The ability of three global plasma assays to recognize thrombophilia

Three global assays, the Calibrated Automated Thrombogram (CAT), the ProC Global (PCG), and the Coagulation Inhibitor Potential (CIP) were performed in frozen plasma samples from 24 normal controls and 24 patients with inherited thrombophilia. Six patients had inherited antithrombin (AT) deficiency; 18 patients had abnormalities in the protein C/S anticoagulant system (protein C deficiency (n=3), protein S deficiency (n=10), homozygous FV Leiden mutation (n=5)). Nine of these twenty four patients carried additionally the heterozygous FV Leiden mutation. All three assays separated the thrombophilia group and the control group (P=0.083 for CAT, P<0.0001 for the other two assays) but there was considerable overlap, particularly in the CAT assay. The CAT assay separated all plasma samples with AT deficiency but was less sensitive to abnormalities in the protein C/S system. In contrast, ProC Global was more sensitive to abnormalities in the protein C system than to AT deficiency. The CIP assay was approximately equally sensitive to defects in both systems. Receiver operator characteristic (ROC) curves confirmed that the ProC Global and the CIP assays performed better than the CAT assay (P=0.0179 and P=0.0003, respectively). With the CIP assay ROC analysis showed that with a sensitivity of 100% the specificity was 87.5%. With the PCG assay, optimal threshold resulted in both a sensitivity and a specificity of 79.2%. Although our material is relatively small, the data suggest that at a cut-off value with a specificity of >80%, the CIP assay should be evaluated as a screening test for severe thrombophilia.     

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.     

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.     

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.     

A prothrombin time-based functional assay of protein S

Protein S activity was measured as the degree of prolongation of a prothrombin time-based clotting assay in which diluted test sample, protein S-depleted plasma previously incubated with Protac to fully activate protein C, bovine thromboplastin and calcium ions are mixed. Assay specificity was first demonstrated by observing that the prolongation of the clotting time was dependent on protein S and was subsequently confirmed by testing plasma samples from patients with conditions known to affect protein S activity. High sensitivity, reproducibility (interassay coefficient of variation lower than 5%) and easy handling of samples and reagents make this assay suitable for screening of congenital and acquired protein S deficiency.     

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.     

Multicentre evaluation of IL Test Free PS: a fully automated assay to quantify free protein S

Deficiency of the anticoagulant vitamin K-dependent protein S (PS) is associated with increased risk of venous thrombosis. In human plasma, PS circulates in two forms: as free protein (free PS) and PS bound to C4b-binding protein (C4BP), a regulator of the complement system. Assays for free PS have higher sensitivity and specificity for protein S deficiency than assays for total protein S. We have extensively evaluated the analytical performance of a novel assay for free PS, the IL Test Free Protein S, which takes advantage of the affinity of C4BP for free PS, and compared its performance to existing methods. IL Test Free Protein S is a rapid, fully automated turbidimetric assay consisting of two reagents: a C4BP coated latex and an anti-PS monoclonal antibody coated latex. The test range, precision and linearity were adequate and the assay tolerated high concentrations of interfering substances of clinical significance. The reference range agreed with previously published studies. The analysis of 903 patient samples belonging to 20 different clinical categories with the new assay yielded free PS results that agreed well with those obtained using the assays established in the participating laboratories. The study demonstrated the IL Test Free Protein S to be rapid, reliable and easy to perform.     

"ProC Global": a functional screening test that predicts recurrent venous thromboembolism

Abnormalities of the Protein C (PC) pathway are found in the majority of patients with thrombophilia. ProC Global is a coagulation assay that reflects the net effect of the PC pathway by measuring the activated partial thromboplastin time (APTT) of patient and control plasma, before and after activation of endogenous PC by Protac, a snake venom. Previous studies have suggested that abnormalities in this test are associated with an increased risk of venous thromboembolism (VTE). A retrospective analysis was performed using frozen plasma samples from 140 patients with confirmed VTE to determine whether an abnormal ProC Global result (in the presence and in the absence of known abnormalities in the PC pathway) is a predictor of initial and recurrent VTE. Patients were tested for the presence of activated protein C resistance, Factor V Leiden, PC and protein S (PS) deficiency, and non-specific inhibitor positivity. Mean ProC Global results were significantly lower in patients with recurrent VTE than in patients without recurrent VTE. The association between abnormal ProC Global result and recurrent VTE showed a strong trend, before (odds ratio, OR 3.6) and after (OR 3.1) exclusion of known thrombophilic abnormalities. Patients with a first episode of idiopathic VTE also expressed significant lower ProC Global results than those with secondary VTE. After exclusion of known PC pathway abnormalities, there was a statistically significant association between abnormal ProC Global and initial idiopathic VTE (p=0.04). These results suggest that ProC Global may serve as a predictor of recurrent VTE and potentially for first episode of idiopathic VTE. ProC Global may help identify patients at increased risk of initial and recurrent VTE.     

Hereditary protein S deficiency and venous thrombo-embolism. A study in three Dutch families

Protein S, a vitamin K-dependent coagulation factor, is involved in the regulation of the anticoagulant activity of activated protein C. Using an immunoradiometric assay for total protein S in plasma we identified 14 patients (7 male and 7 female) in three unrelated Dutch families as fulfilling the criteria for an isolated protein S deficiency. In 9 patients who were not receiving oral anticoagulant treatment the mean total protein S antigen concentration was 0.50 +/- 0.08 U/ml (+/- S.D.) and the calculated free protein S concentration was 0.15 +/- 0.01 U/ml (+/- S.D.). In the five patients who were on oral anticoagulant treatment the mean total protein S antigen was 0.23 +/- 0.05 U/ml (+/- S.D.). Seven of the 14 patients had a history of venous thromboembolism occurring at a mean age of 25 years and often without an apparent cause. Protein S deficiency is inherited as an autosomal dominant trait.     

A randomized cross-over study on the effects of levonorgestrel- and desogestrel-containing oral contraceptives on the anticoagulant pathways

The use of oral contraceptives (OC) causes disturbances of the procoagulant, anticoagulant and fibrinolytic pathways of blood coagulation which may contribute to the increased risk of venous thrombosis associated with OC therapy. Here we report the results of a cycle-controlled randomized cross-over study, in which we determined the effects of so-called second and third generation OC's on a number of anticoagulant parameters. In this study, 28 non-OC using women were randomly prescribed either a second generation (150 microg levonorgestrel/30 microg ethinylestradiol) or a third generation OC (150 microg desogestrel/30 microg ethinylestradiol) and who switched to the other OC after a two month wash out period. The anticoagulant parameters determined were: antithrombin (AT), alpha2-macroglobulin (alpha2-M), alpha1-antitrypsin, protein C inhibitor (PCI), protein C, total and free protein S and activated protein C sensitivity ratios (APC-sr) measured with two functional APC resistance tests which quantify the effect of APC on either the activated partial thromboplastin time (aPTT) or on the endogenous thrombin potential (ETP). During the use of desogestrel-containing OC the plasma levels of alpha2-M, alpha1-antitrypsin, PCI and protein C significantly increased, whereas AT and protein S significantly decreased. Similar trends were observed with levonorgestrel-containing OC, although on this kind of OC the changes in AT, PCI and protein S (which was even slightly increased) did not reach significance. Compared with levonorgestrel, desogestrel-containing OC caused a significant decrease of total (p <0.005) as well as free protein S (p <0.0001) and more pronounced APC resistance in both the aPTT (p = 0.02) and ETP-based (p <0.0001) APC resistance tests. These observations indicate that the activity of the anticoagulant pathways in plasma from users of desogestrel-containing OC is more extensively impaired than in plasma from users of levonorgestrel-containing OC.     

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).     

Determination of plasma protein S--the protein cofactor of activated protein C

Protein S, an important cofactor of activated protein C, and C4b-binding protein were purified from human plasma. Specific antibodies against the purified proteins were raised in rabbits and used for the development of immunologic assays for these proteins in plasma: an immunoradiometric assay for protein S (which measures both free protein S and protein S complexed with C4b-binding protein) and an electroimmunoassay for C4b-binding protein. Ranges for the concentrations of these proteins were established in healthy volunteers and patients using oral anticoagulant therapy. A slight decrease in protein S antigen was observed in patients with liver disease (0.78 +/- 0.25 U/ml); no significant decrease in protein S was observed in patients with DIC (0.95 +/- 0.25 U/ml). Criteria were developed for the laboratory diagnosis of an isolated protein S deficiency.     

Influence of anticoagulants used for blood collection on plasma prothrombin fragment F1 + 2 measurements.

The levels of prothrombin fragment F₁₊₂ were measured by a double antibody radioimmunoassay in blood samples collected into different anticoagulant solutions. We evaluated healthy males between the ages of 42 and 77, asymptomatic patients with hereditary deficiencies of protein C or protein S, and persons receiving tumor necrosis factor infusions. The results in specimens collected in an anticoagulant containing ACD, EDTA, adenosine, and 25 U/ml of heparin (a) were highly correlated with those collected in an anticoagulant containing a synthetic thrombin inhibitor, EDTA, and aprotinin (b). However, in asymptomatic patients with congenital antithrombin III deficiency, we found that the plasma levels of F₁₊₂ in blood collected in anticoagulant (a) were usually substantially higher than those collected in anticoagulant (b). We determined that this phenomenon was not attributable to the venipuncture procedure itself, but rather appears to be due to the action of low concentrations of heparin in the presence of reduced blood levels of antithrombin III. Our data show that the previously documented elevations in plasma F₁₊₂ levels in patients with congenital antithrombin III deficiency appear to be caused by the above in vitro anticoagulant effect, and that this population does not exhibit evidence of a prethrombotic state as defined by the F₁₊₂ assay.     

Functional and immunologic protein S in normal pregnant women and in full-term newborns

Total and free protein S antigen and C4b-binding protein (C4bp) were determined by rocket immuno-electrophoresis, and functional protein S was assayed by a coagulation method, throughout pregnancy and normal puerperium and in a group of normal full-term newborns (FTN). The functional protein S assay is based on a modification of the APTT, using a mixture of test sample, protein S deficient plasma, activated protein C, phospholipids and calcium. This protein S functional assay is specific for protein S since the APTT prolongation by normal plasma was abolished by incubation of plasma with monospecific, rabbit anti-protein S IgG. The ratios of functional protein S/free protein S antigen in healthy men (n = 13) and women (n = 14) were 1.0 +/- 0.13 (mean +/- SD) and 1.03 +/- 0.20, respectively. During pregnancy there is a decrease in functional protein S and a progressive decrease in total and free protein S antigen, with a functional/free protein S ratio of 0.75 +/- 0.28 in the third trimester of pregnancy (n = 16). In early puerperium the functional protein S level was lower than the free protein S antigen level (ratio about 0.5). In the FTN group, the free protein S level was 39% and protein S activity was about 70% that of adults, with a functional/free protein S ratio of 1.84 +/- 0.31. C4bp values were 23.5 +/- 10.3% in the FTN group, and crossed immunoelectrophoresis showed that in this group the major protein S peak corresponded to free protein S.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of oral contraceptives on the anticoagulant activity of protein S in plasma

We determined anticoagulant parameters that depend on protein S function in plasma, i.e. the APC-independent anticoagulant activity of protein S (expressed as pSR) and APC resistance determined with thrombin generation-based tests (expressed as APCsr) as well as plasma levels of total and free protein S and prothrombin in men, women not using oral contraceptives (OC), and in women using second or third generation OC. Thrombin generation in the APC resistance assays was initiated either with factor Xa (Xa-APCsr) or tissue factor (TF-APCsr). The APC-independent anticoagulant activity of protein S was highest in men (pSR=1.69) and gradually decreased from women not using OC (pSR=1.49) via women using second generation (pSR=1.35) to women using third generation OC (pSR=1.27). The pSR correlated inversely with nAPCsr determined with the tissue factor-based APC resistance test (TF-APCsr) but not with nAPCsr determined with the factor Xa-based assay (Xa-APCsr). Multiple linear regression analysis in which sex, OC use, and protein S and prothrombin levels were included as independent variables and the pSR, TF-APCsr or Xa-APCsr as dependent variables indicated that plasma protein S levels poorly predict the pSR and the TF-APCsr, but are the main determinant of the Xa-APCsr. This indicates that OC use alters the expression of protein S activity. This phenomenon can be caused by differences in modulation of the activity of protein S by other plasma proteins that change during OC use or by OC-induced changes in the protein S molecule that impair its anticoagulant activity. Functional impairment of protein S as a result of hormonal influence may, at least in part, contribute to the thrombotic risk of OC users.     

The Ser460Pro mutation in recombinant protein S Heerlen does not affect its APC-cofactor and APC-independent anticoagulant activities

Protein S is a vitamin K-dependent plasma protein that functions as an APC-cofactor, but also exhibits anticoagulant activity in the absence of APC. The Heerlen polymorphism of protein S is characterized by a Ser460Pro substitution and lacks glycosylation at Asn458. It is associated with decreased protein S levels due to selective deficiency of free protein S Heerlen. To understand the lack of thrombotic complications associated with the protein S Heerlen mutation, we compared recombinant protein S Heerlen, wild type (wt) protein S and plasma-derived protein S. wt-Protein S and protein S Heerlen each bound 1:1 to C4BP with dissociation constants of 0.27 and 0.33 nM, respectively. Both wt-protein S and protein S Heerlen, either free or in complex with C4BP, were equally active as prothrombinase inhibitors in the absence of APC. All three protein S preparations stimulated APC-catalyzed inactivation of normal FVa, FVa Leiden and FVIIIa to the same extent. If extrapolated to plasma, it is not likely that the decreased free protein S levels in carriers of the protein S Heerlen mutation are compensated by an increased anticoagulant activity of protein S Heerlen-C4BP complexes. It is possible that an unrecognized plasma factor selectively enhances the anticoagulant activity of protein S Heerlen. If not, the reduction of free protein S levels in heterozygous protein S Heerlen-carriers combined with (low) normal total protein S levels apparently minimally affects the total anticoagulant activity of protein S (APC-cofactor and APC-independent activity) and hence is not associated with increased risk of venous thrombosis.     

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.     

Treatment of hereditary protein C deficiency with stanozolol

Five type I protein C deficient male patients received 5 mg stanozolol b.i.d. during 4 weeks. After four weeks of treatment plasma protein C activity increased from 0.42 to 0.74 U/ml and protein C antigen from 0.49 to 0.75 U/ml. This approximately 1.6 fold increase in plasma protein C was accompanied by an increase in factor II antigen (1.5 fold), factor V activity (1.6 fold), factor X antigen (1.1 fold), antithrombin III antigen (1.3 fold) and heparin cofactor II antigen (1.5 fold), while the concentration of factor VII, factor VIII, and factor IX activity, and of protein S antigen remained unchanged. Prothrombin fragment F1+2, measured in two patients, increased 1.3 fold. In addition to its effect on procoagulant and anticoagulant factors stanozolol had profibrinolytic effects, reflected in an increase in tPA activity and in the concentration of plasminogen. These data indicate that in type I protein C deficient patients stanozolol increases the concentrations of both procoagulant and anticoagulant factors and favours fibrinolysis. The efficacy of stanozolol in preventing thrombotic disease in type I protein C deficient patients, however, remains to be established. During the four weeks of stanozolol treatment no thrombotic manifestations were observed in the protein C deficient patients.     

Analytical considerations for free protein S assays in protein S deficiency

Protein S is an anticoagulant protein that circulates in plasma in complex with C4b-binding protein (C4BP) or in free form. Deficiency of protein S increases the risk of venous thrombosis. Measurement of free protein S, as compared to total levels, has been shown to be superior for prediction of protein S deficiency. We studied the effects of different handling protocols for an immuno- and a ligand (C4BP)-based assay for free protein S. When the assay was performed at 37 degrees C, the levels of free protein S in plasma from protein S deficient patients were approximately twice those obtained at room temperature. The reason for this phenomenon was that plasmas from protein S deficient patients exhibited a time-, temperature-, and dilution-dependent increase in free protein S, which was more pronounced than corresponding dilution of the normal plasma that was used to create the standard curve. These findings demonstrate the importance of assay procedure and sample handling in assays for free protein S.