Plasmin-mediated Fibrinolysis Research Articles

A mathematical model of plasmin-mediated fibrinolysis of single fibrin fibers.

Fibrinolysis, the plasmin-mediated degradation of the fibrin mesh that stabilizes blood clots, is an important physiological process, and understanding mechanisms underlying lysis is critical for improved stroke treatment. Experimentalists are now able to study lysis on the scale of single fibrin fibers, but mathematical models of lysis continue to focus mostly on fibrin network degradation. Experiments have shown that while some degradation occurs along the length of a fiber, ultimately the fiber is cleaved at a single location. We built a 2-dimensional stochastic model of a fibrin fiber cross-section that uses the Gillespie algorithm to study single fiber lysis initiated by plasmin. We simulated the model over a range of parameter values to learn about patterns and rates of single fiber lysis in various physiological conditions. We also used epifluorescent microscopy to measure the cleavage times of fibrin fibers with different apparent diameters. By comparing our model results to the laboratory experiments, we were able to: 1) suggest value ranges for unknown rate constants(namely that the degradation rate of fibrin by plasmin should be ≤ 10 s-1 and that if plasmin crawls, the rate of crawling should be between 10 s-1 and 60 s-1); 2) estimate the fraction of fibrin within a fiber cross-section that must be degraded for the fiber to cleave in two; and 3) propose that that fraction is higher in thinner fibers and lower in thicker fibers. Collectively, this information provides more details about how fibrin fibers degrade, which can be leveraged in the future for a better understanding of why fibrinolysis is impaired in certain disease states, and could inform intervention strategies.

Tissue Factor Pathway Inhibitors as Potential Targets for Understanding the Pathophysiology of Preeclampsia.

Preeclampsia is a hypertensive disorder of pregnancy that causes maternal and perinatal morbidity and mortality worldwide. Preeclampsia is associated with complex abnormalities of the coagulation and fibrinolytic system. Tissue factor (TF) is involved in the hemostatic system during pregnancy, while the Tissue Factor Pathway Inhibitor (TFPI) is a major physiological inhibitor of the TF-initiated coagulation cascade. The imbalance in hemostatic mechanisms may lead to a hypercoagulable state, but prior research has not comprehensively investigated the roles of TFPI1 and TFPI2 in preeclamptic patients. In this review, we summarize our current understanding of the biological functions of TFPI1 and TFPI2 and discuss future directions in preeclampsia research. A literature search was performed from inception to 30 June 2022 in the PubMed and Google Scholar databases. TFPI1 and TFPI2 are homologues with different protease inhibitory activities in the coagulation and fibrinolysis system. TFPI1 is an essential physiological inhibitor of the TF-initiated extrinsic pathway of coagulation. On the other hand, TFPI2 inhibits plasmin-mediated fibrinolysis and exerts antifibrinolytic activity. It also inhibits plasmin-mediated inactivation of clotting factors and maintains a hypercoagulable state. Furthermore, in contrast to TFPI1, TFPI2 suppresses trophoblast cell proliferation and invasion and promotes cell apoptosis. TFPI1 and TFPI2 may play important roles in regulating the coagulation and fibrinolytic system and trophoblast invasion to establish and maintain successful pregnancies. Concentrations of TF, TFPI1, and TFPI2 in maternal blood and placental tissue are significantly altered in preeclamptic women compared to normal pregnancies. TFPI protein family may affect both the anticoagulant (i.e., TFPI1) and antifibrinolytic/procoagulant (i.e., TFPI2) systems. TFPI1 and TFPI2 may function as new predictive biomarkers for preeclampsia and navigate precision therapy.

Identification of substrates of MBL Associated Serine Protease-1 (MASP-1) from human plasma using N-terminomics strategy

MBL Associated Serine Protease-1 (MASP-1) is an abundant enzyme of the lectin complement pathway. MASP-1 cleaves numerous substrates like MASP-2, MASP-3, C2, C3i, fibrinogen, FXIII and prothrombin. It has thrombin-like specificity and can cleave thrombin substrates. Owing to its high concentration and relaxed substrate specificity, MASP-1 has substrates outside the complement system and can influence other proteolytic cascades and physiological processes. The unidentified substrates may assist us to ascertain the role(s) of MASP-1. In this study, we used a high-throughput N-terminomics method to identify substrates of MASP-1 from human plasma. We have identified 35 putative substrates of MASP-1. Among the identified proteins, alpha 2-antiplasmin, alpha-1-acid glycoprotein, antithrombin III, and siglec-6 were demonstrated to be cleaved by MASP-1. We have discussed the physiological relevance of cleavage of these substrates by MASP-1. The expression of Siglec-6 and MASP-1 has been reported in the B cells. Alpha-1-acid glycoprotein cleavage by MASP-1 may occur in the acute phase as it is known to be an inhibitor of platelet aggregation, whereas MASP-1 triggers platelet aggregation. The cleavage alpha2 antiplasmin by MASP-1 implies that MASP-1 may be promoting plasmin-mediated fibrinolysis. Our study supports that MASP-1 may be implicated in thrombosis as well as thrombolysis.

Fibrinogen Mitigates Prion-Mediated Platelet Activation and Neuronal Cell Toxicity.

Prion peptide (PrP) misfolds to infectious scrapie isoform, the β pleat-rich insoluble fibrils responsible for neurodegeneration and fatal conformational diseases in humans. The amino acid sequence 106–126 from prion proteins, PrP(106–126), is highly amyloidogenic and implicated in prion-induced pathologies. Here, we report a novel interaction between PrP(106–126) and the thrombogenic plasma protein fibrinogen that can lead to mitigation of prion-mediated pro-thrombotic responses in human platelets as well as significant decline in neuronal toxicity. Thus, prior exposure to fibrinogen-restrained PrP-induced rise in cytosolic calcium, calpain activation, and shedding of extracellular vesicles in platelets while it, too, averted cytotoxicity of neuronal cells triggered by prion peptide. Interestingly, PrP was found to accelerate fibrin-rich clot formation, which was resistant to plasmin-mediated fibrinolysis, consistent with enhanced thrombus stability provoked by PrP. We propose that PrP-fibrinogen interaction can be clinically exploited further for prevention and management of infectious prion related disorders. Small molecules or peptides mimicking PrP-binding sites on fibrinogen can potentially mitigate PrP-induced cellular toxicity while also preventing the negative impact of PrP on fibrin clot formation and lysis.

Fibrinogen Mitigates Prion-Mediated Platelet Activation and Neuronal Cell Toxicity

Background: Prion peptide (PrP) is known to mediate neurotoxicity and fatal conformational diseases, as well as platelet activation signaling. Fibrinogen is a thrombogenic peptide circulating in abundance in blood. Methods: Interaction between plasma fibrinogen and PrP(106-126) was examined using surface plasmon resonance. To evaluate the effect of this interaction, intracellular calcium, extracellular vesicle release, calpain activity and talin degradation were studied in platelets and cell viability, as well as mitochondrial membrane potential were investigated in neuronal cells. Fibrin-clot formation and fibrinolysis in presence of PrP(106-126) were studied under confocal microscope. Findings: Prior exposure to fibrinogen restrained PrP-induced rise in cytosolic calcium, calpain activation and shedding of extracellular vesicles in platelets while it, too, averted cytotoxicity of neuronal cells triggered by prion peptide. Remarkably, PrP, on the other hand, accelerated fibrin-rich clot formation that was resistant to plasmin-mediated fibrinolysis, thus contributing to enhanced thrombus stability. Interpretation: The interaction between PrP and fibrinogen reverses PrP (106-126)-induced platelet activation and neurotoxicity while prion peptide, on the other hand, alters the morphology of fibrin clot making it more resistant to lysis. This interaction can be exploited to develop small-molecule pharmacological modulators mimicking PrP-binding sites on fibrinogen that may attenuate the adverse effects of prion, on both platelets as well as neuronal cells. Funding Information: This research was supported by Indian Council of Medical Research under CAR, Department of Biotechnology, Science and Engineering Research Board, and a J.C. Bose National fellowship received by D. Dash from the Government of India. Declaration of Interests: None. Ethics Approval Statement: Informed written consent was obtained, strictly as per the recommendations and approval of the Institutional Ethical Committee of Banaras Hindu University. The study methodologies were conformed to the standards set by the Declaration of Helsinki.

Actin is associated with tissue injury in trauma patients and produces a hypercoagulable profile in vitro.

While tissue injury provokes fibrinolysis shutdown in trauma, the mechanism remains elusive. Cellular death causes release of structural proteins, including actin and myosin, which may interact with clot formation and structure. We hypothesized that tissue injury is associated with high circulating actin and that actin produces a hypercoagulable profile with decreased fibrinolysis in vitro. Blood was collected from trauma activation patients at a single Level I trauma center for thrombelastography and proteomics. Proteomic analyses were performed through targeted liquid chromatography coupled with mass spectrometry using isotope-labeled standards for quantification of actin and its endogenous inhibitor gelsolin. Based on the results, we added physiologic concentrations of cytoskeletal G-actin to whole blood from healthy volunteers and analyzed changes in thrombelastography, as well as to plasma and examined clot architecture using confocal microscopy of fluorescently labeled fibrinogen. Overall, 108 trauma patients were included: majority (71%) men, median age of 32.7 years, 66% blunt mechanism, median New Injury Severity Score (NISS) of 41. Compared with patients without severe tissue injury (NISS < 15, n = 10), patients with severe tissue injury (NISS > 15, n = 98) had higher levels of circulating actin (0.0428 vs. 0.0301, p = 0.02). Further, there was a trend toward lower gelsolin levels in patients with fibrinolysis shutdown (0.1844 vs. 0.2052, p = 0.17) and tissue plasminogen activator resistance (0.1676 vs. 0.2188, p = 0.06).Ten healthy volunteers were included in the in vitro experiments (50% male; median age, 31.3 years). Actin significantly increased angle (40.0° to 52.9°, p = 0.002) and decreased fibrinolysis (percent clot lysis 30 minutes after reaching maximum amplitude, 4.0% to 1.6%; p = 0.002), provoking fibrinolytic shutdown in three patients. The addition of actin to control plasma decreased fiber resolvability of fibrin clots, monitored by microscopy, and decreased plasmin-mediated fibrinolysis. Actin increases clot propagation and provokes fibrinolysis shutdown in vitro, through a mechanism of plasmin inhibition. High circulating levels of actin are present in trauma patients with severe tissue injury, suggesting actin contributes to fibrinolysis shutdown in the setting of tissue injury.

Tranexamic acid: A potential treatment option for coronavirus disease 2019

Sir, COVID-19 has multifaceted presentations and majority of the patients are asymptomatic. Nevertheless, there is a subgroup of patients who tend to present with moderate-to-severe disease. Age, gender, socioeconomic status, and comorbidities such as diabetes mellitus, hypertension, cardiovascular disease, chronic lung and kidney disease, history of cancer, smoking, and obesity have been associated with moderate to severe disease.[1] Herein, we report a novel case of an 82-year-old gentleman, with multiple comorbidities including hypertension, coronary artery disease, history of prostate cancer, marginal zone lymphoma, myelodysplastic syndrome, and body mass index of 32 kg/m2, who was tested positive for SARS-CoV-2 reverse transcription-polymerase chain reaction and was predicted to have severe COVID-19 but only presented with mild symptoms with no signs and symptoms of pulmonary involvement or “cytokine storm.” Inflammatory markers including D-dimer, lactate dehydrogenase, C-reactive protein, and ferritin were 587 ng/ml (0–500 ng/ml), 232 U/L (122–222 U/L), 1.1 mg/dl (0–0.8 mg/dl), and 657 ng/ml (24–336 ng/ml), respectively. Chest X-ray showed bilateral infiltrates and computed tomography of the chest showed multifocal ground-glass opacities [Figure 1].Figure 1: Computed tomography of chest showed multifocal ground-glass opacities, more on the peripheral and basal regions, consistent with COVID-19 pictureThis interesting observation may be due to the medication – tranexamic acid (TXA) that he has been taking since October 2019. TXA has anti-inflammatory properties via inhibition of plasmin-mediated activation of complement, monocytes, and neutrophils, as well as modulates various cytokines and cellular immune markers.[2] Draxler et al. have demonstrated a significant reduction in tumor necrosis factor-alpha and interleukin-6 after the administration of the TXA in healthy individuals.[3] Moreover, TXA has also been reported in modulating coagulopathy by direct inhibiting of plasmin-mediated fibrinolysis.[4] The novel observation of potential cytokine storm suppression in this patient on TXA has opened a door for potential use of this medication as part of COVID-19 management. The anti-inflammatory and antifibrinolytic activities of TXA may shed a light in the future treatment for COVID-19. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Biorelevant polyanions stabilize fibrin against mechanical and proteolytic decomposition: Effects of polymer size and electric charge

The release of neutrophil extracellular traps (NETs) containing DNA and histones is an essential mechanism in the neutrophil-mediated innate immunity. In thrombi the polyanionic DNA confers mechanical and lytic resistance to fibrin and heparins interfere with the effects of NET components. Heparins are polyanions used not only as therapeutic agents, but they are also released by mast cells at entry sites of pathogens. Platelets and microorganisms release a different type of polyanions (polyphosphates) of various size (in the range 60–1000 phosphate monomers). With the current study we aimed to evaluate if the stability of fibrin is influenced by the type of polyanion, its molecular size or relative electric charge.Fibrin structure was approached with scanning electron microscopy (SEM) and pressure-driven permeation. An oscillation rheometer was used to investigate viscoelastic properties. Kinetic turbidimetric assays for the generation and dissolution of composite fibrin clots containing unfractionated heparin (UFH), and its partially or fully desulfated derivatives, as well as low molecular-weight heparin (LMWH), pentasaccharide (S5), and polyphosphates composed of 45 (P45), 100 (P100) or 700 (P700) monomers at average.The smaller polyanions P45, P100, LMWH, and S5 accelerated, whereas P700 and UFH retarded clot formation. All polyanions altered the fibrin structure: SEM and clot permeation showed thicker fibers with smaller (LMWH, S5, P700) or larger (UFH, P100) pores. All polyanions stabilized the clots mechanically, but the smaller P45, P100 and LMWH decreased the deformability of fibrin, whereas the large UFH and P700 increased the maximal bearable deformation of clots. Despite the size-dependent structural changes, all heparins caused a 10–15% prolongation of lysis-times with plasmin, and UFH-effects depended on sulfation patterns. The 20–35% prolongation of lysis-times caused by all polyphosphates was a kringle-dependent phenomenon, and was dampened in the presence of 6-aminohexanoate blocking the lysine-binding sites of plasmin.In summary, we found that polyanions of different chemical structure stabilize fibrin clots via size-dependent modulation of fibrin structure and kringle-dependent inhibition of plasmin-mediated fibrinolysis.

Plasmin-mediated fibrinolysis enables macrophage migration in a murine model of inflammation

AbstractEfficient migration of macrophages to sites of inflammation requires cell surface–bound plasmin(ogen). Here, we investigated the mechanisms underlying the deficits of plasmin(ogen)-mediated macrophage migration in 2 models: murine thioglycollate-induced peritonitis and in vitro macrophage migration. As previously reported, macrophage migration into the peritoneal cavity of mice in response to thioglycollate was significantly impaired in the absence of plasminogen. Fibrin(ogen) deposition was noted in the peritoneal cavity in response to thioglycollate, with a significant increase in fibrin(ogen) in the plasminogen-deficient mice. Interestingly, macrophage migration was restored in plasminogen-deficient mice by simultaneous imposition of fibrinogen deficiency. Consistent with this in vivo finding, chemotactic migration of cultured macrophages through a fibrin matrix did not occur in the absence of plasminogen. The macrophage requirement for plasmin-mediated fibrinolysis, both in vivo and in vitro, was negated by deletion of the major myeloid integrin αMβ2-binding motif on the γ chain of fibrin(ogen). The study identifies a critical role of fibrinolysis in macrophage migration, presumably through the alleviation of migratory constraints imposed by the interaction of leukocytes with fibrin(ogen) through the integrin αMβ2 receptor.

Exchange protein directly activated by cAMP plays a critical role in regulation of vascular fibrinolysis

Plasmin-mediated fibrinolysis at the surface of vascular endothelial cells (SVEC) plays a key role in maintaining vascular hemostasis, in which the cAMP pathway participates. After externalization to the SVEC, annexin A2 (ANXA2) serves as a platform for conversion of plasminogen to plasmin. Here we describe a regulatory role of the exchange protein directly activated by cAMP (EPAC) in ANXA2 externalization and vascular fibrinolysis. Knockout of EPAC1 in mice results in a decreased ANXA2 expression on the SVEC associated with increased fibrin deposition and fibrinolytic dysfunction. Reduced levels of EPAC1 are also found in endocardial tissues beneath atrial mural thrombi in patients. Notably, administration of recombinant ANXA2 ameliorates fibrinolytic dysfunction in the EPAC1-null mice. Mechanistically, EPAC1 regulates the SVEC plasminogen conversion depended on ANXA2. EPAC1 promotes tyrosine-23 phosphorylation of ANXA2, a prerequisite for its recruitment to the SVEC. Our data thus reveal a novel regulatory role for EPAC1 in vascular fibrinolysis.

A Novel Enzyme Capture - ELISA for the Direct Determination of Factor XIII Activity

Background: Activated factor XIII (fXIII) mechanically stabilizes fibrin clots by catalyzing the formation of intramolecular crosslinks and retards plasmin-mediated fibrinolysis by incorporating intermolecular α2-antiplasmin (α2AP):fibrin crosslinks. High quality quantitative fXIII activity assays are required for the accurate and timely diagnosis of fXIII deficiency. Currently available high-throughput assays indirectly measure the transglutaminase activity of fXIII via detection of ammonia that is elaborated upon formation of ε-N-(γ-glutamyl)-lysyl crosslinking bonds. Unfortunately, these assays can be confounded by fXIII-independent ammonia producing and consuming reactions present in plasma. We thus set out to develop an assay which directly quantifies the transglutaminase activity. We hypothesized that fXIII could be captured in a microtiter plate with anti-fibrinogen antibodies and, following an activation step, its activity determined in a kinetic fashion via the rate of α2AP incorporation (measured in a subsequent ELISA step).Methods: Co-immunoprecipitation and western blotting techniques were utilized to confirm fXIII/fibrinogen interactions and to determine the presence of basal interactions between fibrinogen and α2AP. Simultaneously, this approach enabled the determination of suitable antibody pairs for use in the enzyme capture-ELISA (EC-ELISA). In microtiter plates, fibrinogen-captured fXIII was activated with excess thrombin, physiologic calcium, and GPRP peptide (to block fibrin polymerization) in the presence of excess α2AP. FXIII activity was stopped with a potent fXIII-inhibitor (iodoacetamide) at predetermined time points. Subsequently, α2AP incorporation was determined with a primary anti-α2AP antibody and secondary HRP-conjugated antibody.Results: Co-immunoprecipitation and western blotting experiments confirmed that both fXIII and α2AP circulate in complex with fibrinogen, as previously reported by others. However, α2AP incorporation significantly increased following incubation with activated factor XIII. Using the EC-ELISA method, we were able to demonstrate that α2AP is incorporated in a manner consistent with first order kinetics.Discussion: These experiments demonstrate the feasibility of an EC-ELISA assay to directly measure fXIII activity. Further work will be required to determine the sensitivity of this assay in test plasmas with varying known quantities of fXIII and how its performance characteristics compare to currently available indirect activity assays. With further optimization, the EC-ELISA method may lead to an improved, high throughput diagnostic assay for clinical use. DisclosuresNo relevant conflicts of interest to declare.

Absence of functional compensation between coagulation factor VIII and plasminogen in double-knockout mice

AbstractPlasminogen deficiency is associated with severely compromised fibrinolysis and extravascular deposition of fibrin. In contrast, coagulation factor VIII (FVIII) deficiency leads to prolonged and excessive bleeding. Based on opposing biological functions of plasminogen and FVIII deficiencies, we hypothesized that genetic elimination of FVIII would alleviate the systemic formation of fibrin deposits associated with plasminogen deficiency and, in turn, elimination of plasminogen would limit bleeding symptoms associated with FVIII deficiency. Mice with single and combined deficiencies of FVIII (F8−/−) and plasminogen (Plg−/−) were evaluated for phenotypic characteristics of plasminogen deficiency, including wasting disease, shortened lifespan, rectal prolapse, and multiorgan fibrin deposition. Conversely, to specifically examine the role of plasmin-mediated fibrinolysis on bleeding caused by FVIII deficiency, F8−/− and F8−/−/Plg−/− mice were subjected to a bleeding challenge. Mice with a combined deficiency in FVIII and plasminogen displayed no phenotypic differences relative to mice with single FVIII or plasminogen deficiency. Plg−/− and F8−/−/Plg−/− mice exhibited the same penetrance and severity of wasting disease, rectal prolapse, extravascular fibrin deposits, and reduced viability. Furthermore, following a tail vein–bleeding challenge, no significant differences in bleeding times or total blood loss could be detected between F8−/− and F8−/−/Plg−/− mice. Moreover, F8−/− and F8−/−/Plg−/− mice responded similarly to recombinant FVIII (rFVIII) therapy. In summary, the pathological phenotype of Plg−/− mice developed independently of FVIII-dependent coagulation, and elimination of plasmin-driven fibrinolysis did not play a significant role in a nonmucosal bleeding model in hemophilia A mice.

Evaluating the potential of endothelial cells derived from human induced pluripotent stem cells to form microvascular networks in 3D cultures

A major translational challenge in the fields of therapeutic angiogenesis and regenerative medicine is the need to create functional microvasculature. The purpose of this study was to assess whether a potentially autologous endothelial cell (EC) source derived from human induced pluripotent stem cells (iPSC-ECs) can form the same robust, stable microvasculature as previously documented for other sources of ECs. We utilized a well-established in vitro assay, in which endothelial cell-coated (iPSC-EC or HUVEC) beads were co-embedded with fibroblasts in a 3D fibrin matrix to assess their ability to form stable microvessels. iPSC-ECs exhibited a five-fold reduction in capillary network formation compared to HUVECs. Increasing matrix density reduced sprouting, although this effect was attenuated by distributing the NHLFs throughout the matrix. Inhibition of both MMP- and plasmin-mediated fibrinolysis was required to completely block sprouting of both HUVECs and iPSC-ECs. Further analysis revealed MMP-9 expression and activity were significantly lower in iPSC-EC/NHLF co-cultures than in HUVEC/NHLF co-cultures at later time points, which may account for the observed deficiencies in angiogenic sprouting of the iPSC-ECs. Collectively, these findings suggest fundamental differences in EC phenotypes must be better understood to enable the promise and potential of iPSC-ECs for clinical translation to be realized.

Human Kunitz-type protease inhibitor engineered for enhanced matrix retention extends longevity of fibrin biomaterials.

Aprotinin is a broad-spectrum serine protease inhibitor used in the clinic as an anti-fibrinolytic agent in fibrin-based tissue sealants. However, upon re-exposure, some patients suffer from hypersensitivity immune reactions likely related to the bovine origin of aprotinin. Here, we aimed to develop a human-derived substitute to aprotinin. Based on sequence homology analyses, we identified the Kunitz-type protease inhibitor (KPI) domain of human amyloid-β A4 precursor protein as being a potential candidate. While KPI has a lower intrinsic anti-fibrinolytic activity than aprotinin, we reasoned that its efficacy is additionally limited by its fast release from fibrin material, just as aprotinin's is. Thus, we engineered KPI variants for controlled retention in fibrin biomaterials, using either covalent binding through incorporation of a substrate for the coagulation transglutaminase Factor XIIIa or through engineering of extracellular matrix protein super-affinity domains for sequestration into fibrin. We showed that both engineered KPI variants significantly slowed plasmin-mediated fibrinolysis invitro, outperforming aprotinin. Invivo, our best engineered KPI variant (incorporating the transglutaminase substrate) extended fibrin matrix longevity by 50%, at a dose at which aprotinin did not show efficacy, thus qualifying it as a competitive substitute of aprotinin in fibrin sealants.

Plasminogen Deficiency Delays the Onset and Protects from Demyelination and Paralysis in Autoimmune Neuroinflammatory Disease.

Multiple sclerosis (MS) is a neuroinflammatory, demyelinating disease of the CNS. Fibrinogen deposition at sites of blood-brain barrier breakdown is a prominent feature of neuroinflammatory disease and contributes to disease severity. Plasminogen, the primary fibrinolytic enzyme, also modifies inflammatory processes. We used a murine model of MS, experimental autoimmune encephalomyelitis (EAE), to evaluate the hypothesis that the loss of plasminogen would exacerbate neuroinflammatory disease. However, contrary to initial expectations, EAE-challenged plasminogen-deficient (Plg-) mice developed significantly delayed disease onset and reduced disease severity compared with wild-type (Plg+) mice. Similarly, pharmacologic inhibition of plasmin activation with tranexamic acid also delayed disease onset. The T-cell response to immunization was similar between genotypes, suggesting that the contribution of plasminogen was downstream of the T-cell response. Spinal cords from EAE-challenged Plg- mice demonstrated significantly decreased demyelination and microglial/macrophage accumulation compared with Plg+ mice. Although fibrinogen-deficient mice or mice with combined deficiencies of plasminogen and fibrinogen had decreased EAE severity, they did not exhibit the delay in EAE disease onset, as seen in mice with plasminogen deficiency alone. Together, these data suggest that plasminogen and plasmin-mediated fibrinolysis is a key modifier of the onset of neuroinflammatory demyelination.SIGNIFICANCE STATEMENT Multiple sclerosis is a severe, chronic, demyelinating disease. Understanding the pathobiology related to the autoreactive T-cell and microglial/macrophage demyelinating response is critical to effectively target therapeutics. We describe for the first time that deficiency of plasminogen, the key fibrinolytic enzyme, delays disease onset and protects from the development of the paralysis associated with a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Administration of a widely used, pharmacologic inhibitor of plasminogen activation, tranexamic acid, also delays the onset of neuroinflammation associated with EAE.

An acidic microenvironment sets the humoral pattern recognition molecule PTX3 in a tissue repair mode

Pentraxin 3 (PTX3) is a fluid-phase pattern recognition molecule and a key component of the humoral arm of innate immunity. In four different models of tissue damage in mice, PTX3 deficiency was associated with increased fibrin deposition and persistence, and thicker clots, followed by increased collagen deposition, when compared with controls. Ptx3-deficient macrophages showed defective pericellular fibrinolysis in vitro. PTX3-bound fibrinogen/fibrin and plasminogen at acidic pH and increased plasmin-mediated fibrinolysis. The second exon-encoded N-terminal domain of PTX3 recapitulated the activity of the intact molecule. Thus, a prototypic component of humoral innate immunity, PTX3, plays a nonredundant role in the orchestration of tissue repair and remodeling. Tissue acidification resulting from metabolic adaptation during tissue repair sets PTX3 in a tissue remodeling and repair mode, suggesting that matrix and microbial recognition are common, ancestral features of the humoral arm of innate immunity.

Inhibition of SERPINE1 Function Attenuates Wound Closure in Response to Tissue Injury: A Role for PAI-1 in Re-Epithelialization and Granulation Tissue Formation

Plasminogen activator inhibitor-1 (PAI-1; SERPINE1) is a prominent member of the serine protease inhibitor superfamily (SERPIN) and a causative factor of multi-organ fibrosis as well as a key regulator of the tissue repair program. PAI-1 attenuates pericellular proteolysis by inhibiting the catalytic activity of both urokinase and tissue-type protease activators (uPA and tPA) effectively modulating, thereby, plasmin-mediated fibrinolysis and the overall pericellular proteolytic cascade. PAI-1 also impacts cellular responses to tissue injury and stress situations (growth, survival, migration) by titering the locale and temporal activation of multimeric cell-surface signaling complexes. This review will describe PAI-1 structure and function and detail the role of PAI-1 in the tissue repair program with an emphasis on cutaneous wound healing.

Shock releases bile acidinducing platelet inhibition and fibrinolysis

BackgroundMetabolites are underappreciated for their effect on coagulation. Taurocholic acid (TUCA), a bile acid, has been shown to regulate cellular activity and promote fibrin sealant degradation. We hypothesize that TUCA impairs whole blood clot formation and promotes fibrinolysis. MethodsTUCA was exogenously added to whole blood obtained from volunteers. A titration from 250 μM–750 μM was used due to biologic relevance. Whole blood mixtures were assayed using thrombelastography for clot strength (maximum amplitude [MA]) and fibrinolysis (LY30) quantification. Tranexamic acid was used to block plasmin-mediated fibrinolysis. Platelet microfluidics were performed. A proteomic analysis was completed on citrated plasma obtained from a shock and resuscitation rat model. ResultsFibrinolysis increased when 750-μM TUCA was added to whole blood (median LY30 0.08–5.7, P = 0.010) and clot strength decreased (median MA of 53.3–43.8, P = 0.010). The addition of tranexamic acid, to a 750-μM TUCA titration, partially reversed the induced fibrinolysis (LY30: without 7.7 versus with 2.7) and the decrease in clot strength (MA: without 48.2 versus with 53.2), but did not reverse the effects to whole blood levels. Platelet function reduced by 50% in the presence of 100-μM TUCA. Rats had a median 52-fold increase in TUCA, after a shock state that stayed elevated after resuscitation. ConclusionsTUCA reduces clot strength and promotes fibrinolysis. The clot strength reduction is attributable to platelet inhibition. This metabolic effect on coagulation warrants further investigation, as localized areas of the body, with high levels of bile acid, may be at risk for postoperative bleeding.

Impact of d-Dimers on the Differential Diagnosis of Acute Chest Pain: Current Aspects Besides the Widely Known.

d-dimers are cleavage products of fibrin that occur during plasmin-mediated fibrinolysis of blood clots. In the emergency department, d-dimer measurement represents a valuable and cost-effective tool in the differential diagnosis of acute chest pain including the main life-threatening entities: acute coronary syndrome, pulmonary embolism, and acute aortic syndrome. Whereas the diagnostic and prognostic values of d-dimer testing in acute coronary syndrome is of less priority, increases of d-dimers are frequently found in venous thromboembolism and acute aortic syndromes, especially acute aortic dissection. As to the high negative predictive value of d-dimer in those disorders, patients with low to intermediate pretest probability may profit in terms of less necessity of further non-invasive or even invasive imaging, simultaneously reducing potential complications and healthcare-related costs. However, because of the low specificity of the different d-dimer tests in contrast to its frequent usage, adequate interpretation is required. Age-related adjustment of d-dimer levels may be used to increase its diagnostic power.

In vivo effect of proton-pump inhibitors on gastric plasmin-dependent fibrinolysis: A study in a porcine model

IntroductionAlkalization of gastric fluids could inhibit plasmin-mediated and/or pepsin-mediated fibrinolysis. We evaluated the gastric antifibrinolytic effect of proton pump inhibitors in a live porcine model. Material and methodsSix pigs were randomly assigned to treatment with proton pump inhibitors vs no treatment. After endoscopic mucosal resection, 8μm sections were incubated on fibrin films. Fibrinolytic activity was assessed through focal lysis time. One-hundred-and-forty-two mucosal sections and 129 submucosal sections were analysed. Twenty-four additional sections were analysed on plates containing tranexamic acid to explore pepsin-mediated fibrinolysis. ResultsFocal lysis times in treated vs control groups were 21.0min vs 21.2min (p=0.39) in the mucosa, and 22.2min vs 20.2min (p=0.56) in the submucosa. No lysis could be seen on the plasmin-inhibited fibrin plates. ConclusionOnly plasmin-mediated fibrinolysis was observed. Proton pump inhibitors had no significant plasmin-dependant antifibrinolytic effect. They may enhance haemostasis through different pathways.