Inhibition of a signaling modality within the gp130 receptor enhances tissue regeneration and mitigates osteoarthritis.

Adult mammals are incapable of multitissue regeneration, and augmentation of this potential may shift current therapeutic paradigms. We found that a common co-receptor of interleukin 6 (IL-6) cytokines, glycoprotein 130 (gp130), serves as a major nexus integrating various context-specific signaling inputs to either promote regenerative outcomes or aggravate disease progression. Via genetic and pharmacological experiments in vitro and in vivo, we demonstrated that a signaling tyrosine 814 (Y814) within gp130 serves as a major cellular stress sensor. Mice with constitutively inactivated Y814 (F814) were resistant to surgically induced osteoarthritis as reflected by reduced loss of proteoglycans, reduced synovitis, and synovial fibrosis. The F814 mice also exhibited enhanced regenerative, not reparative, responses after wounding in the skin. In addition, pharmacological modulation of gp130 Y814 upstream of the SRC and MAPK circuit by a small molecule, R805, elicited a protective effect on tissues after injury. Topical administration of R805 on mouse skin wounds resulted in enhanced hair follicle neogenesis and dermal regeneration. Intra-articular administration of R805 to rats after medial meniscal tear and to canines after arthroscopic meniscal release markedly mitigated the appearance of osteoarthritis. Single-cell sequencing data demonstrated that genetic and pharmacological modulation of Y814 resulted in attenuation of inflammatory gene signature as visualized by the anti-inflammatory macrophage and nonpathological fibroblast subpopulations in the skin and joint tissue after injury. Together, our study characterized a molecular mechanism that, if manipulated, enhances the intrinsic regenerative capacity of tissues through suppression of a proinflammatory milieu and prevents pathological outcomes in injury and disease.

Structure of human factor VIIa-soluble tissue factor with calcium, magnesium and rubidium.

Coagulation factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (GLA) domain, two epidermal growth factor-like (EGF) domains and a protease domain. FVIIa binds three Mg2+ ions and four Ca2+ ions in the GLA domain, one Ca2+ ion in the EGF1 domain and one Ca2+ ion in the protease domain. Further, FVIIa contains an Na+ site in the protease domain. Since Na+ and water share the same number of electrons, Na+ sites in proteins are difficult to distinguish from waters in X-ray structures. Here, to verify the Na+ site in FVIIa, the structure of the FVIIa-soluble tissue factor (TF) complex was solved at 1.8 Šresolution containing Mg2+, Ca2+ and Rb+ ions. In this structure, Rb+ replaced two Ca2+ sites in the GLA domain and occupied three non-metal sites in the protease domain. However, Rb+ was not detected at the expected Na+ site. In kinetic experiments, Na+ increased the amidolytic activity of FVIIa towards the synthetic substrate S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) by ∼20-fold; however, in the presence of Ca2+, Na+ had a negligible effect. Ca2+ increased the hydrolytic activity of FVIIa towards S-2288 by ∼60-fold in the absence of Na+ and by ∼82-fold in the presence of Na+. In molecular-dynamics simulations, Na+ stabilized the two Na+-binding loops (the 184-loop and 220-loop) and the TF-binding region spanning residues 163-180. Ca2+ stabilized the Ca2+-binding loop (the 70-loop) and Na+-binding loops but not the TF-binding region. Na+ and Ca2+ together stabilized both the Na+-binding and Ca2+-binding loops and the TF-binding region. Previously, Rb+ has been used to define the Na+ site in thrombin; however, it was unsuccessful in detecting the Na+ site in FVIIa. A conceivable explanation for this observation is provided.

Enhanced Antifibrinolytic Efficacy of a Plasmin-Specific Kunitz-Inhibitor (60-Residue Y11T/L17R with C-Terminal IEK) of Human Tissue Factor Pathway Inhibitor Type-2 Domain1.

Current antifibrinolytic agents reduce blood loss by inhibiting plasmin active sites (e.g., aprotinin) or by preventing plasminogen/tissue plasminogen activator (tPA) binding to fibrin clots (e.g., ε-aminocaproic acid and tranexamic acid); however, they have adverse side effects. Here, we expressed 60-residue (NH2NAE…IEKCOOH) Kunitz domain1 (KD1) mutants of human tissue factor pathway inhibitor type-2 that inhibit plasmin as well as plasminogen activation. A single (KD1-L17R-KCOOH) and a double mutant (KD1-Y11T/L17R- KCOOH) were expressed in Escherichia coli as His-tagged constructs, each with enterokinase cleavage sites. KD1-Y11T/L17R-KCOOH was also expressed in Pichia pastoris. KD1-Y11T/L17R-KCOOH inhibited plasmin comparably to aprotinin and bound to the kringle domains of plasminogen/plasmin and tPA with Kd of ~50 nM and ~35 nM, respectively. Importantly, compared to aprotinin, KD1-L17R-KCOOH and KD1-Y11T/L17R-KCOOH did not inhibit kallikrein. Moreover, the antifibrinolytic potential of KD1-Y11T/L17R-KCOOH was better than that of KD1-L17R-KCOOH and similar to that of aprotinin in plasma clot-lysis assays. In thromboelastography experiments, KD1-Y11T/L17R-KCOOH was shown to inhibit fibrinolysis in a dose dependent manner and was comparable to aprotinin at a higher concentration. Further, KD1-Y11T/L17R-KCOOH did not induce cytotoxicity in primary human endothelial cells or fibroblasts. We conclude that KD1-Y11T/L17R-KCOOH is comparable to aprotinin, the most potent known inhibitor of plasmin and can be produced in large amounts using Pichia.

Dual Neutral Sphingomyelinase-2/Acetylcholinesterase Inhibitors for the Treatment of Alzheimer's Disease.

We report the discovery of a novel class of compounds that function as dual inhibitors of the enzymes neutral sphingomyelinase-2 (nSMase2) and acetylcholinesterase (AChE). Inhibition of these enzymes provides a unique strategy to suppress the propagation of tau pathology in the treatment of Alzheimer's disease (AD). We describe the key SAR elements that affect relative nSMase2 and/or AChE inhibitor effects and potency, in addition to the identification of two analogs that suppress the release of tau-bearing exosomes in vitro and in vivo. Identification of these novel dual nSMase2/AChE inhibitors represents a new therapeutic approach to AD and has the potential to lead to the development of truly disease-modifying therapeutics.

Plasmin-mediated proteolysis of human factor IXa in the presence of calcium/phospholipid: Conversion of procoagulant factor IXa to a fibrinolytic enhancer.

BACKGROUND: Factor (F) IX/IXa inactivation by plasmin has been studied; however, whether plasmin converts FIXa to a fibrinolytic enhancer is not known. OBJECTIVE: Investigate plasmin proteolysis site(s) in FIXa that inactivates and transforms it into a fibrinolytic enhancer. METHODS: NH2 -terminal sequencing, mass spectrometry analysis, and functional assays. RESULTS: Plasmin in the presence of Ca2+ /phospholipid (PL) rapidly cleaved FIXaß at Lys316↓Gly317 to yield FIXaγ followed by a slow cleavage at Lys413↓Leu414 to yield FIXaδ. FIXaγ/FIXaδ migrated indistinguishably from FIXaß in nondenaturing gel system indicating that C-terminal residues 317-415/317-413 of heavy chain remain noncovalently associated with FIXaγ/FIXaδ. However, as compared with FIXaß, FIXaγ or FIXaγ/FIXaδ (25-75 mixture, 8-hour/24-hour incubation analysis by mass spectrometry) was impaired ~ 10-fold in hydrolyzing synthetic substrate CBS 31.39 (CH3-SO2-D-Leu-Gly-Arg-pNA), ~ 30-fold (~ 5-fold higher Km , ~ 6-fold lower kcat ) in activating FX in a system containing Ca2+ /PL, and ~ 650-fold in a system containing Ca2+ /PL and FVIIIa. Further, FIXaγ or FIXaγ/FIXaδ bound FVIIIa with ~ 60-fold reduced affinity compared with FIXaß. Additionally, in ligand blots, plasminogen or diisopropylfluorophosphate-inhibited plasmin (DIP-plasmin) bound FIXaγ and FIXaδ but not FIXaß. This interaction was prevented by ε-aminocaproic acid or carboxypeptidase B treatment suggesting that plasminogen/DIP-plasmin binds to FIXaγ/FIXaδ through newly generated C-terminal Lys316 and Lys413. Importantly, FIXaγ/FIXaδ mixture but not FIXaγ enhanced tissue plasminogen activator (tPA)-mediated plasminogen activation in a concentration dependent manner. Similarly, FIXaγ/FIXaδ mixture but not FIXaγ enhanced tPA-induced clot lysis in FIX-depleted plasma. CONCLUSION: Plasmin cleavage at Lys316↓Gly317 abrogates FIXaß coagulant activity, whereas additional cleavage at Lys413↓Leu414 converts it into a fibrinolytic enhancer.

Sodium-site in serine protease domain of human coagulation factor IXa: evidence from the crystal structure and molecular dynamics simulations study.

Essentials Consensus sequence and biochemical data suggest a Na+ -site in the factor (F) IXa protease domain. X-ray structure of the FIXa EGF2/protease domain at 1.37 Å reveals a Na+ -site not observed earlier. Molecular dynamics simulations data support that Na+  ± Ca2+ promote FIXa protease domain stability. Sulfate ions found in the protease domain mimic heparin sulfate binding mode in FIXa. SUMMARY: Background Activated coagulation factor IX (FIXa) consists of a γ-carboxyglutamic acid domain, two epidermal growth factor-like (EGF) domains, and a C-terminal protease domain. Consensus sequence and biochemical data support the existence of a Na+ -site in the FIXa protease domain. However, soaking experiments or crystals grown in high concentration of ammonium sulfate did not reveal a Na+ -site in wild-type or mutant FIXa EGF2/protease domain structure. Objective Determine the structure of the FIXa EGF2/protease domain in the presence of Na+ ; perform molecular dynamics (MD) simulations to explore the role of Na+ in stabilizing FIXa structure. Methods Crystallography, MD simulations, and modeling heparin binding to FIXa. Results Crystal structure at 1.37-Å resolution revealed that Na+ is coordinated to carbonyl groups of residues 184A, 185, 221A, and 224 in the FIXa protease domain. The Na+ -site in FIXa is similar to that of FXa and is linked to the Asp189 S1-site. In MD simulations, Na+ reduced fluctuations in residues 217-225 (Na+ -loop) and 70-80 (Ca2+ -loop), whereas Ca2+ reduced fluctuations only in residues of the Ca2+ -loop. Ca2+ and Na+ together reduced fluctuations in residues of the Ca2+ -loop and Na+ -loop (residues 70-80, 183-194, and 217-225). Moreover, we observed four sulfate ions that make salt bridges with FIXa protease domain Arg/Lys residues, which have been implicated in heparin binding. Based upon locations of the sulfate ions, we modeled heparin binding to FIXa, which is similar to the heparin binding in thrombin. Conclusions The FIXa Na+ -site in association with Ca2+ contributes to stabilization of the FIXa protease domain. The heparin binding mode in FIXa is similar to that in thrombin.

Evaluation of an Allosteric BACE Inhibitor Peptide to Identify Mimetics that Can Interact with the Loop F Region of the Enzyme and Prevent APP Cleavage.

The aspartyl protease BACE1 (BACE) has emerged as an appealing target for reduction of amyloid-ß in Alzheimer's disease. The clinical fate of active-site BACE inhibitors may depend on potential side effects related to enzyme and substrate selectivity. One strategy to reduce this risk is through development of allosteric inhibitors that interact with and modulate the Loop F region unique to BACE1. Previously, a BACE-inhibiting antibody (Ab) was shown by co-crystallization to bind and induce conformational changes of Loop F, resulting in backbone perturbations at the distal S6 and S7 subsites, preventing proper binding of a long APP-like substrate to BACE and inhibiting its cleavage. In an effort to discover small Loop F-interacting molecules that mimic the Ab inhibition, we evaluated a peptide series with a YPYF(I/L)P(L/Y) motif that was reported to bind a BACE exosite. Our studies show that the most potent inhibitor from this series, peptide 65007, has a similar substrate cleavage profile to the Ab and reduces sAPPß levels in cell models and primary neurons. As our modeling indicates, it interacts with the Loop F region causing a conformational shift of the BACE protein backbone near the distal subsites. The peptide-bound enzyme adopts a conformation that closely overlays with the crystal structure (PDB: 3R1G) from Ab binding. Importantly, peptide 65007 appears to be BACE substrate and enzyme selective, showing little inhibition of NRG1, PSGL1, CHL1, or Cat D. Thus, peptide 65007 is a promising lead for discovery of Loop F-interacting small-molecule mimetics as allosteric inhibitors of BACE.

Suppression of tau propagation using an inhibitor that targets the DK-switch of nSMase2.

Targeting of molecular pathways involved in the cell-to-cell propagation of pathological tau species is a novel approach for development of disease-modifying therapies that could block tau pathology and attenuate cognitive decline in patients with Alzheimer's disease and other tauopathies. We discovered cambinol through a screening effort and show that it is an inhibitor of cell-to-cell tau propagation. Our in vitro data demonstrate that cambinol inhibits neutral sphingomyelinase 2 (nSMase2) enzyme activity in dose response fashion, and suppresses extracellular vesicle (EV) production while reducing tau seed propagation. Our in vivo testing with cambinol shows that it can reduce the nSMase2 activity in the brain after oral administration. Our molecular docking and simulation analysis reveals that cambinol can target the DK-switch in the nSMase2 active site.

Drug-induced modulation of gp130 signalling prevents articular cartilage degeneration and promotes repair.

OBJECTIVE: Human adult articular cartilage (AC) has little capacity for repair, and joint surface injuries often result in osteoarthritis (OA), characterised by loss of matrix, hypertrophy and chondrocyte apoptosis. Inflammation mediated by interleukin (IL)-6 family cytokines has been identified as a critical driver of proarthritic changes in mouse and human joints, resulting in a feed-forward process driving expression of matrix degrading enzymes and IL-6 itself. Here we show that signalling through glycoprotein 130 (gp130), the common receptor for IL-6 family cytokines, can have both context-specific and cytokine-specific effects on articular chondrocytes and that a small molecule gp130 modulator can bias signalling towards anti-inflammatory and antidegenerative outputs. METHODS: High throughput screening of 170 000 compounds identified a small molecule gp130 modulator termed regulator of cartilage growth and differentiation (RCGD 423) that promotes atypical homodimeric signalling in the absence of cytokine ligands, driving transient increases in MYC and pSTAT3 while suppressing oncostatin M- and IL-6-mediated activation of ERK and NF-κB via direct competition for gp130 occupancy. RESULTS: This small molecule increased proliferation while reducing apoptosis and hypertrophic responses in adult chondrocytes in vitro. In a rat partial meniscectomy model, RCGD 423 greatly reduced chondrocyte hypertrophy, loss and degeneration while increasing chondrocyte proliferation beyond that observed in response to injury. Moreover, RCGD 423 improved cartilage healing in a rat full-thickness osteochondral defect model, increasing proliferation of mesenchymal cells in the defect and also inhibiting breakdown of cartilage matrix in de novo generated cartilage. CONCLUSION: These results identify a novel strategy for AC remediation via small molecule-mediated modulation of gp130 signalling.

A Small Molecule Mimetic of the Humanin Peptide as a Candidate for Modulating NMDA-Induced Neurotoxicity.

Humanin (HN), a 24-amino acid bioactive peptide, has been shown to increase cell survival of neurons after exposure to Aß and NMDA-induced toxicity and thus could be beneficial in the treatment of Alzheimer's disease (AD). The neuroprotection by HN is reported to be primarily through its agonist binding properties to the gp130 receptor. However, the peptidic nature of HN presents challenges in its development as a therapeutic for AD. We report here for the first time the elucidation of the binding site of Humanin (HN) peptide to the gp130 receptor extracellular domain through modeling and the synthesis of small molecule mimetics that interact with the HN binding site on the gp130 receptor and provide protection against NMDA-induced neurotoxicity in primary hippocampal neurons. A brain permeable small molecule mimetic was identified through exploratory medicinal chemistry using microfluidic flow chemistry to facilitate the synthesis of new analogues for screening and SAR optimization.

Interaction of factor V B-domain acidic region with its basic region and with TFPI/TFPI2: Structural insights from molecular modeling studies.

BACKGROUND: Factor V (FV) B-domain contains an acidic region (FV-AR2) and a basic region (FV-BR), which interact with each other and maintain FV in a procofactor form; removal of either region via deletion/proteolysis results in an active FVa molecule. Tissue factor pathway inhibitor type-1 (TFPI) and type-2 (TFPI2) each contain a C-terminus basic segment homologous to FV-BR; this region in TFPI (and predicted in TFPI2) binds to FV-AR2 in platelet FVa (that lacks FV-BR) with high affinity and inhibits FVa function. OBJECTIVES: To understand molecular interactions between FV-AR2 with FV-BR, TFPI-BR and TFPI2-BR. METHODS: Circular dichroism (CD) and molecular modeling approaches. RESULTS AND CONCLUSIONS: CD experiments reveal the presence of ∼20% helical content in both FV-AR2 and FV-BR but each lacks beta-sheet. Predicted structures of FV-AR2 and FV-BR, obtained using threading (I-TASSER), are consistent with the CD data and have compact folds with hydrophobic residues in the interior and charged residues on the surface. Scores from QMEAN and ModFOLD servers indicate a very high probability for each structure to be native. Predicted models of Kunitz domain-3 of TFPI and TFPI2 each with C-terminal basic tail are consistent with known homologous structures. Docking experiments using ClusPro indicate that the acidic groove of FV-AR2 has high shape complementarity to accommodate the conserved basic residues in FV-BR (1002-RKKKK-1006), TFPI-BR (256-RKRKK-260) or TFPI2-BR (191-KKKKK-195). Further, similar electrostatic interactions occur in each case. These models, in the absence of experimentally determined structures, provide a guiding point for proper mutagenesis studies in FV, TFPI and TFPI2.

Quantifying vitamin K-dependent holoprotein compaction caused by differential γ-carboxylation using high-pressure size exclusion chromatography.

This study uses high-pressure size exclusion chromatography (HPSEC) to quantify divalent metal ion (X(2+))-induced compaction found in vitamin K-dependent (VKD) proteins. Multiple X(2+) binding sites formed by the presence of up to 12 γ-carboxyglutamic acid (Gla) residues are present in plasma-derived FIX (pd-FIX) and recombinant FIX (r-FIX). Analytical ultracentrifugation (AUC) was used to calibrate the Stokes radius (R) measured by HPSEC. A compaction of pd-FIX caused by the filling of Ca(2+) and Mg(2+) binding sites resulted in a 5 to 6% decrease in radius of hydration as observed by HPSEC. The filling of Ca(2+) sites resulted in greater compaction than for Mg(2+) alone where this effect was additive or greater when both ions were present at physiological levels. Less X(2+)-induced compaction was observed in r-FIX with lower Gla content populations, which enabled the separation of biologically active r-FIX species from inactive ones by HPSEC. HPSEC was sensitive to R changes of approximately 0.01nm that enabled the detection of FIX compaction that was likely cooperative in nature between lower avidity X(2+) sites of the Gla domain and higher avidity X(2+) sites of the epidermal growth factor 1 (EGF1)-like domain.

The heterodimeric structure of heterogeneous nuclear ribonucleoprotein C1/C2 dictates 1,25-dihydroxyvitamin D-directed transcriptional events in osteoblasts.

Heterogeneous nuclear ribonucleoprotein (hnRNP) C plays a key role in RNA processing. More recently hnRNP C has also been shown to function as a DNA binding protein exerting a dominant-negative effect on transcriptional responses to the vitamin D hormone,1,25-dihydroxyvitamin D (1,25(OH)2D), via interaction in cis with vitamin D response elements (VDREs). The physiologically active form of human hnRNPC is a tetramer of hnRNPC1 (huC1) and C2 (huC2) subunits known to be critical for specific RNA binding activity in vivo, yet the requirement for heterodimerization of huC1 and C2 in DNA binding and downstream action is not well understood. While over-expression of either huC1 or huC2 alone in mouse osteoblastic cells did not suppress 1,25(OH)2D-induced transcription, over-expression of huC1 and huC2 in combination using a bone-specific polycistronic vector successfully suppressed 1,25(OH)2D-mediated induction of osteoblast target gene expression. Over-expression of either huC1 or huC2 in human osteoblasts was sufficient to confer suppression of 1,25(OH)2D-mediated transcription, indicating the ability of transfected huC1 and huC2 to successfully engage as heterodimerization partners with endogenously expressed huC1 and huC2. The failure of the chimeric combination of mouse and human hnRNPCs to impair 1,25(OH)2D-driven gene expression in mouse cells was structurally predicted, owing to the absence of the last helix in the leucine zipper (LZ) heterodimerization domain of hnRNPC gene product in lower species, including the mouse. These results confirm that species-specific heterodimerization of hnRNPC1 and hnRNPC2 is a necessary prerequisite for DNA binding and down-regulation of 1,25(OH)2D-VDR-VDRE-directed gene transactivation in osteoblasts.

Platelets contain tissue factor pathway inhibitor-2 derived from megakaryocytes and inhibits fibrinolysis.

Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C terminus region. The N-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa, and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pM) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry, and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds factor V (FV) and partially B-domain-deleted FV (FV-1033) with K(d) ~9 nM and binds FVa with K(d) ~100 nM. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with K(d) ~36-48 nM and bind FVa with K(d) ~252-456 nM. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the FV B-domain acidic region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (~7 nM) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis.

Decoy plasminogen receptor containing a selective Kunitz-inhibitory domain.

Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in which P2' residue Leu17 (bovine pancreatic trypsin inhibitor numbering) is mutated to Arg selectively inhibits the active site of plasmin with ∼5-fold improved affinity. Thrombin cleavage (24 h extended incubation at a 1:50 enzyme-to-substrate ratio) of the KD1 mutant (Leu17Arg) yielded a smaller molecule containing the intact Kunitz domain with no detectable change in the active-site inhibitory function. The N-terminal sequencing and MALDI-TOF/ESI data revealed that the starting molecule has a C-terminal valine (KD1L17R-VT), whereas the smaller molecule has a C-terminal lysine (KD1L17R-KT). Because KD1L17R-KT has C-terminal lysine, we examined whether it could serve as a decoy receptor for plasminogen/plasmin. Such a molecule might inhibit plasminogen activation as well as the active site of generated plasmin. In surface plasmon resonance experiments, tissue plasminogen activator (tPA) and Glu-plasminogen bound to KD1L17R-KT (Kd ∼ 0.2 to 0.3 µM) but not to KD1L17R-VT. Furthermore, KD1L17R-KT inhibited tPA-induced plasma clot fibrinolysis more efficiently than KD1L17R-VT. Additionally, compared to ε-aminocaproic acid KD1L17R-KT was more effective in reducing blood loss in a mouse liver-laceration injury model, where the fibrinolytic system is activated. In further experiments, the micro(µ)-plasmin-KD1L17R-KT complex inhibited urokinase-induced plasminogen activation on phorbol-12-myristate-13-acetate-stimulated U937 monocyte-like cells, whereas the µ-plasmin-KD1L17R-VT complex failed to inhibit this process. In conclusion, KD1L17R-KT inhibits the active site of plasmin as well as acts as a decoy receptor for the kringle domain(s) of plasminogen/plasmin; hence, it limits both plasmin generation and activity. With its dual function, KD1L17R-KT could serve as a preferred agent for controlling plasminogen activation in pathological processes.

Structural and functional studies of γ-carboxyglutamic acid domains of factor VIIa and activated Protein C: role of magnesium at physiological calcium.

Crystal structures of factor (F) VIIa/soluble tissue factor (TF), obtained under high Mg(2+) (50mM Mg(2+)/5mM Ca(2+)), have three of seven Ca(2+) sites in the γ-carboxyglutamic acid (Gla) domain replaced by Mg(2+) at positions 1, 4, and 7. We now report structures under low Mg(2+) (2.5mM Mg(2+)/5mM Ca(2+)) as well as under high Ca(2+) (5mM Mg(2+)/45 mM Ca(2+)). Under low Mg(2+), four Ca(2+) and three Mg(2+) occupy the same positions as in high-Mg(2+) structures. Conversely, under low Mg(2+), reexamination of the structure of Gla domain of activated Protein C (APC) complexed with soluble endothelial Protein C receptor (sEPCR) has position 4 occupied by Ca(2+) and positions 1 and 7 by Mg(2+). Nonetheless, in direct binding experiments, Mg(2+) replaced three Ca(2+) sites in the unliganded Protein C or APC. Further, the high-Ca(2+) condition was necessary to replace Mg4 in the FVIIa/soluble TF structure. In biological studies, Mg(2+) enhanced phospholipid binding to FVIIa and APC at physiological Ca(2+). Additionally, Mg(2+) potentiated phospholipid-dependent activations of FIX and FX by FVIIa/TF and inactivation of activated factor V by APC. Since APC and FVIIa bind to sEPCR involving similar interactions, we conclude that under the low-Mg(2+) condition, sEPCR binding to APC-Gla (or FVIIa-Gla) replaces Mg4 by Ca4 with an attendant conformational change in the Gla domain ω-loop. Moreover, since phospholipid and sEPCR bind to FVIIa or APC via the ω-loop, we predict that phospholipid binding also induces the functional Ca4 conformation in this loop. Cumulatively, the data illustrate that Mg(2+) and Ca(2+) act in concert to promote coagulation and anticoagulation.

Structural biology of factor VIIa/tissue factor initiated coagulation.

Factor VII (FVII) consists of an N-terminal gamma-carboxyglutamic acid domain followed by two epidermal growth factor-like (EGF1 and EGF2) domains and the C-terminal protease domain. Activation of FVII results in a two-chain FVIIa molecule consisting of a light chain (Gla-EGF1-EGF2 domains) and a heavy chain (protease domain) held together by a single disulfide bond. During coagulation, the complex of tissue factor (TF, a transmembrane glycoprotein) and FVIIa activates factor IX (FIX) and factor X (FX). FVIIa is structurally "zymogen-like" and when bound to TF, it is more "active enzyme-like." FIX and FX share structural homology with FVII. Three structural biology aspects of FVIIa/TF are presented in this review. One, regions in soluble TF (sTF) that interact with FVIIa as well as mapping of Ca2+, Mg2+, Na+ and Zn2+ sites in FVIIa and their functions; two, modeled interactive regions of Gla and EGF1 domains of FXa and FIXa with FVIIa/sTF; and three, incompletely formed oxyanion hole in FVIIa/sTF and its induction by substrate/inhibitor. Finally, an overview of the recognition elements in TF pathway inhibitor is provided.

Engineering kunitz domain 1 (KD1) of human tissue factor pathway inhibitor-2 to selectively inhibit fibrinolysis: properties of KD1-L17R variant.

Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein, and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Full-length TFPI-2 or its isolated first Kunitz domain (KD1) also inhibits plasmin; therefore, it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this study, structure-based investigations and analysis of the serine protease profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2' position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2' residue Leu-17 in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in Escherichia coli, folded, and purified to homogeneity. N-terminal sequences and mass spectra confirmed proper expression of KD1-WT and KD1-L17R. Compared with KD1-WT, the KD1-L17R did not inhibit factor XIa, plasma kallikrein, or factor VIIa/tissue factor. Furthermore, KD1-L17R inhibited plasmin with ∼6-fold increased affinity and effectively prevented plasma clot fibrinolysis induced by tissue plasminogen activator. Similarly, in a mouse liver laceration bleeding model, KD1-L17R was ∼8-fold more effective than KD1-WT in preventing blood loss. Importantly, in this bleeding model, KD1-L17R was equally or more effective than aprotinin or tranexamic acid, which have been used as antifibrinolytic agents to prevent blood loss during major surgery/trauma. Furthermore, as compared with aprotinin, renal toxicity was not observed with KD1-L17R.

An estimate of the numbers and density of low-energy structures (or decoys) in the conformational landscape of proteins.

BACKGROUND: The conformational energy landscape of a protein, as calculated by known potential energy functions, has several minima, and one of these corresponds to its native structure. It is however difficult to comprehensively estimate the actual numbers of low energy structures (or decoys), the relationships between them, and how the numbers scale with the size of the protein. METHODOLOGY: We have developed an algorithm to rapidly and efficiently identify the low energy conformers of oligo peptides by using mutually orthogonal Latin squares to sample the potential energy hyper surface. Using this algorithm, and the ECEPP/3 potential function, we have made an exhaustive enumeration of the low-energy structures of peptides of different lengths, and have extrapolated these results to larger polypeptides. CONCLUSIONS AND SIGNIFICANCE: We show that the number of native-like structures for a polypeptide is, in general, an exponential function of its sequence length. The density of these structures in conformational space remains more or less constant and all the increase appears to come from an expansion in the volume of the space. These results are consistent with earlier reports that were based on other models and techniques.