Utilization of a continuous flow reactor to study the lipoprotein-associated coagulation inhibitor (LACI) that inhibits tissue factor

A microperfusion system containing a glass capillary, the inner surface of which is coated with a phospholipid bilayer containing tissue factor, was used to explore the requirement for factors VIIa and Xa in the complex formed with the lipoprotein-associated coagulation inhibitor (LACI). Various combinations of factors VIIa, Xa, and LACI were perfused together or sequentially at a wall shear rate of 300 sec-1; a final perfusion of factors X and VIIa was performed to evaluate the residual tissue factor activity. Factor Xa concentration at the outlet of the tube was determined using a chromogenic substrate. In the presence of factors VIIa, Xa, and LACI, complete inhibition of tissue factor was observed on both phosphatidylcholine (neutral surfaces) and on phosphatidylserine/phosphatidylcholine (acidic) surfaces; omission of factors Xa or LACI resulted in no inhibition. The absence of factor VIIa in the initial perfusion steps resulted in no inhibition on neutral surfaces whereas about 90% inhibition was observed on acidic surfaces. Initial perfusion with factor Xa, but not LACI, followed by the remaining protein components, resulted in an inhibitory complex. Thus, it appears that a tissue factor:factor Xa:LACI complex can form in the absence of factor VIIa on acidic surfaces; moreover, our data imply a tissue factor binding site for factor Xa, but not for LACI.