The coagulation pathways are initiated by the cell-surface receptor Tissue Factor (TF), which binds the serine proteinase coagulation Factor VIIa (VIIa), resulting in enhanced catalytic function, both amidolytic, towards small pseudo-substrates, and proteolytic, towards macromolecular substrates. Here we implicate Asp44 in TF as a ligand-interactive residue that, in contrast with previously characterized binding residues, is involved in the enhancement of VIIa catalytic function. Whereas charge neutralization by replacement of Asp44 with Asn did not reduce function of human TF, the exchange by Ala resulted in mutants with 8-fold reduced affinity for binding of VIIa. Enhancement of VIIa amidolytic function by TF Ala44 was reduced by 20–25% relative to wild-type and support of proteolytic function was diminished 6-fold indicating that this cofactor residue is significantly enhancing proteolysis of the macromolecular substrate by VIIa. Replacement of Asp44 by Glu, Thr and Arg exhibited a less severe phenotype with an approx. 4-fold reduced affinity for VIIa and a 2–3-fold diminished activation of Factor X. The improved activity of these mutants as compared with the Ala replacement is consistent with functional importance of an extended side chain at this position. The specific influence of the Asp44 exchange on catalytic function of the TF·VIIa complex indicates fine specificity of the TF ligand interface in mediating receptor and cofactor function.
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Research Article|
April 01 1996
Tissue Factor residue Asp44 regulates catalytic function of the bound proteinase Factor VIIa
Curtis R. KELLY;
Curtis R. KELLY
1Departments of Immunology and Vascular Biology, The Scripps Research Institute IMM-17, 10666 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
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John R. SCHULLEK;
John R. SCHULLEK
*
1Departments of Immunology and Vascular Biology, The Scripps Research Institute IMM-17, 10666 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
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Wolfram RUF;
Wolfram RUF
1Departments of Immunology and Vascular Biology, The Scripps Research Institute IMM-17, 10666 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
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Thomas S. EDGINGTON
Thomas S. EDGINGTON
†
1Departments of Immunology and Vascular Biology, The Scripps Research Institute IMM-17, 10666 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
†To whom correspondence should be sent.
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Publisher: Portland Press Ltd
Received:
September 25 1995
Revision Received:
November 06 1995
Accepted:
November 21 1995
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 315 (1): 145–151.
Article history
Received:
September 25 1995
Revision Received:
November 06 1995
Accepted:
November 21 1995
Citation
Curtis R. KELLY, John R. SCHULLEK, Wolfram RUF, Thomas S. EDGINGTON; Tissue Factor residue Asp44 regulates catalytic function of the bound proteinase Factor VIIa. Biochem J 1 April 1996; 315 (1): 145–151. doi: https://doi.org/10.1042/bj3150145
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