Animal Models 2007 SSC Subcommittee Minutes

The focus of the session was to give attendants a review of emerging technology and animal models relevant to thrombosis and hemostasis. The session addressed three themes:

Session 1. Genetic manipulation of hemostasis in mice

Dr. Degen reviewed available mouse strains with altered function of key hemostatic factors, thrombin and fibrinogen. In addition to loss-of-function models knockouts for prothrombin and fibrinogen, several mutations have been introduced into the fibrinogen gene cluster to abolish specific interactions of fibrinogen with integrins that are relevant in inflammation and infection. Novel models included mice with selective expression of Aalpha-chain isoforms (“long” and “short”), and a “non-clottable” form of fibrinogen that lacks the thrombin cleavage sites for fibrinopeptide removal. This model will be instrumental in dissociating functions of fibrinogen, as compared to fibrin. A novel approach was presented to delete the prothrombin gene in a temporally and spatially regulated manner. This model allows an investigator to temporally induce a state of almost complete prothrombin deficiency in adult mice.

Dr. Conway provided an excellent overview of approaches to alter gene functions in a cell type-restricted manner in endothelial cells. He introduced experimental concepts that will allow not only endothelial cell-specific manipulation of gene expression, but in addition target the endothelium of specific organs, in particular the brain. An important aspect of the presentation was to emphasize that several approaches to achieve endothelial cell-selective gene expression/inactivation also affect bone marrow-derived cells.

Dr. Coughlin focused on the role of protease activated receptors in platelet function, i.e. Par4, and gave a comprehensive summary of available data validating Par4 function in platelet-driven hemostasis and thrombosis in mice, as compared to humans.

Dr. Mackman reviewed existing data from the analysis of mice expressing lower or higher amounts of TF in different organs. Findings emphasize the concept of organ-selective functions of tissue factor, and of an organ-selective balance of hemostasis.

Dr. Isermann addressed experimental approaches and use of animal models to study the role of altered hemostais in chronic, as opposed to acute models of disease. This concept was illustrated in the paradigms of atherosclerosis and of the role of the protein C pathway in diabetes. This presentation stressed the value of animal models that introduce –as compared knockout models- more subtle alterations in the hemostatic balance that more closely mimic the situation in human populations.

Session 2. Coagulation – inflammation axis

Dr. Ploplis reviewed published data from a set of transgenic mice expressing various levels of protein C. These animals, as opposed to complete knockouts for protein C, are viable but exhibit numerous derangements causing spontaneous thrombosis and inflammation, and cause pregnancy failure.

Dr. Lupu gave an overview over models of septic inflammation and DIC in Baboons. These models have been exploited to examine the function of the protein C pathway in inflammation. An important outcome of these studies is the recognition that inflammatory disorders such as sepsis with or without DIC comprise several different pathogenic mechanism in different stages of disease. In contrast to mouse models, Baboons can be analyzed with reagents/methods developed for, and applicable to humans, because of evolutionary conservation of proteins and pathways.

Dr. Nichols gave an overview over the use of several pig breeds in research relevant to atherosclerosis, and an update on technological aspects of large vessel function analysis in these animal models.

Session 3. Analytical tools in rodents

Dr. Ruf presented data from experiments in mice using pharmacologic reagents to manipulate coagulation in settings of inflammation (sepsis/LPS); and dissect the role of coagulation and coagulation receptors in hemostasis as compared to cell signaling. An important outcome of these studies in mice was that coagulation activation appears to make only a minor contribution to the inflammatory derangements in mouse models of inflammation triggered by LPS.

Dr. Poncz reviewed strategies to manipulate and target gene expression in murine platelets. Using the paradigm of fVIII gene delivery to platelets and correct hemophilia, analytical methods were discussed to monitor functional outcomes of gene manipulation, such as in vivo imaging of platelet thrombus formation and measurement of bleeding times.

Dr. Smyth could not attend. The theme of her presentation was scheduled to be the use of genetic analysis to delineate novel traits relevant to thrombosis and hemostasis. This topic will be the lead theme for the coming sessions of the Animal model SSC.

The session was extremely well attended. The Chairs / Cochairs came to the consensus that similar sessions with an educational focus on technology will be valuable in future sessions during regular ISTH meeting years.