The first step in the coagulation cascade on intact HL60 cells is exposure of a substrate binding site on tissue factor

Tissue factor (TF) IB the biologic trigger which initiates coagulation in normal and abnormal hemostasis. DiBordered TF regulation may underlie complex pathophyBiologic disturbances such as gran negative sepsis, disseminated intravascular coagulation, and atheroma formation. TF procoagulant activity {PCA) is regulated by anatomic localization of TF on cells distant from the blood, TF pathway inhibitor (TFPI), and encryption of TF PCA on cell-surface membranes. Encryption is defined by the observation that TF is available on cells to bind factor Vila (FVIla), but TF-FVIIa is relatively inactive on unperturbed cells. The activation of encyrpted TF is a calcium/calmodulin-mediated process induced by calcium ionophores (ionomycin or A23187) and blocked by calmodulin inhibitors (calmidaeolium or trifluoperizine). HL60 cells pretreated with phorbol myristate acetate to enhance TF production were employed in this study of TF encryption. Essential lysines on the surface of TF were chemically modified with biotinamidocaproic acid 3sulfo-N-hydroxysuccinimide ester (10 j/M). The rate of inhibition of TF PCA on unperturbed cells was 0.38 min'1. Following ionomycin treatment the rate of inhibition increased by more than two-fold to 0.88 min"1. TFPIfactor Xa (FXa) inhibition of TF-FVIIa on HL60 cell was also determined before and after TF activation. TF-PVIIa on unperturbed cells was only 4% inhibited by TFPI-FXa whereas on ionomycin-stimulated cells the inhibition of TF-FVIIa by TFPI-FXa was 98%. Thus, it appears that the ionophore-induced activation of TF on HL60 cells exposes essential lysines and the binding site for TFPI-FXa. We propose a model of TF encryption which is consistent with these data, mutagenesiB studies, and the crystal structure of TF. In our model TF is a dimer on unperturbed cells. Dimeric TF is encrypted because the essential substrate/TFPI-FXa binding site is buried in the interface between subunits. TF activation is the calcium/calmodulin-induced conversion of dimeric TF to monomeric TF which exposes the essential aubstrate/TFPI-FXa binding site on TF.