Elucidating the Mechanism by Which Protamine Negates Heparin's Antiproliferative Effect on Smooth Muscle Cells

Carmen R. Berg
Work conducted in the laboratory of Elazer R. Edelman, MD, PhD*+
*Harvard-MIT Division of Health, Sciences, and Technology, Massachusetts Institute of Technology, Cambridge MA 02139
+Cardiovascular Division, Department of Medicine, Brigham and Women¹s Hospital, Harvard Medical School, Boston Massachusetts, 02115 USA

Fig.1: Complexes formed from 40 mg/ml heparin sodium and 5 mg/ml protamine sulfate in 5% calf serum/DMEM after 72 hours. 40 µl of 1 mg/ml heparin sodium and 5 ml of 1mg/ml protamine sulfate were added to 1 ml of 5% calf serum/DMEM that had been incubated at 37°C for 24 hours. This photograph is taken after 72 hours of incubation together at 37°C. Fig. 2: Complexes formed from 40 µg/ml heparin sodium and 5 mg/ml protamine sulfate in 5% calf serum/DMEM 72 hours after addition to SMCs. Growth-arrested bovine aortic SMCs (passage 7) were released from Go by the addition of 40 µl of 1 mg/ml heparin sodium and 5 µl of 1 mg/ml protamine sulfate with 1 ml of 5% calf serum/DMEM that had been incubated at 37°C for 24 hours. This photograph is taken after 72 hours of incubation at 37°C. Fig. 3: Complexes formed from preincubated 40 µg/ml heparin sodium and 5 µg/ml protamine sulfate in 5% calf serum/DMEM 72 hours after addition to SMCs. 40 µl of 1 mg/ml heparin sodium and 5 ml of 1mg/ml protamine sulfate were preincubated in 1 ml of 5% calf serum/DMEM at 37°C for 24 hours. This solution was added to growth-arrested bovine aortic SMCs. This photograph is taken after 72 hours of incubation together at 37°C. Fig. 4: Complexes formed from 40 µg/ml heparin sodium and 40 µg/ml protamine sulfate in 5% calf serum/DMEM after 72 hours. 40 µl of 1 mg/ml heparin sodium and 40 µl of 1mg/ml protamine sulfate were added to 1 ml of 5% calf serum/DMEM that had been incubated at 37°C for 24 hours. This photograph is taken after 72 hours of incubation together at 37°C.

Fig. 5: Complexes formed from 40 µg/ml heparin sodium and 40 µg/ml protamine sulfate in 5% calf serum/DMEM 72 hours after addition to SMCs. Growth-arrested bovine aortic SMCs (passage 7) were released from Go by the addition of 40 µl of 1 mg/ml heparin sodium and 40 µl of 1mg/ml protamine sulfate with 1 ml of 5% calf serum/DMEM that had been incubated at 37°C for 24 hours. This photograph is taken after 72 hours of incubation at 37°C. Fig.6: Complexes formed from preincubated 40 mg/ml heparin sodium and 40 mg/ml protamine sulfate in 5% calf serum/DMEM 72 hours after addition to SMCs. 40 µl of 1 mg/ml heparin sodium and 40 µl of 1mg/ml protamine sulfate were preincubated in 1 ml of 5% calf serum/DMEM at 37°C for 24 hours. This solution was added to growth-arrested bovine aortic SMCs. This photograph is taken after 72 hours of incubation together at 37°C.