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Volume 272, Number 37, Issue of September 12, 1997 pp. 23418-23426
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Interaction of Factor IXa with Factor VIIIa
EFFECTS OF PROTEASE DOMAIN Ca²⁺ BINDING SITE, PROTEOLYSIS IN THE AUTOLYSIS LOOP, PHOSPHOLIPID, AND FACTOR X

(Received for publication, December 18, 1996, and in revised form, June 6, 1997)

From the Departments of Medicine, Pathology, and Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63104, and the ^§ Department of Hematology, Emory University School of Medicine, Atlanta, Georgia 30322

We previously identified a high affinity Ca²⁺ binding site in the protease domain of factor IXa involving Glu²³⁵ (Glu⁷⁰ in chymotrypsinogen numbering; hereafter, the numbers in brackets refer to the chymotrypsin equivalents) and Glu²⁴⁵[80] as putative ligands. To delineate the function of this Ca²⁺ binding site, we expressed IX_{wild type} (IX_WT), IX_E235K, and IX_E245V in 293 kidney cells and compared their properties with those of factor IX isolated from normal plasma (IX_NP); each protein had the same M_r and -carboxyglutamic acid content. Activation of each factor IX protein by factor VIIa·Ca²⁺·tissue factor was normal as analyzed by sodium dodecyl sulfate-gel electrophoresis. The coagulant activity of IXa_WT was ~93%, of IXa_E235K was ~27%, and of IXa_E245V was ~4% compared with that of IXa_NP. In contrast, activation by factor XIa·Ca²⁺ led to proteolysis at Arg³¹⁸-Ser³¹⁹[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 absence and presence (in parentheses) of Ca²⁺ was: IXa_NP or IXa_WT 230 µM (78 µM), IXa_E235K 150 µM (145 µM), IXa_E245V 225 µM (240 µM), and autolysis loop cleaved IXa_E245V 330 µM (350 µM). Next, we evaluated the apparent K_d (K_d,app) of interaction of each activated mutant with factor VIIIa. We first investigated the EC₅₀ of interaction of IXa_NP as well as of IXa_WT with factor VIIIa in the 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 half-maximal rate of factor Xa generation. EC₅₀ values for IXa_NP and IXa_WT were similar and are as follows: PL-minus/X-minus (extrapolated), 2.8 µM; PL-minus/X-saturating, 0.25 µM; PLplus/X-minus, 1.6 nM; and PL-plus/X-saturating, 0.09 nM. Further, K_d,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,app values in the absence and presence (in parentheses) of PL were: IXa_NP or IXa_WT, 0.19 µM (0.07 nM); IXa_E235K, 0.68 µM (0.26 nM); IXa_E245V, 2.5 µM (1.35 nM); and autolysis loop-cleaved IXa_E245V, 15.6 µM (14.3 nM). We conclude that (a) PL increases the apparent affinity of factor IXa for factor VIIIa ~2,000-fold, and the substrate, factor X, increases this affinity ~10-15-fold; (b) the protease domain Ca²⁺ binding site increases this affinity ~15-fold, and lysine at position 235 only partly substitutes for Ca²⁺; (c) Ca²⁺ binding to the protease domain increases the S1 reactivity ~3-fold and prevents proteolysis in the autolysis loop; and (d) proteolysis in the autolysis loop leads to a loss of catalytic efficiency with retention of S1 binding site and a further ~8-fold reduction in affinity of factor IXa for factor VIIIa.

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