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Interaction of activated protein C with serpins.

ABSTRACT: The inhibition of activated protein C by six different serine protease inhibitors (serpins) that have arginine residues in the P1 position has been investigated. Micromolar concentrations of C1-inhibitor failed to inhibit the enzyme, and it was inhibited only slowly by antithrombin III with an association rate constant (kass.) of 0.15 M-1.s-1. The kass. values for the other serpins tested (protease nexin I, protein C inhibitor, and mutants of alpha 1-antichymotrypsin and alpha 1-antitrypsin with P1 arginine residues) were at least 1000-fold higher, with P1-Arg-alpha 1-antitrypsin (kass. = 7 x 10(4) M-1.s-1) being the most effective inhibitor. The inhibition with these four serpins appeared to be reversible, with inhibition constants in the nanomolar range. The relatively high value of kass. for protease nexin I (5 x 10(3) M-1.s-1) suggested that it may be involved in the control of activated protein C on the surface of platelets where protein nexin I is present at relatively high concentrations. The value of kass. for protease nexin I, protein C inhibitor and antithrombin III showed a bell-shaped dependence on heparin concentration. At optimal concentrations, heparin accelerated the rate of inhibition by protease nexin I, protein C inhibitor and antithrombin III by 44-, 18- and 13-fold respectively. The kinetic constants for the inhibition of thrombin were also determined, and in all cases the serpins were more effective inhibitors of thrombin. Comparison of the sequences of the active-site regions of activated protein C and thrombin suggested that the more hydrophobic active site of thrombin may be more favourable for interactions with serpins.

SUBMITTER: Hermans JM

PROVIDER: S-EPMC1134845 | biostudies-other | 1993 Oct

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Most members of the serpin family of proteins are potent, irreversible inhibitors of specific serine or cysteine proteinases. Inhibitory serpins are distinguished from members of other families of proteinase inhibitors by their metastable structure and unique suicide-substrate mechanism. Animal serpins exert control over a remarkable diversity of physiological processes including blood coagulation, fibrinolysis, innate immunity and aspects of development. Relatively little is known about the complement of serpin genes in plant genomes and the biological functions of plant serpins. RESULTS: A structurally refined amino-acid sequence alignment of the 14 full-length serpins encoded in the genome of the japonica rice Oryza sativa cv. Nipponbare (a monocot) showed a diversity of reactive-centre sequences (which largely determine inhibitory specificity) and a low degree of identity with those of serpins in Arabidopsis (a eudicot). A new convenient and functionally informative nomenclature for plant serpins in which the reactive-centre sequence is incorporated into the serpin name was developed and applied to the rice serpins. A phylogenetic analysis of the rice serpins provided evidence for two main clades and a number of relatively recent gene duplications. Transcriptional analysis showed vastly different levels of basal expression among eight selected rice serpin genes in callus tissue, during seedling development, among vegetative tissues of mature plants and throughout seed development. The gene OsSRP-LRS (Os03g41419), encoding a putative orthologue of Arabidopsis AtSerpin1 (At1g47710), was expressed ubiquitously and at high levels. The second most highly expressed serpin gene was OsSRP-PLP (Os11g11500), encoding a non-inhibitory serpin with a surprisingly well-conserved reactive-centre loop (RCL) sequence among putative orthologues in other grass species. CONCLUSIONS: The diversity of reactive-centre sequences among the putatively inhibitory serpins of rice point to a range of target proteases with different proteolytic specificities. Large differences in basal expression levels of the eight selected rice serpin genes during development further suggest a range of functions in regulation and in plant defence for the corresponding proteins.

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Interaction of activated protein C with serpins.

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