Human Platelet Phospholipase A2 Activity Research Articles

Stimulation of phospholipase A2 activity in human platelets by trypsin and collagen

Type I collagen enhanced human platelet phospholipase A2 activity whether added to platelet-rich plasma or washed platelets. The stimulatory effect of type I collagen on platelet membrane phospholipase A2 activity was also observed in a cell-free system utilizing platelet membranes. The release of arachidonic acid was enhanced by types I and III but not by type V collagen. The activation of platelet phospholipase A2 by type I collagen was inhibited by soybean trypsin inhibitor and mimicked by trypsin. However, type I collagen addition was not associated with any detectable changes in platelet membrane proteins while trypsin altered many proteins. These results point to acid soluble phospholipase A2 activity of platelets as an enzyme activated by type I collagen.

Inhibition of human platelet phospholipase A2 activity by unsaturated fatty acids.

Phospholipase A2 (PLA2; phosphatide 2-acylhydrolase, EC 3.1.1.4) activity from human platelets increases significantly when the enzyme is separated from an endogenous inhibitor(s). The inhibitor, associated mainly with a particulate fraction, has now been identified as a mixture of unsaturated fatty acids. Treatment of the inhibitor with trypsin, RNase, DNase, or heat did not diminish its inhibitory activity, which was extractable by organic solvents. Incubation of PLA2 with phospholipids or various neutral lipids, including saturated fatty acids, had little or no effect on enzymatic activity. In contrast, unsaturated fatty acids such as palmitoleic acid (16:1), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4), all of which were detected in the particulate fraction, or longer chained unsaturated fatty acids inhibited PLA2 activity by 50% at approximately equal to 5 X 10(-7) M. The level of unsaturated fatty acids in the inhibitor fraction was equivalent to approximately equal to 10(-4) M, apparently sufficient to effectively inhibit PLA2 activity. Methylation of unsaturated fatty acids caused a complete loss of inhibitory activity, and subsequent demethylation restored the activity, suggesting that a free carboxyl group was necessary. Inhibition of PLA2 by unsaturated fatty acids appeared to be noncompetitive. PLA2 absolutely required Ca2+ for activity; the inhibition by unsaturated fatty acids was not reversed by Ca2+. The finding that unsaturated fatty acids are potent inhibitors of PLA2 would explain its generally low activity in human platelet extracts and its marked increase of activity during the course of enzyme purification.

Marked increase of human platelet phospholipase A2 activity in vitro and demonstration of an endogenous inhibitor.

When soluble and particulate fractions of human platelet homogenate were chromatographed on DEAE-cellulose and hydroxylapatite columns, total phospholipase A2 (PLA2; phosphatide 2-acylhydrolase, EC 3.1.1.4) activity increased sharply. PLA2 activity detected in these partially purified preparations was approximately 12 times greater than that associated with the original homogenate. Chromatography of the particulate enzyme on a DEAE-cellulose column yielded one activity peak. Fractions eluted near the activity peak showed a trace of PLA2 activity but inhibited purified PLA2 stoichiometrically, suggesting the presence of an endogenous "inhibitor" in the homogenate. The inhibitor activity was heat-stable, trypsin insensitive, and extractable by chloroform/methanol, and thus appears to be associated with a lipid(s). PLA2 activity was Ca2+ dependent. The crude enzyme was variably stimulated by calmodulin, whereas the purified enzyme was not, suggesting that the effect of calmodulin on the crude enzyme is indirect. Our results suggest that human platelet PLA2 activity reported in the literature may have been underestimated, apparently due to the presence of an endogenous inhibitor.