John Sheehan, MD

Research Interests

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.

Selected Publications

• Sheehan, J.P., Wu, Q.Y., Tollefsen, D.M., and Sadler, J.E. (1993). Mutagenesis of Thrombin Selectively Modulates Inhibition by Serpins Heparin Cofactor II and Antithrombin III: Interaction with the Anion-Binding Exosite Determines Heparin Cofactor II Specificity. J. Biol. Chem. 268:3639-3645 (Abstract)
• Sheehan, J.P. and Sadler, J.E. (1994) Molecular Mapping of the Heparin Binding Exosite of Thrombin. Pro. Natl. Acad. Sci. USA 91:5518-5522 (Abstract)
• Sheehan, J.P., Tollefsen, D.M. and Sadler, J.E. (1994) Heparin Cofactor II is Regulated Allosterically and Not Primarily by Template Effects: Studies with Mutant Thrombins and Glycosaminoglycans. J. Biol. Chem. 269:32747-32751(Abstract)
• Sheehan, J.P. and Lan, H.C. (1998) Phosphorothioate Oligonucleotides Inhibit the Intrinsic Tenase Complex. Blood 92:1617-1625 (Abstract)
• Jagadeeswaran, P., Liu, Y.C., and Sheehan, J.P. (1998) Analysis of Hemostasis in the Zebrafish. Methods Cell. Biol. 59:337-358 (Abstract)
• Jagadeeswaran, P. and Sheehan, J.P. (1999) Analysis of Blood Coagulation in the Zebrafish. Blood Cells, Molecules, and Diseases 25:239-249 (Abstract)
• Jagadeeswaran, P., Sheehan, J.P., Craig, F., and Troyer, D. (1999) Identification and Characterization of Zebrafish Thrombocytes. Br J Haematol. 107(4):731-738) (Abstract)
• Sheehan, J.P. and Phan, T. (2001) Phosphorothioate Oligonucleotides Inhibit the Intrinsic Tenase Complex by an Allosteric Mechanism. Biochemistry 40(16): 4980-4989 (Abstract)
• Sheehan, J.P., Templer, M., Gregory, M., Hanumanthaiah, R., Troyer, D., Phan, T.M., Thankavel, B., and Jagadeeswaran, P. (2001) Demonstration of the Extrinsic Coagulation Pathway in Teleosti: Identification of Zebrafish Coagulation Factor VII. Pro. Natl. Acad. Sci. USA 98:8768-8773 (Abstract)
• Sheehan, J.P., Kobbervig, C.E., and Kirkpatrick, H.M. (2003) Heparin Inhibits the Intrinsic Tenase Complex by Interacting with an Exosite on Factor IXa. Biochemistry 42(38): 11316-11325 (Abstract)
• Yuan, Q.P., Walke, E.N., and Sheehan, J.P. (2005) The Factor IXa Heparin-Binding Exosite is a Cofactor Interactive Site: Mechanism for antithrombin-independent inhibition of intrinsic tenase by heparin. Biochemistry 44(9): 3615-3625 (Abstract)
• Sheehan, J.P. and Walke, E.N. (2006) Depolymerized Holothurian Glycosaminoglycan and Heparin Inhibit the Intrinsic Tenase Complex by a Common Antithrombin-Independent Mechanism. Blood 107(10):3876-82. PMCID: PMC1895295 (Abstract)
• Misenheimer, T.M., Buyue, Y., and Sheehan, J.P. (2007) The Heparin-binding Exosite is Critical to Allosteric Activation of Factor IXa in the Intrinsic Tenase Complex: The role of arginine 165 and factor X. Biochemistry 46(26):7886-95. PMCID: PMC2569175 (Abstract)
• Buyue, Y., Whinna, H., Sheehan, J.P. (2008) The Heparin-Binding Exosite of Factor IXa is a Critical Regulator of Plasma Thrombin Generation and Venous Thrombosis. Blood, 112(8): 3234-3241. [Epub Jul 22, 2008]. PMCID: PMC2569175 (Abstract)
• Buyue, Y., and Sheehan, J.P. (2009) Fucosylated Chondroitin Sulfate Inhibits Plasma Thrombin Generation via Targeting of the Factor IXa Heparin-Binding Exosite. Blood 114 (14) 3092-100 [Epub May 4, 2009]. PMCID: PMC2756211 (Abstract)
• Misenheimer, T.M. and Sheehan, J.P. (2010) The Regulation of Factor IXa by Super-sulfated Low Molecular Weight Heparin. Biochemistry 49(46):9997-100005 [Epub Oct. 27, 2010] PMID: 20945941 (Abstract)
• Matafonov A, Sarilla S, Sun MF, Sheehan JP, Serebrov V, Verhamme IM, Gailani D. (2011) Activation of factor XI by products of prothrombin activation. Blood. 2011 Jul 14;118(2):437-45. Epub 2011 Apr 28 (Abstract)
• Buyue, Y., and Sheehan, J.P. (2012) Low molecular Weight Heparin Inhibits Thrombin Generation via Direct Targeting of Factor IXa: Contribution of the serpin-independent in human plasma. Journal of Thrombosis and Haemostasis Oct;10(10):2086-98 PMCID:  PMC3463736 (Abstract)
• Buyue, Y., Misenheimer, T.M., and Sheehan, J.P. (2013) Low Molecular Weight Heparin Inhibits Thrombin Generation via Direct Targeting of Factor IXa: A Reply to Rebuttal.  Journal of Thrombosis and Haemostasis Mar;11(3):565-6. PMID: 23332108 (Abstract)
• Geng, Y., Verhamme, I.M., Smith, S.A., Cheng, Q., Sun, M., Sheehan, J.P., Morrissey, J.H.,  and Gailani, D. (2013) Factor XI Anion-Binding Sites are required for Productive Interactions with Polyphosphate. Journal of Thrombosis and Haemostasis Nov;11(11):2020-8.  PMID: 24118982 (Abstract)