Allosteric Cysteine Oxidation Does Not Play a Role in Tissue Factor Decryption

Abstract 1174 Tissue factor (TF) has two disulfides in the extracellular domain of the protein. The integrity of one cysteine pair, Cys186–Cys209, has been hypothesized to be essential for an allosteric “decryption” phenomenon regulating TF function, a subject of a lengthy debate. The encryption/decryption hypothesis asserts that cell-surface expressed TF, devoid of procoagulant activity, becomes converted to an active form when Cys186 and Cys209 are oxidized. Unlike a catalytic disulfide bond, which enzymatically mediates thiol-disulfide interchanges in substrate proteins, the hypothesized allosteric bond changes protein structure. The subsequent change in TF conformation is hypothesized to influence the intermolecular interactions between TF, a protease component of the extrinsic factor (F)Xase (FVIIa) and the substrate (FX), leading to enhanced dynamics of FX activation. The conclusions in support of this hypothesis are based on indirect evidence obtained with reagents with oxidizing/reducing properties and mutated recombinant TF. In our study, we determined the status of disulfides in both the recombinant TF1-263 (rTF1-263) and natural placental TF (pTF) by mass spectrometry. We analyzed TF peptides generated by proteolytic trypsin digestion in the non-reduced (NR) and reduced (R) (using10 mM dithiothreitol) TF without alkylation (NA) or with alkylation (A) (using 50 mM iodoacetamide). We found that all four cysteines of the extracellular domain of TF are oxidized in the native TF proteins. There was no evidence for peptides with reduced Cys49 and Cys57 (mass of 1996 Da) in the NR/NA samples of both pTF and rTF1-263. We did, however, observe peptides with mass of 1994 Da corresponding to a peptide with oxidized Cys49 and Cys57. In the R/A samples we identified a peptide (mass of 2110 Da) corresponding to reduced and alkylated peptide containing Cys49 and Cys57. Mass spectrometry analysis of peptides containing Cys186 and Cys209, two cysteines with close proximity to the membrane, revealed that these two residues are also oxidized. We did not observe peptides containing reduced Cys186 (mass of 3060 Da) or Cys209 (mass of 1409 Da) in the NR/NA samples of both pTF and rTF1-263, indicating that they are oxidized. In the R/A samples we identified peptides with mass 3116 Da and 1466 Da corresponding to reduced and alkylated Cys186 and Cys209, respectively. To further confirm that all four cysteines are oxidized, we treated TF with iodoacetamide without prior reduction. We did not detect peptides with alkylated cysteines in the NR/A sample in both pTF and rTF1-263. The mass spectrometry profile was the same as that of the NR/NA sample. The lack of incorporation of iodoacetamide into the NR sample indicates that all four cysteines are quantitatively involved in disulfide bridges. In functional assays, TF with reduced or non-reduced cysteines, with or without alkylation, showed similar activity of the TF/FVIIa complex in either membrane-independent synthetic substrate hydrolysis or membrane-dependent FX activation. The affinity of pTF for FVIIa was not altered by the reduction and alkylation (Kdapp 1.8±0.13 nM NR/NA, 1.8±0.19 nM R/NA, 1.7±0.16 nM R/A). Similarly, the maximum rate of synthetic substrate hydrolysis by the TF/FVIIa complex (FIU/sec) was not significantly affected by the treatment (Vmax 2662 FIU/sec NR/NA, 2434 FIU/sec R/NA and 3038 FIU/sec R/A). In FX activation, reduction and alkylation had little effect on the affinity of the pTF/FVIIa complex for FX (Km 0.9±0.2 μM NR/NA, 1.1±0.1 μM R/NA and 1.1±.2 μM R/A). Furthermore, the treatment only slightly altered the catalytic efficiency of the complex (kcat 7.4±0.06 sec−1 NR/NA, 7.2±0.03 sec−1 R/NA and 6.3±1.04 sec−1 R/A sam). In conclusion, our data indicate that the status of disulfides in the extracellular domain of TF does not contribute to the regulation of FVIIa catalytic function. Disclosures: Mann: Merck, Daiichi Sankyo, Baxter, GTI: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NIH, DOD, Baxter: Research Funding; corn trypsin inhibitor: Patents & Royalties; Haematologic Technologies: Chairman of the Board, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.