Substrate For Kallikrein Research Articles

Subproteome of Lachesis muta rhombeata venom and preliminary studies on LmrSP-4, a novel snake venom serine proteinase.

Background: Lachesis muta rhombeata is one of the venomous snakes of medical importance in Brazil whose envenoming is characterized by local and systemic effects which may produce even shock and death. Its venom is mainly comprised of serine and metalloproteinases, phospholipases A2 and bradykinin-potentiating peptides. Based on a previously reported fractionation of L. m. rhombeata venom (LmrV), we decided to perform a subproteome analysis of its major fraction and investigated a novel component present in this venom.Methods:LmrV was fractionated through molecular exclusion chromatography and the main fraction (S5) was submitted to fibrinogenolytic activity assay and fractionated by reversed-phase chromatography. The N-terminal sequences of the subfractions eluted from reversed-phase chromatography were determined by automated Edman degradation. Enzyme activity of LmrSP-4 was evaluated upon chromogenic substrates for thrombin (S-2238), plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) and upon fibrinogen. All assays were carried out in the presence or absence of possible inhibitors. The fluorescence resonance energy transfer substrate Abz-KLRSSKQ-EDDnp was used to determine the optimal conditions for LmrSP-4 activity. Molecular mass of LmrSP-4 was determined by MALDI-TOF and digested peptides after trypsin and Glu-C treatments were analyzed by high resolution MS/MS using different fragmentation modes. Results:Fraction S5 showed strong proteolytic activity upon fibrinogen. Its fractionation by reversed-phase chromatography gave rise to 6 main fractions (S5C1-S5C6). S5C1-S5C5 fractions correspond to serine proteinases whereas S5C6 represents a C-type lectin. S5C4 (named LmrSP-4) had its N-terminal determined by Edman degradation up to the 53rd amino acid residue and was chosen for characterization studies. LmrSP-4 is a fibrinogenolytic serine proteinase with high activity against S-2302, being inhibited by PMSF and benzamidine, but not by 1,10-phenantroline. In addition, this enzyme exhibited maximum activity within the pH range from neutral to basic and between 40 and 50 °C. About 68% of the LmrSP-4 primary structure was covered, and its molecular mass is 28,190 Da.Conclusions:Novel serine proteinase isoforms and a lectin were identified in LmrV. Additionally, a kallikrein-like serine proteinase that might be useful as molecular tool for investigating bradykinin-involving process was isolated and partially characterized.

New Insights on Moojase, a Thrombin-Like Serine Protease from Bothrops moojeni Snake Venom.

Snake venom serine proteases (SVSPs) are enzymes that are capable of interfering in various parts of the blood coagulation cascade, which makes them interesting candidates for the development of new therapeutic drugs. Herein, we isolated and characterized Moojase, a potent coagulant enzyme from Bothrops moojeni snake venom. The toxin was isolated from the crude venom using a two-step chromatographic procedure. Moojase is a glycoprotein with N-linked glycans, molecular mass of 30.3 kDa and acidic character (pI 5.80–6.88). Sequencing of Moojase indicated that it is an isoform of Batroxobin. Moojase was able to clot platelet-poor plasma and fibrinogen solutions in a dose-dependent manner, indicating thrombin-like properties. Moojase also rapidly induced the proteolysis of the Aα chains of human fibrinogen, followed by the degradation of the Bβ chains after extended periods of incubation, and these effects were inhibited by PMSF, SDS and DTT, but not by benzamidine or EDTA. RP-HPLC analysis of its fibrinogenolysis confirmed the main generation of fibrinopeptide A. Moojase also induced the fibrinolysis of fibrin clots formed in vitro, and the aggregation of washed platelets, as well as significant amidolytic activity on substrates for thrombin, plasma kallikrein, factor Xia, and factor XIIa. Furthermore, thermofluor analyses and the esterase activity of Moojase demonstrated its very high stability at different pH buffers and temperatures. Thus, studies such as this for Moojase should increase knowledge on SVSPs, allowing their bioprospection as valuable prototypes in the development of new drugs, or as biotechnological tools.

Purification and enzymatic characterization of a novel metalloprotease from Lachesis muta rhombeata snake venom

BackgroundLachesis muta rhombeata (Lmr) is the largest venomous snake in Latin America and its venom contains mainly enzymatic components, such as serine and metalloproteases, L-amino acid oxidase and phospholipases A2. Metalloproteases comprise a large group of zinc-dependent proteases that cleave basement membrane components such as fibronectin, laminin and collagen type IV. These enzymes are responsible for local and systemic changes, including haemorrhage, myonecrosis and inflammation. This study aimed the isolation and enzymatic characterization of the first metalloprotease (Lmr-MP) from Lmr venom (LmrV).Methods and resultsLmr-MP was purified through two chromatographic steps and submitted to enzymatic characterization. It showed proteolytic activity on azocasein with maximum activity at pH 7.0–9.0. It was inhibited by EDTA (a metal chelator that removes zinc, which is essential for enzymatic activity) and no effect was observed with PMSF, iodoacetic acid or pepstatin (inhibitors of serine, cysteine and aspartyl proteases, respectively). Ca2+, Mg2+ and Ba2+ ions increased its activity, while Al3+, Cu2+, Ni2+ and Zn2+ inhibited it. Additionally, ZnCl2 showed a dose dependent inhibition of the enzyme. Lmr-MP activity was also evaluated upon chromogenic substrates for plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) showing the highest activity on S-2302. The activity in different solutions (5 mM or 50 mM ammonium bicarbonate, pH 7.8; 0.1% trifluoroacetic acid + 50% acetonitrile; phosphate buffer saline, pH 7.4; 50 mM sodium acetate, pH 4.0 or ammonium acetate pH 4.5) was also evaluated and the results showed that its activity was abolished at acidic pHs. Its molecular mass (22,858 Da) was determined by MALDI-TOF and about 90% of its primary structure was verified by high-resolution mass spectrometry using HCD and ETD fragmentations and database search against the sequence of closely related species. It is a novel enzyme which shared high identity with other snake venom metalloproteases (svMPs) belonging to the P-I group.ConclusionThe purification procedure achieved a novel pure highly active metalloprotease from LmrV. This new molecule can help to understand the metalloproteases mechanisms of action, the Lachesis envenoming, as well as to open new perspectives for its use as therapeutic tools.

The blood fluke Schistosoma mansoni cleaves the coagulation protein high molecular weight kininogen (HK) but does not generate the vasodilator bradykinin

BackgroundSchistosomes are blood dwelling parasitic worms that cause the debilitating disease schistosomiasis. Here we examined the influence of the parasites on their external environment by monitoring the impact of adult Schistosoma mansoni worms on the murine plasma proteome in vitro and, in particular, on how the worms affect the blood coagulation protein high molecular weight kininogen (HK).MethodsFollowing the incubation of adult schistosomes in murine plasma, two-dimensional differential in-gel electrophoresis (2D-DIGE) was conducted to look for changes in the plasma proteome compared with control plasma. A major change to the blood protein kininogen (HK) was observed, and the interaction of Schistosoma mansoni parasite with this protein alone was then investigated by western blot analysis and activity assays. Finally, the generation of bradykinin from HK was monitored using a bradykinin detection kit.ResultsThe most striking change to the plasma proteome concerned HK; while the full-length protein was more abundant in control plasma, carboxyl-terminal truncated forms were more abundant in plasma that contained schistosomes. Incubating parasites in buffer with pure HK followed by Western blot analysis confirmed that human HK is degraded by the worms. The resulting digestion pattern differed from that brought about by kallikrein, a host serine protease that normally acts on HK to release the vasodilator bradykinin. We found that live schistosomes, while digesting HK, do not generate bradykinin nor do they cleave a chromogenic kallikrein substrate. Since the cleavage of HK by the worms is not impeded by the serine protease inhibitor PMSF but is blocked by the cysteine protease inhibitor E64c, we hypothesize that schistosome tegumental cysteine proteases are responsible for HK cleavage.ConclusionsSince proteomic and biochemical studies have revealed that the schistosome tegument contains two cysteine proteases belonging to the calpain family (SmCalp1 and SmCalp2) we conclude that these are likely responsible for the HK cleavage reported here. Schistosome cleavage of HK should help impede blood clotting and inflammation around the worms in vivo and so promote their ease of movement within the vasculature of their hosts.

A kallikrein‐targeting RNA aptamer inhibits the intrinsic pathway of coagulation and reduces bradykinin release

Background Plasma kallikrein is a serine protease that plays an integral role in many biological processes, including coagulation, inflammation, and fibrinolysis. The main function of kallikrein in coagulation is the amplification of activated factor XII (FXIIa) production, which ultimately leads to thrombin generation and fibrin clot formation. Kallikrein is generated by FXIIa-mediated cleavage of the zymogen prekallikrein, which is usually complexed with the non-enzymatic cofactor high molecular weight kininogen (HK). HK also serves as a substrate for kallikrein to generate the proinflammatory peptide bradykinin (BK). Interestingly, prekallikrein-deficient mice are protected from thrombotic events while retaining normal hemostatic capacity. Therefore, therapeutic targeting of kallikrein may provide a safer alternative to traditional anticoagulants with anti-inflammatory benefits. Objectives To isolate and characterize an RNA aptamer that binds to and inhibits plasma kallikrein, and to elucidate its mechanism of action. Methods and Results Using convergent Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we isolated an RNA aptamer that targets kallikrein. This aptamer, Kall1-T4, specifically binds to both prekallikrein and kallikrein with similar subnanomolar binding affinities, and dose-dependently prolongs fibrin clot formation in an activated partial thromboplastin time (APTT) coagulation assay. In a purified in vitro system, Kall1-T4 inhibits the reciprocal activation of prekallikrein and FXII primarily by reducing the rate of FXIIa-mediated prekallikrein activation. Additionally, Kall1-T4 significantly reduces kallikrein-mediated HK cleavage and subsequent BK release. Conclusions We have isolated a specific and potent inhibitor of prekallikrein/kallikrein activity that serves as a powerful tool for further elucidating the role of kallikrein in thrombosis and inflammation.

Modulation of the Plasma Kallikrein-Kinin System Proteins Performed by Heparan Sulfate Proteoglycans.

Human plasma kallikrein-kinin system proteins are related to inflammation through bradykinin. In the proximity of its target cells, high molecular weight kininogen (H-kininogen) is the substrate of plasma kallikrein, which releases bradykinin from H-kininogen. Heparan sulfate proteoglycans (HSPGs) play a critical role in either recruiting kinin precursors from the plasma, or in the assembly of kallikrein-kinin system components on the cell surface. Furthermore, HSPGs mediate the endocytosis and activation of H-kininogen and plasma prekallikrein. In the presence of HSPGs (Chinese hamster ovary cell, CHO-K1, wild type cells) both heparin and heparan sulfate strongly inhibit the H-kininogen interaction with the cell membrane. H-kininogen is internalized in endosomal acidic vesicles in CHO-K1 but not in CHO-745 cells (mutant cells deficient in glycosaminoglycan biosynthesis). The endocytosis process is lipid raft-mediated and is dependent on caveolae. Both types of CHO cells do not internalize bradykinin-free H-kininogen. At pH 7.35, bradykinin is released from H-kininogen on the surface of CHO-745 cells only by serine proteases; however, in CHO-K1 cells either serine or cysteine proteases are found to be involved. The CHO-K1 cell lysate contains different kininogenases. Plasma prekallikrein endocytosis in CHO-K1 cells is independent of H-kininogen, and also prekallikrein is not internalized by CHO-745 cells. Plasma prekallikrein cleavage/activation is independent of glycosaminoglycans but plasma kallikrein formation is more specific on H-kininogen assembled on the cell surface through glycosaminoglycans. In this mini-review, the importance of HSPGs in the regulation of plasma kallikrein-kinin system proteins is shown.

Putative kallikrein substrates and their (patho)biological functions.

Human tissue kallikreins (KLKs) represent the largest contiguous group of protease genes within our genome. All 15 KLK genes co-localize within approximately 260 kb in human chromosome 19q13.3-13.4 (14 640 kb→274 990 kb). They are widely expressed in several tissues and mediate a wide range of critical physiological and pathological processes. Despite the recent developments in KLK research, elucidation of their physiological substrate repertoires remains a largely unfulfilled goal. Phage display, positional scanning and combinatorial peptide library screens have provided some valuable insights into the preferred specificities of these powerful enzymes. More recently, advances in proteomic technologies have enabled more systemic approaches towards identification of KLK substrates in a physiological setting. The advent of degradomic technologies has brought to light several putative physiological substrates and has allowed a deeper appreciation of the in vivo functional roles of KLKs. The aim of this review is to provide an overview of the different techniques that have been utilized towards the elucidation of the substrate specificities of these enzymes and elaborate on their emerging in vivo substrates.

Secretome and degradome profiling shows that Kallikrein-related peptidases 4, 5, 6, and 7 induce TGFβ-1 signaling in ovarian cancer cells

Kallikrein-related peptidases, in particular KLK4, 5, 6 and 7 (4–7), often have elevated expression levels in ovarian cancer. In OV-MZ-6 ovarian cancer cells, combined expression of KLK4–7 reduces cell adhesion and increases cell invasion and resistance to paclitaxel. The present work investigates how KLK4–7 shape the secreted proteome (“secretome”) and proteolytic profile (“degradome”) of ovarian cancer cells. The secretome comparison consistently identified >900 proteins in three replicate analyses. Expression of KLK4–7 predominantly affected the abundance of proteins involved in cell–cell communication. Among others, this includes increased levels of transforming growth factor β-1 (TGFβ-1). KLK4–7 co-transfected OV-MZ-6 cells share prominent features of elevated TGFβ-1 signaling, including increased abundance of neural cell adhesion molecule L1 (L1CAM). Augmented levels of TGFβ-1 and L1CAM upon expression of KLK4–7 were corroborated in vivo by an ovarian cancer xenograft model. The degradomic analysis showed that KLK4–7 expression mostly affected cleavage sites C-terminal to arginine, corresponding to the preference of kallikreins 4, 5 and 6. Putative kallikrein substrates include chemokines, such as growth differentiation factor 15 (GDF 15) and macrophage migration inhibitory factor (MIF). Proteolytic maturation of TGFβ-1 was also elevated. KLK4–7 have a pronounced, yet non-degrading impact on the secreted proteome, with a strong association between these proteases and TGFβ-1 signaling in tumor biology.

Antibodies to Human Factor XII with Antithrombotic Properties

AbstractAbstract 1106The plasma protease factor XIIa (FXIIa) contributes to vascular occlusion in murine thrombosis models, at least partly through activation of factor XI (FXI). While there is good correlation between plasma FXI levels and thrombotic events in humans, the situation is not as clear for FXII (the precursor of FXIIa), suggesting fundamental differences in thrombus formation in mice and humans. To facilitate studies on the effects of FXII/XIIa on thrombus formation, we developed novel inhibitory antibodies to human FXII, designated 9A2 and 15H8, by immunizing FXII-deficient mice with human FXII. Using recombinant human FXII molecules that lack various domains, and chimeras in which specific domains in FXII are replaced with those from the related protein hepatocyte growth factor activator, we determined that 9A2 and 15H8 bind to the FXII/XIIa non-catalytic heavy chain at different sites. 9A2 binds on or near the EGF2 domain, while 15H8 binds to the fibronectin type I and/or kringle domain. These areas have been implicated in FXII binding to polyanionic surfaces. Saturating concentrations of 9A2 or 15H8 reduced FXII activity by 50% and 90%, respectively, in an aPTT assay using normal plasma, while combining the antibodies resulted in >95% inhibition. However, in assays in which clot formation was triggered by adding FXIIa directly to plasma, preincubation of FXIIa with either antibody did not prolong the clotting time. Furthermore, neither antibody had a strong effect in a chromogenic assay of FXI activation by FXIIa, indicating the antibodies interfere with the aPTT assay primarily by inhibiting FXII activation. FXII activation in the aPTT assay is initiated by addition of a polyanion such as silica to the plasma to induce contact activation. In vivo, polymers of inorganic phosphate (polyP) may serve a similar function. Contact activation is triggered in plasma when FXII bound to the polyanion is activated, probably by trace amounts of FXIIa or another protease present in the plasma. Once formed, FXIIa converts the zymogens prekallikrein and FXI to the proteases kallikrein and FXIa, both of which can activate additional FXIIa to amplify the process. In the presence of 9A2 or 15H8, activation of pure FXII in the presence of either silica or polyP was significantly reduced. Interestingly, the antibodies actually potentiated FXII activation by kallikrein or FXIa in the absence of a polyanion. Taken as a whole, these results suggest that binding of 9A2 or 15H8 to FXII results in conformational changes that make FXII a better substrate for kallikrein and FXIa, possibly by mimicking the effect of FXII binding to a polyanion, but that prevent activation of FXII by FXIIa (autoactivation), blunting the overall rate of activation. We tested the effects of 9A2 and 15H8 in a mouse model in which thrombotic occlusion of the carotid artery is induced by exposing the vessel to a 3.5% solution of ferric chloride. Wild type C57Bl/6 mice develop arterial occlusion within 5 to 10 minutes, while FXII-deficient mice are resistant to arterial occlusion. Infusion of human FXII into FXII-deficient mice restores the wild type phenotype. 15H8 prevented thrombus formation in mice reconstituted with human FXII, while 9A2 reduced the rate of thrombotic occlusion by 50%. In an ex vivo flow model, perfusion of human blood through collagen-coated tubes at a shear rate of 300 sec−1 results in tube occlusion by platelet and fibrin rich clot in ∼15 minutes. 15H8 effectively blocked fibrin formation and reduced platelet accumulation, preventing tube occlusion. 9A2 was also effective at preventing clot formation, but there was evidence of some fibrin accumulation over time. In summary, the monoclonal anti-human FXII IgGs 9A2 and 15H8 prevent thrombus formation in whole blood in vivo and ex vivo by interfering with FXII activation. Our data support the hypothesis that pharmacologic inhibition of FXII activation may have therapeutic utility in disorders that are driven or aggravated by the blood contact system. Disclosures:No relevant conflicts of interest to declare.

Biochemical and enzymatic characterization of BpMP-I, a fibrinogenolytic metalloproteinase isolated from Bothropoides pauloensis snake venom

Snake Venom Metalloproteinases (SVMPs) are the most abundant components present in Viperidae venom. They are important in the induction of systemic alterations and local tissue damage after envenomation. In the present study, a metalloproteinase named BpMPI was isolated from Bothropoides pauloensis snake venom and its biochemical and enzymatic characteristics were determined. BpMPI was purified in two chromatography steps on ion exchange CM-Sepharose Fast flow and Sephacryl S-300. This protease was homogeneous on SDS-PAGE and showed a single chain polypeptide of 20kDa under non reducing conditions. The partial amino acid sequence of the enzyme showed high similarity with other SVMPs enzymes from snake venoms. BpMPI showed proteolytic activity upon azocasein and bovine fibrinogen and was inhibited by EDTA, 1,10 phenanthroline and β-mercaptoethanol. Moreover, this enzyme showed stability at neutral and alkaline pH and it was inactivated at high temperatures. BpMPI was able to hydrolyze glandular and tissue kallikrein substrates, but was unable to act upon factor Xa and plasmin substrates. The enzyme did not induce local hemorrhage in the dorsal region of mice even at high doses. Taken together, our data showed that BpMP-I is in fact a fibrinogenolytic metalloproteinase and a non hemorrhagic enzyme.

Postexercise Skeletal Muscle Glucose Transport Is Normal in Kininogen-Deficient Rats

A single exercise bout stimulates skeletal muscle glucose transport (GT) in the absence or presence of insulin. It has been suggested that the kallikrein-kinin system may contribute to exercise effects on both insulin-independent and insulin-dependent GT. Plasma kininogen, a key kallikrein-kinin system component, is a protein substrate for the enzyme kallikrein and the source of the peptide bradykinin. This study aimed to determine whether the postexercise (PEX) increase in insulin-dependent or insulin-independent GT is reduced in rats deficient in plasma kininogen versus normal rats. Male Brown Norway (BN) and Brown Norway Katholiek (BNK; plasma kininogen-deficient) rats were studied. BN and BNK rats were assigned to exercise (4×30-min swim) or sedentary (SED) groups. Rats were anesthetized immediately (0hPEX) or 3 h (3hPEX) after exercise. For 0hPEX and 0hSED rats, one epitrochlearis muscle per rat was used for AMPK phosphorylation and muscle glycogen analyses. The contralateral muscle was incubated with [H]-3-O-methylglucose (3-MG) for GT assay. For 3hPEX and 3hSED rats, one muscle from each rat was incubated without insulin, and the contralateral muscle was incubated with 60 μU·mL insulin, and both muscles were incubated with 3-MG for GT measurement. For 0hPEX versus 0hSED, both BN and BNK rats had greater insulin-independent GT and AMPK phosphorylation with reduced glycogen after exercise. No genotype effects were found 0hPEX. There was a significant main effect of exercise (3hPEX>3hSED) and no interaction between exercise and genotype for basal or insulin-stimulated GT. Plasma kininogen deficiency did not alter insulin-independent GT, AMPK phosphorylation, or glycogen depletion 0hPEX or insulin-dependent GT 3hPEX, suggesting that normal plasma kininogen is not essential for these important exercise effects.

Serine Protease Generation by Oversulfated Chondroitin Sulfate and Contaminants in Heparin: Clinical Implications

We hypothesized that Oversulfated Chondroitin Sulfate (OSCS) and the other contaminants present in contaminated heparin may mediate the generation of several serine proteases. To address this hypothesis, seven batches of recalled heparins linked to adverse patient outcomes were supplemented to freshly drawn, normal human plasma anti‐coagulated with hirudin. The generation of markers of an immune reaction, as well as rate of formation of these enzymes, was measured in biochemically defined assay conditions using chromophore‐conjugated substrates for Kallikrein, Plasma Kininogenase, C1 esterase, Thrombin, Xa and Plasmin. Assays performed with substrates for Kallikrein, Plasma Kininogenase, and C1 esterase showed a time‐dependent generation of these proteases that was strongest in the samples containing purified contaminant OSCS. Of the six contaminated heparins, four preparations showed a strong activation of these proteases which were significantly different (p<0.050) than the control non‐contaminated heparins. Our studies suggest that heparin contaminants are capable of activating multiple serine proteases, which potentially contribute to the pathogenesis of observed adverse clinical events. Classical complement pathway activation is not suggested by this data as a significant increase in C1 esterase activity is noted with all heparins, including purified OSCS.

Localization of Ligand-Binding Exosites In the Catalytic Domain of Factor XIa.

AbstractAbstract 1148Coagulation factor XI (FXI) is a plasma zymogen that is activated to FXIa, the catalytic domain of which contains exosites that interact with its normal macromolecular substrate (FIX), and its major regulatory inhibitor (protease nexin-2 kunitz protease inhibitor, PN2KPI). To localize the catalytic domain residues involved in active site architecture and in various ligand-binding exosites, we aligned the sequence of the FXI catalytic domain with that of the prekallikrein (PK) catalytic domain which is highly homologous (64% identity) in sequence, but functionally very different from FXI. Six distinct regions (R1-R6) of dissimilarity between the two proteins were identified as possible candidates for FXIa-specific ligand binding exosites. FXI/PK chimeric proteins (FXI-R1, FXI-R2, FXI-R3, FXI-R4, FXI-R5, and FXI-R6) containing substitutions with PK residues within the six regions were prepared and characterized. FXIa-R1, R2, R3 displayed enhanced proteolysis after activation suggesting that the residues within R1, R2 and R3 regions may be important to maintain proper folding of the enzyme. Comparisons of amidolytic assays vs. activated partial thromboplastin time assays showed similar activities for all chimeras except FXI-R6, which displayed 60% of the normal amidolytic activity but only 28% of clotting activity suggesting the possibility that the R6 region (autolysis loop) of FXIa may comprise an exosite involved in the interaction with its macromolecular substrate FIX. This hypothesis was further confirmed experiments showing that the proteolytic activation of FIX by FXIa-R6 was significantly impaired compared with that achieved by FXIawt. Although FXIa-R5 and FXIa-R6 were defective (50-60%) in amidolytic assays, these chimeras were very similar to FXIawt in heparin and high molecular weight kininogen binding assays, suggesting that residues within the R5 and R6 regions are involved in active-site architecture. These chimeras were further investigated to determine whether any of them had acquired kallikrein activity. After activation all except FXIa-R4 showed insignificant activity using a kallikrein-specific substrate. FXIa-R4 displayed 87% of the activity of kallikrein using the kallikrein-specific substrate but only 3% of the activity of FXIawt using the FXIa chromogenic substrate. Moreover the cleavage pattern and cleavage rate of high molecular weight kininogen by FXIa-R4 were similar to those achieved by kallikrein but not by FXIawt. Therefore substitutions in the R4 region of FXI with the corresponding residues of PK resulted in loss of activity for the FXIa substrates and gain of activity for the kallikrein substrates suggesting that the R4 region (99-loop) of FXIa plays a role in determining the substrate specificity. From the co-crystal structure of the FXIa catalytic domain with PN2KPI, the residues R3704, Y5901, E98, Y143, I151, and K192 (chymotrypsin numbering) in the FXIa catalytic domain have been identified to be possibly involved in the interactions with its inhibitors. A single mutation comprising Y5901A in the R2 region of FXIa does not affect folding however this mutant displayed resistance to inhibition by PN2KPI indicating that Y5901 is involved in the interaction of FXIa with PN2KPI. In conclusion, these studies of FXI/PK chimeric and mutant proteins implicate residues within the R4 region (99-loop) of FXIa in the determination of amidolytic substrate specificity; residues within the R6 region (autolysis loop) of FXIa in the interaction with the macromolecular substrate, FIX; and the residue Y5901 in the R2 region of FXIa in the interaction of FXIa with PN2KPI. Disclosures:No relevant conflicts of interest to declare.

Proteomic Identification of Novel Plasma Kallikrein Substrates in the Astrocyte Secretome

Plasma kallikrein (PK) is activated during hemorrhage and has been implicated in cerebral vascular permeability and edema. To further characterize the potential effects of PK on the brain that may follow cerebral vascular injury, we have utilized a proteomics approach to search for novel PK substrates in the astrocyte secretome. Extracellular proteins released by astrocytes are critical mediators of cerebral homeostasis, including roles in synapse function and vascular integrity. We identified 1,108 proteins in astrocyte condition medium and 295 of these were annotated as secreted proteins. The total abundance of nine proteins was changed after treatment with PK. Characterization of the secreted proteins revealed low molecular weight fragments for 59 proteins in conditioned media exposed to PK that were not observed in untreated controls. The most striking finding from this study was the appearance of fragmentation of 26 extracellular matrix-associated proteins including collagen isoforms 1-6 and11, nidogen-1 and -2, lysyl oxidase-like protein 1, and matrix metalloproteinase 19 in the presence of PK. We also demonstrated that PK induced the fragmentation of non-matrix proteins, including apolipoprotein E. This report further characterizes the astrocyte secretome and identifies novel potential targets of PK-induced proteolysis that may contribute to its effects on the brain following vascular injury.

Protease Generation by Chondroitin Sulfate, Semisynthetic OSCS Preparations and Contaminants Isolated From Heparin.

Abstract 3133Poster Board III-70The main contaminant in heparins recalled by the FDA in 2008 was reported to be oversulfated chondroitin sulfate (OSCS) (Kishimoto et al 2008). The adverse reactions were reportedly linked to contact activation and the generation of kallikrein. Because of the molecular and biologic heterogeneity of the contaminants isolated from heparin and LMWHs and the potential presence of other carryover contaminants it was hypothesized that heparin contaminants may also activate other proteases such as plasmin, C1 esterase factor Xa (FXa), and thrombin by direct or indirect mechanisms. To investigate the protease activation profile of heparin, porcine chondroitin sulfate (PCS) and OSCS preparations of porcine (POSCS), bovine (BOSCS) shark (SOCS) and squid (SqOSCS) origin along with contaminants isolated from five different recalled batches of heparin were profiled for the generation of kallikrein, plasmin, C1 esterase, FXa, and thrombin. Normal human pooled plasma (NHP) was supplemented with each of the different agents over a concentration range of 100ug/ml to 0.1ug/ml. Specific substrates for kallikrein (Chromozyme PK), plasmin (Chromozyme PL), C1 esterase (S2302), FXa (Chromozyme Xa), and thrombin (Chromozyme Th) were used to measure the generation of specific enzymes. The PCS produced relatively weaker generation of kallikrein, however, the contaminants isolated from the recalled batches of heparin produced a much stronger generation of this enzyme (absorbance 0.45-0.70). The different OSCS preparations produced a lower generation of kallikrein (absorbance .34-.52). While heparin and PCS produced significant activation of C1 esterase, the contaminants produced an even stronger activation of C1 esterase (abs .33-.44). The OSCS preparations from different sources produce relatively weaker activation which was comparable to the starting material. The C1 esterase activation profile was not proportional to the kallikrein activation. The PCS, UFH, and contaminants along with the different OSCS preparations produced varying degrees of plasmin generation. UFH and PCS also produced significant generation of plasmin which was higher than their C1 esterase and prekallikrein generation. With the exception of PCS and UFH, all of the agents produced varying degrees of Xa and thrombin generation. The generation of Xa was higher by all of the OSCS preparations in comparison to the IIa generation. Interestingly each of the OSCS preparations and different contaminants exhibited their own specific protease generation profile. There was no correlation between the kallikrein formation and the generation of other enzymes. These data highly suggest that the molecular composition of OSCS's and the contaminants play a major role in the mediation of the pathophysiologic responses observed with the use of contaminated heparin. The presence of other glycosaminoglycans may influence the protease generation profile of contaminants present in recalled heparins. Moreover, the relative generation of these proteases may also be influenced by the pathologic predisposition of patients where the generation of these proteases may influence the overall pathophysiologic response. DisclosuresNo relevant conflicts of interest to declare.

The assembly and activation of kinin-forming systems on the surface of human U-937 macrophage-like cells

A complex of three plasma proteins, including the high molecular mass kininogen (HK), prekallikrein (PK), and factor XII (FXII), is known to assemble on cell surfaces to release bradykinin-related proinflammatory peptides (kinins). Only recently, the binding of HK to human macrophages was described in the U-937 cell line model. In the present study, the adsorption of the other components of plasma kinin-generating system to these cells was characterized. FXII was found to tightly bind to U-937 cells and was also shown to partially compete with HK for the same binding sites on the macrophage surface. The Mac-1 and gClqR proteins were found to be receptors for FXII on the cell surface. PK indirectly docked to the macrophages via the cell-bound HK and FXII. Within the complex of these proteins assembled on the macrophage, PK could be activated by FXII/FXIIa or independently of this factor, and the active PK effectively released kinins from HK. The cell surface-bound HK could also be the substrate for tissue kallikrein approaching the cell from the bulk fluid. The kinins released at the surface are suggested to induce secondary responses in the macrophages, leading to further propagation of the inflammatory state.

Desmoglein 2 is a substrate of kallikrein 7 in pancreatic cancer

BackgroundIn a previous report we have demonstrated that the chymotryptic-like serine protease kallikrein 7 (KLK7/hK7) is overexpressed in pancreatic cancer. In normal skin, hK7 is thought to participate in skin desquamation by contributing in the degradation of desmosomal components, such as desmogleins. Thus, the ability of hK7 to degrade desmogleins was assessed and the effect of hK7 expression on desmoglein 2 was examined in cultured pancreatic cancer cells.MethodsThe expression of Dsg1, Dsg2, and Dsg3 in pancreatic tissues was examined by immunohistochemistry and their expression in two pancreatic cancer cell lines, BxPC-3 and Panc-1, was determined by western blot analysis. The ability of hK7 to degrade Dsg1 and Dsg2 was investigated using in vitro degradation assays. BxPC-3 cells stably transfected to overexpress hK7 were used to examine the effect of hK7 on cell-surface resident Dsg2.ResultsThe levels of immunoreactive Dsg1 and Dsg2 were reduced in pancreatic adenocarcinomas compared with both normal pancreatic and chronic pancreatitis tissues. Among the desmosomal proteins examined, Dsg2 exhibited robust expression on the surface of BxPC-3 cells. When hK7 was overexpressed in this cell line, there was a significant increase in the amount of soluble Dsg2 released into the culture medium compared with vector-transfected control cells.ConclusionA reduction in the amount of the cell adhesion components Dsg1 and Dsg2 in pancreatic tumors suggests that loss of these desmosomal proteins may play a role in pancreatic cancer invasion. Using in vitro degradation assays, both Dsg1 and Dsg2 could be readily proteolyzed by hK7, which is overexpressed in pancreatic adenocarcinomas. The enforced expression of hK7 in BxPC-3 cells that express significant amounts of Dsg2 resulted in a marked increase in the shedding of soluble Dsg2, which is consistent with the notion that aberrant expression of hK7 in pancreatic tumors may result in diminished cell-cell adhesion and facilitate tumor cell invasion.

The extracellular matrix protein fibronectin is a substrate for kallikrein 7

Kallikrein 7 (hK7), a chymostatin-like serine protease, is overexpressed in pancreatic adenocarcinomas as well as other human cancers. Although it has been demonstrated to participate in normal desquamation by facilitating cell shedding at the skin surface, its role in human malignancies remains unclear. To investigate the ability of hK7 to degrade components of the extracellular matrix (ECM), recombinant hK7 was expressed and purified from cultured mammalian cells. Using a three-step chromatographic purification procedure, recombinant hK7 was obtained that displayed robust proteolytic activity against a fluorogenic peptide substrate following activation by thermolysin. We demonstrate that the active protease is able to cleave fibronectin in a time-dependent manner, but not laminin, using an in vitro degradation assay. These findings indicate that the aberrant expression and secretion of hK7 in human tumors may facilitate metastasis by directly degrading components of the extracellular matrix and may thus play an important role in tumorigenesis.

Cleavage of high-molecular-weight kininogen by elastase and tryptase is inhibited by ferritin

Ferritin is a protein principally known for its role in iron storage. We have previously shown that ferritin can bind high-molecular-weight kininogen (HK). Upon proteolytic cleavage by the protease kallikrein, HK releases the proinflammatory peptide bradykinin (BK) and other biologically active products, such as two-chain high-molecular-weight kininogen, HKa. At inflammatory sites, HK is oxidized, which renders it a poor substrate for kallikrein. However, oxidized HK remains a good substrate for elastase and tryptase, thereby providing an alternative cleavage mechanism for HK during inflammation. Here we report that ferritin can retard the cleavage of both native HK and oxidized HK by elastase and tryptase. Initial rates of cleavage were reduced 45-75% in the presence of ferritin. Ferritin is not a substrate for elastase or tryptase and does not interfere with the ability of either protease to digest a synthetic substrate, suggesting that ferritin may impede HK cleavage through direct interaction with HK. Immunoprecipitation and solid phase binding studies reveal that ferritin and HK bind directly with a Kd of 134 nM. To test whether ferritin regulates HK cleavage in vivo, we used THP-1 cells, a human monocyte/macrophage cell line that has been used to model pulmonary inflammatory cells. We observed that ferritin impedes the cleavage of HK by secretory proteases in stimulated macrophages. Furthermore, ferritin, HK, and elastase are all present in or on alveolar macrophages in a mouse model of pulmonary inflammation. Collectively, these results implicate ferritin in the modulation of HK cleavage at sites of inflammation.

Activation of the SspA Serine Protease Zymogen of Staphylococcus aureus Proceeds through Unique Variations of a Trypsinogen-like Mechanism and Is Dependent on Both Autocatalytic and Metalloprotease-specific Processing

The serine and cysteine proteases SspA and SspB of Staphylococcus aureus are secreted as inactive zymogens, zSspA and zSspB. Mature SspA is a trypsin-like glutamyl endopeptidase and is required to activate zSspB. Although a metalloprotease Aureolysin (Aur) is in turn thought to contribute to activation of zSspA, a specific role has not been demonstrated. We found that pre-zSspA is processed by signal peptidase at ANA(29) downward arrow, releasing a Leu(30) isoform that is first processed exclusively through autocatalytic intramolecular cleavage within a glutamine-rich propeptide segment, (40)QQTQSSKQQTPKIQ(53). The preferred site is Gln(43) with secondary processing at Gln(47) and Gln(53). This initial processing is necessary for optimal and subsequent Aur-dependent processing at Leu(58) and then Val(69) to release mature SspA. Although processing by Aur is rate-limiting in zSspA activation, the first active molecules of Val(69)SspA promote rapid intermolecular processing of remaining zSspA at Glu(65), producing an N-terminal (66)HANVILP isoform that is inactive until removal of the HAN tripeptide by Aur. Modeling indicated that His(66) of this penultimate isoform blocks the active site by hydrogen bonding to Ser(237) and occlusion of substrate. Binding of glutamate within the active site of zSspA is energetically unfavorable, but glutamine fits into the primary specificity pocket and is predicted to hydrogen bond to Thr(232) proximal to Ser(237), permitting autocatalytic cleavage of the glutamine-rich propeptide segment. These and other observations suggest that zSspA is activated through a trypsinogen-like mechanism where supplementary features of the propeptide must be sequentially processed in the correct order to allow efficient activation.