Overview: Horses with severe inflammatory disorders, including pneumonia, colitis, and equine herpes virus infection, often form blood clots that block the circulation and deprive tissues of oxygen. In these diseases, blood clot formation typically occurs in vessels supplying the lungs, intestine, spinal cord, and placenta, and is undetectable by physical examination or diagnostic imaging, such as X-rays and ultrasound. Thus, clots often go unrecognized and horses are not given appropriate treatment until extensive tissue damage (infarction) has occurred. Infarction of affected organs in the above mentioned diseases causes pain and clinical signs of organ failure and is closely associated with mortality. Surviving horses sustain residual tissue injury that often prevents their return to optimal athletic and reproductive performance. Since abnormal clot formation (or thrombosis) complicates many equine inflammatory diseases and directly affects prognosis, veterinarians need a better understanding of why these clots form and better methods of identifying those horses at risk of developing clots. We hypothesize that excessive activation of coagulation occurs in these diseases because of the over-expression of a specific coagulation factor called tissue factor (TF) on white blood cells. Tissue factor, also known as coagulation factor III or tissue thromboplastin, is normally absent from these cells, but it appears on their surface in response to inflammatory or infectious diseases. In this proposal, we plan to test our hypothesis of TF-induced thrombosis in two diseases commonly associated with abnormal clot formation: shipping pneumonia and acute colitis. We can already measure TF activity on equine white cells and are currently developing a novel assay to specifically measure equine TF in plasma. We will use these assays to monitor and hopefully predict the process of clot formation in diseased horses.

Horses affected with shipping pneumonia and acute colitis will be recruited from the Cornell University Hospital for Animals Equine Hospital clientele during a 2-year study period. After obtaining owners' consent, a blood sample will be collected from each affected horse at the same time routine diagnostic samples are collected. Patients will be monitored after admission for evidence of clotting and disease outcome (survival or death).

The results of this project will be applicable for the early detection of horses with inflammatory diseases that are at risk of clotting and, ultimately, the development of new anti-clotting treatment strategies. New drugs aimed at preventing clot formation are critically needed, because currently available treatments (e.g. aspirin or heparin) do not prevent thrombosis and are thus not included in the treatment protocol for these sick horses.

Aim: Measurement of monocyte and plasma tissue factor expression in affected and healthy horses
To determine if TF levels are increased in horses with shipping pneumonia and colitis, we will isolate a particular white blood cell, the monocyte, from equine blood samples. Monocytes are believed to be the primary initiators of abnormal clotting in human patients with inflammatory syndromes, because monocytes express TF on their cell surface after exposure to bacterial toxins. We will measure the amount of cell-surface TF expression on these isolated monocytes using a color-based clot activation assay, already developed in our laboratory. Activated monocytes also shed TF into the plasma, therefore measurements of plasma TF are required for a full assessment of TF's role in clotting. Furthermore, plasma TF values may help us predict which horses are likely to suffer from clots. Available assays used to measure plasma TF in humans do not detect equine TF. Thus, we are in the process of developing an equine-specific plasma TF assay, by generating and using specific antibodies that recognize the horse TF protein. This protein will be obtained through cloning the equine TF gene. We have already cloned a partial fragment of the equine TF gene and are in the process of attaining the complete TF sequence. We will then express and purify recombinant equine TF and immunize rabbits to obtain an antibody specific for horse TF. We predict that monocyte and plasma TF in horses with shipping pneumonia and colitis will be higher than that of healthy control horses. If this is true, we will determine whether novel anti-TF drugs can prevent thrombosis in horses with these and other inflammatory diseases in future clinical trials.