scholarly journals Phosphatidylserine and phosphatidylethanolamine regulate the structure and function of FVIIa and its interaction with soluble tissue factor

2021 ◽  
Vol 41 (2) ◽  
Author(s):  
Tanusree Sengupta ◽  
Tilen Koklic ◽  
Barry R. Lentz ◽  
Rinku Majumder
Keyword(s):  
Tissue Factor ◽  
Proteolytic Activity ◽  
Factor Viia ◽  
Equilibrium Dialysis ◽  
Membrane Binding ◽  
Tryptophan Fluorescence ◽  
Structural Similarity ◽  
Coagulation Cascade ◽  
Factor X ◽  
Gla Domain

Abstract Cell membranes have important functions in many steps of the blood coagulation cascade, including the activation of factor X (FX) by the factor VIIa (FVIIa)-tissue factor (TF) complex (extrinsic Xase). FVIIa shares structural similarity with factor IXa (FIXa) and FXa. FIXa and FXa are regulated by binding to phosphatidylserine (PS)-containing membranes via their γ-carboxyglutamic acid-rich domain (Gla) and epidermal growth-factor (EGF) domains. Although FVIIa also has a Gla-rich region, its affinity for PS-containing membranes is much lower compared with that of FIXa and FXa. Research suggests that a more common endothelial cell lipid, phosphatidylethanolamine (PE), might augment the contribution of PS in FVIIa membrane-binding and proteolytic activity. We used soluble forms of PS and PE (1,2-dicaproyl-sn-glycero-3-phospho-l-serine (C6PS), 1,2-dicaproyl-sn-glycero-3-phospho-ethanolamine (C6PE)) to test the hypothesis that the two lipids bind to FVIIa jointly to promote FVIIa membrane binding and proteolytic activity. By equilibrium dialysis and tryptophan fluorescence, we found two sites on FVIIa that bound equally to C6PE and C6PS with Kd of ∼ 150–160 μM, however, deletion of Gla domain reduced the binding affinity. Binding of lipids occurred with greater affinity (Kd∼70–80 μM) when monitored by FVIIa proteolytic activity. Global fitting of all datasets indicated independent binding of two molecules of each lipid. The proteolytic activity of FVIIa increased by ∼50–100-fold in the presence of soluble TF (sTF) plus C6PS/C6PE. However, the proteolytic activity of Gla-deleted FVIIa in the presence of sTF was reduced drastically, suggesting the importance of Gla domain to maintain full proteolytic activity.

Phospholipid Synergy in Prothrombinase Activity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1175-1175
Author(s):  
Rebecca L Davis-Harrison ◽  
Narjes Tavoosi ◽  
Vincent S. Pureza ◽  
James H. Morrissey
Keyword(s):  
Binding Site ◽  
Tissue Factor ◽  
Blood Clotting ◽  
Factor Viia ◽  
Specific Binding ◽  
Membrane Binding ◽  
Coagulation Cascade ◽  
Membrane Surfaces ◽  
Gla Domain ◽  
Prothrombinase Activity

Abstract Abstract 1175 Most steps in the blood coagulation cascade obligatorily take place on membrane surfaces and are dependent on the exposure of phosphatidylserine (PS). Previous studies from our lab and others have shown that phosphatidylethanolamine (PE) poorly supports clotting reactions by itself, but strongly synergizes with PS to promote several membrane-dependent steps in the blood clotting cascade, although the mechanism for PE-PS synergy has been unclear. We recently put forward a new mechanistic explanation – which we termed the ABC or Anything But Choline hypothesis – for how PS and PE synergize to enhance factor X (fX) activation by the factor VIIa-tissue factor complex (Tavoosi et al., J. Biol. Chem. 286:23247–53, 2011). The membrane contribution to this reaction is dominated by the affinity of fX for the membrane surface; since fX binds to membranes via its gamma-carboxyglutamate-rich (GLA) domain, the ABC hypothesis therefore focuses on the mechanisms by which GLA domains engage the phospholipid bilayer. We identified two main types of GLA domain-phospholipid interactions: a single phospho-L-serine-specific binding site in each GLA domain; and multiple ”phosphate-specific” interactions in which the phosphate groups of non-phosphatidylcholine phospholipids form coordination complexes with the tightly bound calcium ions in GLA domains. In the current study, we test the ABC hypothesis in the context of the prothrombinase complex – i.e., activation of prothrombin by the membrane-bound complex of fXa and factor Va (fVa). Using a variety of approaches including surface plasmon resonance analyses, we measured the contributions of varying phospholipid compositions to the membrane binding affinities of fXa, fVa and prothrombin, as well as to the enzymatic activity of prothrombinase. Our results suggest that phospholipid synergy in prothrombinase activity differs in certain respects from that observed for the factor VIIa-tissue factor complex. Not only did PS synergize with PE for enhancing the activity of prothrombinase, but phosphatidylglycerol (PG) and phosphatidylacid (PA) also synergized with PE, albeit more weakly than with PS (i.e., significantly higher levels of PG or PA in the presence of PE were required to achieve prothrombinase activities comparable to mixtures of PS and PE). In contrast, PE failed to synergize with either PG or PA to support fX activation by the factor VIIa-tissue factor complex. These differences primarily arise from differential membrane binding of the substrates for these two complexes (fX for factor VIIa-tissue factor and prothrombin for prothrombinase). The data suggest that the phospho-L-serine-specific binding site in the GLA domain of prothrombin may not be as stringent as that of fX, as high levels of PG or PA can substitute for PS in membrane binding of prothrombin but not for fX. This study provides further insights into the membrane's role in regulating blood clotting reactions, specifically the binding interactions between GLA domains and membrane surfaces. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Soluble Tissue Factor Mutant Is a Selective Anticoagulant and Antithrombotic Agent

Blood ◽  
1997 ◽  
Vol 89 (9) ◽  
pp. 3219-3227 ◽  
Author(s):  
Robert F. Kelley ◽  
Canio J. Refino ◽  
Mark P. O'Connell ◽  
Nishit Modi ◽  
Pat Sehl ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Arterial Thrombosis ◽  
Factor Viia ◽  
Rabbit Model ◽  
Coagulation Cascade ◽  
Factor X ◽  
Rabbit Plasma ◽  
Extrinsic Pathway ◽  
Antithrombotic Agent ◽  
Thrombotic Disease

Abstract One approach to developing safer and more efficacious agents for the treatment of thrombotic disease involves the design and testing of inhibitors that block specific steps in the coagulation cascade. We describe here the development of a mutant of human tissue factor (TF ) as a specific antagonist of the extrinsic pathway of blood coagulation and the testing of this mutant in a rabbit model of arterial thrombosis. Alanine substitutions of Lys residues 165 and 166 in human TF have been shown previously to diminish the cofactor function of TF in support of factor X (FX) activation catalyzed by factor VIIa (FVIIa). The K165A:K166A mutations have been incorporated into soluble TF (sTF; residues 1-219) to generate the molecule “hTFAA.” hTFAA binds FVIIa with kinetics and affinity equivalent to wild-type sTF, but the hTFAA⋅FVIIa complex shows a 34-fold reduction in catalytic efficiency for FX activation relative to the activity measured for sTF⋅FVIIa. hTFAA inhibits the activation of FX catalyzed by the complex formed between FVIIa and relipidated TF(1-243). hTFAA prolongs prothrombin time (PT) determined with human plasma and relipidated TF(1-243) or membrane bound TF, and has no effect on activated partial thromboplastin time, but is 70-fold less potent as an inhibitor of PT with rabbit plasma. The rabbit homologue of this mutant (“rTFAA”) was produced and shown to have greater potency with rabbit plasma. Both hTFAA and rTFAA display an antithrombotic effect in a rabbit model of arterial thrombosis with rTFAA giving full efficacy at a lower dose than hTFAA. Compared to heparin doses of equal antithrombotic potential, hTFAA and rTFAA cause less bleeding as judged by measurements of the cuticle bleeding time. These results indicate that TF⋅FVIIa is a good target for the development of new anticoagulant drugs for the treatment of thrombotic disease.

Factor VIII-Factor IX Interactions: Molecular Sites Involved in Enzyme-Cofactor Complex Assembly

1999 ◽  
Vol 82 (08) ◽  
pp. 209-217 ◽  
Author(s):  
Patrick Celie ◽  
Joost Kolkman ◽  
Peter Lenting ◽  
Koen Mertens
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Factor Viia ◽  
Three Dimensional ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Factor X ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Factor X Activation

IntroductionThe activation of factor X is one of the steps in the coagulation cascade that is driven by the assembly of an activated serine protease with a membrane-bound cofactor. In the initial phase of coagulation, factor X is activated by the complex of activated factor VII (factor VIIa) and tissue factor. Subsequently, during the so-called propagation phase, factor X activation is catalyzed by the complex of activated factor IX (factor IXa) and activated factor VIII (factor VIIIa). In these complexes, factor VIIa and factor IXa are the factor X-activating enzymes, whereas tissue factor and factor VIIIa serve as non-enzymatic cofactors.1 Factors VIIa and IXa are highly homologous to other cofactor-dependent enzymes, such as activated factor X (factor Xa) and activated protein C, both in amino acid sequence, domain organization, and three-dimensional structure.2 Factor VIIa and IXa further share low or negligible activity towards their natural substrate factor X, unless in complex with their physiological cofactors.Although tissue factor and factor VIIIa serve similar roles as biological amplifiers, they are structurally different. Tissue factor is a small, transmembrane protein with an extracellular part comprising 219 amino acids. Factor VIII is much larger (2,332 amino acids), circulates in plasma, and requires proteolytic processing to exert its biological activity.3 When cofactors are assembled with their respective enzymes, a dramatic increase in enzymatic activity occurs. The underlying molecular mechanism, however, remains poorly understood.During the past few years, remarkable progress has been made in understanding the molecular details of enzyme-cofactor assembly within the coagulation cascade. Crystallography has provided high-resolution structures of tissue factor4 and the various cofactor-dependent coagulation enzymes.2 Moreover, the crystal structure of the factor VIIa—tissue factor complex has been resolved and has allowed the identification of the molecular sites involved in enzyme-cofactor interaction.5,6 Such details are still lacking, however, for the factor IXa—factor VIIIa complex. Current views are derived from three-dimensional models generated by homology modeling based on structurally-related proteins, such as nitrite reductase,7 ceruloplasmin,8 and galactose oxidase.9 Despite their inherent limitations, these models greatly facilitate the interpretation of previous functional studies on factor X activation. As such, the availability of molecular models may be considered an important step toward resolving the structure of the factor IXa—factor VIIIa complex and understanding the role of complex assembly and defects thereof. This chapter provides an overview of the current developments in this field.

scholarly journals Ligand-induced protease receptor translocation into caveolae: a mechanism for regulating cell surface proteolysis of the tissue factor-dependent coagulation pathway.

1996 ◽  
Vol 133 (2) ◽  
pp. 293-304 ◽  
Author(s):  
J R Sevinsky ◽  
L V Rao ◽  
W Ruf
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Cell Biology ◽  
Feedback Inhibition ◽  
Factor Viia ◽  
Coagulation Cascade ◽  
Low Density ◽  
Factor X ◽  
Quaternary Complex ◽  
Enzymatic Complex

The ability to regulate proteolytic functions is critical to cell biology. We describe events that regulate the initiation of the coagulation cascade on endothelial cell surfaces. The transmembrane protease receptor tissue factor (TF) triggers coagulation by forming an enzymatic complex with the serine protease factor VIIa (VIIa) that activates substrate factor X to the protease factor Xa (Xa). Feedback inhibition of the TF-VIIa enzymatic complex is achieved by the formation of a quaternary complex of TF-VIIa, Xa, and the Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI). Concomitant with the downregulation of TF-VIIa function on endothelial cells, we demonstrate by immunogold EM that TF redistributes to caveolae. Consistently, TF translocates from the Triton X-100-soluble membrane fractions to low-density, detergent-insoluble microdomains that inefficiently support TF-VIIa proteolytic function. Downregulation of TF-VIIa function is dependent on quaternary complex formation with TFPI that is detected predominantly in detergent-insoluble microdomains. Partitioning of TFPI into low-density fractions results from the association of the inhibitor with glycosyl phosphatidylinositol anchored binding sites on external membranes. Free Xa is not efficiently bound by cell-associated TFPI; hence, we propose that the transient ternary complex of TF-VIIa with Xa supports translocation and assembly with TFPI in glycosphingolipid-rich microdomains. The redistribution of TF provides evidence for an assembly-dependent translocation of the inhibited TF initiation complex into caveolae, thus implicating caveolae in the regulation of cell surface proteolytic activity.

Tissue factor: beyond coagulation in the cardiovascular system

2009 ◽  
Vol 118 (3) ◽  
pp. 159-172 ◽  
Author(s):  
Alexander Breitenstein ◽  
Giovanni G. Camici ◽  
Felix C. Tanner
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Factor X ◽  
Signalling Molecules ◽  
Smooth Muscle Cell Migration ◽  
Mitogen Activated Protein ◽  
Coronary Syndromes ◽  
Phosphoinositide 3 Kinase

TF (tissue factor) is the main trigger of the coagulation cascade; by binding Factor VIIa it activates Factor IX and Factor X, thereby resulting in fibrin formation. Various stimuli, such as cytokines, growth factors and biogenic amines, induce TF expression and activity in vascular cells. Downstream targets of these mediators include diverse signalling molecules such as MAPKs (mitogen-activated protein kinases), PI3K (phosphoinositide 3-kinase) and PKC (protein kinase C). In addition, TF can be detected in the bloodstream, known as circulating or blood-borne TF. Many cardiovascular risk factors, such as hypertension, diabetes, dyslipidaemia and smoking, are associated with increased expression of TF. Furthermore, in patients presenting with acute coronary syndromes, elevated levels of circulating TF are found. Apart from its role in thrombosis, TF has pro-atherogenic properties, as it is involved in neointima formation by inducing vascular smooth muscle cell migration. As inhibition of TF action appears to be an attractive target for the treatment of cardiovascular disease, therapeutic strategies are under investigation to specifically interfere with the action of TF or, alternatively, promote the effects of TFPI (TF pathway inhibitor).

scholarly journals Multiple roles of the coagulation protease cascade during virus infection

Blood ◽  
2014 ◽  
Vol 123 (17) ◽  
pp. 2605-2613 ◽  
Author(s):  
Silvio Antoniak ◽  
Nigel Mackman
Keyword(s):  
Immune Response ◽  
Tissue Factor ◽  
Viral Infections ◽  
Factor Viia ◽  
Hemorrhagic Fever ◽  
Multiple Roles ◽  
Coagulation Cascade ◽  
Factor X ◽  
Antiviral Protein ◽  
Monkey Model

Abstract The coagulation cascade is activated during viral infections. This response may be part of the host defense system to limit spread of the pathogen. However, excessive activation of the coagulation cascade can be deleterious. In fact, inhibition of the tissue factor/factor VIIa complex reduced mortality in a monkey model of Ebola hemorrhagic fever. Other studies showed that incorporation of tissue factor into the envelope of herpes simplex virus increases infection of endothelial cells and mice. Furthermore, binding of factor X to adenovirus serotype 5 enhances infection of hepatocytes but also increases the activation of the innate immune response to the virus. Coagulation proteases activate protease-activated receptors (PARs). Interestingly, we and others found that PAR1 and PAR2 modulate the immune response to viral infection. For instance, PAR1 positively regulates TLR3-dependent expression of the antiviral protein interferon β, whereas PAR2 negatively regulates expression during coxsackievirus group B infection. These studies indicate that the coagulation cascade plays multiple roles during viral infections.

scholarly journals Substitution of the Gla Domain in Factor X with That of Protein C Impairs Its Interaction with Factor VIIa/Tissue Factor

2007 ◽  
Vol 282 (21) ◽  
pp. 15632-15644 ◽  
Author(s):  
Matthew Ndonwi ◽  
George J. Broze ◽  
Sayeh Agah ◽  
Amy E. Schmidt ◽  
S. Paul Bajaj
Keyword(s):  
Tissue Factor ◽  
Protein C ◽  
Factor Viia ◽  
Factor X ◽  
Gla Domain

Factors Affecting the lnteraction of Tissue Factor/Factor Vll with Factor X in a Heterogeneous Tubular Reactor

1991 ◽  
Vol 65 (02) ◽  
pp. 139-143 ◽  
Author(s):  
Cynthia H Gemmell ◽  
Vincet T Turitto ◽  
Yale Nemerson
Keyword(s):  
Steady State ◽  
Tissue Factor ◽  
Factor Viia ◽  
Transmembrane Protein ◽  
Strong Association ◽  
Tubular Reactor ◽  
Factor Xa ◽  
Phospholipid Bilayer ◽  
Factor Vii ◽  
Factor X

SummaryA novel reactor recently described for studying phospholipiddependent blood coagulation reactions under flow conditions similar to those occurring in the vasculature has been further charactenzed. The reactor is a capitlary whose inner wall is coated with a stable phospholipid bilayer (or two bilayers) containing tissue factor, a transmembrane protein that is required for the enzymatic activation of factor X by factor VIIa. Perfusion of the capillary at wall shear rates ranging from 25 s−1 to 1,200 s−1 with purified bovine factors X and VIIa led to steady state factor Xa levels at the outlet. Assay were performed using a chromogenic substrate, SpectrozymeTMFXa, or by using a radiometric technique. In the absence of Ca2+ or factor VIIa there was no product formation. No difference was noted in the levels of factor Xa achieved when non-activated factor VII was perfused. Once steady state was achieved further factor Xa production continued in the absence of factor VIIa implying a very strong association of factor VIIa with the tissue factor in the phospholipid membrane. In agreement with static vesicle-type studies the reactor was sensitive to wall tissue factor concentration, temperature and the presence of phosphatidylserine in the bilayer.

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