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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 -
Journal of the American Society of... Apr 2024Proteinuria predicts accelerated decline in kidney function in CKD. The pathologic mechanisms are not well known, but aberrantly filtered proteins with enzymatic...
SIGNIFICANCE STATEMENT
Proteinuria predicts accelerated decline in kidney function in CKD. The pathologic mechanisms are not well known, but aberrantly filtered proteins with enzymatic activity might be involved. The urokinase-type plasminogen activator (uPA)-plasminogen cascade activates complement and generates C3a and C5a in vitro / ex vivo in urine from healthy persons when exogenous, inactive, plasminogen, and complement factors are added. Amiloride inhibits uPA and attenuates complement activation in vitro and in vivo . In conditional podocin knockout (KO) mice with severe proteinuria, blocking of uPA with monoclonal antibodies significantly reduces the urine excretion of C3a and C5a and lowers tissue NLRP3-inflammasome protein without major changes in early fibrosis markers. This mechanism provides a link to proinflammatory signaling in proteinuria with possible long-term consequences for kidney function.
BACKGROUND
Persistent proteinuria is associated with tubular interstitial inflammation and predicts progressive kidney injury. In proteinuria, plasminogen is aberrantly filtered and activated by urokinase-type plasminogen activator (uPA), which promotes kidney fibrosis. We hypothesized that plasmin activates filtered complement factors C3 and C5 directly in tubular fluid, generating anaphylatoxins, and that this is attenuated by amiloride, an off-target uPA inhibitor.
METHODS
Purified C3, C5, plasminogen, urokinase, and urine from healthy humans were used for in vitro / ex vivo studies. Complement activation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and ELISA. Urine and plasma from patients with diabetic nephropathy treated with high-dose amiloride and from mice with proteinuria (podocin knockout [KO]) treated with amiloride or inhibitory anti-uPA antibodies were analyzed.
RESULTS
The combination of uPA and plasminogen generated anaphylatoxins C3a and C5a from intact C3 and C5 and was inhibited by amiloride. Addition of exogenous plasminogen was sufficient for urine from healthy humans to activate complement. Conditional podocin KO in mice led to severe proteinuria and C3a and C5a urine excretion, which was attenuated reversibly by amiloride treatment for 4 days and reduced by >50% by inhibitory anti-uPA antibodies without altering proteinuria. NOD-, LRR- and pyrin domain-containing protein 3-inflammasome protein was reduced with no concomitant effect on fibrosis. In patients with diabetic nephropathy, amiloride reduced urinary excretion of C3dg and sC5b-9 significantly.
CONCLUSIONS
In conditions with proteinuria, uPA-plasmin generates anaphylatoxins in tubular fluid and promotes downstream complement activation sensitive to amiloride. This mechanism links proteinuria to intratubular proinflammatory signaling. In perspective, amiloride could exert reno-protective effects beyond natriuresis and BP reduction.
CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER
Increased Activity of a Renal Salt Transporter (ENaC) in Diabetic Kidney Disease, NCT01918488 and Increased Activity of ENaC in Proteinuric Kidney Transplant Recipients, NCT03036748 .
Topics: Humans; Mice; Animals; Urokinase-Type Plasminogen Activator; Plasminogen; Amiloride; Fibrinolysin; Diabetic Nephropathies; Inflammasomes; Mice, Inbred NOD; Proteinuria; Complement Activation; Anaphylatoxins; Fibrosis
PubMed: 38254266
DOI: 10.1681/ASN.0000000000000312 -
Spine Deformity Jul 2022Posterior spinal fusion (PSF) activates the fibrinolytic protease plasmin, which is implicated in blood loss and transfusion. While antifibrinolytic drugs have improved...
PURPOSE
Posterior spinal fusion (PSF) activates the fibrinolytic protease plasmin, which is implicated in blood loss and transfusion. While antifibrinolytic drugs have improved blood loss and reduced transfusion, variable blood loss has been observed in similar PSF procedures treated with the same dose of antifibrinolytics. However, both the cause of this and the appropriate measures to determine antifibrinolytic efficacy during high-blood-loss spine surgery are unknown, making clinical trials to optimize antifibrinolytic dosing in PSF difficult. We hypothesized that patients undergoing PSF respond differently to antifibrinolytic dosing, resulting in variable blood loss, and that specific diagnostic markers of plasmin activity will accurately measure the efficacy of antifibrinolytics in PSF.
METHODS
A prospective study of 17 patients undergoing elective PSF with the same dosing regimen of TXA was conducted. Surgery-induced plasmin activity was exhaustively analyzed in perioperative blood samples and correlated to measures of inflammation, bleeding, and transfusion.
RESULTS
While markers of in vivo plasmin activation (PAP and D-dimer) suggested significant breakthrough plasmin activation and fibrinolysis (P < 0.01), in vitro plasmin assays, including TEG, did not detect plasmin activation. In vivo measures of breakthrough plasmin activation correlated with blood loss (R = 0.400, 0.264; P < 0.01), transfusions (R = 0.388; P < 0.01), and complement activation (R = 0.346, P < 0.05).
CONCLUSIONS
Despite all patients receiving a high dose of TXA, its efficacy among patients was variable, indicated by notable intra-operative plasmin activity. Markers of in vivo plasmin activation best correlated with clinical outcomes. These findings suggest that the efficacy of antifibrinolytic therapy to inhibit plasmin in PSF surgery should be determined by markers of in vivo plasmin activation in future studies.
LEVEL OF EVIDENCE
Level II-diagnostic.
Topics: Antifibrinolytic Agents; Blood Loss, Surgical; Fibrinolysin; Humans; Prospective Studies; Spinal Fusion; Tranexamic Acid
PubMed: 35247191
DOI: 10.1007/s43390-022-00489-6 -
Seminars in Ophthalmology 2017Vitreomacular adhesion (VMA) describes the adhesion of the posterior hyaloid face to the inner retina in any part of the macula. This can arise after incomplete... (Review)
Review
Vitreomacular adhesion (VMA) describes the adhesion of the posterior hyaloid face to the inner retina in any part of the macula. This can arise after incomplete separation of the posterior vitreous cortex from the macula during vitreous liquefaction. While the VMA may resolve spontaneously, a strong and persistent adhesion can lead to a variety of anatomical changes, including vitreomacular traction (VMT) and macular hole (MH). Both conditions can present with metamorphopsia and decreased vision. In cases of symptomatic VMT and full-thickness macular hole, pars plana vitrectomy has long been the standard of care. However, due to the possible surgical complications and need for postoperative care, many have searched for non-surgical options via pharmacologic vitreolysis. Ocriplasmin (Jetrea, Thrombogenics USA, Alcon/Novartis EU) is a recombinant protease approved in October 2012 for the treatment of symptomatic vitreomacular adhesion (VMA). There have been conflicting views on the safety of Ocriplasmin with changes in the ellipsoid zone seen on OCT and changes seen on ERG indicating photoreceptor damage. This publication reviews the efficacy and safety of ocriplasmin injection for VMA based on previously published data.
Topics: Fibrinolysin; Fibrinolytic Agents; Humans; Peptide Fragments; Retinal Diseases; Tissue Adhesions; Treatment Outcome; Vitreous Body; Vitreous Detachment
PubMed: 27786583
DOI: 10.1080/08820538.2016.1228416 -
International Journal of Molecular... May 2023Hemostasis is a delicate balance between coagulation and fibrinolysis that regulates the formation and removal of fibrin, respectively. Positive and negative feedback...
Hemostasis is a delicate balance between coagulation and fibrinolysis that regulates the formation and removal of fibrin, respectively. Positive and negative feedback loops and crosstalk between coagulation and fibrinolytic serine proteases maintain the hemostatic balance to prevent both excessive bleeding and thrombosis. Here, we identify a novel role for the glycosylphosphatidylinositol (GPI)-anchored serine protease testisin in the regulation of pericellular hemostasis. Using in vitro cell-based fibrin generation assays, we found that the expression of catalytically active testisin on the cell surface accelerates thrombin-dependent fibrin polymerization, and intriguingly, that it subsequently promotes accelerated fibrinolysis. We find that the testisin-dependent fibrin formation is inhibited by rivaroxaban, a specific inhibitor of the central prothrombin-activating serine protease factor Xa (FXa), demonstrating that cell-surface testisin acts upstream of factor X (FX) to promote fibrin formation at the cell surface. Unexpectedly, testisin was also found to accelerate fibrinolysis by stimulating the plasmin-dependent degradation of fibrin and enhancing plasmin-dependent cell invasion through polymerized fibrin. Testisin was not a direct activator of plasminogen, but it is able to induce zymogen cleavage and the activation of pro-urokinase plasminogen activator (pro-uPA), which converts plasminogen to plasmin. These data identify a new proteolytic component that can regulate pericellular hemostatic cascades at the cell surface, which has implications for angiogenesis, cancer biology, and male fertility.
Topics: Male; Humans; Fibrinolysis; Fibrinolysin; Glycosylphosphatidylinositols; Serine Proteases; Serine Endopeptidases; Plasminogen; Urokinase-Type Plasminogen Activator; Fibrin; Hemostatics
PubMed: 37298257
DOI: 10.3390/ijms24119306 -
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 -
Journal of Thrombosis and Haemostasis :... May 2022Geographic variability in coagulation across populations and their determinants are poorly understood.
BACKGROUND
Geographic variability in coagulation across populations and their determinants are poorly understood.
OBJECTIVE
To compare thrombin (TG) and plasmin (PG) generation parameters between healthy Tanzanian and Dutch individuals, and to study associations with inflammation and different genetic, host and environmental factors.
METHODS
TG and PG parameters were measured in 313 Tanzanians of African descent living in Tanzania and 392 Dutch of European descent living in the Netherlands and related to results of a dietary questionnaire, circulating inflammatory markers, genotyping, and plasma metabolomics.
RESULTS
Tanzanians exhibited an enhanced TG and PG capacity, compared to Dutch participants. A higher proportion of Tanzanians had a TG value in the upper quartile with a PG value in the lower/middle quartile, suggesting a relative pro-coagulant state. Tanzanians also displayed an increased normalized thrombomodulin sensitivity ratio, suggesting reduced sensitivity to protein C. In Tanzanians, PG parameters (lag time and TTP) were associated with seasonality and food-derived plasma metabolites. The Tanzanians had higher concentrations of pro-inflammatory cytokines, which correlated strongly with TG and PG parameters. There was limited overlap in genetic variation associated with TG and PG parameters between the two cohorts. Pathway analysis of genetic variants in the Tanzanian cohort revealed multiple immune pathways that were enriched with TG and PG traits, confirming the importance of co-regulation between coagulation and inflammation.
CONCLUSIONS
Tanzanians have an enhanced TG and PG potential compared to Dutch individuals, which may relate to differences in inflammation, genetics and diet. These observations highlight the importance of better understanding of the geographic variability in coagulation across populations.
Topics: Adult; Black People; Blood Coagulation; Blood Coagulation Tests; Fibrinolysin; Humans; Inflammation; Netherlands; Tanzania; Thrombin; White People
PubMed: 35102686
DOI: 10.1111/jth.15657 -
Journal of Nephrology Feb 2016Plasmin has recently been reported to be associated with renal fibrosis in experimental models, but its role in human renal diseases is unclear.
BACKGROUND
Plasmin has recently been reported to be associated with renal fibrosis in experimental models, but its role in human renal diseases is unclear.
METHODS
Fifty-seven patients with IgA nephropathy (IgAN) were evaluated retrospectively. Plasmin in their renal biopsy tissues was assessed by in situ zymography using a plasmin-sensitive synthetic peptide, and the relationships between patients' histologic or clinical parameters and their renal plasmin activity [assessed semiquantitatively by calculating the positively stained percentage of the total tubulointerstitial (TI) area] were evaluated.
RESULTS
Plasmin activity was observed almost exclusively in the TI space (mainly in the interstitium and partly in the tubular epithelial cells) and was significantly stronger in patients with TI lesion (tubular atrophy/interstitial fibrosis and tubulointerstitial inflammation) than in those without TI lesion. It was significantly and positively correlated with the global glomerulosclerosis rate and significantly and negatively correlated with estimated glomerular filtration rate not only at the time of renal biopsy but also at the end of the follow-up period. Double stainings for plasmin activity and inflammatory cells, cytokeratin, or α-smooth muscle actin (α-SMA) in selected patients revealed TI infiltration of inflammatory cells, attenuated tubular epithelial expression of cytokeratin, and augmented interstitial expression of α-SMA close to upregulated plasmin activity in the TI space.
CONCLUSIONS
These data suggest that TI plasmin is associated with TI inflammation leading to renal fibrosis, and can cause the decline in renal function seen in patients with IgAN. Reducing plasmin in situ may therefore be a promising therapeutic approach slowing renal fibrogenesis and improving renal function.
Topics: Actins; Adolescent; Adult; Aged; Biomarkers; Biopsy; Disease Progression; Epithelial Cells; Female; Fibrinolysin; Fibrosis; Fluorescent Antibody Technique; Glomerular Filtration Rate; Glomerulonephritis, IGA; Humans; Keratins; Kidney Tubules; Male; Middle Aged; Retrospective Studies; Severity of Illness Index; Young Adult
PubMed: 25971850
DOI: 10.1007/s40620-015-0205-1 -
Hepatology (Baltimore, Md.) Nov 2018The serine protease plasmin degrades extracellular matrix (ECM) components both directly and indirectly through activation of matrix metalloproteinases. Excessive...
The serine protease plasmin degrades extracellular matrix (ECM) components both directly and indirectly through activation of matrix metalloproteinases. Excessive plasmin activity and subsequent ECM degradation cause hepatic sinusoidal fragility and hemorrhage in developing embryos. We report here that excessive plasmin activity in a murine acetaminophen (APAP) overdose model likewise compromises hepatic sinusoidal vascular integrity in adult animals. We found that hepatic plasmin activity is up-regulated significantly at 6 hours after APAP overdose. This plasmin up-regulation precedes both degradation of the ECM component fibronectin around liver vasculature and bleeding from centrilobular sinusoids. Importantly, administration of the pharmacological plasmin inhibitor tranexamic acid or genetic reduction of plasminogen, the circulating zymogen of plasmin, ameliorates APAP-induced hepatic fibronectin degradation and sinusoidal bleeding. Conclusion: These studies demonstrate that reduction of plasmin stabilizes hepatic sinusoidal vascular integrity after APAP overdose. (Hepatology 2018; 00:1-13).
Topics: Acetaminophen; Analgesics, Non-Narcotic; Animals; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Drug Overdose; Fibrinolysin; Fibronectins; Fluorescent Antibody Technique; Immunoblotting; Liver; Male; Mice; Mice, Inbred C57BL; Real-Time Polymerase Chain Reaction
PubMed: 29729197
DOI: 10.1002/hep.30070 -
Journal of Cerebral Blood Flow and... Aug 2014Plasmin, the principal downstream product of tissue-type plasminogen activator (tPA), is known for its potent fibrin-degrading capacity but is also recognized for many... (Review)
Review
Plasmin, the principal downstream product of tissue-type plasminogen activator (tPA), is known for its potent fibrin-degrading capacity but is also recognized for many non-fibrinolytic activities. Curiously, plasmin has not been conclusively linked to blood-brain barrier (BBB) disruption during recombinant tPA (rtPA)-induced thrombolysis in ischemic stroke. This is surprising given the substantial involvement of tPA in the modulation of BBB permeability and the co-existence of tPA and plasminogen in both blood and brain throughout the ischemic event. Here, we review the work that argues a role for plasmin together with endogenous tPA or rtPA in BBB alteration, presenting the overall controversy around the topic yet creating a rational case for an involvement of plasmin in this process.
Topics: Animals; Blood-Brain Barrier; Brain Ischemia; Cerebral Hemorrhage; Fibrinolysin; Fibrinolytic Agents; Humans; Recombinant Proteins; Stroke; Tissue Plasminogen Activator
PubMed: 24896566
DOI: 10.1038/jcbfm.2014.99