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PubMed1985

Gold labeling of thrombin and ultrastructural studies of thrombin-gold conjugate binding by fibrin.

Monodispersed thrombin-gold (T-Au) conjugates were prepared by the absorption of a monolayer (3.8 nm thick) of human alpha-thrombin around individual monodispersed colloidal gold particles (16.5 +/- 1.8 nm). Like free molecular thrombin, T-Au conjugates can cause platelet aggregation, plasma clotting, and the release of fibrinopeptides A and B from fibrinogen. At the same thrombin concentration, T-Au conjugates have only one-tenth the fibrinogen-clotting activity of free thrombin and one-third the amidolytic activity of free thrombin. Hirudin can completely inhibit the fibrinogen-clotting activity of both T-Au conjugates and free thrombin, but can inhibit only half of the amidolytic activity of the conjugates. Diisopropyl fluorophosphonate can completely inhibit the fibrinogen-clotting activity and the amidolytic activity of both T-Au conjugates and free thrombin. T-Au conjugates were further characterized by studying the mechanism of their binding to fibrin and the location of the binding site on fibrin. The results of electron microscopic studies showed that T-Au conjugates, but not albumin-Au conjugates, are bound by fibrin. Increasing T-Au conjugate concentrations are associated with an increase in the number of T-Au conjugates binding to fibrin. At 0.1 microM thrombin, 73% of the T-Au conjugates are bound to branch points of the fibrin network with 27% of the T-Au conjugates present in the fibrin strands. At higher thrombin concentration (e.g., 0.5 microM) the percentage of T-Au conjugates bound to locations other than branch points increases to 62%.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1990

Effect of a synthetic thrombin-inhibitor MD805 on the reaction between thrombin and plasma antithrombin-III.

MD805, a synthetic thrombin-inhibitor, effectively retarded the time-dependent inactivation of thrombin which was generated endogeneously or added exogeneously in human plasma. The kinetical study of the time-dependent inactivation indicated that the type of inhibition was competitive and the obtained Ki of MD805 for thrombin was 3 x 10(-8)M. MD805 also inhibited the formation of thrombin-ATIII complex. These results indicated that the active site of thrombin was involved in the reaction between thrombin and ATIII, and that MD805 competed with ATIII for thrombin in exactly the same manner as it competed with fibrinogen or synthetic peptide substrates. As a result, MD805 would serve as a protective agent for ATIII from being consumed, in addition to its potent thrombin-inhibitory activity without the aid of ATIII. By contrast, heparin accelerated the time-dependent inactivation rate of thrombin and the formation of thrombin-ATIII complex, which indicates that heparin accelerates the consumption of ATIII.

Antithrombin III↗
PubMed1992

Affinity chromatography of platelets on immobilized thrombin: retention of catalytic activity by platelet-bound thrombin.

Radioactivity from I125-labeled human platelets was measured to estimate the extent of binding of platelet surface proteins to immobilized thrombin. 1-3% of the radioactivity was bound with 10-20% of this amount apparently irreversibly bound to the thrombin matrix. Site-specific chemical modification of thrombin with pyridoxal-5'-phosphate, N-bromosuccinimide or tetranitromethane resulted in a variable reduction of the amount of radiolabel bound. When thrombin modified with H-D-PheProArg-chloromethyl ketone (PPACK) was coupled to the matrix, there was no difference in the binding of platelet membrane proteins when compared to a control thrombin preparation while thrombin modified with tosyl-Lys-chloromethyl ketone (TLCK) coupled to the matrix did not bind radiolabel any more effectively than albumin which served as the control. However, when thrombin was modified with PPACK after coupling to the agarose matrix, ability to bind radiolabel was lost. Thrombin bound to platelets remained catalytically active when assayed with a peptide nitroanilide substrate. These results suggest tight binding between thrombin and platelets that is not only not dependent on active site integrity but leaves the bound thrombin catalytically competent.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Systemic vascular effects of thrombin and thrombin receptor activating peptide in rats.

The proteolytic enzyme thrombin activates its receptor by cleavage of a peptide from the extracellular N-terminus. The newly generated N-terminus acts as a tethered ligand to activate the receptor. Receptor-mediated cellular effects of thrombin can be mimicked by synthetic peptides, which correspond to the amino acid sequence of the newly formed N-terminus. The aim of the present study was to investigate vascular effects of thrombin and the thrombin receptor activating peptide (TRAP: SFLLRN) in vitro and in vivo in rats. In precontracted rat aortic rings, both thrombin (0.3, 1, 3 U/ml) and TRAP (1, 3, 10, 20, 40 microM) induced endothelium-dependent relaxant responses. In anaesthetized rats, the mean arterial blood pressure (MAP) was measured continuously in the carotid artery by a pressure transducer. Thrombin and TRAP were administered as intravenous bolus injection via the femoral vein. Thrombin at doses of 3-100 U/kg, as well as TRAP at doses of 0.1-0.6 mg/kg i.v., caused a reversible decrease in MAP. Administration of TRAP at doses of 0.3 and 0.6 mg/kg led to a triphasic response in most of the animals treated (50% and 75%, respectively), i.e. a short drop of MAP was followed by an increase and finally a longer lasting decrease in MAP. Pretreatment with the nitric oxide (NO)-synthase inhibitor N(G)-nitro-L-arginine-methylester (L-NAME) suppressed the dose-dependent vasodilator effects of thrombin. Heparin and hirudin also inhibited the hypotensive response to thrombin. The TRAP-induced triphasic reaction on MAP was not affected by the serotonin antagonists ketanserin and tropisetron, as well as the aminopeptidase inhibitor amastatin. Pretreatment with L-NAME led to an inhibition of hypotension induced by TRAP at 0.1 mg/kg, as well as of the initial transient fall in blood pressure at doses of 0.3 and 0.6 mg/kg. The studies suggest that the thrombin- and TRAP-induced vasodilation in vitro and in vivo is in part due to the release of endothelial NO. In the blood pressure response to TRAP, additional effects seem to be involved.

Animals↗
PubMed1976

Effects of source and concentration of thrombin, and divalent cations, on thrombin time of heparinized plasma.

The effects of the source and concentration of thrombin, and those of divalent cations, on the thrombin time (TT) of heparinized plasma were investigated. A correlation between TT and the heparin concentration was obtained only when the thrombin was of human origin and when it was reconstituted in divalent cation solutions. Relatively small variations in thrombin concentration resulted in marked differences in TT of heparinized plasma. Bovine thrombin gave a very prolonged TT of heparinized plasma compared with human thrombin, though the two thrombins gave identical TT's for non-heparinized control plasma. Divalent cation solution, in which thrombin was reconstituted, had a profound influence on TT of heparin plasma. When thrombin was reconstituted in 0.1 M MnCl2 solution, the TT of a plasma containing 0.5 unit heparin per ml. was the same as that of a plasma containing no heparin. The reliability of the thrombin time test as a means of monitoring heparin anticoagulation must be established by individual laboratories via extensive testing of clinical samples.

Animals↗
PubMed1986

Thrombin specificity with tripeptide chromogenic substrates: comparison of human and bovine thrombins with and without fibrinogen clotting activities.

To assess the thrombin specificity of tripeptide chromogenic substrates, we determined the Michaelis--Menten (Km), catalytic (kcat), and specificity (kcat/Km) constants for S-2238 (H-D-Phe-Pip-Arg-p-nitroanilide), Chromozym-TH (Tos-Gly-Pro-Arg-p-nitroanilide), and Spectrozyme-TH (H-D-hexahydrotyrosyl-Ala-Arg-p-nitroanilide) with high-purity thrombin preparations. Human and bovine alpha-thrombins, prepared by essentially the same procedure, were each greater than 95% in the form of the enzyme (alpha-thrombin) and had a specific fibrinogen-clotting activity greater than 2000 kilo-clotting units per gram of protein (kcu/g). In contrast, human gamma- and bovine beta-thrombins, made by controlled passage of alpha-thrombin through trypsin agarose, were greater than 98% of their respective forms and had fibrinogen-clotting activity less than 1 kcu/g. With the tripeptide chromogenic substrates these four thrombin forms at pH 7.8 and 23 degrees C had Km values of 1.6 to 16 mol/L, kcat values of 35 to 130 s-1, and kcat/Km ratios of 4.7 to 52 L X mol-1 X s-1. Although Km for an individual substrate was slightly higher for bovine than for human alpha-thrombins and although Km values for the nonclotting forms (human gamma- and bovine beta-thrombins) were higher than for clotting forms (alpha-thrombins), we found no major differences among the kinetic values for the three substrates.

Animals↗
PubMed1983

Fate of thrombin and thrombin-antithrombin-III complex adsorbed to a heparinized biomaterial: analysis of the enzyme-inhibitor complexes displaced by plasma.

Heparin covalently-linked to polyvinyl alcohol (PVA) is a biomaterial which is of potential value as a non-thrombogenic coating. 125I-labelled thrombin adsorbed to heparin-PVA beads was not dislodged by phosphate-buffered saline, pH 7.4, although radioactivity was progressively displaced from the adsorbent by fibrinogen-free human plasma. Analysis by gel filtration and affinity chromatography showed that the released radioactivity was distributed between (thrombin-antithrombin-III) complex (approx. 70%) and, probably, (thrombin-alpha-2-macroglobulin) complex (approx. 30%). Less efficient thrombin displacement was obtained by either bovine serum albumin (5% w/v) or antithrombin-III-free human plasma: in the latter case, the dislodged enzyme was presumably associated with alpha-2-macroglobulin. Purified alpha-2-macroglobulin did not displace thrombin from heparin-PVA. The quantity of thrombin displaced by an alpha-2-macroglobulin-free plasma fraction compared well with fibrinogen-free plasma: The eluted enzyme was largely associated with antithrombin-III. Purified antithrombin-III did not displace thrombin from heparin-PVA despite causing greater than 70% inactivation of the bound enzyme. Subsequent treatment with fibrinogen-free plasma dislodged (thrombin-antithrombin-III) at a similar rate to that of bound thrombin. We conclude that plasma contains a component(s) which displaces (thrombin-antithrombin-III) complex from immobilised heparin: presumably this leaves the heparin sites free for further use in enzyme inactivation.

Antithrombin III↗
PubMed1995

Feedback activation of factor XI by thrombin in plasma results in additional formation of thrombin that protects fibrin clots from fibrinolysis.

Recently, an alternative pathway for factor XI activation has been described in which factor XI is activated by thrombin. Patients with a factor XI deficiency bleed mostly from tissues with high local fibrinolytic activity. Therefore, the role of thrombin-mediated factor XI activation in both fibrin formation and fibrinolysis was studied in a plasma system. Clotting was induced by the addition of tissue factor or thrombin to recalcified plasma in the presence or absence of tissue-type plasminogen activator, after which clot formation and lysis were measured using turbidimetry. Thrombin-mediated activation of factor XI was found to take place in plasma under physiologic conditions in the absence of a dextran sulfate-like cofactor. At high tissue factor concentrations, no effect of factor XI was seen on the rate of fibrin formation. Decreasing amounts of tissue factor resulted in a gradually increasing contribution of factor XI to the rate of fibrin formation. In addition, thrombin-mediated factor XI activation resulted in an inhibition of tissue-type plasminogen activator-induced lysis of the clot. This inhibition occurred even at tissue factor concentrations at which no effect of factor XI was observed on fibrin formation. Trace amounts of activated factor XI (1.25 pmol/L, representing 0.01% activation) were capable of completely inhibiting fibrinolysis in our system. The inhibitory effect was found to be mediated by thrombin that is additionally generated in a factor XI-dependent manner via the intrinsic pathway and is capable of protecting the clot against lysis. We also observed that formation of additional thrombin continued after the clot had been formed. We conclude that thrombin-mediated factor XI activation can take place in plasma. The presence of factor XI during coagulation results in the formation of additional thrombin within the clot capable of protecting this clot from fibrinolytic attack. The large amounts of thrombin that are formed by the intrinsic pathway via factor XI may play an important role in the procoagulant and thrombogenic state of clots and may therefore have important clinical and therapeutic implications.

Blood Coagulation↗
PubMed1994

Effects of the synthetic thrombin inhibitor argatroban on fibrin- or clot-incorporated thrombin: comparison with heparin and recombinant Hirudin.

The inhibitory effects of argatroban on clot- or fibrin-bound human thrombin were studied using the thrombin-specific chromogenic substrate S2238 (200 microM). These effects were compared to those of recombinant hirudin (rHV2 Lys 47) and the heparin/antithrombin III complex. Argatroban concentration-dependently inhibited the cleavage of S2238 by a thrombin solution, which had been titrated to give the same change in OD405 nm as fibrin-bound thrombin, with an IC50 of 1.1 microM with 90% inhibition at 8 microM. rHV2 Lys 47 and heparin had IC50 values of 1.2 nM and 0.003 U/ml respectively under these conditions. However, when the compounds were tested against fibrin-bound thrombin, argatroban had an IC50 of 2.8 microM with 65% inhibition at 8 microM, whereas rHV2 Lys 47 had an IC50 of 23 nM (with only 56% inhibition at 200 nM), and heparin had an IC50 of 0.5 +/- 0.38 U/ml (with only 58% inhibition at 5 U/ml); i. e. the two compounds were 19 and 168 times less active against fibrin-bound thrombin than against thrombin in solution. The differences between the inhibitory effects of the compounds against thrombin bound to a plasma clot were even more striking in that the IC50 of argatroban was increased from 1.1 (vs. thrombin in solution) to 2.7 microM, while, although rHV2 Lys 47 and heparin had IC50 values of 2.8 nM and 0.004 U/ml against thrombin in solution, they had little (32% inhibition by 4 microM rHV2 Lys 47) or no effect (even at 5.0 U/ml heparin) against the amidolytic activity of a plasma clot.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombins↗
PubMed1997

Thrombin receptor-activating peptide releases arachidonic acid from human platelets: a comparison with thrombin and trypsin.

The serine proteases thrombin and trypsin are both powerful platelet agonists that act by cleaving the terminal portion of the thrombin receptor and allowing the new C-terminal to auto-stimulate the receptor. Synthetic peptides, termed thrombin receptor-activating peptides (TRAPs), have been shown to mimic many of the effects of thrombin. Here we have compared the effects of inhibitors on platelet aggregation and [14C]-arachidonic acid release in response to thrombin, trypsin and TRAP. Pretreatment of human platelets with BW755C (80 microM), which inhibits both cyclooxygenase and lipoxygenase, blocked trypsin (15-20 nM)- or TRAP (4-6 microM)-induced aggregation, but not thrombin (0.06-0.1 U/ml)-induced aggregation. The protease inhibitor leupeptin (10 micrograms/ml) abolished trypsin-induced aggregation and returned [14C]-arachidonic acid release from [14C]-arachidonic acid-prelabeled platelets to control levels. In contrast, leupeptin did not affect either aggregation or [14C]-arachidonic acid release in platelets stimulated by TRAP. Thrombin-induced aggregation and [14C]-arachidonic acid release were only partially inhibited by leupeptin. These data are consistent with the activation of platelets by both trypsin and TRAP occurring via the proteolytic receptor, whereas thrombin-induced platelet activation appears to occur by a dual mechanism of action. One component of thrombin-induced platelet activation is by a proteolytic action on the moderate-affinity receptor. This effect is sensitive to inhibition by leupeptin and is mimicked by trypsin and TRAP. The other component of thrombin is nonproteolytic and may occur by an action at a high-affinity receptor such as glycoprotein lb.

Arachidonic Acid↗
PubMed1997

Inhibition of thrombin-mediated cellular effects by triabin, a highly potent anion-binding exosite thrombin inhibitor.

Triabin, a 17 kDa protein from the saliva of the assassin bug Triatoma pallidipennis is a potent thrombin inhibitor interfering with the anion-binding exosite of the enzyme. The recombinant protein, produced by the baculovirus/insect cell system, was used to study the inhibitory effect on thrombin-mediated cellular responses. The thrombin (1 nM)-stimulated aggregation of washed human platelets and the rise in cytoplasmic calcium in platelets were inhibited by triabin at nanomolar concentrations. In contrast, the rise in calcium induced by the thrombin receptor-activating peptide (10 microM) was not suppressed by triabin. In isolated porcine pulmonary arteries, preconstricted with PGF 2 alpha thrombin (2 nM) elicited an endothelium-dependent relaxation which was inhibited by triabin in the same concentration range as found for the inhibition of platelet aggregation. Higher concentrations of triabin were required to diminish the contractile response of endotheliumdenuded pulmonary vessels to thrombin (10 nM). In cultured bovine coronary smooth muscle cells, the mitogenic activity of thrombin (3 nM), measured by [3H]thymidine incorporation, was also suppressed by triabin. In all these assays, the inhibitory effect of triabin was dependent on the thrombin concentration used. These studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of thrombin-mediated cellular effects.

Animals↗
PubMed1998

Platelet aggregation induced in vitro by rabbit plasma clot-associated thrombin, and its inhibition by thrombin inhibitors.

The activation of rabbit platelets by rabbit plasma clots, and the inhibition of clot-associated thrombin by heparin:antithrombin III, recombinant hirudin (rHV2Lys47) and argatroban, a low molecular weight thrombin inhibitor, was studied. Plasma clots caused the aggregation of platelets suspended in a plasma-free medium as assessed by single platelet counting, and by scanning electron microscopy (platelet aggregates present on the clot surface). Platelet aggregation, induced by clot-associated thrombin, was inhibited by argatroban with an IC50) of 14 +/- 3 nM compared to an IC50) of 12 +/- 2 nM when human thrombin in solution titrated to give the same decrease in the platelet count as plasma clots was used. rHV2Lys47 also inhibited aggregation induced by clot-associated thrombin with an IC50 of 1.6 +/- 0.4 nM compared to 1.6 +/- 0.5 nM with thrombin in solution. Heparin was less active against clot-associated thrombin (IC50) = 69 +/- 9 mU/ml) than against thrombin in solution (IC50 = 15 +/- 5 mU/ml). This study shows that plasma clot-bound thrombin activates platelets and that direct-acting thrombin inhibitors such as argatroban and rHV2Lys47 are more effective than heparin:antithrombin III in inhibiting this phenomenon.

Animals↗
PubMed1997

Differences between platelet phosphoinositide metabolism stimulated by thrombin or SFLLRN are not accounted for by interaction of thrombin with glycoprotein Ib.

The formation of inositol phosphates was compared in aspirin-treated, washed human platelets suspended in Tyrode's-albumin solution containing 2 mM calcium and stimulated with SFLLRN (thrombin receptor-activating peptide) or thrombin. SFLLRN (20 microM) and thrombin (1 U/ml) resulted in maximal irreversible aggregation and 80-90% secretion of dense granule contents. SFLLRN (50-100 microM) caused larger increases at 10 sec than 20 microM SFLLRN in the formation of inositol trisphosphate (IP3, measured as [3H]inositol label). These increases were not significantly less than those caused by thrombin (1 unit/ml). However, whereas the labeling of IP3 increased from 10-60 sec with thrombin, with SFLLRN it was much less at 60 sec than that at 10 sec. The decrease was not due to degradation of SFLLRN by ectopeptidases, since it was not prevented by amastatin, an inhibitor of ectopeptidases. Degradation of glycoprotein Ib (GPIb) with an O-sialoglycoprotein endopeptidase did not affect the thrombin-stimulated labeling of inositol phosphates, indicating that binding to GPIb is not involved in the sustained thrombin-induced formation of inositol phosphates. The finding that the thrombin-stimulated formation of IP3 was not dependent on Ca2+ in the medium (EGTA added) indicates that the transient SFLLRN-induced formation of IP3 is not due to failure to cause Ca2+ influx. The finding that formation of IP3 was transient in SFLLRN-stimulated platelets, whereas platelet aggregation and secretion were maximal, indicates that the sustained activation of phospholipase C caused by thrombin may have roles related to later processes in which platelets participate.

Blood Platelets↗
PubMed1993

Long-term effects of thrombin require sustained activation of the functional thrombin receptor.

Thrombin is a potent activator of human glomerular epithelial cells (HGEC). Here we compare short-term and long-term effects of thrombin and thrombin receptor agonist peptide (TRAP) which selectively activates the functional thrombin receptor. TRAP, as thrombin, increases intracellular free Ca2+ concentration and acts synergistically with growth factors possessing tyrosine kinase receptors on DNA synthesis. Thrombin induces synthesis of proteins of the fibrinolytic system and cell proliferation if it is present for at least 8 h. TRAP alone does not stimulate protein synthesis and is not mitogenic. However, in the presence of the aminopeptidase inhibitor amastatin all long-term effects of thrombin can be fully mimicked by TRAP. In conclusion, different effects of thrombin and TRAP may be related to the degradation of TRAP by cellular ectoenzymes. The recently cloned thrombin receptor accounts for early intracellular signals and long-term cellular effects that require sustained activation of this receptor.

Anti-Bacterial Agents↗
PubMed1992

Actions of thrombin and thrombin receptor peptide analogues in gastric and aortic smooth muscle: development of bioassays for structure-activity studies.

We have examined the biological activities of thrombin and the thrombin-receptor-related polypeptides, S42FLLRNPNDKYEPF55(TRP42-55), S42FLLRNPND50(TRP42-50), and A42FLLRNPND50(A42-TRP42-50) as well as an arginine-containing basic peptide beginning with the SF motif (SFRGHITR), in rat aortic (RA) rings and in a gastric guinea pig longitudinal (LM) smooth muscle preparation. In the RA preparation, thrombin, as well as the three receptor-related peptides caused a relaxation in tissue that was precontracted with noradrenaline; the basic peptide, SFRGHITR, was inactive either as an agonist or as an antagonist to TRP42-55. In the LM bioassay, which unlike the RA preparation did not persistently desensitize in response to thrombin, all three receptor-related peptides, like thrombin, caused a prompt phasic reproducible contraction. The basic peptide, SFRGHITR, was inactive. In the LM assay, TRP42-55, TRP42-50 and A42-TRP42-55 all caused comparable contractile responses. We conclude that the gastric LM smooth muscle possesses a thrombin receptor and provides a convenient and reliable assay for the activities of thrombin receptor-related peptides. Our data also demonstrated that neither the C-terminal hirudin-related pentapeptide nor the N-terminal serine hydroxyl group are required for the biological activity of the thrombin receptor-derived peptide previously described (TRP42-55). Based on our findings we suggest that only a small portion of the N-terminal sequence of TRP42-55 may be required for thrombin-like biological activity.

Amino Acid Sequence↗
PubMed1997

Effect of activated protein C on thrombin generation and on the thrombin potential in plasma of normal and APC-resistant individuals.

In this paper we describe the effect of activated protein C (APC) on thrombin generation initiated in platelet-poor plasma via the extrinsic or the intrinsic pathway. Thrombin was determined with a specific chromogenic substrate and quantitated by calculating the time integral of the thrombin generation curve, i.e. the endogenous thrombin potential (ETP). Addition of APC to normal plasma after both extrinsic and intrinsic initiation of coagulation resulted in a dose-dependent inhibition of thrombin generation as reflected by the decrease in ETP. Data obtained in intrinsically triggered plasma of normal individuals were subject to large variation. Therefore, the effect of APC on thrombin generation in APC-resistant plasmas was only studied in extrinsically stimulated reaction systems. APC had much less effect on the ETP of plasma from individuals that were heterozygous or homozygous for the mutation Arg506-->Gln506 in factor V (APC resistance). There appears to be a linear relationship between the ETP and the amount of alpha 2-macro-globulin-thrombin complex (alpha 2 M-IIa) that accumulates in plasma during thrombin formation. Since the alpha 2M-IIa complex possesses amidolytic activity, we measured the effect of APC on thrombin generation via the so-called normalized APC sensitivity ratio (APC-sr). The latter was defined as the ratio of the end levels of amidolytic activity of the alpha 2M-IIa complex determined in the presence and absence of 50 nM APC (alpha 2M-IIa + APC/(alpha 2M-IIa - APC) divided by the ratio of a normal plasma pool. Significant differences (P < 0.001) were observed between APC-sr of plasmas from normal individuals (APC-sr: 0.5-1.9, n = 25) and of plasmas from individuals that were heterozygous (APC-sr: 2.1-6.7, n = 17) or homozygous APC resistant (APC-sr: 3.9-5.9, n = 5). There was no overlap between APC-sr of normal plasmas and plasma from individuals, bearing the factor. V mutation. Abnormal APC-sr in certain plasmas (pregnancy, use of oral contraceptives, anticoagulant therapy, protein S deficiency or lupus anticoagulant) were corrected by performing the assay on a plasma sample that was diluted 10-fold in factor V-deficient plasma. Our data show that measurement of the effect of APC on the ETP yields valuable information about the (pro)thrombotic status of plasma (e.g. APC resistance, pregnancy, use of oral contraceptives).

Drug Resistance↗
PubMed1993

Isoproterenol antagonizes endothelial permeability induced by thrombin and thrombin receptor peptide.

We determined whether 1) amino-terminal peptides of the thrombin receptor increase endothelial permeability to a comparable extent as alpha-thrombin does, 2) isoproterenol attenuates the thrombin-induced increase in endothelial permeability by an antagonistic action to that of thrombin or by lowering baseline permeability, and 3) isoproterenol decreases permeability via stimulation of the beta 2-adrenergic receptor. Permeability across monolayers of bovine pulmonary artery endothelial cells (CCL 209) was assessed by the clearance of 125I-labeled albumin. Thrombin receptor peptides increased permeability at 1 microM but required a dose of between 10 and 100 microM to equal the permeability response of 1 microM alpha-thrombin. Dose-response experiments demonstrated that isoproterenol antagonized the action of alpha-thrombin and a thrombin receptor peptide on endothelial permeability and that it lowered baseline permeability. This permeability-decreasing action of isoproterenol occurred via stimulation of the beta 2-adrenergic receptor. Terbutaline, a partial beta 2-agonist, prevented the thrombin-induced permeability, but dobutamine, a partial beta 1-agonist, did not. The active stereoisomer of terbutaline and the racemic form mimicked the action of isoproterenol, but the inactive stereoisomer had no effect. ICI-118,551, a specific beta 2-receptor antagonist, prevented the permeability-decreasing action of isoproterenol, whereas ICI-89,406, a specific beta 1-receptor antagonist, did not. Competitive binding studies of 125I-pindolol with ICI-118,551 or ICI-89,406 demonstrated the presence of beta-adrenergic receptors, predominantly beta 2-receptors, on cell membrane homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗
PubMed1994

Differences in intracellular calcium signaling after activation of the thrombin receptor by thrombin and agonist peptide in osteoblast-like cells.

Thrombin and the thrombin receptor agonist peptide (TRAP) caused a rise in intracellular calcium concentration ([Ca2+]i) in the human osteoblast-like cell line Saos-2. Striking differences in the [Ca2+]i signals elicited by these agonists were revealed. In cell populations, thrombin induced a transient increase in [Ca2+]i while TRAP caused a biphasic [Ca2+]i response consisting of an initial peak and a sustained plateau phase. In individual cells, thrombin mainly caused a single [Ca2+]i transient while TRAP induced repetitive [Ca2+]i spikes. Neither tyrosine phosphorylation, cAMP-dependent phosphorylation, nor pertussis toxin-sensitive G proteins appeared to be involved in thrombin receptor [Ca2+]i signaling in this cell line. However, the sustained [Ca2+]i response caused by TRAP was converted into a transient, thrombin-like response by pretreatment with serine/threonine phosphatase inhibitors. Pretreatment with the phorbol ester phorbol 12-myristate 13-acetate (PMA) abrogated thrombin receptor [Ca2+]i signaling, and TRAP-induced Ca2+ entry was inhibited by the acute treatment with PMA. In contrast, Ca2+ entry stimulated by thapsigargin was not sensitive to agents affecting serine/threonine phosphorylation. The observation that thrombin and TRAP, despite being agonists for a common receptor, induce dissimilar [Ca2+]i responses indicates that binding of TRAP alone is insufficient to fully regulate the thrombin receptor in Saos-2 cells.

Amino Acid Sequence↗