ライフサイエンスコーパス: FVIIIa

1 FVIIIa (Kd approximately 1.7 nM), FVIII((LC)) (Kd approx

2 FVIIIa binding to activated platelets in the presence of

3 FVIIIa cofactor activity measured in the presence of eac

4 d with 125I-labeled FVIII C2 domain, or 125I-FVIIIa, or 125I-FVIII((LC)), or peptides from the C2 dom

5 ns from Lys39, Arg67, and Arg74 to forming a FVIIIa-interactive site.

6 binding interactions with FIXa and FX affect FVIIIa inactivation in vivo.

7 as the FVIII C2 domain is believed to anchor FVIIIa to the phospholipid surface, recent x-ray crystal

8 protease, in the FX-activating complex, and FVIIIa residues 555-561 (homologous to FVa residues 499-

9 ndicating that interactions between FIXa and FVIIIa can increase enzyme affinity when fewer ionic int

10 that this A2 domain sequence of the FVa and FVIIIa cofactors evolved to have different specificity f

11 Both FVIII and FVIIIa binding were specific, saturable, and reversible.

12 The swap variant showed WT-like FVIII and FVIIIa stability, which were markedly reduced for H281A

13 ted active site-modified FIXa, and FVIII and FVIIIa subunits.

14 (EGR-FIXa) on the binding of both FVIII and FVIIIa to activated platelets and show the following: (a

15 es on activated platelets for both FVIII and FVIIIa, emphasizing the validity of a three-receptor mod

16 and affinity parameters in forming FXase and FVIIIa.

17 (n = 484 +/- 59; K(d) = 3.7 +/- 0.31 nM) and FVIIIa interacts with an additional 300-500 sites per pl

18 , in the presence of Ca2+, phospholipid, and FVIIIa, binding of Na+ to FIXa increases its biologic ac

19 APC cleavage site variants (FVIIIa-R336Q and FVIIIa-R562Q) demonstrated that FIXa and FX can protect

20 oximately 20% increased FVIII stability, and FVIIIa did not decay during the time course measured.

21 led near normal values of Km(app) and Kd(app)FVIIIa for all mutants, indicating normal substrate and

22 t A2 dissociation was 3-fold faster for both FVIIIa mutants compared to FVIIIa wild-type.

23 del whereby decay of FXase results from both FVIIIa subunit dissociation and FIXa-catalyzed cleavage,

24 NP-2 is most potent in the presence of both FVIIIa and phospholipids (artificial phospholipid vesicl

25 APC cleavage (FVIIIa-R336Q,R562Q), or both (FVIIIa-R336Q,R562Q/D519V,E665V), in biochemical assays a

26 the interprotein affinity is contributed by FVIIIa subunits other than A3-C1-C2 in the membrane-depe

27 ation (FVIIIa-D519V,E665V) and APC cleavage (FVIIIa-R336Q,R562Q), or both (FVIIIa-R336Q,R562Q/D519V,E

28 ctivated platelet surface or to the cofactor FVIIIa by interfering with the assembly of FX-activating

29 agulant function when bound to its cofactor, FVIIIa.

30 rine protease, FIXa, and a protein cofactor, FVIIIa, assembled on a phospholipid surface.

31 mains are separate subunits in the cofactor, FVIIIa.

32 1818 but not FVIII-N1810C showed a decreased FVIIIa half-life.

33 -exposed in dissociated activated FVIII (dis-FVIIIa), may contribute to interdomain interactions.

34 d in FVIII and exposed to the surface in dis-FVIIIa.

35 Ia variants to probe A2-domain dissociation (FVIIIa-D519V,E665V) and APC cleavage (FVIIIa-R336Q,R562Q

36 To evaluate FVIIIa stability, the FVIII/FV chimeras were activated b

37 es, identified this interface as an extended FVIIIa-interactive patch.

38 ty binding sites for blood coagulation FIXa, FVIIIa, and FX.

39 increases the concentrations (EC50) of FIXa, FVIIIa, and phospholipid vesicles required for half-maxi

40 ndicated that 3 helices are involved in FIXa-FVIIIa assembly.

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

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

43 xcess was not protective of FIXa unless FIXa/FVIIIa interacted prior to ZPI exposure.

44 ed the efficiency of ZPI inhibition of FIXa; FVIIIa in molar excess was not protective of FIXa unless

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

46 of vesicles (K(i) approximately 1.6K(d) for FVIIIa-FIXa).

47 se domain increases the affinity of FIXa for FVIIIa approximately 15-fold.

48 e further increases the affinity of FIXa for FVIIIa fourfold and k(cat) threefold.

49 A model is proposed for FVIIIa and factor IXa assembly within the membrane-bound

50 Thrombin -activated FVIII (FVIIIa) binds to activated platelets with a Kd of 1.7 nM

51 echanisms that downregulate activated FVIII (FVIIIa) to inform disease pathology and therapeutic drug

52 G were released, generating activated FVIII (FVIIIa) with the same primary structure and specific act

53 The activated form of FVIII, FVIIIa, functions as a cofactor for FIXa in catalyzing t

54 FXa also cleaves FVIII/FVIIIa at Arg(336) and Arg(562) resulting in inactivatio

55 However, we know very little about how FVIIIa binding interactions with FIXa and FX affect FVII

56 Ia A2 subunit and Al/A3-Cl-C2 dimer and (ii) FVIIIa inactivation resulting from FIXa-catalyzed proteo

57 dues were mutated, in binding to immobilized FVIIIa A1/A3C1C2 or LC indicated ~4-10-fold increases in

58 of C2 resulted in significant reductions in FVIIIa stability ( approximately 3.6-fold).

59 ing interaction in FVIII that is retained in FVIIIa.

60 have suggested APC plays a marginal role in FVIIIa regulation.

61 for the FVIII MoAb, ESH8) failed to inhibit FVIIIa binding.

62 owever, the capacity for A3-C1-C2 to inhibit FVIIIa-dependent FXa generation in the presence of phosp

63 332 (epitope for FVIII MoAb, ESH4) inhibited FVIIIa binding to platelets, whereas MoAb ESH8 and a C2

64 FIXa), FXase decay is governed by the inter-FVIIIa subunit affinity and residual activity approaches

65 ates the extrinsic (TF:FVIIa) and intrinsic (FVIIIa:FIXa) pathways of coagulation.

66 Fluorescein-labeled FVIIIa was competed much more effectively by C1C2 than C

67 approximately 3,000 for fluorescein-labeled FVIIIa.

68 of FIXa and FX but does not directly mediate FVIIIa binding to the platelet surface.

69 Furthermore, kinetic analysis monitoring FVIIIa inactivation by APC variants at varying FVIIIa su

70 ry structure and specific activity as native FVIIIa.

71 ial rate of decay of FXase containing native FVIIIa increased (0.82 min(-1)) and paralleled the rate

72 a notoriously inefficient enzyme that needs FVIIIa to drive its hemostatic potential, by a mechanism

73 At low reactant concentrations (0.5 nm FVIIIa; 5 nm FIXa), FXase decay is governed by the inter

74 ibits FVIII binding (K(i) = 0.54 nM) but not FVIIIa binding; (b) thrombin and the thrombin receptor a

75 dues 499-505 replaced by residues 555-561 of FVIIIa, which differ at five of seven positions.

76 In the presence and absence of FVIIIa, a 2- to 10-fold reduced V(max) of FX activation

77 vation of FX (in the presence and absence of FVIIIa, respectively): FIXa(N) (0.46 +/- 0.05, 1.40 +/-

78 re as follows in the presence and absence of FVIIIa, respectively: FIXa(N) (0.55 +/- 0.06, 2.9 +/- 0.

79 increases the stoichiometry and affinity of FVIIIa binding to activated platelets only in the presen

80 C1-C2 subunits contribute to the affinity of FVIIIa for FIXa in the membrane-dependent FXase.

81 contribute significantly to the affinity of FVIIIa for FIXa.

82 telets was inhibited, but not the binding of FVIIIa.

83 A3-C1-C2 was an effective competitor of FVIIIa binding to FIXa as judged by inhibition of FXa ge

84 Low concentrations of FVIIIa increased the efficiency of ZPI inhibition of FIX

85 However, the in vivo contribution of FVIIIa inactivation by APC is unexplored.

86 The rate of decay of FVIIIa activity was monitored at 23 degrees C following

87 ithin residues 2303-2332 in the C2 domain of FVIIIa, and an additional site within residues 2248-2285

88 ta also demonstrate that APC inactivation of FVIIIa exceeds FVIII, suggesting differential APC recogn

89 es in rates of FXa-catalyzed inactivation of FVIIIa, which paralleled the rates of proteolysis at Arg

90 e A2 subunit correlates with inactivation of FVIIIa.

91 ; and (d) Annexin V is a potent inhibitor of FVIIIa binding (IC(50) = 10 nM) to activated platelets.

92 Inspection of FVIIIa-specific changes indicated that 3 helices are inv

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

94 residues stabilize the A domain interface of FVIIIa.

95 ecays with time and reflects the lability of FVIIIa.

96 ) and the absence (K(i) = 1.5 x 10(-6) m) of FVIIIa and FX.

97 Data support that both mechanisms of FVIIIa inactivation and FIXa interactions could be lever

98 C) proteolysis are established mechanisms of FVIIIa inactivation.

99 itutions can silence the extended network of FVIIIa-driven allosteric changes.

100 )-catalyzed FX activation in the presence of FVIIIa and phospholipid vesicles, characteristic of a hy

101 n Km both manifested only in the presence of FVIIIa.

102 in the rate of FIXa-catalyzed proteolysis of FVIIIa.

103 educed (~11-fold), whereas the decay rate of FVIIIa due to A2 subunit dissociation was similar to WT

104 d Western blotting, used to monitor rates of FVIIIa inactivation and proteolysis at the primary cleav

105 , suggesting differential APC recognition of FVIIIa relative to FVIII.

106 probe had no effect on the reconstitution of FVIIIa from the A1/A3-C1-C2 dimer and A2 subunit.

107 APC contributes to the in vivo regulation of FVIIIa, which has the potential to be exploited to devel

108 an opposite contribution to the stability of FVIIIa.

109 increases the affinity and stoichiometry of FVIIIa binding to platelets and contributes to the stabi

110 The isolated A2 subunit of FVIIIa interacts weakly with FIXa, and recent modeling s

111 re, these mutations within the A1 subunit of FVIIIa introduce a similar destabilization of the FVIIIa

112 for the isolated A2 and A3-C1-C2 subunits of FVIIIa, suggesting that the A1 subunit of FVIII contains

113 Titration of FVIIIa in FXa generation assays showed that the mutant a

114 ated platelets, and the presence of FVIII or FVIIIa generates a high affinity, low capacity specific

115 t FX activation when either phospholipids or FVIIIa are present, but not in the absence of both facto

116 ependent thrombin generation, but preserving FVIIIa generation by nascent FXa, can support intrinsic

117 Q) demonstrated that FIXa and FX can protect FVIIIa from APC cleavage at Arg562 and Arg336, respectiv

118 with or without protein S, whereas FVIII-QQ/FVIIIa-QQ did not.

119 Here, we investigate this using recombinant FVIIIa variants to probe A2-domain dissociation (FVIIIa-

120 (8- to 26-fold) compared with reconstituting FVIIIa (1.3- to 6-fold) suggesting that the mutations al

121 and phospholipid, with or without saturating FVIIIa, FIXa(Y225P) activated FX with similar K(m) but t

122 The stable FVIIIa described here provides the opportunity to study

123 In the present study, FVIIIa stability and FIXa binding were evaluated in a FV

124 lack of FXa generation via intrinsic tenase (FVIIIa/FIXa) complexes.

125 The FVIIIa A2 subunit bound FIXa with high affinity (Kd = 3.

126 APC bound to FVIII light chain (LC) and the FVIIIa A1/A3C1C2 dimer with equivalent affinity (Kd = 52

127 (i) a weak affinity interaction between the FVIIIa A2 subunit and Al/A3-Cl-C2 dimer and (ii) FVIIIa

128 cate a high affinity interaction between the FVIIIa A2 subunit and FIXa and show a contribution of se

129 To define the FVIIIa domains that mediate platelet interactions, album

130 heavy chain (contiguous A1-A2 domains), the FVIIIa-derived A1/A3-C1-C2 dimer, and the isolated A1 su

131 the serine-protease Factor IXa (FIXa) in the FVIIIa-FIXa complex assembled on the activated platelet

132 a introduce a similar destabilization of the FVIIIa heterotrimer compared to the (ARG)531(HIS) mutati

133 by this residue of approximately 10% of the FVIIIa-FIXa binding energy.

134 p towards understanding the mechanism of the FVIIIa-FIXa complex assembly on the activated platelet s

135 Factor (F)VIII can be activated to FVIIIa by FXa following cleavages at Arg(372), Arg(740),

136 ain and increased the contribution of APC to FVIIIa inactivation.

137 We found that FIXa binding to FVIIIa stabilized the A2 domain and increased the contri

138 d faster for both FVIIIa mutants compared to FVIIIa wild-type.

139 egions 1803-1810 and 1811-1818 contribute to FVIIIa stability.

140 FVa is homologous to FVIIIa, the cofactor for the FIXa protease, in the FX-ac

141 Unlike FVIIIa, the C2 domain did not respond to the presence of

142 using individual APC cleavage site variants (FVIIIa-R336Q and FVIIIa-R562Q) demonstrated that FIXa an

143 IIIa inactivation by APC variants at varying FVIIIa substrate concentration showed ~2.6-4.4-fold incr

144 s thought to regulate activated factor VIII (FVIIIa) cofactor function include A2-domain dissociation

145 Activated factor VIII (FVIIIa) forms a procoagulant complex with factor IXa on

146 a) with its cofactor, activated factor VIII (FVIIIa) is a crucial event in the coagulation cascade.

147 Thrombin-activated factor VIII (FVIIIa) is a heterotrimer with the A2 subunit (amino aci

148 idly dissociates from activated factor VIII (FVIIIa) resulting in a dampening of the activity of the

149 2 domain retention in activated factor VIII (FVIIIa).

150 mportant negative regulator of factor VIIIa (FVIIIa) cofactor activity is A2 subunit dissociation.

151 hances the factor Va (FVa) and factor VIIIa (FVIIIa) inactivating property of activated protein C (AP

152 mined that R338A-FIXa's Kd for factor VIIIa (FVIIIa) was similar to that of wt-FIXa.

153 uiring Ca2+, phospholipid, and factor VIIIa (FVIIIa).

154 ation and APC cleavage contribute to in vivo FVIIIa regulation.

155 vestigate how FIXa responds to assembly with FVIIIa in the presence of phospholipids.

156 ADP is inert; (c) FVa does not compete with FVIIIa or FVIII for functional platelet-binding sites; a

157 y a significant role in its interaction with FVIIIa.

158 as were EC(50) values for interactions with FVIIIa.

159 additional experiments, FIXa with or without FVIIIa activated FX(WT) and FX(PCEGF1) normally, which i

160 wt-FIXa had equal activity, with or without FVIIIa, toward the synthetic substrate, methylsulfonyl-D

161 y, factor IXa (FIXa) binding affinity for WT FVIIIa was significantly reduced in the presence of GMA8

162 The activity of WT FVIIIa was inhibited by both GMA8011 and ESH4, whereas t

163 to A2 subunit dissociation was similar to WT FVIIIa.

164 In plasma-based studies, FVIII-WT/FVIIIa-WT demonstrated dose-dependent sensitivity to APC

165 nized TF-initiated pathway directly yielding FVIIIa-FIXa intrinsic tenase complex may be prohemostati