The C-terminus of tissue factor pathway inhibitor α is required for its interaction with factors V and Va.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that regulates tissue factor (TF)-induced coagulation by inhibiting factor Xa (FXa) and the factor VIIa (FVIIa)/TF complex [1]. Full-length TFPIα (FL-TFPIα) is a 276-residue glycoprotein with an acidic amino-terminus followed by three tandem Kunitz-type domains and a basic carboxy-terminus. In man, approximately 80% of circulating TFPI is tightly bound to plasma lipoproteins (LDL>HDL>VLDL) and lacks the third Kunitz domain (K3) and carboxy-terminus of TFPIα [2]. The remaining TFPI consists of forms that contain the third Kunitz domain, but lack the carboxy-terminus of TFPIα (TFPIα-desCTP), and FL-TFPIα, which is responsible for most of the anticoagulant activity of TFPI in plasma [3,4]. Factor V (FV) is a 330 kDa single chain glycoprotein consisting of two tandem A domains, followed by a B domain, a third A domain, and two C domains. During blood coagulation, FV is proteolytically activated to FVa resulting in the release of the B domain. Duckers et al. showed that TFPIα levels are ∼70% lower in the plasma of patients with severe FV deficiency relative to normal plasma and that the level of TFPIα in normal plasma is reduced by ∼60 - 90% following immuno-precipitation of FV [5]. Using surface plasmon resonance, they demonstrated direct binding of FV to immobilized recombinant FL-TFPIα with half-maximal binding at 13.5 nM FV. TFPIα may also interact with FVa as previous functional studies have shown that FVa enhanced TFPIα inhibition of FXa in the presence of phospholipids and calcium ions [3]. Ligand blot experiments (Figure 1A) showed that both FV and FVa bind FL-TFPIα, whether the FL-TFPIα was expressed in E. coli or mammalian cells. Neither FV nor FVa, however, recognized TFPIα1-252 [3], which lacks the basic carboxy-terminus of TFPIα. Control experiments showed that FXa binds to all the forms of TFPI and that our anti-CTP antibody is selective for FL-TFPIα. The binding of FV(a) to FL-TFPIα was not affected by EDTA (not shown). Thus, FL-TFPIα is bound by both FV and its activated form and this binding requires the carboxyterminus of FL-TFPIα, but not divalent cations or posttranslational modification of FL-TFPIα. Fig. 1 Interaction of TFPI with Factor V and Factor Va Upon size exclusion chromatography of pooled normal plasma (NP) in 0.1 M NaCl, 0.02 M Hepes, pH 7.4 (Figure 1B), three significant peaks of TFPIα are detected by an immunoassay using an anti-K3 domain antibody. The first two peaks, which elute at high apparent molecular weights (>1000 kDa and ∼ 700 kDa, respectively), are also recognized by the anti-CTP antibody, and are consistent with FL-TFPIα bound to other plasma moieties. The third peak elutes at a size consistent with unbound TFPI and is not recognized by the anti-CTP antibody, suggesting that it consists of TFPIα-desCTP. Various individual plasmas showed profiles similar to the elution profile for pooled NP presented in Figure 1B (data not shown), although the relative amplitude of the peaks did show some variability (less than two-fold), perhaps related to the known variability of plasma TFPI levels across individuals [2]. On gel filtration of NP under high salt conditions (1 M NaCl), both TFPIα-desCTP and FL-TFPIα elute at their anticipated sizes of ∼ 40 kDa (data not shown and [6]). The dissociation of FL-TFPIα from the high molecular weight complexes in high salt suggests an ionic interaction, consistent with a role for the charged carboxyterminus of FL-TFPIα. Notably, the substantially carboxyterminal truncated forms of TFPI that circulate tightly bound to plasma lipoproteins remain associated under high salt conditions [2,6,7] and the high molecular weight FL-TFPIα peaks do not co-migrate with LDL (apolipoprotein B) or HDL (apolipoprotein AI) (Figure 1B). Immuno-depletion of FV from plasma reduced the FV levels by >95% and resulted in a significant reduction of the FL-TFPIα level (>80%), as reported by others (5). Gel filtration of the FV-depleted plasma demonstrated a striking reduction in the peaks of FL-TFPIα migrating at high apparent molecular weights (Figure 1B). Immuno-depleted FVII plasma, used as a control, showed a profile similar to normal plasma (data not shown). The plasma constituents with which FL-TFPIα associates in the high molecular weight peaks identified on gel filtration are not known, but FV appears to be involved based on the chromatography profile of FL-TFPIα following FV immunodepletion from plasma.