of the activation of coagulation. The rationale for this is that activated protein C may appear in circulation at an early stage of blood coagulation, when subcoagulant amounts of thrombin are formed. Given the relatively higher half-life of APC: alpha 1AT as compared to that of thrombin:AT-III (TAT) complexes, we hypothesized that APC:alpha 1AT could represent an amplification of the thrombin generated in the first events of coagulation. Using sandwich ELISA's we measured APC: alpha 1AT and TAT complexes as well as complexes of AT-III with its target proteases in normal subjects and in several clinical groups of patients prone to thrombotic episodes, including pregnancy, preeclampsia, hemodialysis, gynecological tumors, diabetes and oral contraceptives. APC: alpha 1AT complex was significantly increased in all clinical groups as compared to normal subjects and showed relatively higher increases than did TAT and ATM complexes in the majority of the groups studied. There was a significant and positive correlation between APC: alpha 1AT and TAT complex levels in the majority of the groups, as well as between TAT and ATM and between APC: alpha 1AT and ATM complex levels. We conclude that APC: alpha 1AT complex can be used as a sensitive marker of prethrombotic states.     

Role of protein S and tissue factor pathway inhibitor in the development of activated protein C resistance early in pregnancy in women with a history of preeclampsia

Pregnancy increases the risk of venous thromboembolism. Particularly in early pregnancy, the thrombosis risk can be attributed to the changes in coagulation. Elevated thrombin generation and resistance to activated protein C (APC) are likely to contribute to the increased thrombosis risk during pregnancy. We studied changes and the determinants of thrombin generation and APC resistance in the first 16 weeks of gestation in women with history of preeclampsia. Additionally, we investigated the influence of pregnancy-induced haemodilution on the coagulation system. We measured thrombin generation, APC resistance and plasma levels of prothrombin, factor V, factor X, protein S and tissue factor pathway inhibitor (TFPI) in 30 non-pregnant and 21 pregnant women at 8, 12 and 16 weeks of gestation. All participants shared a history of a hypertensive complication in the preceding pregnancy. Thrombin generation and APC resistance were higher at eight weeks of pregnancy than in the non-pregnant state, and progressively increased between eight and 16 weeks of gestation. Changes in the TFPI and protein S levels accounted for ~70% of pregnancy-induced APC resistance. Interestingly, a significant correlation (slope 2.23; 95%CI: 1.56 to 2.91; r= 0.58) was observed between protein Stotal or protein Sfree levels and haematocrit. In conclusion, pregnancy induces a decrease of TFPIfree and protein Sfree levels that attenuates the function of the TFPI and protein C systems and results in elevated thrombin generation and increased APC resistance. Besides, our data suggest that pregnancy-dependent haemodilution may contribute to the decreased peripheral protein S levels.     

急性脑血栓形成及其高危人群血浆PC、PS、D-Dimer水平的测定

目的 探讨蛋白Ｃ（ＰＣ）、蛋白Ｓ（ＰＳ）和Ｄ－二聚体（Ｄ－Ｄｉｍｅｒ）对诊断脑血栓形成的血栓前状态意义。方法 采用ＡＰＴＴ法、发色底物法和免疫过滤法检测脑血栓形成急性期、高危人群、对照组的血浆ＰＣ、ＰＳ、Ｄ－Ｄｉｍｅｒ的变化。结果 ３组间ＰＣ、ＰＳ、Ｄ－Ｄｉｍｅｒ水平有非常显著性差异。前两组间ＰＣ、ＰＳ活性的差异无统计学意义，Ｄ－Ｄｉｍｅｒ浓度有显著性差异。结论 脑血栓形成的高危人群处于血栓前状态     

Functional assays for protein C activity and protein C inhibitor activity in plasma

An assay system for protein C (PC) activity and PC-inhibitor in plasma was developed. The assay was based on: (1) binding of PC to wells of a microtiter plate coated with a murine monoclonal anti-PC antibody (C3) that did not interfere with the activity or activation of PC; (2) activation of immobilized PC with Protac C; (3) incubation with or without a source of activated PC inhibitor; and (4) measurement of amidolytic activity using the substrate S-2366. The activity assay was specific for PC and sensitive to less than 1 microliter of plasma or 4 ng PC. Inhibition of activated PC by plasma followed pseudo first order kinetics. Heparin caused a dose dependent increase in the inhibition rate with half maximal stimulation at approximately 3 U/ml and maximal stimulation at heparin concentrations greater than or equal to 10 U/ml. This assay is suitable not only for determination of functional plasma levels of PC and PC inhibitor activities but also for kinetic studies of inhibition of activated PC in complex systems, such as plasma. Studies showed that urokinase interfered with the inhibition of APC by plasma inhibitor(s).     

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).