The Effect Of Physiological Metal Ions On The Procoagulant Enzyme-Cofactor Complexes Of The Extrinsic Pathway Of Coagulation

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.

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).

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.

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.

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