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Production and properties of a factor X-cellulose-binding domain fusion protein.

A fusion protein, FX-CBDCex, which comprises factor X with a cellulose-binding domain (CBDCex) fused to its C-terminus, was produced in BHK cells. It was purified from the culture medium by affinity chromatography on cellulose. FX-CBDCex could be activated to FXa-CBDCex with Russell viper venom. FXa-CBDCex was as active as FXa against a chromogenic substrate and against proteins containing the Ile-Glu-Gly-Arg sequence hydrolysed by FXa. FXa-CBDCex retained its activity when adsorbed to cellulose.

Amino Acid Sequence↗

Glandular kallikrein levels in the rat anterior pituitary during the estrous cycle and pregnancy.

Glandular kallikrein (a trypsin-like serine protease) is a major estrogen-induced protein in the rat anterior pituitary, which appears to be associated with lactotropes. The present study examined glandular kallikrein levels in the anterior pituitary during the rat estrous cycle and pregnancy. After trypsin treatment of anterior pituitary homogenates (to activate latent forms of the enzyme), glandular kallikrein activity was measured by using the chromogenic substrate D-val-leu-arg-p-nitroanilide: 98-95% of the enzymatic activity was immunoprecipitable with glandular kallikrein antiserum. Glandular kallikrein levels did not change significantly during the various phases of the rat estrous cycle. However, a sharp decrease was observed starting on Day 15 of pregnancy and lasting through parturition; levels had almost returned to control values by Day 5 of lactation.

Animals↗

Development of an enzyme-linked immunosorbent assay for goldfish gonadotropin.

An enzyme-linked immunosorbent assay (ELISA) for goldfish gonadotropin (GTH) was developed with the intent of devising a simple, reliable and nonradioisotopic assay for the measurement of GTH in goldfish biological samples. In this assay, soluble GTH of the standards or samples competes with carp GTH (cGTH) immobilized on a solid support (96-well microplate) for the fixation on antibodies to the beta-subunit of carp gonadotropin. The immobilized antigen-antibody complexes are then revealed by the peroxidase-antiperoxidase (PAP) technique. After revelation of the peroxidase activity, the absorbance value of each well is measured with a microplate reader. The cGTH concentration used for coating the wells is 2 ng/ml and the final dilution of the specific antibody is 1:80,000. The assay can be performed within 24 h and can be used over a range of 0.125-4 ng/ml. At about 50% binding, the intra- and interassay coefficients of variation are 5% and 9% respectively. The displacement curves generated by goldfish plasma or pituitary perifusion fractions were strictly parallel to the standard cGTH. In addition, the stimulation by salmon gonadotropin-releasing hormone of pituitary fractions perifused in vitro caused an immediate increase in the GTH measured in the collected fractions, strongly reinforcing the assumption that this assay indeed measures GTH.

Animals↗

Granulosa-thecal cell interactions in the regulation of plasminogen activator activity during ovarian follicular development in the hen.

In the present studies, hen granulosa and thecal cells from the first (F1) and fourth (F4) largest and developing large white follicles (LWF) were cultured alone or cocultured, and plasminogen activator (PA) activity was determined. The PA of the cells (PAc) and the medium (PAm) was measured through use of the chromogenic substrate Val-Leu-Lys-p-nitroanilide, and the total PA (PAt) was calculated. The PA activity of cultured granulosa cells from all stages of follicular development increased with time of culture. Granulosa cell PAc and PAm activity differed with follicular development: the LWF granulosa cells had the highest levels of activity, the F4 had intermediate levels, and the F1 cells had the lowest. Thecal cell PA activity increased during culture but was unaffected by the stage of follicular development. Cocultures of granulosa and thecal cells from F1 follicles exhibited PA activity 2- to 3-fold higher than the sum of the activities of granulosa and thecal cells cultured alone. The PA activity of granulosa cells from LWF was not affected by coculture with thecal cells. Conditioned medium from thecal cells (TCCM) of all stages of follicular development stimulated PAc activity of granulosa cells from F1 and F4 follicles. Conditioned medium from thecal cells of F4 and LWF caused small inhibitory effects on the PAc activity of granulosa cells from LWF. Zymographic analysis of the PA activity of F1 granulosa cell cultures indicates that the enzyme activity is associated with a molecular mass of about 33 kDa, which is consistent with that of urokinase type PA. Thecal cell PA activity was unaffected by granulosa cell-conditioned medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Androgen regulation of an elastase-like protease activity in the seminal vesicle.

The processing of secretory proteins in the guinea pig (GP) seminal vesicle epithelium (SVE) is altered by castration and restored by treatment of animals with androgens. To test the hypothesis that the changes in protein processing are due to changes in the activity of specific proteases, we examined the GPSVE for protease activities capable of cleaving a synthetic elastase substrate, succinyl-alanyl-alanyl-alanyl-p-nitroanilide (Suc(Ala)3pNA). We found that the GPSVE does contain a Suc(Ala)3pNA-cleaving activity that is sensitive to the serine protease inhibitor diisopropylfluorophosphate (DFP) and to the elastase inhibitor elastatinal. Furthermore, the amount of protease activity per milligram of SVE protein is reduced to about 50% of control levels by castration. The activity is completely restored within four days by treatment of castrated animals with androgens, but is not restored by treatment with estradiol, progesterone, or dexamethasone. Although the SVE enzyme did not yield a pattern of specific cleavage products when incubated with a secretory protein substrate in vitro, this enzyme activity was competitively inhibited by a peptide whose primary sequence included the cleavage site used by the processing machinery in vivo.

Amino Acid Sequence↗

Benzidine stain for the histochemical detection of hemoglobin in splinter hemorrhage (subungual hematoma) and black heel.

Minor nail trauma may cause bluish discoloration of the nail, while tangential skin trauma on the heel can result in a so-called black heel. To rule out melanoma in such clinical situations, a biopsy is needed to reveal homogeneous eosinophilic masses deposited under the nail plate or within it (transepidermal elimination). Most dermatopathologists attempt to demonstrate the presence of hemoglobin in these eosinophilic masses with Prussian blue stain, which typically remains negative. In our experience, these traumatically induced blood deposits are always situated in avascular spaces, devoid of degrading phagocytes. Consequently, a histochemical stain for these deposits should be directed specifically toward hemoglobin, not hemosiderin. In the dermatopathologic literature, the various techniques to detect hemoglobin deposits in tissue sections are not well-known. We would like to emphasize benzidine stain, a highly selective and efficient method to demonstrate the presence of hemoglobin deposits in histologic sections. To date, benzidine stain has not been utilized to characterize splinter hemorrhage (subungual hematoma). Of concern, the use of benzidine in histopathology laboratories is restricted because this agent is a known carcinogen, while the non-mutagenic derivative, 3,3',5,5'-tetramethylbenzidine, does not stain histologic sections. Patent blue V, a completely different and less specific agent, stains hemoglobin an intense blue-green.

Benzidines↗

Assay of serum cholinesterase with succinylcholine and propionylthiocholine as substrates.

Serum cholinesterase (E.C. 3.1.1.8) was assayed with succinylcholine as a substrate. The reaction was coupled with choline oxidase and peroxidase in the presence of 4-aminoantipyrine and phenol to produce a red quinone dye that was measured spectrophotometrically. The method requires 25 microliter of sample in a total volume of 1.0 ml. The mean activity for 35 adults of the usual genotype was 74.4 +/- 28 U/l (range 24-125 U/l). Succinylcholine-sensitive individuals had activities below 18 U/l. The same serum samples also were assayed with propionylthiocholine as a substrate. Activities with the two substrates showed a coefficient of correlation of 0.980 (n = 68). However, the method using propionylthiocholine showed more overlap between the activities of succinylcholine-sensitive and insensitive individuals. Assay with succinylcholine thus may offer a more effective method of screening for sensitive individuals, since some escape detection by conventional genotyping with dibucaine and fluoride.

Alcohol Oxidoreductases↗

Expression of heat shock protein 27 (HSP27) in human temporomandibular joint discs of patients with internal derangement.

Heat shock proteins (HSPs) are believed to represent a cellular stress response mechanism that protects intracellular proteins from damaging events. Some studies have demonstrated an enhanced expression of large-molecular-weight HSPs in diseased systematic joints. Small heat shock proteins, and among these HSP27, have been studied to a lesser extent. HSP27 has cytoprotective and biosynthetic functions within chondrocytes, and it is an estrogen-associated protein that is under hormonal modulation. To improve understanding at a molecular level of the pathophysiology of certain temporomandibular joint disorders, the authors carried out this immunohistochemical study to assess the presence of HSP27 in human TMJ discs. Twelve adult human TMJ discs (10 diseased and 2 healthy discs) and 5 TMJ fetal human discs were used in this study. Adult discs and TMJ tissues of human fetuses were fixed in 10% buffered formalin. Samples were then processed for histologic examination. Sections were immunohistochemically stained using the streptavidin-biotin detection method. No reaction product for HSP27 in the discs of fetuses was noted. HSP27 was weakly expressed in normal TMJ discs and highly up-regulated in discs of patients showing new vessel formation and chondroid metaplasia. Any correlation between gender and HSP27 was found in the sample, being the up-regulation of HSP27 related mostly to major histopathological changes. This different pattern of HSP27 immunostaining in human TMJ discs detected in the authors' specimens suggests that the expression of this small HSP is functionally modulated. In fact HSP27 up-regulates in internal derangement specimens with major histopathological changes; on the other hand, it is not expressed or only weakly expressed in TMJ discs of fetuses and normal TMJ discs.

Adult↗

Coagulant and noncoagulant thrombin enzymatic activity on the endothelium.

The present study examines the role of antithrombin III (ATIII) and heparin in the inactivation of thrombin on rabbit aortae in vitro. Thrombin on the endothelium was functionally assayed with a synthetic chromogenic substrate (thrombins) or with fibrinogen (thrombinf), measured as the liberation of fibrinopeptide A. After incubation with thrombin, residual amounts were found on the endothelium. Surface-bound thrombinf constituted approximately one-half of surface-bound thrombins. Displacement of thrombin activity from the thrombin loaded endothelial surface was achieved with Polybrene. The thrombin could be quantitatively recovered in the Polybrene solution and equal amounts of thrombins and thrombinf were found. Incubation of endothelial segments with ATIII resulted in only a minimal uptake of ATIII on the surface but in a significantly increased capacity to inactivate thrombin, whereas incubation of the segments with heparin alone had the opposite effect. The effect of ATIII could be inhibited by prior incubation with Polybrene or by a subsequent incubation with heparin. It is concluded that the vessel wall content of ATIII is one determinant for the capacity to inhibit thrombin and that ATIII is bound to glycosaminoglycans on the vessel wall since it could be displaced by heparin.

Animals↗

Analysis of fibrinogen alpha residues 27-50: interactions with thrombin.

In order to further understand the unusual specificity of thrombin activity, we have examined the interactions of thrombin with fibrinogen alpha-chain residues 27-50 by two complementary approaches. Using genetically engineered constructions, we have synthesized a fusion protein containing fibrinogen A alpha-residues 1-50 and seven variants of this protein with substitutions in alpha-residues 33-41. These fusion proteins were purified and analysed as thrombin inhibitors and substrates. Substitution of Phe35----Leu of Asp40----Gly substantially increased Ki, but all the fusion proteins were equally good substrates when assayed at concentrations equivalent to Ki. These results indicate that Phe35 and Asp40 are important for thrombin binding, but not catalysis. We have also synthesized a peptide analogous to fibrinogen alpha 27-50. This peptide was a competitive inhibitor of thrombin-catalysed cleavage of fibrinogen, but was not an inhibitor of hydrolysis of small chromogenic substrates. These data further indicate that residues alpha 27-50 are important in the thrombin-fibrinogen interaction.

Amino Acid Sequence↗

Development of a chromogenic substrate assay for the determination of hirudin in plasma.

Hirudin is a potent and specific thrombin inhibitor. Since recombinant hirudin is being considered for anticoagulant and antithrombotic therapy we developed a fast and sensitive chromogenic substrate assay for its determination in plasma. The plasma samples (20 microliters) were incubated with 1 ml reagent mixture (0.2 M Tris buffer, 0.025 M NaCl, pH 8.1, containing 0.833 M urea, 0.7 trypsin inhibitor U/ml aprotinin, 100 ng/ml Polybrene and 0.31 NIH U/ml bovine thrombin) for 1 min. Thereafter 100 microliters Chromozym TH (Tos-Gly-Pro-Arg-pNA, 1.9 mM) was added. The change in absorbance/min (delta A/min) was recorded at 405 nm. delta A/min was linear for at least 3 min. The calibration curve was linear at least up to 800 ng hirudin/ml plasma. Intra-assay and inter-assay coefficients of variation were 2.8-3.1% and 5.3-5.8% respectively. The influence of progressive thrombin inhibitors can be neglected because of the short incubation time. Plasma samples can be assayed directly if aprotinin, polybrene and urea are added to the reagent mixture.

Calibration↗

Time dependent release of tissue-type plasminogen activator and plasminogen activator inhibitor into the circulation of pigs during shock.

To investigate short-term activation and inhibition of fibrinolysis during shock, we studied plasma levels of tissue-type plasminogen activator (t-PA) and t-PA inhibition capacity (PAI) in anaesthetized pigs. t-PA in euglobulin fractions of plasma was measured by the conversion of plasminogen to plasmin in the presence of fibrin split products. Plasmin thus generated was measured in a chromogenic substrate assay. PAI was measured as plasma inhibition capacity for human melanoma t-PA. Controls (n = 8) had constant t-PA and PAI for 6 h. Lipopolysaccharide from Salmonella abortus equi in four different doses (n = 9 - 11), or live Escherichia coli (n = 3) induced a transient t-PA increase with peak values at 2 h. PAI decreased to 50% at 2 h and increased to 250% at 6 h. Phorbol myristate acetate (n = 7) induced no change of t-PA or PAI. Dextran sulphate (n = 4) produced a t-PA rise at 30 min, followed by a rapid decline. Endotoxin was an appropriate stimulus for activation and inhibition of fibrinolysis whereas phorbol ester failed to elicit this response.

Animals↗

Simultaneous presence of tissue factor pathway inhibitor (TFPI) and low molecular weight heparin has a synergistic effect in different coagulation assays.

Injection of heparin releases tissue factor pathway inhibitor (TFPI) to the blood and, after heparin neutralization, it has been recently demonstrated that the released TFPI has an anticoagulant activity. Using recombinant TFPI (rTFPI) we have investigated how the simultaneous presence of TFPI and low molecular weight heparin (LMW heparin) affects different coagulation assays. Coagulation was measured using the activated partial thromboplastin time, the prothrombin time and a dilute tissue factor assay. The anticoagulant activity of partly purified plasma TFPI (pTFPI) was much higher than that of TFPI. However, this high anticoagulant activity was unstable, so in order to investigate the effect of pTFPI and LMW heparin we used an inhibitory antibody towards TFPI and looked at the effect of removing TFPI from plasma. When both rTFPI and LMW heparin was added to plasma a synergistic effect was observed in all assays. In the tissue factor dependent coagulation assays, the effect of adding rTFPI or removing pTFPI was more pronounced in the presence of heparin. TFPI plays a significant role in assays where the coagulation time is prolonged for some reason. This may be caused by dilution of tissue factor, by the presence of heparin or by a defect in the coagulation cascade such as that seen in haemophilia.

Blood Coagulation Tests↗

Three-stage chromogenic assay for the analysis of activation properties of factor X by cancer procoagulant.

The cysteine proteinase, cancer procoagulant (CP; EC 3.4.22.26) was isolated from human amnion-chorion and purified by precipitation with polyethylene glycol and either ion exchange or immunoaffinity chromatography. A new, sensitive, three-stage chromogenic assay was developed for determination of CP factor X-activating activity. Using this assay some properties including dose-response, effect of calcium, phospholipid and pH on the activation of factor X by CP was determined. There was an excellent linear correlation (r2 = 0.99) between concentration and the enzymatic activity of CP. The activation of factor X by purified CP was calcium dependent with an optimum calcium concentration of 7 mM. CP was not phospholipid dependent. There was a rather broad pH optimum between pH 6.9 and 7.25 for the activation of factor X by CP.

Blood Coagulation Tests↗

Protein C activation by an activator purified from the venom of Agkistrodon halys halys.

The protein C activator from Agkistrodon halys halys venom was purified 533-fold by ion-exchange chromatography on QAE-Sephadex A-50, affinity chromatography on aprotinin-Sepharose and Mono-Q fast protein liquid chromatography. The purified enzyme is a single chain protein with an apparent molecular weight of 36,000 that activates protein C by proteolytic removal of a small fragment from the heavy chain. The protein C activator exhibited a high amidolytic activity towards the tripeptide substrates D-Pro-Phe-Arg-pNA (S2302) and D-Phe-(pipecolyl)-Arg-pNA (S2238). The activity of the activator was not affected by thiolprotease or metalloprotease inhibitors. The activator was inhibited, however, by benzamidine, Phe-Pro-Arg chloromethyl ketone, p-nitrophenyl p-guanidinobenzoate and soy bean trypsin inhibitor, which classifies the enzyme as a serine protease. The purified protease was capable of activating both human and bovine protein C. Activation of human protein C only occurred at an appreciable rate in a calcium-free reaction medium at low ionic strength. Ca2+ ions inhibited the activation of human protein C with an apparent Ki of 0.8 mM. Addition of NaCl to the reaction medium also strongly inhibited human protein C activation (50% inhibition at 20 mM NaCl). Kinetic analysis of human protein C activation by the venom activator (in a calcium-free medium) revealed an apparent Km for protein C of 0.52 microM and a kcat of 0.17 s-1 at I = 0.05 (kcat/Km = 3.3 x 10(5) M-1 s-1). At I = 0.15 rates of human protein C activation became linear with protein C indicating a strong increase in Km with increasing ionic strength. Activation of bovine protein C was hardly affected by variation of Ca2+ and NaCl concentrations in the reaction medium. The apparent Kis for calcium ion and NaCl inhibition of bovine protein C activation were > 10 mM and 220 mM, respectively. At I = 0.1 and in the absence of Ca2+ ions bovine protein C was activated with a Km of 0.056 microM and a kcat of 0.24 s-1 (kcat/Km = 4.3 x 10(6) M-1 s-1). Our data are indicative for a rather large conformational and/or structural difference between human and bovine protein C at physiological ionic strength.

Amino Acid Sequence↗

Measurement of tissue factor pathway inhibitor in normal and post-heparin plasma.

Assay methods for the detection of both tissue factor pathway inhibitor (TFPI) function (two-stage chromogenic assay) and for TFPI antigen levels (competitive ELISA) have been developed and applied to the measurement of TFPI in normal plasma, in post-heparin plasma and to recombinant TFPI. There was good correlation in TFPI levels, measured using the two methods (r = 0.848; P < 0.001) in the normal plasma samples (n = 21) with the values ranging from 0.6 to 1.4 units per ml relative to a normal reference plasma pool (assigned 1.0 unit per ml). The post-heparin plasma samples were associated with increased levels of both TFPI functional activity and antigen. However, there was poor correlation between the two methods, with an increase in antigen levels greatly exceeding the increase in functional activity. This discrepancy was also found with recombinant TFPI and may reflect the different responses of the two assay methods to lipoprotein-bound TFPI (in the normal plasma reference) and the 'free' TFPI in post-heparin plasma and recombinant TFPI. These findings have implications in the choice of suitable reference materials for the assay of TFPI.

Binding, Competitive↗

Inhibition of clot-bound and free (fluid-phase thrombin) by a novel synthetic thrombin inhibitor (Ro 46-6240), recombinant hirudin and heparin in human plasma.

Clot-bound thrombin remains active and is less accessible to heparin-antithrombin III than fluid-phase thrombin. To determine whether clot-bound human thrombin is more susceptible to inactivation by direct thrombin inhibitors, the activity of a novel synthetic competitive thrombin inhibitor Ro 46-6240, recombinant hirudin and unfractionated heparin were compared with fluid-phase thrombin and clot-bound thrombin. Fibrinopeptide A generated in human plasma was used as an index of thrombin activity. Hirudin was the most potent inhibitor of fluid-phase and clot-bound thrombin. However, Ro 46-6240 inhibited clot-bound thrombin three times more potently than fluid-phase thrombin (IC50 19 vs 56 ng/ml) while hirudin was two times (IC50 8 vs 3 ng/ml) and heparin six times (IC50 1,205 vs 200 ng/ml) less active against clot-bound thrombin compared with fluid-phase thrombin. The relative selectivity for clot-bound thrombin is not a unique property of Ro 46-6240 since two other synthetic thrombin inhibitors tested inhibited clot-bound thrombin more effectively than fluid-phase thrombin and a third was equally active against both forms of thrombin. In contrast, the affinities of two chromogenic substrates were similar for both forms of thrombin. This study shows that direct thrombin inhibitors inhibit clot-bound thrombin more potently than heparin and suggests that an apparent selectivity for clot-bound thrombin can be achieved with some synthetic thrombin inhibitors. Further studies have to show whether the high potency of these direct thrombin inhibitor translates into antithrombotic efficacy in clinical situations with pre-existing clots.

Binding, Competitive↗