Genetically engineered biologically based hemostatic bioassay.

Abstract

Real-time direct measures of hemostatic parameters in vivo are required for optimizing the dynamic delivery of coagulation modifying pharmacotherapies. Typical sensors of physiologic functions in vivo, however, have only a restricted array of sensory inputs, and thus limited capacity to monitor thrombotic and hemostatic activity. To overcome this limitation we have developed a genetically engineered excitable cell line that can be potentially used for an implantable thrombin biosensor. Specifically, we have generated stem cell-derived cardiac myocyte aggregates overexpressing the human thrombin receptor, protease activated receptor-1 (PAR-1), which exploit the inherent electropotential input-output relationship of the cells to detect local changes in thrombin activity. In vitro, the signaling activity of PAR-1 cardiac myocytes was highly responsive to thrombin, inducing a sixfold increase in intracellular cAMP as compared with a twofold increase in control cells. In vivo, the engineered myocytes also detected alterations in local coagulation potential. Specifically, PAR-1 engineered cells implanted in vivo detected local increases in thrombin with a doubling in chronotropic activity compared with a 50% increase in control aggregates. Overall these studies demonstrate the potential of genetic engineering to expand the physiologic signals recognized by excitable cells, and may facilitate the translation of this approach for the real-time monitoring of hemostatic function in vivo.