Mice with Genetic Modifications in Prothrombin Limiting Activation Cleavage Events to Meizothrombin Survive to Adulthood, but Exhibit Alterations in Hemostasis and Thrombus Formation

Abstract 496

Thrombin-mediated proteolysis is the central event not only of hemostasis and thrombosis but also in distinct physiological and pathological contexts such as development, inflammation, cancer biology, and cardiovascular disease. The proteolytic conversion of prothrombin to α-thrombin (fIIa) by the prothrombinase complex occurs through two possible pathways: i) the inactive intermediate termed prethrombin 2, or ii) the proteolytically active two-chain intermediate termed meizothrombin (fIIa^MZ). fIIa^MZ, unlike fIIa, retains the γ-carboxyglutamic acid-rich gla domain and two kringle domains of prothrombin and has distinct catalytic properties relative to fIIa that could be biologically meaningful in vivo, including a diminished capacity to cleave fibrinogen and a significantly increased capacity to activate protein C in the presence of thrombomodulin. Here, the endogenous prothrombin gene was modified in mice to better explore the properties of fIIa^MZin vivo and to test the hypotheses that mice carrying a mutant form of prothrombin with a terminal activation product of fIIa^MZ will: 1) develop to term and survive to adulthood despite an altered hemostatic profile; and 2) exhibit significantly dampened inflammatory responses due to enhanced protein C activation. In order to limit cleavage events to the single site yielding fIIa^MZ, alterations were introduced into the endogenous prothrombin gene resulting in three amino acid substitutions (R157A, R268A, and K281A) located at the P1 positions of potential fXa or autocatalytic cleavage sites. Unlike prothrombin-null mice, mice homozygous for these mutations (hereafter referred to as fII^MZ mice) were found to be present at weaning age and viable into adulthood. Furthermore, unlike mice with a major deficit in either clotting function (e.g., fibrinogen-null) or platelet function (e.g., Gαq-null), fII^MZ females were capable of successfully carrying a litter to term. However, analysis of over 700 progeny generated from crosses of heterozygous fII^MZ/WT mice revealed that only about half of the number homozygous fII^MZ mice expected, based on Mendelian transmission rates, were observed in weaning-age offspring. Complementary studies suggest that the fraction of fII^MZ offspring that fail are lost primarily in utero, but occasional post-partum failures were observed associated with hemorrhagic events. Successful adult fII^MZ mice were found to have similar prothrombin mRNA levels and equivalent fII protein expression to fII^WT mice. Furthermore, fII^MZ animals exhibited normal complete blood cell counts and predictably normal thrombin times, but prolonged PTs and aPTTs. Thrombin generation assays revealed a prolonged time to peak thrombin production, but no significant difference in peak thrombin levels. Consistent with these findings, tail bleeding times in fII^MZ mice were significantly prolonged. None of the fII^MZ mice assayed achieved hemostasis within the 10 minute observation window, whereas fII^WT mice all stopped within 2 minutes of challenge. More sophisticated comparative studies of thrombus formation in mesenteric arterioles following FeCl₃ injury using a real-time intravital microscopy approach established that the time to first thrombus formation in fII^MZ mice was almost twice that of wildtype animals. The majority of the fII^MZ mice failed to occlude the injured vessel within a 25 min observation window, whereas all fII^WT mice successfully formed occlusive thrombi with a mean time of 12.5 minutes. In summary, site-directed alterations of the endogenous prothrombin gene in mice leading to a terminal prothrombin activation product of meizothrombin were found to be compatible with development, growth to adulthood and reproductive success, albeit with modified hemostatic function. Based on the catalytic properties of fIIa^MZ favoring protein C activation, studies are underway to compare APC generation in control and fII^MZ mice and explore the theory that physiological and pathological inflammatory responses will be dampened in fII^MZ mice.