Application of annexin V to measurement of platelete activation

2007 Fiscal Year Final Research Report Summary

• Project/Area Number: 18591793
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Obstetrics and gynecology
• Research Institution: Akita University
• Principal Investigator: SATO Hirokazu Akita University, Akita University School of Medicine, ASSOCIATE PROFESSOR (60272035)
• Co-Investigator(Kenkyū-buntansha): FUJIMOTO Toshio AKITA UNIVERSITY, Akita University School of Medicine, ASSISTANT PROFESSOR (20375249) ; SATO Naoki AKITA UNIVERSITY, Akita University School of Medicine, RESEARCH ASSOCIATE (40447199) ; TANAKA Toshinobu AKITA UNIVERSITY, Akita University School of Medicine, PROFESSOR (40002216)
• Project Period (FY): 2006 – 2007
• Keywords: annexin V / platelete / activation / measurement

Research Abstract

In blood collecting using EDTA or sodium citrate, the chelate of the calcium ion occured so that the binding of annexin V to acidic phospholipid on the surface of blood platelete was interfared. For this reason, the experiments using annexin V were conducted by heparin blood collecting. Although fixation of blood platelets was carried out with formaldehyde for the experiment, activation of the blood platelets by formaldehyde took place. Then, it was thought that preservation of the blood platelets by the fixation using formaldehyde was difficult. Therefoe it was necessary to present an experiment with blood platelets without fixation.
Citrate blood collecting and heparin blood collecting were performed from the gynecology malignant tumor patients, and the D-dimer value was measured using the Vidas assay kit D-dimer 2'. In heparin blood collecting, the value of D-dimer was always higher than that in citrate blood collection, and strong positive correlation of the correlation coefficient 0.998 was shown. As for the gynecology malignant tumor patient, blood coagulation was accelerated as compared with the healthy person. The change of blood coagulation before and after the chemotherapy by an antineoplastic drug was not able to be detected only by change of D-dimer. Value.
The measurement procedure of acidic phospholipid appearing on the activated blood platelet surface was established using Alexa Fluor 488 labeled acidic phospholipid binding protein annexin V. In the experiment, the Alexa Fluor 488 labeled annexin V had higher reproducibility than the FTTC labeled one. Platelet aggregation ability was lower in gynecology malignant tumor patients than in healthy persons. However, no tendancy of platelet aggregation ability measured by common procedures and acidic phosphatide appearance detected by Alexa Fluor 488 labeled annexinV was observed before and after the chemotherapy by an antineoplastic drug at this time.
In this research, it became clear that blood coagulation was accelerating also in gynecology malignant tumor patients. Generally an antineoplastic drug is considered to be the cause of thrombosis indecements. Then, upregulation of the blood coagulation is estimated after the use of an antineoplastic drug. However, apparent upregulation of blood coagulation after chemotherapy was not detected by this method. Then, it seems that the further examination is required. Moreover, even if the annexin V-based method was used, change of platelet aggregation ability was not able to be clarified. Although the appearance of acidic phospholipid on blood platelete was used as one of the marker of platelete activation in this experiment, it is thought that the approach from other viewpoints was also required.