Heparin has long been known to slow the growth of vascular smooth muscle cells. However, the mechanism(s) by which heparin acts has yet to be resolved. The identification of a putative heparin receptor in endothelial cells with antibodies that blocked heparin binding to the cells provided the means to further examine the possible involvement of a heparin receptor in smooth muscle cell responses to heparin. Immunoprecipitation of a smooth muscle cell protein with the anti-heparin receptor antibodies provided evidence that the protein was present in smooth muscle cells. Experiments with the anti-heparin receptor antibodies indicate that the antibodies can mimic heparin in decreasing PDGF induced thymidine and BrdU incorporation. The anti-heparin receptor antibodies were also found to decrease MAPK activity levels after activation similarly to heparin. These results support the identification of a heparin receptor and its role in heparin effects on vascular smooth muscle cell growth.

Vascular cell responses to exogenous heparin have been documented to include decreased vascular smooth muscle cell proliferation following decreased ERK pathway signaling. However, the molecular mechanism(s) by which heparin interacts with cells to induce those responses has remained unclear. Previously characterized monoclonal antibodies that block heparin binding to vascular cells have been found to mimic heparin effects. In this study, those antibodies were employed to isolate a heparin binding protein. MALDI mass spectrometry data provide evidence that the protein isolated is transmembrane protein 184A (TMEM184A). Commercial antibodies against three separate regions of the TMEM184A human protein were used to identify the TMEM184A protein in vascular smooth muscle cells and endothelial cells. A GFP-TMEM184A construct was employed to determine colocalization with heparin after endocytosis. Knockdown of TMEM184A eliminated the physiological responses to heparin, including effects on ERK pathway activity and BrdU incorporation. Isolated GFP-TMEM184A binds heparin, and overexpression results in additional heparin uptake. Together, these data support the identification of TMEM184A as a heparin receptor in vascular cells.For more than 30 years, heparin has been known to specifically bind to cells in the vasculature and alter their physiology in addition to its well recognized function as an anticoagulant. Heparin binds to many proteins, including numerous growth factors, cytokines, coagulation factors, cell adhesion molecules, growth factor receptors, matrix glycoproteins, and others (for a review, see Ref. 1). In fact, heparin and the closely related glycosaminoglycan heparan sulfate (HS), 4 interact with more than 400 proteins (2). Heparin decreases endothelial cell (EC) inflammatory gene expression and slows vascular smooth muscle cell (VSMC) proliferation (reviewed in Ref. 3). Specifically, ECs bind and endocytose heparin (4, 5), which is followed by decreased inflammatory signaling through NF-B (6) and stress kinase activity (7,8). Heparin binding in VSMCs (9) results in decreases in growth factor-induced ERK signaling (10, 11), inhibition of downstream transcription factor activity (12-14), changes in cell cycle inhibitory factors (15), and decreased proliferation (10, 16).Reports of fluorescent heparin uptake into cells, where it modulated transcription factor function (17), and the requirements of HSPGs for basic growth factor delivery to the nucleus (18) indicate that receptor-mediated uptake of heparin or HS may also be critical for some heparin effects. Similarly, shed HSPG syndecan-1 can be taken up by cells and transported to the nucleus, where it alters histone acetylation (19). HS chains are required for uptake, and this uptake can be inhibited by exogenously added heparin. It is likely that the uptake of highly charged heparin and HS chains involves a receptor to manage transport across the membrane. Although many heparin-interacting proteins have been linked to specific functions, a receptor respon...

A rise in the intracellular concentration of Ca2+-ions in human erythrocytes causes the formation of high-molecular-weight membrane protein polymers, cross-linked by gamma-glutamyl-epsilon-lysine side chain bridges. Cross-linking involves proteins at the cytoplasmic side of the membrane (band 4.1, spectrin, and band 3 materials) and the reaction is catalyzed by the intrinsic transglutaminase. This enzyme is regulated by Ca2+-ions and it exits in a latent form in normal cells. The protein polymer, isolated from the membranes of Ca2+-loaded intact human red cells, is heterogeneous in size and may contain as many as 6 moles of gamma-glutamyl-epsilon-lysine cross-links per 100,000 gm of protein. Synthetic compounds, which either compete against the epsilon-lysine cross-linking functionalities of the protein substrates (eg, histamine, aminoacetonitrile, cystamine) or directly inactivate the transamidase (eg, cystamine), inhibit the membrane polymerization reaction in intact human erythrocytes. They also interfere with the Ca2+-induced irreversible shape change from discocyte to echinocyte and inhibit the irreversible loss of membrane deformability. Thus, the transamidase-catalyzed production of gamma-glutamyl-epsilon-lysine cross-links in the membrane may be a common denominator in these cellular manifestations.

The binding of heparin or heparan sulphate to a variety of cell types results in specific changes in cell function. Endothelial cells treated with heparin alter their synthesis of heparan sulphate proteoglycans and extracellular matrix proteins. In order to identify a putative endothelial cell heparin receptor that could be involved in heparin signalling, anti-(endothelial cell) monoclonal antibodies that significantly inhibit heparin binding to endothelial cells were prepared. Four of these antibodies were employed in affinity-chromatographic isolation of a heparin-binding protein from detergent-solubilized endothelial cells. The heparin-binding protein isolated from porcine aortic endothelial cells using four different monoclonal antibodies has an M(r) of 45,000 assessed by SDS/PAGE. The 45,000-M(r) heparin-binding polypeptide is isolated as a multimer. The antibody-isolated protein binds to heparin-affinity columns as does the pure 45,000-M(r) polypeptide, consistent with its identification as a putative endothelial heparin receptor.

Published data provide strong evidence that heparin treatment of proliferating vascular smooth muscle cells results in decreased signaling through the ERK pathway and decreases in cell proliferation. In addition, these changes have been shown to be mimicked by antibodies that block heparin binding to the cell surface. Here we provide evidence that the activity of protein kinase G is required for these heparin effects. Specifically, a chemical inhibitor of protein kinase G, Rp-8-pCPT-cGMS, eliminates heparin and anti-heparin receptor antibody effects on bromodeoxyuridine incorporation into growth factor stimulated cells. In addition, protein kinase G inhibitors decrease heparin effects on ERK activity, phosphorylation of the transcription factor ELK-1, and heparin induced MKP-1 synthesis. Although transient, the levels of cGMP increase in heparin treated cells. Finally, knock down of protein kinase G also significantly decreases heparin effects in growth factor activated vascular smooth muscle cells. Together, these data indicate that heparin effects on vascular smooth muscle cell proliferation depend, at least in part, on signaling through protein kinase G.