Human 253J urinary
carcinoma cells and the F1 (low-metastatic) and F10 (high-metastatic) variants of the B16 murine
melanoma cell line have been shown to activate heparinized human platelets by an
adenosine diphosphate (
ADP)-dependent mechanism based on inhibition by
creatine phosphate/
creatine phosphokinase and the identification of aggregating concentrations (1 to 2 mumol/L) of
ADP in cell-free culture supernatants by high-performance liquid chromatography. Aggregation did not occur in citrated samples, and
hirudin was without effect. Studies were carried out to determine whether extracellular
ADP arose from nonspecific cell damage during cell isolation and manipulation or was a specific process under control of the
tumor cells themselves.
Tumor cell damage during harvesting was shown not to be
a factor because the amounts of
ADP produced by the three cell lines (a) were inversely related to the appearance of lactic
dehydrogenase in the culture supernatants and (b) were similar when measured in confluent monolayers, either in
tumor cells after detachment and resuspension or after crossover studies involving culture in, alternatively,
Hanks' balanced salt solution and minimal essential medium. Metabolic control of
ADP production was indicated by the fact that (a) it was not dependent on cell number, which suggests feedback inhibition; (b) it was reduced 60% when
tumor cells were treated with p-chloromercuribenzene sulfonate; and (c) it was completely abolished in those treated with
iodoacetic acid, which might be expected to increase nonspecific leakage. These studies indicate that
ADP production by these three lines does not arise due to leakage induced by nonspecific membrane damage during cell harvesting and manipulation but is a discrete process under metabolic control of the
tumor cells. Moreover, in B16 murine
melanoma cells the ability to produce
ADP and to support platelet aggregation appears to be unrelated to metastatic potential insofar as identical results were obtained with the F1 and F10 variants.