Basic residues in the 37-loop of activated protein C modulate inhibition by protein C inhibitor but not by alpha(1)-antitrypsin.
The role of lysines 37-39 (chymotrypsin numbering) in the 37-loop of the serine protease activated protein C (APC) was studied by expressing acidic and neutral recombinant APC (rAPC) mutants. Activity of the APC mutants was assessed using human plasma and plasma-purified and recombinant derivatives of protein C inhibitor (PCI; also known as plasminogen activator inhibitor-3) and alpha(1)-antitrypsin, with and without heparin. The catalytic properties of the mutants to small peptidyl substrates were essentially the same as wild-type rAPC (wt-rAPC), yet their plasma anticoagulant activities were diminished. Analysis of the rAPC-protease inhibitor complexes formed after addition of wt-rAPC and mutants to plasma revealed no change in the inhibition pattern by alpha(1)-antitrypsin but a reduction in mutant complex formation by PCI in the presence of heparin. Using purified serpins, we found that inhibition rates of the mutants were the same as wt-rAPC with alpha(1)-antitrypsin; however, PCI (plasma-derived and recombinant forms) inhibition rates of the acidic mutants were slightly faster than that of wt-rAPC without heparin. By contrast, PCI-heparin inhibition rates of the mutants were not substantially accelerated compared to wt-rAPC. The mutants had reduced heparin-binding properties compared to wt-rAPC. Molecular modeling of the PCI-APC complex with heparin suggests that heparin may function not only to bridge PCI to APC, but also to alleviate putative non-optimal intermolecular interactions. Our results suggest that the basic residues of the 37-loop of APC are involved in macromolecular substrate interactions and in heparin binding, and they influence inhibition by PCI (with or without heparin) but not by alpha(1)-antitrypsin, two important blood plasma serpins.
Glasscock
Laura N
LN
Department of Pathology, Center for Thrombosis and Hemostasis, The University of North Carolina School of Medicine, Chapel Hill, NC 27599-7035, USA.
Gerlitz
Bruce
B
Cooper
Scott T
ST
Grinnell
Brian W
BW
Church
Frank C
FC
D001665:Binding Sites; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010949:Plasma / Q000378:metabolism; D011486:Protein C / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018046:Protein C Inhibitor / Q000097:blood / Q000494:pharmacology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D000515:alpha 1-Antitrypsin / Q000494:pharmacology*
D011486:Protein C; D018046:Protein C Inhibitor; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D000515:alpha 1-Antitrypsin; D006493:Heparin; D008239:Lysine