Mechanisms of binding of recombinant extrinsic pathway inhibitor (rEPI) to cultured cell surfaces. Evidence that rEPI can bind to and inhibit factor VIIa-tissue factor complexes in the absence of factor Xa.
Extrinsic pathway inhibitor plays a key role in modulating tissue factor-dependent blood coagulation. We have studied binding of radioiodinated recombinant extrinsic pathway inhibitor (rEPI) to cultured cell surfaces. rEPI in the absence of added reactants bound to a limited extent to three cell lines studied. Binding of rEPI to two cell lines possessing surface tissue factor, but not to a cell line lacking surface tissue factor, was markedly increased in the presence of both factor VIIa and factor Xa, and calcium ions. Moreover, some increased tissue factor-dependent binding was also demonstrated with factor VIIa alone. Binding isotherms of rEPI to factor VIIa-tissue factor obtained with an ovarian carcinoma cell line were hyperbolic. Scatchard plots indicated the following: a Kd value of 4.5 +/- 1.5 nM and 335,000 +/- 84,000 sites/cell when factor Xa was present; a Kd value of 11.9 +/- 3.5 nM and 236,000 +/- 68,000 sites/cell when factor Xa was absent. In functional studies, high concentrations of rEPI, e.g. 27-67.5 nM, were found to inhibit factor VIIa-tissue factor-catalyzed release of activation peptide from tritiated factor IX in the absence of factor Xa. Whereas factor Xa was thus shown not to be required for rEPI to inhibit factor VIIa-tissue factor catalytic activity, its presence markedly enhanced rEPI's inhibitory function. Since the local concentration of extrinsic pathway inhibitor achieved at a site of tissue injury is unknown, the physiologic significance of the observation of extrinsic pathway inhibitor-induced inhibition of factor VIIa-tissue factor activity in the absence of factor Xa is not clear. However, factor Xa-independent inhibition could play a significant role when large doses of rEPI are administered in experimental studies of thrombosis.
Callander
N S
NS
Department of Medicine, University of California, San Diego 92093.
Rao
L V
LV
Nordfang
O
O
Sandset
P M
PM
Warn-Cramer
B
B
Rapaport
S I
SI
D000906:Antibodies; D001345:Autoradiography; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005167:Factor VII / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015942:Factor VIIa / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008074:Lipoproteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D013925:Thromboplastin / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000906:Antibodies; D008074:Lipoproteins; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D005167:Factor VII; D006493:Heparin; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa; D002118:Calcium