Interspecies Loop Grafting in the Protease Domain of Human Protein C Yielding Enhanced Catalytic and Anticoagulant Activity

Abstract

SummaryHuman anticoagulant activated protein C (hAPC) is less potent than the bovine APC (bAPC) molecule and our aims were to elucidate the molecular background for this difference and to create an APC with enhanced anticoagulant activity. In the protease domain of human protein C (hPC), the loop 148 (GWGYHSSREKEAKRN) is four residues longer than the corresponding loop in bovine APC (GWGY RDETKRN). To investigate whether this caused the species difference, the loop in hPC was replaced by the shorter bovine loop, whereas the longer human loop was introduced in bovine protein C. The mutation in hAPC yielded enhanced catalytic activity against chromogenic (4-fold) as well as natural (factors Va and VIIIa) substrates and 2-3-fold increased anticoagulant activity. The opposite effects were obtained with the bovine mutant. As compared to wild-type hAPC, the mutant hAPC was inhibited slightly faster by the protein C inhibitor, whereas the inhibition by α 1-antitrypsin was unaffected by the mutation. A computer model of bAPC was developed in order to analyse further our data. Collectively, our results demonstrate enhanced catalytic efficiency to result from mutagenesis in the loop 148 and show that APC mutant with increased anticoagulant activity can be created. Abbreviations: PC, protein C; APC, Activated protein C. SDS-PAGE, polyacrylamide gel electrophoresis run in the presence of SDS, The chymotrypsinogen nomenclature for APC and thrombin is used throughout the text while the PC numbering is indicated between brackets whenever appropriate. P2, P1 and P1’, P2’.. designate inhibitor residues amino- and carboxy-terminal to the scissile peptide bond, respectively, and S2, S1 and S1’, S2’.. the corresponding subsites of the protease. APTT, activated partial thromboplastin time; rmsd, root mean square deviation. The coordinates of the bAPC model are freely available.

Antithrombotic and anticoagulant effects of wild type and Gla-domain mutated human activated protein C in rats

“…The QGNSEDY-hAPC:B148 variant, which combines the Gla-domain mutation with a loop 148 change that we previously found to be associated with enhanced catalytic and anticoagulant activity [34], was even more active than the QGNSEDY-hAPC variant in the in vitro clotting assays.…”

“…Full length cDNAs for the QGNSEDY-hPC and hPC:B148 introduced in the HindIII and XbaI sites in the pRC/CMV vector were created with recombinant DNA-techniques, as previously described [33][34][35][36]. The mutations in the QGNSEDY variant was H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y, where the first letter preceding the amino-acid number is the one-letter abbreviation for the wild-type amino acid, and the letter after the number refers to the amino acid residue introduced by the mutagenesis [33].…”

“…after removal of the signal sequence and propeptide. In the hPC: B148 mutant, the 148 loop in human protein C, GWGYHSSREKEAKRN (the underlined amino acids correspond to positions 303-310 in the linear sequence, and to 144-149b according to the chymotrypsinogen numbering), was replaced by the four residues shorter corresponding loop in bAPC (GWGYRDETKRN) [34].…”

“…This variant proved to be highly effective as an anticoagulant in human plasma and QGNSEDY-hAPC alone was found to be more potent than WT hAPC together with hPS in a FVa degradation assay [33]. In another recombinant hAPC variant (hAPC:B148), loop 148 in the serine protease domain was exchanged for the corresponding bovine loop, which resulted in a hAPC variant with modestly enhanced catalytic and anticoagulant activity [34]. By combining the mutations in QGNSEDY-hAPC and hAPC:B148 we have now created a new hAPC variant (QGNSEDY-hAPC:B148), which is more effective as an anticoagulant than either of the two original variants.…”

The Role of Factor XIa (FXIa) Catalytic Domain Exosite Residues in Substrate Catalysis and Inhibition by the Kunitz Protease Inhibitor Domain of Protease Nexin 2

“…The autolysis loop (Tyr 143 -Thr 154 ) is a surface-exposed loop structure that has previously been shown to be important for substrate and inhibitor specificity in FIXa (43), FXa (44), FXIa (45), thrombin (46), urokinase-type plasminogen activator (47), and activated protein C (48). In FIXa, residues Arg 143 and Lys 147 have been shown to be involved in substrate (FX) recognition, and Arg 150 has been shown to be involved in interaction with antithrombin when in the heparin-activated conforma- (Fig.…”