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

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

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

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

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

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

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

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

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

15 and affinity parameters in forming FXase and FVIIIa.

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

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

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

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

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

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

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

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

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

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

26 mains are separate subunits in the cofactor, FVIIIa.

27 1818 but not FVIII-N1810C showed a decreased FVIIIa half-life.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

49 ing interaction in FVIII that is retained in FVIIIa.

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

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

52 332 (epitope for FVIII MoAb, ESH4) inhibited FVIIIa binding to platelets, whereas MoAb ESH8 and a C2

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

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

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

56 approximately 3,000 for fluorescein-labeled FVIIIa.

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

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

59 ry structure and specific activity as native FVIIIa.

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

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

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

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

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

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

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

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

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

69 contribute significantly to the affinity of FVIIIa for FIXa.

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

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

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

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

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

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

76 e A2 subunit correlates with inactivation of FVIIIa.

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

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

79 residues stabilize the A domain interface of FVIIIa.

80 ecays with time and reflects the lability of FVIIIa.

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

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

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

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

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

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

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

88 an opposite contribution to the stability of FVIIIa.

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

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

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

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

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

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

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

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

97 (8- to 26-fold) compared with reconstituting FVIIIa (1.3- to 6-fold) suggesting that the mutations al

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

99 The stable FVIIIa described here provides the opportunity to study

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

120 2 domain retention in activated factor VIII (FVIIIa).

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

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

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

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

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

126 y a significant role in its interaction with FVIIIa.

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

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

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

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

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

132 to A2 subunit dissociation was similar to WT FVIIIa.

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