alpha-chymotrypsin and Thrombosis

Topics: Adult; Aged; Anti-Bacterial Agents; Chymotrypsin; Clinical Trials as Topic; Drug Evaluation; Female; Humans; Leg Ulcer; Male; Middle Aged; Placebos; Thrombosis; Trypsin

A novel fibrinolytic enzyme was purified from Lyophyllum shimeji, a popular edible mushroom in Asia. The enzyme was purified using combination of anion exchange chromatography on a Mono Q 5/5 column and size exclusion gel filtration chromatography on Superdex 200 100/300 column. This purification protocol resulted 80.9-fold purification of the enzyme and a final yield of 5.7%. The molecular weight of the purified enzyme was estimated to be 21 kDa by SDS-PAGE and size exclusion gel filtration. The N-terminal amino acid sequence was found to be ITFQSASP, which is dissimilar from that of known fibrinolytic enzymes. The purified enzyme was a neutral protease with an optimal reaction pH and temperature of 8.0 and 37°C, respectively. Enzymatic activity was inhibited by Cu(2+) and Co(2+). It was also significantly inhibited by PMSF and TPCK. Furthermore, it was found to exhibit a higher specificity for S-7388, a well-known chymotrypsin chromogenic substrate, indicating chymotrypsin like serine metalloprotease. The relative fibrinolytic activity of 5 μg purified enzyme have two fold more activity than 1 unit/ml of plasmin on fibrin plate. Furthermore, purified enzyme preferentially hydrolyzed the Aα-chain followed by the Bβ- and γ-chain of fibrinogen, which is precursor of fibrin. Therefore, these data suggests that the fibrinolytic enzyme derived from edible mushroom, L. shimeji, might be useful for thrombolytic therapy and preventing thrombotic disease.

Topics: Agaricales; Amino Acid Sequence; Chromatography, Gel; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Fibrin; Fibrinogen; Fibrinolysis; Hydrogen-Ion Concentration; Metalloproteases; Molecular Weight; Serine; Temperature; Thrombosis

The further optimisation of the novel lead compound CGH752 (Fig. 1) is described. By introducing various substituents into the 6-position of the 3,3-dimethyltetrahydroquinoline (DMTHQS) ring we have been able to favourably affect the in vitro and in vivo activity, and the pharmacokinetics of such compounds. One of the inhibitors synthesised (CGH1484) is bioavailable and shows efficacy in animal models of thrombosis.

Topics: Administration, Oral; Alanine; Animals; Antithrombins; Biological Availability; Chymotrypsin; Drug Design; Fibrinolysin; Kinetics; Molecular Conformation; Molecular Structure; Partial Thromboplastin Time; Structure-Activity Relationship; Sulfonamides; Thiazoles; Thrombosis; Trypsin

Experimental animal models of thrombosis have been established in several species to examine factors responsible for thrombotic disorders in man. One technical facet of all thrombosis models is the need to quantitate cell deposition on thrombogenic surfaces, and this is routinely accomplished with radioisotopic labeling of specific components. Data reported here demonstrate that formalin-fixed thrombi can be hydrolyzed with chymotrypsin allowing recovery and quantitation of platelets and erythrocytes incorporated within the clot. Recovery of platelets from in vitro generated, model thrombi averaged 99 +/- 10% (mean +/- 1 SD; n = 7; range 88-116%) of calculated content; recovery of erythrocytes was 94.1 +/- 1.1% (n = 6) as measured by recovery of cellular hemoglobin after chymotrypsin hydrolysis of clots. Chymotrypsin was also shown to release platelets and erythrocytes from string-bound thrombi generated in vivo with an arterio-venous shunt model in beagle dogs. Platelet recovery from these string clots after chymotrypsin hydrolysis was independently verified with a quantitative Western blot assay of platelet antigens. These data demonstrate that experimental thrombi can be hydrolyzed with chymotrypsin, thereby not only eliminating the need for radioisotopes, but also permitting flow cytometric analysis of cells comprising the thrombus.

Topics: Animals; Blotting, Western; Chymotrypsin; Disease Models, Animal; Dogs; Erythrocyte Count; Flow Cytometry; Hydrolysis; In Vitro Techniques; Platelet Count; Thrombosis

Several approaches to development of systems for directed transport of drugs are illustrated by an example of thrombolytic therapy. Preparation and properties of enzyme derivatives with increased tropism to the thrombus material due to modification of enzymes by fibrinogen or specific antibodies are discussed. The data on directed transport of drugs under the action of the magnetic field and on the selective action of drugs on culture cells are also presented.

Topics: Animals; Antibodies, Monoclonal; Antibody Affinity; Chymotrypsin; Dextrans; Drug Evaluation, Preclinical; Enzymes, Immobilized; Fibrin; Fibrinogen; Fibrinolytic Agents; In Vitro Techniques; Magnetics; Thrombosis; Urokinase-Type Plasminogen Activator

In this report, we describe the anticoagulant and antithrombotic properties of a peptide (residues 1-44) derived from the amino-terminus of the bovine Factor X light chain by limited proteolysis with chymotrypsin, and subsequently purified by QAE-Sephadex chromatography. The effect of Factor X gla-peptide on the activation of human 3H-Factors IX and X was studied using radiometric assays and purified coagulation factors. Factor VIIa-tissue factor catalyzed activation of Factors IX and X was half-maximally inhibited by Factor X gla-peptide at concentrations of 0.8 microM and 0.2 microM, respectively. Factor IXa-VIII catalyzed Factor X activation was half-maximally inhibited at a gla-peptide concentration of 0.5 microM. In addition, thrombin formation by platelets incubated with Factor Xa and prothrombin could be similarly blocked by gla-peptide. Studies with bovine aortic endothelial cells indicated that the Factor X gla-peptide blocked in parallel Factor X binding and activation on the cell surface. Decarboxylation of the peptide by acid heat treatment destroyed its anticoagulant activity. The in vivo anticoagulant potential of native gla-peptide was demonstrated by a rapid prolongation of the PT and APTT following intravenous infusion into a rabbit. In addition, gla-peptide prevented thrombus formation in response to Factors IXa and Xa, but not thrombin, in a Wessler venous stasis model.

Topics: 1-Carboxyglutamic Acid; Animals; Anticoagulants; Aorta; Blood Coagulation; Cattle; Chymotrypsin; Endothelium; Factor IX; Factor X; Partial Thromboplastin Time; Peptide Fragments; Protein Binding; Prothrombin; Rabbits; Thrombosis

Topics: Amylases; Animals; Bandages; Chymotrypsin; Drug Combinations; Empyema; Enzymes, Immobilized; Guinea Pigs; Humans; Intraoperative Care; Lung Abscess; Peptide Hydrolases; Postoperative Care; Postoperative Complications; Rats; Streptokinase; Therapeutic Irrigation; Thrombosis; Trypsin; Wound Healing; Wounds and Injuries

Topics: Adenosine Triphosphate; Adult; Animals; Aorta, Abdominal; Aspirin; Blood Platelets; Chymotrypsin; Dipyridamole; Drug Interactions; Endothelium; Humans; Middle Aged; Platelet Adhesiveness; Platelet Aggregation; Rabbits; Thrombosis

Topics: Animals; Anti-Inflammatory Agents; Blood Coagulation; Chymotrypsin; Disease Models, Animal; Esterases; Fibrinolysin; Injections, Intravenous; Injections, Subcutaneous; Jugular Veins; Ligation; Methods; Phlebography; Pyrazoles; Rabbits; Streptokinase; Thrombin; Thrombophlebitis; Thrombosis; Vena Cava, Inferior

Topics: Chymotrypsin; Deoxyribonuclease I; Fibrinolysin; Fibrinolysis; Glycerol; In Vitro Techniques; Research; Streptodornase and Streptokinase; Streptokinase; Thrombelastography; Thrombosis; Trypsin

Topics: Chymotrypsin; Colorectal Surgery; Drug Combinations; Hemorrhoids; Humans; Postoperative Complications; Rectal Prolapse; Surgical Procedures, Operative; Thrombosis; Trypsin

Topics: Arteriosclerosis; Chymotrypsin; Diabetic Retinopathy; Drug Therapy; Geriatrics; Humans; Retinal Diseases; Retinal Vessels; Thrombosis

Topics: Chymotrypsin; Deoxyribonuclease I; Fibrinolysis; Streptodornase and Streptokinase; Streptokinase; Thromboembolism; Thrombosis; Toxicology; Trypsin

Topics: Chymotrypsin; Humans; Solubility; Thrombosis

Topics: Animals; Arteries; Chymotrypsin; Deoxyribonuclease I; Disease; Dogs; Endopeptidases; Enzyme Precursors; Femoral Artery; Hydrolases; Peptide Hydrolases; Plasminogen; Plasminogen Activators; Solubility; Streptodornase and Streptokinase; Streptokinase; Thrombosis; Trypsin