Immunoradiometric assays for the Ca(II)-dependent and Ca(II)-independent conformation of human protein C

From a rabbit antiserum against human protein C, two subpopulations of antibodies recognizing Ca(II)-dependent and NonCa(II)-dependent epitopes of human protein C respectively were affinity purified for use in solid-phase immunoradiometric assays. The two assays were specific for PC:Ca(II)Ag and PC:NonCa(II)Ag and highly sensitive with a lower limit of detection of 2 ng/ml. The concentration of PC:Ca(II)Ag and PC:NonCa(II)Ag and their ratio in healthy volunteers was 0.91 +/- 0.16 U/ml, 0.95 +/- 0.14 U/ml and 0.97 +/- 0.18. In a group of patients treated with oral anticoagulants the ratio PC:Ca(II)Ag/PC:-NonCa(II)Ag was significantly decreased (to about 0.6) as compared with normal controls suggesting the presence of sub- and noncarboxylated protein C molecules (PIVKA-PC) in plasma of patients on oral anticoagulants. Analysis of plasmas of patients with a hereditary (n = 8) or isolated (n = 4) type II protein C deficiency and a history of thrombo-embolic disease did not reveal patients with a PC:Ca(II)Ag level and a PC:Ca(II)Ag/PC:NonCa(II)Ag ratio below the lower limit of the normal range suggesting that the frequency of genetic protein C variants with defects in the Ca(II)-dependent conformation is low.     

Rabbit polyclonal antibodies against the calcium-dependent conformation of factor IX and their application in solid phase immunoradiometric assays

Two subpopulations of antibodies were isolated from rabbit polyclonal antiserum directed against human factor IX: one against the Ca(II)-dependent conformation of factor IX and one against the Ca(II)-independent conformation of factor IX. The two subpopulations were used for the development of immunoradiometric assays (IRMA's) for factor IX:Ca(II)Ag and factor IX:NonCa(II)Ag respectively. Ranges for the concentration of factor IX:Ca(II)Ag and factor IX:NonCa(II)Ag were established in plasmas of healthy volunteers, patients treated with oral anticoagulants and hemophilia B patients. In the group of patients using oral anticoagulant therapy a progressively reduced ratio of factor IX:Ca(II)Ag to factor IX:NonCa(II)Ag was observed with increasing intensity of oral anticoagulant treatment. Variant factor IX molecules from hemophilia B patients (CRM-, CRM(Red) and CRM+) with a defective Ca(II) binding or defective conformational transition induced by Ca(II) binding, were identified. These defects are absent in variant factor IX molecules from one hemophilia Bm patient and from patients with hemophilia B Leyden.     

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.     

Behavior of protein S during long-term oral anticoagulant therapy

It has recently been reported that a natural anticoagulant, protein S (PS), is depressed during oral anticoagulation. Since more detailed information is required from the clinical standpoint, we measured plasma levels of PS [both total and free (not complexed) PS antigen], C4b-binding protein (C4bp) and other vitamin K-dependent proteins (factors II, VII, IX, X and protein C) in 60 plasma samples from patients on long-term oral anticoagulant therapy with warfarin. Together with the reduction of other vitamin K-dependent plasma proteins, PS decreased during warfarin treatment, being dependent on the intensity of the therapy. A considerable variation in plasma PS levels was also observed among individuals with a similar intensity of anticoagulation. Plasma concentration of C4bp was closely correlated with total PS level, and free PS/total PS ratio was independent of thrombotest values. These findings indicate that long-term oral anticoagulant therapy results in the suppression of the synthesis of PS, and that its reduction is on the whole balanced with C4bp and vitamin K-dependent coagulation factors. It was suggested that the metabolism of C4bp might be regulated by the plasma PS level, although this hypothesis needs further exploration.     

Spectrophotometric assays of prothrombin in plasma of patients using oral anticoagulants

Two spectrophotometric assays for prothrombin have been developed and compared with a one stage coagulant and an immunological assay. One of these assays (called the XAPC assay) uses a combination of factor Xa, phospholipid, Ca2+ and factor V as activator of prothrombin, and measures only normal prothrombin. The second (the ECAR assay) uses Echis carinatus venom as activator. This assay measures both normal prothrombin and PIVKA II (protein induced by vitamin K antagonists/absence). Combination of the results obtained by the XAPC and ECAR assays provides rapid and reliable information on the degree of "subcarboxylation" of prothrombin (oral anticoagulation, vitamin K deficiency). For patients on long term anticoagulant treatment the prothrombin time (Thrombotest) shows better correlation with the ratio prothrombin/prothrombin plus PIVKA II (XAPC/ECAR) than with the factor II concentration. For patients starting the anticoagulant treatment there is no correlation between the Thrombotest time and the XAPC/ECAR ratio. It seems doubtful that (a) spectrophotometric factor II assay(s) will be as useful as the prothrombin time in the control of oral anticoagulation.     

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.     

Immunoradiometric assays for human coagulation factor VII using polyclonal antibodies against the Ca(II)-dependent and Ca(II)-independent conformation

Human coagulation factor VII is a trace plasma protein belonging to the vitamin K-dependent factors. Two specific and sensitive immunoradiometric assays for factor VII were developed using immunopurified rabbit antibodies against the Ca(II)-independent and Ca(II)-dependent conformation of factor VII. Both assays were insensitive to the activation state of factor VII. The distribution of factor VII antigen was studied in 40 healthy individuals and the antigen level in normal plasma was calculated to be 0.52-0.62 micrograms/ml. The two assays were used in a comparative study of factor VII procoagulant activity and factor VII antigen in patients treated with oral anticoagulants.     

Relationship between total prothrombin, native prothrombin and the International Normalized Ratio (INR).

Plasma levels of total prothrombin and fully-carboxylated (native) prothrombin were compared with results of prothrombin time (PT) assays for patients undergoing oral anticoagulant therapy. Mean concentrations of total and native prothrombin in non-anticoagulated patients were 119 +/- 13 micrograms/ml and 118 +/- 22 micrograms/ml, respectively. In anticoagulated patients, INR values ranged as high as 9, and levels of total prothrombin and native prothrombin decreased with increasing INR to minimum values of 40 micrograms/ml and 5 micrograms/ml, respectively. Des-carboxy-prothrombin increased with INR, to a maximum of 60 micrograms/ml. The strongest correlation was observed between native prothrombin and the reciprocal of the INR (1/INR) (r = 0.89, slope = 122 micrograms/ml, n = 200). These results indicated that native prothrombin varied over a wider range and was more closely related to INR values than either total or des-carboxy-prothrombin. Levels of native prothrombin were decreased 2-fold from normal levels at INR = 2, indicating that the native prothrombin antigen assay may be a sensitive method for monitoring low-dose oral anticoagulant therapy. The inverse relationship between concentration of native prothrombin and INR may help in identification of appropriate therapeutic ranges for oral anticoagulant therapy.     

Diagnostic yield of prothrombotic state studies in cryptogenic stroke

OBJECTIVES: Around 30% of ischemic strokes are considered cryptogenic. We analyzed the diagnostic yield of prothrombotic state (PS) studies in patients with an initial cryptogenic stroke (CS). MATERIAL AND METHODS: We prospectively included consecutive CS patients according to the TOAST criteria. PS included plasmatic determinations of antiphospholipid (APL) antibodies and lupus anticoagulant (LA), S (SPd) and C (CPd) protein deficiencies, and genetic analysis of the prothrombin gene mutation (PT G20210A) and the factor V Leiden mutation (FV G1691A). We recorded age, sex and vascular risk factors. RESULTS: From a total of 89 patients (mean age 56.9 +/- 14.3 years, 53% men), we identified 16 PS in 15 patients (16.85%): APL-6, LA-2, SPd-2, CPd-1, PT G20210A -3 and FV G1691A -2. One patient presented an association (APL and PT G20210A). CONCLUSIONS: One of every six patients with initial CS present a PS. Age or sex and conventional cardiovascular risk factors were not related to PS study findings, supporting the relevance of such studies in all patients with an initial CS.     

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.     

L-asparaginase treatment reduces the anticoagulant potential of the protein C system without affecting vitamin K-dependent carboxylation

The changes in plasma levels of the vitamin K-dependent natural anticoagulants protein C (PC) and protein S (PS) and procoagulant factors II, IX and X were evaluated in 8 adult patients during treatment with L-asparaginase (L-ase i.v. 120,000 U/m² over 10 days). PC anticoagulant activity and factor IX, X and II coagulant activity decreased proportionally to their half-lives to a nadir of 50–60% of pretreatment values after 2–5 L-ase infusions, suggesting that inhibition of protein synthesis rather than consumption is the main mechanism responsible for the observed changes. Free PS antigen levels declined at a rate similar to total PS antigen, reaching a nadir of 56% of pretreatment values after 3 L-ase infusions; however, due to C4b-binding protein levels higher than total PS levels (p <0.05), they were constantly lower than the corresponding total PS antigen levels (0.05 < p <0.001). This implicates that total PS antigen levels cannot be taken as an indicator of PS activity. No differences between the antigenic levels and the anticoagulant activities of PC and free PS could be observed suggesting that L-ase does not affect the mechanisms of vitamin K-dependent carboxylation of Gla-residues. The faster rate of decline of PC and PS activities relative to that of factor II may be responsible for the onset of an hypercoagulable state during the early phase of L-ase treatment.     

蛋白C、蛋白S与脑血管病关系的研究

目的:探讨蛋白C(PC)、蛋白S(PS)与脑血管病(CVD)的关系。方法:检测94例缺血性脑血管病(ICVD)患者和26例脑出血(CH)患者的血浆蛋白C:抗原(PC:Ag)、总蛋白S(TPS)和游离蛋白S(FPS)。结果:ICVD患者PC: Ag(17％)、TPS(26．6％)、FPS(23．4％)异常降低者多于对照组(2％)(P＜0．01)。CH患者PC:Ag(19．2％)异常降低者多于对照组(P＜0．05)。结论:17％～26．6％ICVD患者存在PC、PS缺陷;19．2％CH患者存在PC缺陷,多数为继发性缺陷,发现这些患者并区别其为原发性或继发性对明确CVD的病因、治疗和预防有重要意义。     

Inhibitory effect of regucalcin on Ca(2+)-dependent protein kinase activity in rat brain cytosol: involvement of endogenous regucalcin

The effect of regucalcin, a Ca(2+)-binding protein, on Ca(2+)-dependent protein kinase activity in the brain cytosol of rats with different ages (5 and 50 weeks old) was investigated. The addition of calmodulin (10 microg/ml) or dioctanoylglycerol (5 microg/ml) in the enzyme reaction mixture caused a significant increase in protein kinase activity in the presence of CaCl2 (1 mM), indicating that Ca2+ calmodulin or protein kinase C is present in the cytosol. Such an increase was completely prevented by the addition of regucalcin (10(-7) M). Moreover, regucalcin (10(-7) M) significantly inhibited cytosolic protein kinase activity without Ca2+/calmodulin or dioctanoylglycerol addition. Meanwhile, the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the enzyme reaction mixture caused a significant elevation of protein kinase activity, suggesting an inhibitory effect of endogenous regucalcin. Brain cytosolic protein kinase activity was significantly elevated by increasing age (50-week-old rats). Also, regucalcin (10(-7) M) significantly decreased protein kinase activity without Ca(2+) addition in the brain cytosol of aged rats. However, the effect of anti-regucalcin monoclonal antibody (50 ng/ml) in elevating protein kinase activity was not seen in the brain cytosol of aged rats. These results suggest that regucalcin has an inhibitory effect on Ca(2+)-dependent protein kinase activity in rat brain cytosol, and that the effect of endogenous regucalcin may be weakened in the brain cytosol of aged rats.     

Investigation of the interrelationship between coagulation and thrombin-induced EDRF-dependent relaxation in dog coronary artery

Little is known about the possible interrelationships between thrombin-induced EDRF-dependent vascular relaxation and coagulant activity. We have now studied the effects of the anticoagulant zymogen protein C, on EDRF-dependent relaxation in isolated canine coronary arteries. Low concentrations of activated protein C (0.1-30 ng/ml) had no significant effect, but higher concentrations caused relaxation (Emax -39.2 +/- 7.2%; 100-1000 ng/ml). To determine whether relaxation was dependent on coagulation complexes associated with endothelial cell membranes, the coumarin, brodifacoum was given three days before in vitro experiments were carried out in order to inhibit production of active vitamin K1-dependent clotting factors. Brodifacoum (10 mg/kg i.p.) increased prothrombin time from 8.5 +/- 0.24 sec (control), to 46.2 +/- 2.4 sec (p less than 0.05), but had no effect on thrombin-induced relaxation (Emax greater than 90%; ED50 0.026 +/- 0.004 units/ml control; 0.025 +/- 0.004 unit/ml brodifacoum). In the final group of studies, we investigated the effects of the concomitant administration of protein C (1000 ng/ml) and thrombin in vitro. Protein C (1000 ng/ml) increased relaxant sensitivity to thrombin after partial desensitization of the relaxant response by previous thrombin administration, (-60.2 +/- 7.1% thrombin alone; -77.9 +/- 7.2% thrombin + protein C), but had no effect after complete desensitization of the relaxant response. In conclusion, the data appear best explained by protein C and thrombin-induced EDRF dependent relaxation being due to proteolytic actions.     

Modulation of heparin cofactor II function by S protein (vitronectin) and formation of a ternary S protein-thrombin-heparin cofactor II complex

The complement inhibitor S protein, which is identical to the adhesive protein vitronectin, functions as heparin-neutralizing factor by protecting thrombin as well as factor Xa against fast inactivation by antithrombin III. The interference of S protein with glycosaminoglycan-catalyzed inhibition of thrombin by heparin cofactor II was investigated in these studies. S protein significantly counteracted the anticoagulant activity of heparin and pentosan polysulfate but not of dermatan sulfate. In the presence of 0.3 micrograms/ml heparin, 0.5 micrograms/ml pentosan polysulfate, or 2 micrograms/ml dermatan sulfate, S protein induced a concentration-dependent reduction of the inhibition rate of thrombin by heparin cofactor II. This resulted in a decrease of the apparent pseudo first-order rate constants by about 17-fold (heparin), or about 7-fold (pentosan polysulfate), whereas no neutralization of dermatan sulfate was demonstrable at a physiological ratio of S protein to heparin cofactor II. Exposure of the glycosaminoglycan-binding region of S protein by reduction and carboxymethylation of the protein increased the neutralizing activity of S protein towards heparin and pentosan polysulfate. The results of these functional experiments correlated well with the demonstration of direct binding of S protein to both polysaccharides but not to dermatan sulfate. While reduced/carboxymethylated S protein remained also ineffective in neutralizing other dermatan sulfate compounds with varying degree of sulfation, a synthetic highly basic tridecapeptide, representing a portion of the glycosaminoglycan-binding domain of S protein, counteracted their anticoagulant activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thrombophilic abnormalities and recurrence of venous thromboembolism in patients treated with standardized anticoagulant treatment

INTRODUCTION: Whether patients with hereditary or acquired thrombophilia have an increased risk for recurrence of venous thromboembolism (deep vein thrombosis and/or pulmonary embolism) is still controversial. The aim of this study was to evaluate the incidence of recurrence of venous thromboembolism in patients with and without thrombophilic abnormalities treated with standardized anticoagulant treatment. MATERIAL AND METHODS: Database was from a prospective multicenter randomized study aimed at evaluating the long-term clinical benefit of extending to 1 year the 3-month oral anticoagulant treatment after a first episode of idiopathic proximal deep vein thrombosis. The screening for thrombophilia included antithrombin, protein C, protein S deficiencies, resistance to activated protein C and/or factor V R506Q mutation, the mutation 20210GA of the prothrombin gene, hyperhomocysteinemia and antiphospholipid antibodies. The diagnosis of venous thromboembolism recurrence was done by objective tests and adjudicated by a panel unaware of the results of the thrombophilia screening. RESULTS: A screening for thrombophilic abnormalities was performed in 195 patients. Twenty of 57 (35.1%) thrombophilic patients experienced a recurrence of venous thromboembolism as compared with 29 of 138 (21.0%) patients without thrombophilia (HR=1.78, 95% CI 1.002-3.140, p=0.046). The difference in VTE recurrence between patients with and without thrombophilia was accounted for by those who received 3 months of oral anticoagulation (HR=3.21, 95% CI 1.349-7.616, p=0.008). No difference between thrombophilic and non-thrombophilic patients was observed in the time interval from the index episode to recurrent venous thromboembolism (29.1+/-23.9 and 30.6+/-19.8 months, respectively). CONCLUSIONS: Thrombophilic abnormalities are associated with an increased risk of venous thromboembolism recurrence. The role of thrombophilia in the long-term management of venous thromboembolism should be addressed in prospective management studies.     

Protein S activity in patients with heredofamilial protein S deficiency and in patients with juvenile venous thrombosis. Results of a functional method

Using an ACL 300R coagulometer (Instrumentation Laboratory) we assessed the clinical usefulness of a new method to measure PS activity (PS:Act), based on the prolongation of prothrombin time of a mixture of diluted plasma sample, PS depleted plasma previously incubated with Protac for protein C activation, bovine thromboplastin and calcium ions. The results were compared with those from immunological assays. PS:Act was measured in 42 apparently healthy subjects, in 12 patients with hereditary PS deficiency (HPSD group) diagnosed on the basis of immunologic tests and in 48 patients with episodes of juvenile venous thromboembolism at least three months prior to testing (JVTE group). All the HPSD patients had PS:Act below the normal range (< 62%). In JVTE group 9 patients (18.7%) showed abnormal results for PS:Act, 4 (8.3%) had low levels of free PS:Ag; all patients had normal total PS:Ag levels. Levels of antiphospholipid antibodies (immunologic test) were normal in the 9 JVTE patients with low PS:Act. When all the results were considered together (n=102), the correlation coefficient between PS:Act and free PS:Ag was 0.78 (p<0.01).     

Protein C and protein S deficiencies are the most important risk factors associated with thrombosis in Chinese venous thrombophilic patients in Taiwan

The relative risks (odds ratio, OR) of various risk factors for venous thrombophilia, including sex, antithrombin III, protein C (PC), protein S (PS) and plasminogen deficiencies, and C677T homozygous mutation of methylenetetrahydrofolate reductase gene were assessed using age matched (+/-5 years) conditional logistic regression analysis in 116 Chinese venous thrombophilic patients (58 males; 58 females; mean age 47.5+/-17.7 [SD] years) and 125 healthy controls (67 males; 58 females; mean age 45.5+/-15.7 years). None of the patients had prothrombin G20210A and factor V Leiden mutation or an activated PC sensitivity ratio of less than 2. One hundred and five age-matched patients and 105 controls were entered in this analysis. Only PC and PS deficiencies were significantly associated with increased risk for the development of thrombosis with an OR of 10.6 and 6.7, respectively. The findings of this study suggest that PC deficiency and PS deficiency are the most important risk factors for thrombosis in Chinese venous thrombophilic patients.     

Thromboembolic disease--critical evaluation of laboratory investigation.

Previous studies of patients with thromboembolic disease have revealed an association either with hereditary anticoagulant protein deficiencies or with defects in the fibrinolytic system. To obtain a more comprehensive picture and to investigate which analyses are useful in the evaluation of such patients, we have performed an extensive laboratory investigation in 439 individuals with thromboembolic disease. Anticoagulant protein deficiencies were found in 24 patients. Deficiencies of protein C (n = 10) and protein S (n = 9) were most common followed by deficiencies of antithrombin III (n = 3) and plasminogen (n = 2). Six of the nine protein S deficient patients demonstrated a selective deficiency of free protein S with normal total protein S concentrations. To diagnose protein C and S deficiencies among the 201 patients receiving oral vitamin K antagonists, the concentrations of protein C and S were compared with the mean concentration of several other vitamin K-dependent proteins. One protein C and three protein S deficiencies were identified among the treated patients. The number of protein C deficiencies found in this group was significantly lower than the number found among untreated patients. Although fewer protein S deficiencies were also identified among the treated patients, than in the untreated group, the difference was not statistically significant. The results suggest that protein C deficiencies went undetected in the treated group and that oral anticoagulant therapy should be discontinued before efforts to diagnose protein C deficiency are made. We found no cases with heparin cofactor II deficiency. Lupus anticoagulant was present in 10 patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-1 adrenoceptors are decreased in human epileptic foci

Cortical alpha-1 adrenoceptors were measured in tissues obtained from 10 patients immediately following temporal lobectomy for intractable partial epilepsy. At operation each patient exhibited spontaneous spiking restricted to either the anterior (n = 5) or posterior (n = 5) portion of the first two temporal gyri. Control samples were obtained from the nonspiking half of the same gyrus. Receptor-binding assays were performed on isolated cortical membranes using [3H]prazosin. There was a reduction (p less than 0.01) in the receptor density (beta max) of the sites in the epileptic foci without any change in affinity (mean +/- SEM): spiking--beta max, 160.5 +/- 11.3 fmol/mg protein; affinity, 0.17 +/- 0.04 nM; nonspiking--beta max, 218.8 +/- 15.6 fmol/mg protein; affinity 0.17 +/- 0.04 nM. This relative decrease in alpha-1 adrenoceptor density may be the substratum of a noradrenergic hyposensitivity that could contribute to a localized diminution in inhibitory mechanisms in epileptic foci.