The effect of phospholipase C on human blood platelets.

Abstract

A B S T R A C T The effect of phospholipase C (EC 3.1.4.3) on human blood platelets has been studied. Phospholipase C from Bacillus cereus was purified to homogeneity as judged by analytical and sodium dodecyl sulphate disc gel electrophoresis and by immunoelectrophoresis. Human platelets isolated from platelet-rich plasma by gel filtration or by centrifugation and washing were incubated with phospholipase C.A loss of 20-45% of the total platelet phospholipid was observed, whereas 88% was hydrolyzed when platelet homogenates were submitted to identical enzyme treatment. Intact platelets lost 50-75% phosphatidylethanolamine, 20-50% phosphatidylcholine, and 20-25% phosphatidylserine. Sphingomyelin was not a substrate for the enzyme under the conditions used. The platelets contained no detectable endogenous phospholipase C activity.The loss of phospholipid was not accompanied by aggregation of the platelets, nor did the platelets lose their ability to aggregate with ADP or thrombin. Total platelet factor 3 releasable by freezing and thawing was reduced. Measurements of releasable platelet factor 4 and the efflux of serotonin showed that no release reaction was triggered even when up to 45% of the total phospholipid in the platelets was hydrolyzed. When sphingomyelinase was added together with, before, or after phospholipase C, aggregation occurred. Sphingomyelinase alone gave no aggregation. The gel-filtered platelets also aggregated upon addition of purified phospholipase C from Clostridium perfringens. The distribution of phospholipids in the platelet membrane is discussed.

Production of diglyceride from phosphatidylinositol in activated human platelets.

“…The formation of DG from PC, PS, or PE would require the action of a phosphatide phosphodiesterase acting upon one or all of these substrates. Thus far, Ino such activity has been detectable in humain platelets (28 (8,10). The specific activity of DG is slightly (15%) greater than that of PI, which in turn has a higher specific activity than PC, PS, and PE.…”

Purification and some properties of the protein component of tissue thromboplastin from human brain

“…After rechromatography the phospholipids were extracted and separated by t.l.c. as described by Otness & Holm (1976). The isolated phospholipids were scraped off, eluted with chloroform/methanol (2:1, v/v) and evaporated to dryness in a stream of N2.…”

Characterization of arsenic compounds formed by Daphnia magna and Tetraselmis chuii from inorganic arsenate.

“…The yellow-brown fraction was subjected to enzymatic hydrolysis by phospholipase A, C, and D (13)(14)(15). The hydrolysis mixture was extracted with diethyl ether.…”