ISOLATION AND CHARACTERIZATION OF CYSTEINE PROTEINASE IN THROMBOTIC THROMBOCYTOPENIC PURPURA

Thrombotic thrombocytopenic purpura (TTP) is an uncommon disorder char acterized by thrombocytopenia, schistocytic haemolytic anaemia, fever, neurologic lesions, and renal failure, A platelet aggregating factor has been postulated to be responsible for this disorder, but its preci se identity remains debated, Two different groups of investigators hav e provided evidence that the platelet aggregating factor is a cysteine proteinase. We have suggested that it was calpain, whereas others hav e suggested that it was cathepsin L, To help resolve this issue, we ha ve studied the platelet activating activity found in the acute serum s amples from 10 different TTP patients as well as purified calpain and cathepsin L. The TTP activity was measured functionally (platelet sero tonin release assay and casein lysis assay) and antigenically (immunod epletion using anti-calpain and anti-cathepsin L antibodies and antige nic analysis using SDS PAGE), The TTP serum parallelled the activity o f the purified calpain and had optimal pH activity of 7.5. The purifie d cathepsin L activity had optimal activity at low pH (5.5) and activi ty was no longer measurable at pH 7.5. Similarly, a specific cathepsin L inhibitor (Z-Phe-Phe-CHN2) had no effect on the activity of the TTP samples nor on purified calpain, but it did abolish the activity of p urified cathepsin L. The platelet activating activity of the TTP sampl es was detectable in the microparticle pellet following ultracentrifug ation of TTP serum, and could be immunodepleted using antibodies to ca lpain but not to cathepsin L. These studies indicate that the micropar ticle-associated platelet activating factor in TTP corresponds to calp ain.