ライフサイエンスコーパス: FIXa

1 FIXa is a notoriously inefficient enzyme that needs FVII

2 FIXa is a serine protease enzyme involved in the intrins

3 FIXa(Y225P), a Na+ site mutant, was severely impaired in

4 0.5), FIXa(loop1) (19 +/- 4.0, 27 +/- 5.0), FIXa(loop2) (35 +/- 9.0, 65 +/- 12.0), and FIXa(loop3) (

5 14), FIXa(WT) (0.72 +/- 0.08, 3.8 +/- 0.08), FIXa(loop1) (3.2 +/- 0.72, 14.0 +/- 1.60), FIXa(loop2) (

6 Importantly, 4 of 10 FIXa-reactive monoclonal aPLs (including the B2 mAb gene

7 (FIX Delta 88-99,inverted Delta FVII91-102), FIXa(loop2) (FIX Delta 95-109,inverted Delta FVII98-112)

8 (FIX Delta 88-99,inverted Delta FVII91-102), FIXa(loop2) (FIX Delta 95-109,inverted Delta FVII98-112)

9 /- 2.90) relative to FIXa(N) (3.9 +/- 0.11), FIXa(WT) (4.6 +/- 0.17), FIXa(loop3) (4.5 +/- 0.20), and

10 FIX Delta 95-109,inverted Delta FVII98-112), FIXa(loop3) (FIX Delta 111-124,inverted Delta FVII114-12

11 FIX Delta 88-124,inverted Delta FVII91-127), FIXa(loop1) (FIX Delta 88-99,inverted Delta FVII91-102),

12 FIX Delta 88-124,inverted Delta FVII91-127), FIXa(loop1) (FIX Delta 88-99,inverted Delta FVII91-102),

13 ly): FIXa(N) (0.46 +/- 0.05, 1.40 +/- 0.14), FIXa(WT) (0.72 +/- 0.08, 3.8 +/- 0.08), FIXa(loop1) (3.2

14 (N) (3.9 +/- 0.11), FIXa(WT) (4.6 +/- 0.17), FIXa(loop3) (4.5 +/- 0.20), and FIXaR94D (2.2 +/- 0.09)

15 vely: FIXa(N) (0.55 +/- 0.06, 2.9 +/- 0.45), FIXa(WT) (0.80 +/- 0.08, 3.5 +/- 0.5), FIXa(loop1) (19 +

16 5) FIXa amidolytic activity was measured.

17 .45), FIXa(WT) (0.80 +/- 0.08, 3.5 +/- 0.5), FIXa(loop1) (19 +/- 4.0, 27 +/- 5.0), FIXa(loop2) (35 +/

18 , FIXa(loop1) (3.2 +/- 0.72, 14.0 +/- 1.60), FIXa(loop2) (18.4 +/- 1.60, 26.3 +/- 3.40), and FIXa(loo

19 ncreased for FIXa(FVIIEGF2) (43.3 +/- 2.70), FIXa(loop1)(10.9 +/- 2.8), FIXa(loop2) (70.5 +/- 1.60),

20 (43.3 +/- 2.70), FIXa(loop1)(10.9 +/- 2.8), FIXa(loop2) (70.5 +/- 1.60), and FIXa(loop1)G94R (17.1 +

21 to FVa residues 499-506) are recognized as a FIXa binding sequence.

22 The chimeric mutant cleaved a FIXa-specific chromogenic substrate with normal catalyti

23 4) Activity was monitored in a FIXa-based clotting assay.

24 ibited normal amidolytic activities toward a FIXa-specific chromogenic substrate.

25 Following activation, it was found that all FIXa derivatives cleaved the chromogenic substrate CBS 3

26 of FVIII (FVIIIR336I) possessing an altered FIXa cleavage site, showed similar rates of FXase decay

27 intrinsic pathway, and suggest that altering FIXa inhibition could have therapeutic benefits.

28 chain showed that cleavage at the alternate FIXa site (A3 domain) was not inhibitory to FXase.

29 r reactivity of FIXa with AT, we prepared an FIXa/FXa chimera in which the 39-loop of the protease wa

30 , FIXa(loop2) (35 +/- 9.0, 65 +/- 12.0), and FIXa(loop3) (1.1 +/- 0.09, 5.0 +/- 0.90).

31 Delta 95-109,inverted Delta FVII98-112), and FIXa(loop3) (FIX Delta 111-124,inverted Delta FVII114-12

32 a(loop2) (18.4 +/- 1.60, 26.3 +/- 3.40), and FIXa(loop3) (0.7 +/- 0.05, 3.0 +/- 0.15).

33 9 +/- 2.8), FIXa(loop2) (70.5 +/- 1.60), and FIXa(loop1)G94R (17.1 +/- 2.90) relative to FIXa(N) (3.9

34 ts from both FVIIIa subunit dissociation and FIXa-catalyzed cleavage, dependent upon the relative con

35 VII114-127), and point mutants (FIXaR94D and FIXa(loop1)G94R).

36 ve different specificity for binding FXa and FIXa while retaining compatibility as substrate for APC.

37 hly selective and potent factor Xa (FXa) and FIXa inhibitors were identified by simple switch of func

38 t both mechanisms of FVIIIa inactivation and FIXa interactions could be leveraged to enhance FVIII fu

39 IXa(FVIIEGF2), FIXa(loop1), FIXa(loop2), and FIXa(loop1)G94R, whereas FIXa(loop3) and FIXaR94D were n

40 performed in the absence of phospholipid and FIXa.

41 In the present study, FVIIIa stability and FIXa binding were evaluated in a FVIII-N1810C variant, a

42 nteraction between the FVIIIa A2 subunit and FIXa and show a contribution of several residues within

43 In conclusion, IgG anti-FIXa Ab occurred in approximately 30% of APS patients an

44 nal IgG aPLs bound to FIXa and that IgG anti-FIXa Abs in APS patients were significantly higher than

45 st this hypothesis, we searched for IgG anti-FIXa Abs in APS patients.

46 normal controls as the cutoff, the IgG anti-FIXa Abs were present in 11 of 38 (28.9%) APS patients.

47 increase of approximately 20-fold in Kd(app)FIXa and a decrease of approximately 20-fold in Vmax; I9

48 n increase of approximately 5-fold in Kd(app)FIXa and approximately 10-fold decrease in Vmax; and V10

49 n increase of approximately 3-fold in Kd(app)FIXa and approximately 4-fold decrease in Vmax.

50 IIa, compared with native factor IXa (Kd(app)FIXa approximately 1.1 nm, Vmax approximately 12 nm min(

51 A3-C1-C2 subunit and fluorescein-Phe-Phe-Arg-FIXa yielded K(d) values of 52 +/- 10 and 197 +/- 55 nM

52 4, the C-terminal region of A2 thought to be FIXa interactive.

53 Because FIXa is an upstream procoagulant factor, impaired AT reg

54 [NaCl], indicating that interactions between FIXa and FVIIIa can increase enzyme affinity when fewer

55 courses were observed for inhibition of both FIXa in the FXase complex and FXa in the prothrombinase

56 The FVIIIa A2 subunit bound FIXa with high affinity (Kd = 3.9 +/- 1.6 nm) that was s

57 residue 36 is a Glu in both mouse and bovine FIXa and that both proteases are also susceptible to inh

58 and the reciprocal activation of FVII/TF by FIXa(PCGla) was normal; however, both reactions were imp

59 In all cases, FIXa was inhibited by subphysiologic levels of ZPI.

60 We prepared several chimeric FIXa proteins using homologous sequences from factor VII

61 rtant residues, we prepared several chimeric FIXa proteins using homologous sequences from FVII: FIXa

62 affinity binding sites for blood coagulation FIXa, FVIIIa, and FX.

63 ng hemophilia B mice have shown that delayed FIXa inhibition has a major impact on reducing the bleed

64 nity for the enzyme-substrate complex, i.e., FIXa/FVIIIa/ Ca2+/phospholipids/FX complex (Ki' = 6.2 nM

65 = 6.2 nM) than for the enzyme complex, i.e., FIXa/FVIIIa/Ca2+/ phospholipids (Ki = 16.5 nM).

66 Notably, each FIXa or FXa mutant activated FVII and bound to antithrom

67 presence of factor VIIIa increased the EC50,FIXa with an IC50 of 1.5 nM, without affecting the Vmax.

68 nce, whereas there was no effect on the EC50,FIXa.

69 We found that selected FIXa variants (eg, FIXa-V16L) have a prolonged activity half-life in the pl

70 id not respond to the presence of excess EGR-FIXa (45 nM) and FX (1.5 microM) with enhanced binding s

71 ts of FX and active site-inhibited FIXa (EGR-FIXa) on the binding of both FVIII and FVIIIa to activat

72 The presence of EGR-FIXa and FX increases both the number and the affinity o

73 In additional experiments, FIXa with or without FVIIIa activated FX(WT) and FX(PCEG

74 Intact A1/A3-C1-C2 dimer increased Fl-FFR-FIXa anisotropy and bound factor X in a solid phase assa

75 IIIa-like effect on the anisotropy of Fl-FFR-FIXa.

76 f fluorescein-Phe-Phe-Arg factor IXa (Fl-FFR-FIXa; Kd = 42.4 nM), whereas cleavage of factor VIIIa by

77 activation, it combines with activated FIX (FIXa) on anionic membranes to form the intrinsic Xase en

78 rotein C, plasmin, factor VIIa (FVIIa), FIX, FIXa, and FXII.

79 ease in both Vmax and K(m) of activated FIX-(FIXa)-catalyzed FX activation in the presence of FVIIIa

80 ation of: Factors FXa (via TF/VIIa or FIXa), FIXa (via TF/FVIIa or FXIa), thrombin, fibrin, and FXIa.

81 ytometry analysis indicated that fluorescent FIXa exhibits impaired complex formation with only FVIII

82 with a role of the amino acids 1803-1818 for FIXa or A2 domain binding.

83 oteins had similar functional affinities for FIXa (dissociation constant [K(d)] values approximately

84 isplayed 2- to 4-fold decreased affinity for FIXa as compared with WT bA2.

85 as a decreased apparent binding affinity for FIXa.

86 f FVIII, FVIIIa, functions as a cofactor for FIXa in catalyzing the membrane-dependent activation of

87 h Asp194 in serine proteases, was faster for FIXa(Y225P) and addition of Ca2+ overcame this impairmen

88 the functional affinity of DeltaC2 FVIII for FIXa and rates of thrombin activation.

89 its contribute to the affinity of FVIIIa for FIXa in the membrane-dependent FXase.

90 significantly to the affinity of FVIIIa for FIXa.

91 d on phospholipid vesicles was increased for FIXa(FVIIEGF2) (43.3 +/- 2.70), FIXa(loop1)(10.9 +/- 2.8

92 activation (nm FXa min(-1)) was observed for FIXa(FVIIEGF2), FIXa(loop1), FIXa(loop2), and FIXa(loop1

93 nzothiophene template as a lead scaffold for FIXa inhibiton based on its homology with urokinase plas

94 -Ca2+ is catalytically more active than free FIXa.

95 and (ii) FVIIIa inactivation resulting from FIXa-catalyzed proteolysis of the Al subunit.

96 homologous sequences from factor VII (FVII): FIXa(FVIIEGF2) (FIX Delta 88-124,inverted Delta FVII91-1

97 oteins using homologous sequences from FVII: FIXa(FVIIEGF2) (FIX Delta 88-124,inverted Delta FVII91-1

98 Xa min(-1)) was observed for FIXa(FVIIEGF2), FIXa(loop1), FIXa(loop2), and FIXa(loop1)G94R, whereas F

99 pholipid, with or without saturating FVIIIa, FIXa(Y225P) activated FX with similar K(m) but threefold

100 ly 100-fold greater than the K(d) for FVIIIa-FIXa interaction (4.2 +/- 0.6 nM).

101 icles (K(i) approximately 1.6K(d) for FVIIIa-FIXa).

102 s residue of approximately 10% of the FVIIIa-FIXa binding energy.

103 ine-protease Factor IXa (FIXa) in the FVIIIa-FIXa complex assembled on the activated platelet membran

104 ds understanding the mechanism of the FVIIIa-FIXa complex assembly on the activated platelet surface

105 F-initiated pathway directly yielding FVIIIa-FIXa intrinsic tenase complex may be prohemostatic befor

106 FXa generation via intrinsic tenase (FVIIIa/FIXa) complexes.

107 e extrinsic (TF:FVIIa) and intrinsic (FVIIIa:FIXa) pathways of coagulation.

108 Further, interaction of FVIIIa:FIXa(Y225P) was impaired fourfold.

109 Interestingly, RBC-MVs generated FIXa in a prekallikrein-dependent manner.

110 us formation through contact phase-generated FIXa.

111 Here, we generated FIXa variants with altered reactivity to plasma inhibito

112 ass spectrometry (HDX-MS) to investigate how FIXa responds to assembly with FVIIIa in the presence of

113 ogether, the reduced stability plus impaired FIXa interaction of FVIIIC2C2 suggest that the C1 domain

114 probe) validating that substrate binding in FIXa is linked positively to Na+ binding.

115 ges indicated that 3 helices are involved in FIXa-FVIIIa assembly.

116 ative homology analysis revealed that Na+ in FIXa coordinates to the carbonyl groups of residues 184A

117 hand, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and

118 trast, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and

119 Similarly, antithrombin inactivated FIXa derivatives with a similar second-order association

120 the effects of FX and active site-inhibited FIXa (EGR-FIXa) on the binding of both FVIII and FVIIIa

121 In new studies, ZPI inhibited FIXa in the FXase complex.

122 oagulation cascade in selectively inhibiting FIXa would leave hemostasis intact via the extrinsic pat

123 Relative to FXa, AT inhibits FIXa with approximately 40-fold slower rate constant.

124 ntrinsic FX-activating complex that inhibits FIXa bound either to the phospholipid or activated plate

125 n circulation, the biochemical nature of its FIXa cofactor activity, the regulation of activated FVII

126 c domain of activated coagulation factor IX (FIXa) is homologous to those of thrombin and FXa, we hyp

127 assembly of the enzyme-activated factor IX (FIXa) with its cofactor, activated factor VIII (FVIIIa)

128 icated to interact with activated factor IX (FIXa).

129 the homologous protease activated factor IX (FIXa).

130 During blood coagulation, factor IXa (FIXa) activates factor X (FX) requiring Ca2+, phospholip

131 wth factor (EGF2)-like domain of factor IXa (FIXa) are important for assembly of the factor X (FX) ac

132 Interestingly, factor IXa (FIXa) binding affinity for WT FVIIIa was significantly r

133 This loop in factor IXa (FIXa) has 3 basic residues (Arg143, Lys147, and Arg150)

134 -cofactor to the serine-protease Factor IXa (FIXa) in the FVIIIa-FIXa complex assembled on the activa

135 A potent and selective Factor IXa (FIXa) inhibitor was subjected to a series of liver micro

136 Factor IXa (FIXa) is known to have a binding site for heparin that h

137 Factor IXa (FIXa) plays a pivotal role in coagulation by contributin

138 quire occupancy of receptors for factor IXa (FIXa), factor VIII (FVIII), and FX on the activated plat

139 X) activation require binding of factor IXa (FIXa), factor VIII(a) [FVIII(a)], and FX to activated pl

140 interaction of the serpin with factors IXa (FIXa) and Xa (FXa), thereby improving the rate of reacti

141 All of them, including factors IXa (FIXa), FXa/FX, FVa, FVIII, prothrombin, and PS-sensitive

142 as observed for FIXa(FVIIEGF2), FIXa(loop1), FIXa(loop2), and FIXa(loop1)G94R, whereas FIXa(loop3) an

143 etic mechanism is valid in platelet-mediated FIXa-catalyzed FX activation (Ki' = 5.9 nM and Ki = 12.6

144 tropy experiments using fluorescein-modified FIXa confirmed that all FVIII forms interacted with FIXa

145 mmobilized biotinylated active site-modified FIXa, and FVIII and FVIIIa subunits.

146 the 558-565 loop are critical in modulating FIXa enzymatic activity but do not contribute significan

147 Thus, Na+-FIXa-Ca2+ is catalytically more active than free FIXa.

148 reactant concentrations (0.5 nm FVIIIa; 5 nm FIXa), FXase decay is governed by the inter-FVIIIa subun

149 residues of the 39-loop (residues 31-41) of FIXa with corresponding residues of FXa renders the FIXa

150 he protease domain increases the affinity of FIXa for FVIIIa approximately 15-fold.

151 e Na+ site further increases the affinity of FIXa for FVIIIa fourfold and k(cat) threefold.

152 VIII and FX increases 5-fold the affinity of FIXa for the surface of activated platelets, and the pre

153 Occupancy by Na+ increased the affinity of FIXa for the synthetic substrate, whereas occupancy by C

154 onsistent with the elevated concentration of FIXa stabilizing the labile subunit structure of the cof

155 When the concentration of FIXa was increased 10-fold, the initial rate of decay of

156 The serine protease domain of FIXa contains a Ca2+ site and is predicted to contain a

157 crystal structure of the catalytic domain of FIXa indicated that all five basic residues are spatiall

158 epidermal growth factor (EGF)-like domain of FIXa mediates assembly of the FX activating complex.

159 ht basic residues in the catalytic domain of FIXa that can potentially bind to heparin.

160 NP-2 increases the concentrations (EC50) of FIXa, FVIIIa, and phospholipid vesicles required for hal

161 also by murine studies showing formation of FIXa-antithrombin complexes in FXI deficient mice.

162 te FIXa procoagulant function, the impact of FIXa inhibition on thrombin generation and clot formatio

163 and could interfere with AT inactivation of FIXa.

164 I significantly hindered AT inactivation of FIXa.

165 S bind to FIXa and hinder AT inactivation of FIXa.

166 e found to interfere with AT inactivation of FIXa.

167 Unlike inhibition of FXa, inhibition of FIXa did not strictly require protein Z.

168 t whose activity was measured, inhibition of FIXa was investigated five ways.

169 ncreased the efficiency of ZPI inhibition of FIXa; FVIIIa in molar excess was not protective of FIXa

170 (84)-Val(128)) was an effective inhibitor of FIXa binding to platelets in both the presence (K(i) = 0

171 late the cofactor-independent interaction of FIXa with its physiological inhibitor AT and substrate f

172 activated platelets only in the presence of FIXa and FX but does not directly mediate FVIIIa binding

173 ng to activated platelets in the presence of FIXa and FX is closely coupled with rates of F-X activat

174 FVIII variants were activated in presence of FIXa, only FVIII/FV 1811-1818 demonstrated an enhanced d

175 FVIIIa in molar excess was not protective of FIXa unless FIXa/FVIIIa interacted prior to ZPI exposure

176 sulted in a 10-fold reduction in the rate of FIXa-catalyzed proteolysis of FVIIIa.

177 loop contribute to the slower reactivity of FIXa with AT, we prepared an FIXa/FXa chimera in which t

178 olecular basis for the lack of reactivity of FIXa with these plasma inhibitors and discovered that un

179 e results demonstrate that the regulation of FIXa inhibition contributes in a major way to the spatia

180 ocoagulant factor, impaired AT regulation of FIXa might contribute more toward thrombosis than the dy

181 nsights into the physiological regulation of FIXa, enhance our understanding of thrombus formation in

182 g data indicated that five basic residues of FIXa in the following order of importance, Arg(233) > Ar

183 the 39-loop contribute to the resistance of FIXa to inhibition by plasma inhibitors ZPI and TFPI.

184 for restricting the inhibitor specificity of FIXa.

185 e found, we studied the effects of the Ab on FIXa inactivation by AT.

186 t generation of: Factors FXa (via TF/VIIa or FIXa), FIXa (via TF/FVIIa or FXIa), thrombin, fibrin, an

187 template revealed a number of highly potent FIXa inhibitors, though with moderate selectivity agains

188 ies including thrombin generation potential, FIXa binding affinity, Km for FX of FXase complexes, thr

189 Thus, TF synergistically primes FIXa-dependent thrombin generation independently of cofa

190 e substrate FX to FXa by the serine protease FIXa.

191 x (FXase) is comprised of a serine protease, FIXa, and a protein cofactor, FVIIIa, assembled on a pho

192 R338A-FIXa and wt-FIXa had equal activity, with or without FVI

193 R338A-FIXa's increased catalytic efficiency did not result fro

194 Interestingly, R338A-FIXa had reduced affinity for heparin.

195 ffect on activity of either wt-FIXa or R338A-FIXa.

196 Therefore, we propose that R338A-FIXa's increased activity is not due to an allosteric ef

197 functional assays, we determined that R338A-FIXa's Kd for factor VIIIa (FVIIIa) was similar to that

198 HDX-MS was also applied to recombinant FIXa variants that are associated with severe hemophilia

199 old), whereas the anti-C2 antibodies reduced FIXa binding affinity of FVIIIC2C2 variant (~4-fold).

200 er plasma inhibitors are thought to regulate FIXa procoagulant function, the impact of FIXa inhibitio

201 esence and absence of FVIIIa, respectively): FIXa(N) (0.46 +/- 0.05, 1.40 +/- 0.14), FIXa(WT) (0.72 +

202 resence and absence of FVIIIa, respectively: FIXa(N) (0.55 +/- 0.06, 2.9 +/- 0.45), FIXa(WT) (0.80 +/

203 We found that selected FIXa variants (eg, FIXa-V16L) have a prolonged activity

204 Ia-R336Q and FVIIIa-R562Q) demonstrated that FIXa and FX can protect FVIIIa from APC cleavage at Arg5

205 We found that FIXa binding to FVIIIa stabilized the A2 domain and incr

206 ber of residues that potentially contact the FIXa protease domain.

207 s homologous to FVIIIa, the cofactor for the FIXa protease, in the FX-activating complex, and FVIIIa

208 Thus, the inactivation rate of the FIXa chimera by ZPI in the presence of protein Z (PZ), n

209 We conclude that residues 88-109 of the FIXa EGF2 domain mediate binding to platelets and assemb

210 es were aimed at delineating the role of the FIXa Na+ site in macromolecular catalysis.

211 For all of the FIXa proteins, K(m)((app)) values were normal as were EC

212 th corresponding residues of FXa renders the FIXa chimera susceptible to inactivation by both ZPI and

213 ctivity to plasma inhibitors that target the FIXa active site but maintain procoagulant function when

214 Further studies revealed that the FIXa mutant activates factor X with approximately 4-fold

215 ioritized by their binding affinities to the FIXa protein.

216 ns of the benzothiophene template within the FIXa active site by X-ray crystallography and molecular

217 e hypothesized that some aPLs in APS bind to FIXa and hinder AT inactivation of FIXa.

218 an effective competitor of FVIIIa binding to FIXa as judged by inhibition of FXa generation performed

219 patient-derived monoclonal IgG aPLs bound to FIXa and that IgG anti-FIXa Abs in APS patients were sig

220 Phe-1816 of region 1811-1818 contributes to FIXa binding.

221 phospholipid, and FVIIIa, binding of Na+ to FIXa increases its biologic activity by approximately 12

222 the C1 domain resides in close proximity to FIXa in the FXase complex and contributes a critical rol

223 FIXa(loop1)G94R (17.1 +/- 2.90) relative to FIXa(N) (3.9 +/- 0.11), FIXa(WT) (4.6 +/- 0.17), FIXa(lo

224 lar excess was not protective of FIXa unless FIXa/FVIIIa interacted prior to ZPI exposure.

225 were then submitted directly to an in vitro FIXa enzymatic assay.

226 ), FIXa(loop2), and FIXa(loop1)G94R, whereas FIXa(loop3) and FIXaR94D were normal.

227 VIIIa subunits to cofactor association with FIXa were evaluated.

228 or (F) VIIIa forms a number of contacts with FIXa in assembling the FXase enzyme complex.

229 Reconstitution of FVIII with FIXa-cleaved light chain showed that cleavage at the alt

230 nfirmed that all FVIII forms interacted with FIXa.

231 that the mutations altered interaction with FIXa.

232 Phe-1816 contributes to the interaction with FIXa.

233 e about how FVIIIa binding interactions with FIXa and FX affect FVIIIa inactivation in vivo.

234 ctor (F) VIIIa forms a direct interface with FIXa.

235 d A2 subunit of FVIIIa interacts weakly with FIXa, and recent modeling studies have implicated a numb

236 R338A-FIXa and wt-FIXa had equal activity, with or without FVIIIa, toward

237 udin, had no effect on activity of either wt-FIXa or R338A-FIXa.

238 tor VIIIa (FVIIIa) was similar to that of wt-FIXa.

239 nts revealed binding and dissociation of ZPI/FIXa with Kd (app) of 9-12 nm, similar to the concentrat