Both factors Xa and IXa are vitamin-K-dependent serine proteases that consist of disulfide-linked heavy and light chains. The heavy chain contains the serine protease active site, while the light chain contains the GLA (gamma-carboxyglutamic acid) domain, EGF-I (epidermal growth factor-like region) and EGF-II domains. Effect of PS on FXa: We have extensively studied the interaction of factor Xa with 1, 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS). C6PS is a soluble analogue of phosphatidylserine, which is present in platelet membranes; it serves as a model for the effect of membrane-bound PS on factor Xa activity and structure. We located three C6PS binding sites to different domains of factor Xa using a combination of activity, circular dichroism, fluorescence, and equilibrium dialysis measurements, showing that :

  • the Gla domain binds C6PS only in the absence of calcium (kd ∼ 1 mM);

  • a calcium-dependent, regulatory, PS-binding site exists in the EGFN domain when linked by calcium to the Gla domain; and

  • a second calcium-requiring site exists in the EGFC-catalytic domains and shares residues with the substrate recognition site.

We have shown that full functional response to C6PS requires linkage of the Gla, EGFNC, and catalytic domains in the presence of calcium. Recently we have observed that the proteolytic activity of des EGFN factor Xa is not affected in the presence of C6PS, locating more precisely a significant component of the PS-triggered regulatory site to the EGFN domain. Efect of PS on FIXa: We have previously reported that C6PS induces a calcium-dependent conformational change in factor IXa that regulates the amidolytic and proteolytic activities. We have also shown that factor IXa binds 2 molecules of C6PS. Here we have examined the role of the GLA domain in this C6PS regulation of factor IXa using a GLA-domainless variant of factor IXa (GD-IXa). In the absence of the GLA domain, binding to C6PS has no effect on the rate of proteolytic or amidolytic activity of GD-IXa toward both factor X and synthetic substrates. The binding of C6PS to GD-IXa studied using intrinsic tryptophan fluorescence (Kd = ∼50μM) was different from that for native factor IXa (Kd = ∼2μM). The critical micelle concentration (CMC) of C6PS under the conditions of these experiments (∼300μM) was much greater than the C6PS concentrations used in our experiments. We conclude that:

  1. C6PS does bind to GD-IXa, but at a reduced affinity compared to factor IXa;

  2. C6PS does not regulate the amidolytic or proteolytic activities of factor IXa in the absence of GLA domain.

This could be due to two C6PS sites existing in FIXa, with the regulatory site requiring the Gla domain (as observed for FXa), or to a requirement for the Gla domain for the regulatory activity of a single site. Future studies looking at the stoichiometry of binding of C6PS with GD-IXa will help distinguish between these possibilities.

Supported by Supported by NHLBI (HL 072827).

Author notes

Disclosure: No relevant conflicts of interest to declare.