期刊
BIOCHEMISTRY
卷 44, 期 30, 页码 10298-10304出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi050638t
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资金
- NHLBI NIH HHS [R01 HL046213, HL38199, P01 HL064943, R01 HL038199, HL64943, HL76213, HL46213, HL74124, R01 HL070683, R01 HL046213-09, R01 HL070683-01, R01 HL038199-12, R01 HL076213, R01 HL076213-01, P01 HL074124, HL70683] Funding Source: Medline
We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca2+, an ion necessary for cofactor activity [Wakabayashi et al. (2004) J. Biol. Chem. 279, 12677-12684]. Mutagenesis studies showed that replacement of residue Glul 13 with Ala (E113A) yielded a factor VIII point mutant possessing increased specific activity as determined by a one-stage clotting assay. Mutagenesis at this site suggested that substitution with relatively small, nonpolar residues was well tolerated, whereas replacement with a number of polar or charged residues appeared detrimental to activity. Ala substitution resulted in the greatest enhancement, yielding an similar to 2-fold increased specific activity. Time course experiments following reaction with thrombin revealed similar rates of activation and inactivation of E113A as observed for the wild type. Results from factor Xa generation assays showed minimal differences in kinetic parameters and factor IXa affinity for E113A and wild-type factor VIIIa when run in the presence of synthetic phospholipid vesicles, whereas factor VIIIa E113A displayed an similar to 4-fold greater affinity for factor IXa compared with factor Villa wild type in reactions run on the platelet membrane surface. This latter effect may be attributed, in part, to a 2-fold increased affinity of factor Villa E113A for the platelet membrane. Considering that low levels of factors VIIIa and IXa are generated during clotting in plasma, the increased cofactor specific activity observed for E113A factor VIII may result from its enhanced affinity for factor IXa on the physiological membrane.
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