Platelet function is influenced by the platelet thioldisulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1b␣ (GP1b␣). PDI contains two active site dithiols/disulfides. The active sites of 26% of the PDI on resting platelets was in the dithiol form, compared with 81% in the dithiol form on activated platelets. Similarly, GP1b␣ presented one or more free thiols on the activated platelet surface but not on resting platelets. Anti-PDI antibodies increased the dissociation constant for binding of vWF to platelets by ϳ50% and PDI and GP1b␣ were sufficiently close on the platelet surface to allow fluorescence resonance energy transfer between chromophores attached to PDI and GP1b␣. Incubation of resting platelets with anti-PDI antibodies followed by activation with thrombin enhanced labeling and binding of monoclonal antibodies to the N-terminal region of GP1b␣ on the activated platelet surface. These observations indicated that platelet activation triggered reduction of the active site disulfides of PDI and a conformational change in GP1b␣ that resulted in exposure of a free thiol(s).The platelet thiol-disulfide balance is important for platelet function. Perturbation of platelet thiol status effects platelet aggregation and release. The low M r thiol compounds, reduced glutathione (GSH), cysteine, and 6-mercaptopurine, inhibit platelet aggregation induced by several agonists, while the disulfide-bond reducing agents dithiothreitol and ␤-mercaptoethanol promote aggregation (1). In addition, reaction of platelet sulfhydryl groups with the thiol specific compounds, diamide and N-ethylmaleimide, inhibits in vitro aggregation and clot retraction (2-5). These results imply that certain platelet thiol groups are critical for platelet aggregation. Furthermore, the observation that specific depletion of platelet GSH by 1-chloro-2,4-dinitrobenzene only marginally effects platelet aggregability implies that the critical thiol groups are associated with protein (6). In support of this notion, Yamada et al. (7) have shown that the anti-platelet aggregation actions of 2,2Ј-dithiobis(N-2-hydroxypropylbenzamide) are mediated through interaction of the compound with platelet protein thiol groups.Protein-disulfide isomerase (PDI) 1 is a noncovalent homodimer with a subunit molecular mass of 57 kDa that catalyzes thiol-disulfide interchanges that can result in formation, reduction, or rearrangement of protein disulfide bonds. It is generally considered that PDI is important for proper folding and disulfide bonding of nascent proteins in the endoplasmic reticulum (8 -10). PDI also functions as the ␤ subunits of prolyl-4-hyroxylase (11, 12) and the ␤ subunit of triglyceride transfer protein complex (13,14). Bovine aortic endothelial cells (15), rat hepatocytes (16), rat pancreatic cells (17), and human B cells (18,19) secrete PDI which associates with the cell su...

The treatment of homocystinuria that is not responsive to pyridoxine is not usually biochemically or clinically successful, and vascular, ocular, and skeletal complications commonly supervene. Persistent marked homocysteinemia appears to be the most important biochemical disturbance leading to these complications. Ten patients with cystathionine beta-synthase deficiency that was not responsive to pyridoxine and one patient with homocystinuria due to a defect in cobalamin metabolism were treated with 6 g daily of betaine added to conventional therapy, to improve homocysteine remethylation. All patients had a substantial decrease in plasma total homocysteine levels (P less than 0.001) and an increase in total cysteine levels (P less than 0.001). Changes in plasma methionine concentrations were variable. Fasting levels of plasma amino acids became normal in two patients, and in six there was immediate clinical improvement. There were no unwanted effects. We conclude that treatment of homocystinuria that is not responsive to pyridoxine and of disorders of homocysteine remethylation should include betaine in adequate doses to ensure maximum lowering of elevated plasma homocysteine levels.

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Mild homocysteinemia occurs surprisingly often in patients with premature vascular disease. We studied the possible enzymatic sources of this mild hyperhomocysteinemia and the control of homocysteine levels in plasma by treatment of patients with the cofactors and cosubstrates of homocysteine catabolism. We assessed homocysteine metabolism in 131 patients who had premature disease in their coronary, peripheral, or cerebrovascular circulation by using a standard oral methionine-load test Impaired homocysteine metabolism occurred in 28 patients. We assayed levels of the primary enzymes of homocysteine catabolism in cultured skin fibroblast extracts from 15 of these 28 patients. The patients' cystathionine 0-synthase levels (3.68±2.52 nmol/h per milligram of cell protein, mean±SD) were markedly depressed compared with those from 31 healthy adult control subjects (7.61 ±4.49, P<.001). The patients' levels of 5 -methyltetrahydrofolate: homocysteine methyltransferase were normal. While betalne: homocysteine methyltransferase was not expressed in skin fibroblasts, 24-hour urinary betaine and fyiV-dimethylglycine measurements were consistent with normal or enhanced remethylation of homocysteine by betaine: homocysteine methyltransferase in the 13 patients tested. When treated daily with choline and betaine, pyridoxine, or folic acid, there was a normalization of the postmethionine plasma homocysteine level in 16 of 19 patients. Our results indicate that mild homocysteinemia in premature vascular disease may be caused by either a folate deficiency or deficiencies in cystathionine /3-synthase activity. It does not necessarily involve deficiencies of either 5-methyltetrahydrofolate: homocysteine methyltransferase or betaine: homocysteine methyltransferase. Effective treatment regimens are also defined. (Arteriosder Thromb. 1993;13:1253-1260 KEYWORDS • coronary heart disease • 5-methyltetrahydrofolate: homocysteine methyltransferase • cystathionine 0-synthase • betaine: homocysteine methyltransferase

—Despite intense investigation, mechanisms linking the development of occlusive vascular disease with elevated levels of homocysteine (HCY) are still unclear. The vascular endothelium plays a key role in regulating thrombogenesis and thrombolysis. We hypothesized that vascular lesions in individuals with elevated plasma HCY may be related to a dysfunction of the endothelium triggered by HCY. We investigated the effect of HCY on human neutrophil adhesion to and migration through endothelial monolayers. We also examined the effect of HCY on leukocyte adhesion and migration in mesenteric venules of anesthetized rats. We found that pathophysiological concentrations of HCY in vitro induce increased adhesion between neutrophils and endothelial cells. This contact results in neutrophil migration across the endothelial layer, with concurrent damage and detachment of endothelial cells. In vivo, HCY infused in anesthetized rats caused parallel effects, increasing leukocyte adhesion to and extravasation from mesenteric venules. Our results suggest that extracellular H 2 O 2 , generated by adherent neutrophils and/or endothelial cells, is involved in the in vitro endothelial cell damage. The possibility exists that leukocyte-mediated changes in endothelial integrity and function may lead to the vascular disease seen in individuals with elevated plasma HCY.

Objective: To determine the effect of habitual omnivorous and vegetarian diets on folate and vitamin B 12 status and the subsequent effect on homocysteine concentration. Design: Cross-sectional comparison of free-living habitual meat-eaters and habitual vegetarians. Setting: The study was conducted at RMIT University, Melbourne. Subjects: One hundred and thirty-nine healthy male subjects (vegans n 18, ovolacto vegetarians n 43, moderate meat-eaters n 60 and high meat-eaters n 18) aged 20 ± 55 y who were recruited in Melbourne. Outcome measures: Fasting plasma or serum from each subject was analysed for folate, vitamin B 12 and homocysteine concentration. A semi-quantitative Food Frequency Questionnaire was completed by a subset of subjects from each group to determine methionine intake. Results: The two meat eating groups consumed signi®cantly greater levels of methionine (P`0.001). There was no clear trend in plasma folate status between groups, however the plasma vitamin B 12 concentration decreased progressively from the high-meat-eating group to vegans (P`0.05). An inverse trend was observed with plasma homocysteine concentration, with vegans showing the highest levels and high meat eaters the lowest (P`0.05). Conclusions: Dietary methionine intake has no observable effect on plasma homocysteine concentration. In habitual diets, where folate intake is adequate, lowered vitamin B 12 intake from animal foods leads to depleted plasma vitamin B 12 concentration with a concomitant increase in homocysteine concentration. The suggested mechanism is the failure to transfer a methyl group from methyl tetrahydrofolate by vitamin B 12 in the remethylation of homocysteine to methionine.

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