The Factor IXa Second Epidermal Growth Factor (EGF2) Domain Mediates Platelet Binding and Assembly of the Factor X Activating Complex

Wilkinson, Frank H.; Ahmad, Shafeeque; Walsh, Peter N.

doi:10.1074/jbc.m107753200

Cited by 36 publications

(41 citation statements)

References 47 publications

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“…The FXa generation reaction was allowed to proceed at 37°C for 2 min and was stopped by addition of 50 l of stopping buffer (50 mM HEPES (pH 8.1), 175 mM NaCl, and 20 mM EDTA). The amount of FXa generated was determined by using its chromogenic substrate S2765 as described before (3).…”

Section: Methodsmentioning

confidence: 99%

“…However, this contribution is considered to be structural rather than direct, because the segment FIXa variants showed normal FVIIIa binding in surface plasmon resonance experiments (36). Kinetic studies using recombinant FIXa chimeras have consistently shown that residues in the EGF2 domain do not mediate FVIIIa association suggested by both normal kinetic stimulation by FVIIIa and normal K d(app)FVIIIa values (2)(3)(4)6). Thus, the contribution of the non-catalytic domain of FIXa to its interaction with FVIIIa appears to be mainly structural, i.e.…”

Section: Egf2 Domain Of Fixa In Fx Activation On Plateletsmentioning

confidence: 99%

“…4) by multiplying the B max (nanomolar) value for FIXaN by the -fold differences in V max 9 . Data Analysis-FXa generation rates from all reactions described here were fitted to a hyperbolic curve using a non-linear least squares fit as previously standardized (3).…”

Section: Determination Of K Dappfixa and Fx Activation Velocity In Thmentioning

confidence: 99%

“…Previous studies from our laboratory have demonstrated that the zymogen, FIX, binds to a discrete number (n ϳ 250 sites per platelet) of receptors on the surface of activated platelets (K d ϳ 2.5 nM) that can also be occupied by the enzyme, FIXa, and are mediated by residues Gly 4 -Gln 11 within the Gla domain (2)(3)(4)(5)(6). In addition, FIXa, but not FIX, can bind to a site (n ϳ 250 sites per platelet, K d ϳ 0.5 nM) on activated platelets, mediated by residues 88 -109 (disulfide constrained loops 1 and 2) but not by residues 110 -124 (loop 3) within the EGF2 domain (2)(3)(4)(5)(6).…”

mentioning

confidence: 99%

“…In addition, FIXa, but not FIX, can bind to a site (n ϳ 250 sites per platelet, K d ϳ 0.5 nM) on activated platelets, mediated by residues 88 -109 (disulfide constrained loops 1 and 2) but not by residues 110 -124 (loop 3) within the EGF2 domain (2)(3)(4)(5)(6). Occupancy of these binding sites is closely correlated with optimal rate enhancements of FX activation (Ͼ2 ϫ 10 8 -fold) in the presence of FVIIIa, emphasizing the physiological significance of platelet-receptor-mediated coagulation complex assembly (2,6,7).…”

mentioning

confidence: 99%

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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

Yang

Chang

Lin

et al. 2004

Journal of Biological Chemistry

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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.

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Section: Methodsmentioning

confidence: 99%