Activation of the Catalytic Activity of Thrombin for Fibrin Formation by Ultrasound.
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ABSTRACT:
SUBMITTER: Zhao P
PROVIDER: S-EPMC8252103 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cell membrane re-engineering is emerging as a powerful tool for the development of next generation cell therapies, as it allows the user to augment therapeutic cells to provide additional functionalities, such as homing, adhesion or hypoxia resistance. To date, however, there are few examples where the plasma membrane is re-engineered to display active enzymes that promote extracellular matrix protein assembly. Here, we report on a self-contained matrix-forming system where the membrane of human mesenchymal stem cells is modified to display a novel thrombin construct, giving rise to spontaneous fibrin hydrogel nucleation and growth at near human plasma concentrations of fibrinogen. The cell membrane modification process is realised through the synthesis of a membrane-binding supercationic thrombin-polymer surfactant complex. Significantly, the resulting robust cellular fibrin hydrogel constructs can be differentiated down osteogenic and adipogenic lineages, giving rise to self-supporting monoliths that exhibit Young's moduli that reflect their respective extracellular matrix compositions.
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Project description:ObjectiveAcute hyperglycemia on admission for acute coronary syndrome worsens the prognosis in patients with and without known diabetes. Postulated mechanisms of this observation include prothrombotic effects. The aim of this study was to evaluate the effect of elevated glucose levels on blood clotting in acute coronary syndrome patients.Research design and methodsWe studied 60 acute coronary syndrome patients within the first 12 h after pain onset, including 20 subjects with type 2 diabetes, 20 subjects with no diagnosed diabetes but with glucose levels >7.0 mmol/l, and 20 subjects with glucose levels <7.0 mmol/l. We determined generation of thrombin-antithrombin complexes (TATs) and soluble CD40 ligand (sCD40L), a platelet activation marker, at the site of microvascular injury, together with ex vivo plasma fibrin clot permeability and lysis time.ResultsThe acute coronary syndrome patients with no prior diabetes but elevated glucose levels had increased maximum rates of formation and total production of TATs (by 42.9%, P < 0.0001, and by 25%, P < 0.0001, respectively) as well as sCD40L release (by 16.2%, P = 0.0011, and by 16.3%, P < 0.0001, respectively) compared with those with normoglycemia, whereas diabetic patients had the highest values of TATs and sCD40L variables (P < 0.0001 for all comparisons). Patients with hyperglycemia, with no previously diagnosed diabetes, had longer clot lysis time (by approximately 18%, P < 0.0001) similar to that in diabetic subjects, but not lower clot permeability compared with that in normoglycemic subjects.ConclusionsHyperglycemia in acute coronary syndrome is associated with enhanced local thrombin generation and platelet activation, as well as unfavorably altered clot features in patients with and without a previous history of diabetes.
| S-EPMC2494657 | biostudies-literature
Project description:In blood coagulation, thrombin converts fibrinogen into fibrin monomers that polymerize into a clot network. Thrombin also activates Factor XIII by cleaving the R37-G38 peptide bond of the Activation Peptide (AP) segment. The resultant transglutaminase introduces covalent crosslinks into the fibrin clot. A strategy to modify clot architecture would be to design FXIII AP sequences that are easier or more difficult to be thrombin-cleaved thus controlling initiation of crosslinking. To aid in this design process, FXIII V34X (28-41) Activation Peptides were kinetically ranked for cleavage by wild-type thrombin and several anticoagulant mutants. Thrombin-catalyzed hydrolysis of aromatic FXIII F34, W34, and Y34 APs was compared with V34 and L34. Cardioprotective FXIII L34 remained the variant most readily cleaved by wild-type thrombin. The potent anticoagulant thrombins W215A and W215A/E217A (missing a key substrate platform for binding fibrinogen) were best able to hydrolyze FXIII F34 and W34 APs. Thrombin I174A and L99A could effectively accommodate FXIII W34 and Y34 APs yielding kinetic parameters comparable to FXIII AP L34 with wild-type thrombin. None of the aromatic FXIII V34X APs could be hydrolyzed by thrombin Y60aA. FXIII F34 and W34 are promising candidates for FXIII - anticoagulant thrombin systems that could permit FXIII-catalyzed crosslinking in the presence of reduced fibrin formation. By contrast, FXIII Y34 with thrombin (Y60aA or W215A/E217A) could help assure that both fibrin clot formation and protein crosslinking are hindered. Regulating the activation of FXIII is predicted to be a strategy for helping to control fibrin clot architecture and its neighboring environments.
| S-EPMC5600292 | biostudies-literature
Project description:Kallikrein (PKa), generated by activation of its precursor prekallikrein (PK), plays a role in the contact activation phase of coagulation and functions in the kallikrein-kinin system to generate bradykinin. The general dogma has been that the contribution of PKa to the coagulation cascade is dependent on its action on FXII. Recently this dogma has been challenged by studies in human plasma showing thrombin generation due to PKa activity on FIX and also by murine studies showing formation of FIXa-antithrombin complexes in FXI deficient mice. In this study, we demonstrate high-affinity binding interactions between PK(a) and FIX(a) using surface plasmon resonance and show that these interactions are likely to occur under physiological conditions. Furthermore, we directly demonstrate dose- and time-dependent cleavage of FIX by PKa in a purified system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and chromogenic assays. By using normal pooled plasma and a range of coagulation factor-deficient plasmas, we show that this action of PKa on FIX not only results in thrombin generation, but also promotes fibrin formation in the absence of FXII or FXI. Comparison of the kinetics of either FXIa- or PKa-induced activation of FIX suggest that PKa could be a significant physiological activator of FIX. Our data indicate that the coagulation cascade needs to be redefined to indicate that PKa can directly activate FIX. The circumstances that drive PKa substrate specificity remain to be determined.
| S-EPMC7826336 | biostudies-literature
Project description:The thrombin mutant, des-ETW-thrombin, lacking Glu(146), Thr(147), and Trp(148) within a unique insertion loop located at the extreme end of the primary specificity pocket, has been shown previously to exhibit reduced catalytic activity with respect to macromolecular and synthetic thrombin substrates and reduced or enhanced susceptibility to inhibition. Investigation of the hydrolysis of peptidyl p-nitroanilide substrates by des-ETW-thrombin showed increased activity in the presence of heparin and other sulphated glycosaminoglycans. No effect was observed upon the activity of wild-type thrombin. Heparin was found to decrease the K(m) for cleavage of four thrombin-specific substrates by des-ETW-thrombin by 3-4-fold. Similarly, pentosan polysulphate (PPS) decreased the K(m) with these substrates by 8-10-fold. Heparin also increased the rate of inhibition of des-ETW-thrombin by antithrombin III and D-phenylalanyl-prolyl-arginylchloromethane (PPACK). The inhibition of des-ETW-thrombin by a number of thrombin-specific peptide boronic acids also showed significant reduction in the final K(i) in the presence of heparin, due to reduction in the off-rate. A peptide analogue of a sequence of hirudin which binds thrombin tightly to exosite I (fibrinogen recognition site) potentiated the activity of des-ETW-thrombin against peptide p-nitroanilide substrates in a manner similar to heparin. The K(i) for the inhibition of des-ETW-thrombin by p-aminobenzamidine was decreased by these ligands from 9.7 mM to 7.5 mM, 5.1 mM, and 2.5 mM in the presence of heparin, hirudin peptide and PPS respectively, suggesting the increased catalytic activity is due to enhanced access to the primary specificity pocket. The positive influence of these ligands on des-ETW-thrombin was reversed in the presence of ATP or ADP; the latter has previously been shown to inhibit thrombin activity by blocking initial interaction with fibrinogen at exosite 1. Because the effect of heparin and PPS is similar to that of hirudin peptide, it is proposed that the most likely mechanism is that binding at the heparin-binding site (thrombin exosite 2) facilitates interaction at exosite 1 causing a conformational change which partially corrects the defective ground-state binding of the mutant thrombin. Although no effect was observed upon the activity of wild-type thrombin, our findings do provide further evidence of an allosteric property of thrombin which may control the geometry of, and access to, the primary specificity pocket.
| S-EPMC1217199 | biostudies-other
Project description:Blood coagulation is a delicately regulated space- and time-dependent process that leads to the formation of fibrin clots preventing blood loss upon vascular injury. The sensitivity of the coagulation network was previously investigated without accounting for transport processes. To investigate its sensitivity to coagulation factor deficiencies in a spatial reaction-diffusion system, we combined an in vitro experimental design with a computational systems biology model. Clot formation in platelet-free plasma supplemented with phospholipids was activated with identical amounts of tissue factor (TF) either homogeneously distributed (concentration 5 pM, homogeneous model) or immobilized on the surface (surface density 100 pmole/m2, spatially heterogeneous model). Fibrin clot growth and thrombin concentration dynamic in space were observed using video microscopy in plasma of healthy donors or patients with deficiencies in factors (F) II, FV, FVII, FVIII, FIX, FX, or FXI. In the spatially heterogeneous model, near-activator thrombin generation was decreased in FV-, FVII-, and FX-deficient plasma. In the homogeneous model, clotting was not registered in these samples. The simulation and experiment data showed that the coagulation threshold depended on the TF concentration. Our data indicate that the velocity of spatial clot propagation correlates linearly with the concentration of thrombin at the clot wave front but not with the overall thrombin wave amplitude. Spatial clot growth in normal plasma at early stages was neither reaction nor diffusion limited but became diffusion limited later. In contrast, clot growth was always diffusion limited in FV-, FVII-, and FX-deficient plasma and reaction limited in FVIII-, FIX-, and FXI-deficient plasma. We conclude that robustness of the spatially heterogeneous coagulation system was achieved because of the combination of 1) a local high TF surface density that overcomes activation thresholds, 2) diffusion control being shared between different active factors, and 3) an early saturated stimulus-response dependence of fibrin clot formation by thrombin.
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Project description:Ultrasound (US) produces cavitation-induced mechanical forces stretching and breaking polymer chains in solution. This type of polymer mechanochemistry is widely used for synthetic polymers, but not biomacromolecules, even though US is biocompatible and commonly used for medical therapy as well as in vivo imaging. The ability to control protein activity by US would thus be a major stepping-stone for these disciplines. Here, we provide the first examples of selective protein activation and deactivation by means of US. Using GFP as a model system, we engineer US sensitivity into proteins by design. The incorporation of long and highly charged domains enables the efficient transfer of force to the protein structure. We then use this principle to activate the catalytic activity of trypsin by inducing the release of its inhibitor. We expect that this concept to switch "on" and "off" protein activity by US will serve as a blueprint to remotely control other bioactive molecules.
| S-EPMC7839785 | biostudies-literature
Project description:Fibrinogen and fibrin are essential for hemostasis and are major factors in thrombosis, wound healing, and several other biological functions and pathological conditions. The X-ray crystallographic structure of major parts of fibrin(ogen), together with computational reconstructions of missing portions and numerous biochemical and biophysical studies, have provided a wealth of data to interpret molecular mechanisms of fibrin formation, its organization, and properties. On cleavage of fibrinopeptides by thrombin, fibrinogen is converted to fibrin monomers, which interact via knobs exposed by fibrinopeptide removal in the central region, with holes always exposed at the ends of the molecules. The resulting half-staggered, double-stranded oligomers lengthen into protofibrils, which aggregate laterally to make fibers, which then branch to yield a three-dimensional network. Much is now known about the structural origins of clot mechanical properties, including changes in fiber orientation, stretching and buckling, and forced unfolding of molecular domains. Studies of congenital fibrinogen variants and post-translational modifications have increased our understanding of the structure and functions of fibrin(ogen). The fibrinolytic system, with the zymogen plasminogen binding to fibrin together with tissue-type plasminogen activator to promote activation to the active proteolytic enzyme, plasmin, results in digestion of fibrin at specific lysine residues. In spite of a great increase in our knowledge of all these interconnected processes, much about the molecular mechanisms of the biological functions of fibrin(ogen) remains unknown, including some basic aspects of clotting, fibrinolysis, and molecular origins of fibrin mechanical properties. Even less is known concerning more complex (patho)physiological implications of fibrinogen and fibrin.
| S-EPMC5536120 | biostudies-literature
Project description:BackgroundThrombin activates fibrinogen and binds the fibrin E-domain (Kd ~ 2.8 μM) and the splice variant γ'-domain (Kd ~ 0.1 μM). We investigated if the loading of D-Phe-Pro-Arg-chloromethylketone inhibited thrombin (PPACK-thrombin) onto fibrin could enhance fibrin stability.MethodsA 384-well plate thermal shift assay (TSA) with SYPRO-orange provided melting temperatures (Tm) of thrombin, PPACK-thrombin, fibrinogen, fibrin monomer, and fibrin.ResultsLarge increases in Tm indicated that calcium led to protein stabilization (0 vs. 2 mM Ca2+) for fibrinogen (54.0 vs. 62.3 °C) and fibrin (62.3 vs. 72.2 °C). Additionally, active site inhibition with PPACK dramatically increased the Tm of thrombin (58.3 vs. 78.3 °C). Treatment of fibrinogen with fibrin polymerization inhibitor GPRP increased fibrinogen stability by ΔTm = 9.3 °C, similar to the ΔTm when fibrinogen was converted to fibrin monomer (ΔTm = 8.8 °C) or to fibrin (ΔTm = 10.4 °C). Addition of PPACK-thrombin at high 5:1 M ratio to fibrin(ogen) had little effect on fibrin(ogen) Tm values, indicating that thrombin binding does not detectably stabilize fibrin via a putative bivalent E-domain to γ'-domain interaction.ConclusionsTSA was a sensitive assay of protein stability and detected: (1) the effects of calcium-stabilization, (2) thrombin active site labeling, (3) fibrinogen conversion to fibrin, and (4) GPRP induced changes in fibrinogen stability being essentially equivalent to that of fibrin monomer or polymerized fibrin.SignificanceThe low volume, high throughput assay has potential for use in understanding interactions with rare or mutant fibrin(ogen) variants.
| S-EPMC7752828 | biostudies-literature
Project description:Thrombin is an enzyme produced during blood coagulation that is crucial to the formation of a stable clot. Thrombin cleaves soluble fibrinogen into fibrin, which polymerizes and forms an insoluble, stabilizing gel around the growing clot. A small fraction of circulating fibrinogen is the variant γA/γ′, which has been associated with high-affinity thrombin binding and implicated as a risk factor for myocardial infarctions, deep vein thrombosis, and coronary artery disease. Thrombin is also known to be strongly sequestered by polymerized fibrin for extended periods of time in a way that is partially regulated by γA/γ′. However, the role of γA/γ′-thrombin interactions during fibrin polymerization is not fully understood. Here, we present a mathematical model of fibrin polymerization that considered the interactions between thrombin, fibrinogen, and fibrin, including those with γA/γ′. In our model, bivalent thrombin-fibrin binding greatly increased thrombin residency times and allowed for thrombin-trapping during fibrin polymerization. Results from the model showed that early in fibrin polymerization, γ′ binding to thrombin served to localize the thrombin to the fibrin(ogen), which effectively enhanced the enzymatic conversion of fibrinogen to fibrin. When all the fibrin was fully generated, however, the fibrin-thrombin binding persisted but the effect of fibrin on thrombin switched quickly to serve as a sink, essentially removing all free thrombin from the system. This dual role for γ′-thrombin binding during polymerization led to a paradoxical decrease in trapped thrombin as the amount of γ′ was increased. The model highlighted biochemical and biophysical roles for fibrin-thrombin interactions during polymerization and agreed well with experimental observations. Author summary We developed a mathematical model of fibrin polymerization that explicitly incorporated thrombin-fibrin(ogen) interactions, including the γ′ variant of fibrin(ogen). This variant is associated with clots that are more resistant to fibrinolysis and is therefore implicated as a risk factor in cardiovascular disease. However, the underlying mechanism it is not completely understood. We previously modeled γ′-thrombin interactions in a preformed fibrin clot and hypothesized that some thrombin became physically trapped within the clot during its formation (fibrin polymerization). We developed the model in this study to test that hypothesis and determine the role of γ′ during polymerization. The new model showed the plausibility of large amounts of thrombin being trapped within fibrin fibers during polymerization and suggested a dual role for γ′ during polymerization: localization of thrombin to the fibrin(ogen) during an initial early phase of polymerization and sequestration of thrombin during the later phase. The model results suggested a new regulatory mechanism for fibrin polymerization.
| S-EPMC9477365 | biostudies-literature