Proteolysis leads to the appearance of the long form of β3-endonexin in human platelets

After vessel injury, platelets adhere to the subendothelial matrix. Platelet adhesion leads to activation of the platelet integrin αIIbβ3, which then binds to fibrinogen, leading to platelet aggregation. It has been shown that a β3-integrin binding protein, β3-endonexin, can activate the integrin αIIbβ3 expressed in transfected CHO cells. Several isoforms of β3-endonexin are known but it is not clear which isoforms are expressed in platelets and what role they may play during haemostasis. Here, we show that the long form of β3-endonexin (EN-L) can be detected in platelet lysates several hours after thrombus formation, after long-term storage of platelets and after glucose deprivation. After subcellular fractionation, EN-L is found in the detergent insoluble fraction suggesting that it might be associated with the cytoskeleton. EN-L generation is temperature and Ca++ dependent and requires physiological salt concentrations. Proteolysis is responsible for the appearance of EN-L since a calpain inhibitor prevents its formation and the addition of calpain to platelet lysates induces its formation. The appearance of EN-L seems to be linked to apoptotic events occurring during long-term storage of platelets and, possibly, during late steps of haemostasis after thrombus formation.