The Effect Of Physiological Metal Ions On The Procoagulant Enzyme-Cofactor Complexes Of The Extrinsic Pathway Of Coagulation
Authors: Bravo Maria Cristina , McGuinness Catherine G , Gissel Matthew , Orfeo Thomas , Mann Kenneth G. 
Published: October 2019

Abstract

Introduction
The rapid coagulation response to vascular injury is mediated by the formation of three enzyme cofactor complexes (extrinsic tenase, intrinsic tenase and prothrombinase) on membrane surfaces. A common structural feature of these proteases is their GLA domains, each of which requires the binding of divalent metal ions at multiple sites to achieve the conformation necessary for optimal membrane and cofactor binding. Both Ca2+ and Mg2+ ions have been reported to bind to GLA domain sites. However almost all studies kinetically characterizing these complexes have been done in the presence of Ca2+ (2-5 mM) as the sole metal ion, despite the relatively equivalent availability in plasma of both free Ca2+ (∼1.1 mM) and Mg2+ (∼0.6 mM) (Ca2+/Mg2+). A recent study has systematically examined the effects of various Ca2+ concentrations with and without Mg2+ on the membrane binding of activated protein C (APC) and FVIIa and enzymatic activity of APC and the extrinsic tenase complex which were enhanced in Ca2+/Mg2+ relative to Ca2+ alone (Vadivel, K., et al, 2013 JMB). In the current study we compare the effects of plasma levels of Ca2+ and Mg2+ versus Ca2+alone on the catalytic performances of the extrinsic tenase, intrinsic tenase and prothrombinase complexes individually and collectively.

Methods
All experiments were conducted in Hepes buffered saline pH 7.4 containing 0.1% PEG and either 2 mM Ca2+ or 1.1 mM Ca2+/0.6 mM Mg2+ (Ca2+/Mg2+). In closed system experiments, enzyme-cofactor complexes were assembled on phospholipid vesicles composed of a 3:1 ratio of synthetic phosphatidylcholine and phosphatidylserine (PCPS), and zymogen activation monitored via sampling into assay mixtures containing the appropriate chromogenic substrate. In open system experiments complexes were preassembled on PCPS coated capillaries, the zymogen delivered in the flowing phase and the extent of zymogen activation monitored in the effluent as described previously (Haynes, LM., et al, 2011 Biophys J). The combined interaction of the procoagulant enzyme cofactor complexes under both metal ion conditions was studied in a synthetic coagulation proteome monitoring thrombin (IIa) generation as previously described (van’t Veer, C., and Mann, KG, 1997 JBC).

Results
Extrinsic tenase The extrinsic tenase complex had an approximately two-fold higher rate of FXa generation in the presence of Ca2+/Mg2+ (1.78 ±0.05 pM/s) versus Ca2+ alone (0.88 ± 0.02 pM/s) (N=3, p<0.001). Experiments were also conducted in the absence or presence of tissue factor pathway inhibitor (TFPI); in the presence of TFPI and Ca2+/Mg2+ ions the extrinsic tenase complex was three times as catalytically active compared to TFPI and Ca2+ only (N=3, p<0.05). Direct FXa inhibition by TFPI was not significantly different between the Ca2+ and Ca2+/Mg2+ containing buffers. Intrinsic tenase In closed system experiments the intrinsic tenase complex showed impaired FXa generation in the presence of Ca2+/Mg2+ (29.3 ± 3.4 pM/s) compared to Ca2+ alone (51.7 ± 3.0 pM/s) (N=3, p<0.001). Thrombin activation of FVIII was not statistically different between both buffers, however the presence of Ca2+/Mg2+ resulted in a more rapid loss of cofactor activity over Ca2+ alone. Prothrombinase Under flow, IIa generation measured from prothrombinase was not statistically different between both buffers when measured at five different shear rates (100-1000s-1, N≥2). Plasma proteome In the synthetic coagulation proteome (N=4) the presence of Ca2+/Mg2+ led to an average 31% increase in maximum IIa levels compared to Ca2+alone and an average decrease of 1 minute to reach maximum levels.

Conclusions
In summary, the presence of plasma levels of Ca2+ and Mg2+, compared to Ca2+ alone, enhances the initiation phase of the extrinsic pathway of coagulation by enhancing the rate of FXa generation from the extrinsic tenase complex while also impairing the TFPI inhibition of the extrinsic tenase complex. These procoagulant effects are potentially abrogated by the suppressive effects of Ca2+/Mg2+ on the intrinsic tenase complex, potentially caused by the increased rate of spontaneous inactivation of FVIIIa. These results highlight the importance of complementing the assessments of individual enzyme complex systems with studies of the larger complex systems in which they function to identify the net physiologic impact of metal ions that target various components in the coagulation cascade.

Disclosures:
Mann: Haematologic Technologies, Inc: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.