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PubMed1996

Extracellular activities of human granzyme A. Monocyte activation by granzyme A versus alpha-thrombin.

Granzymes, serine proteases located in the granules of cytotoxic T cel ls and NK cells, are essential for induction of target cell apoptosis. However, since cytotoxic cells constitutively secrete a portion of their synthesized granzymes, these proteases could mediate extracellular functions independent of their role in the lytic event. Thrombin, another serine protease, can induce cytokine production in a number of different cell types. In this study, we test the hypothesis that granzymes, like thrombin, can regulate cell-mediated immunity by inducing the production of different cytokines. We show that granzyme A (GA) stimulates IL-6, IL-8, and TNF-alpha production by human PBMC and purified monocytes. In contrast, monocytes exposed to thrombin had enhanced IL-8 production with no induction of IL-6 or TNF-alpha production. However, monocytes exposed to either GA or thrombin had enhanced phagocytic activity. The enzymatic activity of GA and thrombin was required for the induction of cytokine production and for the enhancement of phagocytic activity. The induction of different cytokine profiles by GA vs thrombin suggested that GA activates monocytes via a receptor that was different from the thrombin receptor. This conclusion was strengthened by the fact that GA was incapable of inducing Ca2+ mobilization in insect cells transfected with the thrombin receptor. These results suggest that enzymatically active GA mediates important immunoregulatory functions through signaling pathways that does not involve thrombin receptor activation.

Animals↗
PubMed1997

Thrombin receptor activation results in calcium signaling and protein kinase C-dependent stimulation of DNA synthesis in HEp-2g laryngeal carcinoma cells.

BACKGROUND: Recently, the expression of the "tethered ligand" thrombin receptor in carcinosarcoma and melanoma cells has been shown. However, the role of the thrombin receptor in tumor cell metabolism still is undefined. METHODS: In this article, the "tethered ligand" thrombin receptor was identified on human epidermoid carcinoma cells (HEp-2g cell line) by using immunofluorescence studies with a monoclonal antithrombin receptor antibody and radioligand binding. Furthermore, the effects of alpha-thrombin and thrombin receptor activating peptides (TRAP)-6 on calcium mobilization, protein kinase C (PKC) translocation, and DNA synthesis were estimated. RESULTS: Pharmacologic characterization using [3H]TRAP-6 as a radioligand demonstrated a single class of high affinity binding sites (dissociation constant [KD] = 7.2 +/- 2.2 x 10(-7) M) and a binding capacity of 27 +/- 3.4 fmol/mg protein. The function of these binding sites was demonstrated by alpha-thrombin- and TRAP-6-induced mobilization of free intracellular calcium, and translocation of PKC from cytosol to cell membrane. Moreover, alpha-thrombin and TRAP-6 induced an increase in [3H]thymidine incorporation in HEp-2g cells that could be blocked by the PKC inhibitor bisindolylmaleimide. CONCLUSIONS: To the authors' knowledge, the results of this study demonstrate for the first time functional thrombin receptors in epidermoid carcinoma cells. The thrombin receptor appears to be involved in growth regulation in HEp-2g cells by a PKC-dependent mechanism.

Calcium↗
PubMed1998

Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation.

Production of vascular endothelial growth factor (VEGF) by cancer cells at invasive and metastatic sites is an important aspect of tumor angiogenesis. Although known primarily as a mitogen and a vascular permeability factor (VPF) for endothelial cells, VEGF/VPF has been proposed to induce the expression of procoagulant factors in endothelial cells. In this study, we have explored the ramifications of VEGF induction of tissue factor (TF) in human umbilical vein endothelial cells (HUVECs) and subsequent activation of progelatinase A. Within 3 hr of incubation with VEGF/VPF, endothelial cells accelerate TF generation as measured using chromogenic substrate assays for coagulation factors Xa and thrombin. Incubation of VEGF/VPF-pre-treated cells with prothrombin and factors X, Va, and VIIa at 37 degrees C and subsequent generation of thrombin resulted in activation of secreted endothelial progelatinase A as demonstrated by gelatin zymography. Anti-thrombin III or antibodies to TF inhibited thrombin generation and progelatinase A activation. VEGF/VPF also directly increased HUVEC secretion of interstitial collagenase, tissue inhibitor of metalloproteinases (TIMP-1) and, to a lesser extent, gelatinase A. The effect of thrombin on endothelial proliferation in serum-free media was examined. Thrombin was a growth factor for HUVECs at a lower dose than that required for progelatinase A activation. Whereas TIMP-2 abrogated thrombin-induced progelatinase A activation, it had no significant effect on thrombin-induced endothelial cell growth. We propose that an early step in tumor angiogenesis involves VEGF-induced thrombin generation and increased MMP production with subsequent activation of endothelial progelatinase A and degradation of the underlying basement membrane.

Cell Division↗
PubMed1996

Surface adsorption and fibrinogen interactions with hirudin-thrombin complex.

This study describes the protein interaction properties of hirudin-thrombin complex adsorbed onto a surface. Hirudin-thrombin complex, preadsorbed thrombin bound with hirudin, and thrombin were coated onto the surfaces of glass beads. The activity of the thrombin component adsorbed onto the surface, and hence the ability of hirudin to bind and inactivate thrombin, was determined by measuring the activity of thrombin to cleave fibrinogen to generate fibrin and to cleave a chromogenic substrate, S-2238. Pure thrombin adsorbed on the surface (without hirudin) retained its activity to cleave fibrinogen and cleave S-2238. Hirudin-thrombin complex adsorbed on the surface did not stimulate the activation of fibrinogen, nor did it cleave S-2238.

Biocompatible Materials↗
PubMed1984

Effect of epinephrine on fibrinogen receptor exposure by aspirin-treated platelets and platelets from concentrates in response to ADP and thrombin.

Synergistic effects between agonists on platelet aggregation have long been appreciated. Recently epinephrine was reported to induce maximal aggregation of aspirin-treated platelets when combined with ADP or thrombin, and to increase fibrinogen binding of non-aspirin treated platelets stimulated with low doses of ADP. The present study extends these observations to correlate fibrinogen binding in response to various combinations of ADP, epinephrine, and thrombin with platelet aggregation and 14C-serotonin release using aspirin-treated platelets as well as platelets from stored concentrates. When fresh platelets were stimulated with epinephrine (5 microM) together with either ADP (10 microM) or thrombin (150 mU/ml), fibrinogen binding increased by 180% compared to binding observed in response to ADP or thrombin alone. This was accompanied by enhanced platelet aggregation, but no increase in 14C-serotonin release. While both ADP and epinephrine potentiated the aggregation and fibrinogen binding of stored platelets in response to high doses of thrombin (150 mU/ml), maximal aggregation was achieved only with thrombin (150 mU/ml) and epinephrine (5 microM) in combination. The data thus suggest that 1) epinephrine induces maximal aggregation of aspirin-treated platelets stimulated with thrombin or ADP by significantly enhancing fibrinogen receptor exposure independently of the cyclooxygenase-mediated release reaction; 2) epinephrine stimulates platelets by a mechanism different from that of thrombin or ADP; and 3) as demonstrated by others, the ability of platelets from stored concentrates to aggregate and to bind fibrinogen in response to ADP can be enhanced by epinephrine, and, in addition, these platelets can aggregate and bind fibrinogen maximally when stimulated with combinations of epinephrine and thrombin.

Adenosine Diphosphate↗
PubMed1989

Human thrombin plus 5 per cent ethanolamine oleate injected to sclerose oesophageal varices: a prospective randomized trial.

Fifty cirrhotic Japanese patients with oesophageal varices underwent sclerotherapy in a prospective randomized trial carried out to examine the effects of human thrombin given concomitantly with the sclerosant 5 per cent ethanolamine oleate. The two groups (25 patients each) were comparable with regard to size of the oesophageal varices, and the aetiology and severity of the liver disease. Twenty-five patients, 13 and 12 in the thrombin + and - groups, respectively, had at least one episode of variceal bleeding. The remaining 25 were given prophylactic injections. There was a significantly lower rate of occurrence of bleeding from injection sites when the injection needle was removed at the initial session of sclerotherapy in the thrombin + group, where human thrombin was injected (0.2-0.3 ml, 100-150 units per injection) just before removal of the injection needle. Endoscopy at 1 week after the initial session showed a significantly (P less than 0.05) higher rate of disappearance of red colour signs on varices in the thrombin + group (96 per cent) than in the thrombin - group (72 per cent). Fibrin degradation product E-fraction (FDP-E) values increased 1 h, 1 day and 6 days after the initial session of sclerotherapy in the two groups. The rate of increase in FDP-E values 1 h after sclerotherapy was significantly larger (P less than 0.001) in the thrombin + than in the thrombin - group. There was no clinical sign of disseminated intravascular coagulation. Administration of human thrombin plus a sclerosant seems to be useful and efficacious, especially for patients with huge oesophageal varices.

Endoscopy↗
PubMed2004

Thrombin induces expression of cytokine-induced SH2 protein (CIS) in rat brain astrocytes: involvement of phospholipase A2, cyclooxygenase, and lipoxygenase.

Previously we have reported that thrombin induces inflammatory mediators in brain glial cells (Ryu et al. 2000. J Biol Chem 275:29955). In the present study, we found that thrombin induced a negative regulator of a cytokine signaling molecule, cytokine-induced SH2 protein (CIS), in rat brain astrocytes. In response to thrombin, CIS expression was increased at both the mRNA and protein levels. Although STAT5 is known to regulate CIS expression, thrombin did not activate STAT5, and inhibitors of JAK2 (AG490) and JAK3 (WHI-P97 and WHI-P154) had little effect on thrombin-induced CIS expression. In contrast, cytosolic phospholipase A(2) (cPLA(2)), cyclooxygenase (COX), and lipoxygenase (LO) play a role in CIS expression, since inhibitors of cPLA(2), cyclooxygenase (COX), and LO significantly reduced CIS expression. Reactive oxygen species (ROS) scavengers (N-acetyl-cysteine [NAC] and trolox) reduced thrombin-induced CIS expression, and inhibitors of COX and LO reduced ROS produced by thrombin. Furthermore, prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)), products of COX and LO, respectively, potentiated thrombin-induced CIS expression, indicating that ROS, and PGE(2) and LTB(4) generated by COX and LO, mediate CIS expression. Since interferon-gamma (IFN-gamma)-induced GAS-luciferase activity and tyrosine phosphorylation of STAT1 and STAT3 were lower in CIS-transfected cells compared to control vector-transfected cells, CIS could have anti-inflammatory activity. These data suggest that thrombin-stimulation of ROS and prostaglandin and leukotriene production via the cPLA(2), COX and LO pathways results in CIS expression. More importantly, CIS expression may be a negative feedback mechanism that prevents prolonged inflammatory responses.

Animals↗
PubMed2005

Midazolam suppresses thrombin-induced heat shock protein 27 phosphorylation through inhibition of p38 mitogen-activated protein kinase in cardiac myocytes.

It has been shown that anesthetics have effects of cardiac preconditioning. Heat shock proteins (HSPs) function as molecular chaperone. Among them, HSP27, a low-molecular-weight HSP, abundantly exist in heart. However, the relationship between anesthetics and HSP27 in heart is not yet clarified. We investigated whether thrombin induces or phosphorylates HSP27 in primary cultured mouse myocytes and the effect of midazolam on the thrombin-stimulated HSP27 phosphorylation and the mechanism behind it. Thrombin time dependently phosphorylated HSP27 at Ser-15 and Ser-85 while having no effect on the levels of HSP27. Midazolam markedly suppressed the thrombin-induced phosphorylation of HSP27 at both Ser-15 and Ser-85. Thrombin induced the phosphorylation of p44/p42 MAP kinase and p38 MAP kinase without affecting stress-activated protein kinase/c-Jun N-terminal kinase. In addition, midazolam attenuated the phosphorylation of thrombin-induced p38 MAP kinase but not that of p44/p42 MAP kinase. SB203580 and PD169316, inhibitors of p38 MAP kinase, suppressed the thrombin-induced phosphorylation of HSP27 at both Ser-15 and Ser-85. These results strongly suggest that thrombin induces the HSP27 phosphorylation at least through the p38 MAP kinase activation in cardiac myocytes and that midazolam inhibits the thrombin-induced HSP27 phosphorylation via suppression of p38 MAP kinase activation.

Anesthetics, Intravenous↗
PubMed1986

Formation of protease nexin-thrombin complexes on the platelet surface.

We have recently described a platelet factor that is similar to the fibroblast thrombin inhibitor protease nexin I (PNI). The present manuscript shows that this platelet form of PN (PNp) does not complex [125I]-thrombin that has been blocked at its active site, consistent with the conclusion that it is a thrombin inhibitor. When platelets are incubated with [125I]-thrombin, PNp-[125I]-thrombin complexers accumulate both in the medium and on the platelet surface. In the case of fibroblasts, PNI-[125I]-thrombin complexes that form in solution bind to the cells as a consequence of a receptor-mediated clearance process [Low et al, Proc Natl Acad Sci USA 78:2340, 1981]. We show here that the PNp-[125I]-thrombin complexes that accumulate in platelet-binding incubation medium do not bind to platelets. Thus, the platelet-associated complexes must form by [125I]-thrombin binding to PNp that is associated with the platelet surface. Pretreatment of platelets with heparin markedly increases the number of PNp-[125I]-thrombin complexes that form on platelets. The basis for this increase is unclear. This effect seems incompatible with a heparinlike factor acting as the surface binding site for PNp.

Amyloid beta-Protein Precursor↗
PubMed1978

Binding and internalization of 125I thrombin in chick embryo fibroblasts: possible role in mitogenesis.

Human alpha thrombin acts as a mitogen for cultures of resting chick embryo fibroblasts (CEF) in serum free medium. The use of 125I-labeled thrombin shows that thrombin specifically binds to CEF and that after a lag of approximately 30 to 60 minutes it can not be removed by subsequent exposure to trypsin. The entry of 125I thrombin into the trypsin-insensitive domain is not inhibited to any great extent by excess unlabelled thrombin. The cell-associated thrombin retains its native molecular weight and its catalytic activity toward synthetic amide substrates. It appears to be located in the crude nuclear fraction of homogenized CEF cells. The association of thrombin with CEF is specific, since the non-mitogenic serine protease chymotrypsin is internalized to a much lesser extent than thrombin. The data are discussed in terms of a possible intracellular site for thrombin's mitogenic action.

Cell Nucleus↗
PubMed1992

The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships.

Thrombin is a multifunctional serine proteinase that plays a key role in coagulation while exhibiting several other key cellular bioregulatory functions. The X-ray crystal structure of human alpha-thrombin was determined in its complex with the specific thrombin inhibitor D-Phe-Pro-Arg chloromethylketone (PPACK) using Patterson search methods and a search model derived from trypsinlike proteinases of known spatial structure (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S.R., & Hofsteenge, J., 1989, EMBO J. 8, 3467-3475). The crystallographic refinement of the PPACK-thrombin model has now been completed at an R value of 0.156 (8 to 1.92 A); in particular, the amino- and the carboxy-termini of the thrombin A-chain are now defined and all side-chain atoms localized; only proline 37 was found to be in a cis-peptidyl conformation. The thrombin B-chain exhibits the characteristic polypeptide fold of trypsinlike serine proteinases; 195 residues occupy topologically equivalent positions with residues in bovine trypsin and 190 with those in bovine chymotrypsin with a root-mean-square (r.m.s.) deviation of 0.8 A for their alpha-carbon atoms. Most of the inserted residues constitute novel surface loops. A chymotrypsinogen numbering is suggested for thrombin based on the topological equivalences. The thrombin A-chain is arranged in a boomeranglike shape against the B-chain globule opposite to the active site; it resembles somewhat the propeptide of chymotrypsin(ogen) and is similarly not involved in substrate and inhibitor binding. Thrombin possesses an exceptionally large proportion of charged residues. The negatively and positively charged residues are not distributed uniformly over the whole molecule, but are clustered to form a sandwichlike electrostatic potential; in particular, two extended patches of mainly positively charged residues occur close to the carboxy-terminal B-chain helix (forming the presumed heparin-binding site) and on the surface of loop segment 70-80 (the fibrin[ogen] secondary binding exosite), respectively; the negatively charged residues are more clustered in the ringlike region between both poles, particularly around the active site. Several of the charged residues are involved in salt bridges; most are on the surface, but 10 charged protein groups form completely buried salt bridges and clusters. These electrostatic interactions play a particularly important role in the intrachain stabilization of the A-chain, in the coherence between the A- and the B-chain, and in the surface structure of the fibrin(ogen) secondary binding exosite (loop segment 67-80).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Chloromethyl Ketones↗
PubMed1998

alpha-Thrombin inhibits signal transducers and activators of transcription 3 signaling by interleukin-6, leukemia inhibitory factor, and ciliary neurotrophic factor in CCL39 cells.

We recently demonstrated that, in rat aortic smooth muscle cells, alpha-thrombin stimulated Stat3/SIF-A (signal transducers and activators of transcription 3/sis-inducing factor-A) activity [G. J. Bhat et al. (1997) Hypertension 29(Pt. 2), 356-360]. In the present study, we observed that exposure of CCL39 cells (a Chinese hamster lung fibroblast cell line) to alpha-thrombin resulted in a time-dependent decrease in basal SIF-A activity. We hypothesized that the decrease in basal SIF-A was due to the initiation of an inhibitory pathway, following alpha-thrombin exposure. To test this hypothesis, we determined if alpha-thrombin would inhibit Stat3 and SIF-A activation by interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and ciliary neurotrophic factor (CNTF). In support of this hypothesis, alpha-thrombin inhibited the Stat3/SIF-A response induced by all the above cytokines. The inhibition by alpha-thrombin was concentration dependent, was sensitive to hirudin, and was mimicked by the thrombin receptor agonist peptide. The inhibition did not require the activation of phorbol 12-myristate 13-acetate-sensitive isoforms of protein kinase C and was reversed by pretreatment with the mitogen-activated protein kinase kinase 1 (MAPKK1 or MEK1) inhibitor PD98059. Inhibitory cross talk between alpha-thrombin and IL-6 was also observed in MRC-5 cells, a fibroblast cell line derived from human lung tissue. Thus, we identify a novel alpha-thrombin inhibitory pathway which, acting through a MAPKK1-dependent mechanism, blocks IL-6-, LIF-, and CNTF-induced Stat3/SIF-A activation. This inhibitory cross talk may provide an important regulatory function to modulate gene transcription by these cytokines, during immune and inflammatory responses.

Animals↗
PubMed1999

Formation of PI 3-kinase products in platelets by thrombin, but not collagen, is dependent on synergistic autocrine stimulation, particularly through secreted ADP.

Platelet activation by thrombin or collagen results in secretion and synthesis of several platelet agonists that enhance the responses to the primary agonists (autocrine stimulation). To disclose the effects of thrombin and collagen on the phosphorylation of 3-phosphoinositides per se we incubated platelets with five inhibitors of platelet autocrine stimulation (IAS) that act extracellularly. We found that IAS almost totally blocked thrombin-induced production of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. In contrast, collagen induced massive production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) in the presence of IAS. When testing the effect of each inhibitor individually we found the strongest inhibition of thrombin-induced PtdIns(3,4)P(2) production with the ADP scavenger system CP/CPK. Furthermore, we found a strong synergistic effect between exogenously added ADP and thrombin on production of PtdIns(3,4)P(2). In contrast to the results from 3-phosphorylated phosphoinositides, CP/CPK had little effect on thrombin-induced protein tyrosine phosphorylation. Our results show the importance of autocrine stimulation in thrombin-induced accumulation of 3-phosphorylated phosphoinositides and raise the question as to whether thrombin by itself is capable of inducing PI 3-K activation. In marked contrast to thrombin, collagen per se appears to be able to trigger increased production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3).

Adenosine Diphosphate↗
PubMed1998

Cytoskeletal changes in platelets induced by thrombin and phorbol myristate acetate (PMA).

Changes in shape, and aggregation that accompanies platelet activation, are dependent on the assembly and reorganization of the cytoskeleton. To assess the changes in cytoskeleton induced by thrombin and PMA, suspensions of aspirin-treated,32P-prelabeled, washed pig platelets in Hepes buffer containing ADP scavengers were activated with thrombin, and with PMA, an activator of protein kinase C. The cytoskeletal fraction was prepared by adding Triton extraction buffer. The Triton-insoluble (cytoskeletal) fraction isolated by centrifugation was analysed by SDS-PAGE and autoradiography. Incorporation of actin into the Triton-insoluble fraction was used to quantify the formation of F-actin. Thrombin-stimulated platelet cytoskeletal composition was different from PMA-stimulated cytoskeletal composition. Thrombin-stimulated platelets contained not only the three major proteins: actin (43 kDa), myosin (200 kDa) and an actin-binding protein (250 kDa), but three additional proteins of Mr56 kDa, 80 kDa and 85 kDa in the cytoskeleton, which were induced in by thrombin dose-response relationship. In contrast, PMA-stimulated platelets only induced actin assembly, and the 56 kDa, 80 kDa and 85 kDa proteins were not found in the cytoskeletal fraction. Exposure of platelets to thrombin or PMA induced phosphorylation of pleckstrin parallel to actin assembly. Staurosporine, an inhibitor of protein kinase C, inhibited actin assembly and platelet aggregation induced by thrombin or PMA, but did not inhibit the incorporation of 56 kDa, 80 kDa and 85 kDa into the cytoskeletal fraction induced by thrombin. These three extra proteins seem to be unrelated to the induction of protein kinase C. We conclude that actin polymerization and platelet aggregation were induced by a mechanism dependent on protein kinase C, and suggest that thrombin-activated platelets aggregation could involve additional cytoskeletal components (56 kDa, 80 kDa, 85 kDa) of the cytoskeleton, which made stronger actin polymerization and platelet aggregation more.

Actins↗
PubMed1993

Thrombin promotes aortic endothelial cell spreading and microfilament formation in nonconfluent monolayer cultures.

Thrombin is present at sites of vascular injury. The objective of this study was to determine if thrombin may regulate endothelial repair. To address this our study was designed to determine the effect of thrombin on cell shape and microfilament distribution of porcine aortic endothelial cells in low-density cultures. Since these cells grow as islands of cells, low-density cultures serve as a model for conditions in which reendothelialization occurs from several foci following prominent patchy endothelial denudation. Thrombin incubation for 1, 2, and 4 hr at 0.5, 4, and 8 U/ml caused a significant increase in both the surface area of endothelial cells and the amount of microfilament bundles occupying the cell surface area. The increase in microfilament bundles was more than would be expected from the increased surface area. However, the pattern of distribution of microfilaments and of vinculin adhesion plaques within the cells was not altered by thrombin. Attempts at understanding the molecular mechanisms involved were undertaken. The role of protein kinase C was studied. Phorbol 12-myristate 13-acetate (PMA) caused cell spreading as well but no increase in microfilaments within the first 2 hr of incubation. Some cells showed mild disruption of microfilaments. Following 24 hr incubation with PMA, which reduces protein kinase C, the cells showed retraction and arborization along with thinning and disruption of microfilaments. Removal of PMA and addition of thrombin reversed these changes to normal within 2 hr while washout of PMA on its own did not alter cell shape or microfilaments. Thrombin also partially reversed the effects of H-7, a protein kinase C inhibitor, which on its own also caused cell retraction and disruption of microfilaments. Neomycin sulfate did not alter the thrombin effect suggesting that the participation of the phosphotidylinositol system is not directly required. In conclusion, thrombin promotes endothelial activities which are associated with long-term endothelial repair; however, these effects do not appear to be directly due to the involvement of conventional isoforms of protein kinase C or the phosphotidylinositol system.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Relaxant and contractile responses of porcine pulmonary arteries to a thrombin receptor activating peptide (TRAP).

Recent studies on cloning of the thrombin receptor, which belongs to the family of G-protein-coupled receptors, suggest that thrombin cleaves a peptide from the extracellular N-terminus. A synthetic peptide of 14 amino acids corresponding to the sequence of the newly generated N-terminus was found to possess thrombin-like activity in several cells endowed with thrombin receptors. The relaxant and contractile effects of this thrombin receptor activating peptide (TRAP, Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe) were investigated in porcine pulmonary arteries and compared with the action of thrombin. In PGF2 alpha-precontracted vessels with intact endothelium, TRAP (0.3-10 mumol/l) caused reversible transient and concentration-dependent relaxation which was absent after mechanical removal of the endothelium. Preincubation of the vessels with NG-nitro-L-arginine (200 mumol/l) markedly reduced the relaxation. The TRAP-induced relaxation was associated with an increase in cGMP in the arteries. In comparison to thrombin, TRAP (EC50: 0.8 mumol/l) was less potent by more than three orders of magnitude. In endothelium-denuded pulmonary arteries TRAP (1-20 mumol/l) caused a concentration-dependent contraction which was reversible after washout. The TRAP-induced contractile response was preceded by an increase in generation of inositol 1,4,5-triphosphate (IP3); the peak of IP3 accumulation was reached after 30 s. Compared with the contractile effect of thrombin, that of TRAP was weaker by three of magnitude. The vascular effect of TRAP was not inhibited by the thrombin inhibitors hirudin or heparin while the protein kinase C inhibitor staurosporine (0.1 mumol/l) preferentially inhibited the tonic phase of contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1990

Pharmacological modification of mechanical and electrical responses of frog heart to thrombin.

The pharmacological modification of the thrombin effect on the mechanical and electrical responses of frog heart was examined in the Straub heart preparation and in single ventricular cells. Associated with the positive inotropic action thrombin increases voltage and duration of action potentials of isolated frog ventricular cells. As found by the patch-clamp technique in the cell-attached mode, thrombin stimulates single L-type Ca2+ channels, presumably mediated by a second messenger. The enhancement of contractility by thrombin depends on the proteolytic activity of the enzyme because enzymatically inactivated thrombin has no effect on frog hearts. The positive inotropic effect of thrombin as well as its stimulation of Ca2+ channel currents were inhibited by the protein kinase C blocker 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H7). However, phorbol 12-myristate 13-acetate (PMA), a known stimulator of protein kinase C, was ineffective in stimulating the inotropic action of thrombin. The inhibition of the thrombin-induced enhancement of contractility by indometacin indicates an involvement of arachidonic acid in the action of thrombin on frog heart.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Comparison of the active-site conformations of bovine alpha-thrombin and meizothrombin(desF1) by electron spin resonance.

The active site of the prothrombin activation intermediate meizothrombin(desF1) was probed using several fluorosulfonylphenyl spin labels specific for the active serine hydroxyl of serine proteases. The mobilities of the thrombin species inhibited with the nitroxide spin labels m-IV [4-(2,2,6,6-tetramethyl-piperidine-1-oxyl) -m-(fluorosulfonyl)benzamide] and m-V [3-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl) -m-(fluorosulfonyl)benzamide], which are sensitive to differences between alpha- and gamma-thrombin, were quite similar to that of alpha-thrombin. That is, no major conformational differences between meizothrombin(desF1) and alpha-thrombin were observed in this region of the extended active site. On the other hand, p-IV [4-(2,2,6,6-tetramethyl piperidine-1-oxyl)-p-(fluorosulfonyl)benzamide], p-V [3-(2,2,5,5-tetramethylpyrrolidine-1-oxyl) -p-(fluorosulfonyl)benzamide], and m-VII [N-[m- (fluorosulfonyl)phenyl]-4-N-(2,2,6,6-tetramethyl- piperidine-1-oxyl)urea], which probe an apolar binding region of bovine thrombin, exhibited large differences in mobility between alpha-thrombin and meizothrombin(desF1). The conformational consequences of indole binding to spin-labeled thrombin species demonstrated that both species also possess an indole-binding site. However, the nitroxide mobility changes upon indole binding to the spin-labeled protein derivative were somewhat different between the two thrombin species under study. In addition, the effects of the benzamidine binding were quite similar for each labeled protein. Thus is appears that, while both species posses a fully functional active site, the region in meizothrombin(desF1) probed by spin labels p-IV, p-V, and m-VII, which corresponds to the apolar binding region, differs in conformation from alpha-thrombin.

Animals↗