Blood clotting reactions on nanoscale phospholipid bilayers.
ABSTRACT: Blood clotting reactions, such as those catalyzed by the tissue factor:factor VIIa complex (TF:FVIIa), assemble on membrane surfaces containing anionic phospholipids such as phosphatidylserine (PS). In fact, membrane binding is critical for the function of most of the steps in the blood clotting cascade. In spite of this, our understanding of how the membrane contributes to catalysis, or even how these proteins interact with phospholipids, is incomplete. Making matters more complicated, membranes containing mixtures of PS and neutral phospholipids are known to spontaneously form PS-rich membrane microdomains in the presence of plasma concentrations of calcium ions, and it is likely that blood-clotting proteases such as TF:FVIIa partition into these PS-rich microdomains. Unfortunately, little is known about how membrane microdomain composition influences the activity of blood-clotting proteases, which is typically not under experimental control even in "simple" model membranes. Our laboratories have developed and applied new technologies for studying membrane proteins to gain insights into how blood-clotting protease-cofactor pairs assemble and function on membrane surfaces. This includes using a novel, nanoscale bilayer system (Nanodiscs) that permits assembling blood-clotting protease-cofactor pairs on stable bilayers containing from 65 to 250 phospholipid molecules per leaflet. We have used this system to investigate how local (nanometer-scale) changes in phospholipid bilayer composition modulate TF:FVIIa activity. We have also used detailed molecular-dynamics simulations of nanoscale bilayers to provide atomic-scale predictions of how the membrane-binding domain of factor VIIa interacts with PS in membranes.
Project description:The blood coagulation cascade is initiated when the cell-surface complex of factor VIIa (FVIIa, a trypsin-like serine protease) and tissue factor (TF, an integral membrane protein) proteolytically activates factor X (FX). Both FVIIa and FX bind to membranes via their ?-carboxyglutamate-rich domains (GLA domains). GLA domains contain seven to nine bound Ca(2+) ions that are critical for their folding and function, and most biochemical studies of blood clotting have employed supraphysiologic Ca(2+) concentrations to ensure saturation of these domains with bound Ca(2+). Recently, it has become clear that, at plasma concentrations of metal ions, Mg(2+) actually occupies two or three of the divalent metal ion-binding sites in GLA domains, and that these bound Mg(2+) ions are required for full function of these clotting proteins. In this study, we investigated how Mg(2+) influences FVIIa enzymatic activity. We found that the presence of TF was required for Mg(2+) to enhance the rate of FX activation by FVIIa, and we used alanine-scanning mutagenesis to identify TF residues important for mediating this response to Mg(2+). Several TF mutations, including those at residues G164, K166, and Y185, blunted the ability of Mg(2+) to enhance the activity of the TF/FVIIa complex. Our results suggest that these TF residues interact with the GLA domain of FX in a Mg(2+)-dependent manner (although effects of Mg(2+) on the FVIIa GLA domain cannot be ruled out). Notably, these TF residues are located within or immediately adjacent to the putative substrate-binding exosite of TF.
Project description:In normal hemostasis, the blood clotting cascade is initiated when factor VIIa (fVIIa, other clotting factors are named similarly) binds to the integral membrane protein, human tissue factor (TF). The TF/fVIIa complex in turn activates fX and fIX, eventually concluding with clot formation. Several X-ray crystal structures of the soluble extracellular domain of TF (sTF) exist; however, these structures are missing electron density in functionally relevant regions of the protein. In this context, NMR can provide complementary structural information as well as dynamic insights into enzyme activity. The resolution and sensitivity for NMR studies are greatly enhanced by the ability to prepare multiple milligrams of protein with various isotopic labeling patterns. Here, we demonstrate high-yield production of several isotopically labeled forms of recombinant sTF, allowing for high-resolution NMR studies both in the solid and solution state. We also report solution NMR spectra at sub-mM concentrations of sTF, ensuring the presence of dispersed monomer, as well as the first solid-state NMR spectra of sTF. Our improved sample preparation and precipitation conditions have enabled the acquisition of multidimensional NMR data sets for TF chemical shift assignment and provide a benchmark for TF structure elucidation.
Project description:Seven proteins in the human blood clotting cascade bind, via their GLA (?-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by 3 orders of magnitude. Here we employed nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained l-serine versus d-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins, factor VIIa and activated protein C, than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared with the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.
Project description:Natural phosphatidylserine (PS), which contains two chiral centers, enhances blood coagulation. However, the process by which PS enhanced blood coagulation is not completely understood. An efficient and flexible synthetic route has been developed to synthesize all of the possible stereoisomers of PS. In this study, we examined the role of PS chiral centers in modulating the activity of the tissue factor (TF)-factor VIIa coagulation initiation complex. Full length TF was relipidated with phosphatidylcholine, and the synthesized PS isomers were individually used to estimate the procoagulant activity of the TF-FVIIa complex via a FXa generation assay. The results revealed that the initiation complex activity was stereoselective and had increased sensitivity to the configuration of the PS glycerol backbone due to optimal protein-lipid interactions.
Project description:The molecular mechanism of enhancement of the enzymatic activity of factor VIIa by tissue factor (TF) is not fully understood, primarily because of the lack of atomic models for the membrane-bound form of the TF-FVIIa complex.To construct the first membrane-bound model of the TF-FVIIa complex, and to investigate the dynamics of the complex in solution and on the surface of anionic membranes by using large-scale molecular dynamics (MD) simulations in full atomic detail.Membrane-bound models of the TF-FVIIa complex and the individual factors were constructed and subjected to MD simulations, in order to characterize protein-protein and protein-lipid interactions, and to investigate the dynamics of TF and FVIIa.The MD trajectories reveal that isolated FVIIa undergoes large structural fluctuation, primarily due to the hinge motions between its domains, whereas soluble TF (sTF) is structurally stable. Upon complex formation, sTF restricts the motion of FVIIa significantly. The results also show that, in the membrane-bound form, sTF directly interacts with the lipid headgroups, even in the absence of FVIIa.The first atomic models of membrane-bound sTF-FVIIa, FVIIa and sTF are presented, revealing that sTF forms direct contacts with the lipids, both in the isolated form and in complex with FVIIa. The main effect of sTF binding to FVIIa is spatial stabilization of the catalytic site of FVIIa, which ensures optimal interaction with the substrate, FX.
Project description:Tissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa). The formation of TF-FVIIa complexes on cell surfaces triggers the activation of coagulation cascade and cell signaling. In the present study, we characterized the subcellular distribution of TF and its transport in fibroblasts by dual immunofluorescence confocal microscopy and biochemical methods. Our data show that a majority of TF resides in various intracellular compartments, predominantly in the Golgi. Tissue factor at the cell surface is localized in cholesterol-rich lipid rafts and extensively colocalized with caveolin-1. FVIIa binding to TF induces the internalization of TF. Of interest, we found that TF-FVIIa complex formation at the cell surface leads to TF mobilization from the Golgi with a resultant increase in TF expression at the cell surface. This process is dependent on FVIIa protease activity. Overall, the present data suggest a novel mechanism for TF expression at the cell surface by FVIIa. This mechanism could play an important role in hemostasis in response to vascular injury by increasing TF activity where and when it is needed.
Project description:Tissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa), and the formation of TF-FVIIa complexes on cell surfaces triggers the activation of the coagulation cascade and the cell signaling. Our recent studies have shown that a majority of TF resides in various intracellular compartments, predominantly in the Golgi, and that FVIIa binding to cell surface TF induces TF endocytosis and mobilizes the Golgi TF pool to translocate it to the cell surface. This present study is aimed to elucidate the mechanisms involved in TF endocytosis and its mobilization from the Golgi. Activation of protease-activated receptor 1 (PAR1) and PAR2 by specific peptide agonists and proteases, independent of FVIIa, mobilized TF from the Golgi store and increased the cell surface expression of TF. Blocking PAR2 activation, but not PAR1, with neutralizing antibodies fully attenuated the FVIIa-induced TF mobilization. Consistent with these data, silencing the PAR2 receptor, and not PAR1, abrogated the FVIIa-mediated TF mobilization. In contrast to their effect on TF mobilization, PAR1 and PAR2 activation, in the absence of FVIIa, had no effect on TF endocytosis. However, PAR2 activation is found to be critical for the FVIIa-induced TF endocytosis. Overall the data herein provide novel insights into the role of PARs in regulating cell surface TF expression.
Project description:Essentials Membrane-binding GLA domains of coagulation factors are essential for proper clot formation. Factor X (FX) is specific to phosphatidylserine (PS) lipids through unknown atomic-level interactions. Molecular dynamics simulations were used to develop the first membrane-bound model of FX-GLA. PS binding modes of FX-GLA were described, and potential PS-specific binding sites identified. SUMMARY:Background Factor X (FX) binds to cell membranes in a highly phospholipid-dependent manner and, in complex with tissue factor and factor VIIa (FVIIa), initiates the clotting cascade. Experimental information concerning the membrane-bound structure of FX with atomic resolution has remained elusive because of the fluid nature of cellular membranes. FX is known to bind preferentially to phosphatidylserine (PS). Objectives To develop the first membrane-bound model of the FX-GLA domain to PS at atomic level, and to identify PS-specific binding sites of the FX-GLA domain. Methods Molecular dynamics (MD) simulations were performed to develop an atomic-level model for the FX-GLA domain bound to PS bilayers. We utilized a membrane representation with enhanced lipid mobility, termed the highly mobile membrane mimetic (HMMM), permitting spontaneous membrane binding and insertion by FX-GLA in multiple 100-ns simulations. In 14 independent simulations, FX-GLA bound spontaneously to the membrane. The resulting membrane-bound models were converted from HMMM to conventional membrane and simulated for an additional 100 ns. Results The final membrane-bound FX-GLA model allowed for detailed characterization of the orientation, insertion depth and lipid interactions of the domain, providing insight into the molecular basis of its PS specificity. All binding simulations converged to the same configuration despite differing initial orientations. Conclusions Analysis of interactions between residues in FX-GLA and lipid-charged groups allowed for potential PS-specific binding sites to be identified. This new structural and dynamic information provides an additional step towards a full understanding of the role of atomic-level lipid-protein interactions in regulating the critical and complex clotting cascade.
Project description:The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215-217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215-217 conformation is pivotal to the enhanced activity of the FVIIa variants. We speculate that the allosteric regulation of FVIIa activity by TF binding follows a similar path in conjunction with protease domain N terminus insertion, suggesting a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity.
Project description:Tissue factor (TF) is the cellular receptor for plasma protease factor VIIa (FVIIa), and the TF-FVIIa complex initiates coagulation in both hemostasis and thrombosis. Cell surface-exposed TF is mainly cryptic and requires activation to fully exhibit the procoagulant potential. Recently, the protein disulfide isomerase (PDI) has been hypothesized to regulate TF decryption through the redox switch of an exposed disulfide in TF extracellular domain. In this study, we analyzed PDI contribution to coagulation using an in vitro endothelial cell model. In this model, extracellular PDI is detected by imaging and flow cytometry. Inhibition of cell surface PDI induces a marked increase in TF procoagulant function, whereas exogenous addition of PDI inhibits TF decryption. The coagulant effects of PDI inhibition were sensitive to annexin V treatment, suggesting exposure of phosphatidylserine (PS), which was confirmed by prothrombinase assays and direct labeling. In contrast, exogenous PDI addition enhanced PS internalization. Analysis of fluorescent PS revealed that PDI affects both the apparent flippase and floppase activities on endothelial cells. In conclusion, we identified a new mechanism for PDI contribution to coagulation on endothelial cells, namely, the regulation of PS exposure, where PDI acts as a negative regulator of coagulation.