Phospholipid Breakdown Measurements in Stimulated Platelets

A number of laboratories have been successfiil in obtaining semipurified/purified PLAj preparations fi om a number of sources, including platelets (47-70). These studies have been helpful to understand the molecular size as well as the nature of active sites of these enzymes. Several reports have demonstrated the presence of two substrate specific and distinct etjzymes, including secretory PLA (sPLA,) and cytosolic PL (cPLAj) in platelets. cPL appears to have a molecular mass of 85 kDa that is structurally distinct fi-om that of the mammalian Type II, a 14 kDa non-pancreatic PLAj, which exists in an extracellular form in many tissues, including platelets, inflammatory joint fluid, spleen and placenta (47-70). The human non-pancreatic PLA enzyme purified is also present in platelets and is enriched in rheumatoid synovial fluid. 

The enzyme is calcium-dependent, has a pH optimum of 8-10, and shows a striking preference for substrate presented in the form of Escherichia coli membranes. Using oligonucleotide probes based on amino-terminal sequence data, the corresponding human gene from a genomic DNA library, has been cloned and expressed in animal cells. The protein is secreted from cells in an active form. The deduced amino acid sequence of the human protein consists of 124 amino acids, contains structural features common to all known PLAjS, and has a half-cysteine pattern that is characteristic of the snake venom group II enzyme (60). 

A kinetic analysis of recombinant hs-PLA has shown that this enzyme strongly prefers phosphatidic acid (PA) as a substrate over other phospholipids found in the mammalian plasma membrane including PS, PC and PE (70). The order of preference is PA PE approximately PS PC. It appears that Lys-69 is at least partially involved in the PA specificity of hs-PLAj and Glu-56 in the distinction between PE and PC. These studies suggest that hs-PLA can rapidly hydrolyse PA molecules exposed to the outer layer of cell-derived microvesicles and thereby produce LPA (70). 

In a recent study, a serine-phospholipid-selective phospholipase A has been purified and cloned its cDNA from rat platelets, which secrete two types of phospholipases upon stimulation (71). The purified enzyme from extracellular medium of activated rat platelets, yielded a 55-kDa protein band on SDS-polyacrylamide gel electrophoresis. The presence of active serine residues was confirmed by labeling the 55-kDa protein with pHJDiisopropyl fluorophosphate, an inhibitor of the enzyme. Based on cDNA for the enzyme cloned from a rat megakaryocyte cDNA library, the 456-amino acid 

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