Identification of a specific exosite on activated protein C for interaction with protease-activated receptor 1.
Activated protein C (APC) is a vitamin K-dependent plasma serine protease which down-regulates the clotting cascade by inactivating procoagulant factors Va and VIIIa by limited proteolysis. In addition to its anticoagulant effect, APC also exhibits cytoprotective and antiinflammatory activity through the endothelial protein C receptor-dependent cleavage of protease activated receptor 1 (PAR-1) on endothelial cells. Recent mutagenesis data have indicated that the basic residues of two surface loops including those on 39 and the Ca2+-binding 70-80 loops constitute interactive sites for both factors Va and VIIIa, thereby mediating the interaction of APC specifically with these procoagulant cofactors. The basic residues of both loops have been discovered to be dispensable for the interaction of APC with PAR-1. It is not known if a similar exosite-dependent interaction contributes to the specificity of APC recognition of PAR-1 on endothelial cells. In this study, we have identified two acidic residues on helix-162 (Glu-167 and Glu-170) on the protease domain of APC which are required for the protease interaction with PAR-1, but not for its interaction with the procoagulant cofactors. Thus, the substitution of either Glu-167 or Glu-170 with Ala eliminated the cytoprotective signaling properties of APC without affecting its anticoagulant activity. These mutants provide useful tools for initiating in vivo studies to understand the extent to which the anticoagulant versus antiinflammatory activity of APC contributes to its beneficial effect in treating severe sepsis.
Yang
Likui
L
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Bae
Jong-Sup
JS
Manithody
Chandrashekhara
C
Rezaie
Alireza R
AR
D019943:Amino Acid Substitution; D000893:Anti-Inflammatory Agents / Q000378:metabolism* / Q000627:therapeutic use; D000925:Anticoagulants / Q000378:metabolism* / Q000627:therapeutic use; D001665:Binding Sites / Q000235:genetics; D001777:Blood Coagulation / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D019610:Cytoprotection / Q000187:drug effects / Q000235:genetics; D004730:Endothelium, Vascular / Q000378:metabolism*; D015944:Factor VIIIa / Q000235:genetics / Q000378:metabolism; D015943:Factor Va / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016296:Mutagenesis; D020125:Mutation, Missense; D011485:Protein Binding / Q000235:genetics; D011486:Protein C / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics; D044463:Receptor, PAR-1 / Q000235:genetics / Q000378:metabolism*; D018805:Sepsis / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D012720:Severity of Illness Index; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics
D000893:Anti-Inflammatory Agents; D000925:Anticoagulants; D011486:Protein C; D044463:Receptor, PAR-1; D015943:Factor Va; D015944:Factor VIIIa; D002118:Calcium