Search Results (25)

Viperid snake venoms are notably abundant in metalloproteinases (proteins) (SVMPs), which are primarily responsible for inducing hemorrhage and disrupting the hemostatic process and tissue integrity in envenomed victims. In this study, barnettlysin-III (Bar-III), a hemorrhagic P-III SVMP, was purified from the venom of the Peruvian snake Bothrops barnetti. Bar-III has a molecular mass of approximately 50 kDa and is a glycosylation-dependent functional metalloproteinase. Some biochemical properties of Bar-III, including the full amino acid sequence deduced from its cDNA, are reported. Its enzymatic activity is increased by Ca²⁺ ions and inhibited by an excess of Zn²⁺. Synthetic metalloproteinase inhibitors and EDTA also inhibit its proteolytic action. Bar-III degrades several plasma and ECM proteins, including fibrin(ogen), fibronectin, laminin, and nidogen. Platelets play a key role in hemostasis and thrombosis and in other biological process, such as inflammation and immunity, and platelet activation is driven by the platelet signaling receptors, glycoprotein (GP)Ib-IX-V, which binds vWF, and GPVI, which binds collagen. Moreover, Bar-III inhibits vWF- and convulxin-induced platelet aggregation in human washed platelets by cleaving the recombinant A1 domain of vWF and GPVI into a soluble ectodomain fraction of ~55 kDa (sGPVI). Bar-III does not reduce the viability of cultured endothelial cells; however, it interferes with the adhesion of these cells to fibronectin, vitronectin, and RGD peptides, as well as their migration profile. Bar-III binds specifically to the surface of these cells, and part of this interaction involves α5β1 integrin receptors. These results contribute to a better comprehension of the pathophysiology of snakebite accidents/incidents and could be used as a tool to explore novel and safer anti-venom therapeutics. Full article

The currently available point-of-care hemostasis tests are burdened by criticisms concerning the use of different activators and inhibitors and the lack of dynamic flow. These operating conditions may constitute an impediment to the determination of the patient’s hemostatic condition. Hence, the diffusion of these tests in clinical practice is still limited to specific scenarios. In this work, we present a new method for analyzing the patient’s global hemostasis based on the visualization of the main components of the coagulation process and its computerized quantitative image analysis. The automated “Smart Clot” point-of-care system presents a micro-fluidic chamber in which whole blood flows, without the addition of any activator or inhibitor. In this micro-channel, platelet adhesion, activation and aggregation to the type I collagen-coated surface take place (primary hemostasis), leading to the production of endogenous thrombin on the surface of platelet aggregates and the consequent fibrin mesh and thrombus formation (secondary hemostasis). These observations are verified by inhibiting primary hemostasis with the antiplatelet drugs Indomethacin (−70% on platelet aggregation, −60% on fibrin(ogen) formation) and Tirofiban (complete inhibition of platelet aggregation and fibrin(ogen) formation) and secondary hemostasis with the antithrombin drugs Heparin (−70% on platelet aggregation, −80% on fibrin(ogen) formation) and Lepirudin (−80% on platelet aggregation, −90% on fibrin(ogen) formation). Smart Clot, through a single test, provides quantitative results concerning platelet aggregation and fibrin formation and is suitable for undergoing comparative studies with other coagulation point-of-care devices. Full article

In classical amyloidoses, amyloid fibres form through the nucleation and accretion of protein monomers, with protofibrils and fibrils exhibiting a cross-β motif of parallel or antiparallel β-sheets oriented perpendicular to the fibre direction. These protofibrils and fibrils can intertwine to form mature amyloid fibres. Similar phenomena can occur in blood from individuals with circulating inflammatory molecules (and also some originating from viruses and bacteria). Such pathological clotting can result in an anomalous amyloid form termed fibrinaloid microclots. Previous proteomic analyses of these microclots have shown the presence of non-fibrin(ogen) proteins, suggesting a more complex mechanism than simple entrapment. We thus provide evidence against such a simple entrapment model, noting that clot pores are too large and centrifugation would have removed weakly bound proteins. Instead, we explore whether co-aggregation into amyloid fibres may involve axial (multiple proteins within the same fibril), lateral (single-protein fibrils contributing to a fibre), or both types of integration. Our analysis of proteomic data from fibrinaloid microclots in different diseases shows no significant quantitative overlap with the normal plasma proteome and no correlation between plasma protein abundance and their presence in fibrinaloid microclots. Notably, abundant plasma proteins like α-2-macroglobulin, fibronectin, and transthyretin are absent from microclots, while less abundant proteins such as adiponectin, periostin, and von Willebrand factor are well represented. Using bioinformatic tools, including AmyloGram and AnuPP, we found that proteins entrapped in fibrinaloid microclots exhibit high amyloidogenic tendencies, suggesting their integration as cross-β elements into amyloid structures. This integration likely contributes to the microclots’ resistance to proteolysis. Our findings underscore the role of cross-seeding in fibrinaloid microclot formation and highlight the need for further investigation into their structural properties and implications in thrombotic and amyloid diseases. These insights provide a foundation for developing novel diagnostic and therapeutic strategies targeting amyloidogenic cross-seeding in blood clotting disorders. Full article

Autologous platelet-rich plasma (PRP) preparations are prepared at the point of care. Centrifugation cellular density separation sequesters a fresh unit of blood into three main fractions: a platelet-poor plasma (PPP) fraction, a stratum rich in platelets (platelet concentrate), and variable leukocyte bioformulation and erythrocyte fractions. The employment of autologous platelet concentrates facilitates the biological potential to accelerate and support numerous cellular activities that can lead to tissue repair, tissue regeneration, wound healing, and, ultimately, functional and structural repair. Normally, after PRP preparation, the PPP fraction is discarded. One of the less well-known but equally important features of PPP is that particular growth factors (GFs) are not abundantly present in PRP, as they reside outside of the platelet alpha granules. Precisely, insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) are mainly present in the PPP fraction. In addition to their roles as angiogenesis activators, these plasma-based GFs are also known to inhibit inflammation and fibrosis, and they promote keratinocyte migration and support tissue repair and wound healing. Additionally, PPP is known for the presence of exosomes and other macrovesicles, exerting cell–cell communication and cell signaling. Newly developed ultrafiltration technologies incorporate PPP processing methods by eliminating, in a fast and efficient manner, plasma water, cytokines, molecules, and plasma proteins with a molecular mass (weight) less than the pore size of the fibers. Consequently, a viable and viscous protein concentrate of functional total proteins, like fibrinogen, albumin, and alpha-2-macroglobulin is created. Consolidating a small volume of high platelet concentrate with a small volume of highly concentrated protein-rich PPP creates a protein-rich, platelet-rich plasma (PR-PRP) biological preparation. After the activation of proteins, mainly fibrinogen, the PR-PRP matrix retains and facilitates interactions between invading resident cells, like macrophages, fibroblast, and mesenchymal stem cells (MSCs), as well as the embedded concentrated PRP cells and molecules. The administered PR-PRP biologic will ultimately undergo fibrinolysis, leading to a sustained release of concentrated cells and molecules that have been retained in the PR-PRP matrix until the matrix is dissolved. We will discuss the unique biological and tissue reparative and regenerative properties of the PR-PRP matrix. Full article

Diabetic retinopathy (DR)-associated vision loss is a devastating disease affecting the working-age population. Retinal pathology is due to leakage of serum components into retinal tissues, activation of resident phagocytes (microglia), and vascular and neuronal damage. While short-term interventions are available, they do not revert visual function or halt disease progression. The impact of microglial inflammatory responses on the neurovascular unit remains unknown. In this study, we characterized microglia–vascular interactions in an experimental model of DR. Early diabetes presents activated retinal microglia, vascular permeability, and vascular abnormalities coupled with vascular tortuosity and diminished astrocyte and endothelial cell-associated tight-junction (TJ) and gap-junction (GJ) proteins. Microglia exclusively bind to the neuronal-derived chemokine fractalkine (FKN) via the CX3CR1 receptor to ameliorate microglial activation. Using neuron-specific recombinant adeno-associated viruses (rAAVs), we therapeutically overexpressed soluble (sFKN) or membrane-bound (mFKN) FKN using intra-vitreal delivery at the onset of diabetes. This study highlights the neuroprotective role of rAAV-sFKN, reducing microglial activation, vascular tortuosity, fibrin(ogen) deposition, and astrogliosis and supporting the maintenance of the GJ connexin-43 (Cx43) and TJ zonula occludens-1 (ZO-1) molecules. The results also show that microglia–vascular interactions influence the vascular width upon administration of rAAV-sFKN and rAAV-mFKN. Administration of rAAV-sFKN improved visual function without affecting peripheral immune responses. These findings suggest that overexpression of rAAV-sFKN can mitigate vascular abnormalities by promoting glia–neural signaling. sFKN gene therapy is a promising translational approach to reverse vision loss driven by vascular dysfunction. Full article

Cardiovascular diseases represent the main cause of death. A common feature of cardiovascular disease is thrombosis resulting from intravascular accumulation of fibrin. In the last years, several fibrinolytic enzymes have been discovered in many medicinal or edible mushrooms as potential new antithrombotic agents. This study aimed to compare the fibrin(ogen)olytic activity of crude extracts from the fruiting bodies of four cultivated edible mushrooms: Lentinula edodes, Pleurotus ostreatus, Pleurotus eryngii, and Agrocybe aegerita. Fibrin(ogen)olytic activity was assessed by fibrin plate, spectrophotometric assay and electrophoretic analysis (SDS-PAGE and zymography). The highest activity was detected for P. ostreatus followed by P. eryngii, L. edodes and A. aegerita. Results indicated that enzymes exhibited maximum activity at pH 6–7 and 30–40 °C, respectively. Enzyme activity was inhibited by serine and metalloprotease inhibitors. We proposed a new index called the Specific Fibrin(ogen)olytic Index (SFI), which allows specification of the proportion of the total proteolytic capacity due to the fibrin(ogen)olytic activity. These data suggest that the extracts from fruiting bodies or powdered mushrooms can be used as functional ingredients for the development of new functional foods that may act as thrombolytic agents responding, at the same time, to the increasing demand for safe, healthy and sustainable food. Full article

Although fibrinolytic enzymes and thrombolytic agents help in cardiovascular disease treatment, those currently available have several side effects. This warrants the search for safer alternatives. Several natural cysteine protease preparations are used in traditional medicine to improve platelet aggregation and thrombosis-related diseases. Hence, this study aimed to investigate the effect of ficin, a natural cysteine protease, on fibrin(ogen) and blood coagulation. The optimal pH (pH 7) and temperature (37 °C) for proteolytic activity were determined using the azocasein method. Fibrinogen action and fibrinolytic activity were measured both electrophoretically and by the fibrin plate assay. The effect of ficin on blood coagulation was studied by conventional coagulation tests: prothrombin time (PT), activated partial thromboplastin time (aPTT), blood clot lysis assay, and the κ-carrageenan thrombosis model. The Aα, Bβ, and γ bands of fibrinogen are readily cleaved by ficin, and we also observed a significant increase in PT and aPTT. Further, the mean length of the infarcted regions in the tails of Sprague–Dawley rats was shorter in rats administered 10 U/mL of ficin than in control rats. These findings suggest that natural cysteine protease, ficin contains novel fibrin and fibrinogenolytic enzymes and can be used for preventing and/or treating thrombosis-associated cardiovascular disorders. Full article

Although preclinical research and observer studies on patients with atrial fibrillation concluded that direct oral anticoagulants (DOACs) can protect against dementia like Alzheimer’s disease (AD), clinical investigation towards therapeutical approval is still pending. DOACs target pathological thrombin, which is, like toxic tau and amyloid-ß proteins (Aß), an early hallmark of AD. Especially in hippocampal and neocortical areas, the release of parenchymal Aß into the blood induces thrombin and proinflammatory bradykinin synthesis by activating factor XII of the contact system. Thrombin promotes platelet aggregation and catalyzes conversion of fibrinogen to fibrin, leading to degradation-resistant, Aß-containing fibrin clots. Together with oligomeric Aß, these clots trigger vessel constriction and cerebral amyloid angiopathy (CAA) with vessel occlusion and hemorrhages, leading to vascular and blood–brain barrier (BBB) dysfunction. As consequences, brain blood flow, perfusion, and supply with oxygen (hypoxia) and nutrients decrease. In parenchymal tissue, hypoxia stimulates Aß synthesis, leading to Aß accumulation, which is further enhanced by BBB-impaired perivascular Aß clearance. Aß trigger neuronal damage and promote tau pathologies. BBB dysfunction enables thrombin and fibrin(ogen) to migrate into parenchymal tissue and to activate glial cells. Inflammation and continued Aß production are the results. Synapses and neurons die, and cognitive abilities are lost. DOACs block thrombin by inhibiting its activity (dabigatran) or production (FXa-inhibitors, e.g., apixaban, rivaroxaban). Therefore, DOAC use could preserve vascular integrity and brain perfusion and, thereby, could counteract vascular-driven neuronal and cognitive decline in AD. A conception for clinical investigation is presented, focused on DOAC treatment of patients with diagnosed AD in early-stage and low risk of major bleeding. Full article

We have previously demonstrated that platelet-poor plasma (PPP) obtained from patients with Long COVID/Post-Acute Sequelae of COVID-19 (PASC) is characterized by a hypercoagulable state and contains hyperactivated platelets and considerable numbers of already-formed amyloid fibrin(ogen) or fibrinaloid microclots. Due to the substantial overlap in symptoms and etiology between Long COVID/PASC and ME/CFS, we investigated whether coagulopathies reflected in Long COVID/PASC—hypercoagulability, platelet hyperactivation, and fibrinaloid microclot formation—were present in individuals with ME/CFS and gender- and age-matched healthy controls. ME/CFS samples showed significant hypercoagulability as judged by thromboelastography of both whole blood and platelet-poor plasma. The area of plasma images containing fibrinaloid microclots was commonly more than 10-fold greater in untreated PPP from individuals with ME/CFS than in that of healthy controls. A similar difference was found when the plasma samples were treated with thrombin. Using fluorescently labelled PAC-1, which recognizes glycoprotein IIb/IIIa, and CD62P, which binds P-selectin, we observed hyperactivation of platelets in ME/CFS hematocrit samples. Using a quantitative scoring system, the ME/CFS platelets were found to have a mean spreading score of 2.72 ± 1.24 vs. 1.00 (activation with pseudopodia formation) for healthy controls. We conclude that ME/CFS is accompanied by substantial and measurable changes in coagulability, platelet hyperactivation, and fibrinaloid microclot formation. However, the fibrinaloid microclot load was not as great as was previously noted in Long COVID/PASC. Fibrinaloid microclots, in particular, may contribute to many ME/CFS symptoms, such as fatigue, seen in patients with ME/CFS, via the (temporary) blockage of microcapillaries and hence ischemia. Furthermore, fibrinaloid microclots might damage the endothelium. The discovery of these biomarkers represents an important development in ME/CFS research. It also points to possible uses for treatment strategies using known drugs and/or nutraceuticals that target systemic vascular pathology and endothelial inflammation. Full article

Fibrinolytic enzymes are important components in the treatment of thrombosis-associated disorders. A new bi-functional fibrinolytic enzyme, versiase, was identified from a marine-derived fungus Aspergillus versicolor ZLH-1. The enzyme was isolated from the fungal culture through precipitation with ammonium sulfate at 90% saturation. Additionally, it was further purified by DEAE-based ion-exchange chromatography, with a recovery of 20.4%. The fibrinolytic enzyme presented as one band on both SDS-PAGE and fibrin-zymogram, with a molecular mass of 37.3 kDa. It was elucidated as a member of metalloprotease in M35 family by proteomic approaches. The homology-modeling analysis revealed that versiase shares significant structural homology wuth the zinc metalloendopeptidase. The enzyme displayed maximum activity at 40 °C and pH 5.0. The activity of versiase was strongly inhibited by the metalloprotease inhibitors EDTA and BGTA. Furthermore, versiase hydrolyzed fibrin directly and indirectly via the activation of plasminogen, and it was able to hydrolyze the three chains (α, β, γ) of fibrin(ogen). Additionally, versiase demonstrated promising thrombolytic and anticoagulant activities, without many side-effects noticed. In conclusion, versiase appears to be a potent fibrinolytic enzyme deserving further investigation. Full article

Fibrin forms the structural scaffold of blood clots and has great potential for biomaterial applications. Creating recombinant expression systems of fibrinogen, fibrin’s soluble precursor, would advance the ability to construct mutational libraries that would enable structure–function studies of fibrinogen and expand the utility of fibrin as a biomaterial. Despite these needs, recombinant fibrinogen expression systems, thus far, have relied on the time-consuming creation of stable cell lines. Here we present tests of a transient fibrinogen expression system that can rapidly generate yields of 8–12 mg/L using suspension HEK Expi293^TM cells. We report results from two different plasmid systems encoding the fibrinogen cDNAs and two different transfection reagents. In addition, we describe a novel, affinity-based approach to purifying fibrinogen from complex media such as human plasma. We show that using a high-affinity peptide which mimics fibrin’s knob ‘A’ sequence enables the purification of 50–75% of fibrinogen present in plasma. Having robust expression and purification systems of fibrinogen will enable future studies of basic fibrin(ogen) biology, while paving the way for the ubiquitous use of fibrin as a biomaterial. Full article

Depression is an independent risk factor for cardiovascular disease (CVD). We have previously shown that repeated social defeat (RSD) exaggerates atherosclerosis development by enhancing neutrophil extracellular trap (NET) formation. In this study, we investigated the impact of RSD on arterial thrombosis. Eight-week-old male wild-type mice (C57BL/6J) were exposed to RSD by housing with larger CD-1 mice in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. After confirming depression-like behaviors, mice underwent FeCl₃-induced carotid arterial injury and were analyzed after 3 h. Although the volume of thrombi was comparable between the two groups, fibrin(ogen)-positive areas were significantly increased in defeated mice, in which Ly-6G-positive cells were appreciably co-localized with Cit-H3-positive staining. Treatment with DNase I completely diminished exaggerated fibrin-rich clot formation in defeated mice. Flow cytometric analysis showed that neutrophil CD11b expression before FeCl₃ application was significantly higher in defeated mice than in control mice. In vitro NET formation induced by activated platelets was significantly augmented in defeated mice, which was substantially inhibited by anti-CD11b antibody treatment. Our findings demonstrate that RSD enhances fibrin-rich clot formation after arterial injury by enhancing NET formation, suggesting that NET can be a new therapeutic target in depression-related CVD. Full article

Recently, an inverse relationship between incidence of COVID-19 and seasonal aerosolization of mold spores was demonstrated. Analyses of that relationship suggested mold spores compete with SARS-CoV-2 virions for a receptor on the pulmonary epithelial surface. By inference, the operative receptor was proposed to be Toll-like receptor 4, with surface-localized virions being responsible for symptomatology. In this report, the pathogenesis of COVID-19 is further developed, with a focus on a role for surfactant protein D in the process. This developed proposal provides both mechanistic understanding and suggested treatments of COVID-19. Full article

Data from Chicago confirm the end of flu season coincides with the beginning of pollen season. More importantly, the end of flu season also coincides with onset of seasonal aerosolization of mold spores. Overall, the data suggest bioaerosols, especially mold spores, compete with viruses for a shared receptor, with the periodicity of influenza-like illnesses, including COVID-19, a consequence of seasonal factors that influence aerosolization of competing species. Full article

Toxins from Bothrops venoms targeting hemostasis are responsible for a broad range of clinical and biological syndromes including local and systemic bleeding, incoagulability, thrombotic microangiopathy and macrothrombosis. Beyond hemostais disorders, toxins are also involved in the pathogenesis of edema and in most complications such as hypovolemia, cardiovascular collapse, acute kidney injury, myonecrosis, compartmental syndrome and superinfection. These toxins can be classified as enzymatic proteins (snake venom metalloproteinases, snake venom serine proteases, phospholipases A₂ and L-amino acid oxidases) and non-enzymatic proteins (desintegrins and C-type lectin proteins). Bleeding is due to a multifocal toxicity targeting vessels, platelets and coagulation factors. Vessel damage due to the degradation of basement membrane and the subsequent disruption of endothelial cell integrity under hydrostatic pressure and tangential shear stress is primarily responsible for bleeding. Hemorrhage is promoted by thrombocytopenia, platelet hypoaggregation, consumption coagulopathy and fibrin(ogen)olysis. Onset of thrombotic microangiopathy is probably due to the switch of endothelium to a prothrombotic phenotype with overexpression of tissue factor and other pro-aggregating biomarkers in association with activation of platelets and coagulation. Thrombosis involving large-caliber vessels in B. lanceolatus envenomation remains a unique entity, which exact pathophysiology remains poorly understood. Full article