Chemical cross-linking of activated coagulation factor VII with soluble tissue factor: calcium ions are not essential for full amidolytic activity of the factor VIIa-tissue factor complex after complex formation.

In the previous study involving a yeast expression system, a high molecular mass extracellular domain of human tissue factor (denoted as sTF alpha) with a high content of mannose residues was produced in abundance and 37 kDa sTF beta was obtained in a low yield [Shigematsu et al. (1992) J. Biol. Chem. 267, 21329-21337]. To obtain sTF beta in a high yield, we constructed four kinds of mutant sTF with partial or total replacement of the N-potential glycosylation Asn residues with Ala, and expressed them in yeast. We found that the yield of the beta form of the Asn137-to-Ala mutant (designated as sTF beta NNA) was threefold higher (3 mg/liter) than that of the wild type, suggesting that the replacement of one of the three potential N-glycosylation Asn residues with Ala could be a good way to minimize the addition of mannose repeats. Since it has been reported that calcium ions are required for the effective hydrolysis of peptidyl substrates by the factor VIIa-sTF complex, it is believed to be essential for the expression of full protease activity. Here, we report the enzymatic characterization of a factor VIIa-sTF beta NNA complex cross-linked with a homobifunctional reagent, bis(sulfosuccinimidyl) suberate. The factor VIIa-sTF beta NNA complex cross-linked in the presence of 5 mM calcium ions or 50 mM EDTA was purified. The cross-linked complex did not show factor X activation in the presence of phospholipids. However, it showed essentially the same activity toward peptidyl substrates as before cross-linking, even in the presence of EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)