Tomohide Tsuda

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.

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.

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.

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

Abstract 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°C, the free protein S fraction increased from 0.5 ng/ml (at 4°C) to 7.8 ng/ml, and from 4.5 ng/ml (at 4°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 μg/ml and 6 μg/ml higher at 25°C and 37°C, respectively compared to measurements at 4°C. Measurements of free protein S in plasma using a commercially available assay kit were approximately 0.6 μg/ml higher at 25°C than measurements performed at 4°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°C).

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 β chain of C4b-binding protein (C4BP-β) 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-β 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.

Beneficial effect of EPA (20:5 n-3 PUFA) on preventing venous thromboembolism: a rat tail thrombosis model experiment.

Recent randomized controlled trials on coronary heart disease failed to demonstrate benefits of long chain n-3 polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) [1,2], although EPA and DHA have been recognized to decrease the risk of cardiovascular disease [3]. Herein, however, using a rat tail thrombosis model, we showed a new beneficial effect of EPA in preventing venous thromboembolism. Individuals prone to developing thrombosis have thrombophilia. Among Caucasians, “Activated protein C (APC)-resistance” is the major thrombophilia [4] and “APC-dysfunction” is the major thrombophilia in Japanese and Chinese populations [5]. These thrombophilias both tend to promote thrombosis due to the relatively weak APC anticoagulation reaction to blood coagulation [5,6]. We have examined the effects of phospholipids on blood coagulation and APC anticoagulation reactions, and found phospholipids of phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and phosphatidylethanolamine (PE) to exert complicated effects on the coagulation and anticoagulation reactions. That is, PC strongly and dose-dependently inhibited APC and protein S activities and the FXa-prothrombin reaction but once PC was converted to LPC by lipase, LPC accelerated the FXa-prothrombin reaction [7,8]. Protein S activity was measured as the enhancement in the rate of APC activity in response to adding protein S to the APC-FVa reaction

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.

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.

Development of a new colorimetric method for protein S activity measurement

Development of a reliable method (simple, sensitive and accurate) for protein S measurement is needed (1–4), as the protein C/protein S coagulation regulatory system has been shown to be involved in thrombosis (5–16). This letter describes a new colorimetric method we have recently developed for measuring protein S activity. This method, unlike conventional ones that measure clotting time, measures protein S activity colorimetrically using chromogenic substrates. The principle of this assay system is that protein S activity is calculated by analyzing the correlation between protein S activity and thrombin production rate in the protein C/protein S coagulation regulatory system and the blood clot reaction system reconstructed in vitro (17, 18). Using this method, protein S activity was measured in a variety of samples and their values were compared to those obtained with the conventional method measuring clotting time (19). Human activated protein C (APC), human protein S and human prothrombin, used in the assay, were obtained from Enzyme Research Laboratories, Inc. (South Bend, SD, USA), purified bovine factor Xa (FXa) from New England Biolabs, Inc. (Beverly, MA, USA), purified human factor Va (FVa) from Haematologic Technologies, Inc. (Essex Junction, VT, USA), thrombin chromogenic substrate S-2238 (20) from Chromogenix-Instrumentation Laboratory (Milan, Italy), bovine serum albumin (BSA) from Sigma-Aldrich Co. Ltd. (St. Louis, MS, USA) and porcine liver L-aphosphatidylethanolamine (PE), porcine liver L-aphosphatidylcholine (PC) and bovine brain L-a-phosphatidylserine (PS) from DOOSAN Serdary Research Laboratories (Yongin, Korea). A clot-time

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.