Identification of Residues Asn89, Ile90, and Val107 of the Factor IXa Second Epidermal Growth Factor Domain That Are Essential for the Assembly of the Factor X-activating Complex on Activated Platelets

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, Cys 88 -Cys 99 ; loop 2, Cys 95 -Cys 109 ) 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 ␥-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 K m(app) and K d(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 (K d(app)FIXa ϳ 1.1 nM, V max ϳ 12 nM min ؊1 ), N89A displayed an increase of ϳ20-fold in K d(app)FIXa and a decrease of ϳ20-fold in V max ; I90A had an increase of ϳ5-fold in K d(app)FIXa and ϳ10-fold decrease in V max ; and V107A had an increase of ϳ3-fold in K d(app)FIXa and ϳ4-fold decrease in V max . We conclude that residues Asn 89 , Ile 90 , and Val 107 within loops 1 and 2 (Cys 88 -Cys 109 ) 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.

An ordered sequential mechanism for Factor IX and Factor IXa binding to platelet receptors in the assembly of the Factor X-activating complex

“…FXa generation was initiated by addition of 250 nM FX, was allowed to proceed for 2 min at 37 • C and was stopped by addition of 10 mM EDTA. The amount of FXa generated was determined by using its chromogenic substrate S2765 as described in [29]. Titrations of FIXa proteins at concentrations up to 5 nM were included in the platelet-surface FXa generation assay as controls.…”

“…Competitors (DEGR-FIXa, FVIIa, FVII), at the indicated concentrations, were pre-incubated with normal plasma-derived FIXa (0.1 nM), SFLLRN (5 µM)-activated platelets (5 × 10 7 /ml) and FVIIIa (5 units/ml) at 37 • C for 15 min before addition of 250 nM FX to start the reaction, and FX activation by residual FIXa was allowed to proceed for 2 min and was stopped by the addition of EDTA (10 mM). The amount of FXa generated was determined by using its chromogenic substrate S2765 as described in [29].…”

“…growth factor) domain to bind to an additional ∼ 250 specific binding sites/platelet [21][22][23][24][25][26][27][28][29]. However, the relative functional contributions of these interactions to the assembly of the FXactivating complex are unknown, as is the sequential mechanism required for FIX and FX activation on the platelet surface.…”

Factor IXa as a Target for Pharmacologic Inhibition in Acute Coronary Syndrome

“…Contact between the EGF2 and protease domains of factor IXa may be essential for factor IXa enzymatic activity and for the orderly assembly of the factor X activating complex [13]. The EGF2 domain binds to the surface of activated platelets via two loop structures (loop 1, CyS 99 ; loop 2, CyS 95 –CyS 109 ) promoting factor X‐activating complex assembly [14].…”

“…The presence of factor VIIIa and factor Xa increases, by 5‐fold, the affinity of receptors for factor IXa, that in turn participates in factor X activation. Residues Asn 89 , Ile 90 , and Val 107 within loops 1 and 2 of the EGF‐2 domain of factor IXa may be particularly important for platelet interactions, and for the assembly of a factor X‐activating complex on the surface of activated platelets [14]. The expression of PAR‐1‐stimulated platelet factor IXa binding sites is dependent upon release of calcium from internal stores, producing sustained and pronounced calcium transients [37].…”