SciCombinator

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Concept: Fibrinolysis

168

Persicarin and isorhamnetin were isolated from Oenanthe javanica and their anticoagulant activities were examined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), and the activities of cell-based thrombin and activated factor X (FXa). In addition, the effects of persicarin and isorhamnetin on the expressions of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were tested in tumor necrosis factor-α (TNF-α) activated human umbilical vein endothelial cells (HUVECs). The data obtained showed that persicarin and isorhamnetin both prolonged aPTT and PT significantly and inhibited the activities of thrombin and FXa. In addition, they both inhibited the generations of thrombin and FXa in HUVECs. In accordance with these anticoagulant activities, persicarin and isorhamnetin prolonged in vivo bleeding time and inhibited TNF-α induced PAI-1 production. Furthermore, PAI-1/t-PA ratio was significantly decreased by persicarin. Interestingly, the anticoagulant and profibrinolytic effects of persicarin were greater than those of isorhamnetin, which suggest that the sulfonate group of persicarin positively regulates its anticoagulatory function. Accordingly, our results suggest persicarin and isorhamnetin possess antithrombotic activities and that they could provide bases for the development of new anticoagulant agents.

Concepts: Coagulation, Fibrinolysis, Warfarin, Tissue plasminogen activator, Blood tests, Thrombin, Prothrombin time, Partial thromboplastin time

168

A novel class of small molecule inhibitors for plasminogen activator inhibitor type 1 (PAI-1)(2), represented by AZ3976, was identified in a high through-put screening campaign. AZ3976 displayed an IC(50) value of 26 µM in an enzymatic chromogenic assay. In a plasma clot lysis assay, the compound was active with an IC(50) of 16 µM. Surprisingly, AZ3976 did not bind to active PAI-1, but bound to latent PAI-1 with a K(D) of 0.29 µM at 35 °C and a binding stoichiometry of 0.94, as measured by isothermal calorimetry. Reversible binding was confirmed by surface plasmon resonance (SPR) direct binding experiments. The X-ray structure of AZ3976 in complex with latent PAI-1 was determined to 2.4 Å resolution. The inhibitor was bound in the flexible joint region with the entrance to the cavity located between α-helix D and β-strand 2A. A set of SPR experiments revealed that AZ3976 inhibited PAI-1 by enhancing the latency transition of active PAI-1. Since AZ3976 only had measurable affinity for latent PAI-1, we propose that its mechanism of inhibition is based on binding to a small fraction in equilibrium with active PAI-1, a latent-like pre-latent form, from which latent PAI-1 is then generated more rapidly. This mode of action, with induced accelerated latency transition of active PAI-1 may, together with supporting X-ray data, provide improved opportunities for small molecule drug design in the hunt for therapeutically useful PAI-1 inhibitors.

Concepts: Fibrinolysis, Enzyme inhibitor, Inhibitor, Surface plasmon resonance, Plasminogen activator inhibitor-1, Plasma oscillation

168

The invasive ability of the blood-borne fungal pathogen Cryptococcus neoformans can be enhanced through interactions with host plasma components, such as plasminogen. Previously we showed by in vitro studies that plasminogen coats the surface of C. neoformans and is converted to the active serine protease, plasmin, by host plasminogen activators. Viable, but not formaldehyde- or sodium azide-killed, cryptococcal strains undergo brain microvascular endothelial cell-dependent plasminogen-to-plasmin activation, which results in enhanced, plasmin-dependent cryptococcal invasion of primary bovine brain microvascular endothelial cells and fungal ability to degrade plasmin substrates. In the present work, brain microvascular endothelial cells cultured with viable, but not killed, cryptococcal strains led to significant increases in both urokinase mRNA transcription and cell-associated urokinase protein expression. Soluble urokinase was also detected in conditioned medium from brain microvascular endothelial cells cultured with viable, but not killed, C. neoformans. Exposure of plasminogen pre-coated viable C. neoformans to conditioned medium from strain-matched brain microvascular endothelial cell-fungal co-cultures resulted in plasminogen-to-plasmin activation and plasmin-dependent cryptococcal invasion. siRNA-mediated silencing of urokinase gene expression or the use of specific inhibitors of urokinase activity abrogated both plasminogen-to-plasmin activation on C. neoformans and cryptococcal-brain microvascular endothelial cell invasion. Our results suggest that pathogen exploitation of the host urokinase-plasmin(ogen) system may contribute to C. neoformans virulence during invasive cryptococcosis.

Concepts: DNA, Gene, Cell nucleus, Gene expression, Bacteria, Fibrinolysis, Cryptococcus neoformans, Cryptococcus

148

Thus far, validated whole blood assays used in in vitro fibrinolysis experiments using thromboelastometry (ROTEM) are lacking or have yet to be tested in humans. The objective was first, to establish a standardized modified ROTEM approach to detect both hypo- and hyperfibrinolysis. And second, to perform a technical and clinical validation of the assay.

Concepts: Coagulation, Fibrinolysis, Assay, Standard, Fibrinolytic system, Thromboelastometry

44

Lung injury activates multiple pro-inflammatory pathways, including neutrophils, epithelial, and endothelial injury, and coagulation factors leading to acute respiratory distress syndrome (ARDS). Low-dose methylprednisolone therapy (MPT) improved oxygenation and ventilation in early pediatric ARDS without altering duration of mechanical ventilation or mortality. We evaluated the effects of MPT on biomarkers of endothelial [Ang-2 and soluble intercellular adhesion molecule-1 (sICAM-1)] or epithelial [soluble receptor for activated glycation end products (sRAGE)] injury, neutrophil activation [matrix metalloproteinase-8 (MMP-8)], and coagulation (plasminogen activator inhibitor-1).

Concepts: Pulmonology, Blood, Pneumonia, Acute respiratory distress syndrome, Shock, Pulmonary contusion, Fibrinolysis, Endothelium

29

Background: Trauma is a global disease with over 2.5 million deaths annually from haemorrhage and coagulopathy. Overt hyperfibrinolysis is rare in trauma and associated with massive fatal injuries. Paradoxically, clinical trials suggest a much broader indication for antifibrinolytics. Objective: To determine the incidence and magnitude of fibrinolytic activation in trauma patients and its relationship to clot lysis as measured by thromboelastometry. Methods: Prospective cohort study of 303 consecutive trauma patients admitted between January 2007 and June 2009. Blood was drawn on arrival for thromboelastometry (TEM) and coagulation assays. Follow-up was until hospital discharge or death. TEM hyperfibrinolysis was defined as maximum clot lysis (ML) >15%. Fibrinolytic Activation (FA) measured by plasmin-antiplasmin complex (PAP) and D-dimer levels. Data were collected on demographics, mechanism, severity of injury and baseline vital signs. Outcome Measure(s): 28-day mortality. Secondary: 28-day ventilator-free days and 24-hour transfusion requirement. Results: Only 5% of patients had severe fibrinolysis on TEM, but 57% of patients had evidence of ‘moderate’ fibrinolysis with PAP levels elevated over twice normal (>1500μg/L) without lysis on TEM. TEM only detected clot lysis when PAP levels were increased 30 times normal (p<0.001) and antiplasmin levels were less than 75% of normal. Patients with FA had increased 28-day mortality compared with no FA (12% vs 1%, p<0.001), fewer ventilator-free days and longer hospital stay. Conclusions: FA occurs in the majority of trauma patients and the magnitude of FA correlates with poor clinical outcome. This was not detected by conventional thromboelastometry, an insensitive measure of endogenous fibrinolytic activity. © 2012 International Society on Thrombosis and Haemostasis.

Concepts: Coagulation, Pulmonary embolism, Fibrin, Fibrinolysis, Thrombus, Plasmin, Alpha 2-antiplasmin, Thromboelastometry

27

The factors that contribute to pulmonary embolism (PE), a potentially fatal complication of deep vein thrombosis (DVT), remain poorly understood. Whereas fibrin clot structure and functional properties have been implicated in the pathology of venous thromboembolism and the risk for cardiovascular complications, their significance in PE remain incomplete. Therefore we systematically compared and quantified clot formation and lysis time, plasminogen levels, viscoelastic properties, activated factor XIII crosslinking and fibrin clot structure in isolated DVT and PE subjects. Clots made from plasma of PE subjects showed faster clot lysis times with no differences in lag time, rate of clot formation or maximum absorbance of turbidity as compared to DVT. Differences in lysis times were not due to alterations in plasminogen levels. Compared with DVT, clots derived from PE subjects showed accelerated establishment of viscoelastic properties, documented by a decrease in lag time and an increase in the rate of viscoelastic property formation. The rate and extent of fibrin crosslinking by activated factor XIII were similar between clots from DVT and PE subjects. Evaluation by electron microscopy revealed that plasma fibrin clots from PE subjects exhibited lower fiber density compared to those from DVT subjects. These data suggest that clot structure and functional properties differ between DVT and PE subjects and provide insights into mechanisms that may regulate embolization.

Concepts: Pulmonary embolism, Fibrin, Fibrinolysis, Hematology, Thrombus, Vein, Deep vein thrombosis, Deep vein

27

Ulmus macrocarpa Hance (Ulmaceae) has been used as a traditional oriental medicine for the treatment of edema, mastitis, gastric cancer and inflammation. The aim of this study was to investigate the effects of Ulmus macrocarpa extract (UME) on thrombus formation in vivo, platelet activation ex vivo and fibrinolytic activity in vitro. To identify the antithrombotic activity of UME in vivo, we used an arterial thrombosis model. UME delayed the occlusion time by 13.4 and 13.9 min at doses of 300 and 600 mg/kg, respectively. UME significantly inhibited ex vivo platelet aggregation induced by collagen and adenosine 5'-diphosphate (ADP), respectively, but did not affect the coagulation times following activated partial thromboplastin and prothrombin activation. Therefore, to investigate the antiplatelet effect of UME, the effect of UME on collagen and ADP-induced platelet aggregation in vitro was examined. UME exhibited antiplatelet aggregation activity, induced by ADP and collagen. Furthermore, the fibrinolytic activity of UMEwas investigated. The results showed that UME significantly increased fibrinolysis at 1,000 mg/ml. In conclusion, the results suggested that UME may significantly inhibit artery thrombus formation in vivo, potentially due to antiplatelet activity, and also exhibits potential as a clot‑dissolving agent for thrombolytic therapy.

Concepts: Blood, Thrombosis, Coagulation, Fibrin, Platelet, Fibrinolysis, Thrombus, Thrombolysis

27

Herinase, a new bi-functional fibrinolytic metalloprotease, was purified from a medicinal and edible mushroom Hericium erinaceum. The enzyme was monomeric with a molecular mass of 51 kDa. Analysis of fibrin zymography showed an active band with a similar molecular mass. The N-terminal sequence of herinase VPSSFRTTITDAQLRG was highly distinguished from known fibrinolytic enzymes. Moreover, the enzyme activity was strongly inhibited by EDTA and EGTA, indicating that herinase is a metalloprotease. Herinase exhibited high specificity for the substrate t-PA followed by plasmin. The K m and V max values for H-D-Ile-Pro-Arg-PNA were found to be 4.7 mg and 26.7 U/ml respectively. Similarly, fibrin plate assays revealed that it was able to degrade fibrin clot directly and also able to activate plasminogen. Herinase provoked a rapid degradation of fibrin and fibrinogen α chains and slower degradation of γ chains. It had no activity on the β chains of fibrin and fibrinogen. This result suggests that herinase could possibly contain higher amount of α-fibrinogenase. The activity of herinase was stimulated by metal ions such as Ca(2+), Mg(2+), and Mn(2+), but inhibited by Cu(2+), Fe(2+), and Zn(2+). Herinase exhibited maximum activity at 30 °C and pH 7.0. These results demonstrate that herinase could be a novel fibrinolytic enzyme.

Concepts: Protein, Enzyme, Fibrin, Fibrinolysis, Protease, Plasmin, Edible mushroom, Hericium erinaceus

26

Right atrial thrombus in the setting of a large pulmonary embolus is rare and is associated with serious adverse events. This case report presents the role played by EKOS EkoSonic ultrasound system in successfully treating right atrial thrombus and massive pulmonary embolism.

Concepts: Stroke, Pulmonary embolism, Fibrinolysis, Hematology, Thrombus, Tissue plasminogen activator, Thrombolysis, Embolism