[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Pathologic blood clotting is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Genetic predisposition to thrombosis is still poorly understood, and we hypothesize that there are many additional risk alleles and modifying factors remaining to be discovered. Mammalian models have contributed to our understanding of thrombosis, but are low throughput and costly. We have turned to the zebrafish, a tool for high throughput genetic analysis. Using zinc finger nucleases, we show that disruption of the zebrafish antithrombin III (at3) locus results in spontaneous venous thrombosis in larvae. Though homozygous mutants survive into early adulthood, they eventually succumb to massive intracardiac thrombosis. Characterization of null fish revealed disseminated intravascular coagulation in larvae secondary to unopposed thrombin activity and fibrinogen consumption, which could be rescued by both human and zebrafish at3 cDNAs. Mutation of the human AT3 reactive center loop abolished the ability to rescue, but the heparin-binding site was dispensable. These results demonstrate overall conservation of AT3 function in zebrafish, but reveal developmental variances in the ability to tolerate excessive clot formation. The accessibility of early zebrafish development will provide unique methods for dissection of the underlying mechanisms of thrombosis.
[Show abstract][Hide abstract] ABSTRACT: Deoxycytidine kinase (dCK) is a key enzyme in the nucleoside salvage pathway that is also required for the activation of several anticancer and antiviral nucleoside analog prodrugs. Additionally, dCK has been implicated in immune disorders and has been found to be overexpressed in several cancers. To allow the probing and modulation of dCK activity, a new class of small-molecule inhibitors of the enzyme were developed. Here, the structural characterization of four of these inhibitors in complex with human dCK is presented. The structures reveal that the compounds occupy the nucleoside-binding site and bind to the open form of dCK. Surprisingly, a slight variation in the nature of the substituent at the 5-position of the thiazole ring governs whether the active site of the enzyme is occupied by one or two inhibitor molecules. Moreover, this substituent plays a critical role in determining the affinity, improving it from >700 to 1.5 nM in the best binder. These structures lay the groundwork for future modifications that would result in even tighter binding and the correct placement of moieties that confer favorable pharmacodynamics and pharmacokinetic properties.
[Show abstract][Hide abstract] ABSTRACT: Allosteric conformational changes in antithrombin induced by binding a specific heparin pentasaccharide result in very large increases in the rates of inhibition of factors IXa and Xa, though not of thrombin. These are accompanied by CD, fluorescence and NMR spectroscopic changes. X-ray structures show that heparin binding results in extension of helix D in the region 131-136 with coincident, and possibly coupled, expulsion of the hinge of the reactive center loop (RCL). To examine the importance of helix D extension we have introduced strongly helix-promoting mutations in the 131-136 region of antithombin (YRKAQK to LEEAAE). The resulting variant has endogenous fluorescence indistinguishable from WT antithrombin, yet, in the absence of heparin, shows massive enhancements in rates of inhibition of factors IXa and Xa (114-fold and 110-fold respectively), though not of thrombin, together with changes in near and far UV CD and 1H NMR spectra. Heparin binding gives only ~3-4 fold further rate enhancement, but increases tryptophan fluorescence by ~23% without major additional CD or NMR changes. Variants with subsets of these mutations show intermediate activation in the absence of heparin; again with basal fluorescence similar to WT and large increases upon heparin binding. These findings suggest that in WT antithrombin there are two major complementary sources of conformational activation of antithrombin, probably involving altered contacts of side chains of Tyr 131 and Ala 134 with core hydrophobic residues, while reactive center loop hinge expulsion plays only a minor additional role.
[Show abstract][Hide abstract] ABSTRACT: Serpin protein protease inhibitors inactivate their target proteases through a unique mechanism in which a major serpin conformational change, resulting in a 70Å translocation of the protease from its initial reactive center loop (RCL) docking site to the opposite pole of the serpin, kinetically traps the acyl-intermediate complex. While the initial Michaelis and final trapped acyl-intermediate complexes have been well characterized structurally, the intermediate stages involved in this remarkable transformation are not well understood. To better characterize such intermediate steps, we undertook rapid kinetic studies of the FRET and fluorescence perturbation changes of site-specific fluorophore-labeled derivatives of the serpin, α1-protease inhibitor (α1PI), that report the serpin and protease conformational changes involved in transforming the Michaelis complex to the trapped acyl-intermediate complex in reactions with trypsin. Two kinetically resolvable conformational changes were observed in the reactions, ascribable to i) serpin RCL insertion into sheet A with full protease translocation but incomplete protease distortion followed by ii) full conformational distortion and movement of the protease and coupled serpin conformational changes involving the F helix-sheet A interface. Kinetic studies of calcium effects on the labeled α1PI-trypsin reactions demonstrated both inactive and low activity states of the distorted protease in the final complex that were distinct from the intermediate distorted state. These studies provide new insights into the nature of the serpin and protease conformational changes involved in trapping the acyl-intermediate complex in serpin-protease reactions and support a previously proposed role for helix F in the trapping mechanism.
Journal of Biological Chemistry 09/2013; DOI:10.1074/jbc.M113.510990 · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: α1PDX is an engineered serpin family inhibitor of the proprotein convertase (PC), furin, that exhibits high specificity but limited selectivity for inhibiting furin over other PC family proteases. Here, we characterize serpin B8, a natural inhibitor of furin, together with α1PDX-serpin B8 and furin-PC chimeras to identify determinants of serpin specificity and selectivity for furin inhibition. Replacing reactive center loop (RCL) sequences of α1PDX with those of serpin B8 demonstrated that both the P1-P4 RXXR recognition sequence as well as the P1'-P5' sequence are critical determinants of serpin specificity for furin. Alignments of PC catalytic domains revealed four variable active-site loops whose role in furin reactivity with serpin B8 was tested by engineering furin-PC loop chimeras. The furin 298-300 loop but not the other loops differentially affected furin reactivity with serpin B8 and α1PDX in a manner that depended on the serpin RCL primed sequence. Modeling of the serpin B8-furin Michaelis complex identified serpin exosites in strand 3C close to the 298-300 loop whose substitution in α1PDX differentially affected furin reactivity depending on the furin loop and serpin RCL primed sequences. These studies demonstrate that RCL primed residues, strand 3C exosites and the furin 298-300 loop are critical determinants of serpin reactivity with furin which may be exploited in the design of specific and selective α1PDX inhibitors of PCs.
Journal of Biological Chemistry 06/2013; DOI:10.1074/jbc.M113.462804 · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI), is catalytically activated by its cofactor, protein Z (PZ), to regulate the function of blood coagulation factor Xa on membrane surfaces. The X-ray structure of the ZPI-PZ complex has shown that PZ binds to a unique site on ZPI centered on helix G. In the present study, we show by Ala-scanning mutagenesis of the ZPI-binding interface, together with native PAGE and kinetic analyses of PZ binding to ZPI, that Tyr240 and Asp293 of ZPI are crucial hot spots for PZ binding. Complementary studies with protein Z-protein C chimeras show the importance of both pseudocatalytic and EGF2 domains of PZ for the critical ZPI interactions. To understand how PZ acts catalytically, we analyzed the interaction of reactive loop-cleaved ZPI (cZPI) with PZ and determined the cZPI X-ray structure. The cZPI structure revealed changes in helices A and G of the PZ-binding site relative to native ZPI that rationalized an observed 6-fold loss in PZ affinity and PZ catalytic action. These findings identify the key determinants of catalytic activation of ZPI by PZ and suggest novel strategies for ameliorating hemophilic states through drugs that disrupt the ZPI-PZ interaction.
[Show abstract][Hide abstract] ABSTRACT: The persistence of memory T lymphocytes confers lifelong protection from pathogens. Memory T cells survive and undergo homeostatic proliferation (HSP) in the absence of Ag, although the cell-intrinsic mechanisms by which cytokines drive the HSP of memory T cells are not well understood. In this study we report that lysosome stability limits the long-term maintenance of memory CD8(+) T cell populations. Serine protease inhibitor (Spi) 2A, an anti-apoptotic cytosolic cathepsin inhibitor, is induced by both IL-15 and IL-7. Mice deficient in Spi2A developed fewer memory phenotype CD44(hi)CD8(+) T cells with age, which underwent reduced HSP in the bone marrow. Spi2A was also required for the maintenance of central memory CD8(+) T cell populations after acute infection with lymphocytic choriomeningitis virus. Spi2A-deficient Ag-specific CD8(+) T cell populations declined more than wild-type competitors after viral infection, and they were eroded further after successive infections. Spi2A protected memory cells from lysosomal breakdown by inhibiting cathepsin B. The impaired maintenance of Spi2A-deficient memory CD8(+) T cells was rescued by concomitant cathepsin B deficiency, demonstrating that cathepsin B was a physiological target of Spi2A in memory CD8(+) T cell survival. Our findings support a model in which protection from lysosomal rupture through cytokine-induced expression of Spi2A determines the long-term persistence of memory CD8(+) T cells.
The Journal of Immunology 06/2012; 189(3):1133-43. DOI:10.4049/jimmunol.1003406 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High-molecular weight heparins promote the protein Z-dependent protease inhibitor (ZPI) inhibition of factors Xa (FXa) and XIa (FXIa) by a template mechanism. To map the heparin-binding site of ZPI, the role of basic residues of the D-helix (residues Lys-113, Lys-116, and Lys-125) in the interaction with heparin was evaluated by either substituting these residues with Ala (ZPI-3A) or replacing the D-helix with the corresponding loop of the non-heparin-binding serpin α(1)-proteinase inhibitor (ZPI-D-helix(α1-PI)). Furthermore, both the C-helix (contains two basic residues, Lys-104 and Arg-105) and the D-helix of ZPI were substituted with the corresponding loops of α(1)-proteinase inhibitor (ZPI-CD-helix(α1-PI)). All mutants exhibited near normal reactivity with FXa and FXIa in the absence of cofactors and in the presence of protein Z and membrane cofactors. By contrast, the mutants interacted with heparin with a lower affinity and the ~48-fold heparin-mediated enhancement in the rate of FXa inhibition by ZPI was reduced to ~30-fold for ZPI-3A, ~15-fold for ZPI-D-helix(α1-PI), and ~8-fold for ZPI-CD-helix(α1-PI). Consistent with a template mechanism for heparin cofactor action, ZPI-CD-helix(α1-PI) inhibition of a FXa mutant containing a mutation in the heparin-binding site (FXa-R240A) was minimally affected by heparin. A significant decrease (~2-5-fold) in the heparin template effect was also observed for the inhibition of FXIa by ZPI mutants. Interestingly, ZPI derivatives exhibited a markedly elevated stoichiometry of inhibition with FXIa in the absence of heparin. These results suggest that basic residues of both helices C and D of ZPI interact with heparin to modulate the inhibitory function of the serpin.
[Show abstract][Hide abstract] ABSTRACT: EP217609 is a new dual-action parenteral anticoagulant that combines an indirect factor Xa inhibitor (fondaparinux analog) and a direct thrombin inhibitor (α-NAPAP analog) in a single molecule together with a biotin tag to allow avidin neutralization. EP217609 exhibits an unprecedented pharmacologic profile in showing high bioavailability, long plasma half-life, and potent antithrombotic activity in animals without the complications of thrombin rebound. Here we report the exceptional specificity and selectivity profile of EP217609. EP217609 inhibited thrombin with rapid kinetics (k(on) > 10(7)M(-1)s(-1)), a high affinity (K(I) = 30-40pM), and more than 1000-fold selectivity over other coagulation and fibrinolytic protease targets, comparing favorably with the best direct thrombin inhibitors known. EP217609 bound antithrombin with high affinity (K(D) = 30nM) and activated the serpin to rapidly (k(ass) ∼ 10(6)M(-1)s(-1)) and selectively (> 20-fold) inhibit factor Xa. The dual inhibitor moieties of EP217609 acted largely independently with only modest linkage effects of ligand occupancy of one inhibitor moiety on the potency of the other (∼ 5-fold). In contrast, avidin binding effectively neutralized the potency of both inhibitor moieties (20- to 100-fold). These findings demonstrate the superior anticoagulant efficacy and rapid avidin neutralizability of EP217609 compared with anticoagulants that target thrombin or factor Xa alone.
[Show abstract][Hide abstract] ABSTRACT: Antithrombin, a major anticoagulant, is robustly transported into extravascular compartments where its target proteases are largely unknown. This serpin was previously detected in human milk as complexes with matriptase, a membrane-bound serine protease broadly expressed in epithelial and carcinoma cells, and under tight regulation by hepatocyte growth factor activator inhibitor (HAI)-1, a transmembrane Kunitz-type serine protease inhibitor that forms heat-sensitive complexes with active matriptase. In the current study, we detect, in addition to matriptase-HAI-1 complexes, heat-resistant matriptase complexes generated by nontransformed mammary, prostate, and epidermal epithelial cells that we show to be matriptase-antithrombin complexes. These findings suggest that in addition to HAI-1, interstitial antithrombin participates in the regulation of matriptase activity in epithelial cells. This physiological mechanism appears, however, to largely be lost in cancer cells since matriptase-antithrombin complexes were not detected in all but two of a panel of seven breast, prostate, and ovarian cancer cell lines. Using purified active matriptase, we further characterize the formation of matriptase-antithrombin complex and show that heparin can significantly potentiate the inhibitory potency of antithrombin against matriptase. Second-order rate constants for the inhibition were determined to be 3.9 × 10(3) M(-1)s(-1) in the absence of heparin and 1.2 × 10(5) M(-1)s(-1) in the presence of heparin, a 30-fold increase, consistent with the established role of heparin in activating antithrombin function. Taken together these data suggest that normal epithelial cells employ a dual mechanism involving HAI-1 and antithrombin to control matriptase and that the antithrombin-based mechanism appears lost in the majority of carcinoma cells.
[Show abstract][Hide abstract] ABSTRACT: Protein Z-dependent protease inhibitor (ZPI) is a recently identified member of the serpin superfamily that functions as a cofactor-dependent regulator of blood coagulation factors Xa and XIa. Here we provide evidence that, in addition to the established cofactors, protein Z, lipid, and calcium, heparin is an important cofactor of ZPI anticoagulant function. Heparin produced 20-100-fold accelerations of ZPI reactions with factor Xa and factor XIa to yield second order rate constants approaching the physiologically significant diffusion limit (k(a) = 10(6) to 10(7) M(-1) s(-1)). The dependence of heparin accelerating effects on heparin concentration was bell-shaped for ZPI reactions with both factors Xa and XIa, consistent with a template-bridging mechanism of heparin rate enhancement. Maximal accelerations of ZPI-factor Xa reactions required calcium, which augmented the heparin acceleration by relieving Gla domain inhibition as previously shown for heparin bridging of the antithrombin-factor Xa reaction. Heparin acceleration of both ZPI-protease reactions was optimal at heparin concentrations and heparin chain lengths comparable with those that produce physiologically significant rate enhancements of other serpin-protease reactions. Protein Z binding to ZPI minimally affected heparin rate enhancements, indicating that heparin binds to a distinct site on ZPI and activates ZPI in its physiologically relevant complex with protein Z. Taken together, these results suggest that whereas protein Z, lipid, and calcium cofactors promote ZPI inhibition of membrane-associated factor Xa, heparin activates ZPI to inhibit free factor Xa as well as factor XIa and therefore may play a physiologically and pharmacologically important role in ZPI anticoagulant function.
[Show abstract][Hide abstract] ABSTRACT: The serpins comprise an ancient superfamily of proteins, found abundantly in eukaryotes and even in some bacteria and archea, that have evolved to regulate proteases of both serine and cysteine mechanistic classes. Unlike the thermodynamically determined lock-and-key type inhibitors, such as those of the Kunitz and Kazal families, serpins use conformational change and consequent kinetic trapping of an enzyme intermediate to effect inhibition. By combining interactions of both an exposed reactive center loop and exosites outside this loop with the active site and complementary exosites on the target protease, serpins can achieve remarkable specificity. Together with the frequent use of regulatory cofactors, this permits a sophisticated time- and location-dependent mode of protease regulation. An understanding of the structure and function of serpins has suggested that they may provide novel scaffolds for engineering protease inhibitors of desired specificity for therapeutic use.
Progress in molecular biology and translational science 01/2011; 99:185-240. DOI:10.1016/B978-0-12-385504-6.00005-1 · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The serpin, antithrombin, requires allosteric activation by a sequence-specific pentasaccharide unit of heparin or heparan sulfate glycosaminoglycans to function as an anticoagulant regulator of blood clotting proteases. Surprisingly, X-ray structures have shown that the pentasaccharide produces similar induced-fit changes in the heparin binding site of native and latent antithrombin despite large differences in the heparin affinity and global conformation of these two forms. Here we present kinetic evidence for similar induced-fit mechanisms of pentasaccharide binding to native and latent antithrombins and kinetic simulations which together support a three-step mechanism of allosteric activation of native antithrombin involving two successive conformational changes. Equilibrium binding studies of pentasaccharide interactions with native and latent antithrombins and the salt dependence of these interactions suggest that each conformational change is associated with distinct spectroscopic changes and is driven by a progressively better fit of the pentasaccharide in the binding site. The observation that variant antithrombins that cannot undergo the second conformational change bind the pentasaccharide like latent antithrombin and are partially activated suggests that both conformational changes contribute to allosteric activation, in agreement with a recently proposed model of allosteric activation.
Archives of Biochemistry and Biophysics 12/2010; 504(2):169-76. DOI:10.1016/j.abb.2010.08.021 · 3.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The serpin ZPI is a protein Z (PZ)-dependent specific inhibitor of membrane-associated factor Xa (fXa) despite having an unfavorable P1 Tyr. PZ accelerates the inhibition reaction approximately 2000-fold in the presence of phospholipid and Ca(2+). To elucidate the role of PZ, we determined the x-ray structure of Gla-domainless PZ (PZ(DeltaGD)) complexed with protein Z-dependent proteinase inhibitor (ZPI). The PZ pseudocatalytic domain bound ZPI at a novel site through ionic and polar interactions. Mutation of four ZPI contact residues eliminated PZ binding and membrane-dependent PZ acceleration of fXa inhibition. Modeling of the ternary Michaelis complex implicated ZPI residues Glu-313 and Glu-383 in fXa binding. Mutagenesis established that only Glu-313 is important, contributing approximately 5-10-fold to rate acceleration of fXa and fXIa inhibition. Limited conformational change in ZPI resulted from PZ binding, which contributed only approximately 2-fold to rate enhancement. Instead, template bridging from membrane association, together with previously demonstrated interaction of the fXa and ZPI Gla domains, resulted in an additional approximately 1000-fold rate enhancement. To understand why ZPI has P1 tyrosine, we examined a P1 Arg variant. This reacted at a diffusion-limited rate with fXa, even without PZ, and predominantly as substrate, reflecting both rapid acylation and deacylation. P1 tyrosine thus ensures that reaction with fXa or most other arginine-specific proteinases is insignificant unless PZ binds and localizes ZPI and fXa on the membrane, where the combined effects of Gla-Gla interaction, template bridging, and interaction of fXa with Glu-313 overcome the unfavorability of P1 Tyr and ensure a high rate of reaction as an inhibitor.
[Show abstract][Hide abstract] ABSTRACT: Allosteric activation of antithrombin as a rapid inhibitor of factors IXa and Xa requires binding of a high-affinity heparin pentasaccharide. The currently-accepted mechanism involves removal of a constraint on the antithrombin reactive center loop (RCL) so that the proteinase can simultaneously engage both the P1 arginine and an exosite at Y253. Recent results suggest that this mechanism is incorrect in that activation can be achieved without loop expulsion, while the exosite can be engaged in both low and high activity states. We propose a quite different mechanism in which heparin activates antithrombin by mitigating an unfavorable surface interaction, by altering its nature, and by moving the attached proteinase away from the site of the unfavorable interaction through RCL expulsion.
[Show abstract][Hide abstract] ABSTRACT: Heparin and heparan sulfate glycosaminoglycans allosterically activate the serpin, antithrombin, by binding through a specific pentasaccharide sequence containing a critical 3-O-sulfo group. To elucidate the role of the 3-O-sulfo group in the activation mechanism, we compared the effects of deleting the 3-O-sulfo group or mutating the Lys(114) binding partner of this group on antithrombin-pentasaccharide interactions by equilibrium binding and rapid kinetic analyses. Binding studies over a wide range of ionic strength and pH showed that loss of the 3-O-sulfo group caused a massive approximately 60% loss in binding energy for the antithrombin-pentasaccharide interaction due to the disruption of a cooperative network of ionic and nonionic interactions. Despite this affinity loss, the 3-O-desulfonated pentasaccharide retained the ability to induce tryptophan fluorescence changes and to enhance factor Xa reactivity in antithrombin, indicative of normal conformational activation. Rapid kinetic studies showed that loss of the 3-O-sulfo group affected both the ability of the pentasaccharide to recognize native antithrombin and its ability to preferentially bind and stabilize activated antithrombin. By contrast, mutation of Lys(114) solely affected the preferential interaction of the pentasaccharide with activated antithrombin. These findings demonstrate that the 3-O-sulfo group functions as a key determinant of heparin pentasaccharide activation of antithrombin both by contributing to the Lys(114)-independent recognition of native antithrombin and by triggering a Lys(114)-dependent induced fit interaction with activated antithrombin that locks the serpin in the activated state.
[Show abstract][Hide abstract] ABSTRACT: Serpins form an enormous superfamily of 40–60-kDa proteins found in almost all types of organisms, including humans. Most
are one-use suicide substrate serine and cysteine proteinase inhibitors that have evolved to finely regulate complex proteolytic
pathways, such as blood coagulation, fibrinolysis, and inflammation. Despite distinct functions for each serpin, there is
much redundancy in the primary specificity-determining residues. However, many serpins exploit additional exosites to generate
the exquisite specificity that makes a given serpin effective only when certain other criteria, such as the presence of specific
cofactors, are met. With a focus on human serpins, this minireview examines use of exosites by nine serpins in the initial
complex-forming phase to modulate primary specificity in either binary serpin-proteinase complexes or ternary complexes that
additionally employ a protein or other cofactor. A frequent theme is down-regulation of inhibitory activity unless the exosite(s)
are engaged. In addition, the use of exosites by maspin and plasminogen activator inhibitor-1 to indirectly affect proteolytic
processes is considered.