Heparin rapidly and selectively regulates protein tyrosine phosphorylationin vascular smooth muscle cells

Aberrant vascular smooth muscle cell (VSMC) hyperplasia is the hallmark of atherosclerosis and restenosis seen after vascular surgery. Heparin inhibit s VSMC proliferation in animal models and in cell culture. To test our hypo thesis that heparin mediates its antiproliferative effect by altering phosp horylation of key mitogenic signaling proteins in VSMC, we examined tyrosin e phosphorylation of cellular proteins in quiescent VSMC stimulated with se rum in the presence or absence of heparin. Western blot analysis with anti- phosphotyrosine antibodies shows that heparin specifically alters the tyros ine phosphorylation of only two proteins (42 kDa and 200 kDa). The 200 kDa protein (p200) is dephosphorylated within 2.5 min after heparin treatment w ith an IC50 that closely parallels the IC50 for growth inhibition. Studies using the tyrosine phosphatase inhibitor, sodium orthovanadate, indicate th at heparin blocks p200 phosphorylation by inhibiting a kinase. Phosphorylat ion of p200 is not altered in heparin-resistant cells, supporting a role fo r p200 in mediating the antiproliferative effect of heparin. Purification a nd sequence analysis indicate that p200 exhibits very high homology to the heavy chain of nonmuscle myosin IIA. The 42 kDa protein, identified as mito gen activated protein kinase (MAPK), undergoes dephosphorylation within 15 min after heparin treatment, and this effect is also not seen in heparin-re sistant cells. The identification of only two heparin-regulated tyrosine ph osphoproteins suggests that they may be key mediators of the antiproliferat ive effect of heparin. I Cell Physiol 178:205-215, 1999. (C) 1999 Wiley-Lis s, Inc.