Identification of a heparin-binding protein using monoclonal antibodies that block heparin binding to porcine aortic endothelial cells

Patton, Walter A.; Granzow, C A; Getts, Lori; Thomas, Sunil; Zotter, L M; Gunzel, K A; Lowe-Krentz, Linda J.

doi:10.1042/bj3110461

Cited by 35 publications

(41 citation statements)

References 67 publications

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“…9211 and 9216) were obtained from Cell Signaling Technology (Beverly, MA). HRmAbs were isolated and purified as reported previously (10). Secondary antibodies conjugated to tetramethylrhodamine isothiocyanate (TRITC), Alexa Fluor 488, Alexa Fluor 594, Cy3, and Alexa Fluor 647 (in donkey or bovine with minimal cross-reactivity) were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).…”

Section: Methodsmentioning

confidence: 99%

“…In support of this idea, DUSP1 induction by anti-inflammatory glucocorticoid hormones does increase DUSP1 expression (25,26), and low molecular weight heparin has been reported to decrease peroxide-induced JNK and p38 activity (27). Heparin uptake and many heparin functions likely depend on a heparin/HS receptor.Monoclonal antibodies that block heparin binding to endothelial cells (HRmAbs) are able to mimic heparin responses in VSMCs (10,19,22,24), providing evidence that the protein to which they bind functions as a heparin receptor. The accompanying report identifies TMEM184A as the heparin-interacting protein to which the HRmAbs bind (28).…”

mentioning

confidence: 99%

“…Monoclonal antibodies that block heparin binding to endothelial cells (HRmAbs) are able to mimic heparin responses in VSMCs (10,19,22,24), providing evidence that the protein to which they bind functions as a heparin receptor. The accompanying report identifies TMEM184A as the heparin-interacting protein to which the HRmAbs bind (28).…”

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confidence: 99%

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Heparin Decreases in Tumor Necrosis Factor α (TNFα)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1

Farwell

Kanyi

Hamel

et al. 2016

Journal of Biological Chemistry

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Despite the large number of heparin and heparan sulfate binding proteins, the molecular mechanism(s) by which heparin alters vascular cell physiology is not well understood. Studies with vascular smooth muscle cells (VSMCs) indicate a role for induction of dual specificity phosphatase 1 (DUSP1) that decreases ERK activity and results in decreased cell proliferation, which depends on specific heparin binding. The hypothesis that unfractionated heparin functions to decrease inflammatory signal transduction in endothelial cells (ECs) through heparininduced expression of DUSP1 was tested. In addition, the expectation that the heparin response includes a decrease in cytokine-induced cytoskeletal changes was examined. Heparin pretreatment of ECs resulted in decreased TNF␣-induced JNK and p38 activity and downstream target phosphorylation, as identified through Western blotting and immunofluorescence microscopy. Through knockdown strategies, the importance of heparin-induced DUSP1 expression in these effects was confirmed. Quantitative fluorescence microscopy indicated that heparin treatment of ECs reduced TNF␣-induced increases in stress fibers. Monoclonal antibodies that mimic heparin-induced changes in VSMCs were employed to support the hypothesis that heparin was functioning through interactions with a receptor. Knockdown of transmembrane protein 184A (TMEM184A) confirmed its involvement in heparin-induced signaling as seen in VSMCs. Therefore, TMEM184A functions as a heparin receptor and mediates anti-inflammatory responses of ECs involving decreased JNK and p38 activity.For almost 100 years heparin has been used as an anticoagulant. Specific heparin interactions with proteins important in the anti-clotting system are now well understood. Many heparin binding proteins, including quite a few involved in modulating vascular function, inflammation, and angiogenesis, have been identified (reviewed in Ref. 1). The large number of reports indicating evidence of decreased endogenous heparin and heparan sulfates (HS) 3 in atherosclerosis (in model animals and human disease) led to a proposal that decreases in endogenous heparins might be important in the development of atherosclerosis (2). More recent evidence in support of that hypothesis includes increased heparanase expression in atherosclerosis (reviewed in Ref.3) and increased levels of glycocalyx heparan sulfate in regions of the vasculature where laminar flow decreases the likelihood of atherosclerosis development (4).In addition to heparin, heparin binding proteins typically also bind HS chains on HS proteoglycans (HSPGs). Although the carbohydrate backbones of heparin and HS are identical, modifications and sulfation patterns vary. HS chains have fewer sulfate residues per disaccharide and a lower overall charge, but their widespread expression suggests that HS may provide many in vivo functions identified originally as heparin functions (reviewed in Ref. 5). Heparin binding to growth factors modulates their activity and appears to protect them from degradation...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Heparin Decreases in Tumor Necrosis Factor α (TNFα)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1

Farwell

Kanyi

Hamel

et al. 2016

Journal of Biological Chemistry

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“…50 Although the intracellular location of heparin has not been identified in ECs, some of the internalized heparin is known to proceed to the nucleus of HeLa cells and hepatocytes; and additionally, a putative heparin receptor has been described in ECs. 51 Other specific cellular effects of heparin include the selective inhibition of the mitogenic stimulation of smooth muscle cells by phorbol esters and serum, the suggested mechanism being that heparin selectively represses phorbol ester-inducible activator protein-1-mediated gene expression by interfering with the binding of the heterodimeric Fos-Jun/activator protein-1 transcription factor complex to activator protein-1-like promoter elements. 52 Whether or not heparin can also affect TFPI expression by an interference with the activator protein-1-like consensus sequences present in the 5Ј upstream region of the TFPI gene 53 is a matter of speculation at this moment and requires further investigation.…”

Section: Lupu Et Al Effect Of Heparin On Tfpi In Cultured Ecs 2257mentioning

confidence: 99%

Cellular Effects of Heparin on the Production and Release of Tissue Factor Pathway Inhibitor in Human Endothelial Cells in Culture

Lupu

Poulsen

Roquefeuil

et al. 1999

ATVB

112

113

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Abstract-Tissue factor pathway inhibitor (TFPI), the major downregulator of procoagulant activity of the tissue factor-factor VIIa complex (TF ⅐ FVIIa), is synthesized and constitutively secreted by endothelial cells (ECs). Here we describe the in vitro effects of heparin on the cellular localization, gene expression, and release of TFPI in human ECs in culture. Both unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH; Fragmin) time-dependently induced a significant enhanced secretion of TFPI, paralleled by a redistribution and increase of TFPI on the cell surface and a decrease of intracellular TFPI. Immunogold electron microscopy showed the presence of clusters of TFPI, both on the plasmalemma proper and within cell-surface opened caveolae/enlarged caveolar profiles. Activation of FX by TF ⅐ FVIIa on ECs treated with endotoxin was inhibited by both heparins but to a higher extent by LMWH. Inhibition of protein synthesis by cycloheximide did not reduce the release of TFPI induced by heparin. Long-term incubation (48 hours) resulted in a time-dependent enhanced production of TFPI. After the first 4 to 8 hours, depletion of intracellular TFPI was observed, more significantly with UFH. Northern blot analysis of TFPI mRNA also showed a decrease of the 1.4-kb transcript after 4 hours of incubation with UFH, followed by recovery and an increase over the control level after 24 hours. Incubation of ECs with phorbol ester (PMA) significantly enhanced the secretion of TFPI and increased its activity on the cell surface, probably by preventing invagination of caveolae. Heparin-stimulated release of TFPI decreased significantly in the presence of PMA to a level that was 2.4 times lower than the expected additive value for PMA and UFH separately. Pretreatment of ECs with PMA suppressed a subsequent response to heparin. Altogether, our results suggest that the heparin-induced release of TFPI might involve a more specific mechanism(s) than the previously hypothesized simple displacement of TFPI from the cell surface glycocalyx. We assume that the increased secretion and redistribution of cellular TFPI induced by heparins in ECs in culture can play an important role in the modulation of the anticoagulant properties of the endothelium. Key Words: tissue factor pathway inhibitor Ⅲ human endothelial cells Ⅲ unfractionated heparin Ⅲ low-molecular-weight heparin Ⅲ caveolae E ndothelial cells (ECs) play a central role in the regulation of hemostasis by ensuring the cellular control of both procoagulant and anticoagulant mechanisms. The anticoagulant and profibrinolytic functions predominate in the quiescent state of the endothelium, thus maintaining blood fluidity (for a review, see References 1 and 2). Blood coagulation is initiated when factor VII/VIIa (FVII/VIIa) in plasma gains access to tissue factor (TF) at sites of blood vessel injury, and the resulting TF ⅐ FVIIa complex activates FX to Xa and FIX to IXa, leading to thrombin generation and the formation of a fibrin clot. 3 Although healthy ECs do not express ...

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“…Quantitative measurements of cell motility will be reported separately. Mast cell granules have previously been demonstrated by Azizkhan et al 30 to increase motility of preconfluent endothelial cells. In their experiments, which used a different method of measuring motility, the effect Figure 9.…”

Section: Discussionmentioning

confidence: 89%

Mast Cell Granule Heparin Proteoglycan Induces Lacunae in Confluent Endothelial Cell Monolayers

Lagunoff

Rickard

1999

The American Journal of Pathology

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The addition of rat mast cell granules to confluent bovine pulmonary artery endothelial cell monolayers resulted in the formation of numerous lacunae in the cultures. Several lines of evidence identified heparin proteoglycan as the component of the granule matrix responsible for the effect: presence of the activity in the proteoglycan fraction after chromatography of granule extracts , inhibition of granule activity by digestion with heparinase I , the failure of proteolysis of the proteoglycan fraction with proteinase K to significantly diminish its activity , and the failure of chymase and carboxypeptidase inhibitors to inhibit granule activity. The onset of hole formation was delayed for several hours after granule addition to the culture, and maximal hole formation occurred between 8 and 16 hours and was sustained as long as 24 hours. The lacunae formed by the separation of motile endothelial cells within the monolayer and was not attributable to cell contractile activity or cell loss. Time-lapse video recording showed that the holes were dynamic, individual holes expanding and regressing over a period of hours. Formation of lacunae occurred on gelatin and fibronectin surfaces alike. The presence of active chymase in the granules prevented the action of the proteoglycan. Heparin glycosaminoglycan as distinct from the proteoglycan did not similarly affect the endothelial monolayers but did block the action of granules added subsequently , indicating the likelihood of a heparin-reactive receptor or binding site.

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Identification of a heparin-binding protein using monoclonal antibodies that block heparin binding to porcine aortic endothelial cells

Cited by 35 publications

References 67 publications

Heparin Decreases in Tumor Necrosis Factor α (TNFα)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1

Heparin Decreases in Tumor Necrosis Factor α (TNFα)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1

Cellular Effects of Heparin on the Production and Release of Tissue Factor Pathway Inhibitor in Human Endothelial Cells in Culture

Mast Cell Granule Heparin Proteoglycan Induces Lacunae in Confluent Endothelial Cell Monolayers

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