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J. Biol. Chem., Vol. 277, Issue 8, 5725-5733, February 22, 2002

This Article Full Text Full Text (PDF) All Versions of this Article: 277/8/5725    most recent M107027200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wilkinson, F. H. Articles by Walsh, P. N. Search for Related Content PubMed PubMed Citation Articles by Wilkinson, F. H. Articles by Walsh, P. N. Social Bookmarking                      What's this?

Residues 88-109 of Factor IXa Are Important for Assembly of the Factor X Activating Complex^*

Frank H. Wilkinson^§, Fredda S. London^§, and Peter N. Walsh^§¶

From  The Sol Sherry Thrombosis Research Center, the ^§ Department of Biochemistry, and the ^¶ Department of Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Activated platelets and phospholipid vesicles promote assembly of the intrinsic factor X (FX) activating complex by presenting high-affinity binding sites for blood coagulation FIXa, FVIIIa, and FX. Previous reports suggest that the second epidermal growth factor (EGF)-like domain of FIXa mediates assembly of the FX activating complex (Ahmad, S. S., Rawala, R., Cheung, W. F., Stafford, D. W., and Walsh, P. N. (1995) Biochem. J. 310, 427-431; Wong, M. Y., Gurr, J. A., and Walsh, P. N. (1999) Biochemistry 38, 8948-8960). To identify important residues, we prepared several chimeric FIXa proteins using homologous sequences from FVII: FIXa_FVIIEGF2 (FIX88-124,FVII91-127), FIXa_loop1 (FIX88-99,FVII91-102), FIXa_loop2 (FIX95-109,FVII98-112), FIXa_loop3 (FIX111-124,FVII114-127), and point mutants (FIXaR94D and FIXa_loop1G94R). In the presence and absence of FVIIIa, a 2- to 10-fold reduced V_max of FX activation (nM FXa min¹) was observed for FIXa_FVIIEGF2, FIXa_loop1, FIXa_loop2, and FIXa_loop1G94R, whereas FIXa_loop3 and FIXaR94D were normal. For all of the FIXa proteins, K_m(app) values were normal as were EC₅₀ values for interactions with FVIIIa. However, K_d(app) (in nM) for the FX activating complex assembled on phospholipid vesicles was increased for FIXa_FVIIEGF2 (43.3 ± 2.70), FIXa_loop1(10.9 ± 2.8), FIXa_loop2 (70.5 ± 1.60), and FIXa_loop1G94R (17.1 ± 2.90) relative to FIXa_N (3.9 ± 0.11), FIXa_WT (4.6 ± 0.17), FIXa_loop3 (4.5 ± 0.20), and FIXaR94D (2.2 ± 0.09) suggesting that reduced V_max is a result of impaired complex assembly. These data indicate that residues 88-109 (but not Arg⁹⁴) are important for normal assembly of the FX activating complex on phospholipid vesicles.

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