Protocols to Assess Coagulation Following In Vitro Infection with Hemorrhagic Fever Viruses

Abstract

During the course of infection with a hemorrhagic fever virus (HFV), the checks and balances associated with normal coagulation are perturbed resulting in hemorrhage in severe cases and, in some patients, disseminated intravascular coagulopathy (DIC). While many HFVs have animal models that permit the analyses of systemic coagulopathy, animal infection models do not exist for all HFVs and moreover do not always recapitulate the pathology observed in human tissues. Furthermore, molecular analyses of how coagulation is affected are not always straightforward or practical when using ex-vivo animal-derived samples, thus reinforcing the importance of cell culture studies. This chapter highlights procedures utilizing human umbilical vein endothelial cells (HUVECs) as a model system to evaluate components of the intrinsic (prekallikrein (PK), factor XII (FXII), kininogen, and bradykinin (BK)) and extrinsic (Tissue Factor (TF)) systems. Specifically, protocols are included for the generation of a coculture blood vessel model, plating and infection of HUVEC monolayers and assays designed to measure activation of PK and FXII, cleavage of kininogen, and to measure the expression of TF mRNA and protein.