Alignment of the heparin-activated serpins indicates the presence of two binding sites for heparin: a small high-affinity site on the D-helix corresponding in size to the minimal pentasaccharide heparin, and a longer contiguous low-affinity site extending to the reactive center pole of the molecule. Studies of the complexing of antithrombin and its variants with heparin fractions and with reactive center loop peptides including intermolecular loop-sheet polymers all support a 3-fold mechanism for the heparin activation of antithrombin. Binding to the pentasaccharide site induces a conformational change as measured by circular dichroism. Accompanying this, the reactive center becomes more accessible to proteolytic cleavage and there is a 100-fold increase in the k(ass) for factor Xa but only a 10-fold increase for thrombin, to 6.4 x 10(4) M-1 s-1. To obtain a 100-fold increase in the k(ass) for thrombin requires in addition a 4:1 molar ratio of disaccharide to neutralize the charge on the extended low-affinity site. Full activation requires longer heparin chains in order to stabilize the ternary complex between antithrombin and thrombin. Thus, addition of low-affinity but high molecular weight heparin in conjunction with pentasaccharide gives an overall k(ass) of 2.7 x 10(6) M-1 s-1, close to that of maximal heparin activation.

Immature rat thymocytes readily undergo apoptosis following exposure to many different stimuli, including agents which cause DNA damage, such as the topoisomerase II inhibitor etoposide and irradiation. We have shown previously that cells isolated from the immature rat thymus are resistant to the induction of apoptosis by the DNA-damaging agent cis-diamminedichloroplatinum(II) (cisplatin) (D. L. Evans and C. Dive, Cancer Res., 53:2133-2139, 1993). More than 85% of these thymocytes are quiescent. Here, we demonstrate that following purification of the minority subpopulation of thymocytes that are proliferating, a 2-h exposure to 50 microM cisplatin resulted in rapid apoptosis with 66% apoptotic cells by 12 h. In contrast, purified, nonproliferating thymocytes treated with cisplatin exhibited control levels of apoptosis at 12 h. Both proliferating and nonproliferating thymocytes rapidly underwent apoptosis following continuous exposure to methylprednisolone (10 microM) and etoposide (10 microM). The discrepancy in the levels of apoptosis seen in proliferating and quiescent thymocytes in response to cisplatin could not be attributed to changes in total cellular levels of cisplatin or to the number of DNA-platinum adducts which were determined, respectively, by atomic absorption spectrometry and competitive enzyme-linked immunoadsorbent assay. These results imply that in contrast to engagement of thymocyte apoptosis by methylprednisolone and etoposide, where apoptosis was proliferation independent, cisplatin-induced apoptosis depends on the presence of cells in S and G2-M phases of the cell cycle. Moreover, comparison of etoposide and cisplatin responses in thymocytes suggests that DNA damage per se may not be sufficient to induce apoptosis and that the type of DNA damage is important in this regard.

A major feature of the structure of alpha 1-antitrypsin is a five-stranded A-sheet into which the reactive center loop inserts after cleavage. We describe here the effect of the Z mutation (342Glu to Lys) at the head of the fifth strand of the A-sheet on the mobility of the reactive center loop and hence on the physical properties of the antitrypsin molecule. The mutant Z but not the normal M antitrypsin spontaneously polymerizes at 37 degrees C by a mechanism involving the insertion of the reactive center loop of one molecule into the A-sheet of a second. It is demonstrated that Z antitrypsin polymerized after incubation with 1.0 M guanidinium chloride at 37 degrees C at the same rate as M antitrypsin. Reducing the temperature to 4 degrees C favored the formation of the L-state in M antitrypsin in which the loop is stably incorporated into the A-sheet, but resulted in loop-sheet polymerization in Z antitrypsin. Z, like M antitrypsin, undergoes the S to R transition, but we show that the accompanying change in thermal stability results from loop-sheet polymerization (S) which can be prevented by the insertion of the cleaved strand of the reactive center loop into the A-sheet (R). Z antitrypsin has a reduced association rate constant with neutrophil elastase [(5.3 +/- 0.06) x 10(7) and (1.2 +/- 0.02) x 10(7) M-1 s-1 for M and Z, respectively], but both M and Z antitrypsin had Ki values of less than 5 pM.(ABSTRACT TRUNCATED AT 250 WORDS)

The myxoma and malignant rabbit fibroma poxviruses are lethal tumorigenic viruses of rabbits whose virulence is modulated by the production of a virus-encoded secreted serine proteinase inhibitor, SERP-1. This viral protein was detected in medium harvested from myxoma and malignant rabbit fibroma virus-infected cells, and its inhibitory profile has been characterized by gel and kinetic analysis. SERP-1 forms complexes with and inhibits the human fibrinolytic enzymes plasmin, urokinase, and two-chain tissue-type plasminogen activator (association rate constants 3.4 x 10(4), 4.3 x 10(4), and 3.6 x 10(4) M-1 s-1 respectively). It is also able to inhibit C1S, the first enzyme in the complement cascade with an association rate constant which was unaffected by the addition of heparin (1.3 x 10(3) M-1 s-1). SERP-1 acts as a substrate for and is cleaved by thrombin, porcine trypsin, human neutrophil elastase, porcine pancreatic elastase, thermolysin, subtilisin, bovine alpha-chymotrypsin, and factor Xa. Incubation with kallikrein and cathepsin G had no effect. The structure of SERP-1 has been modeled on other members of the serpin family which revealed the characteristic serpin architecture apart from the absence of the D-helix. Structural analysis and kinetic assays demonstrate that the absence of this region does not prevent inhibitory activity and furthermore allow the identification of cysteine residues involved in internal and intermolecular disulfide bonding.

The serpins are unique among the families of serine proteinase inhibitors in having a reactive centre that is situated on a mobile peptide loop. The structures of three alternative conformations are now known, and it can be deduced that the active form involves the partial insertion of the loop into the A sheet of the molecule. The ability of the loop to move in and out of this sheet has been adapted by e inhibitory activity. Mutations that affect the movement of the loop cause a loss of inhibitory activity, and other mutations can result in an opening of the A sheet and consequent loop-sheet intermolecular polymerization.

Necrosis and apoptosis are two distinct modes of cell death which differ in morphology, mechanism and incidence. Membrane disruptants, respiratory poisons and hypoxia cause ATP depletion, metabolic collapse, cell swelling and rupture leading to inflammation. These are typical features of necrosis. Apoptosis plays a crucial role in embryogenesis and development and is also prevalent in tumours. It is characterised by cell shrinkage, chromatin condensation and systematic DNA cleavage. Apoptotic cells are rapidly engulfed by phagocytes, thus preventing inflammatory reaction to degradative cell contents. In vivo, apoptosis is almost impossible to quantify due to problems of heterogeneity and the short half-life of an apoptotic cell. In vitro, mechanistic studies are further complicated by a late phase of apoptosis where the cell membrane becomes permeable to vital dyes and which occurs in the absence of phagocytes. Here we describe a novel and rapid multiparameter flow cytometric assay which discriminates and quantifies viable, apoptotic and necrotic cells via measurement of forward and side light scatter (proportional to cell diameter and internal granularity, respectively) and the DNA-binding fluorophores Hoechst 33342 and propidium. It is anticipated that mechanistic studies of apoptosis in a variety of cell types will greatly benefit from this mode of analysis.

The serpins are unique among the families of serine proteinase inhibitors in having a reactive centre that is situated on a mobile loop. The structures of three alternative conformations are now known, and it can be deduced that the active form involves the partial insertion of the loop into the A sheet of the molecule. The ability of the loop to move in and out of this sheet has been adapted by evolution to allow the modulation of inhibitory activity. Manipulation of the structure of the loop and of other functional domains in the serpin superfamily enables the production of serpins with tailor-made activities. The ability of the loop to lock in latent conformations or to take part in intermolecular polymerization has implications for the production and stabilization of recombinant serpins. This review has been adapted from Current Opinion in Structural Biology 1992, 2:438–446.

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.