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PubMed1996

Thrombin activates the sarcolemmal Na(+)-H+ exchanger. Evidence for a receptor-mediated mechanism involving protein kinase C.

Thrombin can activate the plasma membrane Na(+)-H+ exchanger in a variety of noncardiac cells. We have studied (1) the effect of thrombin on the activity of the sarcolemmal Na(+)-H+ exchanger in freshly isolated quiescent ventricular myocytes from the adult rat heart and (2) the signaling mechanism(s) underlying any effect. Reverse-transcription polymerase chain reaction analysis revealed thrombin receptor mRNA expression in a myocyte-enriched cell preparation. As an index of Na(+)-H+ exchanger activity, acid efflux rates (JHS) were determined in single myocytes (n = 4 to 11 per group) loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 after two consecutive intracellular acid pulses (induced by transient exposure to 20 mmol/L NH4Cl) in bicarbonate-free medium. At a pHi of 6.9, JH did not change significantly during the second pulse relative to the first in control cells. However, when the second pulse occurred in the presence of 0.2, 1, or 5 U/mL thrombin, JH increased by 30%, 62% (P < .05), and 87% (P < .05), respectively. A hexameric thrombin receptor-activating peptide (SFLLRN) mimicked the effect of thrombin and increased JH by 73% (P < .05) at 25 mumol/L. In contrast, an inactive control peptide (FLLRN) was without effect at 25 mumol/L. In cells pretreated with 100 nmol/L GF109203X or 5 mumol/L chelerythrine (protein kinase C inhibitors), neither 5 U/mL thrombin nor 25 mumol/L SFLLRN produced a significant increase in JH. In the presence of 10 mumol/L HOE-694 (a Na(+)-H+ exchanger inhibitor), pHi did not recover after an acid load, even during exposure to 5 U/mL thrombin or 25 mumol/L SFLLRN, confirming that the Na(+)-H+ exchanger was the primary acid efflux mechanism under the conditions used. Neither 5 U/mL thrombin nor 25 mumol/L SFLLRN affected resting pHi and Ca2+ or background acid loading. We conclude that (1) adult rat ventricular myocytes express a functional thrombin receptor, whose stimulation results in increased activity of the sarcolemmal Na(+)-H+ exchanger, and (2) this effect appears to occur through a protein kinase C-mediated mechanism.

Animals↗
PubMed2005

Characterization and functional activity of thrombin receptors in the human lens.

PURPOSE: To investigate the expression of thrombin receptors in the human lens, the activation of downstream signaling pathways, and the ability of thrombin to regulate lens cell growth. METHODS: Thrombin receptor function in the human lens was determined first by measuring changes in intracellular calcium in response to thrombin and protease-activated receptor-activating peptides (PAR-APs). In the human capsular bag model, cell growth was assessed by phase microscope inspection of the cell coverage of the posterior capsular surface. In the human lens cell line FHL124, it was assessed by [(3)H]thymidine incorporation. Changes in p42/p44 ERK phosphorylation (p-ERK) and protein kinase B (PKB/Akt) phosphorylation (p-Akt) were monitored by Western blot. Reverse transcription-polymerase chain reaction (RT-PCR) applied to isolated lens epithelia and ex vivo capsular bag preparations as well as FHL124 cells determined expression of mRNA for the PARs. RESULTS: Brief exposures to thrombin (10 nM) and PAR1-AP (10 microM) induced an increase in cytosolic calcium in both anterior and equatorial lens cells, but activating peptides for PAR2, -3, and -4 failed to produce responses. Repeated exposure to thrombin produced a significant increase in cell coverage in the capsular bag model and increased [(3)H]thymidine incorporation into FHL124 cells. In the latter, exposure to thrombin (10 nM) and PAR1-AP (10 microM) induced biphasic increases in the phosphorylation of p42/p44 (p-ERK), with peak responses at 20 minutes and 12 hours. Thrombin also produced a 20-fold increase in p-Akt at 12 hours compared with the control, whereas PAR1-AP (10 microM) induced a much smaller response. PAR1-AP did not induce a significant increase in [(3)H]thymidine incorporation and PAR2-AP, PAR3-AP, and PAR4-AP failed to reproduce any of the thrombin-stimulated effects. mPAR1 and -3 were expressed in native lens cells, and this expression was conserved in ex vivo capsular bag preparations as well as in FHL124 cells. CONCLUSIONS: This study identifies thrombin receptors coupled to calcium, ERK, and Akt signaling that modulate growth in native lens tissue and cultured cells, and it appears that the PAR1 subtype is mainly responsible. PAR3 mRNA was also detected, but the receptor itself, if present, was not coupled to the above signaling elements.

Adolescent↗
PubMed2003

Possible roles of thrombin-induced activation of protease-activated receptor 1 in human luteinized granulosa cells.

The presence of thrombin and its receptor, protease-activated receptor 1 (PAR 1), in the ovary suggests that thrombin may regulate ovarian function. In particular, to address the possible role of thrombin in ovulation, a phenomenon displaying mimicry of inflammation, we investigated the effects of thrombin and PAR 1 on the production of inflammation-related substances in human luteinized granulosa cells (LGC). Thrombin stimulated the production of IL-8 and monocyte chemoattractant protein-1 by cultured LGC. The stimulatory effects of thrombin were inhibited by both inhibitors of thrombin (hirudin and PPACK) and a protein kinase C inhibitor (calphostin C). The PAR 1 agonist, SFLLRN, also stimulated the production of IL-8 and monocyte chemoattractant protein-1. Thrombin and SFLLRN stimulated the geletinase activities of LGC, the effect of both being inhibited by hirudin and PPACK. Immunocytochemical study showed that thrombin and SFLLRN induced translocation of nuclear factor kappaB to the nucleus from the cytoplasm in LGC. Expression of PAR 1 mRNA was detected in LGC by RT-PCR analysis. These findings suggest that thrombin plays physiological roles in ovulation by enhancing the production of chemoattractive and gelatinolytic substances by granulosa cells by a mechanism involving PAR 1.

Adult↗
PubMed1995

The response to thrombin of human neutrophils: evidence for two novel receptors.

Human alpha-thrombin was a chemoattractant for human neutrophils yielding a maximal response of similar magnitude to that observed with formyl-Met-Leu-Phe. The observed chemotaxis was not due to stimulation of the proteolytically activated thrombin receptor since: (1) this receptor was not detected by flow cytometry; (2) the inactive thrombin mutant Ser195-->Ala elicited a chemotactic response indistinguishable from that caused by wild-type thrombin; (3) antibodies to the cleavage site of the proteolytically activated receptor did not affect thrombin-induced chemotaxis; (4) a thrombin receptor activating peptide (TRAP) failed to stimulate chemotaxis. These data indicate the existence of a thrombin receptor for neutrophil chemotaxis which is not activated by proteolysis. In addition, although wild-type and ser195-->Ala thrombin did not cause an increase in intracellular Ca2+, a Ca2+ response to TRAP was observed with neutrophils from some donors. The TRAP-induced increase in Ca2+ was reproducible, dose dependent and specific. The use of alanine-substituted peptides demonstrated that the Ca2+ response was due to TRAP stimulation of a receptor other than the proteolytically activated thrombin receptor. Thus, it is necessary to re-evaluate the assumption made in previous studies that responses to TRAP are mediated by the proteolytically activated thrombin receptor.

Adult↗
PubMed1999

Thrombin-induced p65 homodimer binding to downstream NF-kappa B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion.

We investigated the mechanisms by which proinflammatory mediator, thrombin, released during intravascular coagulation and tissue injury, induces ICAM-1 (CD54) expression in endothelial cells. Stimulation of HUVEC with thrombin resulted in dose- and time-dependent increases in ICAM-1 mRNA and cell surface expression and in ICAM-1-dependent endothelial adhesivity toward polymorphonuclear leukocytes. Transient transfection of endothelial cells with ICAM-1 promoter luciferase reporter gene (ICAM-1LUC) constructs indicated that deletion of upstream NF-kappa B site (-533 bases from translation start site) had no effect on thrombin responsiveness, whereas mutation/deletion of downstream NF-kappa B site (-223 bases from the translation start site) prevented the activation of ICAM-1 promoter, indicating that the downstream NF-kappa B site is critical for thrombin inducibility. NF-kappa B-directed luciferase activity increased approximately 3-fold when cells transfected with the plasmid pNF-kappa BLUC containing five copies of consensus NF-kappa B site linked to a minimal adenovirus E1B promoter-luciferase gene were exposed to thrombin, indicating that activation of NF-kappa B was essential for thrombin response. Gel supershift assays demonstrated that thrombin induced binding of NF-kappa Bp65 (Rel A) to downstream NF-kappa B site of the ICAM-1 promoter. Thrombin receptor activation peptide, a 14-amino-acid peptide representing the new NH2 terminus of proteolytically activated receptor-1, mimicked thrombin's action in inducing ICAM-1 expression. These data indicate that thrombin activates endothelial ICAM-1 expression and polymorphonuclear leukocyte adhesion by NF-kappa Bp65 binding to the downstream NF-kappa B site of ICAM-1 promoter after proteolytically activated receptor-1 activation.

Cell Line↗
PubMed2000

Thrombin stimulates cell proliferation in human fibroblast-like synoviocytes in nuclear factor-kappaB activation and protein kinase C mediated pathway.

OBJECTIVE: To examine the effect of thrombin on nuclear factor (NF)-kappaB activation and cell proliferation in synovial cells from patients with rheumatoid arthritis (RA). METHODS: Using cultured human synovial cells from patients with RA, electrophoretic mobility shift assay, [3H]thymidine incorporation assay, and MTT assay were performed. We tested the upregulatory effects of thrombin on NF-kappaB activation and cell proliferation. The effect of thrombin on degradation of IkappaB was analyzed by Western blot. RESULTS: Thrombin transiently induced DNA-binding activity of NF-kappaB, followed by degradation of IkappaBalpha, but not IkappaBbeta1. Moreover, synovial cell proliferation was stimulated by thrombin in a dose dependent manner. The kinetics of synovial cell proliferation induced by thrombin were almost parallel to those of NF-kappaB activation. Supershift analysis revealed that thrombin induced DNA-binding complexes were made up principally of the p65 and p50 Rel family members. Further, protein kinase C inhibitor calphostin C repressed thrombin induced NF-kappaB activation and cell proliferation in synovial cells. CONCLUSION: Thrombin stimulates synovial cell proliferation involved in NF-kappaB activation, at least in part, through a protein kinase C mediated pathway, possibly indicating that thrombin plays an important role in synovial hyperplasia in RA.

Antithrombins↗
PubMed2001

Neuroprotective signal transduction in model motor neurons exposed to thrombin: G-protein modulation effects on neurite outgrowth, Ca(2+) mobilization, and apoptosis.

Thrombin, the ultimate protease in the blood coagulation cascade, mediates its known cellular effects by unique proteolytic activation of G-protein-coupled protease-activated receptors (PARs), such as PAR1, PAR3, and PAR4, and a "tethered ligand" mechanism. PAR1 is variably expressed in subpopulations of neurons and largely determines thrombin's effects on morphology, calcium mobilization, and caspase-mediated apoptosis. In spinal cord motoneurons, PAR1 expression correlates with transient thrombin-mediated [Ca(2+)](i) flux, receptor cleavage, and elevation of rest [Ca(2+)](i) activating intracellular proteases. At nanomolar concentrations, thrombin retracts neurites via PAR1 activation of the monomeric, 21 kDa Ras G-protein RhoA, which is also involved in neuroprotection at lower thrombin concentrations. Such results suggest potential downstream targets for thrombin's injurious effects. Consequently, we employed several G-protein-specific modulators prior to thrombin exposure in an attempt to uncouple both heterotrimeric and monomeric G-proteins from motoneuronal PAR1. Cholera toxin, stimulating Gs, and lovastatin, which blocks isoprenylation of Rho, reduced thrombin-induced calcium mobilization. In contrast, pertussis toxin and mastoparan, inhibiting or stimulating G(o)/G(i), were found to exacerbate thrombin action. Effects on neuronal rounding and apoptosis were also detected, suggesting therapeutic utility may result from interference with downstream components of thrombin signaling pathways in human motor neuron disorders, and possibly other neurodegenerative diseases. Published 2001 John Wiley & Sons, Inc.

Anticholesteremic Agents↗
PubMed1994

Localization and characterization of an alpha-thrombin-binding site on platelet glycoprotein Ib alpha.

Glycoprotein (GP) Ib alpha is required for expression of the highest affinity alpha-thrombin-binding site on platelets, possibly contributing to platelet activation through a pathway involving cleavage of a specific receptor. This function may be important for the initiation of hemostasis and may also play a role in the development of pathological vascular occlusion. We have now identified a discrete sequence in the extracytoplasmic domain of GP Ib alpha, including residues 271-284 of the mature protein, which appears to be part of the high affinity alpha-thrombin-binding site. Synthetic peptidyl mimetics of this sequence inhibit alpha-thrombin binding to GP Ib as well as platelet activation and aggregation induced by subnanomolar concentrations of the agonist; they also inhibit alpha-thrombin binding to purified glycocalicin, the isolated extracytoplasmic portion of GP Ib alpha. The inhibitory peptides interfere with the clotting of fibrinogen by alpha-thrombin but not with the amidolytic activity of the enzyme on a small synthetic substrate, a finding compatible with the concept that the identified GP Ib alpha sequence interacts with the anion-binding exosite of alpha-thrombin but not with its active proteolytic site. The crucial structural elements of this sequence necessary for thrombin binding appear to be a cluster of negatively charged residues as well as three tyrosine residues that, in the native protein, may be sulfated. GP Ib alpha has no significant overall sequence homology with the thrombin inhibitor, hirudin, nor with the specific thrombin receptor on platelets; all three molecules, however, possess a distinct region rich in negatively charged residues that appear to be involved in thrombin binding. This may represent a case of convergent evolution of unrelated proteins for high affinity interaction with the same ligand.

Adenosine Triphosphate↗
PubMed1995

Role of platelets, thrombin, integrin IIb-IIIa, fibronectin and von Willebrand factor on tumor adhesion in vitro and metastasis in vivo.

The role of platelets and thrombin was examined in tumor cell adhesion in vitro and metastasis in vivo. Adhesion of tumor cells to platelets was inhibited by agents inhibiting platelet integrin IIb-IIIa receptor occupancy (MoAb 10E5 and tetrapeptide RGDS) as well as IIb-IIIa ligands (polyclonal antibodies against fibronectin and von Willebrand factor (vWF)). In vivo murine experimental pulmonary metastasis (tail vein injection) could be inhibited by antibody-induced induction of thrombocytopenia and reconstituted by simultaneous injection of human platelets. Preincubation of human platelets with MoAb 10E5 (vs IIb-IIIa) inhibited reconstitution of metastasis. Thrombin activates tumor cell adhesion to platelets by activating platelets as well as tumor cells. Thrombin-activated tumor cells also enhance their adhesion to endothelial cells as well as adhesive ligands fibronectin and vWF. Experimental pulmonary metastasis is enhanced 4-400 fold by preinfusion of thrombin into mice or 10-160 fold by prior treatment of tumor cells with thrombin. The in vitro and in vivo effects of thrombin were mimicked by thrombin receptor activation peptides SFLLRNPNDKYEPF and SFLLRN. Nine of nine tumor cell lines have the seven transmembrane spanning thrombin receptor detected by the polymerase chain reaction. Thus, both platelets and thrombin contribute to experimental tumor metastasis by fostering and enhancing IIb-IIIa integrin platelet interaction with tumor cells. Since many tumor cells generate thrombin, it is proposed that tumor cells may autoactivate a metastatic phenotype.

Amino Acid Sequence↗
PubMed1998

Protease-activated receptor 1 (PAR-1) is required and rate-limiting for thrombin-enhanced experimental pulmonary metastasis.

Thrombin-treated tumor cells induce a metastatic phenotype in experimental pulmonary murine metastasis. Thrombin binds to a unique protease-activated receptor (PAR-1) that requires N-terminal proteolytic cleavage for activation by its tethered end. A 14-mer thrombin receptor activation peptide (TRAP) of the tethered end induces the same cellular changes as thrombin. Four murine tumor cells (Lewis lung, CT26 colon CA, B16F10 melanoma, and CCL163 fibroblasts) contain PAR-1, as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). B16F10 cells did not contain the two other thrombin receptors, PAR-3 and glycoprotein Ib. TRAP-treated B16F10 tumor cells enhance pulmonary metastasis 41- to 48-fold (n = 17). Thrombin-treated B16F10 cells transfected with full-length murine PAR-1 sense cDNA (S6, S7, S14, and S22) enhanced their adhesion to fibronectin 1.5- to 2.4-fold (n = 5, P <.04), whereas thrombin-treated wild-type cells do not. S6 (adhesion index, 1.5-fold) and S14 (index, 2.4-fold) when examined by RT-PCR and Northern analysis showed minimal expression of PAR-1 for S6 over wild-type and considerable expression for S14. Immunohistochemistry showed greater expression of PAR-1 for S14 compared with wild-type or empty-plasmid transfected cells. In vivo experiments with the thrombin-treated S14 transfectant showed a fivefold to sixfold increase in metastases compared with empty-plasmid transfected thrombin-treated naive cells or S6 cells (n = 20, P =.0001 to .02). Antisense had no effect on thrombin-stimulated tumor mass. Thus, PAR-1 ligation and expression enhances and regulates tumor metastasis.

Animals↗
PubMed1998

Contact activation of the plasma coagulation cascade. III. Biophysical aspects of thrombin-binding anticoagulants.

A biophysical model linking fibrin polymerization kinetics (following release from a thrombin-fibrinogen complex), coagulation time, and competitive inhibition of thrombin illustrates the utility of thrombin-binding ligands as anticoagulants in blood collection applications. The resulting mathematical relationship connecting fibrinogen, ligand, and thrombin concentrations was tested against experimentally observed anticoagulation of whole, platelet-poor porcine plasma induced by short, single-stranded DNA oligonucleotides originally found to bind thrombin by screening combinatorial libraries. The thrombin-fibrinogen dissociation constant Ks served as the single adjustable parameter in a least-squares fitting of the model to experimental anticoagulation data. Best-fit Ks values corroborated microM values measured in plasma-free systems, and application of the model to a ligand challenge to the intrinsic pathway of plasma coagulation corroborated nM endogenous thrombin concentrations measured in porcine blood activated by endotoxin insult in vivo. The model fit to data suggests that only about 20% conversion of blood fibrinogen to fibrin is required to coagulate (gel) porcine plasma. This prediction is consistent with the common clinical laboratory observation of latent fibrin formation in "serum" separated from blood before fibrinogen is fully converted to fibrin. It was concluded that the thrombin-binding anticoagulation model was a reasonable simulation of the situation in which an initial bolus of either exogenous or endogenous thrombin is rapidly partitioned between fibrinogen-bound and ligand-bound forms with little or no additional free thrombin created over time.

Animals↗
PubMed2000

Peptide modified gold-coated polyurethanes as thrombin scavenging surfaces.

Thin layers of gold were deposited on polyurethane film and chemisorbed with three peptides having an N-terminal cysteine: Cys-Pro-Arg, Cys-(L)Phe-Pro-Arg, and Cys-(D)Phe-Pro-Arg. The ability of these surfaces to act as thrombin scavengers was evaluated. The peptides are related to the known thrombin inhibitor Phe-Pro-Arg chloromethyl ketone and were shown to have significant thrombin inhibitory activity in solution. Attachment of the peptides to gold was confirmed by water contact angle and X-ray photoelectron spectroscopy measurements. Thrombin adsorption from a buffer and plasma was investigated, and chromogenic substrate assays were carried out for thrombin activity on the surfaces and in the supernatant following adsorption. The data suggest that the peptide-modified surfaces are able to adsorb thrombin with high affinity from a buffer and that thrombin is taken up selectively from plasma. The Cys-(D)Phe-Pro-Arg modified surfaces showed particularly high affinity for thrombin. It was also found that the activity of thrombin adsorbed on the peptide surfaces was inhibited, and inhibition was greatest on the Cys-(D)Phe-Pro-Arg surface. We concluded that the peptide surfaces may have potential as antithrombogenic materials via their ability to scavenge and inhibit thrombin generated as a result of blood-material contact.

Adsorption↗
PubMed1986

Thrombin uptake and inhibition on endothelium and surfaces with a stable heparin coating: a comparative in vitro study.

The endothelial lining and two differently heparin-coated surfaces were compared in vitro regarding thrombin uptake and inhibition. One heparin surface was based on stabilized ionic binding of heparin, the other on covalent binding of partially degraded heparin. Both heparin surfaces have previously been shown to have pronounced thromboresistant properties. The two heparinized polyethylene surfaces and the endothelial surface of segments of the porcine aorta were studied. After exposure to the surfaces, thrombin disappeared from the solution and appeared bound to the surfaces. The disappearance rate of thrombin from the solution was the same on exposure to the endothelium and the covalently bonded heparin surface, but less following exposure to the ionically bonded heparin surface. The thrombin activity appearing on the endothelium was lower than on the heparin surfaces, indicating that the endothelium exerted a slow thrombin inhibiting capacity. On exposure of the thrombin-loaded endothelium to plasma, thrombin was rapidly inhibited. Thrombin bound to the covalently bonded heparin surface was inhibited at a slower rate than on the ionically bonded surface, but still faster than the rate at which free thrombin was inhibited in nonheparinized plasma. It is concluded that the endothelium and stabilized heparin coatings bind thrombin and accelerate its inhibition by plasma.

Animals↗
PubMed1992

Measurement of the rate of thrombin production in human plasma in contact with different materials.

Thrombin production in plasma in contact with various materials was consistent with a first-order autocatalytic model (d[T]/dt = kp[T]; [T] = thrombin concentration, t = time, kp = thrombin production rate constant) since the initial portion of a semilogarithmic plot of thrombin concentration against time was linear. Thrombin concentration was measured in clotting plasma (phospholipid enhanced or platelet-rich plasma) using a fluorogenic substrate (BMCA) by aliquot sampling at various intervals or more conveniently by monitoring cumulative fluorescence. The latter was generated by the action, on BMCA incubated in the clotting plasma, of the thrombin as it was generated. The thrombin concentration was determined from the first derivative of the S-shaped cumulative fluorescence curve. kp was greater for glass (7.92 x 10(-3) cm/s) than for the other materials (polypropylene, polystyrene, polyethylene and PVA; kp approximately 3.1 x 10(-3) cm/s) in plasma with cephalin without flow. A kp for heparin-PVA could not be determined since the thrombin concentration was too low to be quantified. A larger difference between polyethylene and PVA was noted with platelet-rich plasma without flow while lower values (1.0 x 10(-3) cm/s) were noted in a flow system but at a higher surface to volume ratio. The first-order rate constant can be used in simple models relating production of thrombin at a wall of a tube to its mass transfer away from the wall in flowing blood. One such model predicts that the concentration of thrombin at the wall should become infinite at the point in the tube when the mass transfer coefficient equals kp. According to this model, kp on the order of 10(-4) cm/s would be a useful target for a nonthrombogenic material.

Amino Acid Sequence↗
PubMed1987

Relationship of thrombin-stimulated arachidonic acid release and metabolism to mitogenesis and phosphatidylinositol synthesis.

Thrombin and certain prostaglandins are both capable of stimulating the proliferation of cultured cells. Since thrombin stimulates the release and metabolism of arachidonic acid, the precursor of prostaglandins, we examined the relationship between this release and metabolism and the stimulation of cell division in cultured fibroblasts. We also examined the role of prostaglandin synthesis in thrombin-stimulated phosphatidylinositol synthesis. The data in this report demonstrate that the release and metabolism of arachidonic acid are not necessary for thrombin-stimulated cell division. The presence of a low concentration of chymotrypsin prevented thrombin-stimulated arachidonic acid release and metabolism without affecting the stimulation of cell division. Furthermore, thrombin-stimulated cell division occurred in the presence of indomethacin concentrations that prevented cyclooxygenase-mediated metabolism of arachidonic acid. The following experiments showed that thrombin-stimulated phosphatidylinositol synthesis was brought about by a cyclooxygenase-mediated metabolite(s) of arachidonic acid. Indomethacin inhibited the cyclooxygenase-mediated metabolism of arachidonic acid without affecting the thrombin-stimulated release of arachidonic acid. Indomethacin also inhibited thrombin-stimulated phosphatidylinositol synthesis. The dose dependence of this inhibition paralleled the inhibition by indomethacin of cyclooxygenase-mediated metabolism of arachidonic acid. In addition, prostaglandin F2 alpha stimulated phosphatidylinositol synthesis in the presence of indomethacin concentrations which prevented thrombin-stimulated phosphatidylinositol synthesis.

Animals↗
PubMed1990

Neurite outgrowth activity of protease nexin-1 on neuroblastoma cells requires thrombin inhibition.

Protease nexin-1 (PN-1) is a protein proteinase inhibitor recently shown to be identical with the glial-derived neurite-promoting factor or glial-derived nexin. It has been shown to promote neurite outgrowth in neuroblastoma cells and in sympathetic neurons. The present experiments were designed to further test the hypothesis that this activity on neuroblastoma cells is due to its ability to complex and inhibit thrombin. It has been suggested that PN-1:thrombin complexes might mediate the neurite outgrowth activity of PN-1. However, the present studies showed that such complexes, unlike free PN-1, did not promote neurite outgrowth. The neurite outgrowth activity of PN-1 was only detected in the presence of thrombin or serum (which contains thrombin). PN-1 did not affect the rate or extent of neurite outgrowth that occurred when neuroblastoma cells were placed in serum-free medium. Retraction of neurites by thrombin was indistinguishable in cells whose neurites had been extended in the presence or absence of PN-1. The neurite-promoting activity of PN-1 was inhibited by an anti-PN-1 monoclonal antibody, which blocks its capacity to complex serine proteinases. The plasma thrombin inhibitor, antithrombin III, stimulated neurite outgrowth but only when its thrombin inhibitory activity was accelerated by heparin. The neurite outgrowth activity of both antithrombin III and PN-1 corresponded to their inhibition of thrombin. Together, these observations show that PN-1 promotes neurite outgrowth from neuroblastoma cells by inhibiting thrombin and suggest that this depends on the ability of thrombin to retract neurites.

Amyloid beta-Protein Precursor↗
PubMed1992

Thrombin and histamine rapidly stimulate the phosphorylation of the myristoylated alanine-rich C-kinase substrate in human umbilical vein endothelial cells: evidence for distinct patterns of protein kinase activation.

Human alpha-thrombin and histamine each stimulates protein phosphorylation in human umbilical vein endothelial cells (HUVEC). We have identified the most prominent of these phosphoproteins by immunoprecipitation as the human homolog of the widely distributed myristoylated alanine-rich C-kinase substrate (MARCKS). Stimulation by 0.1-10 U/ml of alpha-thrombin produces a time-dependent, sustained (plateau 3-5 min) level of MARCKS phosphorylation. MARCKS phosphorylation requires thrombin catalytic activity but not receptor binding and is also seen in response to stimulation by a peptide, TR (42-55), that duplicates a portion of the thrombin receptor tethered ligand created by thrombin proteolytic activity. One micromolar histamine, like alpha-thrombin, produces sustained phosphorylation of MARCKS (plateau 3-5 min). In contrast, 100 microM histamine results in rapid but transient MARCKS phosphorylation (peak 1-3 min). HUVEC treated with 100 microM histamine for 5 min can be restimulated by alpha-thrombin but not fresh histamine, suggesting that the histamine receptor was desensitized. MARCKS phosphorylation can also be induced by several exogenous protein kinase C (PKC) activators and both alpha-thrombin- and histamine-induced MARCKS phosphorylation are inhibited by the PKC antagonist staurosporine. However, while prolonged PMA pretreatment ablates histamine-induced MARCKS phosphorylation, the ability of thrombin to induce MARCKS phosphorylation is retained. These findings provide evidence for agonist-specific pathways of protein kinase activation in response to thrombin and histamine in HUVEC.

Cells, Cultured↗
PubMed1995

Thrombin activates mitogen-activated protein kinase in primary astrocyte cultures.

Thrombin is known to evoke numerous inflammatory and proliferative responses in a wide variety of its target cells. Recent studies have demonstrated morphoregulatory and mitogenic effects of thrombin on astroglial cells (astrocytes). The present study deals with thrombin-induced activation of mitogen-activated protein (MAP) kinase in primary cultures of rat astrocytes. Treatment of serum-starved astrocytes with thrombin resulted in a rapid activation of tyrosine (Tyr) phosphorylation of a set of proteins including a prominent one with a molecular mass of 42 kDa (p42). The identity of p42 with MAP kinase was confirmed by MAP kinase-immunoreactivity of isolated [i.e., immunoprecipitated with anti-phosphotyrosine (PY) antibodies] p42 and by increased myelin basic protein (MBP) kinase activity present in MAP kinase immunoprecipitates of thrombin-treated cultures. Pertussis toxin (PTX) pretreatment failed to inhibit thrombin stimulation of p42 phosphorylation, indicating the lack of involvement of PTX sensitive G proteins in the mechanism of activation of MAP kinase by thrombin. Chronic exposure of cultures to phorbol 12-myristate 13-acetate to down-regulate PKC resulted in an attenuation of thrombin-induced p42 Tyr phosphorylation, although H-7, a known PKC inhibitor, failed to block thrombin effect. However, staurosporine, a nonspecific protein kinase inhibitor, prevented the activation of p42 phosphorylation. It is concluded that thrombin induces MAP kinase activation in astrocytes by a mechanism involving a staurosporine-sensitive pathway.

Animals↗