Reduced Activity of Protein S in Plasma: A Risk Factor for Venous Thromboembolism in the Japanese Population.

The quantitative assay of protein S can help in rapidly identifying carriers of abnormal protein S molecules through a simple procedure (by determining the total protein S mass, total protein S activity, and protein S-specific activity in blood), without genetic testing. To clarify the relationship between venous thromboembolism (VTE) and protein S-specific activity, and its role in the diagnosis of thrombosis in Japanese persons, the protein S-specific activity was measured and compared between patients with thrombosis and healthy individuals. The protein S-specific activity of each participant was calculated from the ratio of total protein S activity to total protein S antigen level. Plasma samples were collected from 133 healthy individuals, 57 patients with venous thrombosis, 118 patients with arterial thrombosis, and 185 non-thrombotic patients. The protein S-specific activity of one-third of the patients with VTE was below the line of Y = 0.85X (-2 S.D.). Most protein S activities in the plasma of non-thrombotic patients were near the Y = X line, as observed in healthy individuals. In conclusion, it was clearly shown that monitoring protein S activity and protein S-specific activity in blood is useful for predicting the onset and preventing venous thrombosis in at least the Japanese population.

Evaluation of the plasma protein S dynamics during pregnancy using a total protein S assay: Protein S-specific activity decreased from the second trimester.

AIM: The relationship between congenital protein S (PS) deficiency and complications during pregnancy remains unclear, partly due to the difficulty of precisely evaluating the PS level with conventional assays and the physiological decrease of PS during pregnancy. A new PS assay was developed to measure the total PS antigen and activity quantitatively and calculate PS-specific activity. This study aimed to evaluate the plasma PS dynamics during pregnancy using the new PS assay and establish the reference interval for pregnant women. METHODS: A total of 253 pregnant women without a personal or family history of thromboembolism were recruited. Blood samples were obtained in the first, second and third trimesters and at one month post-partum. The total PS antigen, activity, and PS-specific activity were studied. Results were analyzed by the repeated measures single-factor anovas followed by a post-hoc test using Excel Statistics. RESULTS: The mean ± standard deviation (IU/mL) of the total PS antigen levels in the first, second and third trimesters and 1 month post-partum were 0.67 ± 0.12, 0.67 ± 0.09, 0.68 ± 0.11 and 0.92 ± 0.13, respectively. The total PS activity (IU/mL) in the first, second and third trimesters and 1 month post-partum were 0.69 ± 0.14, 0.59 ± 0.10, 0.58 ± 0.12 and 0.87 ± 0.15, respectively. The PS-specific activity was within the normal range of nonpregnant women in the first trimester (1.02 ± 0.10) but decreased in the second and third trimesters (0.88 ± 0.09 and 0.85 ± 0.09, respectively) before increasing in the post-partum period (0.94 ± 0.08). CONCLUSION: The total PS antigen and activity decrease throughout pregnancy, while the PS-specific activity decreases in the second and third trimesters.

Plasma phenotypes of protein S Lys196Glu and protein C Lys193del variants prevalent among young Japanese women.

: Protein S Tokushima (p.Lys196Glu) and two protein C gene variants (p.Arg189Trp, p.Lys193del) are hereditary thrombophilia in Japanese and Chinese populations, respectively; however, their diagnosis by plasma analyses is difficult because of the type II deficiency phenotype. Three gene variant genotypes were examined in young Japanese women (n = 231). Plasma total protein S activity and total protein S antigen levels were measured using a total protein S assay system, protein C and protein S activities by clot-based methods, and protein C and free protein S antigen levels by latex agglutination methods. protein S Tokushima (p.Lys196Glu) and protein C p.Lys193del variants were prevalent among participants with allele frequencies of 1.08 and 0.86%, respectively, whereas any carrier of protein C p.Arg189Trp variant was not identified. The plasma phenotype of the type II deficiency of protein S Tokushima heterozygotes was demonstrated by decreased total protein S activity with a normal total protein S antigen level; however, the protein C activities of protein C p.Lys193del heterozygotes were within reference intervals, whereas their protein C antigen levels were elevated. We compared the diagnostic accuracy of the total protein S activity/total protein S antigen ratio for identifying protein S Tokushima heterozygotes with that of the clot-based protein S activity/free protein S antigen ratio and found that sensitivity and specificity of 100% each was only achieved by the former. Protein S Tokushima and protein C p.Lys193del are prevalent among young Japanese women, and a plasma analysis using the total protein S assay system is more accurate than the clot-based protein S activity/free protein S antigen ratio for diagnosing protein S Tokushima carriers.

Relationship between plasma protein S levels and apolipoprotein C-II in Japanese middle-aged obese women and young nonobese women.

: Protein S, a nonenzymatic cofactor to activated protein C, presents in two forms in plasma, free form and in a complex with C4b-binding protein. The aim of this study was to determine the association of plasma protein S levels with the variables related to cardiovascular disease risk. The relationships between plasma protein S levels with lipids, inflammation markers, and adiposity were first examined on middle-aged obese women (n = 62), then on young nonobese women (n = 160) to verify the findings in the obese women. Total and free protein S antigen levels in middle-aged obese women, approximately half being in a postmenopausal state and suffered from dyslipidemia, correlated negatively with estradiol and positively with triglycerides, total cholesterol, LDL cholesterol, apoA-II, apoB, apoC-II, apoC-III, apoE, hemoglobin A1c, and protein C, whereas there was no correlation with HDL cholesterol, apoA-I, BMI, visceral fat area, blood pressure, or factor VII activity. Multiple linear regression analyses revealed that protein C, apoC-II, and fibrinogen were significant predictors of total protein S antigen levels, accounting for 51.9% of variance, and apoC-II as a singular significant predictor for free protein S antigen levels (12.3% of variance). In young nonobese women, most being normolipidemic, apoC-II was also selected as a significant predictor of total protein S antigen levels, but not of free protein S antigen levels. The positive relationship between plasma protein S levels and apoC-II, a key regulator of triglycerides hydrolysis, may contribute to the pathogenesis of increased concentrations of plasma protein S.

Thrombosis Prediction Based on Reference Ranges of Coagulation-Related Markers in Different Stages of Pregnancy.

INTRODUCTION: Careful monitoring of the hypercoagulable state is required during pregnancy. However, coagulation and fibrinolysis markers are not fully utilized because there are no reference values reflective of coagulation and fibrinolysis dynamics during pregnancy, which differ from the nonpregnant state. METHODS: Changes in antithrombin (AT), fibrinogen (Fbg), prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), soluble fibrin (SF), D-dimer (DD), and protein S (PS) were investigated in healthy pregnant women, and reference ranges in the early, mid, late, and end stages of pregnancy were established. RESULTS: The AT was essentially constant throughout pregnancy. The Fbg, F1+2, TAT, and DD increased significantly as pregnancy progressed. In contrast, SF did not show a significant increase throughout the entire pregnancy period. Total PS antigen and total PS activity showed a corresponding decrease from early gestation. When test data in 3 cases in which deep vein thrombosis or intrauterine fetal death occurred during pregnancy were compared to the established reference ranges, all of the cases had multiple markers with values that exceeded the reference ranges. CONCLUSION: Establishing reference ranges for each week could potentially make it possible to evaluate abnormalities of the coagulation and fibrinolysis systems during pregnancy. Of note, SF might be a useful marker that reflects thrombus formation during pregnancy. Larger-scale studies will be required to establish reference ranges for every gestational week.

Genotyping analysis of protein S-Tokushima (K196E) and the involvement of protein S antigen and activity in patients with recurrent pregnancy loss.

OBJECTIVE: Preston et al. indicated that Protein S (PS) deficiency was associated with stillbirths but not miscarriages. The PS-Tokushima missense variant was reported to serve as a genetic risk factor for deep vein thrombosis in the Japanese population. A previous cross-sectional study showed no increase in the prevalence of PS-Tokushima in patients with recurrent early pregnancy loss or in patients with intra uterine fetal death and/or fetal growth restriction. There has been limited number of prospective studies examining the pregnancy outcome in patients with both a PS deficiency and recurrent pregnancy loss (RPL). We examined the association between PS deficiency, PS-Tokushima and RPL. STUDY DESIGN: The study group consisted of 355 Japanese women with two or more consecutive pregnancy losses and 101 parous women. The frequency of PS-Tokushima and the subsequent live birth rate in relation to a PS deficiency defined as low PS-specific activity (total PS activity/total PS antigen) and the carriage of PS-Tokushima were examined. RESULTS AND CONCLUSIONS: There was no significant difference in the frequency of PS-Tokushima between patients and controls. The 8 patients carriers of PS-Tokushima variant were capable of a subsequent live birth without the use of heparin. There was no significant difference in subsequent live birth rates between patients with low or normal PS-specific activity/PS activity without heparin prophylaxis after excluding miscarriages caused by an abnormal embryonic karyotype using multivariate logistic regression analysis. There was no association between PS-Tokushima and RPL and a PS deficiency or low PS activity was shown not to serve as a reliable clinical predictor of subsequent miscarriage.

New quantitative total protein S-assay system for diagnosing protein S type II deficiency: clinical application of the screening system for protein S type II deficiency.

Venous thromboembolism (VTE) incidence is rising rapidly in Japan with lifestyle westernization and aging. Deficiency of protein S, an important blood coagulation regulator, is a risk factor for VTE. Protein S deficiency prevalence in Asians is approximately 10 times that in Caucasians and that of protein S type II deficiency, associated with the protein S Tokushima mutation (K155E), is quite high in Japan. However, currently available methods for measuring protein S are not precise enough for detection of this deficiency. We developed a novel assay system for precise simultaneous determinations of total protein S activity and total protein S antigen level, using a general-purpose automated analyzer, allowing protein S-specific activity (ratio of total protein S activity to total protein S antigen level) to be calculated. Mean specific activity was 0.99 for samples from healthy individuals but 0.69 or less (mean-3SD) in protein S type II-deficient and warfarin-treated samples, but was 1.0 in an estrogen-treated sample with significantly decreased protein S antigen. Protein S gene analyses in healthy individuals with specific activity 0.69 or less revealed the K155E mutation in all three. These results show our new assay system to be an effective screening tool for protein S type II deficiency. This system can also be used in an automated analyzer, facilitating numerous sample measurements, and is, thus, applicable to regular medical checkups and diagnosing VTE. Such applications would potentially contribute to early detection of protein S type II deficiency, and, thereby, to thrombosis prevention.

Resveratrol, a phytoestrogen found in red wine, down-regulates protein S expression in HepG2 cells.

UNLABELLED: INTRODUATION: Resveratrol, a phytoestrogen present at a high concentration in red wine, has been reported to possess many health benefit effects that are protective against age-related diseases. Protein S (PS), an important anticoagulant factor in the protein C (PC) anticoagulant pathway, is mainly synthesized by hepatocytes, and its plasma level is decreased in high-estrogen conditions such as pregnancy and oral contraceptive use. The aim of this study was to investigate whether resveratrol affects PS expression in HepG2 cells. MATERIALS AND METHODS: The secreted and intracellular levels of PS were determined by an enzyme-linked ligandsorbent assay and Western blotting. The mRNA expressions of PS, PC and ß chain of C4b-binding protein (C4BP-ß) were analyzed by reverse transcription-polymerase chain reaction. The PS gene promotor activities in HepG2 cells transiently expressing estrogen receptor (ER) α were examined by a luciferase reporter assay. RESULTS: Resveratrol dose- and time-dependently down-regulated the PS expression in HepG2 cells at a transcriptional level, resulting in a significant decrease in secreted PS; however, the PC and C4BP-ß mRNA expressions were not affected. This action of resveratrol was not mediated through either the ER signaling or those of mitogen-activated protein kinases and protein kinase C. Piceatannol, a hydroxylated metabolite of resveratrol, and genistein, an isoflavone found in soy products, also down-regulated the PS expression. CONCLUSIONS: Resveratrol down-regulates the PS expression in HepG2 cells in an ER-independent manner, and the two phenolic hydroxyls at carbon-3 and -5 of resveratrol may be involved in this function.

Effect of phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine on the protein C/protein S anticoagulation system.

Phosphatidylserine is known to significantly accelerate the blood coagulation reaction. In a previous communication submitted for publication, we demonstrated that phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine showed effects on the blood coagulation reaction using the factor Xa-prothrombin reaction system, and discuss a new function of membrane phospholipids. The present study examined the role of phospholipids in the blood coagulation regulatory reaction (anticoagulation system), by studying the effects of phospholipids on the protein C/protein S reaction. We have established quantitative methods for measuring activated protein C activity and protein S activity, and used them to measure their activity after the addition of liposomes with different phospholipid compositions. We found that phosphatidylcholine inhibited activated protein C and protein S activities in a dose-dependent manner, as in the factor Xa-prothrombin reaction system. On the other hand, phosphatidylethanolamine and lysophosphatidylcholine showed no effect on activated protein C activity. Phosphatidylethanolamine inhibited and lysophosphatidylcholine accelerated coagulation activity in the factor Xa-prothrombin system, but such effects were not observed in the protein C/protein S reaction system. The coagulation and anticoagulation reactions are exquisitely balanced by thrombin, with a role both as a procoagulant and anticoagulant. Therefore, it is understandable that phosphatidylethanolamine and lysophosphatidylcholine show different effects in the factor Xa-prothrombin and protein C/protein S reaction systems. It appears that coagulation and anticoagulation reactions are co-ordinated and controlled by changes in phospholipid composition of the cellular membrane where the coagulation reaction takes place.

Effect of phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine on the activated factor X-prothrombin system.

Membrane phospholipids are essential in blood coagulation reactions. The importance of negatively changed phosphatidylserine has been shown. The roles of other phospholipids in the blood coagulation system, however, are not clear. This study examined the effects of phosphatidylcholine on the blood coagulation system using liposomes containing varying concentrations of phosphatidylcholine in the presence of phosphatidylserine at a constant concentration. In addition, with phosphatidylserine and phosphatidylcholine at constant concentrations, the effects of phosphatidylethanolamine and lysophosphatidylcholine on the blood coagulation system were examined. Using an in vitro reconstructed system of the activated factor X-prothrombin system, blood coagulation was measured by the rate of thrombin formation after the addition of liposome preparations. The results showed suppression of the system by phosphatidylcholine and phosphatidylethanolamine and acceleration by lysophosphatidylcholine. The results of the present study suggest that the cell membrane, the 'location' of blood coagulation, is one of the regulatory factors, and that changes in phosphatidylcholine content and phospholipid composition of the cell membrane regulate the coagulation reaction.

Dynamic equilibrium between protein S and C4b binding protein is important for accurate determination of free protein S antigen.

Protein S in circulation is in a dynamic equilibrium with C4b binding protein (C4bBP), thus affecting the measurement of free protein S antigen. We addressed the issue of overestimation of the free protein S concentration with current immunoassays due to the dynamic equilibrium and propose a new method for its accurate determination. Our assay system was tested at different reaction temperatures using purified free protein S, protein S-C4bBP complexes, plasma samples, and a commercially available free protein S assay kit. At a reaction temperature of 37 degrees C, the free protein S fraction increased from 0.5 ng/ml (at 4 degrees C) to 7.8 ng/ml, and from 4.5 ng/ml (at 4 degrees C) to 56 ng/ml when the concentration of the assayed protein S-C4bBP complexes was 20 ng/ml and 200 ng/ml, respectively. In plasma samples, free protein S levels were approximately 0.8 microg/ml and 6 pg/ml higher at 25 degrees C and 37 degrees C, respectively compared to measurements at 4 degrees C. Measurements of free protein S in plasma using a commercially available assay kit were approximately 0.6 microg/ml higher at 25 degrees C than measurements performed at 4 degrees C. Dynamic equilibrium between protein S and C4bBP affects the measurement of free protein S antigen. Measurement of free protein S antigen should be performed under conditions where protein S is not dissociated from protein S-C4bBP complexes, as exemplified by assay at low temperature (4 degrees C).

Four missense mutations identified in the protein S gene of thrombosis patients with protein S deficiency: effects on secretion and anticoagulant activity of protein S.

Four missense mutations, G54R, T589I, K155E, and Y595C, were identified in the protein S (PS) gene of the patients with PS deficiency and venous thrombosis. Three patients were heterozygous for the novel mutations, G54R, T589I, and Y595C, while a remaining one patient was homozygous for the K155E mutation, which is known to be a polymorphism in the Japanese population. A family study revealed that the Y595C mutation was associated with a Type I PS deficiency and the K155E mutation with a Type II PS deficiency, while no family study was performed for the patients with the G54R and T589I mutations. To determine whether these four mutations play a causative role in PS deficiency, the four PS mutants and wild-type PS were stably expressed in human embryo kidney (HEK) 293 cells. Pulse-chase experiments showed intracellular degradation and decreased secretion of the Y595C mutant. In the activated protein C (APC) cofactor assays, the specific activity of the K155E mutant decreased to 58% of that of wild-type PS. The APC cofactor activity of the three mutants, G54R, K155E, and T589I, were inhibited by C4b-binding protein (C4BP) with a dose dependency similar to that of wild-type PS. These results indicate that the Y595C and the K155E mutations are responsible for a secretion defect and a decreased anticoagulant activity of PS, respectively. The remaining two mutations, G54R and T589I, however, did not produce any definite abnormality leading to a low plasma PS activity.