ライフサイエンスコーパス: FVa

1 FVa is homologous to FVIIIa, the cofactor for the FIXa p

2 FVa is known to effect a major conformational change to

3 FVa709 (des710-1545), FVa699 (des700-1545), FVa(692 (des693-1545), FVa678 (des679-1545), and FVa658

4 Tightly associated factor V(a) (FVa) and factor X(a) (FXa) serve as the essential prothr

5 c region, which is retained in FXa-activated FVa and platelet FVa.

6 S (C6PS) triggers assembly of a fully active FVa-FXa complex in solution and (2) that 2 molecules of

7 membrane dependence of FXa for high affinity FVa binding.

8 oop on APC of residues 306 to 314 defines an FVa binding site and accounts for much of the difference

9 in this loop in interactions between APC and FVa.

10 romboplastin time (APTT) clotting assays and FVa inactivation assays.

11 rhFVIII inactivation by rAPC by 6.4-fold and FVa inactivation by twofold, whereas membrane-bound FV s

12 Our data suggest that (1) FV and FVa are the physiologically significant substrates for A

13 ive site-labeled with Oregon Green to FV and FVa in the presence of phospholipids is approximately 5,

14 th 5-fluorescein ([5F]FFR-NotD) binds FV and FVa with remarkably high affinity in the absence of phos

15 d that the Arg(306) cleavage sites of FV and FVa(IIa) are distinct substrates for APC.

16 urprisingly modest decrease in bound FXa and FVa with little impact on fibrin formation.

17 s demonstrate that the dimerization site and FVa-binding site are both located in the catalytic domai

18 Because FVa heavy chain binds 2 molecules of C6PS, we conclude t

19 to promote high affinity association between FVa and FXa.

20 -1 bind FV-1033 with K(d) ~36-48 nM and bind FVa with K(d) ~252-456 nM.

21 idues 142-153) has been hypothesized to bind FVa.

22 leted FV (FV-1033) with K(d) ~9 nM and binds FVa with K(d) ~100 nM.

23 Although bound FVa and FXa may have been present on the platelet core a

24 ; second, the inactivation of membrane-bound FVa by APC; and third, the proteolytic inactivation of m

25 litating factor Xa binding to membrane-bound FVa.

26 ysis, demonstrating allosteric regulation by FVa.

27 g site expression, whereas ADP is inert; (c) FVa does not compete with FVIIIa or FVIII for functional

28 raction, we expressed and purified two-chain FVa derivatives that were intracellularly truncated at t

29 nsisting of serine protease FXa and cofactor FVa, anchored to anionic phospholipids on the surface of

30 anisms are activated to produce its cofactor FVa, FXa(I16L) is driven to the protease state and resto

31 B-domain converts it to an active cofactor (FVa) for factor Xa (FXa).

32 e Factor (F) Xa complexed with its cofactor, FVa.

33 phosphoserine present in the plasma-derived FVa heavy chain and was resistant to phosphorylation at

34 ed inactivation than purified plasma-derived FVa on the thrombin-activated platelet surface.

35 dentical to that of purified, plasma-derived FVa.

36 allows distinction between platelet-derived FVa and FVaLeiden subsequent to APC-catalyzed cleavage w

37 chain subunit of purified, platelet-derived FVa contained only a fraction ( approximately 10-15%) of

38 analyses of the APC-cleaved platelet-derived FVa from FW showed a wild-type phenotype, despite the pr

39 duced cleavage, analyses of platelet-derived FVa from JMW demonstrated both normal FVa and FVaLeiden

40 Platelet-derived FVa released from thrombin-activated platelets from a no

41 Purified, platelet-derived FVa was 2-3-fold more resistant to activated protein C-c

42 ed origin of the secretable platelet-derived FVa.

43 ma origin of her secretable platelet-derived FVa.

44 ctivity exhibited by native platelet-derived FVa.

45 shed work, these results define an extensive FVa binding site in the positive exosite of APC that is

46 membranes, the affinity of [5F]FFR-NotD for FVa is similar, but increased approximately 55-fold for

47 w that TFPI-2 is associated with platelet FV/FVa.

48 them, including factors IXa (FIXa), FXa/FX, FVa, FVIII, prothrombin, and PS-sensitive marker Annexin

49 rine (C6PS), binds to discrete sites on FXa, FVa, and prothrombin to alter their conformations, to pr

50 nd the structure of the FXa dimer or the FXa-FVa complex.

51 directly inhibits thrombin generated by FXa/FVa (prothrombinase complex).

52 n a "common" pathway at the level of the FXa/FVa (prothrombinase) complex.

53 e, 0.30 +/- 0.05 and 0.19 +/- 0.04, when FXa/FVa is 1:4, with an increasing FXa and substrate concent

54 assays and APTT assays using purified Gln506-FVa and plasma containing Gln506-FV, it appeared that th

55 Surprisingly, however, FVa(658) exhibited essentially normal kinetic parameters

56 ctivation by alpha-thrombin, factor Va(IIa) (FVa(IIa)).

57 ly contribute to APC cleavage at Arg(506) in FVa and play a small role in the interaction of APC with

58 study the effects of individual cleavages in FVa by APC and the importance of regions near the cleava

59 avage rates at the 2 major cleavage sites in FVa.

60 ssays and in prothrombinase assays measuring FVa residual activity, in agreement with studies of puri

61 A mixture of 50 nM FXa and 50 nM FVa in the presence of 400 muM C6PS yielded both Xa homo

62 erived FVa from JMW demonstrated both normal FVa and FVaLeiden consistent with a plasma-derived origi

63 thesis that efficient inactivation of normal FVa by APC requires cleavage at R306.

64 yzed cleavage and inactivation of FV but not FVa(IIa) at position Arg(306) and that the Arg(306) clea

65 The NotD.FVa.membrane complex activates ProT with K(m)((app)) sim

66 activity of FXa and the cofactor activity of FVa.

67 with functional assays, similar analyses of FVa(IIa), derived from those FV species, revealed near-i

68 d ability to enhance APC-induced cleavage of FVa Arg306.

69 ants were primarily impaired for cleavage of FVa at Arg506.

70 mM Ca(2+) to show that the apparent K(d) of FVa-FXa interaction increased with an increase in FXa co

71 of site-specific fluorescent derivatives of FVa and FXa after laser injury in the mouse cremaster ar

72 ge site for normal efficient inactivation of FVa by APC and supports other studies suggesting that re

73 rs essential for significant inactivation of FVa by APC.

74 nalyses of the APC-catalyzed inactivation of FVa(IIa) in an assay consisting of purified components i

75 and characterized for their inactivation of FVa.

76 is lost upon removal of the acidic region of FVa by thrombin.

77 olved in binding and cleaving at Arg(506) on FVa.

78 Based on FVa inactivation assays and APTT assays using purified G

79 rothrombinase complex by synthetic peptides, FVa residues 493-506 were proposed as a FXa binding site

80 ein C by thrombin and inactivation of plasma FVa by APC are not impaired during moderate hyperhomocys

81 s retained in FXa-activated FVa and platelet FVa.

82 with studies of purified plasma-derived Q506-FVa.

83 mparable with plasma-derived and recombinant FVa.

84 S binding exposes K(351) (part of a reported FVa binding region), K(242) (adjacent to the catalytic t

85 NotD reports FVa and not FV binding by a 3-fold increase in tripeptid

86 lu-Gly-Arg-chloromethyl ketone fully rescued FVa binding.

87 lpha-thrombin, the addition of the resulting FVa(IIa) to the plasma-based APC sensitivity assay produ

88 and R506 provide FXa-binding sites and that FVa cleaved at only R506 retains partial activity.

89 To test the hypothesis that FVa residues 499-505 contribute to FXa binding, we creat

90 idly than activity was lost, suggesting that FVa cleaved at only R506 is partially active.

91 05 contribute to FXa binding, we created the FVa loop swap mutant (designated 499-505(VIII) FV) with

92 n an updated three-dimensional model for the FVa structure, residues 499-505, along with Arg-506, Arg

93 suggest that this A2 domain sequence of the FVa and FVIIIa cofactors evolved to have different speci

94 prothrombin and the C-terminal region of the FVa heavy chain do not contribute in a detectable way to

95 ormed prothrombinase complex containing this FVa mutant had fairly normal kinetic parameters (k(cat)

96 surements and spectroscopic titrations, this FVa loop swap mutant had significantly reduced affinity

97 an inactive procofactor and is activated to FVa by proteolytic removal of a large inhibitory B-domai

98 ion and (2) that 2 molecules of C6PS bind to FVa light chain with one occupying a site in the C2 doma

99 ether dimerization of FXa and its binding to FVa in the presence of C6PS are competitive processes.

100 s that play a significant role in binding to FVa.

101 Xa-I16T), was greatly enhanced when bound to FVa membranes.

102 .Va heterodimers, but no FXa dimers bound to FVa.

103 dues made significant minor contributions to FVa interactions: Lys(191), Lys(192), Asp(214), and Glu(

104 entified that provide major contributions to FVa interactions: Lys(193), Arg(229), and Arg(230).

105 , and FVIIIa residues 555-561 (homologous to FVa residues 499-506) are recognized as a FIXa binding s

106 s show that high affinity binding of NotD to FVa is membrane-independent, unlike the strict membrane

107 that both glycosidase-treated and untreated FVa(IIa) expressed identical cofactor activities and wer

108 In a purified assay using FVa R506Q/R679Q, purified protein S D95A was shown to ha

109 in in the presence and absence of factor Va (FVa) and 5.0 x 10(-5) M phospholipid vesicles are slight

110 nt pathway, where it enhances the factor Va (FVa) and factor VIIIa (FVIIIa) inactivating property of

111 tion of the coagulation cofactors factor Va (FVa) and factor VIIIa.

112 n of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme acti

113 vated protein C (APC) cleavage of Factor Va (FVa) at residues R506 and R306 correlates with its inact

114 quence reported to interfere with factor Va (FVa) binding.

115 factor V (FV) upon activation to factor Va (FVa) by thrombin.

116 teraction of factor Xa (FXa) with factor Va (FVa) forms prothrombinase and drives thrombin formation

117 Platelet- and plasma-derived factor Va (FVa) serve essential cofactor roles in prothrombinase-ca

118 Factor Va (FVa), derived from plasma or released from stimulated pl

119 used by decreased inactivation of factor Va (FVa).

120 ), inactivates blood coagulation factors Va (FVa) and VIIIa.

121 activated protein C (APC), the 499-505(VIII) FVa mutant was inactivated entirely normally by APC.

122 ed dimerization (K(d) ~ 147 nM) and weakened FVa binding (apparent K(d) values of 58, 92, and 128 nM

123 When FVa residual activity was measured after long exposure t

124 neither dimerized nor formed a complex with FVa in the presence of 400 muM C6PS and 5 mM Ca(2+).

125 1.6 +/- 0.3, when FXa is in a 1:1 ratio with FVa but becomes increasingly inverse, 0.30 +/- 0.05 and

126 at 5, 20, and 50 nM FXa while titrating with FVa in the presence of 400 muM C6PS and 3 or 5 mM Ca(2+)