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PubMed1989

Thrombin-treated endothelium primes neutrophil functions: inhibition by platelet-activating factor receptor antagonists.

We have shown previously that fluid phase platelet-activating factor (PAF) can enhance or "prime" polymorphonuclear (PMN) responses to subsequent stimulation with agonists such as formyl-methionine-leucine-phenylalanine (FMLP). Since thrombin induces PAF production in endothelial cells, we tested whether this thrombin-provoked endothelial PAF primes responses of marginated PMNs. Monolayers of human umbilical vein endothelial cells were exposed to either thrombin (0.5-5.0 units/ml) or buffer for up to 5 min and then PMNs were layered on top of the endothelial cells. After a further 5 min incubation, the PMNs were stimulated with a suboptimal concentration of FMLP (10(-7) M), and their superoxide production, elastase release, adhesion to endothelium, and capacity to cause endothelial cell lysis and detachment were assessed. Thrombin pretreatment significantly enhanced each of these FMLP-stimulated neutrophil responses. The extent of this enhancement correlated with both the dose and duration of thrombin treatment of endothelial cells and also the duration of PMN incubation with thrombin-exposed endothelium. Evidence that the augmentation was due to endothelial-derived PAF was obtained as follows: (1) thrombin induced [3H]acetate incorporation into endothelial PAF (assayed in lipid extracts); (2) antithrombin III conjointly inhibited this [3H]acetate uptake and prevented the priming effect of thrombin-treated endothelium on PMN responses; and (3) the PAF receptor antagonist BN52021, when preincubated with PMNs, also effectively blocked the enhancement of PMN responses. We conclude that thrombin stimulation of endothelial cells initiates a sequence of events culminating in the production of PAF--a membrane phospholipid capable of priming marginated PMNs. We suggest that this coagulation-fostered endothelial/PMN interaction may underlie a paracrine response that may potentiate PMN-mediated endothelial injury during sepsis and other thrombin-generating disorders.

Endothelium, Vascular↗
PubMed2006

Preconditioning with thrombin can be protective or worsen damage after endothelin-1-induced focal ischemia in rats.

The serine protease thrombin has shown direct neuroprotective and neurotoxic effects on brain tissue in cerebral ischemia. Previous data suggested that thrombin-induced protection in vivo can be achieved by preconditioning rather than by acute treatment. In the current work, we used a model of mild ischemia to investigate the effects of preischemic intracerebral thrombin injection on neural damage. By intracerebral injection of endothelin-1 in freely moving animals, we achieved middle cerebral artery occlusion (MCAO), and 7 days postischemia we performed histological quantification of the infarct areas. Thrombin was injected as a preconditioning stimulus intracerebrally 7 days or 2 and 3 days before ischemia. For acute treatment, thrombin was injected 20 min before MCAO. Thrombin induced significant neuroprotection when given 7 days before endothelin-1-induced MCAO but was deleterious when given 2 and 3 days before the insult. The deleterious effect was not seen when thrombin was given acutely before ischemia. Our data demonstrate that preconditioning with thrombin can protect against damage or worsen ischemic damage. Its effect depended on the time interval between thrombin injection and insult. A low dose of thrombin did not induce a major deleterious effect in the acute phase of the infarct development after mild transient ischemia.

Animals↗
PubMed1998

Reactivities of the S2 and S3 subsite residues of thrombin with the native and heparin-induced conformers of antithrombin.

A pentasaccharide (PS) fragment of heparin capable of activating antithrombin (AT) markedly accelerates the inhibition of factor Xa by AT, but has insignificant effect on inhibition of thrombin. For inhibition of thrombin, the bridging function of a longer polysaccharide chain is required to accelerate the reaction. To study the basis for the similar reactivity of thrombin with the native or heparin-activated conformers of AT, several residues surrounding the active site pocket of thrombin were targeted for mutagenesis study. Leu99 and Glu192, the variant residues influencing the S2 and S3 subsite specificity of thrombin were replaced with Tyr and Gln. The Tyr60a, Pro60b, Pro60c, and Trp60d residues forming part of the S2 specificity pocket were deleted from the B-insertion loop of the wild-type and Leu99/Glu192 --> Tyr/Gln thrombins. Kinetic studies indicated that the reactivities of all mutants with AT were moderately or severely impaired. Although heparin largely corrected the defect in reactivities, it also markedly elevated the stoichiometries of inhibition with the mutants. Interestingly, PS also accelerated AT inhibition of the mutants 5-68-fold, suggesting that the mutants are able to discriminate between the native and activated conformers of AT. Based on these results and the recent crystal structure determination of AT in complex with PS, a model for thrombin-AT interaction is proposed in which the S2 and S3 subsite residues of thrombin are critical for recognition of the P2 and P3 residues of AT in the native conformation. In the activated conformation, other residues are made accessible for interaction with the protease, and the similar reactivity of thrombin with the native and heparin-activated conformers of AT may be coincidental. The results further suggest that the S2 and S3 subsite residues are crucial in controlling the partitioning of the thrombin-AT intermediate into the alternative inhibitory or substrate pathways of the reaction.

Antithrombins↗
PubMed1995

Crystal structure of human alpha-thrombin complexed with hirugen and p-amidinophenylpyruvate at 1.6 A resolution.

Crystals of human alpha-thrombin complexed with hirugen and the alpha-keto acid thrombin inhibitor APPA (p-amidinophenylpyruvate) that diffract to 1.6 A resolution were obtained by soaking an alpha-thrombin-hirugen crystal in a solution of APPA. The crystal structure was determined using the difference Fourier method and refined to an R factor of 18.7% at 1.6 A resolution. This structure is the highest resolution structure of the thrombin molecule that is currently available. With the exception of the region near Arg77A-Asn78, the structures of the thrombin and hirugen molecules in the ternary complex are similar to those reported for the thrombin-hirugen binary complex. As previously determined for the APPA-trypsin complex, the carbonyl carbon atom of APPA forms a covalent bond with O gamma of Ser195 of thrombin to yield a "transition-state" analog of the tetrahedral intermediate. Comparison of the specificity pocket of the APPA complexes of thrombin and trypsin reveals differences in hydrogen bonding and shows for the first time that the S1 site of thrombin is larger than that of trypsin and as a result thrombin may be able to accommodate a bulkier P1 group than trypsin.

Amino Acid Sequence↗
PubMed2001

Involvement of tyrosine phosphatase PTP1D in the inhibition of interleukin-6-induced Stat3 signaling by alpha-thrombin.

We previously demonstrated that exposure of CCL39 lung fibroblasts to alpha-thrombin inhibits interleukin-6 (IL-6)-induced tyrosine phosphorylation of Stat3 (signal transducers and activators of transcription 3) via activation of mitogen-activated protein (MAP) kinase kinase 1 [Bhat et al. (1998) Arch. Biochem. Biophys. 350, 307-314]. In this study, using CCL39/MRC-5 cells, we investigated if additional signaling intermediates are involved in alpha-thrombin's inhibitory effects on IL-6-induced Stat3 signaling. We also determined if alpha-thrombin inhibits oncostatin M (OSM)-induced Stat3/Stat1, and interferon-gamma (IFN-gamma)-induced Stat1 tyrosine phosphorylation. We demonstrate that, although both IL-6 and OSM belong to the same cytokine family, alpha-thrombin inhibited only the IL-6-induced Stat3 tyrosine phosphorylation. The tyrosine phosphatase PTP1D coprecipitated with Stat3 from alpha-thrombin + IL-6, but not from alpha-thrombin + OSM-treated cells. Pretreatment of cells with a phosphatase inhibitor reversed the inhibitory actions of alpha-thrombin, suggesting a role for PTP1D in alpha-thrombin-mediated inhibition of IL-6-induced Stat3 signaling. Interestingly, alpha-thrombin failed to inhibit OSM- and IFN-gamma-induced Stat1 tyrosine phosphorylation. Cytokine-specific inhibition of the Stat3 signaling involving MAP kinase kinase 1 and PTP1D by alpha-thrombin may play an important role in regulation of gene expression.

Animals↗
PubMed1993

Thrombin enhances T cell proliferative responses and cytokine production.

Human alpha-thrombin, in addition to its procoagulant activity, is a mitogen for fibroblasts and endothelial cells and a chemotactic agent for monocytes. To further understand the complex physiological functions of thrombin, we investigated whether thrombin has any immunoregulatory function with regard to T cell activation. Using highly purified human alpha-thrombin and peripheral blood mononuclear cells (PBMC), we investigated whether thrombin has any effect on cytokine production and/or proliferation induced by different T cell stimuli. At physiological concentrations (1-10 micrograms/ml, 30-300 nM), thrombin significantly enhances T cell proliferation in response to mitogens, superantigens, alloantigens, and anti-CD3 stimulation. Enhanced proliferation was associated with increased IL2 and IL6 production and with an increase in the number of IL2r+ (CD25)-bearing T cells. Thrombin alone was not mitogenic nor did it induce IL2 production or increase the number of IL2r+ T cells. However, PBMC exposed to thrombin alone produced high levels of IL6. Thrombin also enhanced IL2-induced proliferation of murine and human IL2-dependent cell lines. These results suggest that thrombin may play an important role in regulating cell-mediated immunity.

Cytokines↗
PubMed1995

Thermodynamic investigation of hirudin binding to the slow and fast forms of thrombin: evidence for folding transitions in the inhibitor and protease coupled to binding.

Temperature dependent studies of the interaction of the clotting enzyme thrombin with the potent natural inhibitor hirudin reveal a large negative heat capacity change of -1.7(+/- 0.2) kcal/mol per K associated with the formation of the thrombin-hirudin complex, independent of the allosteric state of the enzyme. Binding of N-terminal fragments of hirudin (hir1-49 and hir1-43) is characterized by heat capacity changes of -1.2(+/- 0.1) and -0.9(+/- 0.1) kcal/mol per K, respectively. The magnitude of these heat capacity changes is unprecedented for protease-inhibitor interactions. A thermodynamic analysis based on observed heat capacity and entropy changes predicts that binding is accompanied by substantial coupled folding transitions in both hirudin and thrombin. In the absence of a structure of free thrombin, analysis of differences in the predicted number of residues which fold upon binding hirudin and its fragments leads to the following structural model: three surface loops in thrombin (W60d, W148 and fibrinogen binding loops) are disordered in the free state and fold upon formation of the thrombin-hirudin complex. Molecular dynamics simulations, run over a time scale of 5 ps, are consistent with the hypothesis of large scale coupled folding transitions in both hirudin and thrombin upon formation of the complex. Comparison of the thermodynamics for the interaction of hirudin with the slow and fast forms of thrombin allows dissection of the coupling free energy for allosteric switching. The coupling free energy for the slow-->fast transition increases linearly, in absolute value, with temperature. The coupling enthalpy and entropy terms for hirudin were found to be delta Hoc = 12(+/- 1) kcal/mol and delta Soc = 47(+/- 4) cal/mol per K. Preferential interaction with the fast form is therefore due to the balance of two opposite forces, both quite large in magnitude. The contribution of enthalpic effects opposes the slow-->fast transition and stabilizes binding to the slow form. The contribution of entropic effects favors the slow-->fast transition and stabilizes binding to the fast form. In the physiological temperature range the entropic effects prevail and result in preferential binding of hirudin to the fast form. The region of thrombin recognizing the N-terminal domain of hirudin contains most of the residues that are energetically linked to the slow-->fast transition. This region is part of the "allosteric core" of thrombin and includes the W60d loop, shaping the specificity site S2, and the Na+ binding loop connecting the last two beta-strands of the B chain.

Allosteric Regulation↗
PubMed1997

Human alpha-thrombin inhibition by the highly selective compounds N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester: a kinetic, thermodynamic and X-ray crystallographic study.

Kinetics, thermodynamics and structural aspects of human alpha-thrombin (thrombin) inhibition by newly synthesized low molecular weight derivatives of alpha-azalysine have been investigated. The thrombin catalyzed hydrolysis of N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester (Cbz-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0 degrees C, and analyzed in parallel with that of N-alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). Decarboxylation following the enzymatic hydrolysis of these p-nitrophenyl esters gave the corresponding 1-peptidyl-2(4-aminobutyl) hydrazines (peptidyl-Abh) showing properties of thrombin competitive inhibitors. Therefore, thermodynamics for the reversible binding of D-Phe-Pro-Abh, Cbz-Pro-Abh and Dmc-Abh to thrombin was examined. These results are consistent with the minimum four-step catalytic mechanism for product inhibition of serine proteinases. Eoc-D-Phe-Pro-azaLys-ONp and Eoc-D-Phe-Pro-Abh display a sub-micromolar affinity for thrombin together with a high selectivity versus homologous plasmatic and pancreatic serine proteinases acting on cationic substrates. The three-dimensional structures of the reversible non-covalent thrombin:Eoc-D-Phe-Pro-Abh and thrombin:Cbz-Pro-Abh complexes have been determined by X-ray crystallography at 2.0 A resolution (R-factor = 0.169 and 0.179, respectively), and analyzed in parallel with that of the thrombin:Dmc-azaLys acyl-enzyme adduct. Both Eoc-D-Phe-Pro-Abh and Cbz-Pro-Abh competitive inhibitors are accommodated in the thrombin active center, spanning the region between the aryl binding site and the S1 primary specificity subsite.

Animals↗
PubMed2002

LB30057 inhibits platelet aggregation and vascular relaxation induced by thrombin.

Previous study showed that an amidrazonophenylalanine derivative, LB30057, which has high water solubility, inhibited the catalytic activity of thrombin potently by interaction with the active site of thrombin. In the current investigation, we examined whether LB30057 inhibited platelet aggregation and vascular relaxation induced by thrombin. Treatment with LB30057 to platelet-rich plasma (PRP) isolated from human blood resulted in a concentration-dependent inhibition of thrombin-induced aggregation. Values for IC50 and IC100 were 54 +/- 4 nM and 96 +/- 3 nM, respectively. This inhibition was agonist (thrombin) specific, since IC50 values for collagen and ADP were much greater than those for thrombin. In addition, concentration-dependent inhibitory effects were observed on the serotonin secretion induced by thrombin in PRP. Consistent with these findings, thrombin-induced increase in cytosolic calcium levels was inhibited in a concentration-dependent manner. When LB30057 was treated with aortic rings isolated from rats, LB30057 resulted in a concentration-dependent inhibition of thrombin-induced vascular relaxation. All these results suggest that LB30057 is a potent inhibitor of platelet aggregation and blood vessel relaxation induced by thrombin.

Animals↗
PubMed1984

Catalytic and regulatory functions of N-bromosuccinimide-modified bovine thrombin.

At pH 4.1, bovine thrombin reacts rapidly with N-bromo-succinimide to yield modified enzyme containing oxidized tryptophan residue. Both fibrinogen clotting activity and esterase activity are reduced considerably when three moles of tryptophan residues per mole of thrombin are oxidized, but the Michaelis constants for synthetic substrates are not appreciably altered. Reaction of NBS also results in a decrease in the affinity of thrombin for heparin. The dissociation constant for heparin-thrombin complex is increased by 2.6-fold due to the modification of one tryptophan residue. However, the magnitude of the increase in the dissociation constant remains the same for modified enzymes containing approximately two or three oxidized tryptophan residues. The rate constant for the inactivation of thrombin by antithrombin III is increased by 2.5-fold due to the modification of a single tryptophan residue. This increase in rate constant is not further amplified when more than one tryptophan residue is oxidized. In contrast, in the presence of heparin the rate of inactivation of modified and unmodified thrombins by antithrombin III are not significantly different. Thus, the heparin-sensitized inactivation of thrombin by antithrombin III is affected by the modification of one tryptophan residue. Spectrophotometric titrations of the phenolic hydroxyl groups suggest that the structural environments of tyrosyl groups for both unmodified and modified thrombin containing one oxidized tryptophan residue, are similar. The temperature for half loss of catalytic activity of control and NBS-modified thrombin, containing one oxidized tryptophan, are 52 and 51.5 degrees C respectively. It appears that the one tryptophan residue of thrombin is situated at or close to the binding site of heparin.

Animals↗
PubMed1985

Hirudin insensitive thrombin-stimulated platelet release.

It has been previously proposed that platelet stimulation may involve two platelet-thrombin complexes: an initial platelet-thrombin complex (P-T) which is converted to an activated platelet-thrombin complex (P*-T). By using the release of radioactive serotonin as a measure of thrombin stimulation, we have demonstrated that under appropriate conditions, a hirudin sensitive and a hirudin insensitive complex can be differentiated. At short platelet-thrombin preincubation times (0-2 minutes) at 4 degrees C, in a buffer containing 18.7 mM phosphate, added hirudin almost completely inhibited the release of radioactive serotonin obtained upon subsequent warming to 37 degrees C (only the hirudin sensitive complex exists). If platelets were preincubated with thrombin for longer periods of time (30 minutes), hirudin became less effective in inhibiting the release obtained upon subsequent warming to 37 degrees C. The same results were obtained whether or not the platelets were washed after incubation at 4 degrees C and before warming to 37 degrees C. We postulate that this change in hirudin sensitivity may reflect a slow conversion of the first platelet-thrombin complex (P-T) to an activated platelet-thrombin complex (P*-T) which can undergo release upon warming. This transition appears to be much faster in acetatetris buffer since at short platelet-thrombin incubation times at 4 degrees C, added hirudin had little or no effect on the release obtained upon warming to 37 degrees C. The difference in the ability of hirudin to inhibit thrombin-induced release in the two buffers was shown to depend upon the presence of phosphate and on variations in ionic strength, and not due to a change in the inhibition constant (Ki) for hirudin.

Animals↗
PubMed1985

Fibrin degradation products increase lung transvascular fluid filtration after thrombin-induced pulmonary microembolism.

The effect of fibrin degradation products (FDP) on pulmonary transvascular fluid and protein exchange was examined in the sheep lung lymph fistula preparation. The pulmonary lymph was used to assess changes in pulmonary lymph flow (Qlym) (a measure of net transvascular fluid filtration rate) and the lymph/plasma protein concentration ratio (L/P) (a measure of protein seiving across the microvascular barrier). Studies were made in 3 groups: Control-Thrombin (n = 7) received 96.9 +/- 9.4 U/kg of alpha-thrombin, Control-FDP (n = 6) received infusion of FDP prepared by plasmin digestion of fibrin, and Thrombin-FDP (n = 5) received thrombin (102.0 +/- 7.5 U/kg) and then an FDP infusion was begun at 60 min after the thrombin. In the Control-Thrombin animals, Qlym increased without a change in the L/P after thrombin, indicating an increase in vascular permeability to proteins. In the Control-FDP group, infusion of FDP had no effect on Qlym and L/P. In the Thrombin-FDP group, thrombin increased Qlym with no change in the L/P and the subsequent infusion of FDP further increased Qlym but slightly decreased the L/P, an effect not seen in the Control-FDR group. The results indicate that FDP infusion alone does not alter pulmonary transvascular fluid and protein exchange. However, in the presence of thrombin-induced pulmonary microembolization FDP infusion enhances the net transvascular fluid filtration rate, probably by increasing the capillary hydrostatic pressure.

Animals↗
PubMed1991

Heparin cofactor II significance for the inhibition of thrombin at the injured vessel wall.

The thrombin inhibitory role of antithrombin III (ATIII) and heparin cofactor II (HCII) was studied in vitro using intact and injured rabbit aortae. When intact vessels were loaded with thrombin and then exposed to either heat defibrinogenated human plasma (HDHP) or ATIII the same degree of thrombin inhibition was achieved demonstrating that ATIII was the only plasma component involved in thrombin inhibition on the intact vessel wall. When the media of the vessel wall was loaded with thrombin and then exposed to ATIII or HCII a significantly higher thrombin activity remained on the surface than when it was exposed to defibrinogenated plasma. A mixture of ATIII and HCII resulted in a greater inhibition of thrombin than ATIII or HCII alone. It is concluded that, contrary to what happens on the endothelium, HCII and ATIII inhibit additively thrombin on the injured vessel wall. HCII thus plays an essential role for the inhibition of thrombin at the injured vessel wall. It is also concluded that an additional plasma component participates in thrombin inhibition on the media but its contribution is negligible as compared with ATIII or HCII.

Animals↗
PubMed1987

Activation of phospholipase C in platelets by platelet activating factor and thrombin causes hydrolysis of a common pool of phosphatidylinositol 4,5-bisphosphate.

Despite their physicochemical and mechanistic differences platelet activating factor (or acetylglycerylether phosphorylcholine; AGEPC) and thrombin, both platelet stimulatory agents, induce phosphoinositide turnover in platelets. We therefore investigated the stimulation of the phosphoinositide phosphodiesterase by these agents and questioned whether they evoked hydrolysis of the same or different pools of phosphoinositides. [3H]Inositol-labelled rabbit platelets were challenged with thrombin and/or AGEPC under a variety of protocols, and the phospholipase C mediated production of radioactive inositol monophosphate (IP); inositol bisphosphate (IP2) and inositol trisphosphate (IP3) was used as the parameter. AGEPC (1 X 10(-9) M) caused a transient maximum (5 to 6-fold) increase in [3H]IP3 at 5 s followed by a decrease. Thrombin (2 U/ml) elicited an increase in [3H]IP3 at a much slower rate than AGEPC; 2 fold at 5 s, 5 fold at 30 s and a maximum 6 to 8-fold at 2-5 min. Compared to AGEPC, thrombin stimulated generation of [3H]IP2 and [3H]IP were severalfold higher. When thrombin and AGEPC were added together to platelets there was no evidence for an additive increase in inositol polyphosphate levels except at earlier time points where increases were submaximal. When AGEPC was added at various time intervals after thrombin pretreatment, no additional increases in [3H]IP3 were observed over that maximally seen with thrombin or AGEPC alone. In another set of experiments, submaximal increases (about 1/4 and 1/2 of maximum) in [3H]IP3 were achieved by using selected concentrations of thrombin (0.1 U and 0.3 U, respectively) and then AGEPC (1 X 10(-9) M) was added for 5 s. Once again the increase in [3H]IP3 was close to the maximal level seen with thrombin or AGEPC individually. It is concluded that thrombin and AGEPC differentially activated phosphoinositide phosphodiesterase (phospholipase C) in rabbit platelets and that the stimulation of the phospholipase C by these two stimuli causes IP3 production via hydrolysis of a common pool of phosphatidylinositol 4,5-bisphosphate.

Animals↗
PubMed1987

Thrombin-induced calcium movements in platelet activation.

The thrombin-induced Ca2+ fluxes and their coupling to platelet aggregation of the human platelet were studied using quin2 as a measure of the cytoplasmic Ca2+ concentration [( Ca2+]cyt) and chlorotetracycline (CTC) as a measure of internally sequestered Ca2+. Evidence is given that the CTC fluorescence change is proportional to the free internal Ca2+ concentration in the dense tubular lumen. The intracellular quin2 concentration was 1 mM and analysis showed that it did not perturb the processes reported herein. The value of [Ca2+]cyt at rest and during thrombin activation was analyzed in terms of Ca2+ influx, Ca2+ release, Ca2+ sequestration, and Ca2+ extrusion. Influx was distinguished from internal release by removing extracellular Ca2+ 1 min before thrombin activation. In the presence of 2 mM external Ca2+, the thrombin-induced Ca2+ influx accounts for most of the increase in [Ca2+]cyt (over 80%). Thrombin-induced Ca2+ influx and release have somewhat different EC50 values (0.17 U/ml vs. 0.35 U/ml). The contribution of influx can be inhibited by verapamil, bepridil and Cd2+ (IC50 values of 19 microM, 2 microM and 50 microM). The influx results were analyzed in terms of a thrombin-activated channel. Indomethacin pretreatment experiments suggest that activation of the arachidonic pathway accounts for approx. 50% of the influx-related [Ca2+]cyt elevation. Elevation of [Ca2+]cyt by intracellular release is not inhibited by verapamil or Cd2+ but is inhibited by bepridil with a high IC50 (25 microM). It is only 15-20% inhibited by indomethacin and is thus not dependent on thromboxane A2 formation. The release reaction does not require Ca2+ influx. The rate of thrombin-activated platelet aggregation is shown to have an approximately fourth-power dependence on [Ca2+]cyt with an apparent Km of 0.4 microM. Comparisons of aggregation rates of the partially thrombin-activated vs. fully thrombin-activated, partially verapamil-inhibited conditions suggest that this dependence on [Ca2+]cyt is the major determinant of the aggregation behavior. Analysis shows that calcium influx is the major pathway for elevating [Ca2+]cyt by thrombin when physiological concentrations of external Ca2+ are present.

Aminoquinolines↗
PubMed2006

Influence of thrombin concentration on the mechanical and morphological properties of cell-seeded fibrin hydrogels.

Fibrin is a biopolymer that has been used in a variety of biomaterial, cell delivery and tissue engineering applications. The enzyme thrombin catalyzes the formation of fibrin microfibrils, which form a three-dimensional mesh in which cells can be directly embedded at the time of gel formation. In this study, fibrin hydrogels containing vascular smooth muscle cells were created using varying concentrations of thrombin. Over 7 days in culture, all gels decreased in volume as the fibrin matrix compacted, and the degree of gel compaction increased as thrombin concentration decreased. The material modulus and ultimate tensile stress of the gels also increased with decreasing thrombin concentration. Addition of thrombin to similar constructs made using collagen Type I did not show an effect on gel compaction or mechanical properties, suggesting that these effects were a result of thrombin's action on fibrin polymerization, and not cellular functions. Cell proliferation in fibrin hydrogels was not significantly affected by thrombin addition. Matrix examination using scanning electron microscopy showed increasing fibrin fiber diameters as thrombin concentration decreased. Confocal microscopic imaging of the actin cytoskeleton showed that cell morphology on two-dimensional substrates of fibrin showed marked changes, with higher thrombin concentrations producing cells with longer cellular projections. However, these morphological changes were not as apparent in cells embedded in three-dimensional (3-D) matrices, in which cells exhibited a similar morphology independent of thrombin concentration. These results relate features of the matrix and cellular components of 3-D fibrin constructs to mechanical properties, and contribute to the understanding of structure-function relationships in cell-seeded, 3-D protein hydrogels.

Animals↗
PubMed2001

Thrombin regulates chemokine induction during human retinal pigment epithelial cell/monocyte interaction.

Thrombin, an important clotting factor, extravasates at sites of blood-retina barrier breakdown that is often associated with many retinal diseases. Here we investigated the effects of thrombin on human retinal pigment epithelial (HRPE) cells, monocytes, and HRPE cell/monocyte co-cultures. Thrombin induced secretion and mRNA expression of HRPE interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1). Thrombin also enhanced IL-8 and MCP-1 by HRPE cell/monocyte co-cultures, by apparently enhancing cell-cell contact mechanisms. The thrombin effects on IL-6 secretion were similar to those on chemokine secretion. Thrombin-induced chemokines by co-cultures were inhibited by anti-tumor necrosis factor-alpha (TNF-alpha) antibody, but not by anti-IL-1beta antibody. TNF-alpha was detected in cell lysates of monocytes detached from HRPE cells after co-culture stimulation with thrombin. HRPE cells mainly produced these chemokines. However, thrombin generally potentiated exogenous IL-1beta- and TNF-alpha-induced chemokine production by HRPE cells, monocytes, and co-cultures. Interferon-gamma potentiated chemokine secretion by co-cultures with or without thrombin. Our results indicate that thrombin may cause leukocyte recruitment by inducing HRPE cell and monocyte chemokine and by enhancing HRPE cell/monocyte interactions, in part because of monocyte TNF-alpha induction, suggesting important mechanisms for ocular inflammation during blood-retina barrier breakdown and intra-ocular hemorrhage.

Antibodies↗
PubMed2001

Thrombin activates p38 mitogen-activated protein kinase in vascular smooth muscle cells.

Thrombin is a potent mitogen for vascular smooth muscle cells. However, the signaling pathways by which thrombin mediates its mitogenic response are not fully understood. The ERK (extracellular signal-regulated protein kinase) and JNK (c-Jun N-terminal kinase) members of the mitogen-activated protein kinase (MAPK) family are reported to be activated by thrombin. We have investigated the response to thrombin of another member of the MAPK family, p38 MAPK, which has been suggested to be activated by both stress and inflammatory stimuli in vascular smooth muscle cells. We found that thrombin induced time- and dose-dependent activation of p38 MAPK. Maximal stimulation of p38 MAPK was observed after a 10-min incubation with 1 unit ml(-1) thrombin. GF109203X, a protein kinase C inhibitor, and prolonged treatment with phorbol 12-myristate 13-acetate partially inhibited p38 MAPK activation. A tyrosine kinase inhibitor, genistein, also inhibited p38 MAPK activation in a dose-dependent manner. p38 MAPK activation was inhibited by overexpression of betaARK1ct (beta-adrenergic receptor kinase I C-terminal peptide). p38 MAPK activation was also inhibited by expression of dominant-negative Ras, not by dominant-negative Rac. We next examined the effect of a p38 MAPK inhibitor, SB203580, on thrombin-induced proliferation. SB203580 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. These results suggest that thrombin activates p38 MAPK in a manner dependent on Gbetagamma, protein kinase C, a tyrosine kinase, and Ras, that p38 MAPK has a role in thrombin-induced mitogenic response in the cells.

Animals↗