Components Of Coagulation Cascade Research Articles

Alterations in the kallikrein-kinin system predict death after heart transplant

Heart transplantation remains the definitive treatment for end stage heart failure. Because availability is limited, risk stratification of candidates is crucial for optimizing both organ allocations and transplant outcomes. Here we utilize proteomics prior to transplant to identify new biomarkers that predict post-transplant survival in a multi-institutional cohort. Microvesicles were isolated from serum samples and underwent proteomic analysis using mass spectrometry. Monte Carlo cross-validation (MCCV) was used to predict survival after transplant incorporating select recipient pre-transplant clinical characteristics and serum microvesicle proteomic data. We identified six protein markers with prediction performance above AUROC of 0.6, including Prothrombin (F2), anti-plasmin (SERPINF2), Factor IX, carboxypeptidase 2 (CPB2), HGF activator (HGFAC) and low molecular weight kininogen (LK). No clinical characteristics demonstrated an AUROC > 0.6. Putative biological functions and pathways were assessed using gene set enrichment analysis (GSEA). Differential expression analysis identified enriched pathways prior to transplant that were associated with post-transplant survival including activation of platelets and the coagulation pathway prior to transplant. Specifically, upregulation of coagulation cascade components of the kallikrein-kinin system (KKS) and downregulation of kininogen prior to transplant were associated with survival after transplant. Further prospective studies are warranted to determine if alterations in the KKS contributes to overall post-transplant survival.

The Therapeutic Potential of Anticoagulation in Organ Fibrosis.

Fibrosis, also known as organ scarring, describes a pathological stiffening of organs or tissues caused by increased synthesis of extracellular matrix (ECM) components. In the past decades, mounting evidence has accumulated showing that the coagulation cascade is directly associated with fibrotic development. Recent findings suggest that, under inflammatory conditions, various cell types (e.g., immune cells) participate in the coagulation process causing pathological outcomes, including fibrosis. These findings highlighted the potential of anticoagulation therapy as a strategy in organ fibrosis. Indeed, preclinical and clinical studies demonstrated that the inhibition of blood coagulation is a potential intervention for the treatment of fibrosis across all major organs (e.g., lung, liver, heart, and kidney). In this review, we aim to summarize our current knowledge on the impact of components of coagulation cascade on fibrosis of various organs and provide an update on the current development of anticoagulation therapy for fibrosis.

A Review on Host-Leptospira Interactions: What We Know and Future Expectations

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira spp. It is considered a neglected infectious disease of human and veterinary concern. Our group has been investigating proteins annotated as hypothetical, predicted to be located on the leptospiral surface. Because of their location, these proteins may have the ability to interact with various host components, which could allow establishment of the infection. These proteins act as adherence factors by binding to host receptor molecules, such as the extracellular matrix (ECM) components laminin and glycosaminoglycans to help bacterial colonization. Leptospira also interacts with the host fibrinolytic system, which has been demonstrated to be a powerful tool for invasion mechanisms. The interaction with fibrinogen and thrombin has been shown to reduce fibrin clot formation. Additionally, the degradation of coagulation cascade components by secreted proteases or by acquired surface plasmin could also play a role in reducing clot formation, hence facilitating dissemination during infection. Interaction with host complement system regulators also plays a role in helping bacteria to evade the immune system, facilitating invasion. Interaction of Leptospira to cell receptors, such as cadherins, can contribute to investigate molecules that participate in virulence. To achieve a better understanding of the host-pathogen interaction, leptospiral mutagenesis tools have been developed and explored. This work presents several proteins that mediate binding to components of the ECM, plasma, components of the complement system and cells, to gather research achievements that can be helpful in better understanding the mechanisms of leptospiral-host interactions and discuss genetic manipulation for Leptospira spp. aimed at protein function validation.

Amplified Electrochemical Biosensing of Thrombin Activity by RAFT Polymerization.

As a serine protease, thrombin is a pivotal component in coagulation cascade and has been frequently screened as an informative biomarker for the diagnosis of coagulation disorder-related diseases. Herein, a "signal-on" electrochemical biosensor is described for the highly sensitive and selective detection of thrombin activity, by exploiting a thrombin-specific substrate peptide (Tb peptide) as the recognition element and reversible addition-fragmentation chain transfer (RAFT) polymerization for signal amplification. Specifically, the carboxyl-group-free Tb peptides are self-assembled onto gold electrode surface via the N-terminal cysteine residue and are used for the specific recognition of thrombin molecules. After the proteolytic cleavage of the Tb peptides, the carboxyl-group-containing RAFT agents (4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, CPAD) are tethered to the free carboxyl termini of the truncated peptide fragments via the carboxylate-zirconium-carboxylate chemistry. The subsequent RAFT polymerization leads to the grafting of a polymer chain from each proteolytically cleaved site, enabling the recruitment of a large number of electroactive ferrocene (Fc) tags to the electrode surface when ferrocenylmethyl methacrylate (FcMMA) is used as the monomer. Under optimal conditions, the detection limit of the described thrombin biosensor is as low as 2.7 μU mL-1 (∼0.062 pM), with a linear response over the range of 10-250 μU mL-1 (R2 = 0.997). Results also indicate that the biosensor is highly selective and applicable to the detection of thrombin activity in complex serum samples and the screening of thrombin inhibitors. The described biosensor is low-cost and relatively easy in preparation and thus shows great promise for the highly sensitive and selective detection of thrombin activity.

Hemostatic Abnormalities in Dementia: A Systematic Review and Meta-Analysis.

Alzheimer's disease (AD) is considered the most frequent cause of dementia. It is known that vascular risk factors play an important role in the development and progression of this condition. Alterations in vascular walls represent documented findings in patients with AD and other dementias affecting elderly people. The authors performed a systematic review and meta-analysis, aiming to synthesize observational studies that evaluated how the hemostatic system may contribute to cognitive decline in the elderly, using papers published until April 2018 and as indexed in Medline (PubMed), Scopus, Web of Science, ScienceDirect, Lilacs, Cinahl, PsycINFO, Cochrane Central Register of Controlled Trials, and Cochrane Database of Systematic Reviews. Among 5,278 studies identified, 32 were included in the final synthesis, and these included 485 patients with mild cognitive impairment, 568 with vascular dementia (VD), 1,781 with AD, and 2,855 participants without dementia. AD patients had increased plasma von Willebrand factor (VWF) (standardized mean difference [SMD]: 2.53; 95% confidence interval [CI]: 0.10-4.95), D-dimer (SMD: 0.50; 95% CI: 0.35-0.66), plasminogen activator inhibitor-1 (SMD: 3.34; 95% CI: 1.01-5.67), thrombomodulin (SMD: 1.08; 95% CI: 0.53-1.62), and homocysteine levels (SMD: 0.65; 95% CI: 0.15-1.15). In contrast, the VD group showed increased fibrinogen levels (SMD: 0.77; 95% CI: 0.13-1.41), activated factor VII (SMD: 0.36; 95% CI: 0.05-0.67), factor VIII (SMD: 0.57; 95% CI: 0.22-0.91), VWF (SMD: 2.34; 95% CI: 0.38-4.29), D-dimer (SMD: 1.14; 95% CI: 0.51-1.78), and homocysteine (SMD: 2.17; 95% CI: 1.67-2.68). AD showed an elevation in some markers of endothelial dysfunction, whereas VD presented mostly an involvement of coagulation cascade components.

Probable Roles of Coagulation Cascade and Fibrinolysis System in the Development of Allergic Rhinitis.

Dysregulation of the coagulation cascade and fibrinolysis system may play an etiologic role in many diseases. Allergic diseases such as bronchial asthma, atopic dermatitis, and conjunctivitis are also associated with fibrin accumulation caused by a change in hemostasis. However, only a few studies have dealt with the relationship between allergic rhinitis (AR) and the coagulation system. We investigated the difference of coagulation and fibrinolysis cascade components between an AR mouse model and a control mice. BALB/c mice were sensitized and challenged with ovalbumin. Multiple parameters of coagulation cascade and fibrinolysis system such as factors II, V, VII, X, and XIII; tissue-type plasminogen activator; urokinase-type plasminogen activator (u-PA); plasminogen activator inhibitor-1 (PAI-1); and fibrin were compared between the AR model group and the control group. The symptom scores and eosinophil counts were higher in the AR group than in the control group ( P < .01). The mRNA expression level of u-PA ( P = .040) was significantly lower, and the expression levels of factor II ( P = .038) and factor X ( P = .036) were significantly higher, in the AR group. Immunohistochemical staining revealed that most of the fibrinolysis system and coagulation cascade components were localized to the epithelium, endothelium, and submucosal glands of the nasal mucosa. u-PA was downregulated in the AR group, whereas fibrin deposition was more prominent in the AR group than in the control group. In AR, particular components of the coagulation cascade were increased and fibrinolysis system was decreased compared to normal control. This difference may be associated with the fibrin deposition in the mucosa of AR mouse model.

Risk factors of cerebral ischemia in infants born to mothers with gestational diabetes

Objectives: Gestational diabetes mellitus (GDM) is carbohydrate intolerance that occurs during pregnancy. The present study was arranged to determine the risk of cerebral ischemia (CI) in infants born to mothers with gestational diabetes mellitus and MTHFR gene polymorphism.Material and methods: The study includes 70 pregnant women with GDM, divided into two groups depending on existence of cerebral ischemia (CI) in newborn infants. All patients were tested for coagulation cascade components, polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene and assessed by thromboelastography (TEG).Results: We observed that there was an increased frequency (74.2%) of 1298С MTHFR genotype carriers among women with CI newborns. The state of hypercoagulation according to both coagulation cascade tests and TEG data was diagnosed in women of this group.Conclusions: The results of this study suggest that hyperglycemia may program the development of cerebral ischemia in infants born to women with 1298С MTHFR gene polymorphism.

Coagulation cascade and complement system in systemic lupus erythematosus.

This study was conducted to (1) characterize coagulation cascade and complement system in systemic lupus erythematosus (SLE); (2) evaluate the associations between coagulation cascade, complement system, inflammatory response and SLE disease severity; (3) test the diagnostic value of a combination of D-dimer and C4 for lupus activity. Transcriptomics, proteomics and metabolomics were performed in 24 SLE patients and 24 healthy controls. The levels of ten coagulations, seven complements and three cytokines were measured in 112 SLE patients. Clinical data were collected from 2025 SLE patients. The analysis of multi-omics data revealed the common links for the components of coagulation cascade and complement system. The results of ELISA showed coagulation cascade and complement system had an interaction effect on SLE disease severity, this effect was pronounced among patients with excess inflammation. The analysis of clinical data revealed a combination of D-dimer and C4 provided good diagnostic performance for lupus activity. This study suggested that coagulation cascade and complement system become ‘partners in crime’, contributing to SLE disease severity and identified the diagnostic value of D-dimer combined with C4for lupus activity.

The liver in systemic disease: Sepsis and critical illness.

The liver in systemic disease: Sepsis and critical illness.

Leptospira spp.: Novel insights into host–pathogen interactions

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira spp. It is an important infectious disease that affects humans and animals. The disease causes economic losses as it affects livestock, with decreased milk production and death. Our group is investigating the genome sequences of L. interrogans targeting surface-exposed proteins because, due to their location, these proteins are capable to interact with several host components that could allow establishment of the infection. These interactions may involve adhesion of the bacteria to extracellular matrix (ECM) components and, hence, help bacterial colonization. The bacteria could also react with the host fibrinolytic system and/or with the coagulation cascade components, such as, plasminogen (PLG) and fibrinogen (Fg), respectively. The binding with the first system generates plasmin (PLA), increasing the proteolytic power of the bacteria, while the second interferes with clotting in a thrombin-catalyzed reaction, which may promote hemorrhage foci and increase bacterial dissemination. Interaction with the complement system negative regulators may help bacteria to evade the host immune system, facilitating the invasion. This work compiles the main described leptospiral proteins that could act as adhesins, as PLG and fibrinogen receptors and as complement regulator binding proteins. We present models in which we suggest possible mechanisms of how leptospires might colonize and invade host tissues, causing the disease. Understanding leptospiral pathogenesis will help to identify antigen candidates that would contribute to the development of more effective vaccines and diagnostic tests.

Plasma Fibrinogen Concentrations in Patients with Solid Tumors and Therapeutic Improvements by Combining Anticoagulants or Fibrinolytical Agents

Many of patient’s deaths with solid tumors are caused by altered pathogenesis coagulation cascade components and processes in clinics. Therapeutic actions for inhibiting cancer coagulation complications in clinics are indispensable part of modern cancer therapies. This editorial discusses the relationship between plasma/solid tumor fibrinogen levels and mechanisms of action by anticoagulants and fibrinolytic agents. Revisit old theory with latest hypothetic and therapeutic options. Many updating clinical information between fibrinogen malformation and its specific therapy are highlighted herein.

Staphylococcus aureus manganese transport protein C (MntC) is an extracellular matrix- and plasminogen-binding protein.

Infections caused by Staphylococcus aureus – particularly nosocomial infections - represent a great concern. Usually, the early stage of pathogenesis consists on asymptomatic nasopharynx colonization, which could result in dissemination to other mucosal niches or invasion of sterile sites, such as blood. This pathogenic route depends on scavenging of nutrients as well as binding to and disrupting extracellular matrix (ECM). Manganese transport protein C (MntC), a conserved manganese-binding protein, takes part in this infectious scenario as an ion-scavenging factor and surprisingly as an ECM and coagulation cascade binding protein, as revealed in this work. This study showed a marked ability of MntC to bind to several ECM and coagulation cascade components, including laminin, collagen type IV, cellular and plasma fibronectin, plasminogen and fibrinogen by ELISA. The MntC binding to plasminogen appears to be related to the presence of surface-exposed lysines, since previous incubation with an analogue of lysine residue, ε-aminocaproic acid, or increasing ionic strength affected the interaction between MntC and plasminogen. MntC-bound plasminogen was converted to active plasmin in the presence of urokinase plasminogen activator (uPA). The newly released plasmin, in turn, acted in the cleavage of the α and β chains of fibrinogen. In conclusion, we describe a novel function for MntC that may help staphylococcal mucosal colonization and establishment of invasive disease, through the interaction with ECM and coagulation cascade host proteins. These data suggest that this potential virulence factor could be an adequate candidate to compose an anti-staphylococcal human vaccine formulation.

Pathophysiology of the antiphospholipid syndrome (APS)

Antiphospholipid antibodies (aPL) not only are diagnostic biomarkers of antiphospholipid syndrome (APS), but also exert a pathogenic role in mediating both thrombotic and obstetric variants of the disease. Antibodies reacting against β2 glycoprotein I, in particular those targeting domain I of the molecule, provide the main pathogenic autoantibody subset. In vitro, aPL activate endothelial cells, monocytes, platelets, neutrophils, and complement system; in addition, aPL can interfere with several fluid-phase components of coagulation cascade. In vivo, aPL infusion triggers thrombotic events in animals primed with an additional hit (two-hit theory). With respect to obstetric complications, aPL can react with maternal and foetal sides of placenta, inducing a prothrombotic and proinflammatory phenotype. Interestingly, aPL are per se sufficient to induce pregnancy loss in vivo. However, aPL with similar antigen specificity and titres can mediate different pathogenic mechanisms and be responsible for heterogeneous clinical manifestations of APS.

Novel Pleiotropic Effects of Thrombin in Regulation of Metastatic Potential of Human Rhabdomyosarcoma (RMS) Cells – Identification of Negative PAR1 and PAR3 Receptor Crosstalk.

Abstract 336▪▪This icon denotes an abstract that is clinically relevant.Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of adolescence and childhood that frequently infiltrates bone marrow (BM) to this degrees that it may mimic acute lymphocytic leukemia. We identified chemokines and growth factors (e.g., SDF-1, HGF) that play an important role in RMS metastasis (Blood 2002;100:2597, Cancer Res. 2003;63:7926, Cancer Res. 2007;67:2131). Novel evidence however accumulates that metastatic process for many tumors may be modulated by the components of coagulation cascade (CC) (e.g., thrombin, activated platelets). Thus, we become interested on a role of CC in modulating metastasis of RMS cells. First, we learned that RMS cells express tissue factor (TF) and thus may activate coagulation by generation of thrombin. Thrombin activated in tumor microenvironment activates platelets that release microvesicles. We observed that platelet derived microvesicles (PMV) transfer to RMS cells several platelet integrin receptors (e.g., α2β3) important for RMS cell interaction with endothelium, and thus increase their adhesive potential to endothelial cells. To support this, we noticed that RMS cells covered with PMV showed higher metastatic potential after intravenous injection into immunodeficient SCID mice. We also found that PMV also directly chemoattracted RMS cells and activated MAPKp42/44 and AKT. Next we learned that all 10 human RMS cell lines investigated in our studies express functional PAR1 and PAR3 receptors. To support this, we observed in thrombin stimulated RMS cells phosphorylation of MAPKp42/44 and MAPKp38. To our surprise however, in in vitro experiments thrombin decreased RMS chemotactic response to conditioned media from bone marrow fibroblast and PMVs. Furthermore, we didn't observe any effect of thrombin on proliferation, survival and expression of pro-angiogenic factors in RMS cells. Thrombin also decreased adhesion of RMS cells to fibronectin and bone marrow stroma cells. In contrast PAR1 specific agonist TRAP-6 stimulated proliferation of RMS cells. Different responsiveness to thrombin and TRAP-6 stimulation could be explained by negative modulatory role of PAR3 receptor in response to stimulation by thrombin. Thus, to learn more on a role of PAR1 and PAR3 in RMS proliferation/metastasis we knock-down both receptors by employing shRNA strategy. We observed that PAR1-/- receptor RMS cells that express intact PAR3 cells formed in vivo smaller tumors as compared to unmodified control cells. On the other hand, PAR3-/- RMS cells that express functional PAR1 began to proliferate robust in response to thrombin. In conclusion, we demonstrate that RMS-expressed TF activates prothrombin and that thrombin is a novel, underappreciated, pro-metastatic factor for these cells. Activated in tumor proximity by thrombin, platelets release PMVs that chemoattract and transfer several platelet-derived receptors/adhesion molecules to RMS cells that are crucial for adhesion/interaction with the endothelium. Conversely, by decreasing the responsiveness of RMS cells to local chemoattractants and decreasing adhesiveness of RMS cells, thrombin promotes their release from the primary tumor into circulation. Consequently, RMS cells that are covered by PMVs release into circulation and respond to chemoattractants in distant organs for metastasis. Finally, our data also supports a negative regulatory role of thrombin-PAR3 axis in proliferation of RMS. Disclosures:No relevant conflicts of interest to declare.

The role of hemocytes in the immunity of the spider Acanthoscurria gomesiana

Invertebrates protect themselves against microbial infection through cellular and humoral immune defenses. Since the available information on the immune system of spiders is scarce, the main goal of the present study was to investigate the role of hemocytes and antimicrobial peptides (AMPs) in defense against microbes of spider Acanthoscurria gomesiana. We previously described the purification and characterization of two AMPs from the hemocytes of naïve spider A. gomesiana, gomesin and acanthoscurrin. Here we show that 57% of the hemocytes store both gomesin and acanthoscurrin, either in the same or in different granules. Progomesin labeling in hemocyte granules indicates that gomesin is addressed to those organelles as a propeptide. In vivo and in vitro experiments showed that lipopolysaccharide (LPS) and yeast caused the hemocytes to migrate. Once they have reached the infection site, hemocytes may secrete coagulation cascade components and AMPs to cell-free hemolymph. Furthermore, our results suggest that phagocytosis is not the major defense mechanism activated after microbial challenge. Therefore, the main reactions involved in the spider immune defense might be coagulation and AMP secretion.