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PubMed2006

Specific determination of plasmatic thrombin activity.

Thrombin is the key enzyme of coagulation. Its activity can be determined via fibrinogen Ø fibrin conversion or via cleavage of a chromogenic substrate. The latter method is easier than the first one, but in plasma it is hampered due to unspecific cleavage of the chromogenic substrate by thrombin-like enzymes of hemostasis, especially those of the contact phase. The concentration of the thrombin substrate (HD-CHG-Ala-Arg-pNA) was optimized, using final substrate concentrations of 0 to 5 mM, a final arginine concentration of 1.13 M, and samples of 10 mIU/mL purified thrombin in 7% human albumin or pooled normal citrated plasma without and with EDTA. Twenty microliters pooled normal citrated plasma (frozen/thawed) or factor II-deficient plasma (lyophilized) were incubated with 10 microL 0% to 0.5% Thromborel S (100% = 162 ng/mL tissue factor [TF]) in 6% BSA or with 10 microL 0% (physiol. NaCl) to 50% Pathromtin SL and with 20 microL 25 mM CaCl(2). After 0 to 22 minutes (37 degrees C), 20 microL 1.7 M arginine, pH 8.7 were added. Fifteen microliters 0.9 mM HD-CHGAla-Arg-pNA in 2.3 M arginine, pH 8.6, were added and the increase in absorbance (deltaA) at 405 nm was determined. Thrombin activity was standardized against the (3)A measured for 1 IU/mL thrombin in 7% human albumin (8.8 mA/min RT). The optimal final chromogenic substrate concentration to detect thrombin in this assay system is less than 0.6 mM. Higher substrate concentrations in a plasma milieu result in unspecific cleavage of the substrate. Using final concentrations of chromogenic substrate less than 0.4 mM (the approximate Km- value for thrombin) and final concentrations of arginine greater than 800 mM, in factor II-depleted plasma, when activated either by TF or by the contact phase, there is no significant thrombin generation. The circulating thrombin activity measured in EDTA plasma of 39 healthy donors is 100 +/- 20% of norm (mean value +/- 1 SD; 100% = 5.5 mIU/mL thrombin). This chromogenic assay detects thrombin activity independent of clotting seconds or fibrin mediated turbidity increases. This technique allows to standardize the thrombin activity generated in any biologic system in international thrombin units.

Chromogenic Compounds↗
PubMed2001

Development and current applications of thrombin-specific inhibitors.

Thrombin-specific inhibitors directly diminish thrombin-induced coagulation and cellular activities without the side effects of heparin. Hirudin is the most potent natural thrombin-specific inhibitor. Recombinant hirudins (such as desirudin) have been shown to be effective in the treatment of heparin-induced thrombocytopenia (HIT) and in the prevention of thrombotic complications after hip or knee surgery. The application of recombinant hirudin has been limited mainly by hemorrhagic complications. Synthetic thrombin-specific inhibitors, including oligopeptides, tripeptides and non-peptide low molecular weight (LMW) thrombin inhibitors, have been designed according to their interactions with the active sites of thrombin. Bivalirudin (an anti-thrombin oligopeptide) has been approved for preventing thrombosis in unstable angina patients following angioplasty in adjunct to aspirin. Argotroban (a tripeptide thrombin inhibitor) has been used for the treatment of HIT, peripheral and cerebral thrombotic diseases. The benefit of using thrombin-specific inhibitors alone in acute myocardial infarction or unstable angina remains uncertain. A number of LMW thrombin-specific inhibitors have been developed. Some of them can be administrated orally, and cause less increase in bleeding time than other thrombin inhibitors. The efficacy, safety, stability and oral bioavailability of the thrombin inhibitors may be considerably improved through structural optimization. Most of the LMW thrombin inhibitors are currently being tested in animal models or at early stages of clinical trials. In this review, we will present an overview of recent advances in thrombin-specific inhibitors.

Animals↗
PubMed1985

Uptake and inactivation of thrombin on rabbit aortic endothelium studied with two different substrates.

The endothelium is an important compartment for uptake and inhibition of thrombin. The amount of enzymatically active bound thrombin can be detected with both small synthetic substrates and with aid of fibrinogen as substrate. The present study was designed to investigate the relation between endothelially bound thrombin with amidolytic activity towards a synthetic substrate (S-2238) and thrombin capable of converting fibrinogen by measuring generation of fibrinopeptide A (FPA). The luminal surfaces of rabbit aortae (2 cm2) were exposed in vitro to thrombin (0.625-5.0 NIH units/ml). Thrombin disappeared from the solution and a certain fraction was recovered on the surface. There was a linear relationship between the amount of thrombin on the surface and the concentration of thrombin in the incubation mixture. Approximately one third of the thrombin measured with S-2238 was also able to cleave fibrinogen. After incubation with defibrinogenated plasma almost total inhibition of fibrinogen splitting activity occurred within 30 sec. The inhibition of the amidolytic activity was less complete. When endothelially bound thrombin was exposed to plasma much less FPA was generated than in a fibrinogen solution. A minor fraction of endothelially bound thrombin was inhibited also upon incubation with Tyrode without recovery of any enzymatic activity in the solution. The results indicate that a fraction of thrombin bound to the endothelium has retained enzymatic activity and that a fraction of the enzymatically active thrombin is capable of converting fibrinogen. Inhibition of thrombin enzymatic activity occurs rapidly upon exposure to plasma. The endothelium itself has a minor inhibitory effect also in the absence of plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1980

Heparin inhibits thrombin binding to rabbit thoracic aorta endothelium.

Thrombin binding to freshly prepared sections of rabbit thoracic aorta was studied. After the sections had been exposed to a range of concentrations (0.1 to 3.8 IU/ml) of 125I-labeled thrombin for various periods of time at 37 degrees C, endothelial Häutchen preparations were obtained, and their radioactivity content was determined. Scatchard plot analysis of the data indicated that approximately 5.8 X 10(5) molecules of thrombin associated with each endothelial cell, with a KD of 2.6 X 10(08)M. By incubation with an excess of unlabeled thrombin, 50% of bound 125I-labeled thrombin was displaced from the endothelium in 7.3 min. Exposure of the endothelial surface to heparin (1 to 10 USP U/ml) did not significantly affect subsequent thrombin binding. However, incubation of the aorta in a thrombin solution containing 1 to 10 USP U/ml heparin did reduce enzyme binding to the endothelium by up to 60%. Similarly, the presence of heparin inhibited thrombin binding to the thoracic aorta of exsanguinated rabbits in situ. Endothelium, to which 125I-labeled thrombin was bound, lost 50% to 70% of the bound enzyme when suspended in a solution containing heparin (10 USP U/ml) and compared to the control incubated without heparin. These observations are consistent with the proposal that a major portion of endothelium-bound thrombin may be associated with pericellular heparan sulfate; heparin competes for thrombin with the heparan sulfate sites, and because of its higher affinity for thrombin, heparin displaces bound thrombin from, or inhibits binding of free thrombin by, the endothelium.

Animals↗
PubMed1994

Heparin cofactor II is regulated allosterically and not primarily by template effects. Studies with mutant thrombins and glycosaminoglycans.

Besides its critical role in hemostasis, the serine protease thrombin also participates in wound healing, inflammation, and atherosclerosis. Thrombin is inhibited by the serpins antithrombin and heparin cofactor II (HCiI) in reactions that are accelerated markedly by specific glycosaminoglycans. Following vascular injury, thrombin must be inhibited at both intravascular and extravascular sites that impose different constraints on the recognition of thrombin by these inhibitors. The present study examines the role of anion-binding exosite II of thrombin in the interaction with glycosaminoglycans and HCII. Acceleration of thrombin inhibition by serpins in the presence of glycosaminoglycans is proposed to occur by a template mechanism, in which inhibitor and protease bind simultaneously to the same glycosaminoglycan chain, facilitating their interaction. According to the template model, disruption of protease binding to glycosaminoglycan should significantly reduce acceleration of the inhibition. Specific mutations in exosite II (R89E, R245E, K248E, and K252E) disrupted thrombin binding to both dermatan sulfate and heparin, indicating that both glycosaminoglycans bind to a common site in exosite II. The same mutations markedly decreased the rate constant for thrombin inhibition by antithrombin-heparin (up to 100-fold) but had little effect on the rate constant for thrombin inhibition by HCII-heparin (7-fold maximal reduction) and no effect on the rate constant for thrombin inhibition by HCII-dermatan sulfate. These results are incompatible with a template model for thrombin inhibition by HCII and dermatan sulfate. In the presence of glycosaminoglycan, HCII and antithrombin interact with opposing thrombin exosites and use distinct mechanisms of glycosaminoglycan catalysis. Antithrombin employs a template mechanism that requires heparin to interact with thrombin exosite II, whereas HCII employs an allosteric mechanism that requires thrombin exosite I but is largely independent of exosite II. These findings have potential implications for glycosaminoglycan therapy and for the respective physiologic roles of HCII and antithrombin.

Allosteric Regulation↗
PubMed1995

Constitutive expression and modulation of the functional thrombin receptor in the human kidney.

Thrombin exerts procoagulant effects and has also many cellular effects mediated by cell surface receptors. A functional thrombin receptor from human platelets has been cloned and sequenced. In the present study, by reverse transcription and polymerase chain reaction, using specific primers designed from the thrombin receptor cDNA sequence, we show that the mRNA encoding for this receptor can be amplified from freshly isolated human glomeruli obtained by microdissection of normal kidney cortex. By immunohistochemistry using a specific monoclonal antibody, ATAP2, directed against the extracellular N-terminus of this receptor, we find that this functional thrombin receptor is constitutively expressed in the normal human kidney. The three glomerular cell types, endothelial, mesangial, and epithelial cells, were positively stained, as were the endothelial cells of renal arteries, arterioles, venules, and peritubular capillaries. Occasionally, interstitial cells and smooth muscle cells in the media of renal arteries were also stained. Proximal and distal tubular cells were not stained. By in situ hybridization, using a digoxigenin-labeled cDNA probe specific for thrombin receptor, the thrombin receptor mRNA was found to have the same distribution as the thrombin receptor protein detected by immunohistochemistry. A lighter staining of glomerular endocapillary cells was observed in cases of thrombotic microangiopathy and extracapillary glomerulonephritis, two renal diseases associated with in situ thrombin generation and fibrin formation. In one case of thrombotic microangiopathy, we observed an increase in thrombin receptor mRNA. This suggests that thrombin receptor protein is not always correlated with thrombin receptor mRNA level. Internalization and degradation of thrombin receptor protein have been demonstrated in vitro and could also occur after activation in vivo. This is the first demonstration of the constitutive expression of the functional thrombin receptor in the human kidney. These results suggest that thrombin may exert glomerular and vascular effects within the kidney in normal and in pathological conditions.

Amino Acid Sequence↗
PubMed1976

In vitro and in vivo functions of thrombin-treated platelets.

Thrombin-induced platelet aggregation has been generally believed to be irreversible. However, thrombin-induced aggregation of washed platelets is reversible if fibrin formation is prevented or the fibrin which binds the platelets together is removed from the platelet aggregates. After treatment with high concentrations of thrombin (0.5 units/ml) single platelets can be recovered that have lost practically all of their releasable serotonin and adenine nucleotides. These platelets are able to aggregate upon addition of low concentrations of ADP in the presence of fibrinogen. They aggregate in response to the ionophore A23, 187 in the absence of added fibrinogen, whereas sodium arachidonate-induced aggregation requires added fibrinogen. Thrombin-treated platelets change their shape in response to collagen in the absence of fibrinogen, and will aggregate upon the addition of collagen providing fibrinogen is present. This response to collagen can be blocked with aspirin but not with a mixture of creatine phosphate/creatine phosphokinase. Upon a second exposure to thrombin, thrombin-pretreated platelets do not change their shape and do not undergo aggregation. Thrombin-pretreated platelets will not retract a thrombin-induced fibrin clot unless ADP, sodium arachidonate, the ionophore A23, 187 or collagen are added together with thrombin. The ability of thrombin-treated platelets to adhere to the exposed subendothelial surface of the rabbit aorta is reduced, compared with untreated control platelets. The thrombin-treated platelets shorten the bleeding time of thrombocytopenic rabbits. However, the are not as effective in shortening the bleeding time as normal control platelets. When injected into rabbits with a normal platelet count, the thrombin-treated platelets that circulate after infusion survive for the same length of time as untreated control platelets. These findings indicate that thrombin-induced platelet aggregation with extensive release of granule constituents is not irreversible and that thrombin treatment does not cause irreversible damage of all platelets that would lead to their immediate elimination from the circulation. Furthermore, these platelets can still be haemostatically effective. It is conceivable that platelets that have lost their amine storage granule contents during a release reaction in vivo, such as may occur in certain cases of intravascular coagulation and repeated episodes of thrombosis, may be found in the circulation of man.

Adenosine Diphosphate↗
PubMed1997

Thrombin interaction with platelet GpIB: role of the heparin binding domain.

The platelet membrane glycoprotein Ib (GpIb) has a high affinity binding site for alpha-thrombin whose occupancy is thought to positively modulate the thrombin-induced platelet activation. In this study, aimed at further characterizing the thrombin-GpIb interaction, two thrombin anion exosites referred to as "heparin binding site" (HBS) and "fibrinogen recognition site" (FRS) were investigated as the possible domains involved in GpIb binding. The role of thrombin HBS was explored by performing binding measurements of 125I-alpha-thrombin to purified glycocalicin (GC), the extracytoplasmic portion of GpIb, in the presence of heparin as well as after chemical modifications of the thrombin heparin binding site (thrombin-HBS phosphopyridoxylation). These studies showed that a) thrombin binding to GC could be competitively inhibited by heparin and b) the equilibrium association constant for thrombin-GC interaction was reduced up to ten-fold by chemical modifications at the HBS. On the other hand, the role of FRS in the thrombin-GC interaction could be excluded by other experiments showing that GC in solution could not influence the interaction of alpha-thrombin with two substrates which bind to both the catalytic site and the fibrinogen recognition site: 1) the thrombin receptor peptide 38-60 (TR, L38-E60) and 2) the A alpha-chain of fibrinogen. Altogether these results demonstrated that GC interaction with thrombin involves the enzyme heparin binding site, whereas the fibrinogen recognition site does not play a significant role.

Binding Sites↗
PubMed1997

Thrombin induces apoptosis in cultured neurons and astrocytes via a pathway requiring tyrosine kinase and RhoA activities.

Thrombin activity is a factor in acute CNS trauma and may contribute to such chronic neurodegenerative diseases as Alzheimer's disease. Thrombin is a multifunctional serine protease that catalyses the final steps in blood coagulation. However, increasing evidence indicates that thrombin also elicits a variety of cellular and inflammatory responses, including responses from neural cells. Most recently, high concentrations of thrombin were shown to cause cell death in both astrocyte and hippocampal neuron cultures. The purpose of this study was to determine the mechanisms underlying thrombin-induced cell death. Our data show that thrombin appears to cause apoptosis as evidenced by cleavage of DNA into oligonucleosomal-sized fragments, fragmentation of nuclei, and prevention of death by inhibition of protein synthesis. Synthetic peptides that directly activate the thrombin receptor also induced apoptosis, indicating that thrombin-induced cell death occurred via activation of the thrombin receptor. The signal transduction cascade involves tyrosine and serine/threonine kinases and an intact actin cytoskeleton. Additional study revealed the involvement of the small GTP-binding protein RhoA. Thrombin induced RhoA activity in both astrocytes and hippocampal neurons, and inhibition of RhoA activity with exoenzyme C3 attenuated cell death, indicating that thrombin activation of RhoA was necessary for thrombin-induced cell death. Tyrosine kinase inhibitors blocked thrombin induction of RhoA, indicating that tyrosine kinase activity was required upstream of RhoA. These data suggest a sequential linkage of cellular events from which we propose a model for the second messenger cascade induced by thrombin in neural cells that can lead to apoptosis.

Animals↗
PubMed1998

Characteristics of the interaction between thrombin exosite 1 and the sequence 269-287 [correction of 269-297] of platelet glycoprotein Ibalpha.

The interaction between GPIb and thrombin promotes platelet activation elicited via the hydrolysis of the thrombin receptor and involves structures located on the segment 238-290 within the N-terminal domain of GPIbalpha and the positively charged exosite 1 on thrombin. We have investigated the ability of peptides derived from the 269-287 sequence of GPIbalpha to interact with thrombin. Three peptides were synthesized, including Ibalpha 269-287 and two scrambled peptides R1 and R2 which are comparable to Ibalpha 269-287 with regards to their content and distribution of anionic residues. However, R2 differs from both Ibalpha 269-287 and R1 by the shifting of one proline from a central position to the N-terminus. By chemical cross-linking, we observed the formation of a complex between 125I-Ibalpha 269-287 and alpha-thrombin that was inhibited by hirudin, the C-terminal peptide of hirudin, sodium pyrophosphate but not by heparin. The complex did not form when gamma-thrombin was substituted for alpha-thrombin. Ibalpha 269-287 produced only slight changes in thrombin amidolytic activity and inhibited thrombin binding to fibrin. R1 and R2 also formed complexes with alpha-thrombin, modified slightly its catalytic activity and inhibited its binding to fibrin. Peptides Ibalpha 269-287 and R1 inhibited platelet aggregation and secretion induced by low thrombin concentrations whereas R2 was without effect. Our results indicate that Ibalpha 269-287 interacts with thrombin exosite 1 via mainly electrostatic interactions, which explains why the scrambled peptides also interact with exosite 1. Nevertheless, the lack of effect of R2 on thrombin-induced platelet activation suggests that proline 280 is important for thrombin interaction with GPIb.

Amino Acid Sequence↗
PubMed1998

On the mechanism of thrombin-induced angiogenesis: inhibition of attachment of endothelial cells on basement membrane components.

Human umbilical vein endothelial cells (HUVECs) placed on plastic plates coated with collagen type IV or laminin adhered within 60 min to an extent of about 32 and 39%, respectively. Brief exposure of HUVECs to thrombin caused a marked dose-dependent inhibition of adhesion. Thrombin at 1 IU/ml caused 50% inhibition even after 5 min of exposure of HUVECs. This effect was reversible since reincubation of thrombin-treated HUVECs with fresh growth medium for 15 min restored their ability for attachment. This short-term inhibitory effect of thrombin on the adhesion of HUVECs to extracellular matrix components was specific and depended on the activation of thrombin receptor. Hirudin abolished this effect of thrombin. Similarly, the proteolytically inactive PPACK-thrombin had no effect, but when used in combination with thrombin prevents the inhibitory effect of thrombin. In addition, the thrombin receptor agonist peptide (TRAP) mimicked the effect of thrombin on HUVEC adhesion. The transduction mechanism involved in this action of thrombin seems to be via cAMP, since forskolin or the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine restored the ability of HUVECs that had been exposed to thrombin to adhere. This novel cellular action of thrombin on endothelial cells may represent an important early event in activation of the normally quiescent endothelial cells and initiation of the angiogenic cascade.

Journal Article↗
PubMed1982

Receptor-bound thrombin is not internalized through coated pits in mouse embryo cells.

The localization of thrombin receptors on mouse embryo (ME) cells was examined using electron microscope (EM) immunocytological techniques. ME cells were fixed with formaldehyde, prior to thrombin binding, and thrombin visualized on cell surfaces using affinity-purified antithrombin rabbit antibody and colloidal gold labeled anti-rabbit IgG. Colloidal gold particles were found in clusters on the surface of cells incubated with thrombin. There were approximately seven particles per cluster observed in thin sections with cluster diameters ranging from 70 to 200 nm. These clusters were not observed on cells incubated without thrombin. The total number of particles present on cells incubated with and without thrombin indicate that the colloidal gold labeling is approximately 98% specific for thrombin. Only four colloidal gold particles out of approximately 1,200 were associated with coated pits. Thus the thrombin receptor clusters do not appear to associate with coated membrane regions. To determine whether receptor-bound thrombin was internalized by receptor-mediated endocytosis, ME cells were incubated with 125I-thrombin and examined using EM autoradiography and the trypsin sensitivity of 125I-thrombin which was associated with the cells. In two types of experiments, where thrombin was incubated with cells at 4 degrees C and the temperature increased to 37 degrees C and where initial incubation was at 37 degrees C, the receptor-directed specific internalization proceeded at approximately the same rate as nonspecific internalization. These studies indicate that thrombin that binds to its receptors on ME cells is not rapidly internalized by receptor-mediated endocytosis.

Animals↗
PubMed1993

Normal thrombin binding leads to greater fibrinogen binding and increased platelet aggregation in spontaneously hypertensive rats.

This study was conducted to determine the mechanisms of increased platelet reactivity to thrombin in hypertension. Thrombin induced significantly greater platelet aggregation in spontaneously hypertensive (SHR) than in normotensive (Wistar Kyoto, WKY) rats. Fibrinogen and thrombin binding to platelets was determined using [125I]-fibrinogen and [125I]-thrombin respectively. Increased platelet aggregation in SHR correlated with thrombin-induced greater binding of fibrinogen to SHR than to WKY platelets. However, the number of thrombin receptors (binding sites/platelet) in WKY (19,500 +/- 3,000) and SHR (23,100 +/- 3,000) as well as thrombin dissociation constants were statistically similar in WKY (1.17 +/- 0.2 microM) and SHR (1.62 +/- 0.27 microM) platelets. Fura 2/AM, a fluorescent calcium indicator, loaded platelets were used to quantify the platelet ionized calcium ([Ca2+]i). The [Ca2+]i in unstimulated SHR and WKY platelets was essentially the same. In a calcium poor medium, thrombin-induced a 35% greater increase in [Ca2+]i in SHR than in WKY platelets. These data, taken together with our earlier observations that thrombin induces a significantly greater hydrolysis of phosphoinositide (Thromb. Res. 49, 5-21, 1988), lead us to suggest that thrombin-induced increased generation of inositol 1,4,5-trisphosphate and diacylglycerol induces greater fibrinogen binding and consequently increased aggregation in SHR than WKY platelets. The finding that the thrombin binding isotherms are similar in WKY and SHR platelets suggests that increased platelet sensitivity to thrombin in hypertension may be due to altered signal transduction and not due to changes in the number or affinity of thrombin receptors.

Animals↗
PubMed1985

Affinity labeling of high-affinity alpha-thrombin binding sites on the surface of hamster fibroblasts.

The serine proteinase alpha-thrombin potently stimulates reinitiation of DNA synthesis in quiescent Chinese hamster fibroblasts (CCL39 line). 125I-labeled alpha-thrombin binds rapidly and specifically to CCL39 cells with high affinity (Kd approximately 4 nM). Binding at 37 degrees C was found to remain stable for 6 h or more during which time no receptor down-regulation, ligand internalization and/or degradation could be detected. The structure of alpha-thrombin receptors on CCL39 cells was identified by covalently coupling 125I-alpha-thrombin to intact cells using a homobifunctional cross-linking agent, ethylene glycol bis(succinimidyl succinate). By resolution in sodium dodecyl sulfate polyacrylamide gel electrophoresis we observed the specific labeling of a major alpha-thrombin-binding site of Mr approximately 150 000 revealed as a 125I-alpha-thrombin cross-linked complex of Mr approximately 180 000. Independent of chemical cross-linking, 125I-alpha-thrombin also formed a covalent complex with a minor, 35 000 Mr, membrane component identified as protease nexin. Two derivatives of alpha-thrombin modified at the active site are 1000-fold less than alpha-thrombin for mitogenicity. These two non-mitogenic derivatives bound to cells with similar affinity and maximal binding capacity as native alpha-thrombin, and affinity-labeled the receptor subunit of Mr 150 000. When present in large excess, during incubation of cells with alpha-thrombin, these binding antagonists were ineffective in blocking alpha-thrombin-induced DNA synthesis. These data suggest that the specific 150 000 Mr binding sites that display high affinity for alpha-thrombin do not mediate induction of the cellular mitogenic response.

Affinity Labels↗
PubMed1999

Protease-activated receptor-1 can mediate responses to SFLLRN in thrombin-desensitized cells: evidence for a novel mechanism for preventing or terminating signaling by PAR1's tethered ligand.

The thrombin receptor PAR1 is activated when thrombin cleaves the receptor's amino-terminal exodomain to reveal the new N-terminal sequence SFLLRN which then acts as a tethered peptide ligand. Free SFLLRN activates PAR1 independent of receptor cleavage and has been used to probe PAR1 function in various cells and tissues. PAR1-expressing cells desensitized to thrombin retain responsiveness to SFLLRN. Toward determining the mechanism of such responses, we utilized fibroblasts derived from a PAR1-deficient mouse. These cells were unresponsive to thrombin and SFLLRN and became sensitive to both ligands after transfection with human PAR1 cDNA. Moreover, PAR1-transfected cells responded to SFLLRN after thrombin-desensitization, indicating that signaling of thrombin-desensitized cells to SFLLRN was mediated by PAR1 itself. SFLLRN caused signaling in thrombin-desensitized cells when no uncleaved PAR1 was detectable on the cell surface; however, cleaved PAR1 was present. To determine whether the cleaved receptors could still signal, fibroblasts were transfected with a PAR1 mutant containing a trypsin site/SFLLRN sequence carboxyl terminal to the native thrombin site. These cells retained responsiveness to trypsin after thrombin-desensitization. Conversely, fibroblasts expressing a PAR1 mutant with the trypsin site/SFLLRN sequence amino terminal to the native thrombin site retained responsiveness to thrombin after trypsin-desensitization. This suggests that a population of thrombin-cleaved PAR1 can respond both to exogenous SFLLRN and to a second tethered ligand. In this population, the tethered ligand unmasked by thrombin cleavage must not be functional, suggesting the possibility of a novel mechanism of receptor shutoff involving sequestration or modification of the tethered ligand to prevent or terminate its function.

Amino Acid Sequence↗
PubMed1996

Functional expression of a human thrombin receptor in Sf9 insect cells: evidence for an active tethered ligand.

Desensitization of recombinant human thrombin receptors expressed in Sf9 insect cells was compared with native thrombin receptors in megakaryoblast erythroleukaemia (HEL) cells. Addition of thrombin (2 units/ml) or agonist peptide SFLLRN (10 microM) to HEL cells, or to Sf9 cells infected with recombinant baculovirus containing the thrombin receptor cDNA, produced an increase in the free cytosolic Ca2+ concentration ([Ca2+]i) as measured by fura-2. The response in HEL cells was transient, reflecting a rapid homologous desensitization. In contrast, [Ca2+]i in Sf9 cells expressing the thrombin receptor increased rapidly to a peak value that slowly declined, but remained elevated for at least 12 min following stimulation by thrombin. The sustained [Ca2+]i response to thrombin was not reversed by washout of thrombin or by any subsequent addition of hirudin. Pretreatment of Sf9 cells with either thrombin (2 units/ml) or SFLLRN (10 or 50 microM) for 5 min produced a shift in the ED50 for SFLLRN (added 10 min after washout) from 0.4 microM to 20 and 7 microM, respectively. Thus, desensitization of thrombin receptors expressed in Sf9 cells occurs slowly and reflects a decrease in receptor affinity. The sustained [Ca2+]i response in Sf9 cells stimulated by thrombin may reflect continuous activation by the tethered ligand. To test this hypothesis, the effect of protease treatment during the sustained phase of the response was examined. Addition of either aminopeptidase M or thermolysin reversed the sustained response to SFLLRN, but only thermolysin reversed the sustained response to thrombin. Thermolysin had no effect on the change in [Ca2+]i observed following carbachol stimulation of Sf9 cells expressing the M5 muscarinic receptor. Furthermore, following thermolysin treatment, the cells remained responsive to a subsequent application of SFLLRN. These results demonstrate that the tethered ligand remains active for extended periods of time after thrombin stimulation and suggests that further hydrolysis by extracellular proteases may represent an important mechanism of rapid receptor deactivation.

Amino Acid Sequence↗
PubMed1992

Thrombin receptor activation causes rapid neural cell rounding and neurite retraction independent of classic second messengers.

The protease thrombin is a potent activator of various cell types. Thrombin cleaves and thereby activates its own seven-transmembrane-domain receptor which couples to G proteins. Thrombin also can inhibit neuronal differentiation, supposedly by degrading components of the extracellular matrix. Here we report that active thrombin induces immediate cell rounding and neurite retraction in differentiating N1E-115 and NG108-15 neural cells in serum-free culture. Serum (0.5-5% vol/vol) evokes similar responses, but the cell-rounding and neurite-retracting activity of serum is not attributable to thrombin. Neural cell rounding is transient, subsiding after 10-15 min, and subject to homologous desensitization, whereas retracted neurites rapidly degenerate. Thrombin action is inhibited by cytochalasin, but not colchicine. A novel 14-amino acid peptide agonist of the thrombin receptor fully mimics thrombin's morphoregulatory activity, indicating that thrombin-induced shape changes are receptor-mediated and not secondary to extracellular matrix degradation. Although thrombin receptors couple to phosphoinositide hydrolysis and Ca2+ mobilization, thrombin-induced shape changes appear to depend neither on the Ca2+/protein kinase C- nor the cyclic nucleotide-mediated signal transduction pathways; however, the morphological response to thrombin is blocked by pervanadate, an inhibitor of tyrosine phosphatases, and by broad-specificity kinase inhibitors. Our results suggest that the thrombin receptor communicates to an as-yet-uncharacterized effector to reorganize the actin cytoskeleton and to reverse the differentiated phenotype of neural cells.

Alkaloids↗
PubMed1992

Thrombin-induced expression of endothelial P-selectin and intercellular adhesion molecule-1: a mechanism for stabilizing neutrophil adhesion.

Thrombin-induced expression of endothelial adhesivity toward neutrophils (PMN) was studied using human umbilical vein endothelial cells (HUVEC). HUVEC were challenged with human alpha-thrombin for varying durations up to 120 min, after which the cells were fixed with 1% paraformaldehyde and 51Cr-labeled human PMN were added to determine PMN adhesion. Endothelial adhesivity increased within 15 min after alpha-thrombin exposure, and the response persisted up to 120 min. Expression of endothelial adhesion proteins, P-selectin (GMP-140, PADGEM, CD62), and intercellular adhesion molecule-1 (ICAM-1; CD54) on the endothelial surface was quantitated by increase in the specific binding of anti-P-selectin mAb G1 and anti-ICAM-1 mAb RR1/1 labeled with 125I. P-selectin expression was maximal at 5-15 min alpha-thrombin exposure and decayed to basal levels within 90 min. In contrast, ICAM-1 activity increased at 30 min and remained elevated for 120 min after alpha-thrombin challenge. The initial endothelial adhesivity was dependent on P-selectin expression since PMN adhesion occurring within the first 30 min after alpha-thrombin challenge was inhibited by mAb G1. The later prolonged PMN adhesion was ICAM-1 dependent since this response was inhibited by mAb RR1/1 and to the same degree by the anti-CD18 mAb IB4. Anti-ELAM-1 mAb BB11 had no effect on adhesion of PMN to the alpha-thrombin-challenged cells. The initial P-selectin expression and PMN adhesion responses were reproduced by the 14-amino peptide (SFLLRNPNDKYEPF) (thrombin-receptor activity peptide; TRP-14) which comprised the NH2 terminus created by thrombin's proteolytic action on its receptors. However, TRP-14-induced PMN adhesion was transient, and TRP-14 did not cause ICAM-1 expression. The ICAM-1-dependent PMN adhesion mediated by alpha-thrombin was protein synthesis independent since ICAM-1 expression and PMN adhesion were not inhibited by cycloheximide pretreatment of HUVEC. Moreover, Northern blot analysis indicated absence of ICAM-1 mRNA signal up to 180 min after alpha-thrombin challenge. In conclusion, thrombin-induced endothelial adhesivity involves early- and late-phase responses. The initial reversible PMN adhesion is mediated by rapid P-selectin expression via TRP-14 generation. Thrombin-induced PMN adhesion is stabilized by a protein synthesis-independent upregulation of the constitutive ICAM-1 activity which enables the interaction of ICAM-1 with the CD18 beta 2 integrin on PMN.

Amino Acid Sequence↗