Tissue Factor Activation of Factor X Mediated by Flow

Turitto, Vincent

Abstract

The long-term objective of this research proposal is to develop a better understanding of the role of fluid flow in the regulation of procoagulant activity by vascular cells exposed to flowing blood. Specifically, these studies will examine the ability of mechanical forces generated as a consequence of fluid flow to modulate the activity of the tissue factor (TF)-mediated pathway of blood coagulation. Using precisely designed parallel-plate flow chambers, the effects of fluid shear rate on the expression of TF by cultured human smooth muscle cells will be investigated. Similar studies using a tubular flow reactor whose interior has been coated with a TF-containing phospholipid bilayer will be performed. The levels of TF activity will be assessed from measurements of the production of Factor X. Additionally, the nature of the regulation of TF activity will be examined from measurements of TF mRNA in cultured cells. In order to correlate experimental findings with theoretical considerations, computational routines will be employed to solve the appropriate fluid dynamic and mass transport equations and generate the velocity and shear rate profiles within the flow chambers. The experimental results for the various systems will be compared with theoretical predictions in order to precisely define the nature of the transport of procoagulant proteins to the surface, as well as the variation of the kinetic parameters, Km and Vmax, under differing flow conditions. The dependence of these parameters on flow and the direct activation by flow of tissue factor expressed on vascular cells are of particular interest in this investigation. The combination of these experimental techniques and computational analyses will provide extensive information regarding the mechanism(s) by which fluid flow influences the procoagulant activity of vascular cells and will therefore, contribute to the understanding of TF-mediated blood coagulation under both physiologic and pathophysiologic flow conditions.