Structural and energetic characteristics of the heparin-binding site in antithrombotic protein C

Human activated protein C (APC) is a key component of a natural anticoagula nt system that regulates blood coagulation, rn vivo, the catalytic activity of APC is regulated by two serpins, alpha1-antitrypsin and the protein C i nhibitor (PCI), the inhibition by the latter being stimulated by heparin, W e have identified a heparin-binding site in the serine protease domain of A PC and characterized the energetic basis of the interaction with heparin. A ccording to the counter-ion condensation theory, the binding of heparin to APC is 66% ionic in nature and comprises four to six net; ionic interaction s. To localize the heparin-binding site, five recombinant APC variants cont aining amino acid exchanges in loops 37, 60, and 70 (chymotrypsinogen numbe ring) were created. As demonstrated by surface plasmon resonance, reduction of the electropositive character of loops 37 and 60 resulted in complete l oss of heparin binding. The functional consequence was loss in heparin-indu ced stimulation of APC inhibition by PCI, whereas the PCI-induced APC inhib ition in the absence of heparin was enhanced. Presumably, the former observ ations were due to the inability of heparin to bridge some APC mutants to P CI, whereas the increased inhibition of certain APC variants by PCI in the absence of heparin was due to reduced repulsion between the enzymes and the serpin. The heparin-binding site of APC was also shown to interact with he paran sulfate, albeit with lower affinity. In conclusion, we have character ized and spatially localized the functionally important heparin/heparan sul fate-binding site of APC.