Genetic Elimination of Prothrombin in Adult Mice Results in Fatal Spontaneous Hemorrhage in the Heart and Central Nervous System
Authors: Mullins Eric S. , Kombrinck Keith W. , Talmage Kathryn E. , Shaw Maureen A. , Witte David P. , Ullman Joni M. , Degen Sandra J. , Sun William , Flick Matthew , Degen Jay 
Published: April 2020

Abstract
Thrombin-mediated proteolysis is central to hemostasis, directly controlling both platelet activation and fibrin deposition as well as positively- and negatively-regulating further thrombin generation through the activation of factors XI, VIII, V and protein C. Thrombin also appears to be important in multiple processes distinct from traditional hemostasis, but detailed genetics-based studies have been impeded by the uniform embryonic and perinatal failure of mice with constitutive prothrombin (fII) deficiency. To develop an experimental setting to explore the importance of fII in vascular biology, tissue repair, the inflammatory response and disease processes in adult animals, we have generated mice carrying a conditional prothrombin knockout allele (fIIlox). In the absence of Cre-mediated recombination, homozygous fIIlox/lox mice or compound heterozygous mice carrying one fIIlox allele and one constitutive-null allele were found to carry ~20% and ~10% the normal level of circulating prothrombin, respectively. These fIIlox/lox and fIIlox/− mice exhibited normal developmental and reproductive success, survived well into adulthood and young adults exhibited no appreciable spontaneous bleeding events or other pathologies. However, one-year-old fIIlox/− mice developed modest focal hemosiderin deposits and fibrosis within the heart, consistent with chronic low grade hemorrhage in this tissue. No gross or microscopic pathologies were observed in any other tissue examined in mice carrying an intact conditional knockout allele, regardless of age or gender. The induction of Cre recombinase in adult fIIlox mice using the poly I:C-inducible Mx1-Cre system resulted in the rapid and near-complete recombination of the fIIlox allele within the liver, loss of hepatic fII mRNA, elimination of detectable circulating prothrombin, and profound derangements in coagulation function. The life-expectancy in adults genetically-depleted of prothrombin was found to be very short (generally 5–7 days), and the loss of viability was associated with the development of severe hemorrhagic events within multiple tissues, most prominently in the heart (100% phenotypic penetrance) and brain (~50% phenotypic penetrance). Microscopic analysis of hearts from mice following deletion of the fII allele revealed widespread hemorrhage within the myocardium, particularly in the subepicardial region, focal ischemia and necrosis, neutrophil infiltrates and early granulation tissue. Gross evidence of blood was observed within the pleural cavity in nearly half of Cre-induced fIIlox/− mice at autopsy, and the lack of appreciable hemorrhage within lung tissue favored the heart as the source of this free blood. Less common hemorrhagic events were observed several other tissues, including skeletal muscle, intestines and testes. Examination of brains from mice lacking fII revealed widespread bleeding in the central nervous system, including dural-based hemorrhage and bleeding into both the brain parenchyma and ventricles. These results imply that a robust hemostatic system is essential to limit spontaneous bleeding events in tissues under repetitive mechanical or pulsatile stress. These findings also demonstrate that low levels of fII are compatible with long-term survival in adult mice, affording the ability to examine fII in a diverse spectrum of disease and physiologic processes. As evidence of this concept, mice lacking fII were challenged with S. aureus peritonitis prior to the onset of hemorrhage. Similar to mice lacking fibrinogen, these animals were found to have a profound deficit in their ability to control this infection, pointing to the need for polymerized fibrin for the effective clearance of this bacteria from the peritoneal cavity.