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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
6 S (C6PS) triggers assembly of a fully active FVa-FXa complex in solution and (2) that 2 molecules of
9 oop on APC of residues 306 to 314 defines an FVa binding site and accounts for much of the difference
12 rhFVIII inactivation by rAPC by 6.4-fold and FVa inactivation by twofold, whereas membrane-bound FV s
14 ive site-labeled with Oregon Green to FV and FVa in the presence of phospholipids is approximately 5,
15 th 5-fluorescein ([5F]FFR-NotD) binds FV and FVa with remarkably high affinity in the absence of phos
18 s demonstrate that the dimerization site and FVa-binding site are both located in the catalytic domai
25 ; second, the inactivation of membrane-bound FVa by APC; and third, the proteolytic inactivation of m
28 g site expression, whereas ADP is inert; (c) FVa does not compete with FVIIIa or FVIII for functional
29 raction, we expressed and purified two-chain FVa derivatives that were intracellularly truncated at t
30 nsisting of serine protease FXa and cofactor FVa, anchored to anionic phospholipids on the surface of
31 anisms are activated to produce its cofactor FVa, FXa(I16L) is driven to the protease state and resto
34 phosphoserine present in the plasma-derived FVa heavy chain and was resistant to phosphorylation at
37 allows distinction between platelet-derived FVa and FVaLeiden subsequent to APC-catalyzed cleavage w
38 chain subunit of purified, platelet-derived FVa contained only a fraction ( approximately 10-15%) of
39 analyses of the APC-cleaved platelet-derived FVa from FW showed a wild-type phenotype, despite the pr
40 duced cleavage, analyses of platelet-derived FVa from JMW demonstrated both normal FVa and FVaLeiden
46 shed work, these results define an extensive FVa binding site in the positive exosite of APC that is
47 membranes, the affinity of [5F]FFR-NotD for FVa is similar, but increased approximately 55-fold for
48 f FV to APC, advance our understanding of FV/FVa regulation, and establish a mechanistic framework fo
50 them, including factors IXa (FIXa), FXa/FX, FVa, FVIII, prothrombin, and PS-sensitive marker Annexin
51 rine (C6PS), binds to discrete sites on FXa, FVa, and prothrombin to alter their conformations, to pr
55 e, 0.30 +/- 0.05 and 0.19 +/- 0.04, when FXa/FVa is 1:4, with an increasing FXa and substrate concent
56 n, including prothrombinase (factor Xa [FXa]/FVa), the catalytic complex that directly generates thro
57 PS on their cell membranes, thus generating FVa and FXa binding sites and mediating the formation of
58 assays and APTT assays using purified Gln506-FVa and plasma containing Gln506-FV, it appeared that th
61 ly contribute to APC cleavage at Arg(506) in FVa and play a small role in the interaction of APC with
62 study the effects of individual cleavages in FVa by APC and the importance of regions near the cleava
66 ssays and in prothrombinase assays measuring FVa residual activity, in agreement with studies of puri
68 erived FVa from JMW demonstrated both normal FVa and FVaLeiden consistent with a plasma-derived origi
70 yzed cleavage and inactivation of FV but not FVa(IIa) at position Arg(306) and that the Arg(306) clea
73 with functional assays, similar analyses of FVa(IIa), derived from those FV species, revealed near-i
74 ions, we investigate the membrane binding of FVa and identify the key mechanisms that govern its inte
77 mM Ca(2+) to show that the apparent K(d) of FVa-FXa interaction increased with an increase in FXa co
78 of site-specific fluorescent derivatives of FVa and FXa after laser injury in the mouse cremaster ar
79 isplaces a large portion of the A2 domain of FVa and projects the 654VKCIPDDDEDSYEIFEP670 segment as
80 main of APC engages R506 in the A2 domain of FVa through electrostatic interactions between positivel
81 ate the complex membrane binding dynamics of FVa and provide important insights into the molecular me
82 ge site for normal efficient inactivation of FVa by APC and supports other studies suggesting that re
84 nalyses of the APC-catalyzed inactivation of FVa(IIa) in an assay consisting of purified components i
86 further find that the domain organization of FVa deviates (sometimes significantly) from its crystall
88 c electron microscopy (cryo-EM) structure of FVa has revealed the arrangement of its A1-A2-A3-C1-C2 d
92 rothrombinase complex by synthetic peptides, FVa residues 493-506 were proposed as a FXa binding site
93 ein C by thrombin and inactivation of plasma FVa by APC are not impaired during moderate hyperhomocys
97 S binding exposes K(351) (part of a reported FVa binding region), K(242) (adjacent to the catalytic t
100 lpha-thrombin, the addition of the resulting FVa(IIa) to the plasma-based APC sensitivity assay produ
105 interactions in the coagulation cascade, the FVa-APC interaction has long posed a challenge to struct
106 05 contribute to FXa binding, we created the FVa loop swap mutant (designated 499-505(VIII) FV) with
107 n an updated three-dimensional model for the FVa structure, residues 499-505, along with Arg-506, Arg
108 suggest that this A2 domain sequence of the FVa and FVIIIa cofactors evolved to have different speci
109 prothrombin and the C-terminal region of the FVa heavy chain do not contribute in a detectable way to
110 Here, we report the cryo-EM structure of the FVa-APC complex at 3.15 angstrom resolution in which the
112 ormed prothrombinase complex containing this FVa mutant had fairly normal kinetic parameters (k(cat)
113 surements and spectroscopic titrations, this FVa loop swap mutant had significantly reduced affinity
114 an inactive procofactor and is activated to FVa by proteolytic removal of a large inhibitory B-domai
116 ion and (2) that 2 molecules of C6PS bind to FVa light chain with one occupying a site in the C2 doma
117 ether dimerization of FXa and its binding to FVa in the presence of C6PS are competitive processes.
119 was decreased ~10-fold when FXa was bound to FVa in prothrombinase and a further ~3-4-fold when plasm
122 APC inactivation of FV is slower compared to FVa, although proteolysis occurs at the same sites (Arg(
123 dues made significant minor contributions to FVa interactions: Lys(191), Lys(192), Asp(214), and Glu(
124 entified that provide major contributions to FVa interactions: Lys(193), Arg(229), and Arg(230).
125 , and FVIIIa residues 555-561 (homologous to FVa residues 499-506) are recognized as a FIXa binding s
126 s show that high affinity binding of NotD to FVa is membrane-independent, unlike the strict membrane
127 ing process and characteristics, specific to FVa or common among other membrane proteins, in concert
129 that both glycosidase-treated and untreated FVa(IIa) expressed identical cofactor activities and wer
132 in in the presence and absence of factor Va (FVa) and 5.0 x 10(-5) M phospholipid vesicles are slight
133 nt pathway, where it enhances the factor Va (FVa) and factor VIIIa (FVIIIa) inactivating property of
135 n of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme acti
136 vated protein C (APC) cleavage of Factor Va (FVa) at residues R506 and R306 correlates with its inact
139 teraction of factor Xa (FXa) with factor Va (FVa) forms prothrombinase and drives thrombin formation
143 Platelet- and plasma-derived factor Va (FVa) serve essential cofactor roles in prothrombinase-ca
147 tially regulate APC recognition of FV versus FVa and uncover how FV-short can be protected from this
148 activated protein C (APC), the 499-505(VIII) FVa mutant was inactivated entirely normally by APC.
149 ed dimerization (K(d) ~ 147 nM) and weakened FVa binding (apparent K(d) values of 58, 92, and 128 nM
152 neither dimerized nor formed a complex with FVa in the presence of 400 muM C6PS and 5 mM Ca(2+).
154 1.6 +/- 0.3, when FXa is in a 1:1 ratio with FVa but becomes increasingly inverse, 0.30 +/- 0.05 and
155 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+)