Factors VII and X are the two serine coagulation proteases which engage in the initial interactions leading to prothrombin activation and clot formation via the extrinsic blood coagulation pathway. Factor VII in its activated form, VIIa, is the component of the initial extrinsic pathway macromolecular complex, which includes tissue factor (non- proteolytic cofactor) and calcium, that converts factor X to Xa. Factor VII can be activated by factors IXa, Xa or XIIa, and additionally activates factor IX. Factor VII thus undergoes multiple specific protein-protein interactions. Factor X is pivotal in the blood coagulation mechanisms, being activated at the point of convergence of the extrinsic and intrinsic pathways. In the intrinsic pathway factor Xa is formed through the interaction of factor IXa and factor VIIIa (non-proteolytic cofactor) on phospholipid surface in the presence of calcium. Factor Xa associates with factor Va (non- proteolytic cofactor) and calcium in another membrane surface oriented complex responsible for activation of prothrombin. Thus, factor X participates in three unique macromolecular complexes, suggesting, as for factor VII, the existence of several regions on the surface of the molecule that control its intermolecular associations. Enzyme kinetic and immunologic studies in this laboratory using inhibitory peptides derived from factor VII and factor X primary sequences and antipeptide antibodies strongly suggested specific surface regions on factors VII and X for their multiple types of specific interactions. Interactive sites have been proposed as existing largely in so-called variable regions of factors VII and X which can be identified on the basis of primary sequence homology among serine proteases. Such regions would be expected to contain structural properties conferring specificity for protein-protein interactions. These findings, and observations on dysfunctional inherited variants of actors VII and X for which point mutations have been identified form the background and rationale for this proposal. Site-directed mutagenesis and expression in eukaryotic cells will be used to confirm the association of point/other mutations with dysfunctional forms of factors VII and X. Secondly, site-directed and domain mutagenesis will be used to produce expressed variant species of factor VII and factor X molecules modified within specific sequences thought to participate in their respective activator, cofactor or substrate binding sites. Finally, new generations of maximally effective factor VII- and X-derived inhibitory peptides will be designed. Concepts of molecular modeling and protein folding will be used as tools to further understand how structural perturbations in mutated factors VII and X may affect their various functions. It is anticipated that the findings will contribute to understanding of the structure/function correlates of factors VII and X and lead to design of management of thrombosis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL048319-02
Application #
2224387
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1993-07-01
Project End
1996-06-30
Budget Start
1994-07-01
Budget End
1995-06-30
Support Year
2
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Texas Health Center at Tyler
Department
Biochemistry
Type
Other Domestic Higher Education
DUNS #
City
Tyler
State
TX
Country
United States
Zip Code
75708