Serpin-serine protease binding kinetics: alpha 2-antiplasmin as a model inhibitor.

We have examined in detail the kinetics of binding of the serpin alpha 2-antiplasmin to the serine proteases alpha-chymotrypsin and plasmin. These represent model systems for serpin binding. We find, in contrast to earlier published results with alpha 2-antiplasmin and plasmin, that binding is reversible, and slow binding kinetics can be observed, under appropriate conditions. Binding follows a two-step process with both enzymes, with the formation of an initial loose complex which then proceeds to a tightly bound complex. In the absence of lysine and analogues, equilibrium between alpha 2-antiplasmin and plasmin is achieved rapidly, with an overall inhibition constant (Ki') of 0.3 pM. In the presence of tranexamic acid or 6-aminohexanoic acid, lysine analogues that mimic the effects of fibrin, plasmin binding kinetics are changed such that equilibrium is reached slowly following a lag phase after mixing of enzyme and inhibitor. The Ki' is also affected, rising to 2 pM in the presence of 6-aminohexanoic acid concentrations above 15 mM. Thus extrapolation to the in vivo situation indicates that complex formation in the presence of fibrin will be delayed, allowing a burst of enzyme activity following plasmin generation, but a tight, pseudoirreversible complex will result eventually. Chymotrypsin is more weakly inhibited by alpha 2-antiplasmin, exhibiting an overall Ki' of 0.1 nM, after two-stage complex formation. The inhibition constant for the initial loose complex (Ki) is very similar for both enzymes. The difference in binding strength between the two enzymes is accounted for by the dissociation rate constant of the second step of complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reactive site of human alpha 2-antiplasmin

Human alpha 2-antiplasmin rapidly forms a stable, equimolar complex with either its target enzyme, plasmin, or with trypsin. Perturbation of the inhibitor-trypsin complex results in peptide bond cleavage at the reactive site of the inhibitor with the concomitant release of a small peptide fragment which apparently represents the carboxyl-terminal segment of the inhibitor. Sequence analysis of this fragment, together with that of an overlapping peptide obtained by treatment of native inhibitor with either Staphylococcus aureus V8 proteinase or human neutrophil elastase, yields data which indicate that the reactive site of alpha 2-antiplasmin encompasses a P1-P'1 Arg-Met sequence. However, unlike alpha 1-1-proteinase inhibitor which has a Met residue in the P1-position, oxidation of alpha 2-antiplasmin has no effect on its inhibitory activity toward either plasmin, trypsin, or chymotrypsin, indicating the lesser mechanistic importance of the P'1-residue during enzyme inactivation by this inhibitor.     

Amino-acid sequence of human alpha 2-antiplasmin

The amino-acid sequence of human alpha 2-antiplasmin was determined by Edman degradation of peptides purified from CNBr, tryptic and chymotryptic digests. Of the total sequence of 452 amino acids of mature alpha 2-antiplasmin, as deduced from the cDNA sequence [Holmes et al. (1987) J. Biol. Chem. 262, 1659-1664], 444 residues were identified by amino-acid sequencing. Two differences were found between the peptide and cDNA analyses (Gly instead of Leu at position 10 and Gly instead of Ser at position 369). alpha 2-Antiplasmin contains two disulfide bridges (Cys64-Cys104 and Cys31-Cys113) and four glucosamine-based carbohydrate chains attached to Asn87, Asn256, Asn270 and Asn277. alpha 2-Antiplasmin is homologous with 12 other proteins belonging to the serine protease inhibitor (serpin) superfamily.     

Sulfation of a tyrosine residue in the plasmin-binding domain of alpha 2-antiplasmin

Sulfation of human alpha 2-antiplasmin, the major plasma inhibitor of fibrinolysis, was examined using both protein isolated from human plasma and protein synthesized and biosynthetically labeled with [35S]sulfate by a human hepatoma-derived cell line. Linkage of sulfate to tyrosine was demonstrated by recovery of labeled tyrosine sulfate after base hydrolysis of sulfate-labeled alpha 2-antiplasmin. Analysis by reverse-phase high performance liquid chromatography of peptides released from alpha 2-antiplasmin by cleavage with trypsin or cyanogen bromide indicated that sulfate is linked to a single segment of the protein. A cyanogen bromide peptide corresponding to the sulfate-labeled peptide was prepared from alpha 2-antiplasmin isolated from human plasma. Consistent with the presence of tyrosine sulfate in this peptide, its chromatographic elution was altered by treatment with acid under conditions which release sulfate from a tyrosine residue. No peptide in the total digest of alpha 2-antiplasmin by cyanogen bromide eluted at the position of the peptide following desulfation, suggesting that all of the protein is in a sulfated form. The sequence of the sulfate-containing cyanogen bromide peptide as determined by sequential Edman degradation, amino acid composition, and fast atom-bombardment-mass spectrometry was: Glu-Glu-Asp-Tyr(SO4)-Pro-Gln-Phe-Gly-Ser-Pro-Lys-COOH. This peptide is a segment of the previously identified plasmin-binding domain of alpha 2-antiplasmin.     

Affinity-chromatographic purification of human alpha 2-antiplasmin.

A new simple and efficient purification method for alpha 2-antiplasmin is described that is based on the interaction between alpha 2-antiplasmin and a fragment from elastase-digested plasminogen constituting the three N-terminal triple-loop structures in the plasmin A-chain (LBSI). After a single-step adsorption of the alpha 2-antiplasmin from plasminogen-depleted plasma to LBSI-Sepharose and elution with 6-aminohexanoic acid, an 80-90% pure preparation with a yield of 50-60% is obtained. The major impurity is fibrinogen, which can easily be removed by gel filtration, and, as a result, a homogeneous fully active alpha 2-antiplasmin preparation is obtained that has the same properties as previously described for alpha 2-antiplasmin. Evidence is put forward that a form of alpha 2-antiplasmin with less affinity for the lysine-binding sites in plasminogen may exist, even in unfractionated plasma.     

Current views on structure and function of alpha2-antiplasmin

Data available in literature on basic, primary, rapidly acting alpha 2-antiplasmin (a plasmin inhibitor) are generalized. Methods of its isolation from human blood plasma, properties, structure, kinetics of interaction with plasmin as well as the key role in the fibrinolysis regulation are considered.     

Features of the interaction between alpha2-antiplasmin and plasminogen/plasmin

The kinetic of plasmin, Va1442-plasmin, Lys530-plasmin inhibition reaction by alpha 2-antiplasmin as well as interaction of the inhibitor with different derivatives of the plasminogen and its fragments were studied. It was shown that plasmin, mini- and micro-plasmin activity decreased by 97, 88 and 85%, respectively, for equimolar ratio 1:1 of the inhibitor. The value of the inhibition reached its maximum in 1-2, 5-10 and 10-15 min, respectively. The constants of the complex formation rate were 1.4 x 10(6); 1.7 x 10(5) and 6.2 x 10(4) M-1s-1 for the plasmin, mini- and micro-plasmin with alpha 2-antiplasmin, respectively. Both 10(-2) M 6-aminohexanoic acid and 10(-1) M arginine reduced the complex formation rate between plasmin, mini-plasmin and alpha 2-antiplasmin to the value of the rate reaction between micro-plasmin and inhibitor. alpha 2-Antiplasmin bound with all investigated derivatives and fragments of plasminogen. The amount of inhibitor decreased in the series: plasmin, kringle 1-3, kringle 4, mini-plasminogen, micro-plasminogen. The kringle 1-4 and kringle 5 were determined to control the rate of reaction between enzyme and inhibitor, being not necessary for the inhibition. The comparison of the inhibitor interaction with DPP-plasmin, mini-plasminogen and micro-plasminogen displayed the possibility of the additional region existence in catalytic domain. This region participated in the complex with alpha 2-antiplasmin formation. It is supposed that the multisite interaction between plasmin and alpha 2-antiplasmin provides for the specificity and efficiency the inhibitor action.     

Inhibition of cell surface receptor-bound plasmin by alpha 2-antiplasmin and alpha 2-macroglobulin.

The purpose of this investigation was to characterize the reaction of alpha 2-antiplasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M) with human plasmin bound to rat C6 glioma cells and human umbilical vein endothelial cells (HUVECs). Binding of plasmin (0.1 microM) to C6 cells at 4 degrees C did not cause cell detachment, decrease viability or change cell morphology. The KD and Bmax for the binding of diisopropyl phosphoryl plasmin (DIP-plasmin) to C6 cells were 0.9 microM and 2.6 x 10(6) sites/cell. The dissociation rate constants (koff) for 125I-plasmin were 9.7 x 10(-4) and 4.0 x 10(-4) s-1 at 4 degrees C in the presence and absence of 0.3 microM DIP-plasmin, respectively. Similar constants were determined for 125I-plasminogen and 125I-DIP-plasmin. Neither alpha 2AP nor alpha 2M affected the dissociation of DIP-plasmin. C6 cell-associated 125I-plasmin reacted slowly with alpha 2AP; however, the inhibition rate constants exceeded the koff. alpha 2AP-plasmin complex formed after the plasmin dissociated into solution (reaction pathway 1) and by direct reaction of alpha 2AP with cell-associated enzyme (reaction pathway 2). High concentrations of alpha 2AP favored pathway 2. C6 cell-associated plasmin was also protected from inhibition by alpha 2M. While the same pathways were probably involved in this reaction, alpha 2M was less effective than alpha 2AP as an inhibitor of nondissociated plasmin (pathway 2). When C6 cell-bound plasmin reacted with alpha 2AP, alpha 2AP-plasmin complex was recovered primarily in the medium, suggesting dissociation of complexes formed on the cell surface. Plasmin-receptor dissociation and inhibition experiments were performed at 22 degrees and 37 degrees C, confirming the conclusions of the 4 degrees C studies. Comparable results were also obtained using HUVEC cultures. These studies demonstrate that cell-associated plasmin is protected from inhibition by alpha 2M as well as alpha 2AP. At least two reaction pathways may be demonstrated for the inhibition of plasmin that is initially receptor-bound; however, neither pathway is highly effective, accounting for the "plasmin-protective" activity of the cell surface.     

Inactivation of the serpin alpha(2)-antiplasmin by stromelysin-1.

Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) hydrolyzes the Met(374)-Ser(375) (P3-P2), Glu(416)-Leu(417) and Ser(432)-Leu(433) peptide bonds in human alpha(2)-antiplasmin (alpha(2)-AP), the main physiological plasmin inhibitor. Cleavage is completely abolished in the presence of the MMP inhibitors EDTA or 1,10-phenanthroline. At enzyme/substrate ratio of 1:10 at 37 degrees C, alpha(2)-AP protein cleavage occurs with a half-life of 8 min, and is associated with rapid loss of inhibitory activity towards plasmin with a half-life of 5 min. alpha(2)-AP cleaved by MMP-3 does no longer form a stable complex with plasmin, as shown by SDS-PAGE, and does no longer interact with plasminogen, as shown by crossed immunoelectrophoresis with plasminogen added to the gel. These data are compatible with the removal of a COOH-terminal fragment containing the reactive site peptide bond and the plasmin(ogen)-binding site. In addition, MMP-3 cleaves the Pro(19)-Leu(20) peptide bond in alpha(2)-AP, thereby removing the fibrin-binding site from the inhibitor. A dysfunctional alpha(2)-AP variant (Ala-alpha(2)-AP or alpha(2)-AP Enschede), with an alanine insertion in the reactive site sequence converting it from a plasmin inhibitor into a substrate, was also efficiently cleaved by MMP-3 (half-life of 13 min at 37 degrees C and enzyme/substrate ratio of 1:10). Cleavage and inactivation of alpha(2)-AP by MMP-3 may constitute a mechanism favoring local plasmin-mediated proteolysis.     

Kinetics of the reactions between streptokinase, plasmin and alpha 2-antiplasmin.

Streptokinase reacts very rapidly with human plasmin (rate constant 5.4 S 10(7) M-1 s-1) forming a 1:1 stoichiometric complex which has a dissociation constant of 5 X 10(-11) M. This plasmin-streptokinase complex is 10(5) times less reactive towards alpha 2-antiplasmin than plasmin, the inhibition rate constant being 1.4 X 10(2) M-1 s-1. The loss of reactivity of the streptokinase-plasmin complex towards alpha 2-antiplasmin is independent of the lysine binding sites in plasmin since low-Mr plasmin, which lacks these sites, and plasmin in which the sites have been blocked by 6-aminohexanoic acid, are both equally unreactive towards alpha 2-antiplasmin on reaction with streptokinase. The plasmin-streptokinase complex binds to Sepharose-lysine and Sepharose-fibrin monomer in the same fashion as free plasmin, showing that the lysine binding sites are fully exposed in the complex. Bovine plasmin is rapidly inhibited by human alpha 2-antiplasmin (k1 = 1.6 X 10(6) M-1 s-1) and similarly loses reactivity towards the inhibitor on complex formation with streptokinase (50% binding at 0.4 microM streptokinase).     

On the structure of the stable complex between plasmin and alpha-2-antiplasmin

Rabbits were immunized with a conjugate of leukotriene (LT) C₄ and bovine serum albumin prepared by coupling the single free amino group of the hapten to the protein using gluteraldehyde. Binding of [³H]LTC₄ to the antibodies obtained is inhibited by 50% with 1.5 ng LTC₄. The relative cross-reaction of LTD₄ is 16% and of LTC₄-methyl ester 3.6%. The validity of the radioimmunoassay was demonstrated by comparison with bioassay using the isolated guinea pig ileum. Using the radioimmunoassay it could be shown that endogenous LTC₄ is released in a dose-dependent manner by human polymorphonuclear leucocytes stimulated with the divalent cation ionophore A23187.     

Primary structure of human alpha 2-antiplasmin, a serine protease inhibitor (serpin)

The plasma protein alpha 2-antiplasmin is the main physiological inhibitor of the serine protease plasmin, which is responsible for the dissolution of fibrin clots. We have determined the primary structure of mature human alpha 2-antiplasmin by DNA sequencing of overlapping cDNA fragments prepared from human liver mRNA. cDNA clones were identified by hybridization with a 48-base pair deoxyoligonucleotide probe deduced from the sequence of a 16-amino acid peptide of alpha 2-antiplasmin. Mature human alpha 2-antiplasmin contains 452 amino acids. It is homologous (23-28%) with five other proteins belonging to the serine protease inhibitor (serpin) superfamily. Its reactive site, i.e. the peptide bond cleaved by reaction with its primary target enzyme, plasmin, consists of Arg364-Met365. This dipeptide corresponds to the reactive site Met358-Ser359 of the archetypal serpin, alpha 1-antitrypsin.     

On the molecular interactions between plasminogen-staphylokinase, alpha 2-antiplasmin and fibrin.

The molecular interactions between the plasminogen-staphylokinase complex, alpha 2-antiplasmin and fibrin were studied by measuring the effect of CNBr-digested fibrinogen on the inhibition rate of the plasminogen-staphylokinase complex by alpha 2-antiplasmin. The second-order rate constant for the inhibition of plasminogen-staphylokinase by alpha 2-antiplasmin was 2.7 +/- 0.3.10(6) M-1 s-1 (mean +/- S.D.; n = 7). Addition of CNBr-digested fibrinogen, but not of fibrinogen, resulted in a concentration-dependent reduction of the apparent inhibition rate constant, with a 50 percent reduction at a concentration of 5 nM CNBr-digested fibrinogen. The second-order rate constant for the inhibition of the low-Mr plasminogen-staphylokinase complex (plasminogen lacking the kringle structures comprising the lysine-binding sites) by alpha 2-antiplasmin was about 30-fold lower (9.3 +/- 0.7.10(4) M-1 s-1, mean +/- S.D.; n = 4) than that of plasminogen-staphylokinase and was not affected by addition of CNBr-digested fibrinogen. Inhibition of the plasminogen-staphylokinase complex by the chloromethylketone D-Val-Phe-Lys-Ch2Cl is 9-fold less efficient than that of plasmin (k2/Ki of 700 M-1 s-1 versus 6300 M-1 s-1). Our results confirm and establish that rapid inhibition of plasminogen-staphylokinase by alpha 2-antiplasmin requires the availability of the lysine-binding sites in the plasminogen moiety of the complex. Fibrin, but not fibrinogen, reduces the inhibition rate by alpha 2-antiplasmin by competition for interaction with the lysine-binding site. Protection of the plasminogen-staphylokinase complex bound to fibrin from rapid inhibition by alpha 2-antiplasmin thus appears to contribute to the fibrin-specificity of clot lysis with staphylokinase in a plasma milieu, by allowing preferential plasminogen activation at the fibrin surface, while the free complex is rapidly inhibited in plasma.     

Purification of alpha2-macroglobulin and the construction of immunogenic alpha2-macroglobulin-peptide complexes for use as cancer vaccines

A simple method for purification of immunogenic alpha2-macroglobulin from murine serum is described. Also described is a method for creating alpha2-macroglobulin-antigen complexes using purified alpha2-macroglobulin and peptides derived from tumor cell lysates. The application of such alpha2-macroglobulin-antigen complexes for eliciting cancer immunity is discussed.     

Crystal structure of a MAT alpha 2 homeodomain-operator complex suggests a general model for homeodomain-DNA interactions.

The MAT alpha 2 homeodomain regulates the expression of cell type-specific genes in yeast. We have determined the 2.7 A resolution crystal structure of the alpha 2 homeodomain bound to a biologically relevant DNA sequence. The DNA in this complex is contacted primarily by the third of three alpha-helices, with additional contacts coming from an N-terminal arm. Comparison of the yeast alpha 2 and the Drosophila engrailed homeodomain-DNA complexes shows that the protein fold is highly conserved, despite a 3-residue insertion in alpha 2 and only 27% sequence identity between the two homeodomains. Moreover, the orientation of the recognition helix on the DNA is also conserved. This docking arrangement is maintained by side chain contacts with the DNA--primarily the sugar-phosphate backbone--that are identical in alpha 2 and engrailed. Since these residues are conserved among all homeodomains, we propose that the contacts with the DNA are also conserved and suggest a general model for homeodomain-DNA interactions.     

The suramin-treated rat as a model of mucopolysaccharidosis: reversibility of biochemical and morphological changes in the liver

Rats treated with the trypanocidal drug suramin, a potent inhibitor of several lysosomal enzymes, develop a storage disorder which mimics the features of mucopolysaccharidosis (Constantopoulos et al. 1983). In this paper we have examined the reversibility of the biochemical and pathological changes induced in the liver of the suramin-treated rat. Rats were injected with a single intravenous dose of suramin (250 mg/kg) and allowed to survive for periods of up to 6 months. The liver was examined for suramin content, pathological changes, biochemical storage of glycosaminoglycans (GAGs) and for the blockade of the relevant hydrolytic enzymes. GAG storage in the liver peaked at approximately 14 days after administration of suramin when there was a five-fold increase in the GAG content. Thereafter GAGs decreased in parallel with the fall of suramin concentrations so that within 6 months the content had returned to control levels. The activity of most of the enzymes tested had also returned to control levels within 6 months. The pathological changes which are evident in the liver 1-2 weeks after administration of the drug had diminished considerably within 6 months. These results indicate that significant reversibility of both the biochemical and pathological changes induced by suramin occurs and they support the suitability of the suramin treated rat as a model to assess the value of therapeutic treatments of mucopolysaccharidosis.     

The cytochrome c oxidase-azide-nitric oxide complex as a model for the oxygen-binding site

The complex of cytochrome c oxidase with NO and azide has been studied by EPR at 9.2 and 35 GHz. This complex which shows delta ms = 2 EPR triplet and strong anisotropic signals, due to the interaction of cytochrome a2+3 X NO (S = 1/2) and Cu2+B (S = 1/2), is photodissociable . Its action spectrum is similar to that of cytochrome a2+3 X NO with bands at 430, 560 and 595 nm, but shows an additional band in the near ultraviolet region. The quantum yield of the photodissociation process of cytochrome a2+3 X NO in the metal pair appears to depend on the redox state of CuB. When the photolysed sample was warmed to 77 K, a complex was observed with the EPR parameters of cytochrome a3+3 - N-3 - Cu1 +B (S = 1/2). This process of electron and ligand transfer can be reversed by heating the sample to 220 K. It is suggested that in the triplet species azide is bound to Cu2+B whereas NO is bridged between Cu2+B and the haem iron of the cytochrome a2+3. The complex has a triplet ground state and a singlet excited state with an exchange interaction J = -7.1 cm-1 between both spins. The anisotropy in the EPR spectra is mainly due to a magnetic dipole-dipole interaction between cytochrome a2+3 X NO and Cu2+B. From simulations of the triplet EPR spectra obtained at 9 and 35 GHz, a value for the distance between the nitroxide radical and Cu2+B of 0.33 nm was found. A model of the NO binding in the cytochrome a3-Cu pair shows a distance between the haem iron of cytochrome a3 and CuB of 0.45 nm. It is concluded that the cytochrome a3-CuB pair forms a cage in which the dioxygen molecule is bidentate coordinated to the two metals during the catalytic reaction.