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 Kunitztype protease inhibitor that regulates tissue factor (TF)-induced coagulationby inhibiting factor Xaand theFVIIa–TFcomplex [1].Full-lengthTFPIa (FL-TFPIa) is a 276-residueglycoprotein with an acidic N-terminus followed by three tandem Kunitztype domains and a basic C-terminus. In humans, 80% of circulating TFPI is tightly bound to plasma lipoproteins (LDL > HDL > VLDL), and lacks the third Kunitz domain (K3)andC-terminusofTFPIa [2]. The remainingTFPI consists of forms that contain K3, but lack the C-terminus of TFPIa (TFPIa-desCTP), andFL-TFPIa, which is responsible formost of the anticoagulant activity of TFPI in plasma [3,4]. 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 release of the B domain. Duckers et al. [5] showed that TFPIa levels are 70% lower in the plasma of patients with severe FV deficiency than in normal plasma, and that the level of TFPIa in normal plasma is reduced by 60–90% following immunoprecipitation of FV. Using surface plasmon resonance, they demonstrated direct binding of FV to immobilized recombinant FL-TFPIa, with half-maximal binding at 13.5 nM FV. TFPIa may also interact with FVa, as previous functional studies showed that FVa enhanced TFPIa inhibition of FXa in the presence of phospholipids and calcium ions [3]. Ligand blot experiments (Fig. 1A) showed that both FV and FVa bound FL-TFPIa, whether the FL-TFPIa was expressed in Escherichia coli or in mammalian cells. Neither FV nor FVa, however, recognized TFPIa1–252 [3], which lacks the basic Cterminus of TFPIa. Control experiments showed that FXa binds to all forms of TFPI, and that our anti-C-terminal peptide (CTP) antibody is selective for FL-TFPIa. The binding of FV(a) to FL-TFPIawas not affected by EDTA (not shown). Thus, FL-TFPIa is bound by both FV and its activated form, and this binding requires the C-terminus of FL-TFPIa, but not divalent cations or post-translational modification of FLTFPIa. Upon size exclusion chromatography of pooled normal plasma (NP) in 0.1 M NaCl and 0.02 M Hepes (pH 7.4) (Fig. 1B), three significant peaks of TFPIa were detected by an immunoassay with an anti-K3 antibody. The first two peaks, which eluted at high apparent molecular masses (> 1000 kDa and 700 kDa, respectively), were also recognized by the anti-CTP antibody, and are consistent with FLTFPIa bound to other plasma moieties. The third peak eluted at a size consistent with unbound TFPI, and was not recognized by the anti-CTP antibody, suggesting that it consists of TFPIa-desCTP. Various individual plasmas showed profiles similar to the elution profile for pooled NP presented in Fig. 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 TFPIa-desCTP and FL-TFPIa eluted at their expected sizes of 40 kDa (data not shown and [6]). The dissociation of FL-TFPIa from the high molecular mass complexes in high-salt conditions suggests an ionic interaction, consistent with a role for the charged C-terminus of FL-TFPIa. Notably, the substantially C-terminal-truncated forms of TFPI that circulate tightly bound to plasma lipoproteins remained associated under high-salt conditions [2,6,7] and the high molecular mass FLTFPIa peaks did not comigrate with LDL (apolipoprotein B) or HDL (apolipoprotein AI) (Fig. 1B). Immunodepletion of FV from plasma reduced the FV levels by > 95%, and resulted in a significant reduction in the FLTFPIa level (> 80%), as reported by others [5]. Gel filtration of the FV-depleted plasma demonstrated a striking reduction in the peaks of FL-TFPIa migrating at high apparent molecular masses (Fig. 1B). Immunodepleted FVII plasma, used as a control, showed a profile similar to NP (data not shown). The plasma constituents with which FL-TFPIa associates in the high molecular mass peaks identified on gel Correspondence: George J. Broze Jr, Division of Hematology, Washington University School of Medicine, PO Box 8125, 660 S. Euclid Ave, St Louis, MO 63110, USA. Tel.: +1 314 362 8813; fax: +1 314 362 8811. E-mail: gbroze@dom.wustl.edu.