Mouse recombinant protein C variants with enhanced membrane affinity and hyper-anticoagulant activity in mouse plasma.
Mouse anticoagulant protein C (461 residues) shares 69% sequence identity with its human ortholog. Interspecies experiments suggest that there is an incompatibility between mouse and human protein C, such that human protein C does not function efficiently in mouse plasma, nor does mouse protein C function efficiently in human plasma. Previously, we described a series of human activated protein C (APC) Gla domain mutants (e.g. QGNSEDY-APC), with enhanced membrane affinity that also served as superior anticoagulants. To characterize these Gla mutants further in mouse models of diseases, the analogous mutations were now made in mouse protein C. In total, seven mutants (mutated at one or more of positions P(10)S(12)D(23)Q(32)N(33)) and wild-type protein C were expressed and purified to homogeneity. In a surface plasmon resonance-based membrane-binding assay, several high affinity protein C mutants were identified. In Ca(2+) titration experiments, the high affinity variants had a significantly reduced (four-fold) Ca(2+) requirement for half-maximum binding. In a tissue factor-initiated thrombin generation assay using mouse plasma, all mouse APC variants, including wild-type, could completely inhibit thrombin generation; however, one of the variants denoted mutant III (P10Q/S12N/D23S/Q32E/N33D) was found to be a 30- to 50-fold better anticoagulant compared to the wild-type protein. This mouse APC variant will be attractive to use in mouse models aiming to elucidate the in vivo effects of APC variants with enhanced anticoagulant activity.
Krisinger
Michael J
MJ
Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, University Hospital, Malmö, Sweden.
Guo
Li Jun
LJ
Salvagno
Gian Luca
GL
Guidi
Gian Cesare
GC
Lippi
Giuseppe
G
Dahlbäck
Björn
B
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D002417:Cattle; D002462:Cell Membrane; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010949:Plasma / Q000187:drug effects*; D011486:Protein C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid
D000925:Anticoagulants; D011486:Protein C; D011994:Recombinant Proteins