Circulating and binding characteristics of wild-type factor IX and certain Gla domain mutants in vivo

Abstract

Residue K5 in factor IX ␥-carboxyglutamic acid (Gla) domain participates in binding endothelial cells/collagen IV. We injected recombinant factor IX containing mutations at residue 5 (K5A, K5R) into factor IX-deficient mice and compared their behavior with that of wild-type factor IX. The plasma concentration of factor IX that binds to endothelial cells/collagen IV (recombinant wild type and K5R) was consistently lower than that of the one that does not bind (K5A). Mice treated with wild type or K5R had 79% of the injected factor IX in the liver after 2 minutes, whereas 17% remained in circulation. In mice injected with K5A, 59% of the injected factor IX was found in liver and 31% was found in plasma. When we blocked the liver circulation before factor IX injection, 74% of K5A and 64% of K5R remained in the blood. When we treated the mouse with EDTA after injecting exogenous factor IX, the blood levels of factor IX that bind to endothelial cells/collagen IV increased, presumably because of release from endothelial cell/collagen IV binding sites. In contrast, the levels of the mutants that do not bind were unaffected by EDTA. In immunohistochemical studies, factor IX appears on the endothelial surfaces of mouse arteries after factor IX injection and of human arteries from surgical specimens. Thus, we have demonstrated that factor IX binds in vivo to endothelial cell-collagen IV surfaces. Our results suggest that factor IX Gla-domain mediated binding to endothelial cells/ collagen IV plays a role in controlling factor IX concentration in the blood. IntroductionFactor IX is the zymogen of a serine protease involved in blood coagulation. Activated factor IX (factor IXa) contains 4 identifiable structural domains. Starting from the N-terminus, these are the ␥-carboxyglutamic acid (Gla) domain, the 2 epidermal growth factor-like domains, and the proteolytic domain. Factors IX and IXa bind to a specific site on the surface of cultured vascular endothelial cells. The binding is calcium-dependent, saturable, and reversible in vitro. [1][2][3] Our previous studies on cultured endothelial cells demonstrate that the binding regions in factor IX are in the Gla domain. 4 Furthermore, mutations within the Gla domain, including lysine 5 to alanine (K5A) and valine 10 to lysine (V10K), produced factor IX that did not have measurable affinity for endothelial cells but retained normal clotting activity. In contrast, changing lysine 5 to arginine (K5R) increased factor IX affinity 3-fold for cultured bovine vascular endothelial cells. 5 Cheung et al 6 demonstrated that collagen IV was a strong candidate for the factor IX endothelial cell-associated binding site. Further, results from atomic force scanning microscopy indicate that factor IX molecules bind specifically to 2 sites on collagen IV, 98 and 50 nm from its C-terminus. 7 Although the binding of factor IX to endothelial cells/collagen IV is well documented, little is known about the physiologic significance of binding. The hemophilia B mouse produced using gene-targeting te...

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