In Vivo Anticoagulant and Thrombolytic Activities of a Fibrinolytic Serine Protease (Brevithrombolase) With the k-Carrageenan-Induced Rat Tail Thrombosis Model

SummaryThe anticoagulant effect of selected synthetic inhibitors of thrombin and factor Xa was studied in vitro in commonly used clotting assays. The concentrations of the compounds doubling the clotting time in the various assays were mainly dependent on their thrombin inhibitory activity. Factor Xa inhibitors were somewhat more effective in prolonging the prothrombin time compared to the activated partial thromboplastin time, whereas the opposite was true of thrombin inhibitors.In vivo, in a venous stasis thrombosis model and a thromboplastin-induced microthrombosis model in rats the thrombin inhibitors were effective antithrombotically whereas factor Xa inhibitors of numerically similar IQ value for the respective enzyme were not effective at equimolar dosageThe results are discussed in the light of the different prelequisiles and conditions for inhibition of thrombin and factor Xa in the course of blood clotting.

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Acyl-Enzymes as Thrombolytic Agents in Dog Models of Venous Thrombosis and Pulmonary Embolism

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661069 ◽

1984 ◽

Vol 51 (02) ◽

pp. 248-253 ◽

Cited By ~ 22

Author(s):

R J Dupe ◽

P D English ◽

R A G Smith ◽

J Green

Keyword(s):

Pulmonary Embolism ◽

Side Effects ◽

Venous Thrombosis ◽

Active Site ◽

Acute Pulmonary Embolism ◽

Quantitative Model ◽

Beagle Dog ◽

Thrombosis Model ◽

Derivatives Of

SummaryA quantitative model of venous thrombosis in the beagle dog is described. The model was adapted to permit ageing of isolated experimental clots in vivo. A model of acute pulmonary embolism in this species is also described. In the venous thrombosis model, infusion of streptokinase (SK) or SK-activated human plasmin gave significant lysis but bolus doses of SK. plasmin complex were ineffective. Active site anisoylated derivatives of SK. plasminogen complex, SK-activated plasmin and activator-free plasmin were all active when given as bolus doses in both models. At lytic doses, the acyl-enzymes caused fewer side-effects attributable to plasminaemia than the corresponding unmodified enzymes.

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Phenotypic Analysis of Mice Lacking the Tmprss2-Encoded Protease

Molecular and Cellular Biology ◽

10.1128/mcb.26.3.965-975.2006 ◽

2006 ◽

Vol 26 (3) ◽

pp. 965-975 ◽

Cited By ~ 105

Author(s):

Tom S. Kim ◽

Cynthia Heinlein ◽

Robert C. Hackman ◽

Peter S. Nelson

Keyword(s):

Serine Protease ◽

Serine Proteases ◽

Biological Activities ◽

Type Ii ◽

Phenotypic Analysis ◽

Normal Prostate ◽

Prostate Hyperplasia ◽

Transmembrane Serine Protease

ABSTRACT Tmprss2 encodes an androgen-regulated type II transmembrane serine protease (TTSP) expressed highly in normal prostate epithelium and has been implicated in prostate carcinogenesis. Although in vitro studies suggest protease-activated receptor 2 may be a substrate for TMPRSS2, the in vivo biological activities of TMPRSS2 remain unknown. We generated Tmprss2 −/− mice by disrupting the serine protease domain through homologous recombination. Compared to wild-type littermates, Tmprss2 −/− mice developed normally, survived to adulthood with no differences in protein levels of prostatic secretions, and exhibited no discernible abnormalities in organ histology or function. Loss of TMPRSS2 serine protease activity did not influence fertility, reduce survival, result in prostate hyperplasia or carcinoma, or alter prostatic luminal epithelial cell regrowth following castration and androgen replacement. Lack of an observable phenotype in Tmprss2 −/− mice was not due to transcriptional compensation by closely related Tmprss2 homologs. We conclude that the lack of a discernible phenotype in Tmprss2 −/− mice suggests functional redundancy involving one or more of the type II transmembrane serine protease family members or other serine proteases. Alternatively, TMPRSS2 may contribute a specialized but nonvital function that is apparent only in the context of stress, disease, or other systemic perturbation.

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In Vivo Porcine Aged Deep Vein Thrombosis Model for Testing Ultrasound-based Thrombolysis Techniques

Ultrasound in Medicine & Biology ◽

10.1016/j.ultrasmedbio.2021.08.017 ◽

2021 ◽

Author(s):

Greyson E. Stocker ◽

Jiaqi Shi ◽

Kimberly Ives ◽

Adam D. Maxwell ◽

Paul A. Dayton ◽

...

Keyword(s):

Deep Vein Thrombosis ◽

Vein Thrombosis ◽

Thrombosis Model ◽

Deep Vein

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An Improved In Vivo Rat Tail Vertebra Model for the Study of Trabecular Bone Adaptation

10.1115/imece1999-0430 ◽

1999 ◽

Author(s):

Mark J. Eichler ◽

Chi Hyun Kim ◽

X. Edward Guo

Keyword(s):

Trabecular Bone ◽

Large Scale ◽

Bone Fractures ◽

Mechanical Load ◽

Bone Adaptation ◽

Surgical Trauma ◽

Micro Computed Tomography ◽

Age Related ◽

Rat Tail

Abstract The role of mechanical loading in trabecular bone adaptation is important for the understanding of bone integrity in different loading scenarios such as microgravity and for the etiology of age-related bone fractures. There have been numerous in vivo animal studies of bone adaptation, most of which are related to cortical bone remodeling, aimed at the investigation of Wolff’s Law [4], An interesting experimental model for trabecular bone adaptation has been developed in the rat tail vertebrae [2,3]. This model is attractive for trabecular bone adaptation studies because a controlled mechanical load can be applied to a whole vertebra with minimal surgical trauma, using a relatively inexpensive animal model. In addition, with advanced micro computed tomography (micro-CT) or micro magnetic resonance imaging (micro-MRI) coupled with large scale finite element modeling techniques, it is possible to characterize the three-dimensional (3D) stress/strain environment in the bone tissue close to a cellular level (∼25μm) [1]. Therefore, this in vivo rat tail model has a tremendous potential for quantification of the relationship between mechanical stimulation and biological response in trabecular bone adaptation.

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ROLE OF NITRIC OXIDE (NO) IN CAPSAICIN MEDIATED ANTI-PLATELET ACTIVITY IN IN VITRO, IN VIVO, EX-VIVO MODEL OF PLATELET AGGREGATION ASSAY AND ARTERIAL THROMBOSIS IN RAT: POTENTIAL THERAPEUTIC TARGET?

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2018v10i4.22474 ◽

2018 ◽

Vol 10 (4) ◽

pp. 44

Author(s):

Mihir K Patel ◽

Kiranj K. Chaudagar ◽

Anita A. Mehta

Keyword(s):

Platelet Aggregation ◽

Arterial Thrombosis ◽

Ex Vivo ◽

Platelet Activity ◽

Aggregation Assay ◽

Thrombosis Model ◽

Platelet Aggregation Assay

Objective: Although recent advances in the treatment of congestive heart disease, mortality among patients’ remains a questionable remark. Therefore, we evaluated the role of capsaicin on in vitro and ex vivo platelet aggregation induced by Adenosine Di-Phosphate (ADP) as well as in in vivo thrombosis models and role of NO, KATP was also identified in the capsaicin-induced anti-platelet animal model as well as in vivo model of arterial thrombosis.Methods: According to body weight wistar rats were divided into five groups. Group I and Group II was treated with saline and capsaicin (3 mg/kg, i. v), while animals from Group III were treated with N(ω)-nitro-L-arginine methyl ester (L-NAME) (30 mg/kg, i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group IV animals were treated with glibenclamide (10 mg/kg,i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group V was considered as a positive control and administered clopidogrel (30 mg/kg, p. o). Animals were subjected for in vitro, ex-vivo platelet aggregation assay. ADP (30µM) was utilized as an aggregating agent in these experiments. After these assays; animals of each group were subjected for subaqueous tail bleeding time in a rat model and FeCl3-induced arterial thrombosis model in rats.Results: In ADP-induced in vitro platelet aggregation, a significant reduction in % platelet aggregation was observed at 50µM (64.35±4.641) and 100µM (52.72±4.192) concentration of capsaicin as compared to vehicle control (85.82±3.716). Capsaicin (3 mg/kg, i. v) also showed a significant reduction (49.53±4.075) in ex-vivo ADP-induced platelet aggregation as compared to vehicle control (89.38±2.057). In FeCl3 induced arterial thrombosis model, Capsaicin (3 mg/kg, i. v) exhibited an increase in time to occlusion in this rodent model and presence of the L-NAME and glibenclamide had inhibited the activity of capsaicin.Conclusion: In our study, capsaicin (50 µM, 100µM) exhibited potent anti-platelet activity in ADP-induced platelet aggregation, similarly capsaicin exhibited significant anti-platelet action in the ex-vivo study. Moreover, the presence of L-NAME and glibenclamide inhibited the anti-thrombotic and anti-platelet action of capsaicin. Therefore, it was concluded that NO and KATP may be involved in the anti-thrombotic action of capsaicin.

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Nasal commensal, Staphylococcus epidermidis shapes the mucosal environment to prevent influenza virus invasion through Serpine1 induction

10.1101/2020.08.03.235648 ◽

2020 ◽

Author(s):

Ara Jo ◽

Jina Won ◽

Chan Hee Chil ◽

Jae Young Choi ◽

Kang-Mu Lee ◽

...

Keyword(s):

Influenza Virus ◽

Respiratory Tract ◽

Protease Inhibitor ◽

Serine Protease ◽

Nasal Mucosa ◽

Staphylococcus Epidermidis ◽

Influenza Virus Infection ◽

Serine Protease Inhibitor ◽

Cellular Environment

ABSTRACTOur recent study presented evidence that Staphylococcus epidermidis (S. epidermidis) was the most frequently encountered microbiome component in healthy human nasal mucus and that S. epidermidis could induce interferon (IFN)-dependent innate immunity to control acute viral lung infection. The serine protease inhibitor Serpine1 was identified to inhibit influenza A virus (IAV) spread by inhibiting glycoprotein cleavage, and the current study supports an additional mechanism of Serpine1 induction in the nasal mucosa, which can be regulated through S. epidermidis and IFN signaling. The exposure of in vivo mice to human S. epidermidis increased IFN-λ secretion in nasal mucosa and prevented an increase in the burden of IAV in the lung. S. epidermidis-inoculated mice exhibited the significant induction of Serpine1 in vivo in the nasal mucosa, and by targeting airway protease, S. epidermidis-induced Serpine1 inhibited the intracellular invasion of IAV to the nasal epithelium and led to restriction of IAV spreading to the lung. Furthermore, IFN-λ secretion was involved in the regulation of Serpine1 in S. epidermidis-inoculated nasal epithelial cells and in vivo nasal mucosa, and this was biologically relevant for the role of Serpine1 as an interferon-stimulated gene in the upper airway. Together, our findings reveal that human nasal commensal S. epidermidis manipulates the suppression of serine protease in in vivo nasal mucosa through Serpine1 induction and protects the nasal mucosa from IAV invasion through IFN-λ signaling.IMPORTANCEPreviously, we proved that nasal microbiome could enhance IFN-related innate immune responses to protect the respiratory tract against influenza virus infection. The present study shows a great understanding of the intimate association of S. epidermidis-regulated IFN-lambda induction and serine protease inhibitor in nasal mucosa. Our data demonstrate that S. epidermidis-regulated Serpine1 suppresses the invasion of influenza virus through suppression of airway serine protease at the level of nasal mucosa and impedes IAV spread to the respiratory tract. Thus, human nasal commensal S. epidermidis represents a therapeutic potential for treating respiratory viral infections via the change of cellular environment in respiratory tract.

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Vaccinomics Approach for Designing Potential Peptide Vaccine by TargetingShigellaspp. Serine Protease Autotransporter Subfamily Protein SigA

Journal of Immunology Research ◽

10.1155/2017/6412353 ◽

2017 ◽

Vol 2017 ◽

pp. 1-14 ◽

Cited By ~ 17

Author(s):

Arafat Rahman Oany ◽

Tahmina Pervin ◽

Mamun Mia ◽

Motaher Hossain ◽

Mohammad Shahnaij ◽

...

Keyword(s):

Serine Protease ◽

Bacillary Dysentery ◽

Peptide Vaccine ◽

Human Leukocyte ◽

World Population ◽

Potential Core ◽

Antigenic Protein ◽

Leukocyte Antigen

Shigellosis, a bacillary dysentery, is closely associated with diarrhoea in human and causes infection of 165 million people worldwide per year. Casein-degrading serine protease autotransporter of enterobacteriaceae (SPATE) subfamily protein SigA, an outer membrane protein, exerts both cytopathic and enterotoxic effects especially cytopathic to human epithelial cell type-2 (HEp-2) and is shown to be highly immunogenic. In the present study, we have tried to impose the vaccinomics approach for designing a common peptide vaccine candidate against the immunogenic SigA ofShigellaspp. At first, 44 SigA proteins from different variants ofS. flexneri,S. dysenteriae,S. boydii, andS. sonneiwere assessed to find the most antigenic protein. We retrieved 12 peptides based on the highest score for human leukocyte antigen (HLA) supertypes analysed by NetCTL. Initially, these peptides were assessed for the affinity with MHC class I and class II alleles, and four potential core epitopes VTARAGLGY, FHTVTVNTL, HTTWTLTGY, and IELAGTLTL were selected. From these, FHTVTVNTL and IELAGTLTL peptides were shown to have 100% conservancy. Finally, IELAGTLTL was shown to have the highest population coverage (83.86%) among the whole world population. In vivo study of the proposed epitope might contribute to the development of functional and unique widespread vaccine, which might be an operative alleyway to thwart dysentery from the world.

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Abstract 1033: Simultaneous Clot-targeted Factor Xa Inhibition And Selective Blockade Of Activated GPIIb/IIIa On Platelets Results In Delayed But Potent Antithrombotic Effects Without Bleeding Time Prolongation.

Circulation ◽

10.1161/circ.116.suppl_16.ii_206-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Christoph E Hagemeyer ◽

Steffen U Eisenhardt ◽

Nicole Bassler ◽

Patrick Stoll ◽

Meike Schwarz ◽

...

Keyword(s):

Bleeding Time ◽

Specific Binding ◽

Factor Xa ◽

Single Chain ◽

Occlusion Time ◽

Selective Blockade ◽

Thrombosis Model ◽

Fibrinogen Binding ◽

Antithrombotic Effects

Background: We generated phage-display-derived anti-GPIIb/IIIa single-chain antibodies (e.g. scFv SCE5) that specifically bind to the activated GPIIb/IIIa only and thus specifically block activated platelets only. ScFv SCE5 demonstrates strong antithrombotic potency, comparable to the conformation-unspecific blockers tirofiban and eptifibatide. In contrast bleeding times were not prolonged with scFv SCE5. Here we now use the possibility to add effector molecules using molecular biology methods. The highly potent anticoagulant TAP (tick anticoagulant peptide), which is a direct factor Xa (fXa) inhibitor, was used as an effector molecule. Methods and Results: We genetically fused the activation-specific scFv with TAP, expressed the constructs in E.coli and purified the 39 kDa protein via its Histag binding to Nickel beads. Specific binding of the fusion molecules MA2/SCE5-TAP and strong inhibition of fibrinogen binding was proven in flow cytometry; anti-fXa activity was demonstrated in chromogenic assays. In vivo anticoagulative efficiency was determined by Doppler-flow in a ferric chloride-induced carotid artery thrombosis model in mice. Prolongation in occlusion time with SCE5-TAP was significantly stronger compared to SCE5 alone, recombinant TAP, non-binding mut-scFv-TAP as well as the clinical used drugs enoxaparine and eptifibatide. In contrast to the other anticoagulants tested, bleeding time was not prolonged by SCE5-TAP. Flow experiments studying platelet adhesion on collagen revealed a possible mechanism for the unique finding of a fully normal bleeding time: LIBS exposure on adhering platelets and as such the anticoagulative targeting potency of SCE5-TAP was delayed until considerable layers of platelets were deposited. Conclusions: The combination of activation-specific GPIIb/IIIa blockade and fXa inhibition in one clot-targeted molecule further improves in-vivo antithrombotic efficiency without causing any bleeding time prolongation. The delay of the observed targeting effect may allow a sealing of injuries with platelet layers but may be in time for the prevention of occlusive platelet aggregates. The described blockers represent a new type of highly selective drugs that warrant further clinical development.

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Abstract 54: ML355 in Prevention of Thrombosis in vivo with Minimal Effects on Hemostasis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.54 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Reheman Adili ◽

Katherine Mast ◽

Michael Holinstat

Keyword(s):

Ex Vivo ◽

Platelet Reactivity ◽

Thrombus Formation ◽

Flow Chamber ◽

Human Platelets ◽

Mass Spectrometry Analysis ◽

Vessel Occlusion ◽

Spectrometry Analysis ◽

Thrombosis Model

12-lipoxygenase (12-LOX) has been demonstrated to regulate platelet function, hemostasis, and thrombosis ex vivo , supporting a key role for 12-LOX in regulation of in vivo thrombosis. While pharmacologically targeting 12-LOX in vivo has been a challenge to date, the recent development of the 12-LOX selective inhibitor, ML355, as an effective antiplatelet therapeutic in vivo was assessed. ML355 potently inhibited thrombin and other agonist-induced platelet aggregation ex vivo in washed human platelets and inhibited downstream oxylipin production of platelet 12-LOX as confirmed by Mass spectrometry analysis. Ex vivo flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen was attenuated in human whole blood treated with ML355 to a greater extent compared to aspirin. In vivo , PK assessment of ML355 showed reasonable 12-LOX plasma levels 12 hours following administration of ML355. FeCl 3 -induced injury of the mesenteric arterioles resulted in less stable thrombi in 12-LOX -/- mice and ML355-treated WT mice resulting in impairment of vessel occlusion. Additionally, ML355 dose-dependently inhibited laser-induced thrombus formation in the cremaster arteriole thrombosis model in WT, but not in 12-LOX -/- mice. Importantly, hemostatic plug formation and bleeding following treatment with ML355 were not affected in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model. Our data strongly supports 12-LOX as a key determinant of platelet reactivity in vivo and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapeutics.

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