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L F Brass

Publications and source records attributed to L F Brass.

At least 19 recordsLinked to original sources
PubMed2003

Contact-dependent signaling during the late events of platelet activation.

Signaling events downstream from collagen receptors and G protein-coupled receptors are responsible for the initiation and extension of platelet plug formation. This creates the platelet plug and hopefully results in the cessation of bleeding. It is not, however, all that is required for hemostasis, and growing evidence is emerging that the perpetuation of a stable hemostatic plug requires additional intracellular signaling. At least part of this process is made possible by the persistent close contacts between platelets that can only occur after the onset of aggregation. This review discusses several examples of such signaling mechanisms that help to perpetuate the platelet plug in a contact-dependent manner, including outside-in signaling through integrins, signaling though Eph kinases and ephrins, and the role of CD40L.

Adenosine Triphosphatases↗
PubMed2001

Gene induction by coagulation factor Xa is mediated by activation of protease-activated receptor 1.

Cell signaling by coagulation factor Xa (Xa) contributes to pro-inflammatory responses in vivo. This study characterizes the signaling mechanism of Xa in a HeLa cell line that expresses protease-activated receptor 1 (PAR-1) but not PAR-2, -3, or -4. Xa induced NF-kappaB in HeLa cells efficiently but with delayed kinetics compared to thrombin. This delay caused no difference in gene expression patterns, as determined by high-density microarray analysis. Both proteases prominently induced the angiogenesis-promoting gene Cyr61 and connective tissue growth factor. Inhibition of PAR-1 cleavage abolished MAP kinase phosphorylation and gene induction by Xa, demonstrating that Xa signals through PAR-1 and not through a novel member of the PAR family. Activation of cell surface prothrombin with the snake venom enzyme Ecarin also produced PAR-1-dependent signaling. However, though the response to Ecarin was completely blocked by the thrombin inhibitor hirudin, the response to Xa was not. This suggests that the Xa response is not mediated by locally generated thrombin. The concentration dependence of Xa for PAR-1 activation is consistent with previously characterized Xa-mediated PAR-2 signaling, suggesting that local concentration of Xa on the cell surface, rather than sequence-specific recognition of the PAR scissile bond, determines receptor cleavage. This study demonstrates that PAR-1 cleavage by Xa can elicit the same cellular response as thrombin, but mechanistic differences in receptor recognition may be crucial for specific roles for Xa in signaling during spatial or temporal separation from thrombin generation.

Antithrombins↗
PubMed2001

Evaluation of the substrate specificity of human mast cell tryptase beta I and demonstration of its importance in bacterial infections of the lung.

Human pulmonary mast cells (MCs) express tryptases alpha and beta I, and both granule serine proteases are exocytosed during inflammatory events. Recombinant forms of these tryptases were generated for the first time to evaluate their substrate specificities at the biochemical level and then to address their physiologic roles in pulmonary inflammation. Analysis of a tryptase-specific, phage display peptide library revealed that tryptase beta I prefers to cleave peptides with 1 or more Pro residues flanked by 2 positively charged residues. Although recombinant tryptase beta I was unable to activate cultured cells that express different types of protease-activated receptors, the numbers of neutrophils increased >100-fold when enzymatically active tryptase beta I was instilled into the lungs of mice. In contrast, the numbers of lymphocytes and eosinophils in the airspaces did not change significantly. More important, the tryptase beta I-treated mice exhibited normal airway responsiveness. Neutrophils did not extravasate into the lungs of tryptase alpha-treated mice. Thus, this is the first study to demonstrate that the two nearly identical human MC tryptases are functionally distinct in vivo. When MC-deficient W/W(v) mice were given enzymatically active tryptase beta I or its inactive zymogen before pulmonary infection with Klebsiella pneumoniae, tryptase beta I-treated W/W(v) mice had fewer viable bacteria in their lungs relative to zymogen-treated W/W(v) mice. Because neutrophils are required to combat bacterial infections, human tryptase beta I plays a critical role in the antibacterial host defenses of the lung by recruiting neutrophils in a manner that does not alter airway reactivity.

Amino Acid Sequence↗
PubMed2001

Protease activated receptors: theme and variations.

The four PAR family members are G protein coupled receptors that are normally activated by proteolytic exposure of an occult tethered ligand. Three of the family members are thrombin receptors. The fourth (PAR2) is not activated by thrombin, but can be activated by other proteases, including trypsin, tryptase and Factor Xa. This review focuses on recent information about the manner in which signaling through these receptors is initiated and terminated, including evidence for inter- as well as intramolecular modes of activation, and continuing efforts to identify additional, biologically-relevant proteases that can activate PAR family members.

Amino Acid Sequence↗
PubMed2001

Neutrophil proteases can inactivate human PAR3 and abolish the co-receptor function of PAR3 on murine platelets.

Three members of the protease-activated receptor family, PAR1, PAR3 and PAR4, are activated when thrombin cleaves the receptor N-terminus, exposing a tethered ligand. Proteases other than thrombin can also cleave PAR family members and, depending upon whether this exposes or removes the tethered ligand, either activate or disable the receptor. For example, on human platelets PAR1 is disabled by cathepsin G, although aggregation still occurs because cathepsin G can activate PAR4. The present studies examine the interaction of cathepsin G and a second neutrophil protease, elastase, with PAR3 using two model systems: COS-7 cells transfected with human PAR3 and mouse platelets, which express PAR3 and PAR4, but not PAR1. In contrast to human platelets, cathepsin G did not aggregate murine platelets, and prevented their activation only at low thrombin concentrations. Elastase had no effect on thrombin responses in mouse platelets, but when added to COS cells expressing human PAR3, both cathepsin G and elastase prevented activation of phospholipase C by thrombin. Notably, this inhibition occurred without loss of the binding sites for two monoclonal antibodies that flank the tethered ligand on human PAR3. We therefore conclude that 1) exposure to cathepsin G disables signaling through human PAR3, and prevents murine PAR3 from serving its normal role, which is to facilitate PAR4 cleavage at low thrombin concentrations, 2) elastase disables human, but not murine, PAR3, 3) in contrast to human PAR4, mouse PAR4 will not support platelet aggregation in response to cathepsin G, and 4) the inactivation of human PAR3 by cathepsin G and elastase involves a mechanism other than amputation of the tethered ligand domain. These results extend the range of possible interactions between PAR family members and proteases, and provide further support for species-specific differences in the interaction of these receptors with proteases other than thrombin.

Amino Acid Sequence↗
PubMed2000

The alpha subunits of Gz and Gi interact with the eyes absent transcription cofactor Eya2, preventing its interaction with the six class of homeodomain-containing proteins.

Yeast two-hybrid techniques were used to identify possible effectors for the heterotrimeric G protein G(z) in human bone marrow cells. Eya2, a human homologue of the Drosophila Eya transcription co-activator, was identified. Eya2 interacts with activated Galpha(z) and at least one other member of the Galpha(i) family, Galpha(i2). Interactions were confirmed in mammalian two-hybrid and glutathione S-transferase fusion protein pull-down assays. Regions of Eya2-mediating interaction were mapped to the C-terminal Eya consensus domain. Eya2 is an intrinsically cytosolic protein that is translocated to the nucleus by members of the Six homeodomain-containing family of proteins. Activated Galpha(z) and Galpha(i2) prevent Eya2 translocation and inhibit Six/Eya2-mediated activation of a reporter gene controlled through the MEF3/TATA promoter. Although G proteins are known to regulate the activity of numerous transcription factors, this regulation is normally achieved indirectly via one or more intermediates. We show here a novel functional regulation of a co-activator directly by G protein subunits.

Animals↗
PubMed2000

Loss of signaling through the G protein, Gz, results in abnormal platelet activation and altered responses to psychoactive drugs.

Heterotrimeric G proteins mediate the earliest step in cell responses to external events by linking cell surface receptors to intracellular signaling pathways. G(z) is a member of the G(i) family of G proteins that is prominently expressed in platelets and brain. Here, we show that deletion of the alpha subunit of G(z) in mice: (i) impairs platelet aggregation by preventing the inhibition of cAMP formation normally seen at physiologic concentrations of epinephrine, and (ii) causes the mice to be more resistant to fatal thromboembolism. Loss of G(zalpha) also results in greatly exaggerated responses to cocaine, reduces the analgesic effects of morphine, and abolishes the effects of widely used antidepressant drugs that act as catecholamine reuptake inhibitors. These changes occur despite the presence of other G(ialpha) family members in the same cells and are not accompanied by detectable compensatory changes in the level of expression of other G protein subunits. Therefore, these results provide insights into receptor selectivity among G proteins and a model for understanding platelet function and the effects of psychoactive drugs.

Animals↗
PubMed2000

Thrombin responses in human endothelial cells. Contributions from receptors other than PAR1 include the transactivation of PAR2 by thrombin-cleaved PAR1.

The recent identification of two new thrombin receptors, PAR3 and PAR4, led us to re-examine the basis for endothelial cell responses to thrombin. Human umbilical vein endothelial cells (HUVEC) are known to express PAR1 and the trypsin/tryptase receptor, PAR2. Northern blots detected both of those receptors and, to a lesser extent, PAR3, but PAR4 message was undetectable and there was no response to PAR4 agonist peptides. To determine whether PAR3 or any other receptor contributes to thrombin signaling in HUVEC, PAR1 cleavage was blocked with two selective antibodies and PAR1 activation was inhibited with the antagonist, BMS200261. The antibodies completely inhibited HUVEC responses to thrombin, but BMS200261 was only partly effective, even though separate studies established that the antagonist completely inhibits PAR1 signaling at the concentrations used. Since peptides mimicking the PAR1 tethered ligand domain can also activate PAR2, we asked whether the remaining thrombin response in the presence of the antagonist could be due in part to the intermolecular transactivation of PAR2 by cleaved PAR1. Evidence that transactivation can occur was obtained in COS-7 cells co-expressing PAR2 and a variant of PAR1 that can be cleaved, but not signal. There was a substantial response to thrombin only in cells expressing both receptors. Conversely, in HUVEC, complete blockade of the thrombin response by the PAR1 antagonist occurred only when signaling through PAR2 was also blocked. From these observations we conclude that 1) PAR1 is the predominant thrombin receptor expressed in HUVEC and cleavage of PAR1 is required for endothelial cell responses to thrombin; 2) although PAR3 may be expressed, there is still no evidence that it mediates thrombin responses; 3) PAR4 is not expressed on HUVEC; and 4) transactivation of PAR2 by cleaved PAR1 can contribute to endothelial cell responses to thrombin, particularly when signaling through PAR1 is blocked. Such transactivation may limit the effectiveness of PAR1 antagonists, which compete with the tethered ligand domain rather than preventing PAR1 cleavage.

Cells, Cultured↗
PubMed2000

CXCR4 on human endothelial cells can serve as both a mediator of biological responses and as a receptor for HIV-2.

It has been shown that deletion of the chemokine receptor, CXCR4, causes disordered angiogenesis in mouse models. In the present studies, we examined the distribution and trafficking of CXCR4 in human endothelial cells, tested their responses to the CXCR4 ligand, SDF-1, and asked whether endothelial cell CXCR4 can serve as a cell surface receptor for the binding of viruses. The results show that CXCR4 is present on endothelial cells from coronary arteries, iliac arteries and umbilical veins (HUVEC), but expression was heterogeneous, with some cells expressing CXCR4 on their surface, while others did not. Addition of SDF-1 caused a rapid decrease in CXCR4 surface expression. It also caused CXCR4-mediated activation of MAPK, release of PGI(2), endothelial migration, and the formation of capillary-like structures by endothelial cells in culture. Co-culture of HUVEC with lymphoid cells that were chronically infected with a CD4-independent/CXCR4-tropic variant of HIV-2 resulted in the formation of multinucleated syncytia. Formation of the syncytia was inhibited by each of several different CXCR4 antibodies. Thus, our findings indicate: (1) that CXCR4 is widely expressed on human endothelial cells; (2) the CXCR4 ligand, SDF-1, can evoke a wide variety of responses from human endothelial cells; and (3) CXCR4 on endothelial cells can serve as a receptor for isolates of HIV that can utilize chemokine receptors in the absence of CD4.

Anti-HIV Agents↗
PubMed1999

PAR1 activation initiates integrin engagement and outside-in signalling in megakaryoblastic CHRF-288 cells.

To better understand the means by which cells such as human platelets regulate the binding of the integrin alphaIIbbeta3 to fibrinogen, we have examined agonist-initiated inside-out and outside-in signalling in CHRF-288 cells, a megakaryoblastic cell line that expresses alphaIIbbeta3 and the human thrombin receptor, PAR1. The results show several notable similarities and differences. (1) Activation of PAR1 caused CHRF-288 cells to adhere and spread on immobilized fibrinogen in an alphaIIbbeta3-dependent manner, but did not support the binding of soluble fibrinogen or PAC-1, an antibody specific for activated alphaIIbbeta3. (2) Direct activation of protein kinase C with PMA or disruption of the actin cytoskeleton with low concentrations of cytochalasin D also caused CHRF-288 cells to adhere to fibrinogen. (3) Despite the failure to bind soluble fibrinogen, activation of PAR1 in CHRF-288 cells caused phosphoinositide hydrolysis, arachidonate mobilization and the phosphorylation of p42MAPK, phospholipase A2 and the Rac exchange protein, Vav, all of which occur in platelets. PAR1 activation also caused an increase in cytosolic Ca2+, which, when prevented, blocked adhesion to fibrinogen. (4) Finally, as in platelets, adhesion of CHRF-288 cells to fibrinogen was followed by a burst of integrin-dependent ('outside-in') signalling, marked by FAK phosphorylation and a more prolonged phosphorylation of p42MAPK. However, in contrast to platelets, adhesion to fibrinogen had no effect on Vav phosphorylation. Collectively, these observations show that signalling initiated through PAR1 in CHRF-288 cells can support alphaIIbbeta3 binding to immobilized ligand, but not the full integrin activation needed to bind soluble ligand. This would suggest that there has been an increase in integrin avidity without an accompanying increase in affinity. Such increases in avidity are thought to be due to integrin clustering, which would also explain the results obtained with cytochalasin D. The failure of alphaIIbbeta3 to achieve the high affinity state in CHRF-288 cells was not due to the failure of PAR1 activation to initiate a number of signalling events that normally accompany platelet activation nor did it prevent at least some forms of outside-in signalling. However, at least one marker of outside-in signalling, the augmentation of Vav phosphorylation seen during platelet aggregation, did not occur in CHRF-288 cells.

Blood Platelets↗
PubMed1999

Megakaryocyte precursors, megakaryocytes and platelets express the HIV co-receptor CXCR4 on their surface: determination of response to stromal-derived factor-1 by megakaryocytes and platelets.

Thrombocytopenia is a late complication of human immunodeficiency virus (HIV) infection. The chemokine receptor CXCR4 has been shown to be a co-receptor for lymphocyte-tropic HIV-1 strains. CXCR4 is also a natural receptor for the chemokine SDF-1. We have previously shown that CXCR1 and CXCR2 are present on megakaryocytes and platelets. Although interleukin-8 (IL-8) and other chemokines that bind to these two receptors do not activate platelets, they are able to inhibit megakaryocytopoiesis, presumably through these receptors. We therefore examined whether CXCR4 is present on developing and mature megakaryocytes and on platelets. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated the presence of CXCR4 message. Immature and mature alphaIIbbeta3+ megakaryocytes, and platelets were also positive for CXCR4 by flow cytometric studies using a CXCR4-specific antibody. We then tested whether SDF-1 can affect the biology of these cells. CD34+ cells and immature alphaIIbbeta3+ cells responded to SDF-1 as indicated by Ca2+ mobilization and chemotaxis. However, mature megakaryocytes failed to demonstrate either of these responses, in spite of their continued ability to bind 125I-SDF-1. Further, SDF-1 failed to inhibit megakaryocyte colony growth. Platelets bound 125I-SDF-1 with a K(D) similar to the affinity seen for CXCR4 on other cells, yet SDF-1 did not aggregate washed platelets nor augment aggregation by low-dose ADP or thrombin. SDF-1 also failed to stimulate Ca2+ mobilization, granular release or expression of P-selectin in platelets. Accordingly, although our studies demonstrate that CD34+ precursors, megakaryocytes and platelets all express CXCR4 and bind SDF-1, biological effects were only demonstrable of SDF-1 on CD34+ precursors. The potential biological implications of CXCR4 expression on maturing megakaryocytes and platelets in normal individuals and following HIV infection are discussed.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Identification of CXCR4 domains that support coreceptor and chemokine receptor functions.

The interaction of the chemokine stromal cell-derived factor 1 (SDF-1) with its receptor CXCR4 is vital for cell trafficking during development, is capable of inhibiting human immunodeficiency virus type 1 (HIV-1) utilization of CXCR4 as a coreceptor, and has been implicated in delaying disease progression to AIDS in vivo. Because of the importance of this chemokine-chemokine receptor pair to both development and disease, we investigated the molecular basis of the interaction between CXCR4 and its ligands SDF-1 and HIV-1 envelope. Using CXCR4 chimeras and mutants, we determined that SDF-1 requires the CXCR4 amino terminus for binding and activates downstream signaling pathways by interacting with the second extracellular loop of CXCR4. SDF-1-mediated activation of CXCR4 required the Asp-Arg-Tyr motif in the second intracellular loop of CXCR4, was pertussis toxin sensitive, and did not require the distal C-terminal tail of CXCR4. Several CXCR4 mutants that were not capable of binding SDF-1 or signaling still supported HIV-1 infection, indicating that the ability of CXCR4 to function as a coreceptor is independent of its ability to signal. Direct binding studies using the X4 gp120s HXB, BH8, and MN demonstrated the ability of HIV-1 gp120 to bind directly and specifically to the chemokine receptor CXCR4 in a CD4-dependent manner, using a conformationally complex structure on CXCR4. Several CXCR4 variants that did not support binding of soluble gp120 could still function as viral coreceptors, indicating that detectable binding of monomeric gp120 is not always predictive of coreceptor function.

Amino Acid Sequence↗
PubMed1998

Increased platelet sensitivity to ADP in mice lacking platelet-type 12-lipoxygenase.

Arachidonic acid metabolism is one of several mechanisms culminating in the production of an agonist for platelet activation and recruitment. Although the proaggregatory role of thromboxane A2, a product of the aspirin-inhibitable cyclooxygenase, is well established, relatively little is known regarding the biological importance of arachidonic acid metabolism via the 12-lipoxygenase (P-12LO) pathway to 12-hydro(pero)xyeicosatetraenoic acid. We observed that platelets obtained from mice in which the P-12LO gene has been disrupted by gene targeting (P-12LO-/-) exhibit a selective hypersensitivity to ADP, manifested as a marked increase in slope and percent aggregation in ex vivo assays and increased mortality in an ADP-induced mouse model of thromboembolism. The hyperresponsiveness to ADP is independent of dense granule release, cyclooxygenase-derived eicosanoid synthesis, and protein kinase C activity. The addition of 12-hydroxyeicosatetraenoic acid to P-12LO-/- platelet-rich plasma rescues the hyperresponsive phenotype resulting in a diminished ADP-induced aggregation profile. The enhanced ADP sensitivity of P-12LO-/- mice appears to reveal a mechanism by which a product of the P-12LO pathway suppresses platelet activation by ADP.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1998

Identification and characterization of the CXCR4 chemokine receptor in human T cell lines: ligand binding, biological activity, and HIV-1 infectivity.

The CXCR4 chemokine receptor has been shown to respond to the C-X-C chemokine stromal-derived factor (SDF-1) and has recently been shown to be an important coreceptor for HIV-1 infection. In the present paper we have tested a number of human lymphocyte cell lines, including Jurkat, HUT78, CEM, and Sup-T1 for the presence of CXCR4 receptors. We found that these T cell lines bind SDF-1alpha and SDF-1beta with high affinity. The CXCR4 Ab 12G5 inhibited both SDF-1 binding and HIV-1LAI-mediated fusion of CEM. Scatchard analysis revealed the presence of approximately 150,000 SDF-1alpha-binding sites per cell with a Kd between 5 and 10 nM. Cross-competition experiments using unlabeled SDF-1alpha and SDF-1beta revealed that both chemokines are equally capable of displacing their radiolabeled counterparts. Internalization studies with [125]I-SDF-1alpha revealed that Jurkat cells internalized greater than 90% of the ligand by 2 h at 37 degrees C. SDF-1alpha was also chemotactic for Jurkat cells and caused an increase in the rate of extracellular acidification that was half-maximal at 18 nM SDF-1alpha and could be inhibited by pretreatment with the SDF-1 proteins, pertussis toxin, or the Ab 12G5. Finally, SDF-1alpha also caused an increase in the cytosolic Ca2+ concentration in Sup-T1 cells that was abolished by preincubating the cells with pertussis toxin or PMA and inhibited by the Ab 12G5. This molecular characterization of CXCR4 receptors should prove useful in clarifying receptor interaction with SDF-1 proteins and with HIV-1 glycoprotein, with the ultimate aim of targeting the viral interaction for therapeutic intervention.

Binding Sites↗
PubMed1998

Reaction of mast cell proteases tryptase and chymase with protease activated receptors (PARs) on keratinocytes and fibroblasts.

Protease activated receptors (PARs) compose a family of G protein signal transduction receptors activated by proteolysis. In this study, the susceptibility of PARs expressed on human keratinocytes and dermal fibroblasts to the human mast cell proteases tryptase and chymase was evaluated. PAR activation was measured by monitoring cytosolic [Ca2+] in cells loaded with the fluorescent Ca2+ probe Fura-2. Tryptase produced transient cytosolic Ca2+ mobilization in keratinocytes, but not in fibroblasts. Ca2+ mobilization in keratinocytes required enzymatically active tryptase, demonstrated desensitization, and was blocked by pretreatment of cells with the PAR-2 peptide agonist SLIGKV, trypsin, or the phospholipase inhibitor U73122. Heparin, a GAG that binds to tryptase, stabilizing its functional form, also inhibited tryptase-induced Ca2+ mobilization. The maximal response elicited by tryptase was smaller than that observed upon treatment of keratinocytes with trypsin, a known activator of PAR-2, and keratinocytes made refractory to tryptase by pretreatment with the protease remained responsive to trypsin. Pretreatment of keratinocytes with thrombin, an activator of PAR-1 and -3 (thrombin receptors), had no detectable effect on the tryptase or trypsin responses. These data suggest that in keratinocytes tryptase may be activating a subpopulation of PAR-2 receptors. Treatment of keratinocytes or fibroblasts with human chymase did not produce Ca2+ mobilization, nor did it affect Ca2+ mobilization produced by trypsin. However, chymase pretreatment of fibroblasts rapidly inhibited the ability of these cells to respond to thrombin. Inhibition was dependent on chymase enzymatic activity and was not significantly affected by the presence of heparin. This finding is consistent with studies indicating that PAR-1 may be susceptible to proteases with chymotrypsin-like specificity. These results suggest that the proteases tryptase and chymase secreted from mast cells in skin may affect the behavior of surrounding cells by the hydrolysis of PARs expressed by these cells.

Anticoagulants↗
PubMed1998

Injury-related factors and conditions down-regulate the thrombin receptor (PAR-1) in a human neuronal cell line.

Previous studies have demonstrated that thrombin can induce potent effects on neural cell morphology, biochemistry, and viability. Nearly all of these effects are mediated by proteolytic activation of the thrombin receptor (PAR-1). Mechanisms of PAR-1 regulation in several nonneural cell types have been shown to be novel and cell type specific; however, little is known about PAR-1 regulation in neural cells. In the present study, PAR-1 cell surface expression and regulation were examined in a transformed retinoblast (Ad12 HER 10) cell line using radioiodinated anti-PAR-1 monoclonal antibodies ATAP2, which recognizes intact and cleaved receptors, and SPAN12, which is specific for the intact form of the receptor. Scatchard analysis revealed high-affinity, specific binding to a single affinity class of receptors: K(D) = 3.13 and 5.25 nM, Bmax = 190.1 and 67.8 fmol/mg of protein for 125I-ATAP2 and 125I-SPAN12, respectively. Specificity for PAR-1 was confirmed by demonstrating rapid and near complete decreases for both antibodies following treatment with thrombin or PAR-1 activating peptide (SFLLRN). Differential antibody binding was used to demonstrate rapid and near complete thrombin-induced PAR-1 cleavage and internalization, with protein synthesis-dependent replacement of intact receptors occurring over longer time intervals, but only minimal recycling of cleaved receptors. A variety of factors and conditions were screened for their effects on PAR-1 expression. Significant decreases in PAR-1 expression were induced by the protein kinase C activator phorbol 12-myristate 13-acetate (87% at 3 h), the phospholipid inflammatory mediator lysophosphatidic acid (32% at 3 h), and the injury-related condition hypoglycemia (64 and 100% at 24 h in the absence and presence of dibutyryl cyclic AMP, respectively). The effect of hypoglycemia was shown by RNase protection to be at least partially pretranslational. Finally, thrombin's ability to enhance hypoglycemia-induced cell killing correlated temporally with PAR-1 cell surface expression.

Antibodies, Monoclonal↗
PubMed1998

Differential expression of functional protease-activated receptor-2 (PAR-2) in human vascular smooth muscle cells.

The protease-activated family of G protein-coupled receptors includes PAR-1 and PAR-3, which are activated by thrombin, and PAR-2, which is activated by trypsin and tryptase. PAR-2 has recently been shown to be expressed in human endothelial cells. In the present studies, we have examined the expression of PAR-2 in other cells, particularly vascular smooth muscle, and tested whether the receptors are functional. The results show that PAR-2 is present in human aorta and coronary artery smooth muscle cells, as well as in arteries traversing the walls of the small intestine. It was also detected in human keratinocytes, sweat glands, intestinal smooth muscle, and intestinal epithelium, but not at all in myocardial smooth muscle and only inconsistently in intestinal veins and venules. Activation of aortic smooth muscle cells in culture with PAR-2 peptide agonists caused a transient increase in the cytosolic Ca2+ concentration. In contrast, PAR-2 mRNA could not be detected in saphenous vein smooth muscle cells, and the same cells placed in culture showed little, if any, response to the PAR-2 agonist peptides. These observations show that PAR-2 is widely distributed in human vascular smooth muscle, particularly in arteries. However, this is not a universal finding and at least some venous smooth muscle cells, including those in saphenous veins, apparently do not express the receptor in detectable amounts.

Animals↗