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Titolo:
INTERACTION OF FACTOR IXA WITH FACTOR VIIIA - EFFECTS OF PROTEASE DOMAIN CA2-SITE, PROTEOLYSIS IN THE AUTOLYSIS LOOP, PHOSPHOLIPID, AND FACTOR-X( BINDING)
Autore:
MATHUR A; ZHONG DG; SABHARWAL AK; SMITH KJ; BAJAJ SP;
Indirizzi:
ST LOUIS UNIV,CTR HLTH SCI,DIV BONE MARROW TRANSPLANT ONCOL & HEMATOLST LOUIS MO 63110 ST LOUIS UNIV,SCH MED,DEPT MED ST LOUIS MO 63104 ST LOUIS UNIV,SCH MED,DEPT PATHOL ST LOUIS MO 63104 ST LOUIS UNIV,SCH MED,DEPT BIOCHEM ST LOUIS MO 63110 EMORY UNIV,SCH MED,DEPT HEMATOL ATLANTA GA 30322
Titolo Testata:
The Journal of biological chemistry
fascicolo: 37, volume: 272, anno: 1997,
pagine: 23418 - 23426
SICI:
0021-9258(1997)272:37<23418:IOFIWF>2.0.ZU;2-L
Fonte:
ISI
Lingua:
ENG
Soggetto:
COAGULATION FACTOR-IX; GAMMA-CARBOXYGLUTAMIC ACID; AFFINITY CA2+-BINDING SITE; BLOOD-COAGULATION; TISSUE FACTOR; ACTIVE-SITE; CALCIUM-BINDING; PROTHROMBINASE COMPLEX; HEMOPHILIA-B; HEAVY-CHAIN;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
58
Recensione:
Indirizzi per estratti:
Citazione:
A. Mathur et al., "INTERACTION OF FACTOR IXA WITH FACTOR VIIIA - EFFECTS OF PROTEASE DOMAIN CA2-SITE, PROTEOLYSIS IN THE AUTOLYSIS LOOP, PHOSPHOLIPID, AND FACTOR-X( BINDING)", The Journal of biological chemistry, 272(37), 1997, pp. 23418-23426

Abstract

We previously identified a high affinity Ca2+ binding site in the protease domain of factor IXa involving Glu(235) (Glu(70) in chymotrypsinogen numbering; hereafter, the numbers in brackets refer to the chymotrypsin equivalents) and Glu(245)[80] as putative ligands. To delineatethe 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 M-r 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 IXa(WT) was similar to 93%, of IXa(E235K) wassimilar 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 proteolysis at Arg(318)-Ser(319)[150-151] in the protease domain autolysisloop 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 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 each activated mutant with factor Villa. 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 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 ofactive 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 the 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 (14.3 nM). We conclude that (a) PL increases the apparent affinity of factor IXa for factor VIIIa similar to 2,000-fold, and the substrate, factor X, increases this affinity similar to 10-15-fold; (b) the protease 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) proteolysis in the autolysis loop leads to a loss of catalytic efficiency with retention of S1 binding site and a further similar to 8-fold reduction in affinity of factor IXa for factor VIIIa.

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Documento generato il 23/09/20 alle ore 09:38:55