Protease specificity and heparin binding and activation of recombinant protease nexin I.
Structural and functional properties of alpha-protease nexin I (alpha-PNI) expressed in Chinese hamster ovary cells were studied. All three cysteines were in the reduced form, showing that the potential disulfide bridge between residues Cys117 and Cys131 was not formed. Heparin association rate enhancements were from ka = 8.3 x 10(5) to 0.7-1.6 x 10(9) M-1 s-1 for the interaction of PNI with thrombin, from ka = 5.1 x 10(3) to 3.5 x 10(5) M-1 s-1 for interaction with Factor Xa, and from ka = 2.2 x 10(6) to 1.0 x 10(7) M-1 s-1 for interaction with trypsin; there was no rate enhancement of the plasmin interaction (ka = 1.0 x 10(5) M-1 s-1). The minimal heparin pentasaccharide had no effect on these interactions. Cleavage of the reactive center loop of PNI by three different proteases gave the typical stressed to relaxed change in thermal stability, but unlike with antithrombin III, there was no loss of heparin affinity. A similar difference from antithrombin was that PNI-thrombin complexes retained normal heparin affinity. These results are compatible with a role for protease nexin I as a cell-associated thrombin inhibitor that remains bound to the cell surface even after complexing with the protease, as compared with the role of antithrombin III as a circulating inhibitor of thrombin that becomes activated on binding to the microvasculature and is released on complex formation.
Evans
D L
DL
Department of Haematology, University of Cambridge, United Kingdom.
McGrogan
M
M
Scott
R W
RW
Carrell
R W
RW
D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor; D000818:Animals; D000990:Antithrombin III / Q000378:metabolism; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D004789:Enzyme Activation; D015951:Factor Xa / Q000378:metabolism; D006493:Heparin / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D058255:Protease Nexins; D011485:Protein Binding; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000378:metabolism; D015843:Serpins / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine; D013917:Thrombin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D015843:Serpins; D000990:Antithrombin III; D006493:Heparin; D014357:Trypsin; D013917:Thrombin; D015951:Factor Xa