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The Biochemical Journal Sep 1981Active-site-inhibited plasmin was prepared by inhibition with d-valyl-l-phenylalanyl-l-lysylchloromethane or by bovine pancreatic trypsin inhibitor (Kunitz inhibitor)....
Active-site-inhibited plasmin was prepared by inhibition with d-valyl-l-phenylalanyl-l-lysylchloromethane or by bovine pancreatic trypsin inhibitor (Kunitz inhibitor). Active-site-inhibited Glu-plasmin binds far more strongly to fibrin than Glu-plasminogen [native human plasminogen with N-terminal glutamic acid (residues 1-790)]. This binding is decreased by alpha(2)-plasmin inhibitor and tranexamic acid, and is, in the latter case, related to saturation of a strong lysine-binding site. In contrast, alpha(2)-plasmin inhibitor and tranexamic acid have only weak effects on the binding of Glu-plasminogen to fibrin. This demonstrates that its strong lysine-binding site is of minor importance to its binding to fibrin. Active-site-inhibited Lys-plasmin and Lys-plasminogen (Glu-plasminogen lacking the N-terminal residues Glu(1)-Lys(76), Glu(1)-Arg(67) or Glu(1)-Lys(77))display binding to fibrin similar to that of active-site inhibited Glu-plasmin. In addition, alpha(2)-plasmin inhibitor or tranexamic acid similarly decrease their binding to fibrin. Glu-plasminogen and active-site-inhibited Glu-plasmin have the same gross conformation, and conversion into their respective Lys- forms produces a similar marked change in conformation [Violand, Sodetz & Castellino (1975) Arch. Biochem. Biophys.170, 300-305]. Our results indicate that this change is not essential to the degree of binding to fibrin or to the effect of alpha(2)-plasmin inhibitor and tranexamic acid on this binding. The conversion of miniplasminogen (Glu-plasminogen lacking the N-terminal residues Glu(1)-Val(441)) into active-site-inhibited miniplasmin makes no difference to the degree of binding to fibrin, which is similarly decreased by the addition of tranexamic acid and unaffected by alpha(2)-plasmin inhibitor. Active-site-inhibited Glu-plasmin, Lys-plasmin and miniplasmin have lower fibrin-binding values in a plasma system than in a purified system. Results with miniplasmin(ogen) indicate that plasma proteins other than alpha(2)-plasmin inhibitor and histidine-rich glycoprotein decrease the binding of plasmin(ogen) to fibrin.
Topics: Binding Sites; Electrophoresis, Polyacrylamide Gel; Fibrin; Fibrinolysin; Glutamates; Humans; Lysine; Peptide Fragments; Plasminogen; Protein Binding; Tranexamic Acid; alpha-2-Antiplasmin
PubMed: 6459779
DOI: 10.1042/bj1970619 -
Thrombosis and Haemostasis Feb 2010The formation of platelet-rich thrombi under high shear rates requires both fibrinogen and von Willebrand factor (VWF) as molecular adhesives between platelets. We...
The formation of platelet-rich thrombi under high shear rates requires both fibrinogen and von Willebrand factor (VWF) as molecular adhesives between platelets. We attempted to describe the role of VWF as a potential substrate and modulator of the fibrinolytic system using binding assays, as well as kinetic measurements on the cleavage of fibrin(ogen) and a synthetic plasmin substrate (Spectrozyme-PL). The similar dissociation constants for the binding of plasminogen, plasmin, and active site-blocked plasmin onto immobilised VWF suggest that the primary binding site in plasmin(ogen) is not the active site. The progressive loss of clottability and generation of degradation products during fibrinogen digestion with plasmin were delayed in the presence of VWF at physiological concentrations, while VWF cleavage was not detectable. Determination of kinetic parameters for fibrinogen degradation by plasmin, miniplasmin and microplasmin showed that VWF did not modify the Km, whereas kcat values decreased with increasing VWF concentrations following the kinetic model of non-competitive inhibition. Inhibitory constants calculated for VWF were in the range of its physiological plasma concentration (5.4 mg/ml, 5.7 mg/ml and 10.0 mg/ml for plasmin, miniplasmin and microplasmin, respectively) and their values suggested a modulating role of the kringle 5 domain in the interaction between VWF and (mini)plasmin. VWF had no effect on the amidolytic activity of plasmin on Spectrozyme-PL, or on fibrin dissolution by (mini)plasmin. Our data suggest that VWF, while a poor plasmin substrate relative to fibrinogen, protects fibrinogen against degradation by plasmin preserving its clottability in plasma and its adhesive role in platelet-rich thrombi.
Topics: Blood Coagulation; Cell Adhesion; Fibrinogen; Fibrinolysin; Fibrinolysis; Humans; Kinetics; Peptide Hydrolases; Protein Binding; Thrombosis; von Willebrand Factor
PubMed: 20024497
DOI: 10.1160/TH09-07-0420 -
Kidney International May 1997Transforming growth factor-beta are cytokines with a wide range of biological effects. They play a pathologic role in inflammatory and fibrosing diseases such as... (Review)
Review
Transforming growth factor-beta are cytokines with a wide range of biological effects. They play a pathologic role in inflammatory and fibrosing diseases such as nephrosclerosis. TGF-beta s are secreted in a latent form due to noncovalent association with latency associated peptide (LAP), which is a homodimer formed from the propeptide region of TGF-beta. LAP is disulfide linked to another protein, latent TGF-beta binding protein (LTBP). LTBP has features in common with extracellular matrix proteins, and targets latent TGF-beta to the matrix. Activation of latent TGF-beta can be accomplished in vitro by denaturing treatments, plasmin digestion, ionizing radiation and interaction with thrombospondin. The mechanisms by which latent TGF-beta is activated physiologically are not well understood. Results to date suggest an important role for proteases, particularly plasmin, although other mechanisms probably exist. A general model of activation is proposed in which latent TGF-beta is released from the extracellular matrix by proteases, localized to cell surfaces, and activated by cell-associated plasmin.
Topics: Animals; Carrier Proteins; Cells, Cultured; Endothelium, Vascular; Fibrinolysin; Humans; Intracellular Signaling Peptides and Proteins; Latent TGF-beta Binding Proteins; Transforming Growth Factor beta
PubMed: 9150447
DOI: 10.1038/ki.1997.188 -
Journal of Clinical Pathology.... 1980
Review
Topics: Chemical Phenomena; Chemistry; Enzyme Activation; Fibrinolysin; Fibrinolysis; Humans; Lysine; Plasminogen; Plasminogen Activators
PubMed: 6448871
DOI: No ID Found -
Journal of Thrombosis and Haemostasis :... May 2014Excess fibrin in blood vessels is cleared by plasmin, the key proteolytic enzyme in fibrinolysis. Neurological disorders and head trauma can result in the disruption of...
BACKGROUND
Excess fibrin in blood vessels is cleared by plasmin, the key proteolytic enzyme in fibrinolysis. Neurological disorders and head trauma can result in the disruption of the neurovasculature and the entry of fibrin and other blood components into the brain, which may contribute to further neurological dysfunction.
OBJECTIVES
While chronic fibrin deposition is often implicated in neurological disorders, the pathological contributions attributable specifically to fibrin have been difficult to ascertain. An animal model that spontaneously acquires fibrin deposits could allow researchers to better understand the impact of fibrin in neurological disorders.
METHODS
Brains of plasminogen (plg)- and tissue plasminogen activator (tPA)-deficient mice were examined and characterized with regard to fibrin accumulation, vascular and neuronal health, and inflammation. Furthermore, the inflammatory response following intrahippocampal lipopolysaccharide (LPS) injection was compared between plg(-/-) and wild type (WT) mice.
RESULTS AND CONCLUSIONS
Both plg(-/-) and tPA(-/-) mice exhibited brain parenchymal fibrin deposits that appear to result from reduced neurovascular integrity. Markers of neuronal health and inflammation were not significantly affected by proximity to the vascular lesions. A compromised neuroinflammatory response was also observed in plg(-/-) compared to WT mice following intrahippocampal LPS injection. These results demonstrate that fibrin does not affect neuronal health in the absence of inflammation and suggest that plasmin may be necessary for a normal neuroinflammatory response in the mouse CNS.
Topics: Animals; Astrocytes; Brain; Female; Fibrin; Fibrinolysin; Fibrinolysis; Hippocampus; Inflammation; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Plasminogen; Tissue Plasminogen Activator
PubMed: 24612416
DOI: 10.1111/jth.12553 -
The Journal of Surgical Research Jun 2017Hyperfibrinolysis plays an integral role in the genesis of trauma-induced coagulopathy. Recent data demonstrate that red blood cell lysis promotes fibrinolysis; however,...
BACKGROUND
Hyperfibrinolysis plays an integral role in the genesis of trauma-induced coagulopathy. Recent data demonstrate that red blood cell lysis promotes fibrinolysis; however, the mechanism is unclear. Hemoglobin-based oxygen carriers (HBOCs) have been developed for resuscitation and have been associated with coagulopathy. We hypothesize that replacement of whole blood (WB) using an HBOC results in a coagulopathy because of the presence of free hemoglobin.
MATERIALS AND METHODS
WB was sampled from healthy donors (n = 6). The clotting profile of each citrated sample was evaluated using native thromboelastography. Serial titrations were performed using both HBOC (PolyHeme) and normal saline (NS; 5%, 25%, and 50%) and evaluated both with and without a 75-ng/mL tissue plasminogen activator (tPA) challenge. Tranexamic acid (TXA) was added to inhibit plasmin-dependent fibrinolysis. Fibrinolysis was measured and recorded as lysis at 30 min (LY30), the percentage of clot LY30 after maximal clot strength. Dilution of WB with NS or HBOC was correlated using LY30 via Spearman rho coefficients. Groups were also compared using a Friedman test and post hoc analysis with a Bonferroni adjustment.
RESULTS
tPA-provoked fibrinolysis was enhanced by both HBOC (median LY30 at 5%, 25%, and 50% titrations: 11%, 21%, and 44%, respectively; Spearman = 0.94; P < 0.001) and NS (11%, 28%, and 58%, respectively; Spearman = 0.790; P < 0.001). However, HBOC also enhanced fibrinolysis without the addition of tPA (1%, 4%, 5%; Spearman = 0.735; P = 0.001) and NS did not (1%, 2%, 1%; r = 0.300; P = 0.186. Moreover, addition of TXA did not alter or inhibit this fibrinolysis (WB versus 50% HBOC: 1.8% versus 5.7%, P = 0.04). There was no significant difference in fibrinolysis of HBOC with or without TXA (50% HBOC versus 50% HBOC + TXA: 5.6% versus 5.7%, P = 0.92). In addition, the increased fibrinolysis seen with NS was reversed when TXA was present (WB versus 50% NS: 1.8% versus 1.7%, P = 1.0).
CONCLUSIONS
HBOCs enhance fibrinolysis both with and without addition of tPA; moreover, this mechanism is independent of plasmin as the phenomenon persists in the presence of TXA. Our findings indicate the hemoglobin molecule or its components stimulate fibrinolysis by both tPA-dependent and innate mechanisms.
Topics: Adult; Biomarkers; Blood Substitutes; Fibrinolysin; Fibrinolysis; Healthy Volunteers; Hemoglobins; Humans; Male; Thrombelastography
PubMed: 28601310
DOI: 10.1016/j.jss.2015.04.077 -
Proceedings of the National Academy of... Dec 1987A catalytically active, human microplasmin was produced by incubation of [Lys]plasmin in buffer at pH 11.0 for up to 12 hr. The microplasmin was purified by affinity...
A catalytically active, human microplasmin was produced by incubation of [Lys]plasmin in buffer at pH 11.0 for up to 12 hr. The microplasmin was purified by affinity chromatography that used lysine-Sepharose and soybean trypsin inhibitor-Sepharose columns. It is homogeneous and pure by electrophoretic analysis in NaDodSO4/polyacrylamide gels and by gel filtration on a Superose 12 column. The molecular weight of the microplasmin determined by NaDodSO4 gel electrophoresis is 29,000 and 26,500 under reducing condition, whereas the molecular weight of native plasmin is 76,500. Microplasmin consists mainly of the ligh (B) chain of native human plasmin and possesses one active site per protein molecule when titrated with p-nitrophenyl p'-guanidinobenzoate. Microplasmin hydrolyzes the peptide substrate NH2-D-Val-Leu-Lys-p-nitroanilide (S-2251) with a Km of 0.361 +/- 0.017 mM and a kcat of 40.3 +/- 3.3 s-1 at pH 7.4 and 37 degrees C, whereas native plasmin has a Km of 0.355 +/- 0.002 mM and a kcat of 27.9 +/- 0.3 s-1 under the same conditions.
Topics: Chromatography, Affinity; Fibrinolysin; Humans; Molecular Weight; Peptide Fragments
PubMed: 2960974
DOI: 10.1073/pnas.84.23.8292 -
Blood Sep 1993Fibrin thrombi form at sites of injury, where leukocytes release a variety of oxidants. To determine whether oxidants might affect proteins of the fibrinolytic system,...
Fibrin thrombi form at sites of injury, where leukocytes release a variety of oxidants. To determine whether oxidants might affect proteins of the fibrinolytic system, we examined the effects of various oxidants on plasmin. Plasmin was not inhibited by micromolar concentrations of hypochlorous acid, chloramine T, or H2O2. Neither Fe nor Cu affected plasmin alone or in the presence of H2O2. However, incubation of plasmin with 5 mumol/L Cu(I or II) in the presence of the reducing agent ascorbic acid resulted in a loss of its hydrolytic activity towards proteins as well as towards small synthetic substrates. The addition of EDTA, but not mannitol, prevented its inactivation. Inactivation was prevented by the addition of catalase and accelerated by hydrogen peroxide. Preincubation of plasmin with the competitive inhibitor alpha-N-acetyl-L-lysine methyl ester prevented inactivation by Cu(II) and ascorbate. These results together suggest site-specific oxidation of plasmin's active site. Treatment of the plasminogen activators tissue plasminogen activator and two-chain urokinase-type plasminogen activator, as well as trypsin, neutrophil elastase, and thrombin with Cu(II) and ascorbate resulted in a loss of their amidolytic and proteolytic activity, indicating the general susceptibility of serine proteases to this type of oxidation. Oxidation of the zymogens Glu-plasminogen and single-chain urokinase-type plasminogen activator by Cu(II) and ascorbate resulted in the failure of these molecules to generate active enzymes when treated with plasminogen activators or plasmin, respectively. The active site His residue may be the target of oxidative inactivation, as evidenced by the partial protection afforded plasmin by the addition of Zn(II), histidine, or the platinum derivative, platinum(II) (2,2':6',2"-terpyridine) chloride. Because platelets contain micromolar concentrations of Cu and leukocytes are rich in ascorbate, Cu-dependent site-specific oxidation might play a role in modulating proteolytic events and the life span of thrombi formed at sites of tissue injury.
Topics: Ascorbic Acid; Binding Sites; Copper; Fibrinolysin; Humans; Oxidants; Oxidation-Reduction; Pancreatic Elastase; Plasminogen; Plasminogen Activators; Serine Proteinase Inhibitors; Thrombin; Trypsin
PubMed: 8364203
DOI: No ID Found -
Scientific Reports Aug 2017Despite the common use of thrombolytic drugs, especially in stroke treatment, there are many conflicting studies on factors affecting fibrinolysis. Because of the...
Despite the common use of thrombolytic drugs, especially in stroke treatment, there are many conflicting studies on factors affecting fibrinolysis. Because of the complexity of the fibrinolytic system, mathematical models closely tied with experiments can be used to understand relationships within the system. When tPA is introduced at the clot or thrombus edge, lysis proceeds as a front. We developed a multiscale model of fibrinolysis that includes the main chemical reactions: the microscale model represents a single fiber cross-section; the macroscale model represents a three-dimensional fibrin clot. The model successfully simulates the spatial and temporal locations of all components and elucidates how lysis rates are determined by the interplay between the number of tPA molecules in the system and clot structure. We used the model to identify kinetic conditions necessary for fibrinolysis to proceed as a front. We found that plasmin regulates the local concentration of tPA through forced unbinding via degradation of fibrin and tPA release. The mechanism of action of tPA is affected by the number of molecules present with respect to fibrin fibers. The physical mechanism of plasmin action (crawling) and avoidance of inhibition is defined. Many of these new findings have significant implications for thrombolytic treatment.
Topics: Fibrinolysin; Fibrinolysis; Humans; Kinetics; Models, Theoretical; Tissue Plasminogen Activator
PubMed: 28785035
DOI: 10.1038/s41598-017-06383-w -
American Journal of Physiology. Lung... Nov 2022Cleavage of the furin site in SARS-CoV-2 spike (S) protein accounts for increased transmissibility of COVID-19 by promoting the entry of virus into host cells through...
Cleavage of the furin site in SARS-CoV-2 spike (S) protein accounts for increased transmissibility of COVID-19 by promoting the entry of virus into host cells through specific angiotensin-converting enzyme 2 (ACE2) receptors. Plasmin, a key serine protease of fibrinolysis system, cleaves the furin site of γ subunit of human epithelial sodium channels (ENaCs). Sharing the plasmin cleavage by viral S and host ENaC proteins may competitively inter-regulate SARS-CoV-2 transmissibility and edema resolution via the ENaC pathway. To address this possibility, we analyzed single-cell RNA sequence (scRNA-seq) data sets and found that (encoding urokinase plasminogen activator), (γENaC), and (SARS-CoV-2 receptor) were co-expressed in airway/alveolar epithelial cells. The expression levels of and were significantly higher compared with in healthy group. This difference was further amplified in both epithelial and immune cells in patients with moderate/severe COVID-19 and SARS-CoV-2 infected airway/alveolar epithelial cell lines. Of note, plasmin cleaved the S protein and facilitated the entry of pseudovirus in HEK293 cells. Conclusively, SARS-CoV-2 may expedite infusion by competing the fibrinolytic protease network with ENaC.
Topics: Humans; Spike Glycoprotein, Coronavirus; Angiotensin-Converting Enzyme 2; COVID-19; Furin; Epithelial Sodium Channels; SARS-CoV-2; Fibrinolysin; HEK293 Cells
PubMed: 36193902
DOI: 10.1152/ajplung.00152.2022