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PubMed1992

Molecular cloning of the rat vascular smooth muscle thrombin receptor. Evidence for in vitro regulation by basic fibroblast growth factor.

To study thrombin's receptor-mediated effects on vascular cells, we cloned and characterized a cDNA encoding a rat smooth muscle cell thrombin receptor. A rat aortic smooth muscle (RASM) cell cDNA library was screened with a 500-base pair (bp) sequence from the human thrombin receptor, obtained by polymerase chain reaction (PCR) amplification of cDNA synthesized from human erythropoietic leukemia (HEL) cell mRNA with PCR primers based on the published human thrombin receptor sequence. Clone pRTHR17 contains a 3418-bp insert that includes 50 bp of the 5'-untranslated region and the entire coding and 3'-untranslated regions of the RASM cell thrombin receptor. The sequence of pRTHR17 is 85% similar, at the nucleotide level, and 78% similar, at the deduced amino acid level, to the human thrombin receptor. Although the putative thrombin cleavage and binding sites are present, there are significant differences between the rat and human receptors in their amino-terminal sequences. Detectable signals (consisting of a single band of 3.45 kb) are present by Northern analysis of mRNA from RASM cells, and rat lung, kidney, and testes, but not in aorta or other tissues probed. The results of Southern analysis of rat genomic DNA are consistent with the existence of a single copy of the gene encoding this receptor. The steady state thrombin receptor mRNA level is low in cultured growth-arrested RASM cells and not detectable in rat aorta. To determine whether regulation of the RASM cell thrombin receptor occurs under growth-stimulating conditions, growth-arrested RASM cells were treated with basic fibroblast growth factor (bFGF, recently proposed to be a major mitogen controlling vascular smooth muscle cell growth following injury (Lindner, V., and Reidy, M. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3739-3743)). There was a significant increase in thrombin receptor mRNA following the addition of bFGF. These data demonstrate that: 1) mRNA for a thrombin receptor similar to that reported from human megakaryocyte and hamster fibroblast cell lines is present in proliferating primary culture rat smooth muscle cells, 2) the most significant sequence differences are present in the amino-terminal tail of the thrombin receptor, and 3) the mRNA level for this receptor is regulated under growth-stimulating conditions in vitro.

Amino Acid Sequence↗

PubMed1986

Ultrastructural studies of endothelial and platelet receptor binding of thrombin-colloidal gold probes.

Endothelial cells and platelets are reported to have receptors for alpha-thrombin. To visualize the binding of alpha-thrombin to these cells, we developed a method to label thrombin with colloidal gold. Formed by electrostatic adsorption of thrombin to the negatively charged gold, the resulting probe is stable for weeks and consists of approximately 30 thrombin molecules adsorbed to each 16.5 nm gold particle. The probe retained about 10% of the enzymatic activity (fibrinogen clotting) of the unlabeled native thrombin and 20% of the ability of the native thrombin to aggregate platelets in platelet-rich plasma (PRP). In PRP, approximately 90% of the observed probes were bound to fibrin strands, with the remaining probes (650 per cell) attached to activated platelets. In contrast, washed, paraformaldehyde-fixed human platelets exhibited a marked increase in probe density (4900 per cell). Time-dependent ultrastructural studies (2-240 min) of binding of the thrombin-gold probe to confluent cultures of porcine aortic endothelial cells revealed that the initial binding (7300 probes per cell) occurred randomly at the cell surface. A limited number (25%) of the probes clustered at coated-pit regions and were internalized (60-240 min). The probe induced a limited amount of cellular retraction similar to that achieved with unlabeled thrombin. These results suggest that the thrombin-gold probe is suitable for investigations of the localization of thrombin receptors on cell surfaces and the interaction of thrombin with these receptors during thrombotic events.

Animals↗

PubMed1994

Enhancement of the expression of urokinase-type plasminogen activator from PC-3 human prostate cancer cells by thrombin.

The presence of procoagulants and fibrin deposition have been demonstrated in malignant tumors. Although thrombin, a key enzyme in coagulation, has other various biological functions, the significance of its presence in tumors is not known. We studied the effects of thrombin on the expression of urokinase-type plasminogen activator (uPA) which is known to play a role in tumor invasion, using a human prostate cancer cell line PC-3. Human alpha-thrombin added to cultures of PC-3 produced a dose-dependent and time-dependent increased secretion of uPA that was greatest at 3-6 h after exposure to thrombin. Increase in uPA antigen paralleled the increase in mRNA level, which reached a maximum at 4 h. Thrombin showed the maximum effect on uPA expression at a concentration 1-2 units/ml. Zymography showed that transient exposure to thrombin induced an increase in fibrinolytic activity which could be quenched by anti-uPA antibody. The thrombin receptor-activating peptide also caused an increase in uPA protein and mRNA level, indicating the presence of the same thrombin specific receptor on PC-3 cells as on platelets and endothelial cells. Thrombin did not affect the expression of other components of the plasminogen activation system, tissue-type plasminogen activator and type-1 plasminogen activator inhibitor, and uPA receptor. These results indicate that thrombin increases uPA expression selectively by the stimulation of a functional thrombin receptor on PC-3 cells. Since uPA is known to play a role in pericellular proteolysis of extracellular matrix, thrombin may be involved in the regulation of tumor invasion and metastasis.

Adenocarcinoma↗

PubMed1993

Functional thrombin receptors on human T lymphoblastoid cells.

Human alpha-thrombin stimulated five different T lymphoblastoid cell lines to increase intracellular free Ca2+ concentrations, whereas five B cell lines did not similarly respond. The T cell intracellular free Ca2+ increased rapidly, plateaued within 10 to 20 s, and then declined without any measurable sustained intracellular free Ca2+ elevation. Liberation of inositol trisphosphate peaked within 60 s, and a rapid and sustained activation of protein kinase C was induced. The thrombin-specific inhibitor, hirudin, completely blocked the response to alpha-thrombin. Catalytically inactivated forms of alpha-thrombin failed to stimulate the T cells, whereas trypsin, gamma-thrombin (which lacks fibrinogen clotting activity), or the synthetic 14-amino acid thrombin receptor-activation peptide stimulated T cells, but required approximately 10-, approximately 15-, or approximately 100-fold higher concentrations, respectively, when compared to alpha-thrombin. Stimulation by thrombin receptor-activation peptide was desensitized by alpha-thrombin, and vice versa, but alpha-thrombin did not desensitize stimulation by mAb to the TCR/CD3 Ag. The presence of the receptor on T but not on B cells was confirmed by flow-cytometric analysis using a mAb against the human thrombin receptor. These findings demonstrate functional thrombin receptors on T lymphoblastoid cells that may be capable of activating the cells, independently of an ongoing immune response.

Amino Acid Sequence↗

PubMed1998

T-Cell receptor signaling pathway exerts a negative control on thrombin-mediated increase in [Ca2+]i and p38 MAPK activation in Jurkat T cells: implication of the tyrosine kinase p56Lck.

Activation of the mitogen-activated protein kinase (Erk) and c-Jun terminal kinase is a well-documented mechanism for the seven transmembrane spanning receptors. We have previously shown that thrombin stimulation of the T-leukemic cell line Jurkat induced a transient increase in [Ca2+]i and tyrosine phosphorylation of several cellular proteins. Here, we have analyzed p42-44 MAPK, JNK and p38 MAPK activation using Jurkat T-cell lines deficient in either the tyrosine kinase p56Lck (JCaM1) or the tyrosine phosphatase CD45 (J45.01). Our results demonstrate that p56Lck and CD45 exert a negative control on thrombin-induced p38 MAPK activation and [Ca2+]i release in Jurkat cells. Thrombin receptor expression was identical on the different cell lines as assessed by FACS analysis. Tyrosine phosphorylation of p38 MAPK was drastically increased after thrombin stimulation of JCaM1 or J45.01 cells, as compared with parental cells (JE6.1). P42-44 MAPK and JNK activity also enhanced after thrombin treatment of JE6.1 and JCaM1 cell lines, whereas basal kinase activity was higher in J45.01 cells and was not further stimulated by thrombin. Thrombin and thrombin receptor agonist peptide-induced [Ca2+]i mobilization paralleled p38 MAPK activation in JCaM1 and J45.01 cells. Moreover, reconstitution of J45.01 and JCaM1 cell lines with either CD45 or Lck is accompanied by restoration of a normal thrombin-induced [Ca2+]i response and p38MAPK phosphorylation. These data show that a component of the T-cell receptor signaling pathway exerts a negative control on thrombin-induced responses in Jurkat T cells. Accordingly, we found that thrombin enhanced tyrosine phosphorylation of p56Lck and decreased p56Lck kinase activity in J45.01 cells. Our results are consistent with a negative role for p56Lck on thrombin-induced [Ca2+]i release and p38 MAPK activation in Jurkat T-cell lines.

Calcium↗

PubMed1996

Specific involvement of glypican in thrombin adhesive properties.

We have previously demonstrated that thrombin possesses an active yet cryptic Arg-Gly-Asp (RGD) site which upon exposure induces endothelial cell (EC) adhesion via alpha nu beta 3 integrin [Bar-Shavit et al. (1991): J Cell Biol 112:335]. This was achieved in the presence of cell surface-associated heparan sulfate proteoglycans (HSPG) and exceedingly low concentrations of plasmin [Bar-Shavit et al. (1993): J Cell Biol 123:1279]. A portion of the cell surface-associated HSPG (glypican) is anchored via a covalently linked glycosyl-phosphatidylinositol (PI) residue, which can be released by treatment with glycosyl-PI-specific phospholipase C (PI-PLC). We report here that exposure of either bovine aortic EC, smooth muscle cells (SMC), or wild-type CHO cells to PI-PLC released HSPG involved in the conversion of thrombin to an adhesive molecule. The adhesion-promoting activity of the released HSPG was abolished following treatment with heparinase but not chondroitinase ABC. Incubation of thrombin with heparan sulfate-deficient CHO cells or cells that were pretreated with PI-PLC failed to induce its conversion to an adhesive molecule, indicating that glypican was playing a major role in this conversion. Moreover, affinity-purified glypican, but not syndecan or fibroglycan, elicited efficient conversion of plasmin-treated thrombin into an adhesive molecule. Antibodies raised against the RGD site in thrombin failed to interact with native thrombin, prothrombin, or the RGD site in other adhesive proteins such as vitronectin, fibrinogen, or fibronectin. Anti-thrombin-RGD antibodies which blocked the adhesion-promoting activity of thrombin were also capable of recognizing thrombin that was first incubated with a suboptimal concentration of plasm in in the presence of PI-PLC-released HSPG. Heparin, heparan sulfate, and PI-PLC-released HSPG had no effect on other cellular properties of thrombin such as receptor binding and growth-promoting activity. Altogether we have demonstrated that the heparin binding domain in thrombin plays a specific role in promoting thrombin adhesive properties and that membrane-associated glypican is likely to be the major physiological inducer of this property.

Animals↗

PubMed1986

Growth-promoting effects of esterolytically inactive thrombin on macrophages.

It has been recognized for many years that alpha-thrombin, like other better known mitogens (eg, PDGF, EGF, etc) is capable of initiating proliferation in quiescent cells belonging to the fibroblast family. However, unlike these other peptides, thrombin is a serine protease whose function as a growth stimulator for fibroblasts is intimately linked to its esterolytic activity. Thus, while native alpha-thrombin is capable of evoking DNA synthesis in G0/G1-arrested cells, neither enzymatically inactive thrombin (eg, iPR2P-alpha-thrombin) nor partially degraded thrombin (eg, gamma-thrombin) shares in this capability. Data from our laboratory have shown that thrombin is chemotactic for peripheral blood monocytes and for cells belonging to the monocyte/macrophage family and that this activity is not dependent upon thrombin's enzymatic properties. Our recent findings demonstrate that thrombin also serves as a growth factor for these cells, and this mitogenic capability is independent of esterolytic function and resides in the same region of the molecule as that responsible for chemotaxis. Additionally, by means of techniques such as computer modeling and peptide synthesis, we have now been able to delineate a distinct mitogenic subsite within this chemotactic thrombin sequence. Thus, the sequence in the thrombin B chain that mediates chemotaxis represents a true cell interactive exosite additionally capable of stimulating growth and possibly other biological functions in cells of macrophage/monocyte lineage.

Amino Acid Sequence↗

PubMed2004

Protease nexin-1: a cellular serpin down-regulated by thrombin in rat aortic smooth muscle cells.

Protease nexin-1 (PN-1), a potent inhibitor of serine proteases, is present in vascular cells and forms complexes with thrombin, plasminogen activators, and plasmin. We examined the effect of thrombin on PN-1 expression by rat aortic smooth muscle cells (RASMCs). PN-1 expression was determined by measuring protein and mRNA levels, using respectively immunoblotting and semi-quantitative reverse transcriptase polymerase chain reaction (PCR). Thrombin down-regulated PN-1 expression in a dose- and time-dependent manner. This effect was mediated via the interaction of thrombin with its receptor protease activated receptor (PAR-1) since the peptide thrombin receptor activating peptide (TRAP) reduced PN-1 expression. PN-1 secreted by smooth muscle cells remained essentially associated to cell-surface glycosaminoglycans and was released from the cell surface by heparin. A lower amount of PN-1 was released by heparin from TRAP-stimulated versus unstimulated cells and correlated with a decreased capacity to inhibit thrombin. In addition, the ability to generate peri-cellular plasmin was increased in cells with a low PN-1 expression. Pre-treatment of smooth muscle cells with cycloheximide abolished the reduction of PN-1 expression by thrombin. Furthermore, conditioned media from thrombin-treated cells reproduced the effect of thrombin, suggesting that thrombin acted via the induction of auto/paracrine mediator(s). We observed that fibroblast growth factor-2 (FGF-2)-neutralizing antibodies abolished thrombin effect whereas FGF-2 reproduced it, indicating that FGF-2 is one of the involved mediator. Together, these results indicate that (i) PN-1 modulates the activity of endogenous and exogenous serine proteases in RASMCs, (ii) thrombin down-regulates PN-1 expression and thus may increase its own activity on cells.

Amyloid beta-Protein Precursor↗

PubMed2003

Thrombin contributes to bronchoalveolar lavage fluid mitogenicity in lung disease of the premature infant.

Chronic lung disease of prematurity (CLD) is a common consequence of neonatal respiratory distress syndrome (RDS) and is characterized by pulmonary fibrosis. Increased thrombin activity in the alveolar compartment is associated with pulmonary fibrosis in adults and animals, and contributes to bronchoalveolar lavage (BAL) fluid mitogenicity for fibroblasts. We hypothesized that BAL fluid from infants who develop CLD contains increased mitogenic activity for lung fibroblasts compared to BAL fluid from resolving RDS, and that increased thrombin levels contribute to this activity. Sequential BAL (postnatal days 2-14) was obtained from 37 premature infants who were ventilated for RDS. Twenty-six infants developed CLD, whereas 11 resolved. BAL fluid mitogenic activity was determined in a proliferation assay, using human fetal lung fibroblasts. The contribution of thrombin to mitogenic activity was determined using the thrombin inhibitor PPACK. Furthermore, thrombin levels in BAL fluid were measured using a specific substrate to detect thrombin activity and by measuring thrombin-antithrombin III complex (TATIII). BAL fluid mitogenic activity was comparable between CLD and RDS (CLD, 33% proliferation on day 2 to 41% on day 14; RDS, 21% on day 2 to 54% on day 7). Thrombin inactivation by PPACK completely inhibited mitogenic activity in BAL samples obtained on days 2 and 4 (CLD, P < 0.001 on days 2 and 4; RDS, P < 0.05 on day 4). From day 7 onwards, inhibition of thrombin only partly reduced (P < 0.05) CLD BAL fluid mitogenic activity, indicating that other mitogenic factors contribute as well. Surprisingly, thrombin activity and TATIII were decreased in BAL fluid from CLD compared with RDS patients on days 2 and 4. In conclusion, our study shows that BAL fluid from infants with and without CLD development is equally mitogenic for lung fibroblasts, and that thrombin is a major mitogen in these samples. This suggests that fibroproliferation may occur early in the lungs from infants with both CLD and RDS, and that thrombin contributes to this.

Activin Receptors, Type I↗

PubMed2005

Complement inhibition attenuates brain edema and neurological deficits induced by thrombin.

The present study examined whether thrombin activates the complement cascade in the brain and whether N-acetylheparin, an inhibitor of complement activation, attenuates brain injury induced by thrombin. There were three sets of studies. In the first set, rats had an intracerebral infusion of either five-unit thrombin or a needle insertion. Brains were sampled at 24 hours for Western blot analysis and immuno-histochemistry. In the second set, rats received either five-unit thrombin+saline, five-unit thrombin+25 microg N-acetylheparin or five-unit thrombin+100 microg N-acetylheparin infusion. Brains were sampled 24 hours later for water content measurement. In the third set, rats received either five-unit thrombin+saline or five-unit thrombin+ 100 microg N-acetylheparin. Behavioral tests sensitive to unilateral striatal damage were carried out for two weeks. Western blotting demonstrated that complement C9 and clusterin levels increase 24 hours after thrombin infusion (P < 0.01). Both C9 and clusterin positive cells were found around the injection site. High-dose (100-microg) but not low-dose (25-microg) N-acetylheparin attenuated thrombin-induced brain edema (81.5 +/- 0.4% vs. 83.7 +/- 0.3% in the vehicle, P < 0.05). Behavior was also significantly improved by N-acetylheparin (P < 0.05). In conclusion, thrombin-induced edema formation and neurological deficits were both reduced by N-acetylheparin. This suggests that inhibition may be a novel treatment for the thrombin-induced brain injury that occurs in intracerebral hemorrhage.

Animals↗

PubMed2000

On the mechanism(s) of thrombin induced angiogenesis.

Promotion of tumour progression by thrombin is suggested by several clinical and laboratory observations. A plausible explanation for this effect of thrombin may be related to our previous findings that thrombin is a potent promoter of angiogenesis in the chick chorioallantoic membrane system (CAM) and in the Matrigel system in vivo. In this report we summarise the cellular and molecular actions of thrombin that could be contributing to the activation of angiogenic cascade. Treatment of endothelial cells with thrombin leads to activation of gelatinase A, which may allow for local dissolution of basement membrane, an essential first step of angiogenesis. Similarly thrombin-treated endothelial cells have diminished ability to adhere to collagen type IV and laminin. This new phenotype of endothelial cells can migrate and survive without attachment to extracellular matrix. Thrombin-treatment of endothelial cells increases the vectorial secretion of extracellular matrix proteins, a process essential at the final steps of angiogenesis. In addition, thrombin potentiates the VEGF-induced mitogenesis of endothelial cells. This can be explained by the upregulation of the VEGF receptors (KDR & flt-1) by thrombin treatment. All the aforementioned effects of thrombin are receptor mediated, dose-dependent and require only brief exposure of endothelial cells to thrombin for these actions of thrombin. The transduction mechanisms involved are via protein kinase C (PKC) and MAP-kinase pathways.

Animals↗

PubMed1989

Thrombin-induced platelet aggregation involves an indirect proteolytic cleavage of aggregin by calpain.

5'-p-Fluorosulfonylbenzoyl adenosine (FSBA), a nucleotide analog of ADP, has been shown to inhibit ADP-induced shape change, aggregation and exposure of fibrinogen binding sites concomitant with covalent modification of a single surface membrane polypeptide of Mr 100,000 (aggregin). Since thrombin can aggregate platelets which have been modified by FSBA and are refractory to ADP, we tested the hypothesis that thrombin-induced platelet aggregation might involve cleavage of aggregin. At a low concentration of thrombin (0.05 U/ml), platelet aggregation, exposure of fibrinogen receptors and cleavage of aggregin in FSBA-modified platelets did not occur, indicating ADP dependence. In contrast, incubation of [3H]FSBA-labeled intact platelets with a higher concentration of thrombin (0.2 U/ml) resulted in cleavage of radiolabeled aggregin, aggregation, and exposure of fibrinogen binding sites. Under identical conditions, aggregin in membranes isolated from [3H]FSBA-labeled platelets was not cleaved by thrombin. Thrombin-induced platelet aggregation and cleavage of aggregin were concomitantly inhibited by a mixture of 2-deoxy-D-glucose, D-gluconic acid 1,5-lactone, and antimycin A. These results suggest that thrombin cleaves aggregin indirectly by activating an endogeneous protease. Thrombin is known to elevate intracellular Ca2+ concentration and thereby activates intracellular calcium dependent thiol proteases (calpains). In contrast to serine protease inhibitors, calpain inhibitors including leupeptin, antipain, and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (chelator of Ca2+) inhibited platelet aggregation and cleavage of aggregin in [3H]FSBA-labeled platelets. Leupeptin, at a concentration of 10-20 microM, used in these experiments, did not inhibit the amidolytic activity of thrombin, thrombin-induced platelet shape change, or the rise in intracellular Ca2+. Purified platelet calpain II caused aggregation of unmodified and FSBA-modified platelets and cleaved aggregin in [3H]FSBA-labeled platelets as well as in isolated membranes. The latter is in marked contrast to the action of thrombin on [3H]FSBA-labeled membranes. Thus, thrombin-induced platelet aggregation may involve intracellular activation of calpain which proteolytically cleaves aggregin thus unmasking latent fibrinogen receptors, a necessary prerequisite for platelet aggregation.

Adenosine↗

PubMed1992

Organ-derived microvessel endothelial cells exhibit differential responsiveness to thrombin and other growth factors.

To investigate the relationship between endothelial cells and organ-associated vascular physiology, microvascular endothelial cells were isolated from murine brain, lung, and liver tissues. During culture, these endothelial cells maintained certain differentiated characteristics common to all endothelial cells, but also showed organ-specific characteristics, with distinct patterns of responsiveness to various growth factors. Microvascular endothelial cells from all organs responded to endothelial cell growth factor (ECGF), but lung (LE-1) and brain (MBE-12) endothelial cells showed different responsiveness to thrombin (10-60 nM), combinations of thrombin and ECGF, or thrombin and extracellular matrix. Liver sinusoidal endothelial cells (HSE) were relatively unresponsive to thrombin, but were the most responsive of the endothelial cells to EGF. Endothelial cells isolated from lung and brain, where fluxes in vascular permeability are observed following injury, showed dramatic morphological alterations in response to nanomolar concentrations of thrombin. These cells also exhibited the highest amount of 125I-thrombin binding at these concentrations. Scatchard analysis of 125I-thrombin binding indicated that LE cells have the highest affinity for thrombin, followed by MBE, with HSE exhibiting significantly lower affinity. The binding of 125I-thrombin to these cells was inhibited by the TR-9 monoclonal antibody directed against fibroblast high-affinity thrombin receptors involved in thrombin-stimulated mitogenesis. The results suggest that the differences in growth stimulation observed between organ-derived endothelial cells in response to thrombin, ECGF, and EGF may relate to differential expression of receptors for these factors. These observations demonstrate yet another aspect of the functional heterogeneity of the microvascular endothelium.

Animals↗

PubMed1983

Thrombin generation in normal plasma enriched with purified coagulation factors.

During the intrinsic coagulation of normal platelet-rich plasma only 11% of the prothrombin is converted to thrombin. Complete conversion of prothrombin to thrombin occurs only via the extrinsic pathway (1). Addition of purified prothrombin to normal plasma to double or triple its concentration, doubled or tripled the amount of the generated thrombin as determined by the thrombin elution assay (1), so that the percentage of the proenzyme which was converted to thrombin remained the same. At the same time the activated partial thromboplastin time (APTT) was prolonged. Proportionality of the amount of the generated thrombin to the amount of prothrombin added and a delay in the appearance of thrombin activity was also observed with the thrombin generation test. Normalization of the APTT was observed when factor IX was added together with prothrombin. Addition of factor IX or X to normal plasma shortened the APTT but did not increase the amount of prothrombin which was converted to thrombin as determined by both the thrombin elution assay and the thrombin generation test. Further experiments indicated that (a) more factor X is activated per mg tissue factor than per mg of activated partial thromboplastin and (b) more thrombin is generated per unit of factor Xa in the presence of tissue factor than in the presence of activated partial thromboplastin. Thus, the two pathways differ not only by the mechanism of factor X activation but also by the extent to which prothrombin is activated by factor Xa.

Blood Coagulation↗

PubMed1986

The presence of plasma proteins facilitates the uptake of 125I-thrombin by the rabbit thoracic aorta endothelium in vitro.

Various purified proteins, protein derivatives and two polysaccharides were added individually to a physiological medium in order to effect uptake of 125I-thrombin by the rabbit aorta endothelium. Over a wide range of concentration (0.004-40 mg/ml), the presence of either purified rabbit or bovine albumin during thrombin uptake encouraged an increase (70-110%) in 125I-thrombin binding by the endothelium and subendothelium compared to uptake by aorta segments in the absence of added protein. Pretreatment of aorta segments with albumin before incubation with 125I-thrombin in the absence of albumin did not encourage thrombin uptake to the same extent as having 125I-thrombin and albumin together. Purified human transferrin, rabbit IgG, chicken ovalbumin or denatured bovine casein could replace albumin to produce a similar enhancement of thrombin uptake. Replacing active concentrations of albumin by either reduced-carboxymethylated albumin, defatted albumin, plasmin-treated or thermolysin-treated albumin also caused an increase (50-130%) in thrombin binding, whereas replacement by acid-hydrolysed albumin or with polyglutamic acid was either ineffective or even inhibitory. Lysine-modified or arginine-modified albumins caused a small enhancement (14-32%) and no enhancement of thrombin uptake, respectively. Dextran, at low concentration (0.04-0.4 mg/ml) did not influence thrombin uptake, and at higher concentration (4-40 mg/ml) caused a decrease in uptake by both the endothelium and subendothelial layers. Low concentration of dextran sulphate inhibited thrombin uptake to 20-30% of control values. These data express the importance of accompanying protein in the response of the vascular endothelium during binding of thrombin. The possibility that other protein-cell interactions may be similarly influenced by macromolecular solutes is also discussed.

Animals↗

PubMed1992

Thrombin inactivation and the effects of antithrombin and heparin in a recirculating Langendorff preparation.

A recirculating Langendorff preparation was developed in order to study the effects of antithrombin III (AT) and heparin on thrombin inactivation in the capillary bed. Using this technique, rat hearts were shown to inhibit approximately 50% of the recirculated thrombin, while surface-bound thrombin displayed no enzymatic activity towards recirculated fibrinogen. By displacing thrombin bound to the endothelial surface with Polybrene, thrombin enzymatic activity was found to be equally active against a synthetic chromogenic substrate (S-2238) and fibrinogen. This indicates that thrombin bound to the capillary endothelial surface is not coagulantly active, but, when displaced from the endothelium, its coagulant activity is retained. Hearts pretreated with AT or heparin had no effect on thrombin inhibition or surface-bound thrombin, although there was an uptake of both substances to the heart. These findings are in contrast with the results obtained from large vessel preparations, where pretreatment with AT increased thrombin inhibition and also demonstrated lower levels of surface-bound thrombin, and where heparin eluted endogenous AT from the endothelium. It is concluded that AT plays a more important role in the inhibition of thrombin on large vessels than in the microcapillary system where thrombomodulin is the major inactivator of thrombin.

Animals↗

PubMed2005

Thrombin mediated migration of osteogenic cells.

Given that thrombin is ubiquitously expressed at sites of vascular injury, and that osteogenic cells express receptors for thrombin, we questioned whether thrombin could attract osteogenic cells to a bony wound. Using a scratch wound assay, thrombin stimulated a significant increase in migration of osteogenic cultures of primary marrow cells. This effect was dependent on thrombin proteolytic activity; however, thrombin was unable to stimulate the migration of a more differentiated marrow-derived osteogenic cell line. To better understand the role of thrombin in osteoprogenitor migration, we developed an osteoprogenitor migration assay that combines a modified Boyden chamber with a bone nodule assay. Primary cells that migrated through the transwell filter in the presence of thrombin formed significantly more bone nodules compared to the condition without thrombin. This was not due to proliferation or differentiation effects of thrombin. In contrast, thrombin was unable to stimulate an increase in the number of nodules for the more differentiated osteogenic cell line. Thus, our results suggest that thrombin exhibits differential motogenic effects on osteogenic cells depending on their differentiation state. The cell migration/bone nodule assay described here is the first assay that can be specifically used to examine the effects of factors on the migration of osteoprogenitor cells, particularly those derived from primary populations.

Actins↗

PubMed2000

Inhibition of arterial thrombosis by a peptide ligand of the thrombin receptor.

Thrombin plays an important role in promoting arterial thrombosis by platelet activation and by catalyzing fibrin formation. Use of thrombin inhibitors that block both the platelet-activating and fibrin formation properties of thrombin are associated with hemostasis. This problem might be overcome by developing agents that block only the platelet-activating property of thrombin. Because the platelet-activating property of thrombin is mediated by the thrombin receptor, antagonists of the thrombin receptor might be efficacious and potentially safer with regard to bleeding complications. We investigated whether a peptide ligand (AFLARAA) of the thrombin receptor that blocked alpha-thrombin and thrombin receptor activating peptide-induced platelet aggregation could inhibit thrombosis. A partially occlusive thrombus was generated by application of electric current in rabbit carotid artery. In control animals, the artery was completely occluded within 42+/-12 min after the current was discontinued. When the thrombin receptor activating peptide antagonist was given (100 micromol/kg as an IV bolus followed by 900 micromol/kg infusion for a period of 180 min) starting at the time the current was stopped, blood flow remained patent throughout the infusion period and for an additional 60 min after the infusion was stopped. The antithrombotic effect of the antagonist peptide was not associated with increased bleeding tendency, as judged by the amount of blood adsorbed by a gauze pad placed in a surgical incision extending to the muscle tissue and by a standard template bleeding time. These results indicate that thrombin receptor antagonist peptides can be used as antithrombotic agents.

Animals↗