Animal, Cellular, and Molecular Models of Thrombosis
DRAFT Subcommittee Minutes
23 July 2011
14:05-18:00
Annex Hall 2
Chairman: Timothy Nichols (US)
Co-chairmen: Edward M. Conway (CA), Shaun R. Coughlin (US), Jay L. Degen (US), Cecile Denis (FR), Nigel Mackman (US), Toshiyuki Miyata (JP), Eva-Maria Muchitsch (AT), Susan S. Smyth (US), Hugo ten Cate (NL), Hartmut Weiler (US)
Educational Session
1. Cecil Denis - Rapid in vivo evaluation of haemostatic agents using hydrodynamic injection
Dr. Denis discussed the important methodological issues, strengths and limitations of using hydrodynamic injection to evaluate wild type and mutant hemostatic agents in the circulation (ADAMTS13, VWF, FVIII, FIX, alpha1 antitrypsin), transmembrane proteins (e.g., Siglec-5 in the life cycle of the FVIII-VWF complex), as well as SiRNA applications. This powerful tool is being broadly applied to achieve short-term expression (or reduced expression) of relevant proteins for in vivo characterization and mechanistic studies.
2. Susan Smyth - Models of vascular inflammation. Dr. Smyth discussed the aortic arch ligation model of inducing left ventricular hypertrophy and the associated inflammatory-mediated coronary artery remodeling used in various strains of mice. The important methodological issues were reviewed (e.g. standardization of extent of ligation, inclusion of a sham-operated control). Remarkably, after placement of the ligature, platelets accumulate along the coronary endothelial surface at sites of subendothelial macrophage deposition. Mechanisms mediating these processes are being identified by infusion of anti-platelet Gp1b antibodies and utilization of relevant knock out mice (e.g., Jnx mice with decreased granule secretion in NK, CTL, platelets, and neutrophils). This ongoing work addresses the role of inflammation and coagulation in a very common and debilitating disorder (LVH, diastolic heart failure) that is associated with a significantly increased mortality rate in humans.
3. David Motto - Scanning electron microscopy studies of endothelial injury and thrombus formation in mouse models of thrombosis." Dr. Motto presented an elegant series of SEM taken over the first few minutes following application of ferric chloride (0.37 M, 10%) to murine carotid arteries. Remarkably, endothelium has not sloughed off during these early timepoints and red blood cells appear to be one of the first adherent cells by unknown mechanisms. These RBCs then elongate in the direction of blood flow and form a ruffled boarder at the attaching edge and eventually slough off leaving the ruffled-edge fragment behind. In addition, amorphous (possibly proteinaceous) material begins to envelop the collection of adherent cells and cell fragments. Then, platelets begin to attach to this growing clump of adherent cellular and amorphous debris. This work focuses on understanding the mechanisms that mediate these events and determining their relevance to clinical arterial thrombotic disorders.
Focused talks
1. Tom Knudsen – Characterization of canine coagulation factor VII and its complex formation with tissue factor: canine–human cross-species compatibility. Dr. Knudsen presented the very recent data that validate using canine models to study human FVIIa in vivo. The essential point is that canine TF binds human and canine FVIIa in a comparable manner however human tissue factor does not bind canine FVIIa efficiently. Thus, infusing human or the recently produced canine FVIIa into dogs is a valid approach for the study of TF-FVIIa coagulation events in vivo. The recent and important reports of coating of platelets by canine FVIIa and canine FVII clot and antigen assays were discussed. The role of canine TF in signaling and it comparison to human TF signaling is a future avenue of research.
2. David Lillicrap - Mouse models of FVIII immunogenicity. Dr. Lillicrap presented the rationale for developing these models and exploring the varying immunogenicity noted among FVIII products. Notably, humans with hemophilia A have different prevalence of inhibitors when treatment consists of single donor, pooled plasma, and recombinant FVIII. The goals are to develop an animal model for identifying mechanisms that mediate immunogenicity, to identify mechanisms that mediate tolerance, and to test the immunogenicity of new formulations of FVIII. The relative immunogenicity of FVIII with and without VWF was studied, an array of ~1100 immune response genes has been utilized to characterize this response, and mice with varying degrees of humanization of the immune system have been created to facilitate this work. These tools are acquiring broad utilization in research focused on the development, treatment, and prevention of inhibitor formation in hemophilia A.
3. Nigel Mackman - Measurement of a procoagulant state in mice. Dr. Mackman discussed the role of procoagulant states in mice induced by endotoxin, sepsis, colitis, sickle cell disease, anti-phospholipid antibodies, heparin induced/associated thrombocytopenia, and cancer. The importance of standardized animal models and detection assays between labs was emphasized. Six assays currently available for detecting and characterizing mechanisms that mediate procoagulant states were reviewed: 1. Microparticle-TF activity, 2. The Zymuphen MP activity kit, 3. The calibrated automatic thrombogram (CAT) assay), 4. Enzygnost Thrombin-antithrombin (TAT) assay, 5. Fibrin western blot, and 6. D Dimer (Asserochrome). The method and quality of obtaining blood samples from the animals cannot be overemphasized.
4. Hugo ten Cate - Thrombin and atherosclerosis: effects on plaque morphology. Dr. ten Cate reviewed the essential role of thrombin generation in hemostasis and the pathophysiology in thrombosis. Refinements of mouse models of atherosclerosis are yielding insights into mechanisms that produce a vessel wall localized hypercoagulable state and mediate thromboatherosclerosis, angiogenesis in atherosclerotic plaques, and the role of inflammation and plaque instability in thrombosis complicating atherosclerosis. These refinements will guide future clinical translational research.
5. Fumiaki Banno - Genetic mouse models for evaluating pathophysiological roles of ADAMTS13. Dr. Banno reviewed mouse models of polymorphisms and mutations in ADAMTS13 and their role in thrombosis and TTP. Differential effects of polymorphisms of ADAMTS13 alter the outcome of middle cerebral artery occlusion and reperfusion injury. These models provide the tools to test the hypothesis that hyperactive VWF is present with the different forms of ADAMTS13 and mediate thrombosis.
6. Eva-Maria Muchitsch - Animal models for TTP: Development of a mouse model of TTP for preclinical efficacy testing of rADAMTS13. Dr. Muchitsch reviewed the history of botrocetin, antibody, and sepsis –induced animal models of TTP. The current mouse models with ADAMTS13 knocked out and infused with human recombinant VWF have reproduced the constellation of findings in TTP (save possibly fever). One somewhat unusual finding in mice was heart hemorrhage, a finding reported in some human cases. Current work is focusing on validating the clinical scenarios that recombinant ADAMTS13 will prevent or mollify induction of TTP and will likely provide justification for human clinical trials.
7. Jianglin Fang - Transgenic rabbit models for the study of atherosclerosis. Dr. Fang reviewed the rationale for using transgenic rabbits in atherosclerosis studies: sensitivity to high cholesterol diets, rapid development of atherosclerosis, development of plaques with many human-like features, and the seminal development of the Wantanabe heritable hyperlipidemic (WHHL) rabbit by Dr. Yoshio Watanabe. Transgenic rabbits producing Lp(a) and CRP have been developed by Dr. Fang and provided important tools for determining whether these molecules are causative or consequence of atherogenesis. The rabbit remains an important and powerful tool in atherosclerosis research.
8. Hartmut Weiler - In vivo function of human aPC in mouse models of thrombosis and inflammation. The role of aPC in placental development, sepsis outcome, and preventing thrombosis was reviewed. Dr. Weiler raised the question of whether or not all these effects were sole due to aPC or were other factors involved. He presented data on hyperactivatable aPC and the action of aPC on histones that will be presented later at this meeting.
9. Timothy C. Nichols – Animal Models Committee Business Meeting. Progress in publishing proposed short papers and the development of projects was discussed. Two of three papers proposed at the 56th annual SSC meeting have been published (or accepted for publication) and a third will be ready for submission soon.1-3 The proposed project that would provide a standardized mouse tail bleeding time method was suggested by the manuscript below and the Poncz and Muchitsch laboratories are discussing an approach to this project.
Publications
- Greene TK, Schiviz A, Hoellriegl W, Poncz M, Muchitsch EM. Towards a standardization of the murine tail bleeding model. J Thromb Haemost. 2010;8:2820-2822.
- Owens AP, Lu Y, N. M. Evaluation of the Mouse Model of Ferric Chloride-induced Carotid Arterial Thrombosis. J Thromb Haemost. 2011;Accepted.
- Nichols TC, Franck HWG, Franck C, Raymer RA, Merricks EP. Sensitivity of Whole Blood Clotting Time and Activated Partial Thromboplastin Time for Canine Factor IX. J Thromb Haemost. 2011;To be submitted.
Project being planned: Validation of a standardized mouse tail bleeding time method.
