Phospholipase A2 from plasma of patients with septic shock is associated with high-density lipoproteins and C3 anaphylatoxin: some implications for its functional role

Abstract

Phospholipase A2 (PLA2) activity was purified 12,544-fold with a 13% yield from the plasma of patients diagnosed of septic shock by the sequential use of heparin-agarose affinity chromatography, gel filtration, and reverse-phase f.p.l.c. Gel-filtration chromatography of plasma omitting high-ionic-strength buffer revealed a molecular mass different from that of purified PLA2 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, electroblotted from a SDS-acrylamide gel and with an assessed molecular mass of 19 kDa, showed an identical sequence to that of alpha-chain of human C3 complement component, suggesting the presence in this band of a complex formed by a complement C3-derived anaphylatoxin (C3a)-related fragment and the PLA2 linked side-by-side. Because the preparation of plasma enzyme showed lower activity than the enzyme obtained from fibroblasts transfected with the coding sequence of human group-II PLA2, and because the addition of C3-derived anaphylatoxins from human serum inhibited the activity of this recombinant PLA2, it was considered that C3a-related peptides behave as inhibitors of group-II PLA2. The enzyme showed optimal activity on [14C]oleate-labelled autoclaved E. coli, on synthetic phosphatidylethanolamine, and on [3H]arachidonate-labelled membranes of the monoblast cell line U937, but it did not show any activity on the release of [3H]arachidonate from pre-labelled human polymorphonuclear leukocytes (PMNs). In short, PLA2 from plasma of sepsis patients shows unique associations with other plasma proteins which may influence its functional properties. The association with C3-related peptides shows an inhibitory effect on the enzyme activity, whereas the association with HDL might influence its environment and/or its interaction with cells. The study of the catalytic properties shows a prominent effect on bacterial phospholipids, synthetic phosphatidylethanolamine, 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 PLA2.

Secretory Phospholipase A 2 Elicits Proinflammatory Changes and Upregulates the Surface Expression of Fas Ligand in Monocytic Cells

“…19 Written informed consent was provided by patients. Mouse anti-MAPK monoclonal antibody (mAb) reacting with both p42 and p44 MAPK/ERK (extracellular signal-regulated kinase) was from Zymed Laboratories (San Francisco, Calif).…”

Circulating Inflammatory Mediators in Patients with Fever: Predicting Bloodstream Infection

“…Therefore, sPLA 2 can also be regarded as an acute-phase reactant. sPLA 2 contributes to release of eicosanoids and platelet activating factor, by acting on cell membranes and thereby contributing to tissue damage, and may activate primed neutrophils, but its activity may be inhibited by C3a (14). Alternatively, sPLA 2 and CRP may localize together on damaged plasma membranes, thereby activating complement, so that the elevated C3a level in febrile patients with microbial infection does not necessarily indicate complement activation directly by microbial products (32).…”

“…We have chosen these factors because of interrelationships in the primary nonspecific host response to microbial infection (12,14,32).…”

Cooperation Between Secretory Phospholipase A 2 and TNF-Receptor Superfamily Signaling

“…21 This procedure provides a unique protein band, the identity of which has been ascertained by Edman's N-terminal sequencing. Written informed consent was obtained from patients.…”