It is believed that heparin accelerates factor Xa (FXa) inactivation by antithrombin (AT) by conformationally activating the inhibitor rather than by bridging AT and FXa in a ternary complex (template effect). This is derived from kinetic studies done in the absence of Ca²⁺ or in the presence of EDTA. To test the possibility that the anionic Gla domain of FXa, when not neutralized by Ca²⁺ ions, prevents heparin binding to FXa, the heparin and pentasaccharide dependence of FXa inactivation by AT in both the absence (100 μm EDTA) and presence of Ca²⁺ (2.5 mm) was studied using wild-type FXa and a FXa derivative that lacks the Gla domain (GDFXa). AT inactivated both FXa derivatives similarly in both the absence and presence of Ca²⁺ (k ₂ = 1.7–2.5 × 10³ m ⁻¹ s⁻¹). The active AT-binding pentasaccharide also accelerated the inactivation rates of both derivatives similarly in both the absence and presence of Ca²⁺ (k ₂ = 5.7–8.0 × 10⁵ m ⁻¹ s⁻¹). However, in the presence of an optimum concentration of heparin (∼50 nm) the inactivation rate constant of FXa in the presence of Ca²⁺ (k ₂ = 4.4 × 10⁷ m ⁻¹ s⁻¹) was 13-fold higher than the rate constant in the absence of Ca²⁺ (k ₂ = 3.5 × 10⁶ m ⁻¹ s⁻¹). Heparin acceleration of GDFXa inactivation by AT was rapid and insensitive to the presence or absence of Ca²⁺ (k ₂= 5.1–5.9 × 10⁷ m ⁻¹s⁻¹). The additional cofactor effect of heparin with all FXa derivatives was a bell-shaped curve, which disappeared if the ionic strength of the reaction was increased to ∼0.4. These results suggest that although the major effect of heparin in acceleration of FXa inactivation is through a heparin-induced conformational change in the reactive site loop of AT, the template effect of heparin, nevertheless, contributes significantly to rapid FXa inactivation at physiological Ca²⁺.

Significance: We demonstrate that deletion of thrombomodulin (TM) from endothelial cells confers inflammatory phenotype to TM-deficient ( TM ^−/− ) cells. This is based on the loss of integrity of VE-cadherin at cellular junctions, disrupted basal barrier permeability, and increased expression of cell adhesion molecules in TM ^−/− cells. Proinflammatory phenotype is rescued by reexpression of TM in TM ^−/− cells; however, soluble TM lacked this effect. Interestingly, instead of storage in Weibel–Palade bodies, VWF was secreted in TM ^−/− cells, thereby recruiting platelets to cell surfaces under flow conditions. Increased VWF and inflammatory foci were also observed in lungs of tamoxifen-treated ERcre-TM ^f/f mice. These results suggest cell-bound TM maintains a quiescent phenotype in vascular endothelial cells by regulating expression of procoagulant and proinflammatory molecules.

Activated protein C (APC) requires both Ca²⁺ and Na⁺ for its optimal catalytic function. In contrast to the Ca²⁺-binding sites, the Na⁺-binding site(s) of APC has not been identified. Based on a recent study with thrombin, the 221–225 loop is predicted to be a potential Na⁺-binding site in APC. The sequence of this loop is not conserved in trypsin. We engineered a Gla domainless form of protein C (GDPC) in which the 221–225 loop was replaced with the corresponding loop of trypsin. We found that activated GDPC (aGDPC) required Na⁺ (or other alkali cations) for its amidolytic activity with dissociation constant (K _{d (app)}) = 44.1 ± 8.6 mm. In the presence of Ca²⁺, however, the requirement for Na⁺ by aGDPC was eliminated, and Na⁺ stimulated the cleavage rate 5–6-fold withK _{d (app)} = 2.3 ± 0.3 mm. Both cations were required for efficient factor Va inactivation by aGDPC. In the presence of Ca²⁺, the catalytic function of the mutant was independent of Na⁺. Unlike aGDPC, the mutant did not discriminate among monovalent cations. We conclude that the 221–225 loop is a Na⁺-binding site in APC and that an allosteric link between the Na⁺ and Ca²⁺ binding loops modulates the structure and function of this anticoagulant enzyme.