Membrane-Bound and Soluble Tissue Factor - Fuse and Fire Extinguisher.
Authors: Butenas Saulius , Orfeo Thomas , Brummel-Ziedins Kathleen E. , Mann Kenneth G. 
Published: April 2020

Abstract
A circulating form of tissue factor (TF) has been suggested to have an important role in clot growth, and a soluble TF protein lacking the transmembrane domain (sTF) has been described. We have evaluated the potential role of TF in contributing to clot growth using numerical, synthetic plasma and whole blood models. In these studies we differentiate the influence of membrane-bound (mbTF) and sTF218 on thrombin generation. In previous studies we have shown that thrombin generation consists of an initiation phase (IP), a propagation phase and a termination phase. During the IP, complex enzymes are assembled leading to the robust thrombin generation. Initiation with 5 pM mbTF in numerical simulations yields an IP of thrombin generation spanning ~240 s, a maximum rate of thrombin generation (vmax) of 2 nM/sec and a maximum level of active thrombin (IIamax) of 275 nM. No change in the IP and limited decreases in the vmax and IIamax are observed when the mbTF is electronically nullified 240 s after the initiation of the reaction. No thrombin generation is observed when mbTF is removed at 0 s or 10 s post initiation. In synthetic plasma −2 μM phospholipid and 5 pM mbTF- the IP is 240 s, vmax is 2.6 nM/s and IIamax is 300 nM. Quenching of TF activity at 240 s post initiation, accomplished using a mixture of monoclonal anti-TF and anti-factor VII(a) antibodies, has no effect on these parameters. No thrombin generation is observed over 840 s when antibodies are added to the reaction mixture at 0 s. Contact pathway-inhibited whole blood clotted in 253 s after the addition of 5 pM mbTF. This clotting time was only slightly extended by the addition of antibodies at 60–240 s after the initiation of the reaction. No clot was observed in 1000 s when antibodies were added to blood at 0 s. These data indicate that a functional, mbTF is essential for the initiation of thrombin generation but is not required for the propagation of the process during which factor X activation is largely governed by the factor VIIIa/factor IXa complex. We hypothesized that sTF218 should act as a competitive inhibitor by binding factor VIIa in an inactive complex and effectively decreasing the concentration of the mbTF/factor VIIa complex. In synthetic plasma −2 μM phospholipid and 5 pM mbTF-, the addition of 10 nM TF218 extends the IP by 120 s. A further increase in TF218 to 40 nM prolongs the IP by 220 s. No thrombin generation is observed in the absence of mbTF, with or without 100 nM TF218. The vmax and IIamax during the propagation phase are barely affected by the presence of 10–40 nM TF218 consistent with primary factor Xa generation by the factor VIIIa/factor IXa complex during this phase of the reaction. Similar results were obtained in numerical simulations using TF218 as a competitive inhibitor of factor VIIa/mbTF complex formation. These data indicate that sTF acts as an inhibitor of thrombin generation by inhibiting the formation of the mbTF/factor VIIa complex. Collectively our data identify mbTF as the “fuse” and sTF as the “extinguisher” of the “coagulation explosion”.