CVI Program Unit(s):
Thrombosis / Hemostasis
CVI Research Description:
The studies in my laboratory focus on the molecular mechanisms which are responsible for maintaining normal hemostasis. The generation of thrombin, a key enzyme involved in blood coagulation, is central to this process as inappropriate production can lead to hemorrhage or thrombosis. The enzyme responsible for thrombin generation is prothrombinase, a macromolecular enzyme complex composed of the serine protease FXa, the nonenzymatic cofactor FVa, and calcium ions all bound to a membrane surface. The protein components of prothrombinase circulate in plasma as inactive precursors (factor X and factor V).
Our research is directed toward better understanding
1) how factor Va functions within prothrombinase;
2) how proteolytic activation of the zymogen factor X facilitates the expression of structural determinants (i.e. exosites, metal binding sites, active site, etc.) important to its function in prothrombinase and
3) which structural elements on factor V maintain the protein as an inactive procofactor.
Recombinant variants of factor X and factor V are used along with a combination of equilibrium binding studies employing fluorescence and thermodynamic techniques, kinetic studies of the proteolytic reactions, and protein structural studies to approach these problems. The mechanisms defining these processes are poorly defined, despite their physiologic importance and new insights into these processes may aid in the development of exosite-based FXa or FVa inhibitors capable of attenuating the abnormal hemostatic processes in thrombophilic disease states.
One line description of research:
Our research is directed towards investigating the molecular basis for the expression of functional binding sites on procofactors and zymogens involved in blood coagulation following discrete proteolysis.
Keywords: serine proteinase, recombinant protein expression, blood coagulation, macromolecular enzyme complex.
Description of Research:
The studies in my laboratory focus on the molecular mechanisms which are responsible for maintaining normal hemostasis. The generation of thrombin at the correct time and place is central to this process as inappropriate production can lead to hemorrhage or thrombosis. Our studies are directed toward understanding how discrete proteolysis of zymogen and procofactor proteins involved in blood coagulation relates to the expression of structural determinants (i.e. exosites, metal binding sites, active site, etc..) important to their function. Procofactors and zymogens cannot participate to any significant degree in their respective macromolecular enzymatic complexes. This indicates that proteolytic activation must result in appropriate structural changes that lead to the expression of sites which impart enzyme, substrate and cofactor binding capabilities. Specifically, my laboratory is interested in studying the conversion of factor V to factor Va and factor X to factor Xa (both proteins are part of the macromolecular enzyme complex, prothrombinase) as model systems to provide insight into the molecular mechanisms underlying the expression of macromolecular binding interactions that accompany transitions from the procofactor and zymogen states. These processes are poorly defined, despite their physiologic importance and new insights into these mechanisms may aid in the development of exosite-based FXa or FVa inhibitors capable of attenuating the abnormal hemostatic processes in thrombophilic disease states. Additionally we also interested in defining whether discrete sequences in the FVa heavy chain provide docking surfaces for both thrombin and prothrombin and contributes to mechanisms of FV activation and those underlying the ability of the cofactor to enhance activation by prothrombinase.
A second line of investigation centers on developing a successful gene therapy strategy for hemophilia A by employing novel modifications to the protein cofactor, factor VIII. Factor VIII is a nonenzymatic cofactor in the intrinsic tenase macromolecular enzyme complex which converts factor X to factor Xa. Factor VIII is encoded by a very large gene (>6.0 kb) and thus presents some problems in efficient packaging in certain viral vectors. While truncation of most of the B-domain has proven successful in several applications, the resulting cDNA is still just at the upper limit of what can be accommodated in certain viral vectors. Our laboratory is interested in designing truncated versions of the FVIII gene which can efficiently package into viral vectors and retain biological function. Our initial focus will be to recombinantly express the novel truncated FVIII proteins and biochemically characterize the cofactors then assess the ability of the new gene products to correct the bleeding phenotype in murine models of hemophilia A.
Potential rotation projects:
? To investigate the mechanism by which functional binding sites on FVa and FXa are expressed following discrete proteolysis.
? To determine whether acidic sequences within the heavy chain of FV provide a recognition exosite for both thrombin and prothrombin, which facilitates activation of the procofactor and contributes to cofactor function in prothrombinase.
? To determine whether truncated forms of recombinant factor VIII result in cofactor molecules with unique properties for therapeutic purposes in the treatment of hemophilia A.
- Associate Professor of Pediatrics at University of Pennsylvania School of Medicine (2009– present)
- Assistant Professor of Pediatrics at University of Pennsylvania School of Medicine (2002 – 2009)
- Assistant Professor of Pharmacology at University of Pennsylvania School of Medicine (2002 – 2005)
- Ph.D., Biochemistry, University of Vermont (1998)
- B.A., Biochemistry, Saint Anselm College (1994)
- Camire, R.M.. Prothrombinase Assembly and S1 Site Occupation Restores the Catalytic Activity of FXa Impaired by Mutation at the Sodium-binding Site. J. Biol. Chem.. Vol 277. 2002:37863-70.
- Arruda, V.R., Hagstrom, J.N., Deitch, J., Patterson-Heiman, T., Camire, R.M., Chu, K., Fields, P.A., Herzog, R.W., Couto, L.B., Larson, P.J., High, K.A.. Posttranslational Modifications of Recombinant Myotube-synthesized Human Factor IX.. Blood. Vol 97. 2001:130-138.
- Camire, R.M., Day, G.A., High, K.A.. Prothrombinase assembly and/or S1 site occupation changes the zymogen-like conformation of FXa induced by mutation at the sodium-binding site. XVIII Congress of the International Society on Thrombosis and Haemostasis. Paris, France. (627) . 2001 July:245.
- Camire, R.M., Murphy, S.L., Day, G.A., High, K.A.. Identification and characterization of mutations leading to factor X deficiency. XVIII Congress of the International Society on Thrombosis and Haemostasis, Paris, France.. (8627) . 2001 July:475.
- Camire, R.M., Murphy, S.L., Day, G.A., High, K.A.. Perturbation of the Active Site Cleft in Factor Xa Does Not Influence Macromolecular Substrate Affinity. 43rd Annual Meeting of the American Society of Hematology. Orlando, FL. (2208) . 2001 December.
- Camire, R.M., Larson, P.J., Stafford, D.W., High, K.A.. Enhanced y-Carboxylation of Recombinant Factor X Using a Chimeric Construct Contining the Prothrombin Propeptide.. Biochemistry. Vol 39. 2000:14322-14329.
- Camire, R.M., Day, G.A., High, K.A.. Perturbation of the S1 Specificity Site in Factor Xa does not Influence Macromolecular Substrate Specificity. University of Pennsylvania Cardiovascular Biology Retreat. Bryn Mawr, PA. 2000 April.
- Camire, R.M., Day, G.A., High, K.A.. Prothombinase Assembly Restores the Catalytic Activity of FXa Impaired by Mutation at the Sodium-Binding Site. 42nd Annual Meeting of the American Society of Hematology. San Francisco. (1929) . 2000 December.
- Camire, R.M., Larson, P.J., Stafford, D.W., High, K.A.. Enhanced y-Carboxylation of Recombinant Factor X Using the Prothrombin Propeptide. XVIIth Congress of the International Society on Thrombosis and Haemostasis, Washington, D.C.. (105) . 1999 August:36.
- Camire, R.M., Kalafatis, M., Simioni, P., Girolami, A., Tracy, P.B.. Platelet-derived Factor Va and Factor Va (Leiden) Cofactor Activities are Sustained on the Surface of Activated Platelets Despite the Presence of Activated Protein C.. Blood. Vol 91. 1998:2818-2829.