EssentialsAntithrombin III (AT) binds heparin with higher affinity than AT. A conformation-specific antibody against AT, TPP2009, was made to investigate AT in hemostasis. TPP2009 bound specifically to heparin-AT and greatly reduced the anticoagulant effect of AT. This antibody was effective in elucidating the importance of AT in hemostasis.SummaryBackground Antithrombin III (AT) is an isoform of AT that lacks the post-translational carbohydrate modification at Asn135. This isoform binds heparin with greater affinity than AT, and has been shown to target antithrombotic function to the extracellular vascular endothelial injury site.Objectives To characterize a conformation-specific antibody against AT and begin to investigate the role of AT in maintaining hemostasis.Methods Surface plasmon resonance (SPR), antigen binding and functional assays were conducted to characterize the mode of action of antibodies generated against heparin-bound AT (AT*H) by the use of phage display.Results SPR and binding studies showed that one of the antibodies, TPP2009, bound specifically to AT*H and glycosaminoglycan-associated AT on endothelial cells. In diluted prothrombin and activated factor X (FXa)-induced clotting assays, TPP2009 dose-dependently reduced the anticoagulant effect of heparin in non-hemophilic and FVIII-deficient human plasma, with half-maximal effective concentrations (EC50) of 10.5 nm and 4.7 nm, respectively. In AT-deficient human plasma, TPP2009 dose-dependently inhibited the effects of exogenously added AT and heparin. In purified systems with AT and pentasaccharide, TPP2009 restored > 91% of FXa activity. TPP2009 dose-dependently reversed the effects of heparin in rabbit (EC50, 25.7 nm) and cynomolgus monkey (EC50, 21.5 nm) plasma, but not in mouse plasma. TPP2009 was also effective in partially restoring FXa activity in rabbit and cynomolgus monkey plasma treated with FVIII function-neutralizing antibodies.Conclusions TPP2009 specifically targets a unique conformational epitope on AT*H and blocks AT-mediated anticoagulation. It effectively promotes coagulation in plasma, indicating the importance of AT in hemostasis.

Abstract Gene expression analysis showed that a human mindin homologue, mindin/RG-1, is expressed selectively in prostate tissues and that its expression level is elevated in some prostate tumors. Mindin/RG-1 protein expression is maintained in >80% of prostate cancers metastatic to bone or lymph nodes as well as in locally recurrent tumors in androgen-unresponsive patients. In contrast, mindin/RG-1 expression in other normal tissues is significantly lower than that seen in the prostate. A fully human antibody, 19G9, was generated against mindin/RG-1 protein and was shown to accumulate at high abundance in LNCaP tumor xenografts. Conjugates of this antibody with the chelator CHX-A″-DTPA were generated and radiolabeled with either 111In, 90Y, or 86Y. Small animal positron emission tomography imaging with the 86Y-radiolabeled conjugate showed very specific accumulation of the antibody in LNCaP tumor xenografts with clear tumor delineation apparent at 4 hours. The therapeutic efficacy of [90Y]-CHX-A″-DTPA-19G9 was evaluated in mice bearing LNCaP xenografts. A dose-finding study identified a nontoxic therapeutic dose to be ∼75 μCi. Significant antitumor effects were seen with a single administration of radiolabeled antibody to animals bearing 200 to 400 mm3 tumors. Inhibition of tumor growth was observed in all treated animals over a 49-day period. At 49 days posttreatment, slow tumor growth recurred but this could be prevented for an additional 40-day period by a second administration of a 75 μCi dose at day 49. We conclude that [90Y]-CHX-A″-DTPA-19G9 is a novel antibody conjugate that has considerable promise for therapy of metastatic prostate cancer in androgen-unresponsive patients.

The zona pellucida of mammalian oocytes plays an important role in binding and activation of sperm cells during the molecular events leading to fertilization. The genes coding for the three zona pellucida glycoproteins ZPA, ZPB, and ZPC of various species including mouse, dog, and human have been cloned and sequenced by several groups. However, it has remained a matter of debate as to whether the oocytes alone or in conjunction with the surrounding granulosa cells express and deposit these proteins to form the zona pellucida matrix. Addressing this unresolved issue, we assessed the expression and localization of all three zona pellucida proteins in ovaries of human, cynomolgus monkey and mice using immunohistochemical methods. In addition, oocyte-specific expression of ZPC from the primordial stage onward was confirmed by in situ hybridization. In sections of human ovaries, ZPA, ZPB, and ZPC proteins were immunohistochemically detected in the cytoplasms of primordial oocytes and during later stages of folliculogenesis in the zona pellucida matrices of oocytes. In sections fixed with formalin, a clear homogeneous ring was visible around the oocyte and no staining of granulosa cells was observed. In contrast, staining of ZP proteins was also observed between granulosa cells when Bouin's reagent had been used for tissue fixation. Thus, the original zona pellucida architecture was better preserved by formalin fixation. We further demonstrated that dissolution of the zona pellucida of isolated bovine oocytes occurred after they were exposed to Bouin's reagent. In summary, these results demonstrate that in mice, monkeys and humans, zona proteins are expressed and assembled exclusively by the oocyte and not by the granulosa cells. Previously observed results of ZP expression by an involvement of granulosa cells might therefore be the result of an improper fixation of the tissues leading to the disruption of the zona pellucida. Additionally this study highlights the importance of choosing the correct fixative for immunohistochemistry, not only for the usual reason of retaining antigenicity, but rather to retain the entire architectural structure.

The aim of this study was to investigate the binding interactions of the human progesterone receptor (hPR) with its natural ligand. Therefore, a homology-derived model of the hPR ligand binding domain has been constructed and used to predict residues potentially involved in interactions with progesterone. These residues and the free cysteines have been mutated (in total 13 residues with 15 mutations). All exchanges have been designed to preserve the three-dimensional structure of the protein. With respect to the binding characteristics towards progesterone, the muteins fall into three groups displaying no, reduced, or wildtype-like binding activity.

The activity of vampire bat (Desmodus rotundus) salivary plasminogen activator (D. rotundus PA alpha1) and to a much lesser extent of tissue-type plasminogen activator (t-PA) is stimulated by the presence of fibrin. This cofactor requirement has in the past intuitively been attributed to fibrin binding. We have previously shown that elements of the non-protease domain of D. rotundus PA alpha1 could contribute to fibrin stimulation irrespective of fibrin binding. We now demonstrate that the protease domain of D. rotundus PA alpha1 by itself exhibits fibrin selectivity, i.e. it is 32-fold stimulated by fibrin but only 1.5-fold by fibrinogen. To a lesser extent this fibrin selectivity is also shared by the protease domain of t-PA. Our findings indicate that protein-protein interactions apart from fibrin binding affect the stimulatory mechanism of fibrin on D. rotundus PA alpha1 and t-PA.

The saliva of the blood-eating vampire bat Desmodus rotundus contains plasminogen activators (PAs) that maintain the fluidity of the prey's blood by activating plasminogen and dissolving developing fibrin clots. D. rotundus salivary PAs (DSPAs) are composed of evolutionarily conserved domains reminiscent of human tissue-type PA (tPA), but their catalytic domain lacks a plasmin-sensitive "activation cleavage site". Despite this, all DSPAs are intrinsically active and enormously stimulated in the presence of fibrin. The recombinant catalytic domain of DSPAalpha1 has been crystallized in a covalent complex with Glu-Gly-Arg-chloromethyl ketone and its structure solved at 2.9 A resolution. The structure is similar to that of activated two-chain human tPA. Despite its single-chain status, the activation domain is observed in an enzymatically active conformation, with a functional substrate binding site and active site accommodating the peptidylmethylene inhibitor. The activation pocket, which normally receives the N-terminal Ile16, is occupied by the side chain of Lys156, whose distal ammonium group makes an internal salt bridge with the carboxylate group of Asp194. Lys156 is in a groove shielded from the bulk solvent by the intact "activation loop" (Gln10-Phe21), favoring Lys156-Asp194 salt bridge formation and stabilization of a functional substrate binding site. Together with the characteristic 186 insertion loop, the activation loop could act as a switch, effecting full single-chain enzymatic activity upon binding to fibrin.

The Escherichia coli DNA-binding protein FIS (factor for inversion stimulation) stimulates site-specific recombination reactions catalysed by DNA invertases and is an activator of stable RNA synthesis. To address the question of whether FIS is involved in other cellular processes we have identified and sequenced proteins whose expression pattern is affected by FIS. This has led to the identification of several E. coli genes whose expression in vivo is either enhanced or repressed by FIS. All of these genes encode enzymes or transport proteins involved in the catabolism of sugars or nucleic acids, and their expression is also dependent on the cAMP-CRP complex. In most cases studied the regulation by FIS is indirect and occurs through effects on the synthesis of the respective repressor proteins. We conclude that FIS is a transcriptional modulator involved in the regulation of metabolism in E. coli.

DSPA alpha 1 (Desmodus rotundus salivary plasminogen activator), a plasminogen activator from the saliva of the vampire bat Desmodus rotundus, is an effective thrombolytic agent. An unusual type of posttranslational modification, in which L-fucose is O-glycosidically linked to threonine 61 in the epidermal growth factor domain was found for natural DSPA alpha 1 and its recombinant form isolated from Chinese hamster ovary cells.In the present study a combination of carbohydrate and amino acid composition analysis, amino acid sequencing, and mass spectrometry revealed that the L-fucose is bound to residues 56-68 of DSPA alpha 1. The amino acid sequence of this glycosylation site agreed with the suggested consensus sequence Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys described for other proteins, A new strategy for the identification of the modified amino acid was established. Direct evidence for the occurrence of fucosyl-threonine was obtained by mass spectrometry after digestion of the glycopeptide with a mixture of peptidases. On the basis of these results, DSPA alpha 1 is a suitable model for studying the influence of O-fucosylation on clearance rates, particularly in comparative studies with the identically fucosylated and structurally related tissue plasminogen activator.

The novel plasminogen activator Desmodus rotundus plasminogen activator (DSPA), which exhibits high fibrin specificity and thus an interesting pharmacological profile, was characterized pharmacokinetically in rats and cynomolgus monkeys after i.v. bolus administration of 125I-labelled protein. Furthermore, several toxicokinetic studies with single or repeated administration of unlabelled DSPA were monitored by ELISA and fibrin clot lysis assay (FCLA) to measure antigen and activity levels. The dose range used in both species was 1 to 30 mg/kg. Data were used to describe the pharmacokinetic profile of DSPA in animals. In rats and monkeys DSPA was characterized by long terminal half-lives of 1–2h and 5–8h with bi- or triphasically declining plasma levels. The terminal phase represented a partial area under the curve (AUC) of 42%–57% in monkeys. Total clearance accounted for 6–11 ml/min/kg and approximately 2 ml/min/kg in rats and monkeys, respectively. The volume of distribution in the central compartment was 0.5–0.1 l/kg in both species. Pharmacokinetics were linear and no sex-specific differences were observed. In both species plasma antigen and activity levels, and thus its pharmacokinetics, showed a linear correlation with a slope close to 1 over the dose range of 1–30 mg/kg. The use of 125I-labelled protein only provided limited additional information for the early post-dose phase due to rapid iodine exchange. In terms of distribution in rats, radiolabel indicative for DSPA (i.e. until 30 min post application) was found in the highly perfused organs and tissues. By means of allometric extrapolation a total clearance of approximately 1 ml/min/kg was predicted for humans. DSPA displayed an advantageous pharmacokinetic and pharmacodynamic profile, especially due to its low total clearance, its long terminal half-life, and the complete fibrinolytic activity of antigen present in the plasma, as compared to other established protein fibrinolytics (e.g. t-PA). Animal data encourage the envisaged therapeutic dosage scheme with an i.v. bolus in humans.

DSPA(alpha 1) is a novel plasminogen activator with high fibrin specificity. A sandwich ELISA-system with affinity purified and peroxidase labelled DSPA(alpha 1) antibodies raised in rabbits was developed and exhibited a limit of quantification of 3 ng/ml in undiluted spiked plasma. Accuracy was 98-108% and precision accounted for 3-9.5%. No cross-reactivity with human t-PA or endogenous matrix constituents interfering with assay results was observed.After i.v. administration DSPA(alpha 1) at 1 and 3 mg/kg in cynomolgus monkeys antigen levels in plasma were dose-dependent in both gender and exhibited a triphasic disposition profile with half-lives of 0,04-0,26 h, 0.6-3 h and 4-8.5 h, The mean residence time of DSPA(alpha 1) ranged from 3 to 9 h and total clearance was approximately 2 mg/min/kg independent of sex and dose. These data obtained in monkeys showed a long systemic circulation of the antigen, linear pharmacokinetics and no sex-specific pharmacokinetic differences in the dose range investigated.In conclusion the present ELISA method is suitable for pharmacokinetic studies of DSPA(alpha 1) in animals and man. First investigations in monkeys demonstrated the interesting pharmacokinetic profile of the compound which might be administered by i.v. bolus administration for the treatment of acute myocardial infarction.

The distinguishing characteristic of vampire bat (Desmodus rotundus) salivary plasminogen activators (DSPAs) is their strict requirement for fibrin as a cofactor. DSPAs consist of structural modules known from urokinase (u-PA) and tissue-type plasminogen activator (t-PA) such as finger (F), epidermal growth factor (E), kringle (K), and protease (P), combining to four genetically and biochemically distinct isoenzymes, exhibiting the formulas FEKP (DSPA alpha 1 and alpha 2) and EKP and KP (DSPA beta and DSPA gamma). Only DSPA alpha 1 and alpha 2 bind to fibrin. All DSPAs are single-chain molecules, displaying substantial amidolytic activity. In a plasminogen activation assay, all four DSPAs are almost inactive in the absence of fibrin but strongly stimulated by fibrin addition. The catalytic efficiency (k(cat)/K-m) of DSPA alpha(1) increases 10(5)-fold, whereas the corresponding value of t-PA is only 550. The ratio of the bimolecular rate constants of plasminogen activation in the presence of fibrin versus fibrinogen (fibrin selectivity) of DSPA alpha 1, alpha 2, beta, gamma, and t-PA was found to be 13,000, 6500, 250, 90, and 72, respectively. Whereas all DSPAs are therefore more fibrin dependent and fibrin selective than t-PA, the extent depends on the respective presence of the various domains. The introduction of a plasmin-sensitive cleavage site in a position akin to the one in t-PA partially obliterates fibrin cofactor requirement. Fibrin dependence and fibrin selectivity of DSPAs are accordingly mediated by fibrin binding, which involves the F domain, as yet undefined determinants within the K and P domains, and by the absence of a plasmin-sensitive activation site. These findings transcend the current understanding of fibrin-mediated stimulation of plasminogen activation: in addition to fibrin binding, specific protein-protein interactions come into play, which stabilize the enzyme in its active conformation.

Salivary plasminogen activator from the vampire bat Desmodus rotundus (DSPA α1) is a promising new thrombolytic agent. Continuous growth of a stably transfected, methotrexate amplified, dhfr− CHO cell line yields up to 60 mg l−1 of DSPA α1. Utilizing an engineered baculovirus 10 mg l−1 were produced in batches of Sf 9 insect cells. Recombinant DSPA α1 is purified from both sources using a one-step purification protocol. Although differences in glycosylation were detected, enzymatic activity and fibrin cofactor dependency are unaffected when DSPA α1 derived from the two expression systems is compared.