The binding of 125I-factor Xa to human aortic smooth muscle cell (SMC) monolayers was studied. At 4 degreesC, 125I-factor Xa bound to a single class of binding sites with a dissociation constant value of 3.6+/-0.7 nM and a binding site density of 11,720+/-1,240 sites/cell (n = 9). 125I-factor Xa binding was not affected by factor X, thrombin, or by DX9065, a direct inhibitor of factor Xa, but was inhibited by factor Xa (IC50 = 5.4+/-0.2 nM; n = 9) and by antibodies specific for the effector cell protease receptor 1 (EPR-1), a well-known receptor of factor Xa on various cell types. A factor X peptide duplicating the inter-EGF sequence Leu83-Leu88-(Gly) blocked the binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 = 110+/-21 nM). Factor Xa increased phosphoinositide turnover in SMCs and when added to SMCs in culture was a potent mitogen. These effects were inhibited by DX9065 and by antibodies directed against EPR-1 and PDGF. Increased expression of EPR-1 was identified immunohistochemically on SMCs growing in culture and in SMCs from the rabbit carotid artery after vascular injury. When applied locally to air-injured rabbit carotid arteries, antibodies directed against EPR-1 (100 mug/ artery) strongly reduced myointimal proliferation 14 d after vascular injury (65-71% inhibition, P < 0.01). DX9065 (10 mg/kg, subcutaneous) inhibited myointimal proliferation significantly (43% inhibition, P < 0.05). These findings indicate that SMCs express functional high affinity receptors for factor Xa related to EPR-1, which may be of importance in the regulation of homeostasis of the vascular wall and after vascular injury.

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The binding of [125I]-factor Xa to human umbilical vein endothelial cell (HUVEC) monolayers was studied. At 7 degrees C, [125I]-factor Xa bound to a single class of binding sites with a dissociation constant value of 6.6 +/- 0.8 nM and a binding site density of 57,460 +/- 5,200 sites/cell (n = 3). Association and dissociation kinetics were of a pseudo-first order and gave association and dissociation rate constant values of 0.15 x 10(6) M-1 s-1 and 4.0 x 10(-4) s-1, respectively. [125I]-factor Xa binding was inhibited by factor Xa but was not affected by factor X, thrombin or monoclonal antibodies against factor V, antithrombin-III or tissue factor pathway inhibitor (TFPI) but was inhibited by an antibody specific for the effector cell protease receptor-1 (EPR-1), a well-known receptor of factor Xa on various cell types. [125I]-factor Xa binding to HUVEC was not affected by various inhibitors of factor Xa such as DX 9065, pentasaccharide-antithrombin-III or TFPI. Factor Xa increased intracellular free calcium levels and phosphoinositide turnover in endothelial cells and, when added to HUVEC in culture, factor Xa was a potent mitogen, stimulating an increase in cell number at a 0.3 to 100 nM concentration. HUVEC-bound factor Xa promoted prothrombin activation in the presence of factor Va only. This effect was inhibited by both indirect and direct inhibitors of factor Xa. These findings indicate that HUVEC express functional high affinity receptors for factor Xa, related to EPR-1, which may be of importance in the regulation of coagulation and homeostasis of the vascular wall.

The binding of [125I]-factor Xa to human umbilical vein endothelial cell (HUVEC) monolayers was studied. At 7 degrees C, [125I]-factor Xa bound to a single class of binding sites with a dissociation constant value of 6.6 +/- 0.8 nM and a binding site density of 57,460 +/- 5,200 sites/cell (n = 3). Association and dissociation kinetics were of a pseudo-first order and gave association and dissociation rate constant values of 0.15 x 10(6) M-1 s-1 and 4.0 x 10(-4) s-1, respectively. [125I]-factor Xa binding was inhibited by factor Xa but was not affected by factor X, thrombin or monoclonal antibodies against factor V, antithrombin-III or tissue factor pathway inhibitor (TFPI) but was inhibited by an antibody specific for the effector cell protease receptor-1 (EPR-1), a well-known receptor of factor Xa on various cell types. [125I]-factor Xa binding to HUVEC was not affected by various inhibitors of factor Xa such as DX 9065, pentasaccharide-antithrombin-III or TFPI. Factor Xa increased intracellular free calcium levels and phosphoinositide turnover in endothelial cells and, when added to HUVEC in culture, factor Xa was a potent mitogen, stimulating an increase in cell number at a 0.3 to 100 nM concentration. HUVEC-bound factor Xa promoted prothrombin activation in the presence of factor Va only. This effect was inhibited by both indirect and direct inhibitors of factor Xa. These findings indicate that HUVEC express functional high affinity receptors for factor Xa, related to EPR-1, which may be of importance in the regulation of coagulation and homeostasis of the vascular wall.