Tissue factor residues 157-167 are required for efficient proteolytic activation of factor X and factor VII.

W Ruf,D.J Miles,A Rehemtulla,T.S Edgington

doi:10.1016/s0021-9258(18)41655-9

Abstract

The cell surface receptor tissue factor (TF) initiates coagulation by supporting the proteolytic activation of factors X and IX as well as VII to active serine proteases. Architectural similarity of TF to the cytokine receptor family suggests a strand-loop-strand structure for TF residues 151-174. Site-directed Ala exchanges in the predicted surface loop demonstrated that residues Tyr157, Lys159, Ser163, Gly164, Lys165, and Lys166 are important for function. Addition of side chain atoms at the Ser162 position decreased function, whereas the Ala exchange was tolerated. The dysfunctional mutants bound VII with high affinity and fully supported the catalysis of small peptidyl substrates by the mutant TF.VIIa complex. Lys159-->Ala substitution was compatible with efficient activation of factor X, whereas the Try157-->Ala exchange and mutations in the carboxyl aspect of the predicted loop resulted in diminished activation of factor X. The specific plasma procoagulant activity of all functionally deficient mutants increased 7- to 200-fold upon the supplementation of VIIa suggesting that TF residues 157-167 also provide important interactions that accelerate the activation of VII to VIIa. These data are consistent with assignment of the TF 157-167 region as contributing to protein substrate recognition and cleavage by the TF.VIIa complex.

Highlights

L Y S ' a~r~e important for function
Initiation of the coagulation cascades in vivo is mediated by the cell surface expression of tissue factor (TF)' [1]which serves as the receptor and catalytic cofactor for the serine proteasefactorVIIa(VIIa)as well as a mediator for the autoactivation ofVI1 to VIIa [2]
Upon assembly with TF, VIIa exhibits enhanced catalytic activity evidenced by hydrolysis of small peptidyl [3, 4] as well as protein substrates [3, 5,6,7,8]

Summary

MATERIALS ANDMETHODS

Reagents-Coagulation proteins were purified as described [3]. VIIa was purchased from Novo Nordisk (Gentofte, Denmark), and the functional activityand binding characteristicsof the recombinant protein have previously been described [9]. These data serine) using detergent solubilization and dialysis, as described in areconsistentwith a structurallyalteredmutantprotein detail [17] Both the enzyme-linked immunosorbent assay and clotting assay were quality controlled using cell pellets of a stable cell line expressing wild-type TF. Cell lysate (100 pl), normal or coagulation factor-deficient plasma (50 pl), and 500 nM VIIa or buffer (50p1) were equilibrated a t 37 "C for 1min followed by initiation of the reaction by adding 20 mM CaC12 (100 pl). Preincubation of wild-type and mutantTF with VIIa in thepresence of CaC1, followed by the addition of plasma did not reveal differences compared to the assay where the reaction was started by the addition of Ca'+ This suggests that a slower assembly of VIIa with the TF mutants does not contribute to the functional defect.

Functional activity of TF mutants

TF mutant

RESULTS

Since there was an additional effect when a negative charge