Abstract 645: Heparin’s Effects on Vascular Cells Require Transmembrane Receptor 184A

Introduction: Though heparin has been used in the clinic for decades, molecular mechanism(s) underlying heparin’s functions independent of anti-coagulation are still unclear. Transmembrane protein 184A (TMEM184A) is conserved, yet its physiological and molecular functions remain unknown. There are few studies reporting its expression and potential role in membrane trafficking in exocrine cells, and involvement in signaling during male germ cell differentiation. Sequence analysis reveals that the C terminal domain includes a putative binding site for heparin. We have previously shown that heparin decreases proliferation in vascular smooth muscle cells (VSMCs) by inducing expression of MKP-1 that decreases Elk-1 and ERK activation. Hypothesis: We hypothesized that TMEM184A plays a role in mediating heparin effects in vascular cells. Methods and Results: We observed TMEM184A expression in vascular cells through immunofluorescence and western blotting using three primary antibodies against different regions in TMEM184A, and visualized cells with confocal microscopy. To investigate whether heparin effects were dependent on TMEM184A, VSMCs were electroporated with 20 μg/ml control or TMEM184A shRNA. Control and knockdown VSMCs were treated with 200 μg/ml heparin for 20 min followed by 2 μg/ml platelet-derived growth factor (PDGF). Activated ERK or Elk-1 in the nucleus was compared to untreated controls or cells treated with PDGF alone. Quantitative immunofluorescence of over 100 cells for each treatment from at least three independent experiments showed that heparin treatment prior to 20 min PDGF stimulation significantly decreased active ERK by nearly 50% in control shRNA cells compared to cells treated with PDGF alone (20 min PDGF = 100.0% ± 3.76%, heparin pre-treatment = 55.5% ± 2.20%; p<0.001). In TMEM184A knockdown cells, heparin pre-treatment did not decrease ERK activation (20 min PDGF = 100.0% ± 3.27%, heparin pre-treatment = 109.8% ± 3.06%). Similar results were observed for Elk-1. Heparin also did not decrease proliferation in response to PDGF in knockdown VSMCs as shown with BrdU incorporation assays. Conclusions: Our results provide functional evidence that heparin signaling in VSMCs is mediated at least in part by TMEM184A.