Localization of Phosphatidylserine Binding Sites to Structural Domains of Factor Xa

Arvind Srivastava,Jianfang Wang,Rinku Majumder,Alireza R Rezaie,Johan Stenflo,Charles T Esmon,Barry R Lentz

doi:10.1074/jbc.m105697200

Abstract

Binding of short chain phosphatidylserine (C6PS) enhances the proteolytic activity of factor X(a) by 60-fold (Koppaka, V., Wang, J., Banerjee, M., and Lentz, B. R. (1996) Biochemistry 35, 7482-7491). In the present study, we locate three C6PS binding sites to different domains of factor X(a) using a combination of activity, circular dichroism, fluorescence, and equilibrium dialysis measurements on proteolytic and biosynthetic fragments of factor X(a). Our results demonstrate that the structural responses of human and bovine factor X(a) to C6PS binding are somewhat different. Despite this difference, data obtained with fragments from both human and bovine factor X(a) are consistent with a common hypothesis for the location of C6PS binding sites to different structural domains. First, the gamma-carboxyglutamic acid (Gla) domain binds C6PS only in the absence of Ca(2+) (k(d) approximately 1 mm), although this PS site does not influence the functional response of factor X(a). Second, a Ca(2+)-dependent binding site is in the epidermal growth factor domains (EGF(NC)) that are linked by Ca(2+) and C6PS binding to the Gla domain. This site appears to be the lipid regulatory site of factor X(a). Third, a Ca(2+)-requiring site seems to be in the EGF(C)-catalytic domain. This site appears not to be a lipid regulatory site but rather to share residues with the substrate recognition site. Finally, the full functional response to C6PS requires linkage of the Gla, EGF(NC), and catalytic domains in the presence of Ca(2+), meaning that PS regulation of factor X(a) involves linkage between widely separated parts of the protein.

Highlights

The substantial effects of soluble phosphatidylserine (C6PS1) on the kinetics of prothrombin activation by factor Xa [1] and on the structure of factor Xa, as documented here, indicate that phosphatidylserine (PS) may act as an allosteric regulator of prothrombin activation
In the structure of the analogous serine protease, factor IXa, the EGFN module is bent at the inner-EGFC hinge region to right angles with the EGFC, which is tucked along the catalytic module [8]
The Gla domain is critical for membrane binding and may modulate the structure of the epidermal growth factor (EGF) modules, little is known about the structure of Gla in whole factor Xa

Summary

Introduction

The substantial effects of soluble phosphatidylserine (C6PS1) on the kinetics of prothrombin activation by factor Xa [1] and on the structure of factor Xa, as documented here, indicate that phosphatidylserine (PS) may act as an allosteric regulator of prothrombin activation. PS located on the cytoplasmic face of resting platelet plasma membranes is exposed on the surface of activated platelet vesicles [2, 3] The implication of this PS exposure and of the effect of PS on factor Xa and on its ability to catalyze activation of prothrombin is that PS may act as a second messenger in regulating thrombin formation. Because of the crucial role of thrombin in hemostasis, the exposure of PS may be a crucial regulatory step in blood coagulation In the structure of the analogous serine protease, factor IXa, the EGFN module is bent at the inner-EGFC hinge region to right angles with the EGFC, which is tucked along the catalytic module [8]. SpPCa, Spectrozyme PCa; Gla, N-terminus ␥-carboxyglutamic acid-rich region; EGFN, epidermal growth factor nearest to the N terminus; EGFC, epidermal growth factor nearest the C terminus; Gla-EGFN, Gla domain linked to the epidermal growth factor EGFN; Gla-EGFNC, Gla domain linked to both epidermal growth factors EGFN and EGFC; E2FXa, factor Xa construct lacking both the Gla and the EGFN domains; DEGR, [5-(dimethylamino)-1-napthalenesulfonyl]glutamylycylarginyl chloromethyl ketone; GDFXa, factor Xa construct missing the Gla domain; Y99T, GDFXa mutant in which Tyr-99 is replaced with Thr; CMC, critical micelle concentration

Objectives

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Conclusion