Plasma from patients with vaccine-induced immune thrombotic thrombocytopenia displays increased fibrinolytic potential and enhances tissue-type plasminogen activator but not urokinase-mediated plasminogen activation.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication of adenovirus vector-based COVID-19 vaccines. VITT is associated with markedly raised levels of D-dimer; yet, how VITT modulates the fibrinolytic system is unknown. OBJECTIVES: We aimed to compare changes in fibrinolytic activity in plasma from patients with VITT, patients diagnosed with venous thromboembolism (VTE) after vaccination but without VITT (VTE-no VITT), and healthy vaccinated controls. METHODS: Plasma levels of plasmin-antiplasmin (PAP) complexes, plasminogen, and alpha-2-antiplasmin (α2AP) from 10 patients with VITT, 10 patients with VTE-no VITT, and 14 healthy vaccinated controls were evaluated by enzyme-linked immunosorbent assay and/or Western blotting. Fibrinolytic capacity was evaluated by quantitating PAP levels at baseline and after ex vivo plasma stimulation with 50-nM tissue-type plasminogen activator (tPA) or urokinase for 5 minutes. RESULTS: Baseline PAP complex levels in control and VTE-no VITT individuals were similar but were ∼7-fold higher in plasma from patients with VITT (P < .0001). VITT samples also revealed consumption of α2AP and fibrinogenolysis consistent with a hyperfibrinolytic state. Of interest, VITT plasma produced significantly higher PAP levels after ex vivo treatment with tPA, but not urokinase, compared to the other groups, indicative of increased fibrinolytic potential. This was not due to D-dimer as addition of D-dimer to VTE-no VITT plasma failed to potentiate tPA-induced PAP levels. CONCLUSION: A marked hyperfibrinolytic state occurs in patients with VITT, evidenced by marked elevations in PAP, α2AP consumption, and fibrinogenolysis. An unidentified plasma cofactor that selectively potentiates tPA-mediated plasminogen activation also appears to exist in the plasma of patients with VITT.

Plasminogen activation and plasmin inhibition during in vitro fertilization in bovine: implications for fertilization parameters and early embryo development.

Components of the plasminogen/plasmin system, known to be present in the oocyte, play a key role in maturation and fertilization. The objective of this study was to examine the effect of plasminogen activation and plasmin inhibition by exogenous supplementation of the IVF medium with streptokinase (SK) or ɛ-aminocaproic acid (ε-ACA), respectively, on fertilization parameters and preimplantation embryo development. After in vitro maturation, bovine cumulus-oocyte complexes (COCs) were inseminated in the presence of SK or ε-ACA. The addition of SK to the IVF medium facilitated the adhesion of the spermatozoa to the zona pellucida without affecting the percentages of monospermy. Cleavage rates and blastocyst yield were similar between the SK and Control groups while they were lower with the ε-ACA treatment. Additionally, we found that the expression levels of embryo quality-related genes (SDHA and DNMT3A) could be modified in blastocysts by the addition of SK or ε-ACA during IVF. The results obtained indicate that supplementation of the IVF medium with SK did not greatly alter the embryonic developmental parameters related to embryo quality in blastocysts. Moreover, we noticed that ε-ACA treatment compromises the success of in vitro embryo development, thus highlighting the importance of the plasminogen/plasmin activity during the early stages of embryogenesis in bovine.

Effects of copy number variations on longevity in late-onset Alzheimer's disease patients: insights from a causality network analysis.

Alzheimer's disease (AD), particularly late-onset Alzheimer's disease (LOAD), is a prevalent form of dementia that significantly affects patients' cognitive and behavioral capacities and longevity. Although approximately 70 genetic risk factors linked with AD have been identified, their influence on patient longevity remains unclear. Further, recent studies have associated copy number variations (CNVs) with the longevity of healthy individuals and immune-related pathways in AD patients. This study aims to investigate the role of CNVs on the longevity of AD patients by integrating the Whole Genome Sequencing (WGS) and transcriptomics data from the Religious Orders Study/Memory and Aging Project (ROSMAP) cohort through causality network inference. Our comprehensive analysis led to the construction of a CNV-Gene-Age of Death (AOD) causality network. We successfully identified three key CNVs (DEL5006, mCNV14192, and DUP42180) and seven AD-longevity causal genes (PLGRKT, TLR1, PLAU, CALB2, SYTL2, OTOF, and NT5DC1) impacting AD patient longevity, independent of disease severity. This outcome emphasizes the potential role of plasminogen activation and chemotaxis in longevity. We propose several hypotheses regarding the role of identified CNVs and the plasminogen system on patient longevity. However, experimental validation is required to further corroborate these findings and uncover precise mechanisms. Despite these limitations, our study offers promising insights into the genetic influence on AD patient longevity and contributes to paving the way for potential therapeutic interventions.

The autoactivation of human single-chain urokinase-type plasminogen activator (uPA).

Most serine proteases are synthesized as inactive zymogens that are activated by cleavage by another protease in a tightly regulated mechanism. The urokinase-type plasminogen activator (uPA) and plasmin cleave and activate each other, constituting a positive feedback loop. How this mutual activation cycle begins has remained a mystery. We used hydrogen deuterium exchange mass spectrometry to characterize the dynamic differences between the inactive single-chain uPA (scuPA) and its active form two-chain uPA (tcuPA). The results show that the C-terminal ß-barrel and the area around the new N terminus have significantly reduced dynamics in tcuPA as compared with scuPA. We also show that the zymogen scuPA is inactive but can, upon storage, become active in the absence of external proteases. In addition to plasmin, the tcuPA can activate scuPA by cleavage at K158, a process called autoactivation. Unexpectedly, tcuPA can cleave at position 158 even when this site is mutated. TcuPA can also cleave scuPA after K135 or K136 in the disordered linker, which generates the soluble protease domain of uPA. Plasmin cleaves scuPA exclusively after K158 and at a faster rate than tcuPA. We propose a mechanism by which the uPA receptor dimerization could promote autoactivation of scuPA on cell surfaces. These results resolve long-standing controversies in the literature surrounding the mechanism of uPA activation.

Inactivation of the lysine binding sites of human plasminogen (hPg) reveals novel structural requirements for the tight hPg conformation, M-protein binding, and rapid activation.

Accelerated activation of the human plasminogen zymogen (hPg) to two-chain active plasmin (hPm) is achieved following conformational changes induced by ligand-binding at the lysine-binding sites (LBSs) in four of the five hPg kringle domains. In this manner, pattern D skin-trophic strains of Group A streptococci (GAS), through the expression of surface plasminogen-binding M-protein (PAM), immobilize surface hPg, thereby enabling rapid hPg activation by GAS-secreted streptokinase (SK). Consequently, GAS enhances virulence by digesting extracellular and tight cellular junctional barriers using hPm activity. Many studies have demonstrated the singular importance of the kringle-2 domain of hPg (K2hPg) to PAM-binding using hPg fragments. Recently, we showed, using full-length hPg, that K2hPg is critical for PAM binding. However, these studies did not eliminate any modulatory effects of the non-K2hPg LBS on this interaction. Moreover, we sought to establish the significance of the intramolecular interaction between Asp219 of the LBS of K2hPg and its serine protease domain binding partner, Lys708, to conformational changes in hPg. In the current study, selective inactivation of the LBS of K1hPg, K4hPg, and K5hPg revealed that the LBS of these kringle domains are dispensable for hPg binding to PAM. However, the attendant conformational change upon inactivation of K4hPg LBS increased the affinity of hPg for PAM by an order of magnitude. This finding suggests that the native hPg conformation encloses PAM-binding exosites or sterically hinders access to K2hPg. While simultaneous inactivation of the LBS of K1hPg, K4hPg, and K5hPg inhibited hPg/SK association alongside hPg activation, the replacement of Lys708 generated a slight conformational change that optimally accelerated hPg activation. Thus, we accentuate disparate functions of hPg LBS and conclude, using intact proteins, that K2hPg plays a central role in regulating hPg activation.

Levels of interleukin 1-beta and soluble urokinase plasminogen activation factor in peri-implant sulcular fluid of cement- and screw-retained dental implants.

OBJECTIVE: The present study compared interleukin 1-beta (IL-1ß) and soluble urokinase plasminogen activation factor receptor (suPAR) levels in peri-implant sulcular fluid of patients with cement-retained and screw-retained implants. METHOD AND MATERIALS: Patients with cement-retained and screw-retained implants were included. Demographic data were collected, and implant-related characteristics (geometry, insertion torque, loading and retention protocol, arch location, duration in function, and depth of insertion) were retrieved from records. Modified Plaque Index, crestal bone loss, probing depth, and modified Bleeding Index were measured. suPAR and IL-1ß levels were assessed in peri-implant sulcular fluid. Statistical comparisons were done and correlation between clinicoradiographic parameters and peri-implant sulcular fluid IL-1ß and suPAR were assessed. Statistical significance was judged at P < .05. RESULTS: Clinical and radiographic parameters showed no difference among screw-retained and cement-retained implants. There was no difference peri-implant sulcular fluid volume among patients with cement-retained (0.22 ± 0.00 µL) and screw-retained (0.19 ± 0.005 µL) implants. Levels of IL-1ß and suPAR in patients with cement-retained and screw-retained implants were 50.08 ± 0.6 ng/mL and 44.6 ± 0.08 ng/mL, and 0.28 ± 0.05 ng/mL and 0.22 ± 0.006 ng/mL, respectively. CONCLUSION: Implants with cement-retained or screw-retained restorations are comparable to one another in terms of clinicoradiographic status and demonstrate IL-1ß and suPAR levels within the normal range in the peri-implant sulcular fluid provided oral hygiene is stringently maintained.

Posttranscriptional Regulation of the Plasminogen Activation System by Non-Coding RNA in Cancer.

Various species of non-coding RNAs (ncRNAs) may act as functional molecules regulating diverse biological processes. In cancer cell biology, ncRNAs include RNAs that regulate the expression of oncogenes and tumor suppressor genes through various mechanisms. The urokinase (uPA)-mediated plasminogen activation system (PAS) includes uPA, its inhibitors PAI-1 and PAI-2 and its specific cellular receptor uPAR; their increased expression represents a negative prognostic factor in several cancers. Here, we will briefly describe the main uPA-mediated PAS components and ncRNA species; then, we will review more recent evidence of the roles that ncRNAs may play in regulating the expression and functions of uPA-mediated PAS components in cancer.

Simulated in vitro hypoxic conditions from psoriatic arthritis cartilage change plasminogen activating system urokinase and serpine functionality. Reversal of antiapoptotic protection suggests common homeostatic buffering.

Introduction: Rheumatoid and psoriatic arthritis are both characterised by synovial destruction associated with a higher turnover of the extracellular matrix. In both conditions, inflammatory processes create hypoxic environments which destabilise members of the plasminogen activating system. Aim: Comparing the effect of bioactive concentrations of urokinase (uPA) and serpine (PAI-1) on cellular survival of human fibroblast-like-synoviocytes (HFLS) in rich and hypoxic growth media. Material and methods: Monocultures of HFLS were exposed to bioactive uPA and PAI-1 concentrations in both media conditions for 24, 48 and 72 h. Cellular survival was evaluated with a cell viability assay by spectrum absorbance of formazan reduced WST-8. Results: PAI-1 at 0.1 and 1 µg/ml was found to stimulate cell viability under hypoxic stress at 48 and 72 h of incubation, with the effect increasing from 48 to 72 h. uPA increased cell viability in rich medium at 48 and 72 h of incubation between 5 and 40 ng/l, but was found to reduce viability at 80 ng/l at 24 and 48 h. PAI-1 increased cell viability in the hypoxic stress model, while high concentrations of uPA decreased cell viability in rich medium. Conclusions: The alternative modes of function at extreme concentrations provide a novel description of PAI-1 and uPA activity based on their colocalization and mutual buffering capacity, helping to place these molecules more accurately in the context of arthritic synovial deterioration.

Biochemical and biophysical analysis of the interaction of a recombinant form of Staphylococcus aureus enolase with plasminogen.

Aim: Pathogenic invasion of Staphylococcus aureus is critically dependent on host plasminogen activation. Materials & methods: The pathophysiological implications of the interactions between S. aureus recombinant enolase and host plasminogen were investigated. The effects of mutation and small synthetic peptide inhibitors on interactions were assessed. Results: In vitro, the S. aureus recombinant enolase exists as a catalytically active fragile octamer and a robust dimer. The dimer interacts with the host plasminogen on the S. aureus surface. Conclusion: The interaction of host plasminogen and S. aureus enolase might mediate bacterial adherence to the host, activate the plasminogen with the help of plasminogen activators and prevent α2-antiplasmin-mediated inhibition of plasmin. Incorporating mutant and synthetic peptides inhibited the interactions and their associated pathophysiological consequences.

Giardia duodenalis enolase is secreted as monomer during trophozoite-epithelial cell interactions, activates plasminogen and induces necroptotic damage.

Enolase, a multifunctional protein expressed by multiple pathogens activates plasminogen to promote proteolysis on components of the extracellular matrix, an important event in early host-pathogen interactions. A secreted form of enolase that is released upon the interaction of trophozoites with epithelial cells has been detected in the secretome of G. duodenalis. However, the role of enolase in the host-pathogen interactions remains largely unknown. In this work, the effects of G. duodenalis enolase (Gd-eno) on the epithelial cell model (IEC-6) were analyzed. Firstly, the coding sequence of Giardia enolase was cloned and the recombinant protein used to raise antibodies that were then used to define the localization and role of enolase in epithelial cell-trophozoite interactions. Gd-eno was detected in small cytoplasmic vesicles as well as at the surface and is enriched in the region of the ventral disk of Giardia trophozoites. Moreover, the blocking of the soluble monomeric form of the enzyme, which is secreted upon interaction with IEC-6 cells by the anti-rGd-eno antibodies, significantly inhibited trophozoite attachment to intestinal IEC-6 cell monolayers. Further, rGd-eno was able to bind human plasminogen (HsPlg) and enhanced plasmin activity in vitro when the trophozoites were incubated with the intrinsic plasminogen activators of epithelial cells. In IEC-6 cells, rGd-eno treatment induced a profuse cell damage characterized by copious vacuolization, intercellular separation and detachment from the substrate; this effect was inhibited by either anti-Gd-eno Abs or the plasmin inhibitor ϵ- aminocaproic acid. Lastly, we established that in epithelial cells rGd-eno treatment induced a necroptotic-like process mediated by tumor necrosis factor α (TNF-α) and the apoptosis inducing factor (AIF), but independent of caspase-3. All together, these results suggest that Giardia enolase is a secreted moonlighting protein that stimulates a necroptotic-like process in IEC-6 epithelial cells via plasminogen activation along to TNFα and AIF activities and must be considered as a virulence factor.

bFGF stimulated plasminogen activation factors, but inhibited alkaline phosphatase and SPARC in stem cells from apical Papilla: Involvement of MEK/ERK, TAK1 and p38 signaling.

INTRODUCTION: Basic fibroblast growth factor (bFGF) plays a critical role in odontoblast differentiation and dentin matrix deposition, thereby aiding pulpo-dentin repair and regeneration. OBJECTIVES: The purpose of this study was to clarify the effects of bFGF on plasminogen activation factors, TIMP-1), ALP; and SPARC (osteonectin) expression/production of stem cells from apical papilla (SCAP) in vitro; and the involvement of MEK/ERK, p38, Akt, and TAK1 signaling. METHODS: SCAP were exposed to bFGF with/without pretreatment and co-incubation with various signal transduction inhibitors (U0126, SB203580, LY294002, and 5Z-7-oxozeaenol). The expression of FGF receptors (FGFRs), PAI-1, uPA, p-ERK, p-TAK1, and p-p38 was analyzed via immunofluorescent staining. The gene expression and protein secretion of SCAP were determined via real-time PCR and ELISA. ALP activity was evaluated via ALP staining. RESULTS: SCAP expressed FGFR1, 2, 3, and 4. bFGF stimulated the PAI-1, uPA, uPAR, and TIMP-1 mRNA expression (p < 0.05). bFGF induced PAI-1, uPA, and soluble uPAR production (p < 0.05) but suppressed the ALP activity and SPARC production (p < 0.05) of SCAP. bFGF stimulated ERK, TAK1, and p38 phosphorylation of SCAP. U0126 (a MEK/ERK inhibitor) and 5Z-7-oxozeaenol (a TAK1 inhibitor) attenuated the bFGF-induced PAI-1, uPA, uPAR, and TIMP-1 expression and production of SCAP, but SB203580 (a p38 inhibitor) did not. LY294002, SB203580, and 5Z-7oxozeaenol could not reverse the inhibition of ALP activity caused by bFGF. Interestingly, U0126 and 5Z-7-oxozeaenol prevented the bFGF-induced decline of SPARC production (p < 0.05). CONCLUSION: bFGF may regulate fibrinolysis and matrix turnover via modulation of PAI-1, uPA, uPAR, and TIMP-1, but bFGF inhibited the differentiation (ALP, SPARC) of SCAP. These events are mainly regulated by MEK/ERK, p38, and TAK1. Combined use of bFGF and SCAP may facilitate pulpal/root repair and regeneration via regulation of the plasminogen activation system, migration, matrix turnover, and differentiation of SCAP.

A novel ex vivo approach for measuring plasminogen activation upon established plasma clots.

Background: The fibrinolytic system plays a critical role in maintaining hemostasis. Central to fibrinolysis is the degradation of fibrin by plasmin, produced in the circulation following the activation of plasminogen by plasminogen activators (PAs). Accurately measuring the plasminogen activation rate is vital for the understanding of fibrinolytic processes, particularly in the context of thrombolysis. Yet, due to the insoluble nature of fibrin, in vitro and ex vivo investigations of PA-mediated plasminogen activation have proven challenging. As researchers frequently adopt soluble fibrinogen fragments and/or alter the experimental system beyond what is physiologically relevant, they limit the validation and interpretation of their findings. Here, we present a novel, high-throughput assay for measuring plasminogen activation rates on natural, plasma-derived fibrin that optimally simulates in vivo conditions. Method: Human plasma was used as the source of plasmin(ogen) and fibrin(ogen). "Halo-shaped" plasma clots were produced in a 96-well plate using a thrombin-containing clotting mixture, ensuring that an optically compatible and plasma-free center is maintained in each well. Subsequent additions of a plasmin chromogenic substrate and different PAs were followed by absorbance measurements over time to extract the corresponding enzyme kinetics information. Results and Discussion: Validation experiments demonstrated the capability of our approach to accurately model fibrin-dependent and -independent plasminogen activation as well as sensitively detect variations in plasminogen and fibrinogen plasma levels. Conclusion: This assay allows a straightforward, yet powerful, measurement of plasminogen activation rates on established plasma clots, with the capability of properly assessing fibrin- and non-fibrin-dependent plasminogen activation by various therapeutic PAs.

Role of Plasminogen Activation System in Platelet Pathophysiology: Emerging Concepts for Translational Applications.

Traditionally, platelets have been exclusively considered for their procoagulant and antifibrinolytic effects during normal activation of hemostasis. Effectively, activated platelets secrete coagulation factors, expose phosphatidylserine, and promote thrombin and fibrin production. In addition to procoagulant activities, platelets confer resistance of thrombi to fibrinolysis by inducing clot retraction of the fibrin network and release of huge amounts of plasminogen activator inhibitor-1, which is the major physiologic inhibitor of the fibrinolytic cascade. However, the discovery of multiple relations with the fibrinolytic system, also termed Plasminogen Activation System (PAS), has introduced new perspectives on the platelet role in fibrinolysis. Indeed, the activated membrane surface of platelets provides binding sites on which fibrinolytic enzymes can be activated. This review discusses the evidence of the profibrinolytic properties of platelets through the description of PAS components and related proteins that are contained in or bind to platelets. Our analyses of literature data lead to the conclusion that in the initial phase of the hemostatic process, antifibrinolytic effects prevail over profibrinolytic activity, but at later stages, platelets might enhance fibrinolysis through the engagement of PAS components. A better understanding of spatial and temporal characteristics of platelet-mediated fibrinolysis during normal hemostasis could improve therapeutic options for bleeding and thrombotic disorders.

Roles of the tissue-type plasminogen activator in immune response.

The COVID-19 pandemic has once again brought to the forefront the existence of a tight link between the coagulation/fibrinolytic system and the immunologic processes. Tissue-type plasminogen activator (tPA) is a serine protease with a key role in fibrinolysis by converting plasminogen into plasmin that can finally degrade fibrin clots. tPA is released in the blood by endothelial cells and hepatocytes but is also produced by various types of immune cells including T cells and monocytes. Beyond its role on hemostasis, tPA is also a potent modulator of inflammation and is involved in the regulation of several inflammatory diseases. Here, after a brief description of tPA structure, we review its new functions in adaptive immunity focusing on T cells and antigen presenting cells. We intend to synthesize the recent knowledge on proteolysis- and receptor-mediated effects of tPA on immune response in physiological and pathological context.

Longitudinal Assessment of Cytokine Expression and Plasminogen Activation in Hantavirus Cardiopulmonary Syndrome Reveals Immune Regulatory Dysfunction in End-Stage Disease.

Pathogenic New World orthohantaviruses cause hantavirus cardiopulmonary syndrome (HCPS), a severe immunopathogenic disease in humans manifested by pulmonary edema and respiratory distress, with case fatality rates approaching 40%. High levels of inflammatory mediators are present in the lungs and systemic circulation of HCPS patients. Previous studies have provided insights into the pathophysiology of HCPS. However, the longitudinal correlations of innate and adaptive immune responses and disease outcomes remain unresolved. This study analyzed serial immune responses in 13 HCPS cases due to Sin Nombre orthohantavirus (SNV), with 11 severe cases requiring extracorporeal membrane oxygenation (ECMO) treatment and two mild cases. We measured viral load, levels of various cytokines, urokinase plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1). We found significantly elevated levels of proinflammatory cytokines and PAI-1 in five end-stage cases. There was no difference between the expression of active uPA in survivors' and decedents' cases. However, total uPA in decedents' cases was significantly higher compared to survivors'. In some end-stage cases, uPA was refractory to PAI-1 inhibition as measured by zymography, where uPA and PAI-1 were strongly correlated to lymphocyte counts and IFN-γ. We also found bacterial co-infection influencing the etiology and outcome of immune response in two cases. Unsupervised Principal Component Analysis and hierarchical cluster analyses resolved separate waves of correlated immune mediators expressed in one case patient due to a sequential co-infection of bacteria and SNV. Overall, a robust proinflammatory immune response, characterized by an imbalance in T helper 17 (Th17) and regulatory T-cells (Treg) subsets, was correlated with dysregulated inflammation and mortality. Our sample size is small; however, the core differences correlated to survivors and end-stage HCPS are instructive.

Circulating Forms of Urokinase-Type Plasminogen Activator Receptor in Plasma Can Predict Recurrence and Survival in Patients with Urothelial Carcinoma of the Bladder.

Urothelial carcinoma of the bladder is a highly aggressive disease characterised by a very heterogeneous clinical outcome. Despite cystectomy, patients still have a high recurrence risk and shortened survival. Urokinase-type plasminogen activator receptor (uPAR) is present in tumour tissue specimens from patients with urothelial carcinoma. The different uPAR forms in blood are strong prognostic markers in other cancer types. We investigate the presence of different uPAR forms in tumour tissue and test the hypothesis that preoperative plasma levels of the uPAR forms predict recurrence free survival, cancer specific survival, and overall survival in patients treated with cystectomy for urothelial carcinoma. Using Western blotting we analyse neoplasia and adjacent benign-appearing urothelium from randomly selected patients for the presence of intact and cleaved uPAR forms. Prospectively collected preoperative plasma samples from 107 patients who underwent radical cystectomy for urothelial carcinoma are analysed. The different uPAR forms are measured by time-resolved fluorescence immunoassays. uPAR in tumour tissue from patients with urothelial carcinoma is demonstrated in both an intact and cleaved form. The different uPAR forms in plasma are all significantly associated with both recurrence free survival, cancer specific survival, and overall survival, high concentrations predicting short survival. uPAR (I) has the strongest association with a HR of 2.56 for overall survival. In the multivariable survival analysis uPAR (I) is significantly associated with cancer specific survival and overall survival.

Fibrinolysis: A Primordial System Linked to the Immune Response.

The fibrinolytic system provides an essential means to remove fibrin deposits and blood clots. The actual protease responsible for this is plasmin, formed from its precursor, plasminogen. Fibrin is heralded as it most renowned substrate but for many years plasmin has been known to cleave many other substrates, and to also activate other proteolytic systems. Recent clinical studies have shown that the promotion of plasmin can lead to an immunosuppressed phenotype, in part via its ability to modulate cytokine expression. Almost all immune cells harbor at least one of a dozen plasminogen receptors that allows plasmin formation on the cell surface that in turn modulates immune cell behavior. Similarly, a multitude of pathogens can also express their own plasminogen activators, or contain surface proteins that provide binding sites host plasminogen. Plasmin formed under these circumstances also empowers these pathogens to modulate host immune defense mechanisms. Phylogenetic studies have revealed that the plasminogen activating system predates the appearance of fibrin, indicating that plasmin did not evolve as a fibrinolytic protease but perhaps has its roots as an immune modifying protease. While its fibrin removing capacity became apparent in lower vertebrates these primitive under-appreciated immune modifying functions still remain and are now becoming more recognised.

Tissue-Type Plasminogen Activator and Tenecteplase-Mediated Increase in Blood Brain Barrier Permeability Involves Cell Intrinsic Complement.

Background: Tissue-type plasminogen activator (t-PA) has been the mainstay of therapeutic thrombolysis for patients with acute ischaemic stroke (AIS). However, t-PA can cause devastating intracerebral hemorrhage. t-PA can also influence the CNS in part by modulation of BBB permeability. Complement activation also occurs after AIS and has also been reported to increase BBB permeability. The complement components, C3 and C5, can also be activated by t-PA via plasmin formation and cell intrinsic complement may be involved in this process. Tenecteplase (TNK-tPA) is a t-PA variant with a longer plasma half-life, yet the ability of TNK-tPA to modulate the BBB and complement is less clear. Aim: To evaluate the effect of C5 and C5a-receptor 1 (C5aR1) inhibitors on t-PA- and TNK-tPA-mediated opening of the BBB. Methods: We used an in vitro model of the BBB where human brain endothelial cells and human astrocytes were co-cultured on the opposite sides of a porous membrane assembled in transwell inserts. The luminal (endothelial) compartment was stimulated with t-PA or TNK-tPA together with plasminogen, in the presence of PMX205 (a non-competitive C5aR1 antagonist), Avacopan (a competitive C5aR1 antagonist) or Eculizumab (a humanized monoclonal inhibitor of human C5). BBB permeability was assessed 5 and 24 h later. Immunofluorescence was also used to detect changes in C5 and C5aR1 expression in endothelial cells and astrocytes. Results: PMX205, but not Avacopan or Eculizumab, blocked t-PA-mediated increase in BBB permeability at both the 5 and 24 h time points. PMX205 also blocked TNK-tPA-mediated increase in BBB permeability. Immunofluorescence analysis revealed intracellular staining of C5 in both cell types. C5aR1 expression was also detected on the cell surfaces and also located intracellularly in both cell types. Conclusion: t-PA and TNK-tPA-mediated increase in BBB permeability involves C5aR1 receptor activation from cell-derived C5a. Selective inhibitors of C5aR1 may have therapeutic potential in AIS.

Embelin ameliorated sepsis-induced disseminated intravascular coagulation intensities by simultaneously suppressing inflammation and thrombosis.

Disseminated intravascular coagulation (DIC), an acute syndrome of systemic thrombus formation in microvasculatures throughout the body, can be induced by severe infections, e.g. sepsis. Anticoagulants are clinically used to alleviate the intensities of DIC. However, anticoagulants only reduce the thrombus formation but have negligible effects on the inflammatory conditions. We previously reported embelin, a natural product, as an inhibitor of plasminogen activator inhibitor-1 (PAI-1), suggesting the potent antithrombotic property. In this study, we used three thrombotic mice models to confirm the antithrombotic property of embelin. By combining the anti-inflammatory and the antithrombotic properties, we proposed embelin as a potent therapeutic agent for sepsis-induced DIC, which involves both inflammation and thrombosis. In a lipopolysaccharides-induced septic mice model, embelin not only significantly ameliorated the inflammation levels, but also effectively reduced the pulmonary hemorrhages and the micro-thrombi formations in lung. In contrast, low-molecular-weight-heparin, an anticoagulant, only moderately ameliorated the pulmonary hemorrhages and thrombotic obstructions, but had non-measurable effect on the inflammatory conditions. In addition, embelin alleviated the dysregulation of the global coagulation in septic mice, but did not affect the global coagulation in normal mice. Our current study demonstrates the antithrombotic property of embelin and the potency of the treatment or prevention of syndromes combining inflammation and thrombosis, e.g. sepsis-induced DIC.

Influence cationic and anionic PAMAM dendrimers of low generation on selected hemostatic parameters in vitro.

BACKGROUND: Polyamidoamine (PAMAM) dendrimers are a new class of monodisperse polymers that are used for drug delivery in systemic administrations. The influence of PAMAM dendrimers on components of the blood coagulation system has been extensively studied, but their effect on the activity of the fibrinolysis system has not been studied to date. METHODS: The effect of cationic (G1-G3) and anionic (G1.5-G3.5) PAMAM dendrimers on the conformation and function of the main components of the coagulation and fibrinolysis systems was comparatively studied. Changes in overall plasma hemostatic potential, thrombin generation, prothrombin time, thrombin and tPA activities, the fluorescence of fibrinogen and plasminogen, zeta potential, polymerization of fibrinogen, and activation of plasminogen were analyzed to assess coagulofibrinolytic mechanisms of influence of the charge of the dendrimers. RESULTS: Cationic dendrimers increased prothrombin time, suppressed thrombin generation in plasma, and changed the conformation and coagulability of fibrinogen, while anionic dendrimers did not have such effects. Anionic dendrimers slightly reduced tPA activity and altered plasminogen conformation much more strongly than the cationic dendrimers. Plasminogen activation by tPA was strongly inhibited by anionic dendrimers and weakly stimulated by cationic dendrimers. All these effects were enhanced with increasing generation and concentration of the dendrimers. CONCLUSIONS: PAMAM-NH2 dendrimers inhibit the extrinsic activation pathway of the coagulation system and alter the conformation and function of fibrinogen. PAMAM-COOH dendrimers change the conformation of plasminogen and inhibit its activation by tPA. This study gives new insight into the effect of anionic PAMAM dendrimers on the activity of the fibrinolytic system. For intravenous applications, the antifibrinolytic effect of anionic PAMAM dendrimers of generation ≥G2.5 should be considered.