Development of a microplate coagulation assay for Factor V in human plasma

Thrombosis Journal

Original Basic Research

Derek Tilley¹  John A Samis²  Irina Levit³ 

[1] Applied Bioscience, Faculty of Science, University of Ontario Institute of Technology, L1H7K4, Oshawa, ON, Canada;Applied Bioscience, Faculty of Science, University of Ontario Institute of Technology, L1H7K4, Oshawa, ON, Canada;Faculty of Health Sciences, University of Ontario Institute of Technology, L1H7K4, Oshawa, ON, Canada;Faculty of Health Sciences, University of Ontario Institute of Technology, L1H7K4, Oshawa, ON, Canada;

关键词: Thrombin;    Disseminate Intravascular Coagulation;    Disseminate Intravascular Coagulation;    Clot Formation;    Factor Versus;   

DOI  :  10.1186/1477-9560-9-11

 received in 2011-04-03, accepted in 2011-06-28,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundFactor V (FV) in its activated form, FVa, is a critical regulator of thrombin generation during fibrin clot formation. There is a need of a simple, fast, and inexpensive microplate-based coagulation assay to measure the functional activity of FV in human plasma. The objective of this study was to develop a microplate-based assay that measures FV coagulation activity during clot formation in human plasma, which is currently not available.MethodsThe FV assay requires a kinetic microplate reader to measure the change in absorbance at 405nm during fibrin formation in human plasma. The FV assay accurately measures the time, initial rate, and extent of fibrin clot formation in human plasma.ResultsThe FV microplate assay is simple, fast, economical, sensitive to approx 24-80pM, and multiple samples may be analyzed simultaneously. All the required materials are commercially available. Standard curves of time or initial rate of fibrin clot formation vs FV activity in the 1-stage assay (Without activation by thrombin) may be used to measure FV activity in samples of human plasma. The assay was used to demonstrate that in nine patients with disseminated intravascular coagulation (DIC), the FV 1-stage, 2-stage (With activation by thrombin), and total (2-stage activity - 1-stage activity) activities were decreased, on average, by approximately 54%, 44%, and 42%, respectively, from prolonged clot times when compared to normal pooled human reference plasma (NHP). The results indicate that the FV in the DIC patient plasmas supported both a delayed and slower rate of fibrin clot formation compared with NHP; however, the extent of fibrin clot formation in the DIC patients remained largely unchanged from that observed with NHP.ConclusionsThe FV microplate assay may be easily adapted to measure the activity of any coagulation factor using the appropriate factor-deficient plasma and clot initiating reagent. The microplate assay will find use in both research and clinical laboratories to provide measurement of the functional coagulation activity of FV in human plasma.

【 授权许可】

Unknown   
© Tilley et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.