Activation of the fibrinogen receptor on human platelets exposed to alpha chymotrypsin

Relationship with a major proteolytic cleavage at the carboxyterminus of the membrane glycoprotein IIb heavy chain


Correspondence to D. Pidard, INSERM U. 150, Hôpital Saint-Louis, 1, Avenue Claude Vellefaux, F-75010 Paris, France


The serine proteinase alpha chymotrypsin from bovine pancreas (CT) is known to expose fibrinogen binding sites on the surface of human platelets in the absence of cell activation and granular secretion. This is accompanied by the appearance of membrane-bound chymotryptic fragments of both glycoprotein (GP) IIb and GPIIIa, the two subunits of the platelet fibrinogen receptor, the GPIIb-IIIa complex. However, no clear relationship between discrete proteolytic event(s) within GPIIb-IIIa and fibrinogen-binding-site expression has yet been established. We have now evaluated the proteolysis of GPIIb-IIIa by CT by Western blot analyses using a panel of polyclonal and monoclonal antibodies against GPIIb or GPIIIa. The different proteolytic events were then correlated with the kinetics of the expression of active fibrinogen binding sites on platelets, as measured through the binding of 125I-labelled purified fibrinogen and to the capacity of CT-treated platelets to aggregate. Treatment of platelets with CT at 22°C resulted in the expression of fibrinogen binding sites prior to cleavage of GPIIIa (Mr∼ 90000) into a previously described, major membrane-bound fragment with Mr 60000. In contrast, fibrinogen receptor expression closely paralleled a proteolytic cleavage at the carboxy terminus of the GPIIb heavy chain (Mr∼ 120000), which was converted into a faster migrating species with Mr∼ 115000. This proteolysis resulted in the release of a soluble peptide with an expected molecular mass of less than 3.7 kDa. Quantitation of this peptide using a competitive immunoenzymatic assay, confirmed that its release from the platelet surface correlated with the expression of fibrinogen binding sites and aggregability. When platelets were exposed to CT at 37°C, a prompt increase in fibrinogen binding sites and platelet aggregability was observed, whereas the GPIIb heavy chain was rapidly converted into the carboxy-terminal-cleaved form. However, incubation at 37°C for longer than 10 min resulted in extensive and simultaneous degradation of both the GPIIb heavy and light chains and of GPIIIa, with the latter being converted into the 60-kDa fragment. These later events were associated with a sharp decline of platelet aggregability and a reduction in the number of fibrinogen binding sites. These data allow us to propose that an early and limited proteolytic processing of the GPIIb component of the platelet fibrinogen receptor is associated with a shift of this receptor complex into a state which expresses specific binding sites for fibrinogen. Further cleavage of GPIIIa to generate the 60-kDa fragment results in loss of receptor activity.