Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Platelet microbicidal protein (PMP) is released from platelets in response to thrombin stimulation. PMP is known to possess in vitro bactericidal activity against Staphylococcus aureus and viridans group streptococci. To determine whether PMP is active against other intravascular pathogens, we evaluated its potential fungicidal activity against strains of Candida species and Cryptococcus neoformans. Anionic resin adsorption and gel electrophoresis confirmed that the fungicidal activity of PMP resided in a small (approximately 8.5-kDa), cationic protein, identical to previous studies of PMP-induced bacterial killing (M.R. Yeaman, S.M. Puentes, D.C. Norman, and A.S. Bayer, Infect. Immun. 60:1202-1209, 1992). When assayed over a 180-min period in vitro, the susceptibilities of these fungi to PMP varied considerably. Generally, Candida albicans strains (mean survival, 33.5% +/- 6.9% [n = 6]) as well as isolates of Candida glabrata (mean survival, 50.8% +/- 2.9% [n = 2]) were the most susceptible to killing by PMP, while Candida guillermondii and Candida parapsilosis were relatively resistant to PMP-induced killing. Compared with C. albicans, C. neoformans was relatively resistant to the fungicidal activity of PMP, with a mean survival among the isolates studied of 77.4% +/- 12.4% (n = 6). Against C. albicans, PMP-induced fungicidal activity was time dependent (range, 0 to 180 min), PMP concentration dependent (range, 10 to 150 U/ml), and inversely related to the fungal inoculum (range, 5 x 10(3) to 1 x 10(5) CFU/ml). Scanning electron microscopy of PMP-exposed C. albicans and C. neoformans cells revealed extensive surface damage and collapse, suggesting that the site of PMP fungicidal action may directly or indirectly involve the fungal cell envelope.
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PMID:Thrombin-induced rabbit platelet microbicidal protein is fungicidal in vitro. 846 Sep 23

The influence of two different E. coli asparaginase (ASP) preparations on fibrinolytic proteins in childhood ALL was recently reported, demonstrating a clearly significant association between ASP activity and haemostatic changes. Since the Bayer preparation is no longer available for treatment of large series of patients with ALL, the present study was designed to prospectively evaluate coagulation and fibrinolytic changes in leukaemic children receiving different doses of Medac ASP, which is now available for treatment of childhood ALL. Leukaemic children in whom ASP Medac was administered at 3 d intervals in a two-step dose reduction (5000 IU/m2, n = 10; 2500 IU/m2, n = 15) were compared with children who had received Bayer ASP 10,000 IU/m2 in the same time schedule in a former randomized trial; at the same venipuncture, blood samples for coagulation studies were obtained before each ASP administration together with serum samples for pharmacokinetic monitoring. Compared with Bayer ASP 10,000 IU/m2, patients receiving Medac ASP 5000 IU/m2 showed significantly decreased values of fibrinogen, plasminogen, and alpha 2-antiplasmin, along with significantly enhanced thrombin generation. Improvement occurred in children treated with 2500 IU/m2 Medac ASP; alpha 2-antiplasmin and D-dimer no longer differed from the Bayer group. Since both patient groups showed complete asparagine depletion during the course of ASP administration, the lower dosage of 2500 IU/m2 administered at 3 d intervals should guarantee the specific metabolic therapy for ALL, leading to depletion of the circulating pool of asparagine.
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PMID:Changes in coagulation and fibrinolysis in childhood ALL: a two-step dose reduction of one E. coli asparaginase preparation. 885 48

Recent in vitro studies have demonstrated that rabbit platelets release a small, cationic antimicrobial protein in response to thrombin stimulation under physiological conditions (M. R. Yeaman, S. M. Puentes, D. C. Norman, and A. S. Bayer, Infect. Immun. 60:1202-1209, 1992). This observation prompted our present investigation, focused on determining the array of antimicrobial proteins contained within rabbit platelets and their in vitro activity against common bloodstream pathogens. A group of small (6.0- to 9.0-kDa), cationic proteins with in vitro antimicrobial activity was purified from whole and thrombin-stimulated rabbit platelets by gel filtration and reversed-phase high-performance liquid chromatography. Purified proteins in micromolar concentrations (10 to 40 microg/ml) exerted in vitro microbiostatic and/or microbicidal activities against Staphylococcus aureus, Escherichia coli, and Candida albicans in a dose-dependent manner. The antimicrobial activities of proteins purified from rabbit platelet acid extracts were generally inversely related to pH, with maximal activity observed at pH 5.5. In contrast, the predominant protein isolated from thrombin-stimulated rabbit platelets, though biochemically and microbiologically similar to proteins extracted by acid, exhibited antimicrobial activities which were modestly enhanced at pH 7.2 compared with pH 5.5. Amino acid compositional analyses in combination with molecular mass determinations suggest that the majority of these proteins are distinct molecules not derived from a single common precursor. Collectively, these data indicate that rabbit platelets contain proteins which exert potent in vitro antimicrobial activity against bacterial and fungal pathogens which commonly invade the bloodstream. Moreover, several of these proteins were released from platelets stimulated with thrombin under physiological conditions and exerted potent antimicrobial activities in physiological pH ranges. These observations support the hypothesis that platelets serve an important role in host defense against infection, via localized release of antimicrobial proteins in response to stimuli associated with tissue injury or microbial colonization.
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PMID:Purification and in vitro activities of rabbit platelet microbicidal proteins. 903 12

Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide generated from rabbit platelets when they are exposed to thrombin in vitro. It has potent microbicidal activity against a broad spectrum of bacterial and fungal pathogens, including Staphylococcus aureus. Previous in vitro studies involving whole staphylococcal cells and planar lipid bilayers (as artificial bacterial membrane models) suggested that membrane permeabilization by tPMP-1 is voltage dependent (S.-P. Koo, M. R. Yeaman, and A. S. Bayer, Infect. Immun. 64:3758-3764, 1996; M. R. Yeaman, A. S. Bayer, S. P. Koo, W. Foss, and P. M. Sullam, J. Clin. Investig. 101:178-187, 1998). Thus, the aims of the present study were to specifically characterize the electrophysiological events associated with membrane permeabilization by tPMP-1 by using artificial planar lipid bilayer membranes. We assessed the influence of transmembrane voltage polarity and magnitude on the initiation and modulation of tPMP-1 membrane permeabilization at various concentrations of tPMP-1 (range, 1 to 100 ng/ml) added to the cis side of the membranes. The incidence of membrane permeabilization induced by tPMP-1 at all of the concentrations tested was more frequent at -90 mV than at +90 mV. It is noteworthy that membrane permeabilization due to 1-ng/ml tPMP-1 was successfully initiated at -90 mV but not at +90 mV. Further, the mean onset times of induction of tPMP-1 activity were comparable under the various conditions. Modulation of ongoing membrane permeabilization was dependent on voltage and tPMP-1 concentration. Membrane permeabilization at a low tPMP-1 concentration (1 ng/ml) was directly correlated with trans-negative voltages, while a higher tPMP-1 concentration (100 ng/ml) induced conductance which was more dependent on trans-positive voltages. Collectively, these data indicate that the mechanism of tPMP-1 microbicidal activity at the bacterial cytoplasmic membrane may involve distinct induction and propagation stages of membrane permeabilization which, in turn, are modulated by transmembrane potential, as well as peptide concentration.
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PMID:Membrane permeabilization by thrombin-induced platelet microbicidal protein 1 is modulated by transmembrane voltage polarity and magnitude. 1022 10

Platelet activation results in changes in a number of cell surface molecules including an increase in P-Selectin (CD62P) that may be rapidly and conveniently measured by immunofluorescent flow cytometry. The ADVIA 120 (Bayer) is a new system that facilitates more accurate measurement of platelet volume and in addition provides an approximate measure of the mean refractive index (RI) of the platelets reported as mean platelet component (MPC) concentration. We were interested to determine whether changes in MPC might reflect changes in platelet activation status. To investigate this, the platelet CD62P expression, determined by flow cytometry, and change in MPC, measured on the ADVIA 120 system, was first examined in vitro after stimulation of EDTA anticoagulated whole blood with submaximal concentrations of bovine thrombin in the presence or absence of the thromboxane synthase inhibitor, Ridogrel. Thrombin produced a dose-dependent increase in platelet CD62P expression and a decrease in MPC that could be inhibited by Ridogrel at physiological concentrations. In the second set of experiments, blood from 20 normal controls was collected into both EDTA and sodium citrate (SC) anticoagulants. Within 30 min of venesection and again at 3 h post-venesection after storage at room temperature, the platelet MPC and CD62P expression were determined. Platelets in all samples with both anticoagulants showed very low levels of CD62P expression when first analysed. At 3 h there was a small increase in CD62P expression on platelets in whole blood anticoagulated with SC, but a significant (P < 0.001) increase was observed on platelets anti-coagulated with EDTA. A negative correlation was found between the change in MPC of the platelets and the increase in the mean fluorescence intensity (MFI) (r = -0.69, P < 0.001, n = 20) and the percentage (r = -0.72, P < 0.001, n = 20) of CD62P positive platelets at 3 h in blood anticoagulated with EDTA. We conclude that a reduction in MPC as measured by the ADVIA 120 may be used to detect anticoagulant induced, as well as thrombin stimulated, in vitro platelet activation in blood anticoagulated with EDTA. Further, we conclude that platelet activation is negligible for up to 3 h in sodium citrate anticoagulated whole blood.
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PMID:Use of mean platelet component to measure platelet activation on the ADVIA 120 haematology system. 1051 12

Platelet microbicidal proteins (PMPs) are small, cationic peptides which possess potent microbicidal activities against common bloodstream pathogens, such as Staphylococcus aureus. We previously showed that S. aureus strains exhibiting resistance to thrombin-induced PMP (tPMP-1) in vitro have an enhanced capacity to cause human and experimental endocarditis (T. Wu, M. R. Yeaman, and A. S. Bayer, Antimicrob. Agents Chemother. 38:729-732, 1994; A. S. Bayer et al., Antimicrob. Agents Chemother. 42:3169-3172, 1998; V. K. Dhawan et al., Infect. Immun. 65:3293-3299, 1997). However, the mechanisms mediating tPMP-1 resistance in S. aureus are not fully delineated. The S. aureus cell membrane appears to be a principal target for the action of tPMP-1. To gain insight into the basis of tPMP-1 resistance, we compared several parameters of membrane structure and function in three tPMP-1-resistant (tPMP-1(r)) strains and their genetically related, tPMP-1-susceptible (tPMP-1(s)) counterpart strains. The tPMP-1(r) strains were derived by three distinct methods: transposon mutagenesis, serial passage in the presence of tPMP-1 in vitro, or carriage of a naturally occurring multiresistance plasmid (pSK1). All tPMP-1(r) strains were found to possess elevated levels of longer-chain, unsaturated membrane lipids, in comparison to their tPMP-1(s) counterparts. This was reflected in corresponding differences in cell membrane fluidity in the strain pairs, with tPMP-1(r) strains exhibiting significantly higher degrees of fluidity as assessed by fluorescence polarization. These data provide further support for the concept that specific alterations in the cytoplasmic membrane of S. aureus strains are associated with tPMP-1 resistance in vitro.
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PMID:In vitro resistance of Staphylococcus aureus to thrombin-induced platelet microbicidal protein is associated with alterations in cytoplasmic membrane fluidity. 1081 10

The cationic molecule thrombin-induced platelet microbicidal protein 1 (tPMP-1) exerts potent activity against Staphylococcus aureus. We previously reported that a Tn551 S. aureus transposon mutant, ISP479R, and two bacteriophage back-transductants, TxA and TxB, exhibit reduced in vitro susceptibility to tPMP-1 (tPMP-1(r)) compared to the parental strain, ISP479C (V. Dhawan, M. R. Yeaman, A. L. Cheung, E. Kim, P. M. Sullam, and A. S. Bayer, Infect. Immun. 65:3293-3299, 1997). In the current study, the genetic basis for tPMP-1(r) in these mutants was identified. GenBank homology searches using sequence corresponding to chromosomal DNA flanking Tn551 mutant strains showed that the fourth gene in the staphylococcal mnh operon (mnhABCDEFG) was insertionally inactivated. This operon was previously reported to encode a Na(+)/H(+) antiporter involved in pH tolerance and halotolerance. However, the capacity of ISP479R to grow at pH extremes and in high NaCl concentrations (1 to 3 M), coupled with its loss of transmembrane potential (DeltaPsi) during postexponential growth, suggested that the mnh gene products are not functioning as a secondary (i.e., passive) Na(+)/H(+) antiporter. Moreover, we identified protein homologies between mnhD and the nuo genes of Escherichia coli that encode components of a complex I NADH:ubiquinone oxidoreductase. Consistent with these data, exposures of tPMP-1-susceptible (tPMP-1(s)) parental strains (both clinical and laboratory derived) with either CCCP (a proton ionophore which collapses the proton motive force) or pieracidin A (a specific complex I enzyme inhibitor) significantly reduced tPMP-induced killing to levels seen in the tPMP-1(r) mutants. To reflect the energization of the gene products encoded by the mnh operon, we have renamed the locus sno (S. aureus nuo orthologue). These novel findings indicate that disruption of a complex I enzyme locus can confer reduced in vitro susceptibility to tPMP-1 in S. aureus.
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PMID:Transposon disruption of the complex I NADH oxidoreductase gene (snoD) in Staphylococcus aureus is associated with reduced susceptibility to the microbicidal activity of thrombin-induced platelet microbicidal protein 1. 1635 37

Alterations in hemostasis have frequently been observed in children with acute lymphoblastic leukemia. Thrombotic events are well documented in patients receiving L-asparaginase as a single agent or in combination with other chemotherapeutic drugs. The present prospective, randomized study evaluated the effect of two different L-asparaginase preparations, native Escherichia coli L-asparaginase (Crasnitin; Bayer AG, Leverkusen, Germany; n = 10) and L-asparaginase derived from Erwinia chrysanthemi (Erwinase; Porton Pruducts, London, UK; n = 10) on the changes in parameters concerning hypercoagulability. Patients were randomized to receive a total of eight doses of 10,000 IU/m2 L-asparaginase intravenously with intervals of 3 days during induction therapy. Before starting L-asparaginase treatment all patients had already demonstrated an increased thrombin generation shown by the elevated levels of prothrombin fragment 1+2 and thrombin antithrombin III, presumably due to therapy with prednisone, daunorubicin and vincristine. A significant decrease in alpha2-antiplasmin and plasminogen levels was measured in the E. coli L-asparaginase but not in Erwinase-treated patients. Increased thrombin generation combined with a decrease in alpha2-antiplasmin and plasminogen levels may lead to a state of increased risk for thrombosis due to a delay in fibrin elimination in E. coli L-asparaginase-treated patients only.
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PMID:Changes in hypercoagulability by asparaginase: a randomized study between two asparaginases. 1647 96

In order to describe the haemostatic role of a variation in inhibitor reactivity with different factor VIII (FVIII) concentrates, we have compared inhibitor titres against a panel of FVIII concentrates and correlated titre with the capacity to inhibit thrombin generation. Three plasma-derived concentrates were tested in vitro in mixing experiments with inhibitor plasmas from 11 patients with severe haemophilia A: Fanhdi, which contains von Willebrand factor (VWF) with a final ratio of approximately 1:1 (VWF IU per IU FVIII:C); Haemate-P with a ratio of 2.5:1 and Hemofil-M containing only trace amounts of VWF. In addition, the recombinant FVIII concentrate Kogenate Bayer containing no VWF was included. Inhibitor titres and the capacity to generate thrombin were measured. A statistically significant difference in measured titres was found with the highest titres recorded against Hemofil-M. The inhibitor titres needed to inhibit 50% maximum thrombin generation were the lowest for Kogenate Bayer and the highest and similar for Fanhdi and Haemate-P with intermediate titres needed for inhibition of Hemofil-M. In this study, the thrombin generation assay provides additional indications for the role of VWF in the treatment of patients with inhibitors. The VWF-containing concentrates Fanhdi and Haemate-P, added to FVIII-deficient plasma with the presence of inhibitor, generate more thrombin than do the purified concentrates Hemofil-M and Kogenate Bayer.
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PMID:Impact of different inhibitor reactivities with commercial factor VIII concentrates on thrombin generation. 1788 Apr 65

We and others have previously shown that inhibitor containing plasma from patients with congenital haemophilia A sometimes reacts less with von Willebrand factor (VWF) containing concentrates compared with highly purified plasma-derived or recombinant factor VIII (FVIII) concentrates. To further substantiate the haemostatic role of a variation in inhibitor reactivity with different FVIII concentrates, we compared the inhibitor titres from 11 plasma samples against a panel of FVIII concentrates and correlated titre with the capacity to inhibit thrombin generation. Three plasma-derived concentrates were tested: Fandhi (Grifols) which contains VWF with a final ratio of approximately 1 (VWF IU per IU FVIII:C); Haemate (CSL Behring) with a ratio of 2.5 and Haemofil M (Baxter), a monoclonal antibody-purified concentrate containing only trace amounts of VWF. In addition, the recombinant FVIII concentrate Kogenate Bayer (Bayer) containing no VWF was included in the panel. A statistically significant difference in measured titres against the four concentrates was found. The inhibitor titre needed to inhibit 50% maximum thrombin generation was lowest for Kogenate Bayer and highest and similar for Fandhi and Haemate. This study confirms the results from previous research regarding variation of inhibitor reactivity against different concentrates and further shows that the VWF containing concentrates Fandhi and Haemate added to FVIII-deficient plasma with the presence of inhibitor generate more thrombin than do the purified concentrates Haemofil M and Kogenate Bayer. A further interesting aspect could be that bypass therapy may have an increased efficacy when infused together with FVIII concentrates containing VWF. However, the clinical implications of all these findings in vitro need to be established.
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PMID:VWF/FVIII complex and the management of patient with inhibitors: from laboratory to clinical practice. 1807 1


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