Coagulation factor Va is an actin filament binding and cross-linking protein

1995 ◽  
Vol 73 (1-2) ◽  
pp. 105-112 ◽  
Author(s):  
Emilia Furmaniak-Kazmierczak ◽  
Michael E. Nesheim ◽  
Graham P. Côté
Keyword(s):  
Actin Filament ◽  
Light Chain ◽  
Actin Filaments ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Actin Binding ◽  
Cross Linking ◽  
Factor V ◽  
Proteolytic Activation ◽  
Factor Va

Bovine coagulation cofactor factor Va is shown to bind to filaments of skeletal muscle actin with a dissociation constant of 40–50 nM in the presence of 50 mM NaCl. At saturation, approximately one molecule of factor Va was bound for every two actin molecules. The binding of factor Va to F-actin was inhibited by increasing ionic strength, being approximately 20-fold weaker at 150 mM NaCl. Addition of factor Va dramatically increased both the low-speed sedimentation and the low-shear viscosity of actin filament solutions, indicating that factor Va cross-links actin filaments. Factor Va also bound to actin filaments saturated with myosin. The isolated 74-kilodalton light chain of factor Va displayed actin binding and cross-linking properties indistinguishable from those of intact factor Va. The procofactor factor V bound weakly to F-actin, indicating that proteolytic activation is required to uncover the actin binding sites within the light chain domain. Actin filaments had only a slight inhibitory effect on the prothombinase activity of the factor Va – factor Xa – phospholipid complex. Since high concentrations of actin filaments can be exposed to the circulation when cells are damaged, the interaction of factor Va with actin may be of physiological relevance.Key words: blood coagulation, factor V, actin.

scholarly journals Platelet coagulation factor Va: the major secretory platelet phosphoprotein

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2180-2190
Author(s):  
MD Rand ◽  
M Kalafatis ◽  
KG Mann
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Activated Protein C ◽  
Coagulation Factor ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Factor V ◽  
Platelet Factor ◽  
Factor Va ◽  
Plasma Factor

Platelet-derived coagulation factor Va is the primary secreted substrate for a thrombin-stimulation-dependent platelet kinase. Human platelet factor Va, consisting of a molecular weight (M(r)) 105,000 heavy chain and an M(r) 74,000 light chain, incorporates phosphate in at least two sites on the light chain. Phosphorylated factor Va represents 50% of the secreted protein-associated phosphate. This modification occurs exclusively at serine residues and is inhibited by H-7 and staurosporine, which suggests a protein kinase C (PKC)-mediated event. Purified plasma factor V and Va are phosphorylated in the light chain region by rat brain PKC. The activity of platelet factor Va in prothrombinase on platelets is not altered when phosphorylation is inhibited by staurosporine. Plasma-derived factor Va in the presence of thrombin stimulated platelets is phosphorylated on both the heavy chain and the light chain. Plasma factor V and factor Va heavy chain phosphorylation occurs without light chain phosphorylation in the presence of added 32P gamma-ATP and non-stimulated or collagen- stimulated platelets or casein kinase II. This differential phosphorylation of factor Va heavy and light chain shows two independent platelet kinase activities that act on factor Va. The heavy chain factor V/Va kinase activity is similar to casein kinase II, which we have demonstrated previously to act on factor Va and accelerate activated protein C inactivation of the cofactor. Our data show platelet-dependent phosphorylation of platelet and plasma factor V and Va resulting in significant covalent modifications of the cofactor. These modifications may play a role in directing the extracellular distribution of factor V and factor Va.

scholarly journals Bundling of actin filaments by alpha-actinin depends on its molecular length.

1990 ◽  
Vol 110 (6) ◽  
pp. 2013-2024 ◽  
Author(s):  
R K Meyer ◽  
U Aebi
Keyword(s):  
Smooth Muscle ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Molar Ratio ◽  
Cross Linking ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Chicken Gizzard ◽  
Molecular Length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.

Gene duplication of coagulation factor V and origin of venom prothrombin activator in Pseudonaja textilis snake

2005 ◽  
Vol 93 (03) ◽  
pp. 420-429 ◽  
Author(s):  
Thi Nguyet Minh Le ◽  
Md Abu Reza ◽  
Sanjay Swarup ◽  
R. Manjunatha Kini
Keyword(s):  
Cleavage Site ◽  
Activated Protein C ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Venom Gland ◽  
Factor V ◽  
Gene Encoding ◽  
Origin And Evolution ◽  
Functional Sites ◽  
Factor Va

SummaryThe origin and evolution of venom toxins is a mystery that has evoked much interest. We have recently shown that pseutarin C, a prothrombin activator from Pseudonaja textilis venom, is structurally and functionally similar to mammalian coagulation factor Xa – factor Va complex. Its catalytic subunit is homologous to factor Xa while the nonenzymatic subunit is homologous to factor Va. P.textilis therefore has two parallel prothrombin activator systems: one expressed in its venom gland as a toxin and the other expressed in its liver and released into its plasma as a haemostatic factor. Here we report the complete amino acid sequence of factor V (FV) from its liver determined by cDNA cloning and sequencing. The liver FV shows 96% identity to pseutarin C nonenzymatic subunit. Most of the functional sites involved in its interaction with factor Xa and prothrombin are conserved. However, many potential sites of post-translational modifications and one critical cleavage site for activated protein C are different. The absence of the latter cleavage site makes pseutarin C nonenzymatic subunit resistant to inactivation and enhances its potential as an excellent toxin. By PCR and real-time quantitative analysis, we show that pseutarin C nonenzymatic subunit gene is expressed specifically in the venom gland at ~280 fold higher than that of FV gene in liver. These two are thus encoded by two separate genes that express in a highly tissue-specific manner. Our results imply that the gene encoding pseutarin C nonenzymatic subunit was derived by the duplication of plasma FV gene and they have evolved to perform distinct functions.

scholarly journals Activation of Bovine Blood Coagulation Factor V, A Prerequisite for it of Bind Both Prothrombin and Factor Xa.

1977 ◽  
Author(s):  
M.C. Guillin ◽  
A. Bezeaud ◽  
J.P. Freeman ◽  
C.M. Jackson
Keyword(s):  
Factor Xa ◽  
Coagulation Factor ◽  
Factor V ◽  
Factor Va ◽  
Bovine Plasma ◽  
Coagulation Factor V ◽  
Russell’S Viper ◽  
Blood Coagulation Factor V ◽  
Bovine Factor ◽  
Plasma Heparin

It is known that prior to bind bovine prothrombin and to become fully functional, bovine Factor V must itself be “activated” by either thrombin or an enzyme isolable from Russell’s viper venom. The purpose of this work was to determine if Factor V activation is also required in order for it to bind bovine Factor Xa.This has been investigated by measuring the binding of both “native” (unactivated) Factor V and Factor V activated by the Russell’s viper venom activating enzyme, to a column of agarose-bound Factor Xa. The experiments were also performed using diisopropylfluorophosphate (DFP) inhibited Factor Xa covalently bound to agarose. Both purified bovine Factor V (Va) and bovine plasma were used and gave the same results. In order to prevent initiation of clotting in bovine plasma, heparin wad added to the plasma to promote inactivation of Factor Xa by antithrombin III.The results indicate that Factor V activation is a prerequisite for it to bind Factor Xa ; Factor Va binds both Factor Xa and DFP inhibited Factor Xa, unmodified Factor V does not.These experiments suggest that Factor V may not participate in prothrombin activation at all, until after some thrombin has been formed. If this is so, an alternate pathway by which the first thrombin is generated must be considered and may be proposed to be simply that involving Factor Xa, phospholipid and Ca2+ alone.

Identification of the Multimerin Binding Region within the C2 Domain of Human Factor V Using Constructs Generated by Alanine-Scanning and Site-Directed Mutagenesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 124-124
Author(s):  
Samira B. Jeimy ◽  
Rachael A. Woram ◽  
Nola Fuller ◽  
Mary Anne Quinn-Allen ◽  
Gerard Nicolaes ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Light Chain ◽  
Coagulation Factor ◽  
Procoagulant Activity ◽  
Site Directed Mutagenesis ◽  
Factor V ◽  
C2 Domain ◽  
Wild Type ◽  
Factor Va ◽  
Covalently Linked

Abstract Activated coagulation factor V is a key non-enzymatic cofactor that is an essential component of the prothrombinase complex. In blood, much of the procoagulant factor V is stored in platelets, as a complex with the α-granule protein multimerin, for activation-induced release during clot formation. Presently, the molecular nature of multimerin - factor V binding has not been determined, although multimerin is known to interact with the light chain of factor V and Va. Using modified enzyme-linked immunoassays and recombinant factor V constructs, we previously found that discontinuous regions in the C2 domain of factor V were important for binding multimerin, and that these regions overlapped with areas in factor V important for its procoagulant function. Specifically, four (S2183T, W2063A/W2064A, K2060Q/K2061Q, K2060Q/K2061Q/W2063A/ W2064A) full-length, site-directed C2 mutants, and 12 (W2063A, W2064A (W2063, W2064)A, R2074A (R2072, R2074)A (K2101, K2103, K2104)A, L2116A (K2157, H2159, K2161)A, R2171A, R2174A, E2189A (R2187, E2189)A) B domain deleted, charge to alanine constructs had significantly reduced multimerin binding (p< 0.01), relative to the corresponding wild-type. In the present study, we evaluated multimerin-factor V binding with a new assay that used affinity purified, recombinant multimerin immobilized onto microtitre wells to test the binding of recombinant factor V constructs. Because results from the new binding assays were in agreement on the regions of the C2 domain important for multimerin binding, the new assay was used to examine the effect of thrombin on factor V-multimerin binding. Thrombin exposure led to significant dissociation of preformed multimerin-factor V complexes (p<0.01). In addition, thrombin cleaved factor Va had significantly reduced multimerin-binding in assays using antibodies against the factor Va heavy chain and light chain (p<0.01). Recently, our lab identified that platelets contain forms of factor V covalently linked to multimerin via cysteine 1085 in the factor V B-domain. After recombinant factor V was activated by thrombin, there was no detectable binding of the liberated B-domain to multimerin (p<0.001). Nonetheless, the B domain of factor V appeared to enhance factor V binding to multimerin, as factor V constructs synthesized without the B-domain had reduced multimerin binding even after conversion to factor Va, compared to wild-type factor V. Based on the overlap between multimerin-binding and procoagulant, PS binding regions in the C2 domain of factor V, we assessed the effect of multimerin on factor V procoagulant activity in one stage and two stage prothrombinase assays. However, multimerin did not neutralize factor V procoagulant activity when tested in molar excess. Our study indicates that multimerin binding of factor V is modulated by conformational changes in factor V upon activation, and that the factor V B-domain may function to enhance binding to multimerin. The dissociation of multimerin-factor V complexes by thrombin suggests multimerin might be important for delivering and localizing factor V onto platelets, prior to prothrombinase assembly.

scholarly journals Platelet coagulation factor Va: the major secretory platelet phosphoprotein

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2180-2190 ◽  
Author(s):  
MD Rand ◽  
M Kalafatis ◽  
KG Mann
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Activated Protein C ◽  
Coagulation Factor ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Factor V ◽  
Platelet Factor ◽  
Factor Va ◽  
Plasma Factor

Abstract Platelet-derived coagulation factor Va is the primary secreted substrate for a thrombin-stimulation-dependent platelet kinase. Human platelet factor Va, consisting of a molecular weight (M(r)) 105,000 heavy chain and an M(r) 74,000 light chain, incorporates phosphate in at least two sites on the light chain. Phosphorylated factor Va represents 50% of the secreted protein-associated phosphate. This modification occurs exclusively at serine residues and is inhibited by H-7 and staurosporine, which suggests a protein kinase C (PKC)-mediated event. Purified plasma factor V and Va are phosphorylated in the light chain region by rat brain PKC. The activity of platelet factor Va in prothrombinase on platelets is not altered when phosphorylation is inhibited by staurosporine. Plasma-derived factor Va in the presence of thrombin stimulated platelets is phosphorylated on both the heavy chain and the light chain. Plasma factor V and factor Va heavy chain phosphorylation occurs without light chain phosphorylation in the presence of added 32P gamma-ATP and non-stimulated or collagen- stimulated platelets or casein kinase II. This differential phosphorylation of factor Va heavy and light chain shows two independent platelet kinase activities that act on factor Va. The heavy chain factor V/Va kinase activity is similar to casein kinase II, which we have demonstrated previously to act on factor Va and accelerate activated protein C inactivation of the cofactor. Our data show platelet-dependent phosphorylation of platelet and plasma factor V and Va resulting in significant covalent modifications of the cofactor. These modifications may play a role in directing the extracellular distribution of factor V and factor Va.

Factor Va And Platelet Cytoskeletons

1981 ◽  
Author(s):  
G P Tuszynski ◽  
P N Walsh ◽  
A Koshy ◽  
J Piperno ◽  
B White
Keyword(s):  
Calcium Ionophore ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Common Mechanism ◽  
Factor V ◽  
Platelet Suspension ◽  
Final Level ◽  
Chromogenic Assay ◽  
Factor Va ◽  
Triton X

We have recently shown that the triton X-100 insoluble cytoskeletons from thrombin-activated platelets contain coagulation Factor Va (Tuszynski, G.P. et al. J. Cell. Biol. 87, 219, 1980). Factor Va activity was measured by two independent techniques, a one-stage clotting assay, and a two- stage chromogenic assay utilizing purified Factor Xa, prothrombin, and the chromogenic substrate S-2238. Further analysis of these cytoskeletons by SDS-PAGE and by immunodiffusion revealed the presence of fibrin. These findings suggest that a common mechanism may link alpha granular proteins to the platelet cytoskeleton. To investigate this mechanism, platelet cytoskeletons prepared from platelets treated under various conditions were assayed for Factor Va activity. Both the rate of Factor Va appearance and the final level of activity associated with the cytoskeleton were diminished 30-50% in platelets that were treated with aspirin (500μM) or with indomethacin (20μM). When secretion of Factor V, serotonin and nucleotides was inhibited greater than 90% by incubation of a platelet suspension with 2-de- oxyglucose (30μM), gluconolactone (40μM) and antimycin-A (9μM), cytoskeletons prepared from this platelet suspension contained less than 10% Factor Va activity of controls. The rate of appearance of cytoskeletal Factor Va activity increased with increasing thrombin concentration while the final level remained constant. Calcium ionophore, A-23187 (1μM), released Factor V but less than 2% was associated with the cytoskeleton suggesting that Factor Va, the activated from of Factor V, was required for binding. We conclude that prior secretion of Factor V and its further activation by thrombin are necessary for the association of coagulation Factor Va with the cytoskeleton. These results are consistent with the hypothesis that released Factor Va becomes associated with the cytoskeleton via cell surface components.

ISOLATION AND CHARACTERIZATION OF cDNA CLONES FOR HUMAN FACTOR V

1987 ◽  
Author(s):  
B Dahlbäck ◽  
A LundWall
Keyword(s):  
Human Factor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Cdna Clones ◽  
Factor V ◽  
Single Chain ◽  
Factor Va ◽  
Isolation And Characterization ◽  
Terminal Fragment ◽  
Coagulation Factor V

Coagulation factor V is a single chain, 330 kDa glycoprotein functioning as a cofactor to factor Xa in the activation of prothrombin. Thrombin cleaves factor V into four major fragments, out of which the N-terminal (105kDa) and the C-terminal (71-74kDa) fragments together constitute the active factor V species. To isolate cDNA clones a λ-gt 11 liver library was screened with a polyclonal, monospecific antiserum against human factor V. Four positive clones (two "weak", Aland A2 and two "strong", A3 and A4) were identified and isolated. Al(0.7kb), A2 (1.25kb) and A4 (0.85kb) reacted strongly with an antiserum against the 105 kDa, N-terminal fragment (heavy chain of factor Va), whereas A3 (1.25kb) gave the best signal with an antiserum against the 71-74 kDa, C-terminal fragment (light chain of factor Va). A1 hybridized with A2 and A4, whereas A2 only hybridized with Al. A3, which did not hybridize to any of the other clones, was used to rescreen the library and 9 positive clones (Bl-9) were isolated. B9 (3kb) coded for the entire C-terminal factor V fragment and the 3' noncoding sequence. B8 (1.8kb) partially overlapped B9 but extented the 5' sequence with 0.8kb. In a third screening round Al was used in combination with B8 and a 1.1 kb clone (CIO) was identified which hybridized to both. C10 did not hybridize with A2. The following overlapping cDNA clones can be orderedfrom the 5´end: A2-A1-C10-B8-B9 and together they cover 6 kb of coding sequence

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