Activation of platelets by collagen is mediated through a tyrosine kinase-dependent pathway that is associated with phosphorylation of the Fc receptor γ chain, the tyrosine kinase syk, and phospholipase Cγ2 (PLCγ2). We recently described a collagen-related triple-helical peptide (CRP) with the sequence GCP*(GPP*)GCP*G (single letter amino acid code: P* = hydroxyproline; Morton et al, Biochem J 306:337, 1995). The cross-linked peptide is a potent stimulus of platelet activation but, unlike collagen, does not support α2β1-mediated, Mg2+-dependent adhesion, suggesting that its action is independent of the integrin α2β1 . This finding suggests the existence of a platelet receptor other than α2β1 that underlies activation. In the present study, we show that CRP stimulates tyrosine phosphorylation of the same pattern of proteins in platelets as collagen, including syk and PLCγ2. Protein tyrosine phosphorylation induced by CRP is not altered in the absence of Mg2+ or the presence of monoclonal antibodies (MoAbs) to the integrin α2β1 (MoAb 6F1 and MoAb 13), conditions that prevent the interaction of collagen with the integrin. In contrast, phosphorylation of syk and PLCγ2 by collagen is partially reduced by MoAb 6F1 and MoAb 13 or by removal of Mg2+. This may reflect a direct role of α2β1 in collagen-induced signaling events or an indirect role in which the integrin facilitates the binding of collagen to its signaling receptor. The results show an α2β1-independent pathway of platelet activation by CRP that involves phosphorylation of syk and PLCγ2. This pathway appears to contribute to platelet activation by collagen.

The tumour suppressor activity of p53 in vivo can be subject to pressure from the physiological stress of hypoxia and we report on the development of a cell system to define the p53-dependent stages in the adaptation of cells to hypoxia. p53 +/+ cells exposed to hypoxia exhibited a transient arrest in G2/M, but escaped from this checkpoint and entered a long-term G 0 /G 1 arrest. By contrast, isogenic p53-null cells exposed to hypoxic conditions exhibited a 6-10-fold higher level of apoptosis, suggesting that p53 acts as a survival factor under limiting oxygen concentrations. Surprisingly, hypoxia-dependent growth arrest in p53 +/+ cells did not result in either p21 WAF1 protein levels. Hypoxia inhibited 5-FU-induced p53-dependent cell death and attenuated p53 phosphorylation at the ATM and CK2/FACT phosphorylation sites. Although anoxia activates the p53 response, hypoxia silences the p53 transactivation pathway and identifies a physiological signalling model to study mechanisms of p53 inactivation under hypoxic conditions.