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Blood Nov 2016In this issue of , Marcos-Contreras and colleagues present convincing evidence that mechanistically links hyperfibrinolysis, typically seen as a bleeding risk, with...
In this issue of , Marcos-Contreras and colleagues present convincing evidence that mechanistically links hyperfibrinolysis, typically seen as a bleeding risk, with increased brain endothelial permeability through plasmin-mediated cleavage of high-molecular-weight kininogen (HMWK) to bradykinin (BK). This study establishes plasmin as a key effector of what might be termed “hemovascular dysfunction”: a pathological state of blood enzymatic activity resulting in vascular structural and functional disruption. Their findings point the way toward improved treatment of patients with pharmacologically (stroke and myocardial infarction) or pathologically activated fibrinolysis (trauma and surgery) through selective blockade of bradykinin activity. Combined blockade of bradykinin and plasmin activation may provide additional therapeutic benefits in hemorrhagic shock by reducing tissue edema in resuscitation while enhancing hemostasis.
Topics: Fibrinolysin; Humans
PubMed: 27856467
DOI: 10.1182/blood-2016-09-735720 -
Translational Stroke Research Feb 2015Plasmin, a direct fibrinolytic, shows a significantly superior hemostatic safety profile compared to recombinant tissue plasminogen activator (rtPA), the only...
Plasmin, a direct fibrinolytic, shows a significantly superior hemostatic safety profile compared to recombinant tissue plasminogen activator (rtPA), the only FDA-approved thrombolytic for the treatment of acute ischemic stroke. The improved safety of plasmin is attributed to the rapid inhibition of free plasmin by endogenous plasmin inhibitors present in very high concentrations (1 μM). However, this rapid inhibition prevents the intravenous (IV) administration of plasmin. In emergency situations, catheter-based local administration is not practical. There is a need for an alternative technique for IV administration of plasmin. A possible solution is the encapsulation of plasmin in echogenic liposomes (ELIP) for protection from inhibitors until ultrasound (US)-triggered release at the clot site. ELIP are bilayer phospholipid vesicles with encapsulated gas microbubbles. US induces oscillation and collapse of the gas bubbles, which facilitates ELIP rupture and delivery of the encapsulated contents. Plasmin-loaded ELIP (PELIP) were manufactured and characterized for size, gas and drug encapsulations, and in vitro thrombolytic efficacy using a human whole blood clot model. Clots were exposed to PELIP with and without exposure to US (center frequency 120 kHz, pulse repetition frequency 1667 Hz, peak-to-peak pressure of 0.35 MPa, 50 % duty cycle). Thrombolytic efficacy was calculated by measuring the change in clot width over a 30-min treatment period using an edge detection MATLAB program. The mean clot lysis obtained with PELIP in the presence of US exposure was 31 % higher than that obtained without US exposure and 15 % higher than that obtained with rtPA treatment (p < 0.05).The enhanced clot lysis is attributed to the US-mediated release of plasmin from the liposomes.
Topics: Fibrinolysin; Fibrinolytic Agents; Humans; Liposomes; Microbubbles; Thrombolytic Therapy; Thrombosis; Ultrasonics
PubMed: 25411015
DOI: 10.1007/s12975-014-0376-4 -
The Journal of Experimental Medicine Apr 1953Human complement is inactivated by plasmin, the proteolytic enzyme of plasma or serum active at or near neutrality. The addition of streptokinase to human serum, which...
Human complement is inactivated by plasmin, the proteolytic enzyme of plasma or serum active at or near neutrality. The addition of streptokinase to human serum, which converts plasminogen to plasmin, also causes the inactivation of complement components C'2 and C'4 and varying amounts of C'1. C'3 is the most resistant to inactivation by plasmin. Chloroform-activated human plasmin and bovine plasmin also destroy these components of complement, but are less effective than the streptokinase-activated enzyme. The inactivation of complement by the addition of streptokinase to human serum is inhibited by high hydrogen ion concentrations, low temperature, and elevated ionic strength. The inactivation of the components of complement in various fractions of serum is influenced by the available plasminogen and the content of plasmin inhibitors in these fractions. Certain similarities are pointed out between the components of complement and the factors in the plasmin system and between the inactivation of the components of complement by antigen-antibody reactions, by specific agents, and by plasmin. The possible significance of these relationships in immune hemolysis and complement fixation, and the possible role of the plasmin system in the instability of complement and the development of anticomplementary properties in serum are discussed.
Topics: Animals; Cattle; Complement System Proteins; Fibrinolysin; Hemolysis; Humans; Hydrogen-Ion Concentration; Peptide Hydrolases
PubMed: 13052820
DOI: 10.1084/jem.97.4.573 -
Journal of Thrombosis and Haemostasis :... Apr 2023Fibrinolysis is a series of enzymatic reactions that degrade insoluble fibrin. Plasminogen activators convert the zymogen plasminogen to the active serine protease... (Review)
Review
Assays to quantify fibrinolysis: strengths and limitations. Communication from the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee on fibrinolysis.
Fibrinolysis is a series of enzymatic reactions that degrade insoluble fibrin. Plasminogen activators convert the zymogen plasminogen to the active serine protease plasmin, which cleaves and solubilizes crosslinked fibrin clots into fibrin degradation products. The quantity and quality of fibrinolytic enzymes, their respective inhibitors, and clot structure determine overall fibrinolysis. The quantity of protein can be measured by antigen-based assays, and both quantity and quality can be assessed using functional assays. Furthermore, variations of commonly used assays have been reported, which are tailored to address the role(s) of specific fibrinolytic factors and cellular elements (eg, platelets, neutrophils, and red blood cells). Although the concentration and/or activity of a protein can be quantified, how these individual components contribute to the overall fibrinolysis outcome can be challenging to determine. This difficulty is due to temporal changes within and around the thrombi during the clot breakdown, particularly the fibrin matrix structure, and composition. Furthermore, terms such as "fibrinolytic activity/potential," "plasminogen activation," and "plasmin activity" are often used interchangeably despite having different definitions. The purpose of this review is to 1) summarize the assays measuring fibrinolysis activity and potential, 2) facilitate the interpretation of data generated by these assays, and 3) summarize the strengths and limitations of these assays.
Topics: Humans; Fibrinolysis; Fibrinolysin; Thrombosis; Plasminogen; Fibrin; Serine Proteases; Communication
PubMed: 36759279
DOI: 10.1016/j.jtha.2023.01.008 -
The Journal of Clinical Investigation Jul 1981An enzyme-linked differential antibody immunosorbent assay has been developed for the quantification of alpha2-plasmin inhibitor-plasmin and alpha2-macroglobulin-plasmin...
An enzyme-linked differential antibody immunosorbent assay has been developed for the quantification of alpha2-plasmin inhibitor-plasmin and alpha2-macroglobulin-plasmin complexes. In this method the inhibitor-plasmin complex is bound to a surface by an inhibitor-specific antibody, and the plasmin bound to the inhibitor is quantified by a second antibody, rabbit antiplasminogen F(ab')2, labeled with alkaline phosphatase. The hydrolysis of p-nitrophenyl phosphate by the alkaline phosphatase is expressed in femtomoles of plasminogen per milliliter, by reference to a standard plasminogen curve. Inhibitor-enzyme complexes were generated in plasma by the addition of plasmin or of urokinase. The concentration of plasmin added was well below the plasma concentration of alpha2-plasmin inhibitor (1 microM) or of alpha2-macroglobulin (3.5 microM), so that neither inhibitor would be fully saturated with enzyme. Under these conditions increasing amounts of plasmin generated an increase in both alpha2-plasmin inhibitor-plasmin and alpha2-macroglobulin-plasmin complexes. Varying amounts of plasmin were incubated with each of the purified inhibitors in the concentration found in plasma, and the complexes. Varying amounts of plasmin were incubated with each of the purified inhibitors in the concentration found in plasma, and the complexes that formed were quantified by immunoassay. These studies made it possible to quantify the distribution of plasmin between the two inhibitors in plasmin or urokinase-treated plasma. In plasmin-treated plasma, 10% or less of the plasmin bound to both inhibitors was in complex with alpha2-macroglobulin. In contrast, between 19 and 51% of the plasmin generated in urokinase-activated plasma was bound to alpha2-macroglobulin. Thus, major changes in the distribution of plasma were observed, according to whether plasmin was added to plasma or whether plasminogen was activated endogenously. The pattern of inhibitor plasmin complexes generated in vivo by the therapeutic infusion of urokinase was similar to that found for urokinase-activated plasma. 23 normal individuals had low levels of alpha2-plasmin inhibitor-plasmin complexes, whereas six patients with laboratory evidence for disseminated intravascular coagulation demonstrated a 16- to 35-fold increase in he concentration of these complexes. These data indicated that a useful new probe for the study of the fibrinolytic enzyme system had been developed.
Topics: Antifibrinolytic Agents; Disseminated Intravascular Coagulation; Enzyme Inhibitors; Fibrinolysin; Humans; Immune Sera; Immunoenzyme Techniques; Immunoglobulin G; Plasminogen; Pulmonary Embolism; Urokinase-Type Plasminogen Activator; alpha-2-Antiplasmin; alpha-Macroglobulins
PubMed: 6166634
DOI: 10.1172/jci110253 -
Frontiers in Cellular and Infection... 2013The ability to take advantage of plasminogen and its activated form plasmin is a common mechanism used by commensal as well as pathogenic bacteria in interaction with... (Review)
Review
The ability to take advantage of plasminogen and its activated form plasmin is a common mechanism used by commensal as well as pathogenic bacteria in interaction with their respective host. Hence, a huge variety of plasminogen binding proteins and activation mechanisms exist. This review solely focuses on the genus Streptococcus and, in particular, on the so-called non-activating plasminogen binding proteins. Based on structural and functional differences, as well as on their mode of surface linkaging, three groups can be assigned: M-(like) proteins, surface displayed cytoplasmatic proteins with enzymatic activities ("moonlighting proteins") and other surface proteins. Here, the plasminogen binding sites and the interaction mechanisms are compared. Recent findings on the functional consequences of these interactions on tissue degradation and immune evasion are summarized.
Topics: Animals; Bacterial Proteins; Carrier Proteins; Fibrinolysin; Host-Pathogen Interactions; Humans; Immune Evasion; Plasminogen; Protein Binding; Streptococcus
PubMed: 24319673
DOI: 10.3389/fcimb.2013.00085 -
Journal of Thrombosis and Haemostasis :... Apr 2015The plasmin(ogen) and complement systems are simultaneously activated at sites of tissue injury, participating in hemostasis, wound healing, inflammation and immune... (Comparative Study)
Comparative Study
BACKGROUND
The plasmin(ogen) and complement systems are simultaneously activated at sites of tissue injury, participating in hemostasis, wound healing, inflammation and immune surveillance. In particular, the C3 proteolytic fragment, iC3b, and its degradation product C3dg, which is generated by cleavage by factor I (FI) and the cofactor complement receptor CR1, are important in bridging innate and adaptive immunity. Via a thioester (TE) bond, iC3b and C3dg covalently tag pathogens, modulating phagocytosis and adaptive immune responses.
OBJECTIVE
To examine plasmin-mediated proteolysis of iC3b, and to evaluate the functional consequences, comparing the effects with products generated by FI/CR1 cleavage of iC3b.
METHODS
Dose-dependent and time-dependent plasmin-mediated cleavage of iC3b were characterized by analytical gel electrophoresis. The properties of the resultant TE bond-containing fragments on phagocytosis and induction of pro-inflammatory cytokines were measured in cell culture systems.
RESULTS
At low concentrations, plasmin effectively cleaves iC3b, but at numerous previously undescribed sites, giving rise to novel C3c-like and C3dg-like moieties, the latter of which retain the TE bond. When attached to zymosan or erythrocytes and exposed to THP-1 macrophages, the C3dg-like proteins behave almost identically to the bona fide C3dg, yielding less phagocytosis as compared with the opsonin iC3b, and more macrophage secretion of the pro-inflammatory cytokine, IL-12.
CONCLUSION
Plasmin cleavage of iC3b provides a complement regulatory pathway that is as efficient as FI/CR1 but does not require a cellular cofactor.
Topics: Animals; Cell Line; Complement Activation; Complement C3 Convertase, Alternative Pathway; Complement C3b; Fibrinolysin; Fibrinolysis; Humans; Immunity, Innate; Inflammation Mediators; Interleukin-12; Macrophages; Peptide Fragments; Phagocytosis; Proteolysis; Rabbits; Signal Transduction; Time Factors
PubMed: 25556624
DOI: 10.1111/jth.12837 -
Journal of Thrombosis and Haemostasis :... Sep 2019Excessive, plasmin-mediated fibrinolysis augments bleeding and contributes to death in some patients. Current therapies for fibrinolytic bleeding are limited by modest...
BACKGROUND
Excessive, plasmin-mediated fibrinolysis augments bleeding and contributes to death in some patients. Current therapies for fibrinolytic bleeding are limited by modest efficacy, low potency, and off-target effects.
OBJECTIVES
To determine whether an antibody directed against unique loop structures of the plasmin protease domain may have enhanced specificity and potency for blocking plasmin activity, fibrinolysis, and experimental hemorrhage.
METHODS
The binding specificity, affinity, protease cross-reactivity and antifibrinolytic properties of a monoclonal plasmin inhibitor antibody (Pi) were examined and compared with those of epsilon aminocaproic acid (EACA), which is a clinically used fibrinolysis inhibitor.
RESULTS
Pi specifically recognized loop 5 of the protease domain, and did not bind to other serine proteases or nine other non-primate plasminogens. Pi was ~7 logs more potent in neutralizing plasmin cleavage of small-molecule substrates and >3 logs more potent in quenching fibrinolysis than EACA. Pi was similarly effective in blocking catalysis of a small-molecule substrate as α -antiplasmin, which is the most potent covalent inhibitor of plasmin, and was a more potent fibrinolysis inhibitor. Fab or chimerized Fab fragments of Pi were equivalently effective. In vivo, in a humanized model of fibrinolytic surgical bleeding, Pi significantly reduced bleeding to a greater extent than a clinical dose of EACA.
CONCLUSIONS
A mAb directed against unique loop sequences in the protease domain is a highly specific, potent, competitive plasmin inhibitor that significantly reduces experimental surgical bleeding in vivo.
Topics: Aminocaproic Acid; Animals; Antibodies, Monoclonal, Humanized; Antibody Affinity; Antifibrinolytic Agents; Binding, Competitive; Catalytic Domain; Cross Reactions; Drug Evaluation, Preclinical; Female; Fibrinolysin; Fibrinolysis; Hemorrhage; Humans; Mice; Mice, Inbred C57BL; Models, Molecular; Protein Conformation; Protein Domains; Random Allocation; Recombinant Fusion Proteins; Species Specificity; Substrate Specificity
PubMed: 31136076
DOI: 10.1111/jth.14522 -
American Journal of Physiology.... Feb 2016The purpose of this study was to determine whether trauma-induced coagulopathy is due to changes in 1) thrombin activity, 2) plasmin activity, and/or 3) factors that...
The purpose of this study was to determine whether trauma-induced coagulopathy is due to changes in 1) thrombin activity, 2) plasmin activity, and/or 3) factors that stimulate or inhibit thrombin or plasmin. Sprague-Dawley rats were anesthetized with 1-2% isoflurane/100% oxygen, and their left femoral artery and vein were cannulated. Polytrauma included right femur fracture, and damage to the small intestines, the left and medial liver lobes, and right leg skeletal muscle. Rats were then bled 40% of blood volume. Plasma samples were taken before trauma, and at 30, 60, 120, and 240 min. Polytrauma and hemorrhage led to a significant fall in prothrombin levels. However, circulating thrombin activity did not change significantly over time. Antithrombin III and α2 macroglobulin fell significantly by 2 h, then rose by 4 h. Soluble thrombomodulin was significantly elevated over the 4 h. Circulating plasmin activity, plasminogen, and D-dimers were elevated for the entire 4 h. Tissue plasminogen activator (tPA) was elevated at 30 min, then decreased below baseline levels after 1 h. Plasminogen activator inhibitor-1 was significantly elevated at 2-4 h. Neither tissue factor pathway inhibitor nor thrombin activatable fibrinolysis inhibitor changed significantly over time. The levels of prothrombin and plasminogen were 30-100 times higher than their respective active enzymes. Polytrauma and hemorrhage in rats lead to a fibrinolytic coagulopathy, as demonstrated by an elevation in plasmin activity, D-dimers, and tPA. These results are consistent with the observed clinical benefit of tranexamic acid in trauma patients.
Topics: Animals; Antithrombin III; Blood Coagulation; Femoral Fractures; Fibrinolysin; Fibrinolysis; Hemorrhage; Intestine, Small; Liver; Male; Multiple Trauma; Muscle, Skeletal; Plasminogen; Prothrombin; Rats; Rats, Sprague-Dawley; Thrombin; alpha-Macroglobulins
PubMed: 26632604
DOI: 10.1152/ajpregu.00401.2015 -
Acta Biomaterialia Mar 2022Fibrinolysis is the enzymatic digestion of fibrin, the primary structural component in blood clots. Mechanisms of fibrin fiber digestion during lysis have long been...
Fibrinolysis is the enzymatic digestion of fibrin, the primary structural component in blood clots. Mechanisms of fibrin fiber digestion during lysis have long been debated and obtaining detailed structural knowledge of these processes is important for developing effective clinical approaches to treat ischemic stroke and pulmonary embolism. Using dynamic fluorescence microscopy, we studied the time-resolved digestion of individual fibrin fibers by the fibrinolytic enzyme plasmin. We found that plasmin molecules digest fibers along their entire lengths, but that the rates of digestion are non-uniform, resulting in cleavage at a single location along the fiber. Using mathematical modeling we estimated the rate of plasmin arrival at the fiber surface and the number of digestion sites on a fiber. We also investigated correlations between local fiber digestion rates, cleavage sites, and fiber properties such as initial thickness. Finally, we uncovered a previously unknown tension-dependent mechanism that pulls fibers apart during digestion. Taken together these results promote a paradigm shift in understanding mechanisms of fibrinolysis and underscore the need to consider fibrin tension when assessing fibrinolytic approaches. STATEMENT OF SIGNIFICANCE: We developed a method for interrogating lysis of individual fibrin fibers, enabling the time-resolved observation of individual fiber digestion for the first time. Our results resolve longstanding disagreements about fibrinolytic processes and reveal previously unknown mechanisms that also play a role. Also, we developed the first microscale mathematical model of plasmin-fibrin interaction, which predicts the number of plasmin molecules on each fiber and can serve as a framework for investigating novel therapeutics.
Topics: Fibrin; Fibrinolysin; Fibrinolysis; Humans; Thrombosis
PubMed: 35007782
DOI: 10.1016/j.actbio.2022.01.006