Variations of protein C in uremic hemodialysed patients

Protein C has been measured by three different assays (antigenic, amidolytic and chronometric) in 27 end-stage renal insufficient patients before and after hemodialysis. Protein C levels have been compared with other coagulation inhibitors (antithrombin III, protein S) and fibrinolytic parameters. Baseline anticoagulant activity of protein C has been found impaired in eight cases whereas other inhibitors were normal. In four cases, both anticoagulant and antigenic levels were low. In one case, amidolytic method could also found a low activity. Hemodialysis leads to an increase of protein C activity and antigen level. Heparinemia after hemodialysis does not interfere with the chronometric measurement of protein C anticoagulant activity. Total protein level, hematocrit, protein S and antithrombin III are also elevated after hemodialysis. Baseline fibrinolytic parameters are normal and remain unchanged after hemodialysis. The clinical relevance of such modifications is discussed.     

Protein C assays in uremia

Protein C was determined in 42 patients with terminal uremia and 20 healthy controls in three different ways 1) anticoagulant activity 2) amidolytic activity 3) antigen level. Protein C anticoagulant activity was markedly decreased in uremia, but was partly normalized during hemodialysis treatment, whereas the amidolytic activity and antigen level of protein C were normal and without changes during dialysis. The activities and antigen levels of factor II and X were normal before and after hemodialysis. In anticoagulated patients we found a good correlation between prothrombin levels and protein C levels determined with three different assays. We did not find any evidence for a defect carboxylation of protein C as the cause for the defective protein C in uremia. The BaCl2 precipitation in the Protein C anticoagulant assay was incomplete both in uremia and in controls but without differences between the two groups. In vitro addition of urea and creatinine did not decrease protein C activity. The cause of the defective protein C in uremia is still not known but it might contribute to thromboembolic complications.     

Assay of protein c in human plasma: Comparison of amidolytic, coagulation, and immunochemical assays

We studied functional protein C activity, both anticoagulant and amidolytic, as well as protein C antigen in 30 normal subjects, several members of a family with congenital protein C deficiency, 18 patients with severe preeclampsia, 27 patients with coronary heart disease, including 15 patients with myocardial infarction and 12 with angor pectoris, 20 patients on stable oral anticoagulant therapy (thrombotest values: 3–12%) and three patients with disseminated intravascular coagulation. Protein C values measured by the coagulant assay were compared to those obtained with amidolytic and immunochemical assays. In all the groups studied, the activity assays (amidolytic and coagulant) correlated significantly with each other as well as with the immunochemical assay. In patients on oral anticoagulant therapy the coagulant assay gave lower protein C values than amidolytic and immunochemical assays. A good correlation was found between immunological and amidolytic protein C assays (r=0.90, p < 0.001), immunological and coagulant protein C assays (r=0.93, p < 0.001), and amidolytic and coagulant protein C assays (r=0.95, p < 0.001) in all the samples studied without including the protein C values of patients on oral anticoagulant therapy. These results allow us to recommend the functional protein C coagulant assay in patients on stable oral anticoagulant therapy because only this assay evaluates the “in vivo” protein C function in these patients.     

Dysfunctional activated protein C (PC Cádiz) in a patient with thrombotic disease

The partial characterization of a dysfunctional protein C (PC), provisionally named "PC Cádiz", in a 45-year-old male patient suffering from recurrent venous thrombosis is described. The only defect found in laboratory assays for haemostasis and hepatic function was a half normal level of both amidolytic and anticoagulant protein C activity, measured by different functional assays that use thrombin-thrombomodulin complex and a snake venom to activate protein C. Protein C antigen was always found to be within normal levels. Two young daughters of the propositus were found to have the same defect. Double-crossed immunoelectrophoresis, performed in the presence and absence of Ca2+ in the first dimension, showed no clear differences between patient and control PC. PC adsorption to barium salts was also found to be normal. Measurement of the PC activation peptide in the barium citrate eluates after PC activation showed no significant differences between patient and 10 normal controls, the concentration of this peptide being very similar to that of PC zymogen in the same eluates before PC activation. These results indicate that this abnormal PC is able to be normally activated by thrombin-thrombomodulin complex but does not exhibit serine protease activity, probably due to a defect in the PC molecule near the active site center.     

Low levels of the anticoagulant activity of protein C in patients with chronic renal insufficiency: an inhibitor of protein C is present in uremic plasma.

Twenty-nine of 54 uremic patients had low levels of protein C measured as anticoagulant activity, contrasting with normal levels measured as amidolytic activity or antigenic concentration. We demonstrate that this discrepancy is due to the presence of a soluble plasma inhibitor that interferes specifically with the anticoagulant activity of activated protein C. The inhibitor does not interfere with other coagulation assays. It is resistant to diisopropylfluorophosphate, high temperatures and repeated freezing and thawing. It can be dissociated from protein C by anti-protein C antibodies or by dialysis in vitro and in vivo. It binds to positively charged resins and can be eluted with high salt concentrations without losing its inhibitory capacity. The inhibitory effect is correlated with plasma creatinine levels and fluctuates with time.     

Hereditary dysfunctional protein C molecules (type II): assay characterization and proposed classification

Protein C (PC) deficiency is among the increasing number of recognized causes of hereditary thrombotic disease. Two types of PC deficiency have been described: 1) Type I, which is characterized by a concomitant decrease in PC activity and antigen, and 2) Type II, characterized by disproportionately low activity compared to antigen (i.e. a dysfunctional molecule). To date, only a small number of Type II patients have been described. This study was undertaken to evaluate a number of dysfunctional PC molecules by comparing PC clotting and amidolytic activities with antigen levels. For these studies, an automated PTT-based clotting PC assay was developed. This assay was sensitive to 1% of a normal plasma pool, specific, accurate, and reproducible (+/- 12%). A good correlation (r = 0.918) of the clotting activity to antigen was found in normal individuals and Type I heterozygous and homozygous patients. To classify Type II PC deficient patients, the antigen, amidolytic and clotting PC levels were compared in ten affected families. The clotting activities were decreased in all affected members, whereas the antigen levels were within the normal limits. In four of the 10 families, the amidolytic activity was normal and similar to the antigen levels. This suggests that in certain families, defects in the PC molecule occur in regions not associated with amidolytic functions. From these studies, the molecular basis of Type II PC deficiency is varied and complex, involving different functional domains of the PC molecule. Therefore, we have suggested a nomenclature algorithm for Type II PC deficiency based on the location of the defect within the specific domains of the PC molecule.     

Abnormal propeptide processing resulting in the presence of two abnormal species of protein C in plasma: characterization of the dysfunctional protein C Padua3 (protein C(R-1L/propeptide)).

A heterozygous G-->T transversion at position 1388 of the protein C (PC) gene which predicted the substitution of Arg(-1) to a Leu (PC(R-1L)) was identified in a thrombophilic patient. The PC(R-1L) was purified from the patient's plasma by immunoaffinity chromatography using Ca++-independent and Ca++-dependent monoclonal antibodies. NH2-terminal sequencing of the light chain of PC(R-1L) revealed two amino acid sequences: one was identical to the complete propeptide sequence of PC, while the other matched the normal PC light chain sequence elongated by one amino acid (Leucine at position 1). Activated PC(R-1L/propeptide) exhibited normal amidolytic and impaired anticoagulant activity. Thus, the substitution of a Leu for an Arg at position -1 of PC shifts the propeptidase cleavage site by one amino acid. In addition, in PC(R-1L/propeptide) the propeptide cleavage at Lys(-2) is less efficient since approximately 60% of PC variant molecules present in patient's plasma retained the entire propeptide. Our findings suggest that depending on the specific amino acid substitution at position-1, PC can be secreted in plasma containing the entire propeptide attached to the light chain. Impaired interaction of elongated APC molecules with a membrane-surface and/or factor Va which is the physiological substrate for APC, is manifested in vivo by thrombophilia.     

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.     

Functional assays of protein C: comparison of two snake-venom assays with two thrombin assays

Protein C (PC) activities measured by two thrombin-based assays have been compared with those obtained by two assays based on snake venom activation of plasma PC followed by measurement of both the amidolytic and anticoagulant activities of activated PC. This study indicates that snake venom assays gave results similar to those of the thrombin assays in 20 healthy subjects, in 16 patients with DIC and in 15 patients with congenital PC deficiency. There was, however, some degree of misclassification of normals and congenitally-deficient patients, with only the clotting snake venom assay resulting in no misclassifications. In 15 patients stabilized on warfarin treatment and in 17 with liver disease, the clotting snake venom assay gave significantly lower values than the other assays, so that it might prove to be more sensitive than the other assays to these defects.     

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).     

A new hereditary abnormal protein C (protein C Yonago) with a dysfunctional Gla-domain.

A familial abnormal protein C most probably with the dysfunctional Gla domain was found in a 60-year-old man with recurrent thrombosis. Namely, the anticoagulant activity as measured by the APTT method and the antigen level by an ELISA utilizing a calcium-dependent antibody were reduced to nearly half of normal, 43.5% and 2.1 micrograms/ml (normal range: 2.8-5.0 micrograms/ml), respectively. On the other hand, the amidolytic activity determined on a synthetic chromogenic substrate, S-2366, and the total antigen measured by an ELISA utilizing a polyclonal antibody were both in the normal range, 74.1% and 83% of normal, respectively. Crossed immunoelectrophoresis showed more anodal migration than the normal control in the presence of calcium ions, and adsorption of protein C to barium citrate was insufficient. These data altogether indicated that a half population of protein C in the patient's plasma was dysfunctional in the Gla domain or its related structures. Four other members of his immediate family were found to have the same abnormality of protein C, although they had been all asymptomatic. We thus conclude that the dysfunctional protein C is hereditary, and that the abnormalities noted in several tests are most likely due to a structural defect residing in the Gla or its related regions. We hereby designate this abnormal protein C as protein C Yonago.     

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.     

Protein C Sapporo (protein C Glu 25 --> Lys): a heterozygous missense mutation in the Gla domain provides new insight into the interaction between protein C and endothelial protein C receptor

Interaction of the gamma-carboxyglutamic acid (Gla) domain of protein C with endothelial protein C receptor (EPCR) is a critical step for efficient activation of protein C, though interactions by mutants in the Gla domain of protein C with EPCR have been rarely evaluated. We identified a 44-year-old Japanese woman with a history of recurrent thromboembolism as an inherited missense mutation, the first such case reported in Japan, which involved a protein C Gla 25 mutation. Total protein C antigen and Gla protein C antigen levels in the proband were normal. Protein C activity measured with an anticoagulant assay was reduced, whereas that measured with an amidolytic assay was normal. She was therefore phenotypically diagnosed as type IIb protein C deficiency. Direct sequencing of the PCR fragments revealed a heterozygous G to A transition at nucleotide position 1462 in exon 3, which predicted an amino acid substitution of Glu 25 by Lys. Her mother and one son were also heterozygous for this mutation. A molecular dynamics simulation of Gla 25-->Lys/EPCR complex in water suggested that the affinity between the molecules was decreased compared to the wild type Gla domain/EPCR complex. Since Gla 25 has been shown to play an important role in protein C function, not only in membrane phospholipid binding but also in binding to EPCR, our findings provide new insight into the mechanism by which the Glu 25-->Lys mutation induces type IIb protein C deficiency in individuals.     

An international collaborative study on the performance of protein C antigen assays. Report of the ICTH subcommittee on protein C. International Committee on Thrombosis and Haemostasis

An international collaborative study was undertaken to evaluate the performance and specificity of protein C antigen (PC) assays. Thirteen lyophilized plasma samples were distributed among 17 laboratories and analysed with 24 methods. No statistically significant results were obtained with the different methods in plasmas containing only the protein C zymogen. ELISA's, RIA's and IRMA's were found to be more sensitive than the electro-immunoassay. In plasmas of patients on oral anticoagulant treatment ELISA methods tend to give lower PC antigen levels than the electro-immunoassay. Complexes between activated protein C (APC) and the protein C inhibitor (PCI), when present in plasma together with PC zymogen, are detected with 100% efficiency in the electro-immunoassay, with 50% efficiency in the ELISA, and with less than 10% efficiency in the RIA or in assays using monoclonal antibodies against PC. Mean coefficient of variation was calculated to be 22%, and could be reduced - especially in case of the ELISA by normalisation. Within laboratory variation was calculated to be 11.7% and between laboratory variation 17.8%.     

Inhibition of human factor VIIa-tissue factor activity by placental anticoagulant protein

Previous studies indicated that human placental anticoagulant protein, a member of the lipocortin family, prolonged the clotting time of normal plasma when clotting was induced by brain thromboplastin or by kaolin in the presence of cephalin and calcium. Using a two-stage amidolytic assay to assess factor X activation and a tritiated peptide release assay to assess factor IX activation, we have examined the ability of purified preparations of placental anticoagulant protein (Mr = 36.5 kDa) to inhibit the activation of either factor X or factor IX by a complex of human factor VIIa-tissue factor. Placental anticoagulant protein markedly inhibits factor X and factor IX activation by factor VIIa-tissue factor in a non-competitive manner with Ki values of 40 nM and 70 nM, respectively. Placental anticoagulant protein had no effect on factor Xa amidolytic activity, and its inhibitory activity was not diminished by prior incubation with antibody raised against partially purified plasma extrinsic pathway inhibitor. Binding of placental anticoagulant protein to phospholipid vesicles, crude tissue factor and purified, relipidated human brain tissue factor apoprotein was observed only in the presence of calcium ions. These results indicate that placental anticoagulant protein is a potent factor VIIa-tissue factor inhibitor and suggests that its mechanism of action involves binding to the phospholipid portion of the tissue factor lipoprotein.     

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.     

Anticoagulant and antithrombotic properties of synthetic sulfated bis-lactobionic acid amides

Sulfated bis-lactobionic acid amides, a new class of polyanions with heparin-like properties, were synthesized and their antithrombotic and anticoagulant activities were determined. Compared to heparin and Fragmin^R, a low molecular weight heparin, the substances exhibited moderate to low anticoagulant activities in aPTT, thrombin clotting time and Heptest assays. In amidolytic assays no anti-Ila activity and only exceedingly low anti-Xa activity was observed. The antithrombotic activity of the bis-lactobionic acid amides was determined using two thrombosis models. In rabbit and rat models thrombi were induced by a combination of endothelial damage and reduction of blood flow or only by endothelial damage. At least one of the bis-lactobionic acid amides (LW 10082) exhibited a considerable antithrombotic activity which was similar to low molecular weight heparin.     

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.     

Recombinant human protein C: comparative functional studies with human plasma protein C

Protein C (PC) is the central protein in a major antithrombotic regulatory mechanism. Hereditary deficiencies of PC are associated with thrombosis. Therapeutic PC replacement may be an important treatment if pure functional human protein C is available in sufficient quantity. Human PC has been produced on a commercial scale using recombinant techniques. To study the functional properties of recombinant protein C (r-PC), we undertook a comparative investigation of the basic properties of r-PC and plasma protein C (n-PC). Both were isolated by immunopurification methods. Protac C activation proceeded at the same rate and kinetics for both forms. With thrombin-thrombomodulin (T-TM) activation, r-PC is significantly better than the activation of n-PC (for r-PC: Kcat/Km = 378 vs. n-PC: Kcat/Km = 35). No difference in the anticoagulant (aPTT prolongation) or profibrinolytic activities (inactivation of PAI-1 and PAI-3) were observed between activated r-PC and n-PC. Based on these functional studies, recombinant protein C has similar properties to the plasma form of protein C. However, T-TM activation of r-PC occurs faster than the n-PC. The mechanism is unknown, but may be due to the presence of larger amounts of single chain protein C which exists in a conformation more rapidly activated by the T-TM complex.