Differential regulation of alpha and beta chains of C4b-binding protein during acute-phase response resulting in stable plasma levels of free anticoagulant protein S.

Regulation of C4b-binding protein (C4BP) isoforms during acute phase and its relationship to the plasma concentration of free protein S was elucidated. An assay for beta chain containing C4BP (C4BP beta+) was developed and the concentrations of total C4BP, C4BP beta+, total, free, and bound protein S were measured in patients with acute-phase response. Even though total C4BP was increased to 162% (mean value) of controls, the corresponding value of C4BP beta+ was only 122%. In the acute-phase group, total protein S was increased to the same extent as C4BP beta+ (mean value of 124%), whereas free protein S was not decreased. In controls, total and bound protein S correlated with total C4BP and C4BP beta+. However, in the acute-phase group, the correlation between bound protein S and total C4BP was lost, although the correlation between C4BP beta+ and protein S remained. The present results suggest stable levels of free protein S during acute phase to be the result of differential regulation of C4BP alpha- and beta-chain expression, and the concentration of free protein S to be the resulting molar excess of protein S over C4BP beta+. This mechanism ensures functional levels of free anticoagulant protein S despite high levels of C4BP.

Thrombomodulin in pediatric cardiac surgery.

In 30 consecutive children with congenital heart disease scheduled for pediatric cardiac operations, thrombomodulin, protein C, free protein S, and thrombin-antithrombin complex were measured by enzyme-linked immunosorbent assay after the induction of anesthesia (baseline value), and then before, during, and after cardiopulmonary bypass until the first postoperative day. The patients were divided prospectively into two groups: children weighing less than 10 kg (group 1; n = 15) and those weighing more than 10 kg (group 2; n = 15). At baseline, the plasma concentration of thrombomodulin was significantly higher in the children in group 1 than in those in group 2 (83.1 +/- 11.0 ng/mL versus 29.2 +/- 12.1 ng/mL). During cardiopulmonary bypass, the thrombomodulin level was reduced in both groups without showing any significant group differences. Five hours after cardiopulmonary bypass and on the first postoperative day, the thrombomodulin level exceeded normal values only in the children weighing less than 10 kg. In both groups, the protein C levels were already below normal at the beginning of the study. The baseline protein S concentration was higher in the smaller children (80% +/- 18%) than in the larger children (66% +/- 11%). It was reduced by cardiopulmonary bypass in both groups; however, postoperatively it did not return to normal in group 1 (45.1% +/- 10%). Plasma levels of the thrombin-antithrombin complex were similar in both groups, with a marked increase at the end of cardiopulmonary bypass, and returned to near-normal levels by 5 hours after bypass.(ABSTRACT TRUNCATED AT 250 WORDS)

New direct assay of free protein S antigen using two distinct monoclonal antibodies specific for the free form.

Monoclonal antibodies (mAbs) specific for free protein S and devoid of reactivity with protein S-C4b-BP complexes, have been produced. A one-step sandwich-type enzyme-linked immunoassay (ELISA) has been developed with two mAbs reacting with distinct epitopes of free protein S. F(ab')2 fragments from mAb 15C4 were coated on microplates and mAb 34G2 conjugated with horseradish peroxidase (HRP) was added immediately before diluted plasma. The presence of calcium in the sample diluent prevented dissociation of complexes during the assay. This assay was specific as demonstrated by good recovery of purified protein S added to plasma and the lack of influence of C4B-BP-protein S complexes. Thus, addition of increasing amounts of purified C4B-BP to human citrated plasma induced a dose-dependent decrease of free protein S. The assay was sensitive, allowing measurement of 5-500 ng/ml of free protein S, with a detection threshold of 2 ng/ml. It was also reproducible with inter-assay and intra-assay variation coefficients of 2.5-5.1% and 3.1-5.0%, respectively. Thus, this new ELISA of free protein S antigen in plasma has the advantages of being fast, accurate and reproducible. It appears to be extremely useful for routine studies as no preliminary treatment of plasma is required.

Clinical applications of a direct assay of free protein S antigen using monoclonal antibodies. A study of 59 cases.

A new one-step ELISA using two monoclonal antibodies specific for distinct epitopes of the free form of protein S (ELISA-m) has been developed for the direct measurement of free protein S in untreated plasma. This assay has been compared with the classic method using polyclonal antibodies to protein S (ELISA-p). The latter method has the drawback of requiring PEG precipitation of plasma which is time-consuming, difficult to perform with accuracy and therefore poorly reproducible in most laboratories. Results of both ELISAs were compared with those of a functional assay. In 30 normal subjects, there was an excellent correlation between ELISA-m and ELISA-p (r = 0.95) as well as between ELISA-m and the functional assay (r = 0.96). In twelve patients with a congenital deficiency, the levels of free protein S antigen were similarly decreased with ELISA-m and ELISA-p and in good agreement with those of protein S activity. In 20 patients with miscellaneous inflammatory diseases, the levels of free proteins S were normal with good correlation between both ELISAs and PS activity, despite high levels of C4bBP-protein S complexes. As expected, in 15 dicoumarol-treated patients, there was a significant and parallel decrease of free protein S antigen with both ELISAs, with even lower levels of protein S activity. In 14 patients with liver cirrhosis, the mean values for free protein S antigen were normal using both assays, but with wide extreme values, whereas protein S activity was significantly lower.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of coagulation factor activation during cardiopulmonary bypass with a new monoclonal antibody.

Antithrombin-III (AT) is a key inhibitor of blood coagulation that neutralizes activated serine esterases by forming covalent modified complexes (ATm). A new monoclonal antibody directed against short-lived AT-activated serine protease complexes provides a means of measuring subclinical coagulation activity during cardiopulmonary bypass (CPB). Twelve patients undergoing CPB for coronary artery bypass grafting were studied and AT, ATm, D-dimers (DD), and several other coagulation and fibrinolytic markers were measured during the surgical procedure. There were decreases in AT, factors V, II, X, IX, protein S (total and free), C4b-binding protein, thrombomodulin, and platelets counts, whereas heparin, ACT, thrombospondin, plasminogen activator inhibitor (PAI-1), and tissue plasminogen activator (tPA) increased. ATm and the percentage of ATm available (ATm/AT) showed a peak during CPB. These results demonstrate that during CPB, the use of heparin produces an equilibrium involving increased coagulation activation and consumption in association with increased fibrinolysis. The equilibrated consumption of both coagulation and fibrinolytic factors leads to low levels of all factors after cardiac surgery. The ATm assay allows assessment of the differential effects of CPB and surgical trauma on coagulation activation. It is speculated that ATm levels may be useful in monitoring the consumption of coagulation factors.

Haemostatic variables in patients with unstable angina.

To assess the contribution of thrombus formation in the pathogenesis of unstable angina, we employed the recently developed assays of small fragments which reflect the degree of activation of various components of the haemostatic system. Such haemostatic measurements were undertaken in patients with unstable angina (n = 47) from the time of their admission to the coronary care unit (CCU) at 8-h intervals in the first 24 h and then daily for a total of 5 days. The results obtained were compared with healthy control values. Patients exhibited lower ATIII, prolongation of the APTT and TT, but not PT or the reptilase time, which is a consequence of heparinization. There was significant elevation of fibrinogen, factor VIII:C, von Willebrand factor:antigen and von Willebrand factor:ristocetin cofactor throughout the study period. There was also evidence of thrombin generation as indicated by the elevated levels of fibrinopeptide A (FPA) and thrombin-antithrombin complexes. The platelet release proteins, beta-thromboglobulin (BTG) and platelet factor 4 (PF4), were markedly elevated in the first 2 days and dropped gradually thereafter. The fibrinolytic inhibitor, plasminogen activator inhibitor (PAI), levels were elevated throughout. Proteins C and S, plasminogen and alpha 2-antiplasmin remained unchanged. It was concluded that in patients with unstable angina, there is significant activation of the clotting system and inhibition of fibrinolysis which confirms the existence of a tendency towards thrombus formation in patients with unstable angina.

Prothrombotic states in young people with idiopathic stroke. A prospective study.

BACKGROUND AND PURPOSE: Although 4% of cerebral infarcts in the young can be attributed to hematologic disturbances that predispose to thrombosis, the frequency of cerebral infarcts caused by prothrombotic states is not known. Recently, the association between cerebral infarction and deficiencies of elements of the natural anticoagulant system has been recognized. METHODS: Thirty-six consecutive patients under 40 years of age with cerebral infarction of undetermined cause were prospectively studied. Quantitation of natural anticoagulants was done at least 3 months after the cerebral infarction. The following activity tests were performed, all by the chromogenic method: antithrombin III, protein C, plasminogen, tissue plasminogen activator, and inhibitor of tissue plasminogen activator. Protein S was quantified by the Laurell rocket method. All patients underwent a complete cardiological examination, including two-dimensional echocardiography, as well as four-vessel cerebral angiography. Some patients were also studied by transesophageal echocardiography. RESULTS: Of 36 patients, 17 were male, with a mean age of 28 years. Mean age for women was 25 years. Nine patients (25%; 5 women, 4 men) had a deficiency of one natural anticoagulant and constituted group I. In these patients, isolated protein S deficiency was detected in five cases (13.8%); in one case, we observed the association between protein S deficiency and antiphospholipid antibodies; and deficiency of protein C was seen in one case (2.7%), of antithrombin III in one case (2.7%), and of plasminogen in one case (2.7%). Instances of cerebral infarction without natural anticoagulant deficiency (group II) included 12 women and 15 men. There were no differences in clinical and radiological findings between the two groups. CONCLUSIONS: Considering the importance of prothrombotic state, especially caused by deficiency of protein S, in the development of cerebral infarcts, we suggest that it should be looked for in every young patient affected by this pathological entity and in whom no etiologic factors can be determined.

Enhanced thrombin generation in children with sickle cell disease.

Recent studies suggest that increased activity of the coagulation system, measured with sensitive assays for activation markers, may be important in the pathogenesis of vascular occlusion in sickle cell disease (SCD). Since most of these studies were carried out in adult patients and SCD is an inherited disorder with severe morbidity even in childhood, we decided to determine the activity of the coagulation system in children with SCD. In a prospective study markers of thrombin generation as well as coagulation inhibitors were investigated in 16 homozygous SCD patients and 16 age-matched control children. Significantly increased plasma concentrations of the prothrombin fragment F1+2 and of thrombin-antithrombin III (TAT) complexes were found in SCD patients. The levels of protein C activity and total and free protein S were significantly reduced in SCD patients as compared with control values. Plasma AT III levels were not different in the two groups. We conclude that, in children with SCD, evidence of enhanced thrombin generation is present, which may in part be due to reduced levels of the inhibitors proteins C and S. The clinical relevance of this coagulation imbalance has to be demonstrated.

[Vasoactive prostanoids and inhibitors of blood coagulation in pregnancy-induced hypertension]. / Vasoaktive Prostanoide und Inhibitoren der Gerinnung bei schwangerschaftsbedingter Hypertonie.

The aim of the present study was to investigate the occurrence of changes in the plasma levels of vasoactive prostanoids and inhibitors of blood coagulation in normal pregnancy and in cases of pregnancy induced hypertension. Levels of the coagulation inhibitors antithrombin III, protein C, Protein S as well as the prostaglandin metabolites thromboxane B2 and 6-oxo-prostaglandin F1 alpha were measured between 13 and 37 weeks gestation in 36 primigravidae. In 8 of the examined patients persistently raised blood pressure values of 140/90 and above were measured after 20 weeks of gestation. Our results indicated that an imbalance of vasoactive prostanoids may precede the appearance of clinical symptoms of PIH. The determination of coagulation factors before blood pressure is elevated has no predictive value regarding the later development of PIH. The reduced levels of protein C associated with our PIH group are considered to be the result of an activated coagulation followed by consumption of clotting factors. Reduced measured levels of protein S in normotensive as well as hypertensive pregnancies offer an explanation for the increased risk of thromboembolic disease. This increased susceptibility to thromboembolic disorders is further enhanced by the altered balance between the platelet aggregator and vasoconstrictor thromboxane A2 and its antagonist prostacyclin.

Plasma protein S-antigen (PS:Ag) in selected disease states.

Protein S is a vitamin K-dependent glycoprotein acting as a cofactor for activated protein C and thereby exerting an antithrombotic effect. When compared to values recorded in the 10 healthy normal weight normolipidemic control subjects (80.1% +/- 5.16; mean +/- SEM), plasma protein S-antigen (PS:Ag) level was found to be significantly (p < 0.01) decreased in the 11 patients with decompensated cirrhosis of the liver (54.72% +/- 4.89) and in the 12 surgical patients in critical condition (59.2 +/- 4.96), while obviously (p < 0.001) increased plasma levels were noted in the group including 20 overweight and hyperlipidemic subjects (113% +/- 3.1). Since the low PS:Ag level was associated with a decreased serum cholinesterase (CHE) activity, while both plasma PS:Ag and serum CHE activity were increased in overweight and hyperlipidemic subjects it is considered that impaired or respectively enhanced hepatic protein synthesis is at least partially responsible for changes affecting this antithrombotic plasma protein.

Hereditary protein S deficiency in a large New Jersey kindred.

PURPOSE: Protein S is a vitamin K-dependent anticoagulant protein that serves as a cofactor for activated protein C. Deficiency of protein S has been associated with recurrent thrombotic events. To characterize better the risks of thrombosis in protein S deficiency, we studied 62 members in a large kindred. METHOD: All members were evaluated by a thorough clinical history. Plasma samples were assayed for total protein S antigen and protein S activity. Upper and lower extremity venous duplex examinations were performed in the majority of adult members. RESULT: Twenty-six (40%) of the 62 family members were classified as deficient on the basis of either low total protein S antigen levels or low protein S functional activity. Five members deficient in protein S had 16 venous thrombotic events. In all members the onset of thrombotic events occurred after 19 years of age, with a tendency for recurrence. Three lower extremity deep venous thromboses that had been occult previously were first diagnosed on surveillance duplex scanning. Only one member whose protein S level was not deficient had a single episode of superficial thrombophlebitis. CONCLUSION: Our findings in this large kindred confirm an autosomal-dominant inheritance pattern. Thrombotic events occurred after the age of 19 years in affected individuals and tended to be recurrent. The diagnosis of protein S deficiency is based on functional and immunologic plasma assays. In this study venous duplex scanning proved to be a useful diagnostic adjuvant.

Resistance to activated protein C in nine thrombophilic families: interference in a protein S functional assay.

Nine thrombophilic patients who had had previous diagnoses of functional protein S deficiency were reinvestigated. The functional protein S assays gave dose-response curves that were not parallel to those of the reference plasma. The same pattern was true for approximately half of the first-degree relatives of the propositi. When protein S was extracted from the plasma of the patients by immunoabsorption, it had a normal ratio of functional activity to immunologic concentration. Restriction fragment length polymorphism analysis, informative in one family, showed no linkage between the protein S gene marker and the abnormal behavior of the protein S functional assay. All the propositi and 23/36 first-degree relatives were resistant to the prolongation of activated partial thromboplastin time induced by activated protein C. Furthermore, there was striking concordance in all patients and relatives between the abnormal pattern of the protein S functional assay and resistance to activated protein C. We conclude that a plasma-based functional protein S assay is sensitive to activated protein C resistance and this may lead to spuriously low results in the assay. In agreement with the results of others, this study indicates that resistance to activated protein C is a frequent hemostatic defect in selected thrombophilic populations.

Comparison of functional assays for protein S: European collaborative study of patients with congenital and acquired deficiency.

Four functional assays for protein S were evaluated by 4 different laboratories, each center using its own method. The aim of this study was to compare these different assays and to establish a relationship with results of immunological assays of total and free protein S antigen and C4bBP. The same plasma samples were distributed to each center and tested in blind. In 47 normal subjects, there was no significant difference between the 4 functional assays, with mean values ranging from 93 to 100%. These values were in good agreement with those of free and total protein S antigen. In 34 patients with a quantitative congenital deficiency of protein S the mean values of protein S activity were decreased with the 4 assays, ranging from 25 to 40%. Free protein S antigen was reduced to a similar extent, whereas total antigen was either normal or decreased. The correlation of protein S activity with free protein S antigen was satisfactory for 3 methods, with coefficients of correlation varying from 0.84 to 0.92 whereas it was only 0.70 in one lab. When total protein S antigen was reduced, protein S activity was decreased in all the patients with the 4 assays. In contrast when total protein S antigen was normal an important overlap of protein S activity between normals and patients was observed in one lab with 12 patients misclassified. In 8 patients with a functional defect, results of protein S activity differed substantially according to the assay used and about half of these patients were misclassified.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein S levels during the normal menstrual cycle and during estrogen therapy for premature ovarian failure.

Protein S levels have been reported to be decreased in pregnancy and with oral contraceptive use. This study monitored the effects of estrogen shifts on protein S levels. Four patients with premature ovarian failure were treated with either oral or transdermal patch estrogen replacement. Blood drawn on days 1, 14, and 28 of therapy was analyzed for estradiol, estrone, free and total protein S, and C4b-binding protein (C4b-BP) levels. Similar studies were performed on six normally cycling control patients and seven postmenopausal women. In healthy females, total levels of protein S fell from 22.1 +/- 0.73 micrograms/mL on day 1 to 19.2 +/- 1.29 micrograms/mL on day 14 (p < 0.023). Free protein S levels declined from 6.45 +/- 0.70 micrograms/mL to 5.59 +/- 0.69 micrograms/mL (p < 0.016). C4b-BP levels did not change during the normal menstrual cycle. Baseline total protein S (44.1 +/- 7.0 micrograms/mL) and C4b-BP (193 +/- 18%) levels were elevated in patients with premature ovarian failure. On oral therapy, there was a strong, negative correlation (r = -0.979, p < 0.021) between C4b-BP and estradiol levels. C4b-BP levels did not change in patients with the patch. Both estrogen therapies produced similar declines (44 to 26 micrograms/mL) in total protein S levels. In all cases, total protein S levels changed as a reciprocal function of estradiol. C4b-BP (128 +/- 6.5%) and total protein S (32.2 +/- 3.0 micrograms/mL) levels were higher in postmenopausal women than in nonmenopausal females. Free protein S levels in postmenopausal women (9.6 +/- 0.6 micrograms/mL) were normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Early prognostic factors in ischemic stroke. The role of protein C and protein S.

BACKGROUND AND PURPOSE: Protein C and protein S have been found reduced in some patients with ischemic cerebrovascular diseases, but the relevance of this finding for prognosis is unsettled. METHODS: In 43 consecutive patients admitted over 6 months for acute ischemic stroke, protein C and free protein S were evaluated on admission, and at 2 and 6 months after stroke. Excluded were all patients with known causes liable to reduce the levels of protein C, free protein S, or both. RESULTS: In 14% of patients, abnormally low levels of protein C were found at entry. In comparison to the remaining sample, this group had a significantly lower initial score on the Barthel and Canadian neurological scales, a higher prevalence of emboligenic cardiac diseases, and had a higher mortality at 6 months. No statistical difference was found for the other vascular risk factors. Eight patients (18.4%) had abnormally low levels of free protein S at entry. In comparison to the remaining sample, there was no statistical difference in the severity scores, prevalence of emboligenic cardiac diseases, mortality, or vascular risk factors. CONCLUSIONS: These findings suggest that low levels of protein C in the acute stroke reflect the massive activation of coagulation factors and are predictive of adverse outcome, whereas the significance of low levels of free protein S remains to be clarified.

Hemostatic and metabolic effects of lowering the ethinyl-estradiol dose from 30 mcg to 20 mcg in oral contraceptives containing desogestrel.

The metabolic and hemostatic effects of two oral contraceptives containing 150 mcg desogestrel and 20 mcg ethinyl-estradiol (EE) (MERCILON) or 30 mcg EE (MARVELON) were compared in order to examine the effect of reducing the EE dose in contraceptive pills. Forty-nine women participated in this randomized study during 6 cycles. In both groups, there was a significant increase in triglycerides, HDL-cholesterol and apoprotein A1; the same increase was observed for SBP and CBG. Slight and transient variations of fasting blood glucose levels were seen in the 30 mcg EE group and in the two groups for fasting insulin levels. The increase in renin substrate was significantly higher with the 30 mcg EE than with the 20 mcg EE pill. In both groups, plasminogen increased significantly, but antithrombin III, total and free protein S and fibrinogen decreased significantly only in women taking the 30 mcg EE pill, whereas there was no significant change in the 20 mcg EE group. Reducing the dose of EE in oral contraceptives from 30 mcg to 20 mcg minimizes their impact on renin substrate and hemostatic parameters.

Decreased protein S activity in sickle cell disease.

In sickle cell disease (SCD), vaso-occlusion is a complex process involving cellular, vascular and humoral factors and possibly thrombotic events. We studied three physiological inhibitors of the coagulation system, antithrombin III (AT III), protein C (PC) and protein S (PS), in three groups of subjects: 27 homozygous patients observed either in crisis or in a steady state, 23 heterozygous patients and 30 healthy subjects. PS study included the measurement of total and free PS antigen, PS activity and C4bBP antigen. In heterozygous subjects the results were similar to those of controls, but in homozygous subjects abnormalities of PS and to a lesser extent PC were observed. Values of PC were extremely variable with 10 cases lower than the normal range (2 SD of the mean) and 17 others within this range. In all cases total PS antigen was slightly reduced (77 +/- 18%, M +/- SD) with a more marked decrease of free antigen (59 +/- 17%) and normal values of C4bBP. Levels of PS activity were greatly reduced and lower than those of free antigen with a mean ratio of PS activity to free antigen of 0.6. These abnormalities were associated with significantly high concentrations of fibrinogen D-dimers. PS deficiency in SCD may be at least partly due to adsorption of free PS to aminophospholipids abnormally expressed on sickle cells membranes, microvesicles and activated platelets, while the discrepancy between PS activity and free antigen could reflect proteolytic inactivation of PS by traces of thrombin.