Plakalbumin, α1-antitrypsin, antithrombin and the mechanism of inflammatory thrombosis

Abstract

An old puzzle in protein biochemistry concerns the ready conversion of ovalbumin, by proteolysis, to the much more stable derivative, plakalbumin. Ovalbumin is now known to belong to the serpin superfamily, most of which are serine proteinase inhibitors. We report here studies of two such members of the family, the human plasma proteins alpha 1-antitrypsin and antithrombin, and show that they undergo a similar change in stability on selective proteolysis. This change, which is accompanied by a loss of inhibitory activity, can best be considered as an irreversible molecular transition from a native stressed (S) conformation, to a more ordered relaxed (R) form. The maintenance of the native S conformation, and hence the maintenance of inhibitory activity, is critically dependent on the integrity of an exposed loop of polypeptide. We propose that the susceptibility of this peptide loop to proteolytic cleavage gives it an incidental role as a physiological switch which allows the inactivation of individual inhibitors by specific proteolysis. The vulnerability of this exposed loop in each inhibitor also explains the pathological action of a number of venoms and toxins. In particular, the demonstration here of the cleavage of antithrombin, by leukocyte elastase, explains an observed change in blood coagulation that accompanies severe inflammation and which can result in fatal thrombosis.

Identification of hydrophobic fragments of α1‐antitrypsin and Cl protease inhibitor in human bile, plasma and spleen

“…. am round to be insensitive to treatment with elastase from human neutrophils [4], but is cleaved by proteases from Staphylococcus aureus [q and elastase from Psrudontotzas aeruginosu [ l8] and macrophages [6]. If the inactivation peptide in human bile is generated by its natural target protease, elastase, this would have a greater possibility to cleave within the exposed region of ol,-antitrypsin in vivo than previously detected in vitro.…”

“…Such cleavages can be mediated by endogenous serpin target proteases [&8], and by non-target proteases from both venom and bacterial toxins [7-g]. This mechanism has therefore been proposed to be involved in bacterial pathogenicity [7], and in inflammation [3,4], but an in vivo physiological production of these fragments has, to our knowledge, not been reported. We now identify such an inactivation peptide from or,-antitrypsin, recovered in the phospholipid fraction of human bile.…”

Structural aspects of serpin inhibition

“…The cleavage at the binding loop of cllP1 by chymotrypsinogen A [37] facilitates crystallization, probably by allowing a normally flexible binding loop to be incorporated into existing secondary structures, a transition often referred to as the S+R (strained+relaxed) transition [38,39]. Cleaved ollPI* is folded into a highly ordered structure, with threep-sheets (A-C), nine a-helices (A-I) and six helical turns ( Fig.…”

“…Ovalbumin has no known inhibitory function and does not undergo the S-+R transition [38,39] characteristic of the inhibitors. Plakalbumin, a cleaved ovalbumin, was therefore expected to provide a model for the overall structure of intact serpins, particularly for B-sheet A.…”

Crystal structure of bovine antithrombin III

“…Carrell (Carrell, 1982) suggested that the cleaved molecule may be more stable than the native one. Nevertheless, the N-terminus domain of antithrombin is intact in our crystals; this domain, which includes the heparin binding site, contains more residues than that of aI-AT.…”