Original basic research

Analysis of blood coagulation in mice: pre-analytical conditions and evaluation of a home-made assay for thrombin-antithrombin complexes

Dirkje W Sommeijer¹ , René van Oerle³ , Pieter H Reitsma¹ , Janneke J Timmerman¹ , Joost CM Meijers² , Henri MH Spronk³ and Hugo ten Cate³

¹  Laboratory for Experimental Internal Medicine, Academic Medical Center of Amsterdam, Amsterdam, The Netherlands

²  Department of Vascular Medicine, Academic Medical Centre of Amsterdam, Amsterdam, The Netherlands

³  Laboratory for Clinical Thrombosis and Haemostasis, Department of Internal Medicine, and Cardiovascular Research Institute Maastricht, University of Maastricht, The Netherlands

author email corresponding author email

Thrombosis Journal 2005, 3:12doi:10.1186/1477-9560-3-12

Published:	22 August 2005

Abstract

Background

The use of mouse models for the study of thrombotic disorders has gained increasing importance. Methods for measurement of coagulation activation in mice are, however, scarce. The primary aim of this study was to develop a specific mouse thrombin-antithrombin (TAT) ELISA for measurement of coagulation activation and to compare it with two commercially available assays for human TAT complexes. In addition, we aimed to improve methods for mouse plasma anticoagulation and preparation.

Methods and results

First, for the measurement of TAT-complexes in plasma a mouse specific TAT-ELISA was developed using rabbit polyclonal antibodies raised against mouse thrombin and rat antithrombin, respectively. This ELISA detected an increase in TAT levels in a mouse model of endotoxemia. Two commercial human TAT ELISAs appeared to be less specific for mouse thrombin-rat antithrombin complexes.

Second, to prevent clotting of mouse blood sodium citrate was either mixed with blood during collection in a syringe or was injected intravenously immediately prior to blood collection. Intravenous sodium citrate completely inhibited blood coagulation resulting in plasma with consistently low TAT levels. Sodium citrate mixed with blood during collection resulted in increased TAT levels in 4 out of 16 plasma samples. Third, heparinase was added to plasma samples after in vivo injection of different heparin doses to test its neutralizing effect. Heparinase neutralized up to a 20 U of heparin/mouse and resulted in accurate APTT and factor VIII determinations.

Conclusion

These procedures and reagents for plasma preparation and coagulation testing will improve studies on thrombotic disorders in mice.