Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Our studies of alpha-thrombin as a growth factor have led to the development of a synthetic peptide (p508) that in vitro competes with thrombin for binding to high affinity receptors, and enhances mitogenic activity. To determine if this peptide could be used to accelerate wound closure in vivo, full thickness 6 mm dermal biopsy wounds on the dorsal skin of anesthetized rats were treated with p508 peptide, thrombin or PBS as control. At day 7, the p508 treated wound areas were 20% to 50% smaller than either thrombin or PBS treated wound sites. This suggests that p508 enhances aspects of wound healing, and avoids the normal in vivo regulatory mechanisms of intact thrombin.
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PMID:A synthetic peptide representing the thrombin receptor-binding domain enhances wound closure in vivo. 136 52

Because fibrinolysis is now recognized as an important factor in hypercoagulable states, we have developed and characterized an easily performed, rapid, and quantitative screening test that assesses a patient's fibrinolytic activity. This modification of the dilute whole blood clotting time (DWBCT) counts the number of intact erythrocytes released from the clot formed in samples obtained before and after the application of a venous occlusion cuff. Samples were corrected for the plasma volume changes that occurred during venous occlusion. This test was performed on nine healthy volunteers. Specimens were diluted 1:1 with PBS, rapidly clotted with thrombin and incubated at 37 C. Starting thirty minutes after the thrombin was added and then at twenty minute intervals until 110 minutes, the number of RBCs released from the clot were counted using a Coulter S Plus counter. There were consistently more RBCs released at each time period after venous occlusion (p less than 0.001). Aliquots were also obtained for measuring PAI activity and TPA levels. PAI activity was lower post-cuff at every point (p less than 0.001). TPA level was higher at every point (p less than 0.001) post-cuff. The addition of exogenous TPA, activated protein C, or anti-PAI antibodies increased the amount of clot lysis; while the addition of anti-TPA antibodies and EACA each prevented the post-cuff increase. Unlike the euglobulin lysis time (ELT) this modified DWBCT (mDWBCT) measures the patients intact fibrinolytic system, including PAI and erythrocytes, in a quantitative fashion. Unlike either the ELT or the DWBCT the mDWBCT can be performed within two hours, so results are rapidly available for clinical decisions. These studies have demonstrated an easily performed, inexpensive, quantitative screening test of a patient's overall fibrinolytic system that reacts appropriately to pharmacologic manipulations.
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PMID:A modified quantitative whole blood clot lysis method for general laboratory analysis of fibrinolysis. 211 74

Prokallikrein was activated by trypsin and by alpha-chymotrypsin, but not by proteases, such as plasmin, thrombin, urokinase, carboxypeptidase B, papain, elastase, pepsin, and cathepsin D. Moreover, rat fresh serum did not activate prokallikrein. Maximum activation of prokallikrein by trypsin was obtained at the concentration of 10 micrograms to 1 mg per ml in PBS and that by alpha-chymotrypsin was at the concentration of 5 mg per ml. The enzymic properties of trypsin-activated and alpha-chymotrypsin-activated kallikreins were identical with those of active kallikrein in the kidney.
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PMID:Activation of prokallikrein in the rat kidney by proteases. 637 43

Flow cytometry (FC) provides a reproducible investigation of cell surface antigens on platelets. The aim of this study was to elaborate appropriate protocols and to compare them with other techniques that have already been published. (1) Venipuncture with tubes containing citrate was better for the preservation of the antigenicity than using ACD tubes. The isolated platelets could not be completely distinguished from detritus and protein aggregates. Therefore a platelet concentration between 10(7) and 10(8)/ml measurement buffer was necessary to obtain a sufficient resolution by FC. (2) Isolation methods using either differential centrifugation or diluted Ficoll-Hypaque as a flotation medium provided platelets of equal purity. The method with Ficoll-Hypaque resulted in a higher number of isolated platelets than differential centrifugation. The demonstration of platelets and their antigens in whole blood without isolation gave good results provided the platelets were not activated. Activation of platelets with 1 NIH-U thrombin/l resulted in the loss of a part of the highly activated platelets because of their aggregation. (3) Comparing different concentrations of paraformaldehyde in PBS, fixation with 1% for 15 min provided the best antigen preservation for most of the antigens investigated. Isolation induced platelet activation. In order to avoid this effect, the whole anticoagulated blood was fixed with 1% paraformaldehyde for 15 min immediately after venipuncture. Then the platelets were isolated using diluted Ficoll-Hypaque. In this way, systemic activation of platelets can be detected with antibodies against glycoproteins which are translocated from the alpha-granules or lysosomes to the cell membrane. These activation markers can be determined on immediately fixed platelets (already in the whole blood) without any interference due to unspecific activation caused by the isolation procedure. (4) Platelet treatment with citric acid at pH 3, in order to remove the antigenicity of HLA-class I molecules, was sensitive to immediate fixation with paraformaldehyde in the whole blood. Fixation after isolating the platelets made it possible to demonstrate antigen stripping, and the free heavy chain, devoid of the beta 2-microglobulin, could be clearly demonstrated. (5) Using standardization beads, the average number of antigenic sites per platelet could be determined for the investigated specificities. It was shown that antibodies which have been directly conjugated or biotinylated and combined with streptavidin-phycoerythrin yielded similar results in terms of the number of antigenic binding sites while unconjugated antibodies in combination with FITC-conjugated anti-mouse-IgG led to overestimation of antigenic binding sites.
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PMID:Standardization of the flow cytometric determination of HLA class I antigens, 'platelet-specific' glycoproteins and activation markers. 776 17

The anticoagulant activity of albumin-heparin conjugates covalently immobilized on carboxylated polystyrene beads was determined before and after exposure to different plasma/PBS dilutions using a thrombin inhibition assay, a FXa inhibition assay, and a modified aPTT assay. Exposure of albumin-heparin modified surfaces (alb-hep surfaces) to plasma dilutions resulted in surfaces with a lower anticoagulant activity than surfaces which were not exposed to plasma dilutions. The reduction of the activity increased up to +/- 80% when the surfaces were exposed to solutions containing more than 70% plasma. Alb-hep surfaces incubated in plasma which was preexposed to heparin-Sepharose retained 30% of their initial activity. These observations were attributed to non-specific adsorption of plasma proteins onto the surface and to interaction of heparin binding proteins with the immobilized heparin. Both processes result in a decreased accessibility of the immobilized heparin and thus in a reduced anticoagulant activity displayed by the heparinized surface. Identification of adsorbed proteins with SDS gel electrophoresis and immunoblotting revealed that many different proteins were present at the heparinized surface. Only small differences were observed between the gel electrophoresis pattern of adsorbed proteins obtained from heparinized surfaces and from a surface containing immobilized albumin.
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PMID:The effect of protein adsorption on the anticoagulant activity of surface immobilized heparin. 863 81

To clarify the release properties of anti-cancer drugs from fibrin glue, a study was performed using several anti-cancer drugs with remarkably different physical properties. Concentrated fibrinogen, fibronectin, and coagulation factor XIII were prepared from healthy human plasma according to the cryoprecipitate method. Fibrin glue containing anti-cancer drugs was prepared as follows; the cryoprecipitate was mixed with each anti-cancer drug and aprotinin, then thrombin was added. These glues were incubated in PBS containing plasminogen and urokinase at 37 degrees C for seven days, and the medium was then sampled several times after centrifugation. The drug concentration in each sample was measured using HPLC. Fibrin glue without aprotinin was quickly hemolyzed and disappeared after 2--4 h. That with aprotinin was only slightly hemolyzed and more than half remained after 7 days. Mitomycin C and fluorouracil were quickly released from the glue regardless of the presence or absence of aprotinin. However, enocitabine was gradually released from glue with aprotinin although quickly released from that without. The rate of release of each drug from the glue with aprotinin correlated well with its hydrophobicity. Thus, to establish a sustained release system using fibrin glue, one should use the more lipophilic anti-cancer drugs and a fibrinolytic enzyme inhibitor.
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PMID:Novel drug delivery system using autologous fibrin glue--release properties of anti-cancer drugs. 1072

We have previously shown that atherosclerotic apolipoprotein E-deficient (apoE(-/-)) x LDL receptor-deficient (LDLR(-/-)) mice develop myocardial infarction when exposed to hypoxic stress. This study was performed to assess the role of thrombin and thrombosis in this process. ApoE(-/-) x LDLR(-/-) mice were fed a cholesterol-rich diet for 8 mo and were then subjected to hypoxic stress while receiving isoflurane anesthesia. One group received a bolus dose (5.6 micromol/kg) of the thrombin inhibitor melagatran, and control animals received PBS 10 min before the hypoxic stress. The mice were exposed to 10 min of hypoxia followed by normoxia. Ten minutes after the stress, Alzet pumps delivering melagatran (20 nmol x kg x (-1)min(-1)) or PBS were implanted, and the mice were allowed to recover for 48 h. The cardiac response was analyzed by histology, immunohistochemistry, and serum troponin T assay. All animals showed reversible ECG changes as a sign of ischemia during hypoxic stress, and 50% developed infarctions afterward as judged by troponin T levels. The group that received thrombin inhibitor had significantly lower troponin T and smaller myocardial infarctions than the PBS-treated group. These data show that thrombin generation is an important pathogenetic factor and suggest that coronary thrombosis is involved in myocardial infarction in atherosclerotic mice. Exposure of atherosclerotic mice to hypoxia leads to myocardial infarction through a two-phase pathway in which acute transient ischemia is followed by thrombin-dependent, irreversible, myocardial ischemia and myocardial cell death.
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PMID:Thrombin inhibitor reduces myocardial infarction in apoE-/- x LDLR-/- mice. 1503 Nov 24

Aptamer-based microarrays for the quantitation of multiple protein analytes have been developed. A multiplex aptamer microarray was generated by printing two RNA aptamers (anti-lysozyme and anti-ricin) and two DNA aptamers (anti-IgE and anti-thrombin) on to either streptavidin (SA) or neutravidin (NA)-coated glass slides. However, substantial optimization was required in order to ensure the simultaneous function of the aptamer:analyte pairs. The effects of protein labeling, assay buffer, surface coating, and immobilization chemistry and orientation were investigated. A single buffer (PBS buffer containing 5 mM MgCl2 and 0.1% Tween 20) was found to work well with all the aptamers, even though this was not the buffer originally used in their selection, while neutravidin-coated slides yielded a lower detection limit, wider detection range, and more uniform background than streptavidin-coated slides. Incubation with Cy3-labeled proteins yielded sensitive, target-specific, and dose-dependent responses to each protein. Target protein concentrations as low as 72 pg/mL (5 pM, lysozyme), 15 ng/mL (0.5 nM, ricin), 1.9 ng/mL (0.01 nM, IgE), and 170 ng/mL (5 nM, thrombin) could be detected. These results show that aptamer arrays can potentially be used with numerous proteins in parallel, furthering the notion that aptamer arrays may be useful in proteomics.
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PMID:Optimization of aptamer microarray technology for multiple protein targets. 1772 65

Humans demonstrate limited spontaneous endothelialization of prosthetic bypass grafts. However the local application of growth factors to prosthetic grafts or to injured blood vessels can provide an immediate effect on endothelialization. Novel chimeric proteins combining potent angiogens with extracellular matrix binding domains may localize to exposed matrices and provide sustained activity to promote endothelial regeneration after vascular interventions. We have ligated a thrombin-resistant mutant of fibroblast growth factor (FGF)-1 (R136K) with a collagen binding domain (CBD) in order to direct this growth factor to sites of exposed vascular collagen or selected bioengineered scaffolds. While FGF-1 and R136K are readily attracted to a variety of matrix proteins, R136K-CBD demonstrated selective and avid binding to collagen approximately 4x that of FGF-1 or R136K alone (P<0.05). The molecular stability of R136K-CBD was superior to FGF-1 and R136K. Its chemotactic activity was superior to R136K and FGF-1 (11+/-1% vs. 6+/-2% and 4+/-1%; P<0.01). Its angiogenic activity was similar to R136K and significantly greater than control by day 2 (P<0.01). After day 3, FGF-1-treated endothelial cell's (EC) sprouts had regressed back to levels insignificant compared to the control group (P=0.17), while both R136K and R136K-CBD continued to demonstrate greater sprout lengthening as compared to control (P<0.0002). The mitogenic activity of all growth factors was greater than control groups (20% PBS); in all comparisons (P<0.0001). This dual functioning angiogen provides proof of concept for the application of designer angiogens to matrix binding proteins to intelligently promote endothelial regeneration of selected matrices.
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PMID:Construction and characterization of a thrombin-resistant designer FGF-based collagen binding domain angiogen. 1795 Apr 55

Kallikrein is a multitalented enzyme in hemostasis and inflammation. Normally, kallikrein is formed in intrinsic hemostasis and activates factor XII. A total of 10 microL of 0 to 100 microg/mL human plasma kallikrein in 6% human albumin-PBS were incubated with 90 microL 111.1 microg/mL prothrombin in 6% human albumin in absence and presence of 23 mM Ca(++). After 0 to 64 minutes (37 degrees C), 100 microL of 2.5 M arginine, pH 9, were added. Fifty microliters of 0.72 mM HD-CHG-Ala-Arg-pNA in 1.36 M arginine were added and increase in absorbance at 405 nm was determined. Within 8 minutes (37 degrees C), 1 microg/mL kallikrein, ie, 2.5% of the normal plasmatic prekallikrein concentration, generates approximately 3 mIU/mL thrombin in absence and 27 mIU/mL thrombin in presence of Ca(++). Kallikrein can directly activate prothrombin; there is a shortcut in the intrinsic hemostasis system that generates catalytic amounts of thrombin without following the known intrinsic clotting pathway.
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PMID:Kallikrein activates prothrombin. 1816 May 76


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