Loss of Clotting Function in Mice Expressing a Mutant Form of Fibrinogen Lacking the α Chain Thrombin Cleavage Site
Authors: Flick Matthew , Ullman Joni M. , Palumbo Joseph S. , Mullins Eric S. , Kombrink Keith W , Talmage Kathryn E. , Shaw Maureen A. , Lord Susan T. , Degen Jay , Gorkun Oleg V 
Published: April 2020

The conversion of soluble fibrinogen to an insoluble fibrin matrix following thrombin cleavage of the Aα and Bβ chains is critical to hemostasis. However, even soluble fibrinogen holds potential biological significance apart from fibrin polymer formation; for example, fibrinogen contributes significantly to overall blood rheology and can engage a number of integrins (e.g., αIIbβ3), enzymes (e.g., fXIII) and matrix proteins (e.g. fibronectin). To provide a system to study mice carrying fibrinogen but no capacity for fibrin formation, we employed a gene-targeting strategy to eliminate the Aα chain thrombin cleavage site and replace it with a sequence recognized by the highly-selective alternate protease, enterokinase (P6-P1: ADDDDK). Based on the prevailing view that thrombin cleavage of the Aα chain is essential to polymer formation, we hypothesized that this mutant fibrinogen, termed FibEK, would be locked in the soluble form in vivo, but would be readily clotted in vitro by exogenous enterokinase. The FibEK allele was transmitted through the germline and supported the expected level of mRNA expression and plasma protein production. Like previously established fibrinogen-null mice, FibEK mice uniformly developed to term. However, survival beyond the perinatal period was vastly superior in mice carrying fibrinogen-EK relative to fibrinogen-null animals in the same (C57Bl/6) genetic background; ~90% of FibEK mice survive to adulthood, whereas less than 30% of fibrinogen-null mice survive the perinatal period. Consistent with our initial hypothesis, FibEK plasma was found to be unclottable following the addition of excess exogenous murine thrombin at 37°C and complementary fibrinopeptide release assays by HPLC revealed that murine thrombin is completely incapable of releasing fibrinopeptide A (FpA) from fibrinogen-EK. Murine thrombin did support quantitative FpB release from fibrinogen-EK, albeit at a slower rate than wild-type fibrinogen. No thrombin-induced fibrin polymer formation could be appreciated by standard turbidity assays in incubation mixtures containing FibEK plasma at 37°C, even after 24 hrs. In contrast, wild-type plasma supported rapid polymer formation, with turbidity peaking within minutes of thrombin addition. Interestingly, despite the complete absence of FpA release, both plasma and purified fibrinogen-EK supported the formation of small and irregular clots (characterized by thin fibrils in scanning EM) following long incubations at reduced temperatures (i.e., 24 hr at 22 °C). Unlike fibrinogen-null mice, platelet-rich plasma prepared from FibEK mice supported robust ADP-induced platelet aggregation. However, FibEK animals essentially phenocopy fibrinogen-null mice in their inability to efficiently clear the microbial pathogen S. aureus. Following intraperitoneal infection with 109 CFU of S. aureus, control mice successfully cleared &gt;99% of the bacteria within 1 hour, whereas the number of bacteria retrieved within peritoneal lavage fluid of both FibEK and FibAα−/− mice remained similar to the original input CFU. In summary, FibEK mice provide a unique opportunity to further explore the biochemistry of thrombin-mediated clot formation in vitro and in vivo. These animals will also be a vital tool in defining the specific contribution of fibrin matrices to broad array of physiological and pathological processes in vivo.