title: Notecarin D binds human factor V and factor Va with high affinity in the absence of membranes.
abstract: Notecarin D (NotD) is a prothrombin (ProT) activator in the venom of the tiger snake, Notechis scutatus, and a factor Xa (FXa) homolog. NotD binds specifically to the FXa binding site expressed on factor V (FV) upon activation to factor Va (FVa) by thrombin. NotD active site-labeled with 5-fluorescein ([5F]FFR-NotD) binds FV and FVa with remarkably high affinity in the absence of phospholipids (K(D) 12 and ≤ 0.01 nm, respectively). In the presence of membranes, the affinity of [5F]FFR-NotD for FVa is similar, but increased ∼55-fold for FV. Binding of FXa active site-labeled with Oregon Green to FV and FVa in the presence of phospholipids is ∼5,000- and ∼80-fold weaker than [5F]FFR-NotD, respectively. NotD reports FVa and not FV binding by a 3-fold increase in tripeptide substrate hydrolysis, demonstrating allosteric regulation by FVa. The NotD·FVa·membrane complex activates ProT with K(m)((app)) similar to prothrombinase, and ∼85-fold weaker without membranes. Active site-blocked NotD exhibits potent anticoagulant activity in plasma thrombin generation assays, representing inhibition of productive prothrombinase assembly and possible disruption of FXa inhibition by the tissue factor pathway inhibitor. The results show that high affinity binding of NotD to FVa is membrane-independent, unlike the strict membrane dependence of FXa for high affinity FVa binding.
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2561.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Newell-Caito
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2561.
authors.forename: Malabika
authors.initials: M
authors.lastname: Laha
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2561.
authors.forename: Anthony C
authors.initials: AC
authors.lastname: Tharp
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2561.
authors.forename: Jonathan I
authors.initials: JI
authors.lastname: Creamer
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2561.
authors.forename: Hong
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2561.
authors.forename: Ashoka A
authors.initials: AA
authors.lastname: Maddur
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, 6200MD Maastricht, The Netherlands.
authors.forename: Guido
authors.initials: G
authors.lastname: Tans
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2561. Electronic address: paul.bock@vanderbilt.edu.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Bock
mesh_terms: D016880:Anisotropy; D001777:Blood Coagulation; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism; D004546:Elapid Venoms / Q000737:chemistry*; D005165:Factor V / Q000737:chemistry*; D015943:Factor Va / Q000737:chemistry*; D015951:Factor Xa / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D010455:Peptides / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding
chemical_list: D004546:Elapid Venoms; D010455:Peptides; D010743:Phospholipids; C472032:notecarin D; D015943:Factor Va; D005165:Factor V; D015951:Factor Xa