ライフサイエンスコーパス: FIXa

1 FIXa is a serine protease enzyme involved in the intrins

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

3 0.5), FIXa(loop1) (19 +/- 4.0, 27 +/- 5.0), FIXa(loop2) (35 +/- 9.0, 65 +/- 12.0), and FIXa(loop3) (

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

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

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

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

8 /- 2.90) relative to FIXa(N) (3.9 +/- 0.11), FIXa(WT) (4.6 +/- 0.17), FIXa(loop3) (4.5 +/- 0.20), and

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

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

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

12 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

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

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

15 5) FIXa amidolytic activity was measured.

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

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

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

19 (43.3 +/- 2.70), FIXa(loop1)(10.9 +/- 2.8), FIXa(loop2) (70.5 +/- 1.60), and FIXa(loop1)G94R (17.1 +

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

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

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

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

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

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

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

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

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

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

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

31 9 +/- 2.8), FIXa(loop2) (70.5 +/- 1.60), and FIXa(loop1)G94R (17.1 +/- 2.90) relative to FIXa(N) (3.9

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

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

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

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

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

37 performed in the absence of phospholipid and FIXa.

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

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

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

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

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

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

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

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

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

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

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

49 4, the C-terminal region of A2 thought to be FIXa interactive.

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

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

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

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

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

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

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

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

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

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

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

61 = 6.2 nM) than for the enzyme complex, i.e., FIXa/FVIIIa/Ca2+/ phospholipids (Ki = 16.5 nM).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

78 as a decreased apparent binding affinity for FIXa.

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

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

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

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

83 significantly to the affinity of FVIIIa for FIXa.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

101 us formation through contact phase-generated FIXa.

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

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

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

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

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

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

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

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

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

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

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

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

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

115 the homologous protease activated factor IX (FIXa).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

146 and could interfere with AT inactivation of FIXa.

147 I significantly hindered AT inactivation of FIXa.

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

149 e found to interfere with AT inactivation of FIXa.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

165 for restricting the inhibitor specificity of FIXa.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

189 ioritized by their binding affinities to the FIXa protein.

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

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

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

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

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

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

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

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

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

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

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

201 VIIIa subunits to cofactor association with FIXa were evaluated.

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

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

204 nfirmed that all FVIII forms interacted with FIXa.

205 that the mutations altered interaction with FIXa.

206 Phe-1816 contributes to the interaction with FIXa.

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

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

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

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

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

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