PHOSPHOLIPASE A(2) FROM PLASMA OF PATIENTS WITH SEPTIC SHOCK IS ASSOCIATED WITH HIGH-DENSITY-LIPOPROTEINS AND C3 ANAPHYLATOXIN - SOME IMPLICATIONS FOR ITS FUNCTIONAL-ROLE

Phospholipase A(2) (PLA(2)) activity was purified 12 544-fold with a 1 3% yield from the plasma of patients diagnosed of septic shock by the sequential use of heparin-agarose affinity chromatography, gel filtrat ion, and reverse-phase f.p.l.c. Gel-filtration chromatography of plasm a omitting high-ionic-strength buffer revealed a molecular mass differ ent from that of purified PLA(2) and co-elution with apolipoprotein A- I peaks, which suggests its association with high-density lipoproteins (HDL). N-terminal analysis of the enzyme activity protein band, elect roblotted from a SDS-acrylamide gel and with an assessed molecular mas s of 19 kDa, showed an identical sequence to that of alpha-chain of hu man C3 complement component, suggesting the presence in this band of a complex formed by a complement C3-derived anaphylatoxin (C3a)-related fragment and the PLA(2) linked side-by-side. Because the preparation of plasma enzyme showed lower activity than the enzyme obtained from f ibroblasts transfected with the coding sequence of human group-II PLA( 2), and because the addition of C3-derived anaphylatoxins from human s erum inhibited the activity of this recombinant PLA(2), it was conside red that C3a-related peptides behave as inhibitors of group-II PLA(2). The enzyme showed optimal activity on [C-14]oleate-labelled autoclave d E. coli, on synthetic phosphatidylethanolamine, and on [H-3]arachido nate-labelled membranes of the monoblast cell line U937, but it did no t show any activity on the release of [H-3]arachidonate from pre-label led human polymorphonuclear leukocytes (PMNs). In short, PLA(2) from p lasma of sepsis patients shows unique associations with other plasma p roteins which may influence its functional properties. The association with C3-related peptides shows an inhibitory effect on the enzyme act ivity, whereas the association with HDL might influence its environmen t and/or its interaction with cells. The study of the catalytic proper ties shows a prominent effect on bacterial phospholipids, synthetic ph osphatidylethanolamine, and membranes from U937 monoblasts, but not on synthetic phosphatidylcholine or on PMNs, even when these cells were maintained in culture to allow spontaneous apoptosis and became a good substrate for pancreatic type PLA(2).