Abstract
Activated platelets promote intrinsic factor X-activating complex assembly by presenting high affinity, saturable binding sites for factor IXa mediated by two disulfide-constrained loop structures (loop 1, Cys88-Cys99; loop 2, Cys95-Cys109) within the second epidermal growth factor (EGF2) domain. To identify amino acids essential for factor X activation complex assembly, recombinant factor IXa point mutants in loop 1 (N89A, I90A, K91A, and R94A) and loop 2 (D104A, N105A, and V107A) were prepared. All seven mutants were similar to the native factor IXa by SDS-PAGE, active site titration, and content of gamma-carboxyglutamic acid residues. Kinetic constants obtained by either titrating factor X or factor VIIIa on SFLLRN-activated platelets or phospholipid vesicles revealed near normal values of Km(app) and Kd(app)FVIIIa for all mutants, indicating normal substrate and cofactor binding. In a factor Xa generation assay in the presence of activated platelets and cofactor factor VIIIa, compared with native factor IXa (Kd(app)FIXa approximately 1.1 nm, Vmax approximately 12 nm min(-1)), N89A displayed an increase of approximately 20-fold in Kd(app)FIXa and a decrease of approximately 20-fold in Vmax; I90A had an increase of approximately 5-fold in Kd(app)FIXa and approximately 10-fold decrease in Vmax; and V107A had an increase of approximately 3-fold in Kd(app)FIXa and approximately 4-fold decrease in Vmax. We conclude that residues Asn89, Ile90, and Val107 within loops 1 and 2 (Cys88-Cys109) of the EGF2 domain of factor IXa are essential for normal interactions with the platelet surface and for the assembly of the factor X-activating complex on activated platelets.
Highlights
Coagulation factor IX (FIX)1 is a vitamin-K-dependent serine protease zymogen that plays a pivotal role in blood coagulation
To identify essential residues in the disulfide-constrained loops 1 and 2 of the EGF2 domain (Cys88-Cys109) that are important for platelet-mediated FIXa/FVIIIa complex assembly, we utilized the primary amino acid sequence alignment within the disulfide-constrained loops 1 and 2 of the EGF2 domain (Fig. 1) to identify “candidate” residues that are highly conserved among species and different from those in FVII, because residues in FVII molecules have been shown to be ineffective in mediating platelet surface-mediated FX activation complex assembly [2, 6]
Recombinant FIXa point mutants in loop 1 (N89A, I90A, K91A, and R94A) and loop 2 (D104A, N105A, and V107A) were prepared to study the contributions of these residues to FX activation complex assembly on activated platelets
Summary
Coagulation factor IX (FIX) is a vitamin-K-dependent serine protease zymogen that plays a pivotal role in blood coagulation. “Candidate” residues, most likely to be involved in mediating FX activation on the surface of activated platelets, were selected for alanine-scanning mutagenesis on the basis of: 1) surface exposure of amino acid side chains by x-ray crystallography; 2) conservation among species; and 3) dissimilarity with those in a homologous protein FVII, which does not participate in the assembly of the intrinsic FX-activating complex and does not bind to activated platelets with high affinity [9] Based on these criteria, we selected and prepared recombinant FIXa point mutants in loop 1 (N89A, I90A, K91A, and R94A) and loop 2 (D104A, N105A, and V107A) of the EGF2 domain to. The present results support the conclusion that residues Asn, Ile, and Val107 within loops 1 and 2 (Cys88-Cys109) of the EGF2 domain of FIXa are essential for normal interactions with the platelet surface and for the assembly of the FX-activating complex on activated platelets
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