scholarly journals Glycosylation of human proteinase-activated receptor-2 (hPAR2): role in cell surface expression and signalling

2002 ◽  
Vol 368 (2) ◽  
pp. 495-505 ◽  
Author(s):  
Steven J. COMPTON ◽  
Sabrina SANDHU ◽  
Suranga J. WIJESURIYA ◽  
Morley D. HOLLENBERG
Keyword(s):  
Cell Surface ◽  
Western Blot ◽  
Molecular Mass ◽  
Cell Line ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mutant Cell ◽  
Surface Expression ◽  
Receptor Expression ◽  
Cell Surface Expression

We have analysed the role of N-linked glycosylation in regulating human proteinase-activated receptor-2 (hPAR2) expression and function. Epitope-tagged wild-type hPAR2 (wt-hPAR2) or hPAR2 that lacked glycosylation sequons (following site-directed mutagenesis) in either the N-terminus [hPAR2N30A (Asn30→Ala)], extracellular loop 2 [ECL2; hPAR2N222Q (Asn222→Gln) or hPAR2N222A (Asn222→Ala)] or both (hPAR2N30A,N222A or hPAR2N30A,N222Q) were expressed in the Chinese-hamster ovary (CHO) fibroblast cell line, Pro5. Western blot analysis of wt-hPAR2 showed mature wt-hPAR2 to have a molecular mass of 55—100kDa, and 33—48kDa following N-glycosidase F deglycosylation. FACS analysis and immunocytochemistry of the wt-hPAR2 and PAR2 mutant cell lines revealed that removal of both glycosylation sequons decreases (50% of wt-hPAR2) cell surface expression. Western blot analysis indicated that both N-linked sites are glycosylated. In functional studies, hPAR2N30A displayed a selective and significant increase in sensitivity towards tryptase. Interestingly, hPAR2N222A displayed a loss in sensitivity towards all PAR2 agonists tested. However, further analysis revealed receptor sensitivity to alanine mutations in this domain, as the more conservative substitution hPAR2N222Q displayed no change in response to PAR2 agonists. hPAR2N30A,N222Q displayed increased sensitivity towards tryptase, but a loss in sensitivity towards trypsin and the synthetic peptide SLIGRL-NH2, although this loss in sensitivity towards trypsin and SLIGRL-NH2 was secondary to changes in cell-surface expression. Finally, expression of sialic-acid-deficient wt-hPAR2 in the CHO Lec2 glycosylation-deficient mutant cell line, showed a 40kDa loss in molecular mass, in addition to a marked and selective increase in sensitivity towards tryptase. We conclude that hPAR2 N-linked glycosylation and sialylation regulates receptor expression and/or signalling.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

scholarly journals Regulation of cell-surface receptors for human interferon-γ on the human histiocytic lymphoma cell line U937

1991 ◽  
Vol 274 (3) ◽  
pp. 775-780 ◽  
Author(s):  
D S Finbloom
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Specific Radioactivity ◽  
Receptor Expression ◽  
Cell Surface Expression ◽  
Cell Surface Receptors ◽  
Minor Role ◽  
Ifn Gamma ◽  
Surface Receptors ◽  
High Specific Radioactivity

Interferon-gamma (IFN gamma) binds to high-affinity receptors on monocytes and is rapidly internalized. This study investigates the ability of the human monocyte-like cell line, U937, to regulate the cell-surface expression of the IFN gamma receptor (IFN gamma R) during endocytosis of ligand. Recombinant IFN gamma was radiolabelled to high specific radioactivity with Bolton-Hunter reagent and used to enumerate IFN gamma R on treated U937 cells. Cells which had internalized IFN gamma for up to 3 h displayed maximal levels of IFN gamma R at all time points tested after all unlabelled IFN gamma had been acid-stripped from the cell at pH 2.78. Therefore there was no evidence of down-modulation of the receptor. After trypsin treatment of the IFN gamma R, the cells were able to synthesize and insert into the cell membrane up to 1000 IFN gamma R molecules/h after a 60 min lag. Since biosynthesis played a minor role during the first 30 min of endocytosis, I examined other possibilities to explain the lack of down-modulation of the receptor. A solubilized-receptor assay revealed the presence of an intracellular pool of receptors equal to about 25% of the number of cell surface receptors. Using trypsin to differentiate between intracellular and surface receptors, I observed that 43% of those receptors that were internalized after a 30 min exposure to IFN gamma (580 molecules) could be recycled back to the plasma membrane. In addition, equal rates of receptor decay (t1/2 = 5 h) were observed in the presence of cycloheximide with or without IFN gamma. All the data taken together suggest that during the first 30 min of endocytosis both the expression of an intracellular source of receptor and recycling of internalized receptors contribute to maintain optimal receptor expression.

CD39/NTPDase1 Variants Identified in Human Neutrophils Regulate Antithrombotic Activity.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3088-3088 ◽  
Author(s):  
Kim E. Olson ◽  
Dianne Pulte ◽  
Marinus Johan Broekman ◽  
Ashley E. Olson ◽  
Joan Drosopoulos ◽  
...  
Keyword(s):  
Cell Surface ◽  
Atpase Activity ◽  
Western Blot ◽  
Transmembrane Domain ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Human Neutrophils ◽  
Platelet Activity ◽  
Total Rna ◽  
Cos Cells

Abstract Blood-borne cellular elements expressing ectonucleotidase activity have been shown to regulate platelet activation and recruitment in response to agonists. In particular, exposure of a platelet releasate to isolated neutrophils (PMN) results in loss of its platelet activating activity in a subsequent assay (Valles et al, J Clin Invest1993, 92:1357–1365). Whereas expression of CD39 on vascular endothelial cells has been well characterized, expression on leukocytes has been less well studied. Freshly prepared lymphocyte and PMN cell populations were evaluated for both cell surface expression of CD39 and ectonucleotidase activity. FACS analysis showed that 98% of PMN were positive for CD39 compared to only 20% of lymphocytes. In addition, neutrophils stained more intensely, indicating the presence of a higher quantity of cell surface-expressed CD39. Interestingly, neutrophils exhibited only 1/3 of the ATPase and 1/2 of the ADPase activities of the same number of lymphocytes, although the latter are thought to have greater antithrombotic capacity. RT-PCR products from total RNA isolated from lymphocytes and PMN were sequenced. This revealed alternately spliced CD39 mRNA species present in PMN at levels equal to that of CD39 mRNA. In contrast, lymphocytes, which showed much higher levels of CD39 mRNA, expressed these variants at much lower levels. RACE analyses of cDNAs generated from total RNA demonstrated two CD39 gene-derived mRNAs. Each was comprised of an alternate 3′ segment lacking the C-terminal transmembrane domain, and distinguished by an internal deletion. Myc- and Flag-tagged constructs expressed in COS cells resulted in cell surface expression of the respectively tagged variants (immunocytochemistry, western blot analyses of plasma membrane preparations). Membrane preparations assayed for enzyme activity revealed no apyrase activity for either molecule expressed alone or together. Co-transfection of CD39 with equal amounts of either construct singly or in combination resulted in a 30-50% decrease in ATPase activity compared to CD39 alone. Similarly, CD39 co-expressed with either construct alone lost 75–90% of its ADPase activity. Unexpectedly, co-transfection of CD39 with both variants together resulted in a 20–40% increase in ADPase activity. Glutaraldehyde cross-linking of membrane preparations from triply transfected COS cells followed by immunoprecipitation and western blot analyses demonstrated the presence of all three species in higher order complexes. Thus, both variants can simultaneously associate with CD39, generating hetero-multimers with altered substrate preference and catalytic efficiency compared to CD39 tetramers. These observations add to our understanding of the regulation of ectonucleotidase activity at the cell surface. The balanced expression of CD39 and its two identified variants may underlie the anti-platelet activity of neutrophils previously reported. The finding that association of CD39 with either construct alone results in near complete loss of ADPase activity with only partial diminution of ATPase activity suggests a possible etiology for a pro-thrombotic phenotype.

Metalloproteinases Are Involved in Lipopolysaccharide– and Tumor Necrosis Factor-–Mediated Regulation of CXCR1 and CXCR2 Chemokine Receptor Expression

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2173-2185 ◽  
Author(s):  
Masud H. Khandaker ◽  
Gordon Mitchell ◽  
Luoling Xu ◽  
Joseph D. Andrews ◽  
Rajkumari Singh ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Cell Surface ◽  
Tumor Necrosis ◽  
Inflammatory Responses ◽  
Proteinase Inhibitors ◽  
Surface Expression ◽  
Receptor Expression ◽  
Cell Surface Expression ◽  
Metalloproteinase Inhibitors ◽  
Necrosis Factor

The neutrophil-specific G-protein–coupled chemokine receptors, CXCR1 and CXCR2, bind with high affinity to the potent chemoattractant interleukin-8 (IL-8). The mechanisms of IL-8 receptor regulation are not well defined, although previous studies have suggested a process of ligand-promoted internalization as a putative regulatory pathway. Herein, we provide evidence for two distinct processes of CXCR1 and CXCR2 regulation. Confocal microscopy data showed a redistribution of CXCR1 expression from the cell surface of neutrophils to internal compartments after stimulation with IL-8, whereas stimulation with bacterial lipopolysaccharide (LPS) or tumor necrosis factor- (TNF-) did not induce CXCR1 internalization but instead mediated a significant loss of membrane-proximal CXCR1 staining intensity. To investigate whether proteolytic cleavage was the mechanism responsible for LPS- and TNF-–induced downmodulation of IL-8 receptors, we tested a panel of proteinase inhibitors. The downmodulation of CXCR1 and CXCR2 by LPS and TNF- was most dramatically inhibited by metalloproteinase inhibitors; 1,10-phenanthroline and EDTA significantly attenuated LPS- and TNF-–induced loss of CXCR1 and CXCR2 cell surface expression. Metalloproteinase inhibitors also blocked the release of CXCR1 cleavage fragments into the cell supernatants of LPS- and TNF-–stimulated neutrophils. In addition, while treatment of neutrophils with LPS and TNF- inhibited IL-8 receptor–mediated calcium mobilization and IL-8–directed neutrophil chemotaxis, both 1,10-phenanthroline and EDTA blocked these inhibitory processes. In contrast, metalloproteinase inhibitors did not affect IL-8–mediated downmodulation of CXCR1 and CXCR2 cell surface expression or receptor signaling. Thus, these findings may provide further insight into the mechanisms of leukocyte regulation during immunologic and inflammatory responses.

scholarly journals Palmitoylation of human proteinase-activated receptor-2 differentially regulates receptor-triggered ERK1/2 activation, calcium signalling and endocytosis

2011 ◽  
Vol 438 (2) ◽  
pp. 359-367 ◽  
Author(s):  
Andrew Botham ◽  
Xiaodan Guo ◽  
Yu Pei Xiao ◽  
Alyn H. Morice ◽  
Steven J. Compton ◽  
...  
Keyword(s):  
Cell Line ◽  
Mutant Cell ◽  
Calcium Signalling ◽  
Surface Expression ◽  
Cysteine Residue ◽  
Receptor Internalization ◽  
Cell Surface Expression ◽  
Extracellular Signal ◽  
Proteinase Activated Receptors ◽  
Kinase Phosphorylation

hPAR2 (human proteinase-activated receptor-2) is a member of the novel family of proteolytically activated GPCRs (G-protein-coupled receptors) termed PARs (proteinase-activated receptors). Previous pharmacological studies have found that activation of hPAR2 by mast cell tryptase can be regulated by receptor N-terminal glycosylation. In order to elucidate other post-translational modifications of hPAR2 that can regulate function, we have explored the functional role of the intracellular cysteine residue Cys361. We have demonstrated, using autoradiography, that Cys361 is the primary palmitoylation site of hPAR2. The hPAR2C361A mutant cell line displayed greater cell-surface expression compared with the wt (wild-type)-hPAR2-expressing cell line. hPAR2C361A also showed a decreased sensitivity and efficacy (intracellular calcium signalling) towards both trypsin and SLIGKV. In stark contrast, hPAR2C361A triggered greater and more prolonged ERK (extracellular-signal-regulated kinase) phosphorylation compared with that of wt-hPAR2 possibly through Gi, since pertussis toxin inhibited the ability of this receptor to activate ERK. Finally, flow cytometry was utilized to assess the rate and extent of receptor internalization following agonist challenge. hPAR2C361A displayed faster internalization kinetics following trypsin activation compared with wt-hPAR2, whereas SLIGKV had a negligible effect on internalization for either receptor. In conclusion, palmitoylation plays an important role in the regulation of PAR2 expression, agonist sensitivity, desensitization and internalization.

scholarly journals Implication of Hypoxia-Inducible Factor-1 (HIF-1) As a New Therapeutic Target in JAK2V617F Positive Myeloproliferative Neoplasms (MPN)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4318-4318 ◽  
Author(s):  
Julian Baumeister ◽  
Nicolas Chatain ◽  
Annika Hubrich ◽  
Caroline Küstermann ◽  
Stephanie Sontag ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Line ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Research Funding ◽  
Myeloproliferative Neoplasms ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Protein Levels

Abstract Myeloproliferative neoplasms (MPN) are a heterogeneous group of malignancies including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The JAK2V617F mutation can be found in 90% of PV and approximately 50% of ET and PMF patients. Hypoxia-inducible factors (HIFs) are master transcriptional regulators of the response to decreases in cellular oxygen levels. Unveiling the function of deregulated HIF-1 signaling in normal and malignant hematopoiesis was the aim of several recent publications, highlighting the importance of HIF-1 for the maintenance of leukemic stem cells (LSCs) in acute and chronic myeloid leukemia (AML/CML). In a JAK2V617F knock-in mouse model and in patients, JAK2V617F was shown to induce the accumulation of reactive oxygen species (ROS) in the hematopoietic stem cell compartment, leading to a stabilization of HIF-1α protein. Further, aberrant STAT5 and PI3K/AKT/mTOR signaling induced HIF-1α expression on the transcriptional and translational level. Ruxolitinib treatment inhibited growth and reduced the expression of HIF-1α and its target gene VEGF in the JAK2V617F human erythroleukemia cell line HEL. In several leukemic cell lines constitutive expression of HIF-1α was reported, even under normoxic conditions. However, it still remains unknown whether HIF-1α plays a role in JAK2V617F positive MPN. In this study, we investigated the role HIF-1α signaling in JAK2V617F positive MPN in vitro. We retrovirally transduced the murine bone marrow cell line 32D with JAK2V617F or JAK2WT. Western blot analysis revealed significant increases in HIF-1α protein levels in JAK2V617F positive cells compared to JAK2WT controls after cultivation in normoxic conditions and this effect was abrogated by treatment with the JAK1/JAK2 inhibitor ruxolitinib. Inhibition of HIF-1, binding to hypoxia response elements (HRE), by low doses of echinomycin (1 nM), significantly impaired proliferation and survival. Using an Annexin-V/7-AAD flow cytometry assay apoptosis was found to be selectively induced in JAK2V617F positive, but not JAK2WT cells after echinomycin treatment. Additionally, BrdU/7-AAD cell cycle analysis revealed that only JAK2V617F positive cells were significantly arrested in G0/1 phase. These findings were consistent with shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F transduced 32D cells in presence but not the absence of HIF-2 antagonist 2. Inhibition of HIF-2 was necessary due to a compensatory increase of HIF-2α protein levels, shown by Western Blot analysis, counteracting HIF-1α-KD mediated effects. We isolated PBMCs and BMMNCs from JAK2V617F positive patients or healthy controls using Ficoll density gradient centrifugation. Echinomycin significantly abrogated the colony formation ability alone and in combination with ruxolitinib. In vitro treatment with echinomycin significantly decreased cell number and viability of 8 JAK2V617F positive BMMNC samples (4 PV, 3 PMF, 1 preMF; p[1nM]=0.0169, p[5nM]=0.0009) and 7 PBMC samples (6 PV, 1 PMF; p[1nM]=0.0156, p[5nM]=0.0156) in a dose-dependent manner. In contrast, PBMCs from 6 healthy donors were unaffected by the treatment. The same effect was observed in heterozygous and homozygous iPS cell-derived progenitors from JAK2V617F positive PV patients, whereas JAK2WT cells were unaffected by the treatment. Collectively, our data indicate that targeting HIF-1 might represent a novel therapeutic approach in classical Philadelphia-chromosome-negative MPN. Disclosures Brümmendorf: Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy.

scholarly journals The PAR2 signal peptide prevents premature receptor cleavage and activation

2019 ◽  
Author(s):  
Belinda Liu ◽  
Grace Lee ◽  
Jiejun Wu ◽  
Janise Deming ◽  
Chester Kuei ◽  
...  
Keyword(s):  
Cell Surface ◽  
Signal Peptide ◽  
Synthetic Peptide ◽  
Surface Expression ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Peptide Ligand ◽  
Protease Activated Receptors ◽  
N Terminus

AbstractUnlike closely related GPCRs, protease-activated receptors (PAR1, PAR2, PAR3, and PAR4) have a predicted signal peptide at their N-terminus, which is encoded by a separate exon, suggesting that the signal peptides of PARs may serve an important and unique function, specific for PARs. In this report, we show that the PAR2 signal peptide, when fused to the N-terminus of IgG-Fc, effectively induced IgG-Fc secretion into culture medium, thus behaving like a classical signal peptide. The presence of PAR2 signal peptide has a strong effect on PAR2 cell surface expression, as deletion of the signal peptide (PAR2ΔSP) led to dramatic reduction of the cell surface expression and decreased responses to trypsin or the synthetic peptide ligand (SLIGKV). However, further deletion of the tethered ligand region (SLIGKV) at the N-terminus rescued the cell surface receptor expression and the response to the synthetic peptide ligand, suggesting that the signal peptide of PAR2 may be involved in preventing PAR2 from intracellular protease activation before reaching the cell surface. Supporting this hypothesis, an Arg36Ala mutation on PAR2ΔSP, which disabled the trypsin activation site, increased the receptor cell surface expression and the response to ligand stimulation. Similar effects were observed when PAR2ΔSP expressing cells were treated with protease inhibitors. Our findings indicated that these is a role of the PAR2 signal peptide in preventing the premature activation of PAR2 from intracellular protease cleavage before reaching the cells surface. The same mechanism may also apply to PAR1, PAR3, and PAR4.

scholarly journals Mytoxin B and Myrothecine A Induce Apoptosis in Human Hepatocarcinoma Cell Line SMMC-7721 via PI3K/Akt Signaling Pathway

Molecules ◽  
2019 ◽  
Vol 24 (12) ◽  
pp. 2291 ◽  
Author(s):  
Huiliang Song ◽  
Yi Fu ◽  
Dan Wan ◽  
Wenjing Xia ◽  
Fengwei Lyu ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Line ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Akt Signaling ◽  
Akt Signaling Pathway ◽  
Hepatocarcinoma Cell ◽  
Apoptotic Protein ◽  
Human Hepatocarcinoma Cell Line ◽  
Hepatocarcinoma Cell Line

Trichothecene macrolides comprise a class of valuable leading compounds in developing anticancer drugs, however, there are few reports concerning their anticancer mechanisms, especially the anticancer mechanism of the 10,13-cyclotrichothecane derivatives that are found mainly in symbiotic fungi. In vitro anticancer activity of two trichothecene macrolides mytoxin B and myrothecine A against the human hepatocarcinoma cell line SMMC-7721 was investigated in the present study. MTT assay showed that mytoxin B and myrothecine A inhibited the proliferation of SMMC-7721 cells in dose- and time-dependent manners. Annexin V-FITC/PI dual staining assay revealed that mytoxin B and myrothecine A both could induce SMMC-7721 cells apoptosis in a dose-dependent manner. The decreased expression level of anti-apoptotic protein Bcl-2 and the increased expression level of pro-apoptotic protein Bax were observed apparently in Western blot analysis. The reduced ratio of Bcl-2/Bax further confirmed the apoptosis-inducing effect of mytoxin B and myrothecine A on SMMC-7721 cells. Moreover, the expression levels of caspases-3, -8, and -9, and cleaved caspases-3, -8, and -9 were all upregulated in both mytoxin B and myrothecine A-treated cells in Western blot analysis, which indicated that both compounds might induce SMMC-7721 cells apoptosis through not only the death receptor pathway but also the mitochondrial pathway. Finally, mytoxin B and myrothecine A were found to reduce the activity of PI3K/Akt signaling pathway that was similar to the effect of LY294002 (a potent and specific PI3K inhibitor), suggesting that both mytoxin B and myrothecine A might induce SMMC-7721 cells apoptosis via PI3K/Akt pathway.

Plasmin Proteolysis of Endothelial Cell and Vessel Wall Associated Tissue Factor Pathway Inhibitor

2001 ◽  
Vol 86 (09) ◽  
pp. 923-928 ◽  
Author(s):  
Paul Stalboerger ◽  
Carmelo Panetta ◽  
Robert Simari ◽  
Noel Caplice
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Cell Surface ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Vessel Wall ◽  
Anticoagulant Activity ◽  
Vascular Cells ◽  
Normal Vessel

SummaryPlasmin is an important protease that mediates clot fibrinolysis and vessel wall extracellular matrix proteolysis. Recently, in vitro studies have suggested that plasmin can cleave and inactivate recombinant TFPI, a major inhibitor of TF-mediated coagulation. We hypothesized that such an interaction may occur in vascular cells expressing TFPI, or in the vessel wall, with implications for thrombolysis. In a series of experiments, we examined the effects of plasmin on cell surface and extracellular matrix (ECM) associated TFPI in endothelial cells (EC) in culture and on EC and smooth muscle cells (SMC) in the vessel wall. Plasmin (0.2 μM) decreased cell surface and matrix associated TFPI activity in cultured endothelial cells by 77 ± 5 % and 69 ± 6% respectively (p < 0.01). Plasminogen, the proenzyme form of plasmin had no such effect on cell surface TFPI or matrix TFPI. Cell surface TFPI antigen measured by fluorescence activated cell sorter (FACS) was also significantly reduced by plasmin. Proteolysis of conditioned medium TFPI was suggested by loss of a ~45kD TFPI on Western Blot analysis following plasmin treatment. Plasmin also proteolysed a ~45kD TFPI protein in the intact ECM of EC, an effect which was inhibited by preincubation with aprotinin, a plasmin inhibitor. Incubation of similar concentrations of plasmin, with homogenates of normal vessel decreased a ~45kD TFPI immunoreactive band on Western blot analysis. Plasmin also decreased surface TFPI activity on frozen sections of normal vessel as measured by an amidolytic assay. Finally, plasmin treatment of atherosclerotic plaque sections caused complete removal of TFPI immunoreactivity associated with luminal EC and intimal SMC, when compared to control treated plaque (n = 3). Together these data suggest that plasmin proteolyses the majority of EC-associated (surface and matrix) TFPI and may remove TFPI from the luminal surface and intima of the vessel wall. TFPI proteolysis in cultured EC was associated with significant reduction in TFPI anticoagulant activity. These data provide evidence that plasmin degradation of TFPI occurs in vascular cells and in the vessel wall and may have implications for rethrombosis following thrombolysis in vivo.

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