Factor IXa Protease Helix-330 (Region 1):A Critical Site for Factor VIIIa Interaction And A Site-Specific Target for Coagulation Inhibitors.
Inventor: Dr. S. Paul Bajaj
Blood Factor IX is a vitamin K-dependent protein that participates in the middle phase of the intrinsic as well as the extrinsic coagulation cascade. Factor IX may be activated to the multi-domain active serine protease factor IXa by factor XIa or by factor VIIa. Factor IXa thus formed activates factor X. For a physiologically significant rate, this reaction requires the cofactor, factor VIIIa, in addition to calcium and the phospholipid membrane surface provided by platelets.
The protease domain of factor IXa is believed to provide the primary specificity for binding to factor VIIIa. Calcium binding to the protease domain in factor IXa and the consequent protection of the 318-319 peptide bond from proteolysis stabilizes a segment in the factor IXa domain that directly interacts with VIIIa. To explore this interaction further, studies were carried out on the role of the surface exposed factor IXa protease helix-330 (ie. Region 1) site. The helix-330 amino acid structure is highly conserved and is identical in factor IX from all species but it is different from other homologous blood coagulation serine proteases. Interestingly, point mutations in eight of the nine residues of helix-330 are reported to cause hemophilia B.
When point mutants were constructed in the factor IXa helix-330, it was striking that all of the variants exhibited impaired binding affinity for factor VIIIa, both in the presence and absence of phospholipid. Thus, the factor IXa protease helix-330 (Region 1) provides a critical site for binding factor VIIIa. It has been reported that residues 330-338 are also involved in the binding of factor IXa to factor X.
Detailed sequence knowledge of the factor IXa protease helix-330 (Region 1) permitted the design of polypeptides (and derivatives thereof such as peptidomimetics or nonpeptdiomemetics) of from 3 to 20 contiguous amino acids which comprise the critical amino acids of the factor IXa protease helix-330 (Region 1) site. Such polypeptide constructs, which do not activate factor X but bind to Factor VIIIa, effectively block binding of factor VIIIa to factor IXa protease helix-330 (Region 1), are effective inhibitors of blood coagulation. A method for screening agents for their ability to bind to factor VIIIa and block the interaction of factor VIIIa with factor IXa protease helix-330 (Region 1) is described. Method for detecting factor VIIIa in blood samples and for treating patients with thrombosis are also provided.
Factor IXa protease helix-330 (Region 1), which plays a critical role in blood coagulation, provides an novel opportunity for the design and development of site-specific coagulation inhibitors. Various polypeptides (and various derivatives) inhibit blood coagulation by effectively binding to and blocking the factor VIIIa site which binds with factor IXa protease helix-330 (Region 1). Such helix-330 type compositions have significant potential as coagulation inhibitors useful for treatment of patients having cardiovascular disorders (thrombosis, atherosclerosis, and restenosis). An in vitro assay for screening potential drug candidates for their ability to block the helix-330 binding site of factor VIIIa is also available.
Keywords: thrombosis, hemostasis, factor IX, factor VIII, factor X, cardiovascular disease, restenosis
Patent: US Patent Application 09/584,866
License: Collaborative research and licensing opportunities are available.
Reference Number: SLU-1009