Ecotin is a potent inhibitor of the contact system proteases factor XIIa and plasma kallikrein

Ulmer, Jana S.; Lindquist, Robert N.; Dennis, Mark S.; Lazarus, Robert A.

doi:10.1016/0014-5793(95)00466-m

Cited by 47 publications

(32 citation statements)

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“…coagulation cascade and other enzymes, such as kallikrein, fiddler crab collagenase, and granzyme B (Chung et al, 1983;Seymour et al, 1994;Tsu et al, 1994;McGrath et al, 1995). The reactive site of ecotin was determined to be Met 84 for its complexes with chymotrypsin, trypsin, porcine pancreatic elastase, factor Xa, factor XIIa, and plasma kallikrein (McGrath et al, 1991b;Seymour et al, 1994;Ulmer et al, 1995). Although the specificity of substratelike canonical serine protease inhibitors is determined mostly by their P1 residue that fits into the binding pocket of the cognate protease, the P1 reactive site methionine residue of ecotin was proved not to be crucial for its specificity toward target proteases.…”

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confidence: 99%

Crystal structure analyses of uncomplexed ecotin in two crystal forms: Implications for its function and stability

et al. 1996

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Ecotin, a homodimeric protein composed of 142 residue subunits, is a novel serine protease inhibitor present in Escherichia coli. Its thermostability and acid stability, as well as broad specificity toward proteases, make it an interesting protein for structural characterization. Its structure in the uncomplexed state, determined for two different crystalline environments, allows a structural comparison of the free inhibitor with that in complex with trypsin. Although there is no gross structural rearrangement of ecotin when binding trypsin, the loops involved in binding trypsin show relatively large shifts in atomic positions. The inherent flexibility of the loops and the highly nonglobular shape are the two features essential for its inhibitory function. An insight into the understanding of the structural basis of thermostability and acid stability of ecotin is also provided by the present structure.

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confidence: 99%

Crystal structure analyses of uncomplexed ecotin in two crystal forms: Implications for its function and stability

et al. 1996

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“…Recently, ecotin, a 142-residue protein from Escherichia coli , has been shown to potently inhibit plasma kallikrein with a K i of ϳ160 pM (6); however, ecotin is not totally selective and also potently inhibits Factor XIIa, Factor Xa, and human leukocyte elastase (7). Bovine pancreatic trypsin inhibitor (BPTI, aprotinin) is a well studied member of the Kunitz domain family of serine protease inhibitors that moderately inhibits plasma kallikrein with K i of ϳ30 nM (8 -10).…”

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Potent and Selective Kunitz Domain Inhibitors of Plasma Kallikrein Designed by Phage Display

Dennis¹,

Herzka²,

Lazarus³

1995

Journal of Biological Chemistry

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Phage displaying APPI Kunitz domain libraries have been used to design potent and selective active site inhibitors of human plasma kallikrein, a serine protease that plays an important role in both inflammation and coagulation. Selected clones from two Kunitz domain libraries randomized at or near the binding loop (positions 11-13, 15-19, and 34) were sequenced following five rounds of selection on immobilized plasma kallikrein. Invariant preferences for Arg at position 15 and His at position 18 were found, whereas His, Ala, Ala, and Pro were highly preferred residues at positions 13, 16, 17, and 19, respectively. At position 11 Pro, Asp, and Glu were favored, while hydrophobic residues were preferred at position 34. Selected variants, purified by trypsin affinity chromatography and reverse phase high performance liquid chromatography, potently inhibited plasma kallikrein, with apparent equilibrium dissociation constants (K i *) ranging from ϳ75 to 300 pM. From sequence and activity data, consensus mutants were constructed by site directed mutagenesis. One such mutant, KALI-DY, which differed from APPI at 6 key residues (T11D, P13H, M17A, I18H, S19P, and F34Y), inhibited plasma kallikrein with a K i * ‫؍‬ 15 ؎ 14 pM, representing an increase in binding affinity of more than 10,000-fold compared to APPI. Similar to APPI, the variants also inhibited Factor XIa with high affinity, with K i * values ranging from ϳ0.3 to 15 nM; KALI-DY inhibited Factor XIa with a K i * ‫؍‬ 8.2 ؎ 3.5 nM. KALI-DY did not inhibit plasmin, thrombin, Factor Xa, Factor XIIa, activated protein C, or tissue factor⅐Factor VIIa. Consistent with the protease specificity profile, KALI-DY did not prolong the clotting time in a prothrombin time assay, but did prolong the clotting time in an activated partial thromboplastin time assay >3.5-fold at 1 M.

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“…Natural anticoagulant proteins displaying anti-FXIIa activity were also reported, e.g. from leguminous plants [21], hematophagous insects [22-24], helminth parasites [25] and bacteria [26]. Again, despite their proven efficacy, all these proteins were generally not selective over blood coagulation proteases.…”

Section: Discussionmentioning

confidence: 99%

COU254, a specific 3-carboxamide-coumarin inhibitor of coagulation factor XII, does not protect mice from acute ischemic stroke

Kraft

Schwarz

Pochet

et al. 2010

Exp & Trans Stroke Med

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BackgroundAnticoagulation is an important means to prevent from acute ischemic stroke but is associated with a significant risk of severe hemorrhages. Previous studies have shown that blood coagulation factor XII (FXII)-deficient mice are protected from pathological thrombus formation during cerebral ischemia without bearing an increased bleeding tendency. Hence, pharmacological blockade of FXII might be a promising and safe approach to prevent acute ischemic stroke and possibly other thromboembolic disorders but pharmacological inhibitors selective over FXII are still lacking. In the present study we investigated the efficacy of COU254, a novel nonpeptidic 3-carboxamide-coumarin that selectively blocks FXII activity, on stroke development and post stroke functional outcome in mice.MethodsC57Bl/6 mice were treated with COU254 (40 mg/kg i.p.) or vehicle and subjected to 60 min transient middle cerebral artery occlusion (tMCAO) using the intraluminal filament method. After 24 h infarct volumes were determined from 2,3,5-Triphenyltetrazoliumchloride(TTC)-stained brain sections and functional scores were assessed. Hematoxylin and eosin (H&E) staining was used to estimate the extent of neuronal cell damage. Thrombus formation within the infarcted brain areas was analyzed by immunoblot.ResultsInfarct volumes and functional outcomes on day 1 after tMCAO did not significantly differ between COU254 pre-treated mice or untreated controls (p > 0.05). Histology revealed extensive ischemic neuronal damage regularly including the cortex and the basal ganglia in both groups. COU254 treatment did not prevent intracerebral fibrin(ogen) formation.ConclusionsCOU254 at the given concentration of 40 mg/kg failed to demonstrate efficacy in acute ischemic stroke in this preliminary study. Further preclinical evaluation of 3-carboxamide-coumarins is needed before the antithrombotic potential of this novel class of FXII inhibitors can be finally judged.

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Ecotin is a potent inhibitor of the contact system proteases factor XIIa and plasma kallikrein

Cited by 47 publications

References 28 publications

Crystal structure analyses of uncomplexed ecotin in two crystal forms: Implications for its function and stability

Crystal structure analyses of uncomplexed ecotin in two crystal forms: Implications for its function and stability

Potent and Selective Kunitz Domain Inhibitors of Plasma Kallikrein Designed by Phage Display

COU254, a specific 3-carboxamide-coumarin inhibitor of coagulation factor XII, does not protect mice from acute ischemic stroke

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