Structural changes in factor VIIa induced by Ca²⁺ and tissue factor studied using circular dichroism spectroscopy

Abstract

Factor VIIa (fVIIa) is composed of four discrete domains, a y-carboxyglutamic acid (G1a)-containing domain, two epidermal growth factor (EGF)-like domains, and a serine protease domain, all of which appear to be involved, to different extents, in an optimal interaction with tissue factor (TF). All except the second EGF-like domain contain at least one Ca2+ binding site and many properties of fVIIa, e.g., TF and phospholipid binding and amidolytic activity, are Ca2+-dependent. A CD study was performed to characterize and locate the conformational changes in fVIIa induced by Ca'+ and TF binding. In addition to intact fVIIa, derivatives lacking the Gla domain or the protease domain were used. Assignment of the Ca2+-induced changes in the far-UV region of the fVIIa spectrum to the Gla domain could be made by comparing the CD spectra obtained with these fVIla derivatives. The changes primarily appeared to reflect a Ca*+-induced ordering of a-helices existing in the apo state of fVIIa. This was corroborated by models of the apo and Ca2+ forms of fVIIa constructed on the basis of known structures of homologous proteins. Far-UV spectra of the Gla domain of fVIIa, obtained as difference spectra between fVIIa derivatives, were very similar to those of isolated Gla peptides from other vitamin K-dependent plasma proteins. The near-UV CD spectrum of fVIIa was dominated by aromatic residues residing in the protease domain and specific bands affected by Ca2+ were indicative of tertiary structural alterations. The formation of a NIIa:TF complex led to secondary structural changes that appeared to be restricted to the catalytic domain, possibly shedding light on the mechanism by which T F induces an enhancement of fVIIa catalytic activity.

Incorporation of an Active Site Inhibitor in Factor VIIa Alters the Affinity for Tissue Factor

“…(Fig. 7) were similar to the previously published spectra of FVIIa (23). However, a few minor differences were found between the FVIIa and FFR-FVIIa spectra.…”

“…The residual FVIIa activity in FFR-FVIIa was Ͻ0.1% when measured in an FVIIa-specific amidolytic assay. Des-(1-44)-FVIIa and des-(1-44)-FFR-FVIIa (19), factors X (FX) (20) and Xa (FXa) (21), human brain TF apoprotein (22), sTF (23), and polyclonal rabbit anti-human TF IgG (3) were prepared as described previously. TF apoprotein was reconstituted into 60% phosphatidylcholine, 40% phosphatidylserine vesicles as described (25).…”

“…8). Since the light chain of FVIIa has been shown not to contribute to the near-UV CD spectrum of FVIIa (23), this indicates that changes in the tertiary structure affecting the environment of Tyr residues have occurred in the protease domain as a result of incorporation of the inhibitor.…”

Tissue factor–mediated endocytosis, recycling, and degradation of factor VIIa by a clathrin-independent mechanism not requiring the cytoplasmic domain of tissue factor

“…Recombinant human VIIa, 20 VIIa blocked in the active site with phenylalanyl-phenylalanyl-arginyl chloromethyl ketone (FFR-VIIa), 21 recombinant sTF, 22 monospecific polyclonal rabbit antihuman TF immunoglobulin G (IgG), 23 and recombinant full-length TFPI 24 were prepared as previously described.…”

Mechanism of the Ca2+-induced Enhancement of the Intrinsic Factor VIIa Activity

“…Active site-inhibited FVIIa variants were obtained by incubation with the inhibitor PhePheArg-chloromethylketone essentially as described (26). Soluble TF (sTF, residues 1-209) was produced as described (27). Antithrombin (AT) was from Hematologic Technologies (Essex Junction, VT).…”