Multifunctional interaction of CihC/FbpC orthologs of relapsing fever spirochetes with host-derived proteins involved in adhesion, fibrinolysis, and complement evasion.

Introduction: Relapsing fever (RF) remains a neglected human disease that is caused by a number of diverse pathogenic Borrelia (B.) species. Characterized by high cell densities in human blood, relapsing fever spirochetes have developed plentiful strategies to avoid recognition by the host defense mechanisms. In this scenario, spirochetal lipoproteins exhibiting multifunctional binding properties in the interaction with host-derived molecules are known to play a key role in adhesion, fibrinolysis and complement activation. Methods: Binding of CihC/FbpC orthologs to different human proteins and conversion of protein-bound plasminogen to proteolytic active plasmin were examined by ELISA. To analyze the inhibitory capacity of CihC/FbpC orthologs on complement activation, a microtiter-based approach was performed. Finally, AlphaFold predictions were utilized to identified the complement-interacting residues. Results and discussion: Here, we elucidate the binding properties of CihC/FbpC-orthologs from distinct RF spirochetes including B. parkeri, B. hermsii, B. turicatae, and B. recurrentis to human fibronectin, plasminogen, and complement component C1r. All CihC/FbpC-orthologs displayed similar binding properties to fibronectin, plasminogen, and C1r, respectively. Functional studies revealed a dose dependent binding of plasminogen to all borrelial proteins and conversion to active plasmin. The proteolytic activity of plasmin was almost completely abrogated by tranexamic acid, indicating that lysine residues are involved in the interaction with this serine protease. In addition, a strong inactivation capacity toward the classical pathway could be demonstrated for the wild-type CihC/FbpC-orthologs as well as for the C-terminal CihC fragment of B. recurrentis. Pre-incubation of human serum with borrelial molecules except CihC/FbpC variants lacking the C-terminal region protected serum-susceptible Borrelia cells from complement-mediated lysis. Utilizing AlphaFold2 predictions and existing crystal structures, we mapped the putative key residues involved in C1r binding on the CihC/FbpC orthologs attempting to explain the relatively small differences in C1r binding affinity despite the substitutions of key residues. Collectively, our data advance the understanding of the multiple binding properties of structural and functional highly similar molecules of relapsing fever spirochetes proposed to be involved in pathogenesis and virulence.

Development of a chemiluminescence assay for tissue plasminogen activator inhibitor complex and its applicability to gastric cancer.

BACKGROUND: Venous thromboembolism (VTE), is a noteworthy complication in individuals with gastric cancer, but the current diagnosis and treatment methods lack accuracy. In this study, we developed a t-PAIC chemiluminescence kit and employed chemiluminescence to detect the tissue plasminogen activator inhibitor complex (t-PAIC), thrombin-antithrombin III complex (TAT), plasmin-α2-plasmin inhibitor complex (PIC) and thrombomodulin (TM), combined with D-dimer and fibrin degradation products (FDP), to investigate their diagnostic potential for venous thrombosis in gastric cancer patients. The study assessed variations in six indicators among gastric cancer patients at different stages. RESULTS: The t-PAIC reagent showed LOD is 1.2 ng/mL and a linear factor R greater than 0.99. The reagents demonstrated accurate results, with all accuracy deviations being within 5%. The intra-batch and inter-batch CVs for the t-PAIC reagent were both within 8%. The correlation coefficient R between this method and Sysmex was 0.979. Gastric cancer patients exhibited elevated levels of TAT, PIC, TM, D-D, FDP compared to the healthy population, while no significant difference was observed in t-PAIC. In the staging of gastric cancer, patients in III-IV stages exhibit higher levels of the six markers compared to those in I-II stages. The ROC curve indicates an enhancement in sensitivity and specificity of the combined diagnosis of four or six indicators. CONCLUSION: Our chemiluminescence assay performs comparably to Sysmex's method and at a reduced cost. The use of multiple markers, including t-PAIC, TM, TAT, PIC, D-D, and FDP, is superior to the use of single markers for diagnosing VTE in patients with malignant tumors. Gastric cancer patients should be screened for the six markers to facilitate proactive prophylaxis, determine the most appropriate treatment timing, ameliorate their prognosis, decrease the occurrence of venous thrombosis and mortality, and extend their survival.

The clinical significance of TAT, PIC, TM, and t-PAIC in vascular events of BCR/ABL-negative myeloproliferative neoplasms.

Predicting the likelihood vascular events in patients with BCR/ABL1-negative myeloproliferative neoplasms (MPN) is essential for the treatment of the disease. However, effective assessment methods are lacking. Thrombin-antithrombin complex (TAT), plasmin-α2- plasmininhibitor complex (PIC), thrombomodulin (TM), and tissue plasminogen activator-inhibitor complex (t-PAIC) are the new direct indicators for coagulation and fibrinolysis. The aim of this study was to investigate the changes of these four new indicators in thrombotic and hemorrhagic events in BCR/ABL1-negative MPN. The study cohort of 74 patients with BCR/ABL negative myeloproliferative disorders included essential thrombocythemia, polycythemia vera, and primary myelofibrosis (PMF). A panel of 4 biomarkers, including TAT, PIC, TM, and t-PAIC were determined using Sysmex HISCL5000 automated analyzers, whereas fibrin/fibrinogen degradation products (FDP), D-dimer and Antithrombin III (ATIII) were analyzed using Sysmex CS5100 coagulation analyzer. A total of 24 (32.4%) patients experienced thrombotic events and hemorrhagic events occurred in 8 patients (10.8%). Compared to patients without hemorrhagic-thrombotic events, patients with thrombotic events had higher fibrinogen (FIB) level, FDP level and lower ATIII activity, while patients with hemorrhagic events had lower white blood cell count and hemoglobin level, higher FDP level (P < 0.05). Patients with a JAK2V617F mutation were more likely to experience thrombotic events (P < 0.05). In addtion, patients with thrombotic events had higher TAT, PIC, TM, and t-PAIC levels than patients without hemorrhagic-thrombotic events (P < 0.05), whereas patients with hemorrhagic events had a lower median value in TAT and TM (no statistical difference, P > 0.05). Patients with higher TAT, TM and t-PAIC were more likely to experience thrombotic events (P < 0.05), and only TAT was positively correlated with thrombotic events (Spearman r =0.287, P = 0.019). TAT, PIC, TM, and t-PAIC combined with ATIII and FDP have a certain value for predicting thrombosis in patients with BCR/ABL1-negative MPN. These 6 parameters are worth further exploration as predictive factors and prognostic markers for early thrombotic events.

Optimization of plasma-based BioID identifies plasminogen as a ligand of ADAMTS13.

ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13, regulates the length of Von Willebrand factor (VWF) multimers and their platelet-binding activity. ADAMTS13 is constitutively secreted as an active protease and is not inhibited by circulating protease inhibitors. Therefore, the mechanisms that regulate ADAMTS13 protease activity are unknown. We performed an unbiased proteomics screen to identify ligands of ADAMTS13 by optimizing the application of BioID to plasma. Plasma BioID identified 5 plasma proteins significantly labeled by the ADAMTS13-birA* fusion, including VWF and plasminogen. Glu-plasminogen, Lys-plasminogen, mini-plasminogen, and apo(a) bound ADAMTS13 with high affinity, whereas micro-plasminogen did not. None of the plasminogen variants or apo(a) bound to a C-terminal truncation variant of ADAMTS13 (MDTCS). The binding of plasminogen to ADAMTS13 was attenuated by tranexamic acid or ε-aminocaproic acid, and tranexamic acid protected ADAMTS13 from plasmin degradation. These data demonstrate that plasminogen is an important ligand of ADAMTS13 in plasma by binding to the C-terminus of ADAMTS13. Plasmin proteolytically degrades ADAMTS13 in a lysine-dependent manner, which may contribute to its regulation. Adapting BioID to identify protein-interaction networks in plasma provides a powerful new tool to study protease regulation in the cardiovascular system.

Dysregulated fibrinolysis and plasmin activation promote the pathogenesis of osteoarthritis.

Joint injury is associated with risk for development of osteoarthritis (OA). Increasing evidence suggests that activation of fibrinolysis is involved in OA pathogenesis. However, the role of the fibrinolytic pathway is not well understood. Here, we showed that the fibrinolytic pathway, which includes plasminogen/plasmin, tissue plasminogen activator, urokinase plasminogen activator (uPA), and the uPA receptor (uPAR), was dysregulated in human OA joints. Pharmacological inhibition of plasmin attenuated OA progression after a destabilization of the medial meniscus in a mouse model whereas genetic deficiency of plasmin activator inhibitor, or injection of plasmin, exacerbated OA. We detected increased uptake of uPA/uPAR in mouse OA joints by microPET/CT imaging. In vitro studies identified that plasmin promotes OA development through multiple mechanisms, including the degradation of lubricin and cartilage proteoglycans and induction of inflammatory and degradative mediators. We showed that uPA and uPAR produced inflammatory and degradative mediators by activating the PI3K, 3'-phosphoinositide-dependent kinase-1, AKT, and ERK signaling cascades and activated matrix metalloproteinases to degrade proteoglycan. Together, we demonstrated that fibrinolysis contributes to the development of OA through multiple mechanisms and suggested that therapeutic targeting of the fibrinolysis pathway can prevent or slow development of OA.

Identification of plasminogen-binding sites in Streptococcus suis enolase that contribute to bacterial translocation across the blood-brain barrier.

Streptococcus suis is an emerging zoonotic pathogen that can cause invasive disease commonly associated with meningitis in pigs and humans. To cause meningitis, S. suis must cross the blood-brain barrier (BBB) comprising blood vessels that vascularize the central nervous system (CNS). The BBB is highly selective due to interactions with other cell types in the brain and the composition of the extracellular matrix (ECM). Purified streptococcal surface enolase, an essential enzyme participating in glycolysis, can bind human plasminogen (Plg) and plasmin (Pln). Plg has been proposed to increase bacterial traversal across the BBB via conversion to Pln, a protease which cleaves host proteins in the ECM and monocyte chemoattractant protein 1 (MCP1) to disrupt tight junctions. The essentiality of enolase has made it challenging to unequivocally demonstrate its role in binding Plg/Pln on the bacterial surface and confirm its predicted role in facilitating translocation of the BBB. Here, we report on the CRISPR/Cas9 engineering of S. suis enolase mutants eno261, eno252/253/255, eno252/261, and eno434/435 possessing amino acid substitutions at in silico predicted binding sites for Plg. As expected, amino acid substitutions in the predicted Plg binding sites reduced Plg and Pln binding to S. suis but did not affect bacterial growth in vitro compared to the wild-type strain. The binding of Plg to wild-type S. suis enhanced translocation across the human cerebral microvascular endothelial cell line hCMEC/D3 but not for the eno mutant strains tested. To our knowledge, this is the first study where predicted Plg-binding sites of enolase have been mutated to show altered Plg and Pln binding to the surface of S. suis and attenuation of translocation across an endothelial cell monolayer in vitro.

Pharmacokinetic-Pharmacodynamic Modelling in Hemophilia A: Relating Thrombin and Plasmin Generation to Factor VIII Activity After Administration of a VWF/FVIII Concentrate.

BACKGROUND: Hemophilia A patients are treated with factor (F) VIII prophylactically to prevent bleeding. In general, dosage and frequency are based on pharmacokinetic measurements. Ideally, an alternative dose adjustment can be based on the hemostatic potential, measured with a thrombin generation assay (TGA), like the Nijmegen hemostasis assay. OBJECTIVE: The objective of this study was to investigate the predicted performance of a previously developed pharmacokinetic-pharmacodynamic model for FVIII replacement therapy, relating FVIII dose and FVIII activity levels with thrombin and plasmin generation parameters. METHODS: Pharmacokinetic and pharmacodynamic measurements were obtained from 29 severe hemophilia A patients treated with pdVWF/FVIII concentrate (Haemate P®). The predictive performance of the previously developed pharmacokinetic-pharmacodynamic model was evaluated using nonlinear mixed-effects modeling (NONMEM). When predictions of FVIII activity or TGA parameters were inadequate [median prediction error (MPE) > 20%], a new model was developed. RESULTS: The original pharmacokinetic model underestimated clearance and was refined based on a two-compartment model. The pharmacodynamic model displays no bias in the observed normalized thrombin peak height and normalized thrombin potential (MPE of 6.83% and 7.46%). After re-estimating pharmacodynamic parameters, EC50 and Emax values were relatively comparable between the original model and this group. Prediction of normalized plasmin peak height was inaccurate (MPE 58.9%). CONCLUSION: Our predictive performance displayed adequate thrombin pharmacodynamic predictions of the original model, but a new pharmacokinetic model was required. The pharmacodynamic model is not factor specific and applicable to multiple factor concentrates. A prospective study is needed to validate the impact of the FVIII dosing pharmacodynamic model on bleeding reduction in patients.

C5a-C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms.

Recent studies have indicated the involvement of neutrophil-mediated inflammatory responses in the process leading to intracranial aneurysm (IA) rupture. Receptors mediating neutrophil recruitment could thus be therapeutic targets of unruptured IAs. In this study, complement C5a receptor 1 (C5AR1) was picked up as a candidate that may cause neutrophil-dependent inflammation in IA lesions from comprehensive gene expression profile data acquired from rat and human samples. The induction of C5AR1 in IA lesions was confirmed by immunohistochemistry; the up-regulations of C5AR1/C5ar1 stemmed from infiltrated neutrophils, which physiologically express C5AR1/C5ar1, and adventitial fibroblasts that induce C5AR1/C5ar1 in human/rat IA lesions. In in vitro experiments using NIH/3T3, a mouse fibroblast-like cell line, induction of C5ar1 was demonstrated by starvation or pharmacological inhibition of mTOR signaling by Torin1. Immunohistochemistry and an experiment in a cell-free system using recombinant C5 protein and recombinant Plasmin indicated that the ligand of C5AR1, C5a, could be produced through the enzymatic digestion by Plasmin in IA lesions. In conclusion, we have identified a potential contribution of the C5a-C5AR1 axis to neutrophil infiltration as well as inflammatory responses in inflammatory cells and fibroblasts of IA lesions. This cascade may become a therapeutic target to prevent the rupture of IAs.

Purification and Properties of a Plasmin-like Marine Protease from Clamworm (Perinereis aibuhitensis).

Marine organisms are a rich source of enzymes that exhibit excellent biological activity and a wide range of applications. However, there has been limited research on the proteases found in marine mudflat organisms. Based on this background, the marine fibrinolytic enzyme FELP, which was isolated and purified from clamworm (Perinereis aibuhitensis), has exhibited excellent fibrinolytic activity. We demonstrated the FELP with a purification of 10.61-fold by precipitation with ammonium sulfate, ion-exchange chromatography, and gel-filtration chromatography. SDS-PAGE, fibrin plate method, and LC-MS/MS indicated that the molecular weight of FELP is 28.9 kDa and identified FELP as a fibrinolytic enzyme-like protease. FELP displayed the maximum fibrinolytic activity at pH 9 (407 ± 16 mm2) and 50 °C (724 ± 27 mm2) and had excellent stability at pH 7-11 (50%) or 30-60 °C (60%), respectively. The three-dimensional structure of some amino acid residues of FELP was predicted with the SWISS-MODEL. The fibrinolytic and fibrinogenolytic assays showed that the enzyme possessed direct fibrinolytic activity and indirect fibrinolysis via the activation of plasminogen; it could preferentially degrade Aα-chains of fibrinogen, followed by Bß- and γ-chains. Overall, the fibrinolytic enzyme was successfully purified from Perinereis aibuhitensis, a marine Annelida (phylum), with favorable stability that has strong fibrinolysis activity in vitro. Therefore, FELP appears to be a potent fibrinolytic enzyme with an application that deserves further investigation.

The effect of plasmin-mediated degradation on fibrinolysis and tissue plasminogen activator diffusion.

We modify a three-dimensional multiscale model of fibrinolysis to study the effect of plasmin-mediated degradation of fibrin on tissue plasminogen activator (tPA) diffusion and fibrinolysis. We propose that tPA is released from a fibrin fiber by simple kinetic unbinding, as well as by "forced unbinding," which occurs when plasmin degrades fibrin to which tPA is bound. We show that, if tPA is bound to a small-enough piece of fibrin that it can diffuse into the clot, then plasmin can increase the effective diffusion of tPA. If tPA is bound to larger fibrin degradation products (FDPs) that can only diffuse along the clot, then plasmin can decrease the effective diffusion of tPA. We find that lysis rates are fastest when tPA is bound to fibrin that can diffuse into the clot, and slowest when tPA is bound to FDPs that can only diffuse along the clot. Laboratory experiments confirm that FDPs can diffuse into a clot, and they support the model hypothesis that forced unbinding of tPA results in a mix of FDPs, such that tPA bound to FDPs can diffuse both into and along the clot. Regardless of how tPA is released from a fiber, a tPA mutant with a smaller dissociation constant results in slower lysis (because tPA binds strongly to fibrin), and a tPA mutant with a larger dissociation constant results in faster lysis.

Perioperative treatment with tranexamic acid in melanoma (PRIME): protocol for a Danish multicentre randomised controlled trial investigating the prognostic and treatment-related impact of the plasminogen-plasmin pathway.

INTRODUCTION: Inflammation is a hallmark of cancer and is involved in tumour growth and dissemination. However, the hallmarks of cancer are also the hallmarks of wound healing, and modulating the wound inflammatory response and immune contexture in relation to cancer surgery may represent effective targets of therapies.Repurposing anti-inflammatory drugs in a cancer setting has gained increasing interest in recent years. Interestingly, the known and thoroughly tested antifibrinolytic drug tranexamic acid reduces the risk of bleeding, but it is also suggested to play important roles in anti-inflammatory pathways, improving wound healing and affecting anti-carcinogenic mechanisms.As a novel approach, we will conduct a randomised controlled trial using perioperative treatment with tranexamic acid, aiming to prevent early relapses by >10% for patients with melanoma. METHODS AND ANALYSIS: Design: investigator-initiated parallel, two-arm, randomised, blinded, Danish multicentre superiority trial. PATIENTS: ≥T2 b melanoma and eligible for sentinel lymph node biopsy (n=1204).Project drug: tranexamic acid or placebo. TREATMENT: before surgery (intravenous 15 mg/kg) and daily (peroral 1000 mg x 3) through postoperative day 4. PRIMARY OUTCOME: relapse within 2 years after surgery.Primary analysis: risk difference between the treatment arms (χ2 test). SECONDARY OUTCOMES: postoperative complications, adverse events and survival.Inclusion period: summer 2023 to summer 2026. ETHICS AND DISSEMINATION: The trial will be initiated during the summer of 2023 and is approved by the National Committee on Health Research Ethics, the Danish Medicine Agency, and registered under the Data Protection Act. The study will be conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice. Patients included in the study will adhere to normal Danish treatment protocols and standards of care, and we expect only mild and temporary side effects. Positive and negative results will be published in peer-reviewed journals, with authorships adhering to the Vancouver rules. TRIAL REGISTRATION NUMBER: NCT05899465; ClinicalTrials.gov Identifier.

Amiloride Reduces Urokinase/Plasminogen-Driven Intratubular Complement Activation in Glomerular Proteinuria.

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 .

Genotype-dependent N-glycosylation and newly exposed O-glycosylation affect plasmin-induced cleavage of histidine-rich glycoprotein (HRG).

Histidine-rich glycoprotein (HRG) is an abundant plasma protein harboring at least three N-glycosylation sites. HRG integrates many biological processes, such as coagulation, antiangiogenic activity, and pathogen clearance. Importantly, HRG is known to exhibit five genetic variants with minor allele frequencies of more than 10%. Among them, Pro204Ser can induce a fourth N-glycosylation site (Asn202). Considerable efforts have been made to reveal the biological function of HRG, whereas data on HRG glycosylation are scarcer. To close this knowledge gap, we used C18-based LC-MS/MS to study the glycosylation characteristics of six HRG samples from different sources. We used endogenous HRG purified from human plasma and compared its glycosylation to that of the recombinant HRG produced in Chinese hamster ovary cells or human embryonic kidney 293 cells, targeting distinct genotypic isoforms. In endogenous plasma HRG, every N-glycosylation site was occupied predominantly with a sialylated diantennary complex-type glycan. In contrast, in the recombinant HRGs, all glycans showed different antennarities, sialylation, and core fucosylation, as well as the presence of oligomannose glycans, LacdiNAcs, and antennary fucosylation. Furthermore, we observed two previously unreported O-glycosylation sites in HRG on residues Thr273 and Thr274. These sites together showed more than 90% glycan occupancy in all HRG samples studied. To investigate the potential relevance of HRG glycosylation, we assessed the plasmin-induced cleavage of HRG under various conditions. These analyses revealed that the sialylation of the N- and O-glycans as well as the genotype-dependent N-glycosylation significantly influenced the kinetics and specificity of plasmin-induced cleavage of HRG.

Plasmin-cleaved von Willebrand factor as a biomarker for microvascular thrombosis.

ABSTRACT: von Willebrand factor (VWF) is an essential contributor to microvascular thrombosis. Physiological cleavage by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) limits its prothrombotic properties, explaining why ADAMTS13 deficiency leads to attacks of microthrombosis in patients with thrombotic thrombocytopenic purpura (TTP). We previously reported that plasminogen activation takes place during TTP attacks in these patients. Furthermore, stimulation of plasminogen activation attenuates pathogenesis in preclinical TTP models in vivo. This suggests that plasmin is an endogenous regulator of VWF thrombogenicity, in particular when ADAMTS13 falls short to prevent microvascular occlusions. VWF cleavage by plasmin is biochemically distinct from cleavage by ADAMTS13. We hypothesized that plasmin-cleaved VWF (cVWF) holds value as a biomarker of microvascular thrombosis. Here, we describe the development of a variable domain of heavy-chain-only antibody (VHH)-based bioassay that can distinguish cVWF from intact and ADAMTS13-cleaved VWF in plasma. We validate this assay by tracking cVWF release during degradation of microthombi in vitro. We demonstrate that endogenous cVWF formation takes place in patients with TTP during acute attacks of thrombotic microangiopathy but not in those in remission. Finally, we show that therapeutic plasminogen activation in a mouse model of TTP amplifies cVWF formation, which is accompanied by VWF clearance. Our combined findings indicate that cVWF is released from microthrombi in the context of microvascular occlusion.

Comparative analysis of hyperfibrinolysis with activated coagulation between amniotic fluid embolism and severe placental abruption.

Amniotic fluid embolism (AFE) and placental abruption (PA) are typical obstetric diseases associated with disseminated intravascular coagulation (DIC). AFE is more likely to be complicated with enhanced fibrinolysis than PA. AFE may have an additional mechanism activating fibrinolytic cascade. We aimed to compare the coagulation/fibrinolysis factors among AFE, PA, and peripartum controls. We assessed AFE cases registered in the Japanese AFE Registry, and PA cases complicated with DIC (severe PA) and peripartum controls recruited at our hospital. The following factors in plasma were compared: prothrombin fragment 1 + 2 (PF1 + 2), plasmin α2-plasmin inhibitor complex (PIC), tissue factor (TF), tissue plasminogen activator (tPA), annexin A2 (AnnA2), total thrombin activatable fibrinolysis inhibitor (TAFI) including its activated form (TAFIa), and plasminogen activator inhibitor-type 1 (PAI-1). PF1 + 2 and PIC were markedly increased in both AFE (n = 27) and severe PA (n = 12) compared to controls (n = 23), without significant difference between those disease groups; however, PIC in AFE showed a tendency to elevate relative to PF1 + 2, compared with severe PA. AFE had significantly increased tPA and decreased total TAFI levels compared with severe PA and controls, which might be associated with further plasmin production in AFE and underlie its specific fibrinolytic activation pathway.

Urinary tissue factor (uTF), tissue factor pathway inhibitor (TFPI) and plasmin as novel biomarkers in early diagnosis of lupus nephritis.

Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease, with multi systematic affection. Lupus nephritis (LN) is the most frequent cause of renal damage in SLE patients with variable presentations that may progress to end stage renal failure. Coagulation disorders are frequently reported in SLE and LN with higher mortality rates. Renal biopsy is an invasive process, and the existing indicators for LN diagnosis and activity are unreliable. New urinary biomarkers with significant validity, safety, and accuracy are the current focus of most studies. Our study sought to assess the value of urinary tissue factor (uTF), tissue factor pathway inhibitor (TFPI), and plasmin as biomarkers for the early identification and detection of LN and its activity. This was a cross-sectional study, included 100 subjects (80 SLE patients, and 20 healthy controls), they were recruited from the Internal Medicine department, Rheumatology and Nephrology units and outpatient's clinics at Assiut University hospital between the period of 2020 and 2022. All patients underwent full history taking, clinical evaluation, and activity scoring calculation and laboratory investigations. The results showed that the best diagnostic accuracy of LN was observed with TFPI (90% accuracy, sensitivity 80% and specificity 95% with p <0.001 at cutoff point of >193.2 ng/ml), followed by uTF (75.4% overall accuracy at cut off point of >12.6 ng/ml, sensitivity 90% and specificity 68% with p < 0.001) and plasmin (70.3% accuracy at cut off point of >30.5 ng/ml, sensitivity 55% and specificity 78% with p < 0.001). Urinary TFPI was the best predictor of LN occurrence with odd ratio of 4.34, (p < 0.001). In conclusion urinary TFPI could be used as a diagnostic marker for LN with high accuracy and an early predictor of LN.

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.

Identification of potent anti-fibrinolytic compounds against plasminogen and tissue-type plasminogen activator employing in silico approaches.

The zymogen protease Plasminogen (Plg) and its active form plasmin (Plm) carry out important functions in the blood clot disintegration (breakdown of fibrin fibers) process. Inhibition of plasmin effectively reduces fibrinolysis to circumvent heavy bleeding. Currently, available Plm inhibitor tranexamic acid (TXA) used for treating severe hemorrhages is associated with an increased incidence of seizures which in turn were traced to gamma-aminobutyric acid antagonistic activity (GABAa) in addition to having multiple side effects. Fibrinolysis can be suppressed by targeting the three important protein domains: the kringle-2 domain of tissue plasminogen activator, the kringle-1 domain of plasminogen, and the serine protease domain of plasminogen. In the present study, one million molecules were screened from the ZINC database. These ligands were docked to their respective protein targets using Autodock Vina, Schrödinger Glide, and ParDOCK/BAPPL+. Thereafter, the drug-likeness properties of the ligands were evaluated using Discovery Studio 3.5. Subsequently, we subjected the protein-ligand complexes to molecular dynamics simulation of 200 ns in GROMACS. The identified ligands P76(ZINC09970930), C97(ZINC14888376), and U97(ZINC11839443) for each protein target are found to impart higher stability and greater compactness to the protein-ligand complexes. Principal component analysis (PCA) implicates, that the identified ligands occupy smaller phase space, form stable clusters, and provide greater rigidity to the protein-ligand complexes. Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis reveals that P76, C97, and U97 exhibit better binding free energy (ΔG) when compared to that of the standard ligands. Thus, our findings can be useful for the development of promising anti-fibrinolytic agents.Communicated by Ramaswamy H. Sarma.

Characterization and Usefulness of Clot-Fibrinolysis Waveform Analysis in Critical Care Patients with Enhanced or Suppressed Fibrinolysis.

INTRODUCTION: Recently, clot-fibrinolysis waveform analysis (CFWA), which is a coagulation and fibrinolysis global assay based on assessing the activated partial thromboplastin time with tissue-type plasminogen activator, was developed. This study aimed to investigate the characteristics of CFWA using plasma samples from patients in the critical care unit. MATERIALS AND METHODS: The fibrinolysis times using CFWA were measured in 298 plasma samples. These samples were divided into three groups based on the reference interval (RI) of fibrinolysis time using CFWA: shortened group, less than RI; within group, within RI; prolonged group, more than RI. The coagulation and fibrinolysis markers, including D-dimer, plasmin-α2 plasmin inhibitor complex (PIC), fibrin monomer complex (FMC), plasmin-α2 plasmin inhibitor (α2-PI), plasminogen (Plg), and fibrinogen (Fbg) were analyzed and compared among the three groups. RESULTS: The FMC level decreased in the order of shortened, within, and prolonged groups, and the decrease was statistically significant among all three group pairs. The opposite tendency was observed for Fbg and fibrinolysis-related markers of α2-PI and Plg, and significant differences were recognized in all pair comparisons except for between within and prolonged groups in Plg. The mean values of the fibrinolysis markers D-dimer and PIC in all three groups were higher than the cut-off values, and the PIC value differed significantly between the within and prolonged groups. CONCLUSION: The fibrinolysis reaction was detected in all three groups, but the status differed. CFWA has the potential to reflect the fibrinolysis status in one global assay.