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The Biochemical Journal Dec 2010Cell activation by stressors is characterized by a sequence of detectable phenotypic cell changes. A given stimulus, depending on its strength, induces modifications in...
Cell activation by stressors is characterized by a sequence of detectable phenotypic cell changes. A given stimulus, depending on its strength, induces modifications in the activity of membrane phospholipid transporters and calpains, which lead to phosphatidylserine exposure, membrane blebbing and the release of microparticles (nanoscale membrane vesicles). This vesiculation could be considered as a warning signal that may be followed, if the stimulus is maintained, by cell detachment-induced apoptosis. In the present study, plasminogen incubated with adherent cells is converted into plasmin by constitutively expressed tPA (tissue-type plasminogen activator) or uPA (urokinase-type plasminogen activator). Plasmin formed on the cell membrane then induces a unique response characterized by membrane blebbing and vesiculation. Hitherto unknown for plasmin, these membrane changes are similar to those induced by thrombin on platelets. If plasmin formation persists, matrix proteins are then degraded, cells lose their attachments and enter the apoptotic process, characterized by DNA fragmentation and specific ultrastructural features. Since other proteolytic or inflammatory stimuli may evoke similar responses in different types of adherent cells, the proposed experimental procedure can be used to distinguish activated adherent cells from cells entering the apoptotic process. Such a distinction is crucial for evaluating the effects of mediators, inhibitors and potential therapeutic agents.
Topics: Animals; Apoptosis; Blood Platelets; Blotting, Western; CHO Cells; Cell Adhesion; Cell Survival; Cricetinae; Cricetulus; Fibrinolysin; Humans; In Situ Nick-End Labeling; Kinetics; Microscopy, Electron; Plasminogen; Tissue Plasminogen Activator
PubMed: 20846121
DOI: 10.1042/BJ20100561 -
Annals of the New York Academy of... 1999
Review
Topics: Animals; Drug Resistance, Neoplasm; Fibrinolysin; Humans; Neoplasms, Experimental
PubMed: 10667229
DOI: 10.1111/j.1749-6632.1999.tb09426.x -
Bioorganic & Medicinal Chemistry Letters May 2016In this letter we report the design and synthesis of a series of plasmin inhibitors, which share the amino acid-based linker with limited free rotation between the...
In this letter we report the design and synthesis of a series of plasmin inhibitors, which share the amino acid-based linker with limited free rotation between the hydantoin moiety and the benzimidazole scaffold. Our studies led to potent plasmin inhibitors and yielded important new insights into their structure-activity relationship for binding to the active site of plasmin.
Topics: Amino Acids; Benzimidazoles; Fibrinolysin; Hydantoins; Hydrophobic and Hydrophilic Interactions
PubMed: 27009905
DOI: 10.1016/j.bmcl.2016.03.047 -
Annals of the New York Academy of... Aug 1957
Topics: Fibrinolysin
PubMed: 13479034
DOI: 10.1111/j.1749-6632.1957.tb42627.x -
Blood Nov 2015Coagulation factor XIIIa (FXIIIa) is a transglutaminase that covalently cross-links fibrin and other proteins to fibrin to stabilize blood clots and reduce blood loss. A...
Coagulation factor XIIIa (FXIIIa) is a transglutaminase that covalently cross-links fibrin and other proteins to fibrin to stabilize blood clots and reduce blood loss. A clear mechanism to describe the physiological inactivation of FXIIIa has been elusive. Here, we show that plasmin can cleave FXIIIa in purified systems and in blood. Whereas zymogen FXIII was not readily cleaved by plasmin, FXIIIa was rapidly cleaved and inactivated by plasmin in solution (catalytic efficiency = 8.3 × 10(3) M(-1)s(-1)). The primary cleavage site identified by mass spectrometry was between K468 and Q469. Both plasma- and platelet-derived FXIIIa were susceptible to plasmin-mediated degradation. Inactivation of FXIIIa occurred during clot lysis and was enhanced both in plasma deficient in fibrinogen and in plasma treated with therapeutic levels of tissue plasminogen activator. These results indicate that FXIIIa activity can be modulated by fibrinolytic enzymes, and suggest that changes in fibrinolytic activity may influence cross-linking of blood proteins.
Topics: Factor XIII; Fibrinolysin; Fibrinolysis; Humans; Proteolysis; Tissue Plasminogen Activator
PubMed: 26359437
DOI: 10.1182/blood-2015-07-650713 -
The Journal of Biological Chemistry Sep 1991Actin has been found to bind to plasmin's kringle regions, thereby inhibiting its enzymatic activity in a noncompetitive manner. We, therefore, examined its effect upon...
Actin has been found to bind to plasmin's kringle regions, thereby inhibiting its enzymatic activity in a noncompetitive manner. We, therefore, examined its effect upon the conversion of plasminogen to plasmin by tissue plasminogen activator. Actin stimulated plasmin generation from both Glu- and Lys-plasminogen, lowering the Km for activation of Glu-plasminogen into the low micromolar range. Accelerated plasmin generation did not occur in the presence of epsilon-amino caproic acid or if actin was exposed to acetic anhydride, an agent known to acetylate lysine residues. Actin binds to tissue plasminogen activator (t-Pa) (Kd = 0.55 microM), at least partially via lysine-binding sites. Actin's stimulation of plasmin generation from Glu-plasminogen was inhibited by the addition of aprotinin and was restored by the substitution of plasmin-treated actin, indicating the operation of a plasmin-dependent positive feedback mechanism. Native actin binds to Lys-plasminogen, and promotes its conversion to plasmin even in the presence of aprotinin, indicating that plasmin's cleavage of either actin or plasminogen leads to further plasmin generation. Plasmin-treated actin binds Glu-plasminogen and t-PA simultaneously, thereby raising the local concentration of t-PA and plasminogen. Together, but not separately, actin and t-PA prolong the thrombin time of plasma through the generation of plasmin and fibrinogen degradation products. Actin-stimulated plasmin generation may be responsible for some of the changes found in peripheral blood following tissue injury and sepsis.
Topics: Actins; Amino Acid Sequence; Animals; Electrophoresis, Polyacrylamide Gel; Fibrinolysin; Kinetics; Molecular Sequence Data; Rabbits; Tissue Plasminogen Activator
PubMed: 1832675
DOI: No ID Found -
The Biochemical Journal Mar 1992The amidolytic activity of plasmin with the chromogenic substrate H-D-valyl-L-leucyl-L-lysine p-nitroanilide (S-2251) is stimulated by oleic acid in a dose-dependent and...
The amidolytic activity of plasmin with the chromogenic substrate H-D-valyl-L-leucyl-L-lysine p-nitroanilide (S-2251) is stimulated by oleic acid in a dose-dependent and saturable fashion. The activity of plasmin on S-2251 in the presence of oleic acid followed a sigmoidal kinetic pattern, with an almost 4-fold stimulation of activity at 60 microM-oleic acid. Half-maximal stimulation occurred at an oleic acid level of 19.5 microM. The amino acid analogue 6-aminohexanoic acid (AHA), which is known to bind to lysine-binding sites in plasmin, suppressed the stimulatory effect of oleic acid in a concentration-dependent manner; at 0.3 mM-AHA, about 70% of the oleic acid-dependent enhancement of plasmin activity was abolished. The l/v versus 1/[S] plot for plasmin changed in the presence of oleic acid from a linear to a non-linear curve, suggesting positive co-operativity. 14C-labelled oleic acid bound to plasmin, and the bound ligand was displaced by an excess of unlabelled oleic acid. Oleic acid also produced a marked (40-fold) stimulation of the plasminogen-dependent cleavage of S-2251 by urokinase. A half-maximal effect on plasminogen activation was obtained at 40 microM-oleic acid. The present findings suggest that the ability of oleic acid to stimulate plasmin activity and to enhance the conversion of plasminogen to plasmin depends on the interaction of oleic acid with specific lysine-binding sites in plasmin.
Topics: Aminocaproic Acid; Drug Interactions; Electrophoresis; Fibrinolysin; Humans; Kinetics; Oleic Acid; Oleic Acids; Plasminogen; Stimulation, Chemical; Urokinase-Type Plasminogen Activator
PubMed: 1532491
DOI: 10.1042/bj2820863 -
Blood Coagulation & Fibrinolysis : An... Apr 2007Stabilized, active plasmin is a novel thrombolytic for direct delivery to clots. Although it is known that protease inhibitors in plasma inhibit plasmin, the amount of...
Stabilized, active plasmin is a novel thrombolytic for direct delivery to clots. Although it is known that protease inhibitors in plasma inhibit plasmin, the amount of plasmin that can be added to plasma/blood before free plasmin is observed is not clear. Determination of free plasmin activity in plasma using chromogenic substrates represents a challenge due to false-positive signals from plasmin entrapped by alpha2-macroglobulin. Size-exclusion chromatography was used to separate the plasmin-alpha2-macroglobulin complex from uninhibited, free plasmin. In this in-vitro study, exogenous plasmin is effectively inhibited up to 2.4 micromol/l after 5-min incubation with plasma at 37 degrees C. Initially, plasmin was consumed predominantly by alpha2-antiplasmin up to 1.2 micromol/l plasmin. Following exhaustion of alpha2-antiplasmin, plasmin was further consumed by alpha2-macroglobulin up to 2.4 micromol/l plasmin added to human plasma. Whole human blood was found to have an increased inhibitory capacity over that of plasma; free plasmin activity could be measured only above 3.8 micromol/l added plasmin. In conclusion, several mechanisms exist that control plasmin activity in human blood; in addition to alpha2-antiplasmin and alpha2-macroglobulin, blood cells contribute to the inhibition of exogenously administered plasmin. These in-vitro results indicate that doses of plasmin up to approximately 12 mg/kg in humans can be completely inactivated by blood.
Topics: Blood Cells; Fibrinolysin; Humans; Plasma; alpha-2-Antiplasmin; alpha-Macroglobulins
PubMed: 17413760
DOI: 10.1097/MBC.0b013e32808738b4 -
Journal of the Indian Medical... Jun 1961
Topics: Fibrinolysin; Humans
PubMed: 13692170
DOI: No ID Found -
The Biochemical Journal May 2005Human CPN (carboxypeptidase N) is a tetrameric plasma enzyme containing two glycosylated 83 kDa non-catalytic/regulatory subunits that carry and protect two active...
Human CPN (carboxypeptidase N) is a tetrameric plasma enzyme containing two glycosylated 83 kDa non-catalytic/regulatory subunits that carry and protect two active catalytic subunits. Because CPN can regulate the level of plasminogen binding to cell surface proteins, we investigated how plasmin cleaves CPN and the consequences. The products of hydrolysis were analysed by activity assays, Western blotting, gel filtration and sequencing. When incubated with intact CPN tetramer, plasmin rapidly cleaved the 83 kDa subunit at the Arg457-Ser458 bond near the C-terminus to produce fragments of 72 and 13 kDa, thereby releasing an active 142 kDa heterodimer, and also cleaved the active subunit, decreasing its size from 55 kDa to 48 kDa. Further evidence for the heterodimeric form of CPN was obtained by re-complexing the non-catalytic 72 kDa fragment with recombinant catalytic subunit or by immunoprecipitation of the catalytic subunit after plasmin treatment of CPN using an antibody specific for the 83 kDa subunit. Upon longer incubation, plasmin cleaved the catalytic subunit at Arg218-Arg219 to generate fragments of 27 kDa and 21 kDa, held together by non-covalent bonds, that were more active than the native enzyme. These data show that plasmin can alter CPN structure and activity, and that the C-terminal 13 kDa fragment of the CPN 83 kDa subunit is a docking peptide that is necessary to maintain the stable active tetrameric form of human CPN in plasma.
Topics: Amino Acid Sequence; Fibrinolysin; Humans; Hydrolysis; Lysine Carboxypeptidase; Molecular Sequence Data; Protein Structure, Quaternary; Protein Subunits
PubMed: 15617514
DOI: 10.1042/BJ20041471