hirudin and leupeptin

Previous research in our laboratory indicated that prothrombin and other coagulation enzymes required to activate prothrombin to thrombin are synthesized by the cornea and that apoptotic human corneal stromal cells can provide a surface for prothrombin activation through the intrinsic and extrinsic coagulation pathways. The purpose of the work reported here is to study the role of thrombin activity in the regulation of matricellular protein Cyr61 (CCN1) produced by wounded phenotype human corneal stromal fibroblasts and myofibroblasts.. Stromal cells from human donor corneas were converted to defined wounded phenotype fibroblasts and myofibroblasts with fetal bovine serum, followed by basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGFβ-1), respectively, and stimulated with varying concentrations (0-10.0 units (U)/ml) of thrombin from 1-7 h. Cyr61 transcript levels were determined using reverse transcriptase-PCR (RT-PCR) and quantitative PCR (qPCR) while protein forms were analyzed using western blot data. Protease activities were characterized via protease class-specific inhibitors and western blot analysis. Thrombin activity was quantified using the fluorogenic peptide Phe-Pro-Arg-AFC. Protease-activated receptor (PAR) agonist peptides-1 and -4 were used to determine whether cells increased Cyr61 through PAR signaling pathways. The PAR-1 antagonist SCH 79797 was used to block the thrombin cleavage of the receptor. PCR data were analyzed using MxPro software and western blot data were analyzed using Image Lab™ and Image J software. Student. In cultured human corneal stromal fibroblasts and myofibroblasts, thrombin regulates Cyr61 through two mechanisms: 1) thrombin increases the Cyr61 expression at the message and protein levels, and 2) thrombin increases the activation of a leupeptin-sensitive protease that stimulates the cleavage of Cyr61 into N- and C-terminal domain populations in or near the thrombospondin type-1 domain. Generation of Cyr61 peptides during corneal injury stimulation may reveal additional functions of the protein, which modulate corneal wound healing activities or decrease activities of the full-length Cyr61 form.

Topics: Alternative Splicing; Cell Differentiation; Corneal Stroma; Culture Media, Conditioned; Cysteine-Rich Protein 61; Fibroblast Growth Factor 2; Fibroblasts; Hirudins; Humans; Leupeptins; Myofibroblasts; Primary Cell Culture; Proteolysis; Pyrroles; Quinazolines; Receptors, Proteinase-Activated; RNA, Messenger; Signal Transduction; Stromal Cells; Thrombin; Transforming Growth Factor beta1

We investigated the regulation of arachidonic acid liberation catalyzed by group-IV cytosolic phospholipase A2 (cPLA2) in human platelets upon stimulation with thrombin through interaction with protease-activated receptor-1 (PAR-1) or glycoprotein Ib. Leupeptin, a protease inhibitor, completely inhibited thrombin-induced arachidonic acid liberation and Ca2+ mobilization, with inhibition of its protease activity. However, preincubation with thrombin in the presence of leupeptin potentiated Ca2+ ionophore-induced arachidonic acid liberation. The preincubation did not affect the intracellular Ca2+ level or cPLA2 activity in response to ionomycin. Human leukocyte elastase, which cleaves glycoprotein Ib, did not inhibit the enhancement of arachidonic acid liberation by thrombin in the presence of leupeptin. However, the effect of thrombin with leupeptin was abolished by a peptide corresponding to residues 54-65 of hirudin (hirudin peptide), which impairs the binding of thrombin to PAR-1. Furthermore, Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, which binds to platelets but has no protease activity, also enhanced Ca2+ ionophore-induced arachidonic acid liberation. In contrast, trypsin with leupeptin did not mimic the effect of thrombin with leupeptin, and furthermore trypsin-induced arachidonic acid liberation was insensitive to hirudin peptide. On the basis of the present results, we suggest that thrombin may accelerate cPLA2-catalyzed arachidonic acid liberation through non-proteolytic action toward PAR-1 but not toward glycoprotein Ib in co-operation with the proteolytic action leading to Ca2+ mobilization.

Topics: Amino Acid Chloromethyl Ketones; Antithrombins; Arachidonic Acid; Blood Platelets; Calcium; Enzyme Activation; Hirudins; Humans; Ionomycin; Ionophores; Leukocyte Elastase; Leupeptins; Peptide Fragments; Phospholipases A; Phospholipases A2; Platelet Glycoprotein GPIb-IX Complex; Receptor, PAR-1; Receptors, Thrombin; Thrombin; Trypsin

The production of nerve growth factor (NGF) in peripheral organs may play a role in the pathophysiology of hypertension and in obstructive disorders of the bladder outlet. We have been examining the cellular processes of NGF delivery and secretion in smooth muscle. NGF secretion from vascular smooth muscle cells (VSMCs) cultured from genetically hypertensive (WKHT), hyperactive (WKHA), and a control Wistar rat strain were assayed using a two-site ELISA of the culture media. Bladder smooth muscle cells (BSMCs) from the Wistar strain were also studied. The serine protease, thrombin, increased NGF secretion from all types of VSMCs but had no effect on Wistar BSMCs. The thrombin-mediated increase in NGF secretion was prevented by actinomycin D and cycloheximide, suggesting that RNA transcription and protein synthesis are required. The effect of thrombin was additive with a phorbol ester-induced elevation in NGF secretion rates from 4 to 6 h and was attenuated by a 24-h downregulation of protein kinase C. These results suggest that extracellular protease activity may regulate NGF secretion in smooth muscle. Thrombin may act in response to vascular injury, increasing NGF secretion from VSMCs, initiating VSMC migration, and preparing the VSMCs for reinnervation following an insult.

Topics: Animals; Aorta; Aprotinin; Cells, Cultured; Hirudins; Leupeptins; Muscle, Smooth; Muscle, Smooth, Vascular; Nerve Growth Factors; Protease Inhibitors; Protein Kinase C; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Wistar; Thrombin; Trypsin; Urinary Bladder

Enzyme inhibitors have been compared with conventional anticoagulants for the flow cytometric analysis of live leucocytes in whole blood. At 18 to 20 degrees C in vitro, PPACK (60 microM), hirudin (10 units ml-1), leupeptin (20 microg ml-1) and aprotinin (50 microg ml-1) inhibited blood clotting for 4 h or more, whereas PMSF (8 mg ml-1) or AEBSF (5 mg ml-1) inhibited clotting for only 20 or 40 min, respectively. When labelled with CD11b antibodies and analysed immediately ex vivo at 4 degrees C, the percentages of lymphocytes, monocytes, and polymorphs which stained positively and their mean fluorescence intensities were similar, irrespective into which anticoagulant blood was collected. Less than 1.5-fold increases in expression occurred on monocytes and polymorphs when blood anticoagulated with enzyme inhibitors or conventional anticoagulants was kept at 4 degrees C, or when blood anticoagulated with citrate, heparin, or hirudin was kept at 18 to 20 degrees C for 1 h before labelling and analysis, whereas approximately 2-fold increases in expression occurred in blood kept with K3EDTA, leupeptin, or aprotinin and more than 3-fold increases in blood kept with AEBSF or PPACK at 18 to 20 degrees C for 1 h. Further studies showed that leupeptin could be used effectively as the anticoagulant when investigating functional responses of live leucocytes in whole blood samples by flow cytometry and for the isolation of leucocytes with minimal modulation of adhesion molecules.

Topics: Adult; Amino Acid Chloromethyl Ketones; Anticoagulants; Aprotinin; Blood; Blood Cell Count; Citrates; Flow Cytometry; Hirudins; Humans; L-Selectin; Leukocytes; Leukocytes, Mononuclear; Leupeptins; Macrophage-1 Antigen; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phenylmethylsulfonyl Fluoride; Sulfones

Thrombin induces a number of physiological responses in several types of cells. To determine the action of thrombin in the vein, the electrophysiological and mechanical effects of thrombin were studied in rat portal vein smooth muscle cells. Ca2+ channel currents were recorded using the whole-cell patch-clamp technique. Thrombin had both inhibitory and stimulatory effects on the Ca2+ channel current. The inhibitory effect was reversed on washout of thrombin, whereas the stimulatory effect was maintained after thrombin was removed. Thrombin (1 unit/ml) produced a reversible decrease of 27.3 +/- 3.3% (n = 12) in the current amplitude and a sustained increase of 71.2 +/- 12.9% (n = 20). The thrombin-induced inhibition of Ca2+ channel current was blocked by the thrombin inhibitor hirudin and by the protease inhibitor leupeptin. The stimulatory effect of thrombin was inhibited by hirudin, by intracellular application of guanosine 5'-O-(beta-thio)diphosphate, and by antiphophatidylinositide antibodies but not by pertussis toxin. The thrombin-induced enhancement of the Ca2+ channel current amplitude was not observed when the current was previously stimulated by phorbol 12,13-dibutyrate. This suggests that the inhibitory effect of thrombin was related to its proteolytic activity and that the stimulatory effect involved activation of a pertussis toxin-insensitive GTP-binding protein, phosphatidylinositide hydrolysis, and protein kinase C activation. Both thrombin effects occurred in the same concentration range (0.001-10 units/ml). The thrombin-induced contraction of portal vein strips was completely inhibited by isradipine, and thrombin did not produce an increase in cytosolic [Ca2+], measured by indo-1 fluorescence in cells clamped at -50 mV, sufficient to activate Ca(2+)-dependent chloride current.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcium Channels; GTP-Binding Proteins; Guanosine Diphosphate; Hirudins; In Vitro Techniques; Isradipine; Leupeptins; Membrane Potentials; Muscle, Smooth, Vascular; Portal Vein; Rats; Rats, Wistar; Thionucleotides; Thrombin; Vasoconstriction

Mouse NB2a/dl neuroblastoma cells elaborate axonal neurites in response to various chemical treatments including dibutyryl cyclic AMP and serum deprivation. Hirudin, a specific inhibitor of thrombin, initiated neurite outgrowth in NB2a/dl cells cultured in the presence of serum; however, these neurites typically retracted within 24 h. The cysteine protease inhibitors leupeptin and N-acetyl-leucyl-leucyl-norleucinal (CI; preferential inhibitor of micromolar calpain but also inhibits millimolar calpain) at 10(-6) M considerably enhanced neurite outgrowth induced by serum deprivation, but could not induce neuritogenesis in the presence of serum. A third cysteine protease inhibitor, N-acetyl-leucyl-leucyl-methional (CII; preferential inhibitor of millimolar calpain but also inhibits micromolar calpain), had no detectable effects by itself. Cells treated simultaneously with hirudin and either leupeptin, CI, or CII elaborated stable neurites in the presence of serum. Cell-free enzyme assays demonstrated that hirudin inhibited thrombin but not calpain, CI and CII inhibited calpain but not thrombin, and leupeptin inhibited both proteases. These results imply that distinct proteolytic events, possibly involving more than one protease, regulate the initiation and subsequent elongation and stabilization of axonal neurites. Since the addition of exogenous thrombin or calpain to serum-free medium did not modify neurite outgrowth, the proteolytic events affected by these inhibitors may be intracellular or involve proteases distinct from thrombin or calpain.

Topics: Animals; Axons; Blood; Calpain; Endopeptidases; Hirudins; Leupeptins; Neuroblastoma; Osmolar Concentration; Protease Inhibitors; Thrombin; Tumor Cells, Cultured