A cathepsin-like cysteine proteinase proaggregating activity in thrombotic thrombocytopenic purpura

The platelet aggregating activity (PAA) of serum and plasma from thrombotic thrombocytopenic purpura (TTP, eight patients) and haemolytic uraemic syndrome (HUS, three patients) was investigated. Sample-induced aggregation of washed platelets and the parallel 3H-serotonin release were studied both in the presence and the absence of purified von Willebrand factor (vWF) or defibrinated cryoprecipitate, according to Kelton et al (1987). All the samples but two were positive in the test, vWF being indispensable for aggregation in all but two of the active cases. Samples from five patients during remission were ineffective under the same conditions, as were 14 controls from healthy donors. An inhibition study using four cysteine proteinase inhibitors (1 mM iodoacetamide, 0.5 mM E-64, 0.1 mM antipain and 0.1 mM leupeptin) and two serine proteinase inhibitors (200 U/ml aprotinin and 0.2 U/ml hirudin) confirmed that TTP/HUS's PAA behaved enzymatically as a cysteine proteinase, as described (Murphy et al, 1987a). These results were confirmed on a protein preparation obtained from pooled plasmas after Al(OH)3 adsorption, ammonium sulphate precipitation, binding to and elution from a p-(chloromercuri)benzoate (PCMB) affinity resin. To further characterize this activity we investigated whether it was a calcium-dependent neutral protease or a lysosomal cathepsin by testing the sensitivity to a series of peptidyl diazomethyl ketones, synthetic inhibitors specifically designed for cysteine proteinases. We confirm here the presence of a circulating platelet aggregating cysteine proteinase in TTP/HUS and provide evidence for the first time that this activity behaves like a lysosomal cathepsin.