Gonzalo Izaguirre

Molecular mechanisms of antithrombin-heparin regulation of blood clotting proteinases. a paradigm for understanding proteinase regulation by serpin family protein proteinase inhibitors

Serpin family protein proteinase inhibitors regulate the activity of serine and cysteine proteinases by a novel conformational trapping mechanism that may itself be regulated by cofactors to provide a finely-tuned time and location-dependent control of proteinase activity. The serpin, antithrombin, together with its cofactors, heparin and heparan sulfate, perform a critical anticoagulant function by preventing the activation of blood clotting proteinases except when needed at the site of a vascular injury. Here, we review the detailed molecular understanding of this regulatory mechanism that has emerged from numerous X-ray crystal structures of antithrombin and its complexes with heparin and target proteinases together with mutagenesis and functional studies of heparin-antithrombin-proteinase interactions in solution. Like other serpins, antithrombin achieves specificity for its target blood clotting proteinases by presenting recognition determinants in an exposed reactive center loop as well as in exosites outside the loop. Antithrombin reactivity is repressed in the absence of its activator because of unfavorable interactions that diminish the favorable RCL and exosite interactions with proteinases. Binding of a specific heparin or heparan sulfate pentasaccharide to antithrombin induces allosteric activating changes that mitigate the unfavorable interactions and promote template bridging of the serpin and proteinase. Antithrombin has thus evolved a sophisticated means of regulating the activity of blood clotting proteinases in a time and location-dependent manner that exploits the multiple conformational states of the serpin and their differential stabilization by glycosaminoglycan cofactors.

Localization of an Antithrombin Exosite That Promotes Rapid Inhibition of Factors Xa and IXa Dependent on Heparin Activation of the Serpin

We have previously shown that exosites in antithrombin outside the P6-P3′ reactive loop region become available upon heparin activation to promote rapid inhibition of the target proteases, factor Xa and factor IXa. To identify these exosites, we prepared six antithrombin-α1-proteinase inhibitor chimeras in which antithrombin residues 224-286 and 310-322 that circumscribe a region surrounding the reactive loop on the inhibitor surface were replaced in 10-16-residue segments with the homologous segments of α1-proteinase inhibitor. All chimeras bound heparin with a high affinity similar to wild-type, underwent heparin-induced fluorescence changes indicative of normal conformational activation, and were able to form SDS-stable complexes with thrombin, factor Xa, and factor IXa and inhibit these proteases with stoichiometries minimally altered from those of wild-type antithrombin. With only one exception, conformational activation of the chimeras with a heparin pentasaccharide resulted in normal ∼100-300-fold enhancements in reactivity with factor Xa and factor IXa. The exception was the chimera in which residues 246-258 were replaced, corresponding to strand 3 of β-sheet C, which showed little or no enhancement of its reactivity with these proteases following pentasaccharide activation. By contrast, all chimeras including the strand 3C chimera showed essentially wild-type reactivities with thrombin after pentasaccharide activation as well as normal full-length heparin enhancements in reactivity with all proteases due to heparin bridging. These findings suggest that antithrombin exosites responsible for enhancing the rates of factor Xa and factor IXa inhibition in the conformationally activated inhibitor lie in strand 3 of β-sheet C of the serpin.

Residues Tyr253 and Glu255 in Strand 3 of β-Sheet C of Antithrombin Are Key Determinants of an Exosite Made Accessible by Heparin Activation to Promote Rapid Inhibition of Factors Xa and IXa

We previously showed that conformational activation of the anticoagulant serpin, antithrombin, by heparin generates new exosites in strand 3 of β-sheet C, which promote the reaction of the inhibitor with the target proteases, factor Xa and factor IXa. To determine which residues comprise the exosites, we mutated strand 3C residues that are conserved in all vertebrate antithrombins. Combined mutations of the three conserved surface-accessible residues, Tyr253,Glu255, and Lys257, or of just Tyr253 and Glu255, but not any of these residues alone, was sufficient to reproduce the exosite defects of a strand 3C antithrombin-α1-proteinase inhibitor chimera in reactions of the heparin-activated variants with both factor Xa and factor IXa. Importantly, the exosite-defective antithrombins bound heparin with nearly wild-type affinities, and the heparin-activated mutants showed near normal reactivities with thrombin, a protease that does not utilize the exosite. Mutation of the conserved but partially buried strand 3C residue, Gln254, the reactive loop P6′ residue, Arg399, which interacts with Glu255, or a residue proposed to constitute the exosite from modeling studies, Glu237, all produced minimal effects on antithrombin reactivity with thrombin, factor Xa, and factor IXa in the absence or presence of heparin. Together, these results indicate that Tyr253 and Glu255 are key exosite determinants responsible for promoting the reactions of conformationally activated antithrombin with both factor Xa and factor IXa.

Tyrosine Phosphorylation of α-Actinin in Activated Platelets

The integrin αIIbβ3 mediates tyrosine phosphorylation of a 105-kDa protein (pp105) in activated platelets. We have partially purified a 105-kDa tyrosine-phosphorylated protein from platelets stimulated with phorbol 12-myristate 13-acetate and obtained the sequence of an internal 12-mer peptide derived from this protein. The sequence was identical to human α-actinin sequences deposited in the Swiss Protein Database. α-Actinin, a 105-kDa protein in platelets, was subsequently purified from activated platelets by four sequential chromatographic steps. Fractions were analyzed by Western blotting and probed with α-actinin and anti-phosphotyrosine antibodies. The distribution of α-actinin and pp105 overlapped throughout the purification. Furthermore, in the course of this purification, a 105-kDa tyrosine-phosphorylated protein was only detected in fractions that contained α-actinin. The purified α-actinin protein was immunoprecipitated with antibodies to phosphotyrosine in the absence but not in the presence of phenyl phosphate. α-Actinin resolved by two-dimensional gel electrophoresis of activated platelet lysates was recognized by the antibodies to phosphotyrosine, whereas pretreatment of the platelets with bisindolylmaleimide, a protein kinase C inhibitor that prevents tyrosine phosphorylation of pp105, inhibited the reactivity of the antibodies to phosphotyrosine with α-actinin. Taken together, these data demonstrate that a fraction of α-actinin is tyrosine-phosphorylated in activated platelets.

Metabolism of retinaldehyde and other aldehydes in soluble extracts of human liver and kidney.

Purification and characterization of enzymes metabolizing retinaldehyde, propionaldehyde, and octanaldehyde from four human livers and three kidneys were done to identify enzymes metabolizing retinaldehyde and their relationship to enzymes metabolizing other aldehydes. The tissue fractionation patterns from human liver and kidney were the same, indicating presence of the same enzymes in human liver and kidney. Moreover, in both organs the major NAD+-dependent retinaldehyde activity copurified with the propionaldehyde and octanaldehyde activities; in both organs the major NAD+-dependent retinaldehyde activity was associated with the E1 isozyme (coded for byaldh1 gene) of human aldehyde dehydrogenase. A small amount of NAD+-dependent retinaldehyde activity was associated with the E2 isozyme (product of aldh2 gene) of aldehyde dehydrogenase. Some NAD+-independent retinaldehyde activity in both organs was associated with aldehyde oxidase, which could be easily separated from dehydrogenases. Employing cellular retinoid-binding protein (CRBP), purified from human liver, demonstrated that E1 isozyme (but not E2 isozyme) could utilize CRBP-bound retinaldehyde as substrate, a feature thought to be specific to retinaldehyde dehydrogenases. This is the first report of CRBP-bound retinaldehyde functioning as substrate for aldehyde dehydrogenase of broad substrate specificity. Thus, it is concluded that in the human organism, retinaldehyde dehydrogenase (coded for by raldH1gene) and broad substrate specificity E1 (a member of EC 1.2.1.3aldehyde dehydrogenase family) are the same enzyme. These results suggest that the E1 isozyme may be more important to alcoholism than the acetaldehyde-metabolizing enzyme, E2, because competition between acetaldehyde and retinaldehyde could result in abnormalities associated with vitamin A metabolism and alcoholism.

Mechanism by Which Exosites Promote the Inhibition of Blood Coagulation Proteases by Heparin-activated Antithrombin

Heparin activates the serpin, antithrombin, to inhibit its target blood-clotting proteases by generating new protease interaction exosites. To resolve the effects of these exosites on the initial Michaelis docking step and the subsequent acylation and conformational change steps of antithrombin-protease reactions, we compared the reactions of catalytically inactive S195A and active proteases with site-specific fluorophore-labeled antithrombins that allow monitoring of these reaction steps. Heparin bound to N,N′-dimethyl-N-(acetyl)-N′-(7-nitrobenz-3-oxa-1,3-diazol-4-yl)ethylenediamine (NBD)-fluorophore-labeled antithrombins and accelerated the reactions of the labeled inhibitor with thrombin and factor Xa similar to wild type. Equilibrium binding of NBD-labeled antithrombins to S195A proteases showed that exosites generated by conformationally activating antithrombin with a heparin pentasaccharide enhanced the affinity of the serpin for S195A factor Xa minimally 100-fold. Moreover, additional bridging exosites provided by a hexadecasaccharide heparin activator enhanced antithrombin affinity for both S195A factor Xa and thrombin at least 1000-fold. Rapid kinetic studies showed that these exosite-mediated enhancements in Michaelis complex affinity resulted from increases in kon and decreases in koff and caused antithrombin-protease reactions to become diffusion-controlled. Competitive binding and kinetic studies with exosite mutant antithrombins showed that Tyr-253 was a critical mediator of exosite interactions with S195A factor Xa; that Glu-255, Glu-237, and Arg-399 made more modest contributions to these interactions; and that exosite interactions reduced koff for the Michaelis complex interaction. Together these results show that exosites generated by heparin activation of antithrombin function both to promote the formation of an initial antithrombin-protease Michaelis complex and to favor the subsequent acylation of this complex.

Tissue Distribution of Human Aldehyde Dehydrogenase E3 (ALDH9): Comparison of Enzyme Activity with E3 Protein and mRNA Distribution

The tissue distribution of the E3 isozyme of human aldehyde dehydrogenase has been investigated by three methods: enzyme activity assay employing betaine aldehyde as substrate, Western blotting employing E3 isozyme-specific antibodies, and Northern blotting using a human liver E3 cDNA as probe. All three methods showed that E3 isozyme was universally distributed among all tissues tested. The highest levels of the E3 isozyme activity were found in liver, adrenal gland, and kidney. These same tissues also showed highest levels of the E3 protein via the Western blot. This distribution is consistent with the possible physiological role of E3 isozyme in the synthesis of the osmolyte, betaine, and the neurotransmitter, GABA. Northern blot analysis, however, differed from that of enzyme assay and the Western blot in that it showed highest mRNA levels in skeletal and heart muscles, which had low enzyme activities and E3 protein levels.

Methylglyoxal as substrate and inhibitor of human aldehyde dehydrogenase: comparison of kinetic properties among the three isozymes.

Methylglyoxal was demonstrated to be a substrate for the isozymes E1, E2 and E3 of human aldehyde dehydrogenase. Pyruvate was the product from the oxidation of methylglyoxal by the three isozymes. At pH 7.4 and 25 degrees C, the major and minor components of the E3 isozyme catalyzed the reaction with Vmax of 1.1 and 0.8 mumol NADH min-1 mg-1 protein, respectively, compared to 0.067 and 0.060 mumol NADH min-1 mg-1 protein for the E1 and E2 isozymes, respectively. The E2 isozyme had a K(m) for methylglyoxal of 8.6 microM, the lowest compared to 46 microM for E1 and 586 and 552 microM for the major and minor components of the E3 isozyme, respectively. Both components of the E3 isozyme showed substrate inhibition by methylglyoxal, with Ki values of 2.0 mM for the major component and 12 mM for the minor component at pH 9.0. Substrate inhibition by methylglyoxal was not observed with the E1 and E2 isozymes. Methylglyoxal strongly inhibited the glycolaldehyde activity of the E1 and E2 isozymes. Mixed-type models of inhibition were employed as an approach to calculate the inhibition constants, 44 and 10.6 microM for E1 and E2 isozymes, respectively.

Engineering Functional Antithrombin Exosites in α1-Proteinase Inhibitor That Specifically Promote the Inhibition of Factor Xa and Factor IXa

We have previously shown that residues Tyr-253 and Glu-255 in the serpin antithrombin function as exosites to promote the inhibition of factor Xa and factor IXa when the serpin is conformationally activated by heparin. Here we show that functional exosites can be engineered at homologous positions in a P1 Arg variant of the serpin α1-proteinase inhibitor (α1PI) that does not require heparin for activation. The combined effect of the two exosites increased the association rate constant for the reactions of α1PI with factors Xa and IXa 11–14-fold, comparable with their rate-enhancing effects on the reactions of heparin-activated antithrombin with these proteases. The effects of the engineered exosites were specific, α1PI inhibitor reactions with trypsin and thrombin being unaffected. Mutation of Arg-150 in factor Xa, which interacts with the exosite residues in heparin-activated antithrombin, abrogated the ability of the engineered exosites in α1PI to promote factor Xa inhibition. Binding studies showed that the exosites enhance the Michaelis complex interaction of α1PI with S195A factor Xa as they do with the heparin-activated antithrombin interaction. Replacement of the P4-P2 AIP reactive loop residues in the α1PI exosite variant with a preferred IEG substrate sequence for factor Xa modestly enhanced the reactivity of the exosite mutant inhibitor with factor Xa by ∼2-fold but greatly increased the selectivity of α1PI for inhibiting factor Xa over thrombin by ∼1000-fold. Together, these results show that a specific and selective inhibitor of factor Xa can be engineered by incorporating factor Xa exosite and reactive site recognition determinants in a serpin.

Conformational activation of antithrombin by heparin involves an altered exosite interaction with protease

Background: Exosites are known to mediate heparin allosteric activation of antithrombin. Results: Mutagenesis revealed that an exosite differentially contributes to antithrombin reactivity with factors Xa/IXa in unactivated and heparin-activated states. Conclusion: Heparin allosteric activation of antithrombin results from alterations in an exosite interaction with protease induced by core conformational changes. Significance: The findings support our recently proposed model of antithrombin allosteric activation. Heparin allosterically activates antithrombin as an inhibitor of factors Xa and IXa by enhancing the initial Michaelis complex interaction of inhibitor with protease through exosites. Here, we investigate the mechanism of this enhancement by analyzing the effects of alanine mutations of six putative antithrombin exosite residues and three complementary protease exosite residues on antithrombin reactivity with these proteases in unactivated and heparin-activated states. Mutations of antithrombin Tyr253 and His319 exosite residues produced massive 10–200-fold losses in reactivity with factors Xa and IXa in both unactivated and heparin-activated states, indicating that these residues made critical attractive interactions with protease independent of heparin activation. By contrast, mutations of Asn233, Arg235, Glu237, and Glu255 exosite residues showed that these residues made both repulsive and attractive interactions with protease that depended on the activation state and whether the critical Tyr253/His319 residues were mutated. Mutation of factor Xa Arg143, Lys148, and Arg150 residues that interact with the exosite in the x-ray structure of the Michaelis complex confirmed the importance of all residues for heparin-activated antithrombin reactivity and Arg150 for native serpin reactivity. These results demonstrate that the exosite is a key determinant of antithrombin reactivity with factors Xa and IXa in the native as well as the heparin-activated state and support a new model of allosteric activation we recently proposed in which a balance between attractive and repulsive exosite interactions in the native state is shifted to favor the attractive interactions in the activated state through core conformational changes induced by heparin binding.