John P Sheehan, MD
600 Highland Avenue, Box: 5669
My laboratory is interested in molecular mechanisms of coagulation and thrombosis, with emphasis on regulation of the coagulation serine proteases, and identification of novel targets for antithrombotic therapy (Figure 1). Currently, the laboratory is focused on regulation of the intrinsic tenase complex (factor IXa-factor VIIIa) (Figure 2). In vitro and ex vivo modeling of tissue factor-initiated blood coagulation suggests that this enzyme complex is the rate-limiting step for thrombin generation in response to injury. Animal studies suggest that targeting this enzyme complex may improve the bleeding risks associated with antithrombotic therapy. The activated form of factor IX is a protease that is poorly reactive with substrates and inhibitors in solution, yet undergoes a dramatic 106-fold enhancement in catalytic efficiency for factor X upon incorporation into the intrinsic tenase complex. The regulation of this remarkable enzyme within the intrinsic tenase complex is being addressed by a combination of site-directed mutagenesis, enzymatic and biochemical characterization of mutant proteases; evaluation of effects on plasma thrombin generation and injury models in hemophilia B mice. We have identified a critical cofactor interactive site involved in protease activation, which regulates the stability of the enzyme complex, and represents a novel molecular target for antithrombotic therapy (Figure 3). This novel target is being pursued by a combination of high throughput screening of chemical libraries and molecular modeling approaches. The results of these studies are relevant to understanding the therapeutic efficacy of heparin, the development of novel antithrombotic strategies, and the design of improved replacement therapy for hemophilia B.
My laboratory is also interested in the zebrafish as a genetic model for mammalian hemostasis(Figure 4). The zebrafish is a powerful model for vertebrate genetics, allowing saturation mutagenesis with phenotypic screening and specific gene knockdown approaches. The major hemostatic pathways are conserved between zebrafish and humans, which allows for identification of novel hemostatic genes and functions. We have identified the zebrafish homologue for human protein C, a protease with both anticoagulant and anti-inflammatory properties. Treatment with activated protein C demonstrates a survival advantage for selected patients with sepsis, however, the potential benefits and mechanisms for the anti-inflammatory effects of this protease remain controversial. We are characterizing the activities of the zebrafish homologue both in vitro, and in inflammation models in the zebrafish. Identifying the downstream effectors of activated protein C is important for understanding the interface between hemostatic and inflammatory pathways.
"Molecular models of the procoagulant factor VIIIa-factor IXa complex." J Thromb Haemost. 2005 Sep;3(9):2044-56.
NIH T32 HL007899, Principal Investigator, Research Training in Hematology, 07/1/03-1/31/2019
Access to Insight Basic Research Grant 2011, Novo Nordisk 11/01/11-10/31/12
Bayer Hemophilla Special Project Award, Bayer Corporation. 10/01/11-9/30/13