Our previous observations provided evidence that a bioactive substance (BAS) with
inhibitory effect on platelet aggregation and with inotropic activity on smooth muscle
preparations is present in aortic wall of rats treated previously with indomethacin.
The ability to inhibit platelet aggregation was used for monitoring its purification
and partial characterization. The original sample was extracted by rinsing aortic
rings (1.5 mg dried rings) in buffer Krebs (300 μl) for 40 minutes at room temperature.
The substance was purified by gel filtration (Bio-gel P-30, Sephadex G-100, Sephadex
G-75) and ion exchange chromagra-phy (DEAE cellulose). In the last method, salt gradient
elution was performed. Further purification by Sephadex G-50 resulted in removal of
90% of the contaminating substances without a loss of inhibitory activity. The main
peak of both chromatographic procedures was analyzed on SDS-PAGE and PAGE with denaturing
solvents. The substance was evident by Coomassie Brilliant Blue and periodic acid
Schiff staining.
In order to determine if the BAS was susceptible to proteolysis, an aliquot of the
original sample (29 ug total protein) was incubated with trypsin (final concentration
0.3 μg/ml) and with chymotrypsin (final concentration 3 μg/ml). The BAS activity was
not detected. An aliquot of the same original sample was incubated with neuraminidase
(final concentration 1.2 units). The BAS activity was detected.
The substance appeared to be stable for at least 18 hours at room temperature and
2 hours at 37°C, In addition it was stable over a pH range between 6.8 to 8.6, showing
an anionic behaviour.
The protein concentration of this substance determined by the method of Lowry was
1 μg/ml
Partial characterization supports the conclusion that the substance present in aortic
wall of rats is a homogeneous protein, which has a molecular size estimated at 55-65
kDa.
