A mouse bleeding model to study oral anticoagulants

Monroe, Dougald M.; Hoffman, Maureane

doi:10.1016/j.thromres.2014.03.003

Cited by 18 publications

(16 citation statements)

References 18 publications

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“…In humans, mean plasma concentrations of all 3 inhibitors are in the range of 100-600 nM 30,31 . Similar concentrations of dabigatran and 10-100 fold higher concentrations of rivaroxaban have been shown to impair clot formation following tail bleeds in mice 32,33 . Although difficult to predict, the low polarity of the 3 molecules leads us to estimate that the molecules will be poorly absorbed from the media and result in similar tissue levels to those seen in humans 34 .…”

Section: Zebrafish Thrombin Activity Is Inhibited By Direct Oral Antimentioning

confidence: 91%

Disruption of the kringle 1 domain of prothrombin leads to late onset mortality in zebrafish

Grzegorski

Liu

et al. 2020

Sci Rep

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The ability to prevent blood loss in response to injury is a conserved function of all vertebrates. Complete deficiency of the central clotting enzyme prothrombin has never been observed in humans and is incompatible with postnatal life in mice, thus limiting the ability to study its role in vivo. Zebrafish are able to tolerate severe hemostatic deficiencies that are lethal in mammals. We have generated a targeted genetic deletion in the kringle 1 domain of zebrafish prothrombin. Homozygous mutant embryos develop normally into the mid-juvenile stage but demonstrate complete mortality by 2 months of age primarily due to internal hemorrhage. Mutants are unable to form occlusive venous and arterial thrombi in response to endothelial injury, a defect that was phenocopied using direct oral anticoagulants. Human prothrombin engineered with the equivalent mutation exhibits a severe reduction in secretion, thrombin generation, and fibrinogen cleavage. Together, these data demonstrate the conserved function of thrombin in zebrafish and provide insight into the role of kringle 1 in prothrombin maturation and activity. Understanding how zebrafish are able to develop normally and survive into early adulthood without thrombin activity will provide important insight into its pleiotropic functions as well as the management of patients with bleeding disorders.Maintaining blood flow in a closed circulatory system requires a delicate balance between pro-and anticoagulant factors. Disequilibrium of these factors in either direction can lead to pathology. In response to vascular injury, the balance shifts towards coagulation in an effort to stabilize blood clots and prevent exsanguination. Critical to this clot stabilization is the activation of prothrombin, a vitamin K-dependent clotting factor, to form the central clotting enzyme, thrombin 1 . Thrombin cleaves soluble fibrinogen into fibrin monomers, which then polymerize to form the insoluble fibrin clot 2 . Additionally, thrombin interacts with protease activated receptors (PARs) on the surface of various cells, including platelets, which makes it the most potent endogenous agonist of primary hemostasis 3 . Human prothrombin (F2) variants have been linked to both thrombophilia and bleeding diatheses. The most common variant (~2% in European populations) 4 is a guanine to adenine transition in the 3' untranslated region at position 20210 that leads to increased plasma prothrombin levels and a 2-3 fold elevated risk of deep vein thrombosis 5,6 . Congenital prothrombin deficiencies result in a bleeding diathesis, but are rare. Acquired deficiencies due to liver failure or vitamin K deficiency are more common 7,8 .Structurally, prothrombin consists of six domains: signal peptide, propeptide, Gla, kringle 1, kringle 2, and serine protease; a domain composed of a light and heavy chain 1 . Following translation, the propeptide targets the protein for post-translational gamma-carboxylation of the glutamic acid residues within the Gla domain. This vitamin K-dependent process is necessary...

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Section: Zebrafish Thrombin Activity Is Inhibited By Direct Oral Antimentioning

confidence: 91%

Disruption of the kringle 1 domain of prothrombin leads to late onset mortality in zebrafish

Grzegorski

Liu

et al. 2020

Sci Rep

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“…This study utilized the saphenous vein model, which had been used previously to measure the bleeding times in HemA mice . In these studies, untreated HemA mice did not form clots within 15 minutes.…”

Section: Methodsmentioning

confidence: 99%

“…HemA mice (n = 5/condition) were subjected to injury by puncture of the saphenous vein, as described previously. [12][13][14] In this model, the initial clot was disturbed for observation of the secondary (rFVIII-dependent) bleeding times and clot structure, ( Figure 4A). Bleeding times in the HemA mice were greatly prolonged, but reduced by rFVIII ( Figure 4B).…”

Section: Effect Of Fviii Replacement On Hema Wound Clot Structure Amentioning

confidence: 99%

Clot stability as a determinant of effective factor VIII replacement in hemophilia A

Leong

Chernysh

et al. 2017

Research and Practice in Thrombosis and Haemostasis

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Summary Background Factor VIII (FVIII) replacement is standard of care for patients with hemophilia A (HemA); however, patient response does not always correlate with FVIII levels. We hypothesize this may be in part due to the physical properties of clots and contributions of fibrin, platelets, and erythrocytes, which may be important for hemostasis. Objective To understand how FVIII contributes to effective hemostasis in terms of clot structure and mechanical properties Patients/Methods In vitro HemA clots in human plasma or whole blood were analyzed using turbidity waveform analysis, confocal microscopy, and rheometry with or without added FVIII. In vivo clots from saphenous vein puncture in wild-type and HemA mice with varying FVIII levels were examined using scanning electron microscopy. Results FVIII profoundly affected HemA clot structure and physical properties; added FVIII converted the open and porous fibrin meshwork and low stiffness of HemA clots to a highly branched and dense meshwork with higher stiffness. Platelets and erythrocytes incorporated into clots modulated clot properties. The clots formed in the mouse saphenous vein model contained variable amounts of compressed erythrocytes (polyhedrocytes), fibrin, and platelets depending on the levels of FVIII, correlating with bleeding times. FVIII effects on clot characteristics were dose-dependent and reached a maximum at ~25% FVIII, such that HemA clots formed with this level of FVIII resembled clots from unaffected controls. Conclusions Effective clot formation can be achieved in HemA by replacement therapy, which alters the architecture of the fibrin network and associated cells, thus increasing clot stiffness and decreasing clot permeability.

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“…As previously described , mice were anesthetized with isoflurane throughout all procedures. The hair on the ventral side of both hindlimbs was removed.…”

Section: Methodsmentioning

confidence: 99%

Involvement of the heparanase procoagulant domain in bleeding and wound healing

Crispel

Ghanem

Attias

et al. 2017

Journal of Thrombosis and Haemostasis

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Background Heparanase, which is known to be involved in angiogenesis and metastasis, was shown to form a complex with tissue factor (TF) and to enhance the generation of activated factor X (FXa). Our study demonstrated that peptides derived from TF pathway inhibitor (TFPI)-2 impeded the procoagulant effect of heparanase, and attenuated inflammation, tumor growth, and vascularization. Aims To identify the procoagulant domain in the heparanase molecule, and to evaluate its effects in a model of wound healing that involves inflammation and angiogenesis. Methods Twenty-four potential peptides derived from heparanase were generated, and their effect was studied in an assay of FXa generation. Peptides 14 and 16, which showed the best procoagulant effect, were studied in a bleeding mouse model and in a wound-healing mouse model. Results Peptides 14 and 16 increased FXa levels by two-fold to three-fold, and, at high levels, caused consumption coagulopathy. The TFPI-2-derived peptides explored in our previous study were found to inhibit the procoagulant effect induced by peptides 14 and 16. In the bleeding model, time to clot formation was shortened by 50% when peptide 14 or peptide 16 was topically applied or injected subcutaneously. In the wound-healing model, the wound became more vascular, and its size was reduced to one-fifth as compared with controls, upon 1 week of exposure to peptide 14 or peptide 16 applied topically or injected subcutaneously. Conclusions The putative heparanase procoagulant domain was identified. Peptides derived from this domain significantly shortened bleeding time and enhanced wound healing.

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A mouse bleeding model to study oral anticoagulants

Cited by 18 publications

References 18 publications

Disruption of the kringle 1 domain of prothrombin leads to late onset mortality in zebrafish

Disruption of the kringle 1 domain of prothrombin leads to late onset mortality in zebrafish

Clot stability as a determinant of effective factor VIII replacement in hemophilia A

Involvement of the heparanase procoagulant domain in bleeding and wound healing

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