| Deficiency of the blood coagulation factor IXa (flXa) results in hemophilia B, while its overactivity can lead to thrombotic complications. Understanding the molecular mechanisms of flXa regulation can lead to novel drugs for hemophilia and thrombosis. Heparin binding to flXa modulates the active site to increase inhibition by the Kunitz-type inhibitor basic pancreatic trypsin inhibitor (BPTI). This study's main focus was to elucidate what region(s) of BPTI are required to "sense" heparin-flXa. BPTI, with a mutated stabilizing loop region, (BPTI-KPISL) was constructed and found to inhibit flXa similar to BPTI but with reduced ability to sense heparin-flXa. For easier production of mutants, a histidine tag was added to BPTI (HisBPTI). It was discovered that HisBPTI could be captured onto a phospholipid surface that could bind and inhibit coagulation proteases. These results have implications with respect to designing inhibitors that recognize the heparin-flXa complex, and generation of antithrombogenic artificial surfaces. |
