Regulation of factor Xa in vitro in human and mouse plasma and in vivo in mouse. Role of the endothelium and plasma proteinase inhibitors.

Abstract

A B S T R A C T The regulation of human Factor Xa was studied in vitro in human and mouse plasma, and in vivo in mouse. In human plasma, '251-Factor Xa bound to a1-proteinase inhibitor, antithrombin III, and a2-macroglobulin in a ratio of 4.9:1.9:1 as determined by gel electrophoresis and by adsorption to IgG-(antiproteinase inhibitor)-Sepharose beads. The distribution of Factor Xa in mouse plasma was similar. The clearance of Factor Xa in mice was rapid (50% clearance in 3 min) and biphasic. a1-Proteinase inhibitor-trypsin, even at a 2,000-fold molar excess, failed to inhibit the clearance of Factor Xa, while a2-macroglobulin-trypsin inhibited only the later phase of clearance. The plasma clearance of diisopropylphosphoryl-Factor Xa was more rapid than native Factor X. (50% clearance in 2.5 min), and the clearance was blocked by diisopropylphosphoryl-thrombin. Electrophoresis experiments confirmed that by 2 min after injection into the murine circulation, 90% of the bound Factor Xa was on a2-macroglobulin, in marked contrast to the in vitro results. Organ distribution studies at 3 and 15 min with 125I_ Factor X. demonstrated that the majority of radioactivity was in the liver, with significant radioactivity also present in lung and kidney. Autopsies performed 30 s after injection of 125I-Factor Xa also demonstrated significant binding to the aorta and vena cava. These studies indicate that Factor Xa binds to specific thrombin-binding sites on endothelial cells, and that this binding alters its proteinase inhibitor specificity. Factor Xa binds to a2-macroglobulin in vivo, whereas the predominant in vitro inhibitor of Factor X. is a,-proteinase inhibitor.

Regulation of factor IXa in vitro in human and mouse plasma and in vivo in the mouse. Role of the endothelium and the plasma proteinase inhibitors.

“…Human prothrombin and Factor X were prepared by the method of Miletich et al (10). Human a-thrombin (specific activity 2,700 U/mg), DIP-thrombin, Factor Xa, and DIP-Factor Xa were prepared as previously described (8,11,12). Diisopropylfluorophosphate (20 mM) failed to inhibit Factor IXa, even after reaction for 6 or 24 h; this is consistent with previous studies (6).…”

“…However, thrombin bound to thrombomodulin activates protein C at least 100-fold faster than does free thrombin (34). From the data obtained to date (2,3,8,33), there is no evidence that thrombomodulin is the binding site for Factor Xa or Factor IXa, and it seems from the present work that there are at least two classes of Factor IXa binding sites. From all these observations it seems likely that there are several classes of endothelial binding sites that bind the vitamin K-dependent proteinases.…”

“…SDS-polyacrylamide gel electrophoresis of plasma samples was performed on 5% slab gels according to the method of Weber and Osborn (29). 25 (8). The beads were characterized by incubating 50 ,ul of the resultant IgG-Sepharose preparations with 1-500 jg '25I-labeled human a2M, a1PI, and ATIII, and mouse ATIII, or with the corresponding '251-proteinase inhibitor-trypsin complex in 500 Ml of 50 mM Tris-HCI, 0.15 M NaCl, 60 mg/ml bovine serum albumin, pH 7.40, at 37°C for 2 h with constant mixing in a series of saturation curves.…”

“…Plasma elimination studies of '25I-Factor IX and '25I-Factor IX. (0.5 to I ug) alone or in the presence of large molar excesses of unlabeled proteins were performed in CD-1 female mice, as previously described (8). To examine the distribution of Factor IX.…”

“…These results were compared with those obtained from mouse plasma, and the in vivo regulation of Factor IXa was then studied using our previously described mouse model. We have recently shown that Factor Xa binds to an endothelial cell thrombin-binding site; this binding alters the proteinase inhibitor specificity of Factor Xa such that a2M is the primary in vivo inhibitor of Factor Xa, whereas a1PI is the primary in vitro inhibitor of Factor Xa (8). In light of these results, the endothelial binding of Factor IXa and the possible role ofthis binding in the regulation ofFactor IXa were examined in vivo in our mouse model.…”