Pergamon Press

Lige Sci ces Vol . 16, pp . 1703-1710 Printed M U.A .A .

SPECIFIC CONDITIONS FOR ENHANCEMENT OF LIPOPROTEIN LIPASE ACTIVITY BY PLATELETS Peter Nilsson-Ehle+ , Arlene S . Garfinkel and Michael C . Schot: Medical Service and Research, Veterans Administration Wadsworth Hospital Center, Los Angeles, Calif . 90073 and Dept . of Medicine, UCLA School of Medicine, Los Angeles, Calif . 90024 (Received in final form May 12, 1975) Summary Homogenates of human blood platelets, but not of red blood cells, have been found to stimulate lipoprotein lipase activity only when assayed against an emulsified triglyceride substrate sonicated for a short period of time . The degree of stimulation was inversely related to the time of sonication of the substrate . Using chylomicrons as substrate no stimulation of lipoprotein lipase activity by platelet homogenate was seen . Lipoprotein lipase (LPL) regulates the rate and pattern of triglyceride assimilation into adipose and other tissues (1) . Since the physiological site of action of this enzyme presumably is on or in the capillary endothelium (1), much interest has focused on possible enzyme modifiers present in the blood stream including blood cells (2,3) . A recent report from this laboratory has described stimulation of LPL activity by blood platelets, the activating principle being heat-labile, nondialyzable, partially resistant to papain, and present mainly in the particulate fraction of platelet homogenates (4) . Subsequent experiments have shown the effect of platelets on LPL activity to be variable, ranging from stimulation to slight inhibition depending upon the experimental conditions . The data in this communication indicate that enhancement of lipase activity by platelet homogenates is dependent upon physical-chemical properties of the substrate emulsion . Addition of platelet homogenate to artifical substrates sonicated for short periods of time caused a large and consistent enhancement of LPL activity . Since no stimulation of LPL activity by platelet homogenates was observed using a properly sonicated triglyceride emulsion or chylomicrons as substrate, it is uncertain whether there are any physiological or pathophysiological implications of this phenomenon . +Postdoctoral Trainee supported by American Heart Association Greater Los Angeles Affiliate Grant 492-IG2 . 1703

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Lipoprotein Lipase and Platelets

Vol . 16, No . 11

Materials and Methods Cell Homogenates - Platelets were sedimented at 2,400 x g for 10 min and homogenates prepared as described previously (4) . The final homogeneous suspensions contained the membranes from about 10 10 platelets per ml ; the protein content was 1 .4 mg/ml as determined by the method of Lowry at al . (5) . Red blood cell homogenates were prepared from red blood cell concentrates of 50 ml human blood . These cells were sedimented at 100 x g for 10 min, washed, frozen, thawed, and homogenized (4) . The homogenates were finally washed three times with distilled water to remove hemoglobin . The final suspensions ap eared homogeneous and contained the membranes from about 10 0 cells per ml . The protein concentration was 0 .1 mg/ml . Chylomicron Preparation - Rabbit chyle was obtained by thoracic duct cannulation as described by Rudel at _al . (6) . Chyle was collected for 10 h after feeding a fat rich meal (6) followed by 1 mCi [ 3 H]oleic acid given by gastric tube . The chyle was centrifuged for 15 h at 100,000 x g at 16 0 C and the upper turbid layer collected . This chylomicron suspension had a triglyceride concentration of 4 .25 Umoles/ml (triglyceride determination according to van Handel and Zilversmit (7)) and a specific activity of 0 .97 x 106 cpm/umole triglyceride . Thin layer chromatography of a chloroform-methanol extract of the suspension showed that >96% of the radioactivity was associated with the triglyceride fraction . Substrate Preparations - Chylomicron substrates were prepared by mixing the chylomicron suspensions with 0 .2M TRIS-HC1-buffer (pH 8 .0) and/or serum . Emulsified triolein substrates, stabilized with lysolecithin and preincubated with serum, were prepared as described previously (8) . The serum concentration used has been shown to provide maximal activation of LPL activity in this assay system (8) . The substrates were sonicated in 3 ml batches for various periods of time (15 sec - 4 min) with the microtip of a Branson W185 Sonifier Cell Disruptor at a setting of 2 .5 . In order to monitor the energy output from the sonicator probe, we measured the gram force produced under these conditions . With the probe immersed in a beaker of water on a top loading balance, a deflection of the balance of 1 .5 g was observed . Although all substrates were opaque, those sonicated less than one min contained visible small lipid droplets . It has been shown earlier (8) that 4 min sonication produces an emulsion with homogeneous particle size (984 . The main contaminant (1 .51) was associated with the diglyceride fraction . Unlabeled triglyceride (from NuChek Prep ., Elysian, Minn .) and lysolecithin (from Sigma Chem . Co ., St . Louis, Mo .) were pure as judged from thin layer chromatography . Crystalline bovine serum albumin was obtained from Sigma Chem . Co ., and heparin (160 U/mg) from Calbiochem, Lajolla, Calif . [9,10(n)- 3 H]oleic acid was purchased from New England Nuclear, Boston, Mass . Results The effect of platelet homogenate on LPL activity was markedly influenced by the preparation of the substrate emulsion (Fig . 1) . With a substrate sonicated for 4 min there was a slight inhibition of LPL activity by high concentrations of platelet homogenate . Fig . 1 Influence of Substrate Preparation on the Effect of Platelet Homogenate on Lipoprotein Lipase Activity .

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Varying amounts of platelet homogenate were preincubated for 5 min at room temperature with 0 .05 al of an extract of acetone-ether powder of adipose tissue in a total volume of 0 .1 al . Enzymatic activity was assayed using 0.1 ml of four different triolein emulsions, sonicated for 4 min (A), 1 .5 min (B), 45 sec (C) and 15 sec (D) . Control activities (1001) were for substrate A, 20 .2 mU/mlt e, 14 .6 mU/mlj C, 9 .6 all/ml ; and D, 3 .8 mU/ml enzyme . Each value represents the mean of two determinations . Platelet homogenate alone had negligible lipolytic activity (

Specific conditions for enhancement of lipoprotein lipase activity by platelets.

Pergamon Press Lige Sci ces Vol . 16, pp . 1703-1710 Printed M U.A .A . SPECIFIC CONDITIONS FOR ENHANCEMENT OF LIPOPROTEIN LIPASE ACTIVITY BY PLATEL...
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