Protein disulphide isomerase mediates platelet aggregation and secretion

Platelet surface thiols and disulphides play an important role in platelet responses. Agents that reduce disulphide bonds expose the fibrinogen recept or in platelets and activate the purified glycoprotein (GP) IIbIIIa recepto r. Protein disulphide isomerase (PDI), an enzyme that rearranges disulphide s bonds, is found on the platelet surface where it is catalytically active. We investigated the role of PDI in platelet responses using (1) rabbit ant i-PDI IgG specific for PDI, (2) a competing substrate (scrambled ribonuclea se A), and (3) the PDI inhibitor, bacitracin. Fab fragments of the rabbit a nti-PDI IgG inhibited platelet responses to the agonists tested (ADP and co llagen), whereas Fab fragments prepared identically from normal rabbit IgG had no inhibitory effect. Scrambled ribonuclease A blocked platelet aggrega tion and secretion, but native ribonuclease A did not. When biphasic platel et aggregation was examined using platelets in citrated plasma, the princip le effect of bacitracin was on second phase or irreversible aggregation res ponses and the accompanying secretion. Using flow cytometry and an antibody specific for activated GPIIbIIIa (PAC-1), the rabbit anti-PDI Fab fragment s substantially inhibited activation of GPIIbIIIa when added before, but no t after, platelet activation. In summary, we have demonstrated that protein disulphide isomerase mediates platelet aggregation and secretion, and that it activates GPIIbIIIa, suggesting this receptor as the target of the enzy me.