Further characterization of platelet-aggregating cysteine proteinase activity in thrombotic thrombocytopenic purpura.

Circulating platelet-aggregating activities have been described by several authors. In a previous study we have confirmed the presence in serum and plasma from patients with thrombotic thrombocytopenic purpura (TTP) of a platelet aggregating cysteine proteinase (PACP). This activity differed in that, among the class of thiol enzymes, it showed characteristics of a lysosomal cathepsin rather than of a cytoplasmic calpain. To further investigate the enzymatic properties of this PACP we have designed a study to evaluate the dependence of the activity on Ca++. Calcium-dependence is a property of calpains but not of cathepsins. A proteolytic assay was set up and conducted with and without Ca++. The release of acid-soluble peptide from denatured human globin promoted by TTP samples and standards was fluorimetrically measured. The results show that TTP samples were equally active with and without Ca++ similar to standard papain (the cathepsin B-like proteinase), whereas standard calpain only acted with Ca++. An inhibition study performed by the proteolytic assay confirmed the same pattern of sensitivity of PACP to a series of specific cystein proteinase inhibitors previously shown by the proaggregating assay. The enzymatic behaviour of PACP of TTP resembled a lysosomal (cathepsins) rather than a cytoplasmic (calpains) cysteine proteinase.