Interaction of factor IXa with factor VIIIa. Effects of protease domain Ca2+ binding site, proteolysis in the autolysis loop, phospholipid, and factor X.
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 IXwild type (IXWT), IXE235K, and IXE245V in 293 kidney cells and compared their properties with those of factor IX isolated from normal plasma (IXNP); each protein had the same Mr and gamma-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 IXaWT was approximately 93%, of IXaE235K was approximately 27%, and of IXaE245V was approximately 4% compared with that of IXaNP. In contrast, activation by factor XIa.Ca2+ led to proteolysis at Arg318-Ser319[150-151] in the protease domain autolysis loop of IXaE245V with a concomitant loss of coagulant activity; this proteolysis was moderate in IXaE235K and minimal in IXaWT or IXaNP. Interaction of each activated mutant with an active site probe, p-aminobenzamidine, was also examined; the Kd of interaction in the absence and presence (in parentheses) of Ca2+ was: IXaNP or IXaWT 230 microM (78 microM), IXaE235K 150 microM (145 microM), IXaE245V 225 microM (240 microM), and autolysis loop cleaved IXaE245V 330 microM (350 microM). Next, we evaluated the apparent Kd (Kd,app) of interaction of each activated mutant with factor VIIIa. We first investigated the EC50 of interaction of IXaNP as well as of IXaWT 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 IXaNP or IXaWT concentration that yielded a half-maximal rate of factor Xa generation. EC50 values for IXaNP and IXaWT were similar and are as follows: PL-minus/X-minus (extrapolated), 2.8 microM; PL-minus/X-saturating, 0.25 microM; PLplus/X-minus, 1.6 nM; and PL-plus/X-saturating, 0.09 nM. Further, Kd,app of binding of active site-blocked factor IXa to factor VIIIa was calculated from its ability to inhibit IXaWT in the Tenase assay. Kd,app values in the absence and presence (in parentheses) of PL were: IXaNP or IXaWT, 0. 19 microM (0.07 nM); IXaE235K, 0.68 microM (0.26 nM); IXaE245V, 2.5 microM (1.35 nM); and autolysis loop-cleaved IXaE245V, 15.6 microM (14.3 nM). We conclude that (a) PL increases the apparent affinity of factor IXa for factor VIIIa approximately 2,000-fold, and the substrate, factor X, increases this affinity approximately 10-15-fold; (b) the protease domain Ca2+ binding site increases this affinity approximately 15-fold, and lysine at position 235 only partly substitutes for Ca2+; (c) Ca2+ binding to the protease domain increases the S1 reactivity approximately 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 approximately 8-fold reduction in affinity of factor IXa for factor VIIIa.
Mathur
A
A
Department of Medicine, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Zhong
D
D
Sabharwal
A K
AK
Smith
K J
KJ
Bajaj
S P
SP
D015055:1-Carboxyglutamic Acid / Q000032:analysis; D001550:Benzamidines / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation; D015949:Factor IXa / Q000235:genetics / Q000378:metabolism*; D015944:Factor VIIIa / Q000378:metabolism*; D015942:Factor VIIa / Q000494:pharmacology; D005170:Factor X / Q000494:pharmacology; D015945:Factor XIa / Q000494:pharmacology; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D010446:Peptide Fragments / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017421:Sequence Analysis
D001550:Benzamidines; D010446:Peptide Fragments; D010743:Phospholipids; D015055:1-Carboxyglutamic Acid; C006500:4-aminobenzamidine; D015944:Factor VIIIa; D005170:Factor X; D015942:Factor VIIa; D015949:Factor IXa; D015945:Factor XIa; D002118:Calcium