Proteolytic cleavage of the β1 subunit of platelet α2β1 integrin by the metalloproteinase jararhagin compromises collagen-stimulated phosphorylation of pp72(syk)

Abstract

Early signaling events in the stimulation of platelets by collagen include the tyrosine phosphorylations of FcR γ-chain, pp72(syk) and phospholipase Cγ2. These events are dependent on the main platelet collagen receptor, α2β1 integrin (glycoprotein Ia-IIa complex). We recently found that jararhagin, a 52-kDa snake venom metalloproteinase, selectively inhibits collagen-induced platelet secretion and aggregation in parallel with the cleavage of the β1 subunit of the α2β1 integrin. The present study demonstrates that jararhagin also interferes with collagen-induced phosphorylation of the protein-tyrosine kinase pp72(syk). This effect is not observed when the platelet aggregation response to collagen is inhibited by two venom RGD-containing disintegrins, contortrostatin and echistatin. These disintegrins inhibit platelet aggregation through their high affinity binding to the platelet α(IIb)β3 integrin (glycoprotein IIb-IIIa complex). We also show that mild stimulation by ADP of jararhagin-treated platelets, but not of platelets treated with the RGD-containing disintegrins, restores the collagen-induced platelet aggregation. ADP also restored both pp72(syk) and pleckstrin phosphorylation of jararhagin-treated platelets in response to collagen, presumably via interaction of collagen with ADP-activated α(IIb)β3 integrin. Thus, RGD-containing disintegrins do not interfere with agonist-induced pp72(syk) phosphorylation but inhibit aggregation through occupancy of the α(IIb)β3 integrin. Conversely, jararhagin affects early platelet signaling events in response to collagen through its effects on the α2β1 integrin without interfering with the function of the α(IIb)β3 integrin. Our demonstration that the degradation of the β1 subunit of α2β1 by jararhagin results in the loss of pp72(syk) phosphorylation, suggests that this subunit is critically involved in collagen-induced platelet signaling.