Xiuri Jin

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.

A Simple Search of TM Segments in Polytopic Membrane Protein Using Matrix - Assisted Laser DesorptionIonization Time - of - Flight Mass Spectrometry

Using both high performance liquid chromatography (HPLC) and amino acid sequencing (AAS), we previously analyzed band 3 TM peptide-segments that make up the transmembrane protein structure. However, the HPLC/AAS combination method was highly time-consuming. Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry is used to obtain accurate molecular weight information for proteins/peptides simply and sensitively. We applied the MALDI-TOF mass spectrometry technique to search for TM segments in membrane proteins. In combination with trypsin cleavages after alkali treatments (pH12 or 13) and sample preparation using organic solvents for MALDI-TOF mass spectrometry, we determined the TM segments of band 3 and glycophorin A in erythrocyte membrane. The method can be applied to other polytopic membrane proteins in erythrocyte membrane.

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.