A flow cytometric method for determining the binding of coagulation factor X to monocytes in whole human blood.
It is known that inflammation affects the coagulation pathway, but the mechanisms are not clear. Because a persistent inflammatory condition is associated with several chronic diseases, including cardiovascular disorders, there is intense interest in determining if and how chronic inflammation contributes to a hypercoagulable state. One pathway by which inflammation interacts with coagulation is via monocyte binding and activation of coagulation Factor X (FX). Upon activation, monocytes express the alphaMbeta2 integrin CD11b/CD18, which has a binding site for the plasma protein FX. Binding is followed by the cleavage of FX into its activated form Xa(FXa) which, in turn, is responsible for the conversion of prothrombin to thrombin. To assess the contribution of this pathway, a straightforward assay in whole blood is needed for studies of inflammation-induced coagulation and thrombosis. The current assay for FXa binding requires isolation of the monocytes and measurement of bound FXa activity with a chromogenic substrate. Harvesting a sufficient number of monocytes for analysis requires a relatively large blood sample. In addition, it is known that the process of isolating neutrophils and monocytes from whole blood induces an upregulation of CD11b. Thus, the measurement process itself causes an artifact in receptor expression resulting in an overestimate of true state of monocyte activation and FX binding. To address these limitations, we developed a flow cytometric assay to directly measure the binding of FX to monocytes in whole blood. In this report we describe the methods of the procedure in detail and apply the procedure to demonstrate a significant increase of both monocyte CD11b expression and FXa binding when human blood samples were activated with the endotoxin, lipopolysaccharide in-vitro.
Le Guyader
Alexandre
A
Section of Cardiovascular and Thoracic Surgery and The Sarver Heart Center, University of Arizona Health Sciences Center, P.O. Box 245071, Tucson, Arizona 85724, USA.
Davis-Gorman
Grace
G
Copeland
Jack G
JG
McDonagh
Paul F
PF
D039481:CD11b Antigen / Q000032:analysis; D005170:Factor X / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D009000:Monocytes / Q000378:metabolism*
D039481:CD11b Antigen; D005170:Factor X