ROLE OF THE A(-BINDING TO PROTEIN-C INHIBITOR() HELIX IN HEPARIN)

Interactions between proteins and heparin(-like) structures involve el ectrostatic forces and structural features. Based on charge distributi ons in the linear sequence of protein C inhibitor (PCI), two positivel y charged regions of PCI were proposed as possible candidates for this interaction. The first region, the A(+) helix, is located al the N-te rminus (residues 1-11), whereas the second region, the H helix, is pos itioned between residues 264 and 280 of PCI. Competition experiments w ith synthetic peptides based on the sequence of these regions demonstr ated that the H helix has the highest affinity for heparin. In contras t to previous observations we found that the A(+) helix peptide compet ed for the interaction of PCI with heparin, but its affinity was much lower than that of the H helix peptide. Recombinant PCI was also used to investigate the role of the A(+) helix in heparin binding. Full-len gth (wild-type) rPCI as well as an A(+) helix deletion mutant of PCI ( rPCI-Delta 2-11) were expressed in baby hamster kidney cells and both had normal inhibition activity with activated protein C and thrombin. The interaction of the recombinant PCIs with heparin was investigated and compared to plasma PCI. The A(+) helix deletion mutant showed a de creased affinity for heparin in in hibition reactions with activated p rotein C and thrombin, but had similar association constants compared to wild-type rPCI. The synthetic A(+) helix peptide competed with rPCI -Delta 2-11 for binding to heparin. This indicated that the interactio n between PCI and heparin is fairly non-specific and that the interact ion is primarily based on electrostatic interactions. In summary, our data suggest that the H helix of PCI is the main heparin binding regio n of PCI, but the A(+) helix increases the overall affinity for the PC I-heparin interaction by contributing a second positively charged regi on to the surface of PCI.