Binding of factors IX and IXa to cultured vascular endothelial cells.

Abstract

Factor IX and its activated form IXa have been found to bind to confluent cultured bovine aortic and human umbilical vein endothelial cells. Binding of bovine factors IX and IXa to the bovine endothelial cells was saturable and specific and reached a plateau in 75 min at 40C and 30 min at 370C. Binding was half-maximal at a total factor IX or IXa concentration of 2.3 ± 0.2 nM. At 4TC, a maximum of 42 (1) and plasminogen activator (2, 3) and by the action of a specific cellmembrane component termed "thrombomodulin," which binds thrombin and alters its substrate specificity (4-6). Potential clotpromoting activities of endothelial cells have received little attention. We hypothesized that the initial reactions of thrombosis occur on the endothelial cell surface and asked how the clot-promoting activity is localized. One possible mechanism would be through the binding of factor IX to endothelial cells, if bound factor IXa (the activated form of factor IX) were active in promoting activation of later zymogens which promote thrombin formation. This paper reports reversible, calcium-dependent binding of both factor IX, the zymogen, and the serine protease, factor IXa, to cultured endothelial cells. Bound factor IXa retains its coagulant activity and appears to be more active bound than in solution.MATERIALS AND METHODS Preparation and Radiolabeling of Coagulation Factors. Human factor IX was isolated from human plasma by the method of W. Kisiel (7). Tritiated bovine factor IX (bovine [3H]IX) used in initial studies was a gift of Y. Nemerson (Mt. Sinai School of Medicine), and bovine factor IX used in later studies was aThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. (9). Cleavage of factor IX reached a plateau when 43-52% of the total initial radioactivity remained in solution with 5% CC13COOH. Purified proteins were analyzed by NaDodSO4/ polyacrylamide gel electrophoresis (14). The reduced and unreduced forms of bovine and human factor IX were homogeneous on 10% NaDodSO4/polyacrylamide gel electrophoresis (see electrophoresis of reduced bovine [3H]IX in Fig. 1A). The major peak migrated identically with the unlabeled protein, and a variable earlier peak containing from 0.5% to 2% of the total counts was thought to represent aggregated factor IX produced during radiolabeling. NaDodSO4/polyacrylamide gel electrophoresis of bovine [3H]IXa without reduction shows two major peaks and two minor peaks that comprise <2% of the total radioactivity (Fig. 1B). The minor peaks represent aggregated protein (higher molecular weight) and residual unactivated factor IX. The earlier major peak migrates identically with unlabeled factor IXa, and the later peak is presumably the activation peptide. Factor IXa used in these experiments consists of two disulfide-linked polypeptide chains from which the activation peptide has been cleaved [factor IXao (11)].Human fact...

Endothelial cells stimulated with tumor necrosis factor-alpha express varying amounts of tissue factor resulting in inhomogenous fibrin deposition in a native blood flow system. Effects of thrombin inhibitors.

“…In addition, individual cells could have been in different phases ofthe cell cycle, which might affect tissue factor expression as suggested by Andoh et al (44). Alternatively, independent of tissue factor expression, the endothelial cells might show variable procoagulant activity because they differ in the number or in the activity of their binding sites for the coagulation Factors IX and X (45)(46)(47).…”

The Factor IX γ-Carboxyglutamic Acid (Gla) Domain Is Involved in Interactions between Factor IX and Factor XIa

“…Amino acids 3-11 in the FIX Gla domain are critical for high affinity binding of the protein to aortic endothelial cells (57). It appears that this interaction is caused by binding of FIX to collagen IV and not phospholipid in the cell membrane (58).…”

An endothelial cell-dependent pathway of coagulation.

“…Bovine aortic endothelial cells (BAEC) were obtained from 3-month-old calves and grown in culture as described (11). They were characterized by the presence of von Willebrand factor antigen (35) and angiotensin-converting enzyme activity (36 For experiments using platelets, blood was drawn from healthy volunteers who had not taken any medication for 2 weeks by the protocol described by Van Rijn et al (37).…”

“…Recently, participation of endothelial cells in procoagulant reactions has received more attention. Aortic endothelium, both cultured and native, can bind factors IX and IXa (11)(12)(13). Cell-bound factor IX can be activated by both intrinsic and extrinsic pathways of coagulation and cell-bound factor IXa in the presence of factor VIII can activate factor X (14).…”