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Previous work with cultured mammalian cells and perfused laboratory animals suggested to us that hydrolysis of homocysteine thiolactone was catalyzed in these systems. We confirmed this finding by measuring the sulfhydryl-releasing activity of cultured endothelial cells from human umbilical arteries in homocysteine thiolactone solution, pH 7.4, 37°C. The reaction was vigorous and stereospecific and showed saturation kinetics (K a values for L-and D,Lhomocysteine thiolactone were 3.9 and 8 2 mmol/1, respectively, and V,^ values were 10.75 and 10.1 /imol/min/10* cells, respectively). L-Homocysteine thiolactone was quantitatively converted to homocysteine, as measured by amino acid analysis. Human serum also accelerated the elimination of homocysteine thiolactone, although in this process, the majority of the newly formed sulfhydryl-containing product was precipitable by sulfosalicylic acid, indicating likely homocysteinylation of serum proteins. However, approximately 38% of the sulfhydryl-containing product was not precipitated, and because thiolactone elimination stereospecifically favored the L-enantiomer, a possible subsidiary role for serum-catalyzed hydrolysis of the thiolactone was suggested. No homocysteine thiolactone could be found in serum samples from six patients with acute myocardial infarction, three patients with cystathionine 0-synthase deficiency, and six normal subjects. Thus, humans have active vascular systems for elimination of homocysteine thiolactone, a process that could be responsible for an absence of the compound in serum. (Arteriosclerosis and Thrombosis 1991;ll:663-670) H omocysteine thiolactone has been widely used experimentally for perfusing mammals in attempts to elicit homocysteinemia and the atherogenesis expected to result from it (References 1-7 and L.A. Harker, personal communication). Thiolactone has also been used as a substitute for homocysteine in cell culture experiments, particularly in studying its effects on vascular endothelial cells. -13 In both these experimental situations, there have been the tacit assumptions that first, homocysteine thiolactone will undergo spontaneous hydrolysis to homocysteine (Figure 1) and second, this spontaneous hydrolysis will occur rapidly and the resulting concentration of homocysteine occurring in

SummaryPlatelet-derived growth factor (PDGF) is a potent chemotactic and mitogenic factor implicated to play important roles in a variety of normal and pathophysiologic settings. We investigated PDGF receptor expression on human megakaryocytes and several megakaryocytic cell lines (CHRF, DAMI, Meg-01, M-07e) using enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunocytochemical staining. Both PDGF receptor subtypes were identified on CHRF, DAMI, and Meg-01 cells by ELISA; PDGF beta-receptor levels exceeded alpha-receptor levels. Flow cytometry revealed that beta-receptor levels on CHRF and DAMI cells exceeded those on Meg-01 cells, and that M-07e expressed neither receptor. Immunocytochemical staining confirmed these findings and determined that bone marrow megakaryocytes also expressed PDGF receptors. Exposure of megakaryocytes to PDGF-BB dramatically induced the expression of the immediate-early gene, c-fos, within 30 min. Moreover, PDGF-BB significantly stimulated CHRF proliferation and colony formation. The present study demonstrates the presence of functional PDGF receptors on human megakaryocytes and their ability to mediate a mitogenic response.