Peptidyl Chloromethyl Ketone Research Articles

A Single Cleavage Reaction in the Proteolytic Activation of Factor V to the Cofactor Is Rate-Limiting in a Step That Is Selectively Dependent on Anion Binding Exosite 2 of Thrombin

Human factor V (hFV) is a procofactor with a domain organization of A1-A2-B-A3-C1-C2. It requires proteolytic activation before it can assemble within the prothrombinase complex and function as a cofactor. Activation by thrombin results from cleavages following Arg709, Arg1018 and Arg1545 to release the B domain in two fragments and form heterodimeric factor Va (FVa, A1-A2/A3-C1-C2). While A1-A2/A3-C1-C2 is the prototypic active cofactor, variable cofactor function is attributed to partially cleaved species. Several studies have proposed that meizothrombin (mIIa), an intermediate produced during prothrombin activation, is also an efficient activator of membrane-bound hFV and possibly responsible for FVa formation early in the clotting process. However, this is inconsistent with the proposed importance of anion binding exosite 2 (ABE2) for hFV activation by thrombin which is occluded by the covalently attached propiece in mIIa. We have studied the kinetics of hFV cleavage using a quantitative two-color immunoblotting approach to simultaneously and unambiguously identify intermediates and products containing A1-A2 and/or A3-C1-C2. We have also compared hFV cleavage by thrombin by cleavage with a stable mIIa variant and by mIIa lacking both the 4-carboxyglutamic acid and kringle 1 domains (mIIa-desF1) in the presence of membranes. Addition of 0.1 nM thrombin yielded quantitative cleavage of hFV (50 nM) in ~6 minutes, producing products consistent with cleavage at Arg709 and/or Arg1018. However, the C-terminal intermediate beginning at 1019 but uncleaved at Arg1545 persisted for more than 90 minutes. A3-C1-C2, arising from cleavage at Arg1545, appeared at a ~20-fold slower rate than the A1-A2 species. Thus, for the reaction with thrombin, cleavage at Arg1545 represents the rate limiting step in the conversion of hFV to heterodimeric FVa. Similar rates of hFV consumption and appearance of species cleaved at Arg709 and/or Arg1018 were seen with mIIa. However, cleavage at Arg1545 to produce A3-C1-C2 was substantially slower, by a factor of ~100, than A1-A2 formation. Equivalently large differences in the rates of A1-A2 and A3-C1-C2 formation were evident in the absence of membranes except the rate of all cleavages by mIIa was further reduced. Because the results obtained with mIIa were replicated with mIIa-desF1, the data imply that it is covalent occlusion of ABE2 in these mIIa variants that underlies their selectively impaired ability to cleave at Arg1545 and produce heterodimeric Va but does not affect cleavage at Arg709 or Arg1018. The binding of Xa to activated FV, essential for membrane-dependent prothrombinase assembly, is a prerequisite for cofactor function. We correlated hFV cleavage with the development of cofactor function by binding measurements using Xa inactivated with a fluorescent probe covalently tethered to the active site with a peptidyl chloromethylketone. For hFV cleavage by thrombin, Xa binding and the assembly of prothrombinase proceeded unexpectedly slowly and correlated with cleavage at Arg1545, widely separated in time from rapid cleavage at Arg709 and/or Arg1018. Therefore, slow cleavage at Arg1545 dictates both the formation of heterodimeric FVa and the ability of the product to bind Xa with high affinity to form prothrombinase. Only a small fraction of Xa binding developed within an hour in hFV cleavage reactions initiated with mIIa in comparison to the same concentration of thrombin. Our findings indicate that ABE2 in thrombin variants plays an essential role in the rate-limiting cleavage at Arg1545 required for the formation of heterodimeric FVa and for the assembly of prothrombinase. Occlusion of ABE2 by the covalently linked propiece in mIIa variants greatly impairs their ability to activate hFV to the active cofactor by selectively decreasing the rate of cleavage at Arg1545. The surprising and near absolute need for cleavage at Arg1545 to produce cofactor is consistent with new biochemical and structural evidence implicating an essential role played by an acidic sequence immediately N-terminal to Arg1545 in concert with a basic sequence C-terminal to Arg1018 within the B domain in restricting the binding of Xa to the incompletely cleaved procofactor and preventing cofactor function. DisclosuresKrishnaswamy:Novo Nordisk: Consultancy; Janssen: Consultancy, Research Funding; Portola: Research Funding.

A Proteolytic Site-Directed Affinity Label to Inhibit the Human ATP-Dependent Protease Caseinolytic Complex XP.

Human caseinolytic protease component X and P (hClpXP) is a heterooligomeric ATP-dependent protease. The hClpX subunit catalyzes ATP hydrolysis whereas the hClpP subunit catalyzes peptide bond cleavage. In this study, we generated a peptidyl chloromethyl ketone (dansyl-FAPAL-CMK) that inhibited the hClpP subunit through alkylation of the catalytic His122, which was detected by LC-MS. This inhibitor is composed of a peptide sequence derived from a hydrolyzed peptide product of a substrate cleaved by hClpXP. Binding of FAPAL positions the electrophilic chloromethyl ketone moiety near His122 where alkylation occurs. Dansyl FAPAL-CMK exhibits selectivity for hClpXP over other ATP-dependent proteases such as hLon and the 26S proteasome and abolishes hClpXP activity in HeLa cell lysate. Using the fluorogenic peptide substrate FR-Cleptide as reporter, we detected biphasic inhibition time courses; this supports a slow-binding, time-dependent, covalent inhibition mechanism that is often found in active-site directed affinity labels. Because this inhibitor reacts only with hClpXP but not hLon or the proteasome, it has the potential to serve as a chemical tool to help validate endogenous protein substrates of hClpXP in cell lysate, thereby benefiting investigation of the physiological functions of hClpXP in different cell types or tissue samples.

Recombinant and native TviCATL from Trypanosoma vivax: Enzymatic characterisation and evaluation as a diagnostic target for animal African trypanosomosis

African animal trypanosomosis (nagana) is caused by tsetse-transmitted protozoan parasites. Their cysteine proteases are potential chemotherapeutic and diagnostic targets. The N-glycosylated catalytic domain of Trypanosoma vivax cathepsin L-like cysteine protease, rTviCATLcat, was recombinantly expressed and purified from culture supernatants while native TviCATL was purified from T. vivax Y486 parasite lysates. Typical of Clan CA, family C1 proteases, TviCATL activity is sensitive to E-64 and cystatin and substrate specificity is defined by the S2 pocket. Leucine was preferred in P2 and basic and non-bulky, hydrophobic residues accepted in P1 and P3 respectively. Reversible aldehyde inhibitors, antipain, chymostatin and leupeptin, with Arg in P1 and irreversible peptidyl chloromethylketone inhibitors with hydrophobic residues in P2 inhibited TviCATL activity. TviCATL digested host proteins: bovine haemoglobin, serum albumin, fibrinogen and denatured collagen (gelatine) over a wide pH range, including neutral to slightly acidic pH. The recombinant catalytic domain of TviCATL showed promise as a diagnostic target for detecting T. vivax infection in cattle in an indirect antibody detection ELISA.

Assembly of Prothrombinase on Endothelial Cells: Receptor-Mediated or Phospholipid-Driven?

Membrane binding by factors Xa and Va plays an essential role in facilitating their interaction to yield membrane-bound prothrombinase. This concept is backed by a large body of biochemical work using synthetic phospholipid vesicles typically composed of 25% phosphatidylserine (PS) and 75% phosphatidylcholine (PC). However, it remains uncertain whether kinetic and thermodynamic findings with these membranes can be extrapolated to explain the assembly of prothrombinase on cell surfaces relevant to coagulation in the vasculature. We examined the binding of fluorescent derivatives of Xa and Va to endothelial cells cultured in flow chambers using confocal imaging of fluorescence intensity. Cell-bound factor Xa was imaged using Alexa488 or Alexa532 covalently linked to the active site with a peptidyl chloromethyl ketone. Minimal fluorescence was observed on the endothelial surface without intentional activation of the cells by thrombin. Following thrombin activation, total fluorescence of bound Xa in the absence of added Va was marginally different from the unactivated cells but increased 16-fold when excess Va was present. Heterogeneity was evident in the distribution of fluorescence over the surface of the cells that supported Xa binding. Thus, the binding of Xa requires the presence of Va and is distributed in a heterogeneous fashion on the activated endothelial cell surface. Factor Va bound to the endothelial cell was assessed using a derivative singly labeled at Cys539 with Alexa488 or Alexa532. Detection of bound Va also required prior activation by thrombin. The same robust signal for bound but heterogeneously distributed fluorescence was observed following activation both in the presence or absence of added Xa. Together the data indicate that Xa binding to the activated endothelial cell requires bound Va whereas Va binding is unaffected by the presence of Xa, implying a receptor-like role for Va. This is in marked contrast to the behavior on membranes containing 25% PS to which either Xa or Va can bind singly with good affinity. More definitive studies of prothrombinase assembly were conducted using donor fluorescence lifetime imaging on the cells using XaAlexa488 as donor and VaAlexa532 as acceptor. XaAlexa488 bound in the presence of unlabeled Va exhibited an average lifetime of 3.1 ns which was decreased to 2.1 ns in the presence of VaAlexa532. Equivalent lifetimes and energy transfer efficiency were measured using 25% PS containing membranes. Although Xa binding to the cells shows a near-absolute requirement for Va, the resulting cell-bound prothrombinase is comparable to that assembled on synthetic vesicles. An explanation could lie in the binding constraints associated with the exposure of limiting amounts of PS on the activated cell surface expected to disproportionately affect the binding of Xa and Va to membranes. These constraints were replicated using membrane bilayers supported on glass microspheres (lipospheres) containing either 0% or 5% PS balanced by PC and analysis of binding by flow cytometry. Background levels of binding were observed for all approaches in the absence of PS. Va binding was studied using a constant number of lipospheres and increasing concentrations of VaAlexa488 in the presence of different concentrations of unlabeled Xa. Mean fluorescence per 5% PS containing liposphere increased saturably with increasing concentrations of VaAlexa488 and was unaffected by Xa. The binding curves were consistent with a nM dissociation constant. In contrast, background levels of Xa were bound when monitored using varying concentrations of XaAlexa488 in the absence of Va. A family of saturable curves was obtained at different fixed concentrations of unlabeled Va, signifying binding with nM affinity, with amplitude proportional to the concentration of Va. Thus, 5% PS containing lipospheres can replicate the paradoxical receptor-like role for Va in prothrombinase assembly on activated endothelial cells. The binding behavior on lipospheres could be fully accounted for by the markedly decreased affinity of Xa for 5% PS membranes and the major contribution of linked interactions in the stabilization of membrane-bound prothrombinase. Despite appearances to the contrary, a model developed from binding studies with pure phospholipids can be generalized to provide a thermodynamic accounting for the peculiarities of prothrombinase assembly on the endothelial cell. DisclosuresNo relevant conflicts of interest to declare.

Imaging of Coagulation Reactions During Thrombus Formation In Vivo with Novel Fluorescent Protein Derivatives

AbstractAbstract 817Significant gaps remain in the understanding of how blood cells and the vasculature differentially support coagulation enzyme complex function leading to regulated thrombus formation in vivo. These gaps partly result from the lack of analytic tools with appropriate sensitivity for incisive conclusions. Here we have combined state of the art confocal fluorescent microscopy, established mouse models, and unique recombinant clotting factors as direct imaging probes to advance our understanding of this process. Hemophilic mice typically do not form stable thrombi following microscopic laser injury in the cremaster model. However, thrombus formation in either hemophilia A (HA) or hemophilia B (HB) mice can be restored by infusion of factor Va. Such rescue implies the assembly of prothrombinase and the formation of sufficient thrombin to permit thrombus formation. We employed this injury model using HB mice as a platform to assess the localization of site-specific fluorescent derivatives of Va and Xa in the growing thrombus. We have prepared a FV variant (Va-810SYA) that is constitutively Va-like with three free cysteines mutated while leaving a single free cysteine at position 539. This site was labeled with Alexa488-maleimide without affecting cofactor function. We also employed Xa bearing Alexa488 or Alexa647 tethered to the active site with a peptidyl chloromethyl ketone. The resulting Alexa488-EGR-Xa or Alexa647-EGR-Xa derivatives are inactive but retain the ability to assemble into prothrombinase. Co-infusion of Alexa555-labeled anti-CD41 antibody to visualize platelets and Alexa488-Va-810SYA (15 μg; n = 4 mice; 20 injuries) produced a robust signal indicating the accumulation of Va-810SYA and platelets at the site of microscopic laser injury. Similarly, infusion of Va-810SYA (15 μg) with Alexa488-EGR-Xa (2 μg) into HB mice (n = 3; 19 injuries) revealed a strong signal denoting the ability to directly image Xa at the site of vascular damage. Because thrombus formation in these animals requires the infusion of Va and the assembly of prothrombinase, at least part of the Va and Xa visualized in the vicinity of the thrombus must be functional rather than adventitiously bound. Similar results were obtained in wild type mice (n = 3; 21 injuries) using Alexa488-Va-810SYA or Alexa647-EGR-Xa indicating that these findings are not peculiar to HB animals. More detailed measurements were pursued by three-dimensional (3-D) confocal imaging analysis 4 min post-injury with fluorescent derivatives of Va, Xa, platelets or fibrin. As expected, platelets and fibrin were limited to the site of microscopic injury. Factors Va and Xa were also found with platelets and fibrin; however these proteins surprisingly were also distributed away from the thrombus on vascular surfaces. These results suggest that surfaces other than platelets, such as the endothelium, can accommodate the assembly and function of Va and Xa. To test this, HB mice (n=3; 15 injuries) were infused with Va or Xa along with integrilin to inhibit platelet function. Consistent with prior results in PAR4−/− mice, the reduction of platelets at the site of microscopic injury with integrilin had no obvious effect on fibrin deposition. Despite this reduction in platelets, the accumulation of Va or Xa bound in the vicinity of the injury site was not significantly altered providing an explanation for the unchanged levels of fibrin. These unexpected results question the predominant role ascribed to activated platelets in supporting the assembly and function of procoagulant enzyme complexes. Overall these studies indicate that site-specific fluorescent derivatives of Va and Xa can be visualized with appropriate sensitivity and represent powerful tools to establish their spatial distribution at the site of laser injury in the mouse cremaster model. Our approach provides new biological insights into the integrated behavior of the system of enzymic reactions and their modulation by blood cells which lead to thrombus formation in vivo. Disclosures:Camire:Pfizer: Patents & Royalties, Research Funding.

Fluorescence Resonance Energy Transfer Studies of Prothrombin Recognition by Prothrombinase.

Prothrombinase, the membrane bound complex of factors Xa and Va, catalyzes thrombin formation by the cleavage of prothrombin at R320 followed by R271. The two cleavage sites are expected to be ~40Å apart in prothrombin. Essentially ordered cleavage arises from exosite tethering of prothrombin to the enzyme which facilitates engagement of the R320 site but precludes docking of the R271site to the active site of the catalyst. Prior cleavage at R320 appears necessary for efficient active site engagement by the R271 site. The physical bases for these observations have been investigated using fluorescence resonance energy transfer to assess the disposition of the 271 site to the active site of the catalyst. Expression and purification of a prothrombin variant containing C in place of R271 (IIC271) surprisingly yielded a product lacking free cysteines. A free thiol could be rescued following treatment with reduced glutathione and could be modified with PEG or Alexa532 maleimides yielding ~1 mol reagent incorporated /mol protein. Characterization of IIC271 modified with Alexa532 maleimide (IIC271*) established that the probe was covalently contained only in those fragments bearing residue 271. IIC271* could be cleaved normally by prothrombinase at R320 yielding a product with the expected catalytic activity. Thus, thiol rescue permits selective modification of the engineered 271 site without obvious deleterious effects. Fluorescence studies employed prothrombinase assembled using Xa containing Alexa488 covalently linked to the active site with a peptidyl chloromethylketone as donor and IIC271* as acceptor. Increasing concentrations of IIC271* yielded a large and saturable decrease in donor fluorescence. This decrease could be reversed by the addition of excess unlabelled prothrombin, was not observed in the absence of membranes and could be eliminated by the addition of EDTA. Changes in donor fluorescence exhibit the features expected for energy transfer arising from the binding of prothrombin to prothrombinase. Titrations with increasing concentrations of IIC271* yielded Kd = 40.7 ± 13.5 nM and n = 1.12 ± 0.23 mol IIC271*/mol prothrombinase. Agreement between the Kd measured using active site blocked enzyme and the previously measured Km further substantiates the primary role played by exosite binding in determining substrate affinity. Maximal energy transfer efficiency inferred from donor quenching (79.3 ± 1.7%) was confirmed by measurements of excited state lifetime. Based on the measured Forster distance (57.4 Å), this value indicates that the acceptor at the 271 site in IIC271* is ~46 Å away from the donor at the active site of Xa within prothrombinase. IIC271* was cleaved by ecarin at R320 to yield meizothrombin (mIIaC271*), stabilized by covalent inactivation and re-purified. Similar experiments performed with mIIaC271* as acceptor yielded substantial differences in the extent of donor quenching in comparison to IIC271*. Thus, major changes are evident at the 271 site following cleavage at R320. We conclude that exosite-dependent binding of prothrombin to prothrombinase constrains the substrate such that the 271 site is distant from and cannot efficiently engage the active site of the catalyst. This explains the inability of prothrombinase to efficiently cleave at R271 in prothrombin. Proteinase formation following initial cleavage at R320 reorients the 271 site with respect to the active site of prothrombinase. Our findings offer a physical explanation for how prothrombinase is able to discriminate between the cleavage sites in prothrombin resulting in the ordered cleavage of the two distant sites within the substrate.

Active Site-independent Recognition of Substrates and Product by Bovine Prothrombinase: A FLUORESCENCE RESONANCE ENERGY TRANSFER STUDY

The conversion of prothrombin to thrombin is catalyzed by prothrombinase, an enzyme complex composed of the serine proteinase factor Xa and a cofactor protein, factor Va, assembled on membranes. Kinetic studies indicate that interactions with extended macromolecular recognition sites (exosites) rather than the active site of prothrombinase are the principal determinants of binding affinity for substrate or product. We now provide a model-independent evaluation of such ideas by physical studies of the interaction of substrate derivatives and product with prothrombinase. The enzyme complex was assembled using Xa modified with a fluorescent peptidyl chloromethyl ketone to irreversibly occlude the active site. Binding was inferred by prethrombin 2-dependent perturbations in the fluorescence of Oregon Green(488) at the active site of prothrombinase. Active site-independent binding was also unequivocally established by fluorescence resonance energy transfer between 2,6-dansyl tethered to the active site of Xa and eosin tethered to the active sites of either thrombin or meizothrombin des fragment 1. Comparable interprobe distances obtained from these measurements suggest that substrate and product interact equivalently with the enzyme. Competition established the ability of a range of substrate or product derivatives to bind in a mutually exclusive fashion to prothrombinase. Equilibrium dissociation constants obtained for the active site-independent binding of prothrombin, prethrombin 2, meizothrombin des fragment 1 and thrombin to prothrombinase were comparable with their affinities inferred from kinetic studies using active enzyme. Our findings directly establish that binding affinity is principally determined by the exosite-mediated interaction of either the substrate, both possible intermediates, or product with prothrombinase. A single type of exosite binding interaction evidently drives affinity and binding specificity through the stepwise reactions necessary for the two cleavage reactions of prothrombin activation and product release.

Mouse ADAM33: two splice variants differ in protein maturation and localization.

We compared the tissue mRNA prevalence and protein maturation of two splice variants of mouse ADAM33, a metalloprotease implicated in airway hyperresponsiveness. These variant cDNAs, designated 914 (alpha) and 906 (beta), encode membrane-bound forms that differ primarily in 26 residues (exon 17) between the cysteine-rich and epidermal growth factor-like domains. Proteins of approximately 120 and 103 kD, detectable by anti-ADAM33 antibodies, were expressed in 914-transfected HEK293 cells. The time-dependent appearance of the approximately 100-kD form and its inhibition by a peptidyl chloromethylketone, or the calcium ionophore, A23187, indicated that this was mature ADAM33, which was processed by a furin-like convertase. One form, approximately 110 kD, was detected in 906-transfected cell lysates. Trypsin and biotinylation treatment of transfected cells demonstrated that all of the mature approximately 100-kD, a minority of the approximately 120-kD pro-form, and none of the 906-expressed 110-kD form localized to the cell surface. The mature form was resistant to endoglycosidase H(f). The approximately 110-kD form was endoglycosidase H(f)-sensitive, indicating retention proximal to the trans-Golgi, consistent with a lack of maturation. Quantitation of transcripts demonstrated that those containing exon 17 predominate, whereas those lacking exon 17 are negligible in the mouse lung, although detectable at low levels in mouse testis, heart, and brain. Thus, potential dominant-negative effects exerted by the nonprocessed 906-encoded beta splice variant are unlikely to occur in mouse lung.

Processing of the Ebola virus glycoprotein by the proprotein convertase furin.

In the present study, we have investigated processing and maturation of the envelope glycoprotein (GP) of Ebola virus. When GP expressed from vaccinia virus vectors was analyzed by pulse-chase experiments, the mature form and two different precursors were identified. First, the endoplasmic reticulum form preGPer, full-length GP with oligomannosidic N-glycans, was detected. preGPer (110 kDa) was replaced by the Golgi-specific form preGP (160 kDa), full-length GP containing mature carbohydrates. preGP was finally converted by proteolysis into mature GP1,2, which consisted of two disulfide-linked cleavage products, the amino-terminal 140-kDa fragment GP1, and the carboxyl-terminal 26-kDa fragment GP2. GP1,2 was also identified in Ebola virions. Studies employing site-directed mutagenesis revealed that GP was cleaved at a multibasic amino acid motif located at positions 497 to 501 of the ORF. Cleavage was blocked by a peptidyl chloromethylketone containing such a motif. GP is cleaved by the proprotein convertase furin. This was indicated by the observation that cleavage did not occur when GP was expressed in furin-defective LoVo cells but that it was restored in these cells by vector-expressed furin. The Reston subtype, which differs from all other Ebola viruses by its low human pathogenicity, has a reduced cleavability due to a mutation at the cleavage site. As a result of these observations, it should now be considered that proteolytic processing of GP may be an important determinant for the pathogenicity of Ebola virus.

Purification and characterization of an arginine-specific peptidase from ragweed (Ambrosia artemisiifolia) pollen.

Ragweed (Ambrosia artemisiifolia) is clinically the most important source of seasonal aeroallergens, as it is responsible for the majority and most severe cases of hay fever (allergic rhinitis). Extracts from pollen grains have been shown to contain numerous proteins with various functions, including a novel serine proteolytic enzyme with chymotrypsin-like specificity that has been previously described (J. Biol. Chem. 1996; 271:26227-26232). We now report the isolation and properties of a second, trypsin-like enzyme with a molecular mass near 80 kD, from ragweed pollen extracts. This enzyme has a blocked N-terminus, a pH optimum near 9.0, and requires Ca2+ for stability and activity, but not reducing agents. The enzyme is inhibited by diisopropyl fluorophosphate, a general serine class proteinase inhibitor, and more specifically by N-p-tosyl-L-lysine chloromethyl ketone. Activity toward protein substrates was not detected, but various synthetic substrates and small biologically active peptides were efficiently cleaved, with a strong preference for Arg in the P1 position and either Arg or Gly in the P2 position. This specificity was confirmed through inhibition studies with both peptidyl chloromethyl ketone and organophosphate inhibitors. Significantly, atrial natriuretic peptide and angiotensin 2, whose degradation would amplify kinin activity and influence inflammatory diseases of the respiratory tract and nasal passages, were also rapidly hydrolyzed. Thus, the ragweed pollen endopeptidase may be involved in the inactivation of regulatory neuropeptides during pollen-initiated allergic reactions.

Purification and Characterization of a Novel Endopeptidase in Ragweed (Ambrosia artemisiifolia) Pollen

Ragweed (Ambrosia artemisiifolia), the major cause of late summer hay fever (allergic rhinitis) in the United States and Canada, is clinically the most important source of the seasonal aeroallergens. A novel endopeptidase was extracted from the pollen of this plant and purified by a series of column chromatographic steps. It has a molecular mass of 82 kDa according to gel filtration and SDS-polyacrylamide gel electrophoresis and a pH optimum near 9.0, and its activity is unaffected by chelating or reducing agents. A 17-amino acid amino-terminal sequence of this protein showed no similarity with any other proteases. The enzyme was inhibited by diisopropyl fluorophosphate, a general serine class inhibitor, and more specifically N-p-tosyl-L-phenylalanine chloromethyl ketone, a chymotrypsin-like proteinase inhibitor. Various synthetic substrates were efficiently cleaved with a strong preference for Phe in the P1 and P3 position and Pro in the P2 position. This specificity was confirmed through inhibition studies with both peptidyl chloromethyl ketone and organophosphate inhibitors. In addition to synthetic substrates, the neuropeptides, vasoactive intestinal peptide and substance P, which are required for normalized lung functions, were also rapidly hydrolyzed. Activity toward protein substrates was not detected with the exception of the inactivation of alpha-1-proteinase inhibitor, which occurred through cleavage within the reactive site loop. These results indicate that the purified enzyme is a novel endopeptidase, which may be involved in both the degradation of neuropeptides and the inactivation of protective proteinase inhibitors during pollen-initiated allergic reactions.

Identification of the Reactive Histidine of Cucumisin, a Plant Serine Protease: Modification with Peptidyl Chloromethyl Ketone Derivative of Peptide Substrate

A radioactive peptidyl chloromethyl ketone derived from substrates of cucumisin, 3H-labeled Z-Ala-Ala-Pro-Phe-chloromethyl ketone (3H-ZAAPFCK), was synthesized. When cucumisin was incubated with a 100-fold molar excess of 3H-ZAAPFCK for 16 h, 98% of the cucumisin activity was inhibited and about 0.93 mol of 3H-ZAAPFCK was incorporated in 1 mol of cucumisin. The 3H-ZAAPFCK-modified cucumisin was reduced and pyridylethylated, and then digested by trypsin. The radioactive peptide fragment was isolated and its amino acid sequence was determined. The radioactive fragment contained 32 amino acid residues and the sequence around the labeled residue was found to be -Asp-Thr-Asn-Gly-(His)-Gly-Thr-His-Thr-Ala-. This sequence is analogous to that around the reactive site histidine residue of the subtilisin family.

Evidence That Meizothrombin Is an Intermediate Product in the Clotting of Whole Blood

Meizothrombin is an intermediate that is produced during the conversion of prothrombin to thrombin in systems composed of purified factor Xa and factor Va that are quantitatively assembled on an anionic phospholipid surface. The biological significance of this intermediate has recently been challenged by the apparent absence of meizothrombin during clotting of sodium citrate-anticoagulated plasma. We analyzed the formation of thrombin during coagulation of nonanticoagulated, unchilled, minimally manipulated whole blood in glass tubes. The process was stopped at 0, 3, 5, and 7 minutes by the addition of biotinylated peptidyl chloromethyl-ketone active-site labeling reagents. Plasma/serum was separated by centrifugation, and labeled species were extracted by immunoadsorption with a polyclonal anti-prothrombin antibody. The purified prothrombin-derived species were separated by SDS-polyacrylamide gradient gel electrophoresis and visualized on a chemiluminescent avidin blot. Meizothrombin appeared as an intermediate product of this reaction and persisted with some increase through the 7-minute time point. We also observed incorporation of the active-site label into a species of lower molecular weight consistent with the B1 chain of beta- and/or gamma-thrombin. These degraded forms of thrombin have not been previously demonstrated in a biologically relevant preparation. Our data clearly establish the generation of meizothrombin as an intermediate product of thrombin generation during whole-blood clotting. The data also represent the first experimental evidence for the generation of beta- and gamma-thrombin in a biologically relevant environment and time scale.

Proteolytic processing of human cytomegalovirus glycoprotein B (gpUL55) is mediatedby the human endoprotease furin

Inhibition of endoproteolytic cleavage of glycoprotein B (gB; gpUL55) of human cytomegalovirus was achieved by treatmentof infected fibroblasts with decanoyl peptidyl chloromethyl ketone (decRVKR-CMK), which inhibits the action of cellular subtilisin-like endoproteases with the amino acid recognition motif R × K/R R. Uncleaved gB precusor molecules of 160 kDa that were accumulated were endoglycosidase H resistant, suggesting that correct cellular transport occurred in the presence of the drug. The inhibitor also prevented endoproteolytic gB processing in CV-1 cells infected with a recombinant vaccinia virus-gB construct (VVgB). Evidence for direct involvement of the ubiquitous subtilisin-like endoprotease furin in gB cleavage was obtained from the observation that coinfection of CV-1 cells with WgB and a recombinant vaccinia-human furin construct reestablished endoproteolytic activity which was normally absent late after infection with WgB alone.

Radioiodination of the active site of tissue plasminogen activator: A method for radiolabeling serine proteases with tyrosylprolylarginyl chloromethyl ketone

Tyrosylprolylarginyl chloromethyl ketone (YPRck)is a radioiodinatable inhibitor that irreversibly binds the active site of tissue plasminogen activator (WA). A two-step reaction is employed where (1) the YPRck reagent is iodinated and (2) the 125I-YPRck is reacted with the tPA sample; therefore the oxidative effects of conventional protein iodination are avoided. Using fibrin binding as a probe of native tPA binding function, YPRck labeling was shown to be superior to other types of surface iodination. 125I-YPRck was prepared at a high specific radioactivity; i.e., one 125I per 3.5 molecules of peptidyl chloromethyl ketone. Labeled YPRck formed a one to one covalent, sodium dodecyl sulfate stable, complex with tPA resulting in a preparation of 10 mCi per milligram protein, which corresponded to an incorporation ratio of 1:3.5 ( 125I-YPRck:tPA). Both one-chain and two-chain forms of tPA reacted with YPRck. Radiolabeling tPA with 125I-YPRck occurred in a time-dependent manner with half-maximal incorporation at approximately 30 min under the conditions employed in these studies. The pH optimum for the reaction of tPA with 125I-YPRck was 7.4. Solutions of tPA at less than 1 μg/ml were efficiently labeled with 125I-YPRck, thus allowing the quantitation of functional protease by incorporation of radiolabel. Significantly, 125I-YPRck specifically labeled tPA in cell culture supernatants after transient transfection of cells with plasmid DNA containing the gene for tPA. Other serine proteases were tested for their relative reactivity with 125I-YPRck. Thrombin and Factor Xa incorporated 125I-YPRck to higher levels than two-chain tPA; whereas plasmin, urokinase, and other plasma proteases were not as efficiently radiolabeled. The use of 125I-YPRck allows rapid and specific radiolabeling of a large number of tPA samples in a nondenaturing environment with a known localization of the radiolabeling reagent.

Cooperative activation of human factor IX by the human extrinsic pathway of blood coagulation

The activation of human coagulation factor IX by human tissue factor.factor VIIa.PCPS.Ca2+ (TF.VIIa.PCPS.Ca2+) and factor Xa.PCPS.Ca2+ enzyme complexes was investigated. Reactions were performed in a highly purified system consisting of isolated human plasma proteins and recombinant human tissue factor with synthetic phospholipid vesicles (PCPS: 75% phosphatidylcholine (PC), 25% phosphatidylserine (PS)). Factor IX activation was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, [3H]factor IX activation peptide assay, colorimetric substrate thiobenzyl benzyloxycarbonyl-L-lysinate (Z-Lys-SBzl) hydrolysis, and specific incorporation of a fluorescent peptidyl chloromethyl ketone. Factor IX activation by the TF.VIIa.PCPS.Ca2+ enzyme complex was observed to proceed through the obligate non-enzymatic intermediate species factor IX alpha. The simultaneous activation of human coagulation factors IX and X by the TF.VIIa.PCPS.Ca2+ enzyme complex were investigated. When factors IX and X were presented to the TF.VIIa complex, at equal concentrations, it was observed that the rate of factor IX activation remained unchanged while the rate of factor X activation slowed by 45%. When the proteolytic cleavage products of this reaction were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was observed that the intermediate species factor IX alpha was generated more rapidly when factor X was present in the reaction mixture. When factor IX was treated with factor Xa.PCPS in the presence of Ca2+, it was observed that factor IX was rapidly converted to factor IX alpha. The activation of factor IX alpha by the TF.VIIa.PCPS.Ca2+ complex was evaluated, and it was observed that factor IX alpha was activated more rapidly by the TF.VIIa.PCPS.Ca2+ complex than was factor IX itself. These data suggest that factors IX and X, when presented to the TF.VIIa.PCPS.Ca2+ enzyme complex, are both rapidly activated and that factor Xa, which is generated in the initial stages of the extrinsic pathway, participates in the first proteolytic step in the activation of factor IX, the generation of factor IX alpha.

Thromboxane and cysteinyl-leukotriene formation are differentially activated in spontaneously clotting whole human blood in vitro

We have previously demonstrated that clotting of whole human blood in vitro not only triggers the production of thromboxane (TX) B 2 but is also accompanied by formation of 5-lipoxygenase-derived cysteinyl-leukotrienes (LT). In order to further characterize the mechanisms leading to activation of the cysteinyl-LT production, we have now investigated the effects of thrombin on cysteinyl-LT as well as TXB 2 formation in whole human blood. Addition of exogenous human α-thrombin (0.1 – 3.0 U/ml) to whole human blood incubated in vitro led to a concentration- and time-dependently increased release of TXB 2 into the serum samples. The serum contents of cysteinyl-LT were, however, not significantly affected. Inactivation of endogenously generated thrombin by inhibitors such as recombinant hirudin (HBW 023, 0.43–1.43 μM) or the peptidyl chloromethyl ketone, D-Phe-Pro-Arg-CH2Cl (1.0 – 100 μM) concentration- and time-dependently inhibited the release of TXB2 into the serum or plasma samples. In contrast, however, serum contents of cysteinyl-LT remained unchanged. The identity of immunoreactive material was confirmed by thin-layer chromatography of immunoreactive TXB 2 and by reversed phase HPLC of immunoreactive cysteinyl-LT. As expected, washed human platelets stimulated with α-thrombin were identified as the major source of TXB 2 generation but purified monocytes were also found to release some TXB 2 upon α-thrombin stimulation. Release of TXB 2 by isolated human polymorphonuclear leukocytes (PMN) was negligible in the presence of this stimulus. None of the cells which are known to possess 5-lipoxygenase activity such as PMN or monocytes did release neither cysteinyl-LT nor LTB4 upon stimulation with human α-thrombin up to 10 U/ml. These data demonstrate that TXB 2 production and cysteinyl-LT formation are differentially activated in spontaneously clotting whole human blood in vitro, the former being dependent on endogenously generated thrombin the latter being dependent on a stimulus yet to be identified.

Inhibition of endoproteolytic cleavage of cytomegalovirus (HCMV) glycoprotein B by palmitoyl-peptidyl-chloromethyl ketone

Endoproteolytic cleavage of glycoprotein B (gB) of human cytomegalovirus (HCMV) is inhibited by palmitoylated peptidyl-chloromethyl ketone (palFAKR-CEK) at concentrations above 30 μ M. Inhibitor treatment of HCMV-infected human fibroblasts neither interfered with exposure of gB on the plasma membrane, detected by surface membrane immunostaining, nor reduced production of intracellular infectious viral progeny. Release of infectious virus, on the other hand, was impaired.

Active Site-Specific Immunoassays

This study describes a process by which serine proteases that contain an S-1 arginine subsite and active site histidine may be inactivated and subsequently quantitated using a combination of peptidyl chloromethylketone chemistry and immune recognition technology. Active site labeling and inactivation of proteases is attained by modification of the active site histidine with a peptidyl chloromethylketone. In the specific illustrations demonstrated, we used the compound biotinyl-epsilon-aminocaproyl-phenylalanylprolylarginyl chloromethylketone. This reagent reacts quantitatively and specifically with the active site histidine of a wide variety of proteases that are elaborated in the coagulation and fibrinolytic system. The inactivated enzyme(s) may be quantitated by combinations of antiprotein antibodies and avidin binding technology using the biotin moiety on the peptide inhibitor. We have demonstrated the capability of capture of inactivated enzyme products directly on to solid-phase avidin with subsequent quantitation of bound protein using specific antibodies. In the converse system we have captured specific proteases using antiprotein antibodies in the solid phase and have quantitated bound enzyme by using avidin. Subsequent detection and quantitation has been achieved using the enzymatic activity of horseradish peroxidase conjugated either to the antibody or to avidin. Both types of assays are feasible, with avidin capture being the preferred mode when enzyme is evaluated in the presence of excess zymogen, as would be common in the evaluation of most blood-clotting enzymes. Assays are illustrated for tissue plasminogen activator, plasmin, thrombin, factor Xa, and activated protein C, which can measure protease concentrations as low as 50 pmol/L. Specific applications of the assays are provided in studies of the activation of prothrombin by the prothrombinase complex and of factor X with Russell's viper venom factor X activator. These assays measure the mass of active site present in the reaction mixture and are relatively independent of subspecies of enzyme or the environment in which the activity is generated. These assay systems provide powerful tools for elucidating product-precursor relationships in multienzyme feedback reactions involving zymogen activation.

Active site-specific immunoassays

This study describes a process by which serine proteases that contain an S-1 arginine subsite and active site histidine may be inactivated and subsequently quantitated using a combination of peptidyl chloromethylketone chemistry and immune recognition technology. Active site labeling and inactivation of proteases is attained by modification of the active site histidine with a peptidyl chloromethylketone. In the specific illustrations demonstrated, we used the compound biotinyl- epsilon-aminocaproyl-phenylalanylprolylarginyl chloromethylketone. This reagent reacts quantitatively and specifically with the active site histidine of a wide variety of proteases that are elaborated in the coagulation and fibrinolytic system. The inactivated enzyme(s) may be quantitated by combinations of antiprotein antibodies and avidin binding technology using the biotin moiety on the peptide inhibitor. We have demonstrated the capability of capture of inactivated enzyme products directly on to solid-phase avidin with subsequent quantitation of bound protein using specific antibodies. In the converse system we have captured specific proteases using antiprotein antibodies in the solid phase and have quantitated bound enzyme by using avidin. Subsequent detection and quantitation has been achieved using the enzymatic activity of horseradish peroxidase conjugated either to the antibody or to avidin. Both types of assays are feasible, with avidin capture being the preferred mode when enzyme is evaluated in the presence of excess zymogen, as would be common in the evaluation of most blood-clotting enzymes. Assays are illustrated for tissue plasminogen activator, plasmin, thrombin, factor Xa, and activated protein C, which can measure protease concentrations as low as 50 pmol/L. Specific applications of the assays are provided in studies of the activation of prothrombin by the prothrombinase complex and of factor X with Russell's viper venom factor X activator. These assays measure the mass of active site present in the reaction mixture and are relatively independent of subspecies of enzyme or the environment in which the activity is generated. These assay systems provide powerful tools for elucidating product-precursor relationships in multienzyme feedback reactions involving zymogen activation.