Mechanical prophylaxis is a heparin-independent risk for anti-platelet factor 4/heparin antibody formation after orthopedic surgery.

Platelet-activating antibodies, which recognize platelet factor 4 (PF4)/heparin complexes, induce spontaneous heparin-induced thrombocytopenia (HIT) syndrome or fondaparinux-associated HIT without exposure to unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). This condition mostly occurs after major orthopedic surgery, implying that surgery itself could trigger this immune response, although the mechanism is unclear. To investigate how surgery may do so, we performed a multicenter, prospective study of 2069 patients who underwent total knee arthroplasty (TKA) or hip arthroplasty.

Generation of N-acylphosphatidylethanolamine by members of the phospholipase A/acyltransferase (PLA/AT) family.

Bioactive N-acylethanolamines (NAEs), including N-palmitoylethanolamine, N-oleoylethanolamine, and N-arachidonoylethanolamine (anandamide), are formed from membrane glycerophospholipids in animal tissues. The pathway is initiated by N-acylation of phosphatidylethanolamine to form N-acylphosphatidylethanolamine (NAPE). Despite the physiological importance of this reaction, the enzyme responsible, N-acyltransferase, remains molecularly uncharacterized.

Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes.

A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1.

Characterization of the human tumor suppressors TIG3 and HRASLS2 as phospholipid-metabolizing enzymes.

Tazarotene-induced protein 3 (TIG3) and HRAS-like suppressor family 2 (HRASLS2) exhibit tumor-suppressing activities and belong to the lecithin retinol acyltransferase (LRAT) protein family. Since Ca(2+)-independent N-acyltransferase and H-rev107 (another tumor suppressor), both of which are members of the LRAT family, have been recently reported to possess catalytic activities related to phospholipid metabolism, we examined possible enzyme activities of human TIG3 and HRASLS2 together with human H-rev107. The purified recombinant proteins of TIG3, HRASLS2, and H-rev107 functioned as phospholipase (PL) A(1/2) in a Ca(2+)-independent manner with maximal activities of 0.

cDNA cloning and characterization of human and mouse Ca(2+)-independent phosphatidylethanolamine N-acyltransferases.

The formation of N-acylphosphatidylethanolamine by N-acylation of phosphatidylethanolamine (PE) is the initial step in the biosynthetic pathway of bioactive N-acylethanolamines, including the endocannabinoid anandamide and the anti-inflammatory substance N-palmitoylethanolamine. We recently cloned a rat enzyme capable of catalyzing this reaction, and referred to the enzyme as Ca(2+)-independent N-acyltransferase (iNAT). Here we report cDNA cloning and characterization of human and mouse iNATs.

Amino acid residues crucial in pH regulation and proteolytic activation of N-acylethanolamine-hydrolyzing acid amidase.

N-Acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme which hydrolyzes bioactive N-acylethanolamines, including anandamide and N-palmitoylethanolamine. NAAA shows acidic pH optimum in terms of both catalytic activity and maturation by specific proteolysis. However, molecular mechanism involved in this characteristic pH dependency remained unclear.

Discovery and characterization of a Ca2+-independent phosphatidylethanolamine N-acyltransferase generating the anandamide precursor and its congeners.

N-Acylphosphatidylethanolamines (NAPEs) are precursors of bioactive N-acylethanolamines, including the endocannabinoid anandamide. In animal tissues, NAPE is formed by transfer of a fatty acyl chain at the sn-1 position of glycerophospholipids to the amino group of phosphatidylethanolamine (PE), and this reaction is believed to be the principal rate-limiting step in N-acylethanolamine synthesis. However, the Ca2+-dependent, membrane-associated N-acyltransferase (NAT) responsible for this reaction has not yet been cloned.

Molecular characterization of N-acylethanolamine-hydrolyzing acid amidase, a novel member of the choloylglycine hydrolase family with structural and functional similarity to acid ceramidase.

Bioactive N-acylethanolamines, including anandamide (an endocannabinoid) and N-palmitoylethanolamine (an anti-inflammatory and neuroprotective substance), are hydrolyzed to fatty acids and ethanolamine by fatty acid amide hydrolase. Moreover, we found another amidohydrolase catalyzing the same reaction only at acidic pH, and we purified it from rat lung (Ueda, N., Yamanaka, K.

Biosynthesis of anandamide and N-palmitoylethanolamine by sequential actions of phospholipase A2 and lysophospholipase D.

Anandamide (an endocannabinoid) and other bioactive long-chain NAEs (N-acylethanolamines) are formed by direct release from N-acyl-PE (N-acyl-phosphatidylethanolamine) by a PLD (phospholipase D). However, the possible presence of a two-step pathway from N-acyl-PE has also been suggested previously, which comprises (1) the hydrolysis of N-acyl-PE to N-acyl-lysoPE by PLA1/PLA2 enzyme(s) and (2) the release of NAEs from N-acyllysoPE by lysoPLD (lysophospholipase D) enzyme(s). In the present study we report for the first time the characterization of enzymes responsible for this pathway.

Molecular characterization of a phospholipase D generating anandamide and its congeners.

Anandamide (N-arachidonoylethanolamine) is known to be an endogenous ligand of cannabinoid and vanilloid receptors. Its congeners (collectively referred to as N-acylethanolamines) also show a variety of biological activities. These compounds are principally formed from their corresponding N-acyl-phosphatidylethanolamines by a phosphodiesterase of the phospholipase D-type in animal tissues.

Activation of N-acylethanolamine-releasing phospholipase D by polyamines.

N-acylethanolamines including anandamide (an endogenous ligand of cannabinoid receptors) are biosynthesized from N-acyl-phosphatidylethanolamine (PE) by a phosphodiesterase of the phospholipase D type. The enzyme partially purified from the particulate fraction of rat heart hydrolyzed N-palmitoyl-PE to N-palmitoylethanolamine with a specific activity of 50 nmol/min per mg protein at 37 degrees C in the presence of 10 mM CaCl2. We found that the enzyme was highly activated in dose-dependent manner by polyamines like spermine, spermidine, and putrescine.