INTERACTION OF FACTOR IXA WITH FACTOR VIIIA - EFFECTS OF PROTEASE DOMAIN CA2-SITE, PROTEOLYSIS IN THE AUTOLYSIS LOOP, PHOSPHOLIPID, AND FACTOR-X( BINDING)

We previously identified a high affinity Ca2+ binding site in the prot ease domain of factor IXa involving Glu(235) (Glu(70) in chymotrypsino gen numbering; hereafter, the numbers in brackets refer to the chymotr ypsin equivalents) and Glu(245)[80] as putative ligands. To delineate the function of this Ca2+ binding site, we expressed IXwild type (IXWT ), IXE235K, and IXE245V in 293 kidney cells and compared their propert ies with those of factor IX isolated from normal plasma (IXNP); each p rotein had the same M-r and gamma-carboxyglutamic acid content. Activa tion of each factor IX protein by factor VIIa . Ca2+. tissue factor wa s normal as analyzed by sodium dodecyl sulfate-gel electrophoresis. Th e coagulant activity of IXa(WT) was similar to 93%, of IXa(E235K) was similar to 27%, and of IXa(E245V) was similar to 4% compared with that of IXa(NP). In contrast, activation by factor XIa . Ca2+ led to prote olysis at Arg(318)-Ser(319)[150-151] in the protease domain autolysis loop of IXa(E245V) with a concomitant loss of coagulant activity; this proteolysis was moderate in IXa(E235K) and minimal in IXa(WT) or IXa( NP). Interaction of each activated mutant with an active site probe, p -aminobenzamidine, was also examined; the K-d of interaction in the ab sence and presence (in parentheses) of Ca2+ was: IXa(NP) or IXa(WT) 23 0 mu M (78 mu M), IXa(E235K) 150 mu M (145 mu M), IXa(E245V) 225 mu M (240 mu M), and autolysis loop cleaved IXa(E245V) 330 mu M (350 mu M). Next, we evaluated the apparent K-d (K-d,K-app) of interaction of eac h activated mutant with factor Villa. We first investigated the EC50 o f interaction of IXa(NP) as well as of IXa(WT) with factor VIIIa in th e presence and absence of phospholipid (PL) and varying concentrations of factor X. At each factor X concentration and constant factor VIIIa , EC50 was the free IXa(NP) or IXa(WT) concentration that yielded a ha lf-maximal rate of factor Xa generation. EC50 values for IXa(NP) and I Xa(WT) were similar and are as follows: PL-minus/X-minus (extrapolated ), 2.8 mu M; PL-minus/X-saturating, 0.25 mu M; PL-plus/X-minus, 1.6 nM ; and PL-plus/X-saturating, 0.09 nM. Further, K-d,K-app of binding of active site-blocked factor IXa to factor VIIIa was calculated from its ability to inhibit IXa(WT) in the Tenase assay. K-d,K-app values in t he absence and presence (in parentheses) of PL were: IXa(NP) or IXa(WT ), 0.19 mu M (0.07 nM); IXa(E235K), 0.68 mu M (0.26 nM); IXa(E245V), 2 .5 mu M (1.35 nM); and autolysis loop-cleaved IXa(E245V), 15.6 mu M (1 4.3 nM). We conclude that (a) PL increases the apparent affinity of fa ctor IXa for factor VIIIa similar to 2,000-fold, and the substrate, fa ctor X, increases this affinity similar to 10-15-fold; (b) the proteas e domain Ca2+ binding site increases this affinity similar to 15-fold, and lysine at position 235 only partly substitutes for Ca2+; (c) Ca2 binding to the protease domain increases the S1 reactivity similar to 3-fold and prevents proteolysis in the autolysis loop; and (d) proteo lysis in the autolysis loop leads to a loss of catalytic efficiency wi th retention of S1 binding site and a further similar to 8-fold reduct ion in affinity of factor IXa for factor VIIIa.