Effect of Homocysteine and Homocystine on Platelet and Vascular Arachidonic Acid Metabolism

Pediatr. Res. 16: 490-493 (1982) Effect of Homocysteine and Homocystine on Platelet and Vascular Arachidonic Acid Metabolism JANET E. GRAEBER,"~'JEFFREY H. SLOTT, RODNEY E. ULANE, JOSEPH D. SCHULMAN, A N D MARIE J. STUART Neonatal and Pediatric Medicine Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland [J.E.G., R.E. U., J. D.S.] and Department of Pediatrics, SUNY, Upstate Medical Center, Syracuse, New York [J.H.S., M.J.S.], USA Summary Normal hemostasis depends in part on the balance achieved between proaggregatory and prothrombotic platelet thromboxane A*, measured as its stable end-product thromboxane Bz (TXBz), and vascular prostacyclin (PGI~),which inhibits platelet aggregation and is antithrombotic. Cvstathionine-Bsvnthase deficiencv is characterized by a high frequkncy of thro1;lbokmbolic disease. w e therefore studied, in vitro, the effects of homocysteine and related compounds on platelet TXBp and vascular PGIz formation. In paired samples of platelet rich plasma, which had been preincubated with L-homocystine(1 mM), mean production of the two platelet cyclooxygenase products, TXBz and 12-hydroxy-5, 8,lO-heptadecatrienoic acid increased significantly from control levels 113.6% + 1.9 to 19.8% + 2.1 ( P < 0.02) TXBz and 29.8% + 4.2 to 39.4% 4.1 ( P < 0.01) HHTI. In the presence of D,Lhomocysteine (1 mM), mean platelet TXBz and 12-hydroxy-5,8,10heptadecatrienoic acid production was also significantly increased 112.7% +- 1.5 to 16.9% + 1.5 ( P < 0.01) TXBz and 27% + 4 to 31% + 4.1 ( P < 0.02) HHTI. Cystine, cysteine, or methionine (1 mM) did not have similar effects in this test system. Homocysteine and homocystine were without effect on the synthesis of vascular PGIz by umbilical artery segments [control, 0.22 +- 0.03 to 0.21 + 0.03 ng/mg with D,L-homocysteineand 0.20 +- 0.04 control to 0.19 f 0.04 ng/mg with D,L-homocystinel. A homocyst(e)ine-induced increase in platelet thromboxane production in the absence of an increase in vascular prostacyclin, if present in vivo, may contribute to the vascular thromboses characteristic of human homocystinemias (homocystinurias). + The homocystinurias occur as a result of genetically determined defects in the metabolism of homocysteine (15). The most common etiology, cystathionine-P-synthase deficiency, results in a decrease in the rate of conversion of homocysteine to cystathionine and is generally accompanied by hyper&ethionine&ia. Deficiencies in the remethylation of homocysteine to methionine cause homocystinemia accompanied by hypomethioninemia but are much less common. In all these disorders, accumulated homocysteine is oxidized to homocystine, which is found in excessive amounts in blood and urine. Cystathionine-P-synthase dificiency is characterized by ectopia lentis, skeletal deformities, central nervous system abnormalities and a high frequency of thromboembolic disease. Atherosclerosis and occlusion of major vessels such as myocardial, cerebral, renal and pulmonary arteries and veins may occur as early as the first decade, often with fatal results (15). Studies on other disorders with a high incidence of thrombotic complications suggest that normal heGostasis depends in part on the balance achieved between proaggregatory and prothrombotic platelet thromboxane A2 and vascular prostacyclin, which inhibits platelet aggregation and is thus antithrombotic (14). We therefore undertook an in vitro study of the effects of homocysteine and related compounds on platelet thromboxane and vascular prostacyclin formation. MATERIALS AND METHODS Evaluation of platelet arachidonic acid metabolism after incubation in vitro with homocystine, homocysteine, cystine, cysteine, or methionine. Blood samples were obtained after informed consent from control subjects using a two-syringe technique and 9 volumes of blood to 1 volume citrate-phosphate-dextrose solution. Plateletrich plasma (PRP) was obtained by centrifugation of the samples at 200 X g for 20 min. PRP from each control was divided into three aliquots. Hank's balanced salt solution (HBSS) was added to one aliquot as a control, whereas L-homocystine or D,L-homocysteine (1 mM final concentration in HBSS) was added to each of the other two platelet aliquots. All samples were incubated at 37"C, at pH 7.4 for 45 min. Each patient's platelets served as their own control in order to evaluate the effects of the test compounds. In a second set of experiments, PRP from controls was divided into four aliquots and incubated with HBSS alone, or L-cystine, D,L-cysteine,or L-methionine (I mM in HBSS) as described above. To assess platelet conversion of [14C]-arachidonicacid to metabolites by the lipoxygenase and cyclooxygenase enzymes, the platelets were washed (16) after the period of incubation, and resuspended in HBSS (37°C) containing 0.5 mM calcium chloride at a concentration of 5 x 10' platelets per ml. ['4C]-~rachidonic acid (specific activity 56.5 mCi/mM) made up as a sodium salt in 0.01 M Tris buffer, pH 7.4, was then added to a 1 ml platelet suspension (final concentration of arachidonate per sample was 6 yM). Aggregation was monitored in a Payton dual channel aggregometer. After 6 min, the samples were added to extraction vials containing 10 ml of absolute ethanol, then &luted, acidified with 1 N HCI to a pH of 3.3 and extracted into diethyl ether. Separation of [14C]-arachidonicacid from thromboxane Bz (TXB2) was performed by thin layer chromatography of the free acids on silica gel G with diethyl ether:methanol:acetic acid (13553, v/v) as eluting solvent. Thromboxane Bz (TXBZ) standard (kindly supplied by Dr. John E. Pike, Upjohn Co., Kalamazoo, MI) was also applied to each plate. The plates were then scanned on a thinlayer radiochromatogram scanner (Berthold, Wildbad, W. Germany) and the silica gel corresponding to the TXBz peak scraped from the glass and counted in a scintillation counter. The remaining arachidonate and metabolites on the silica gel plates were extracted into 2 ml ether, then methylated using diazomethane, and separated on a thin layer chromatogram (silica gel B) using the organic layer of isooctane:water:ethyl acetate (2:2: 1) as mobile 49 1 ARACHIDONIC ACID METABOLISM phase (5). The thin layer plates were scanned using a Berthold each of the six experiments. When the lipoxygenase product radiochromatogram scanner to determine the location of individ- HETE was measured in the presence or absence of L-homocystine, ual peaks. The thin layer plates were also subjected to autoradiog- no significant mean differences were observed (38.3% f 4.2 and raphy to improve the definition of 12-hydroxy-5,8, 10-heptade- 41% f 4.9, respectively). catrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic As shown in Figure 3 similar results were observed in eight acid (HETE) as depicted in Figure 1. Areas corresponding to paired experiments in the presence of 1 mM D,L-homocysteine. individual peaks (...truncated)