Are Erk, Btk, and PECAM-1 major players in GPIb signaling? The challenge of unraveling signaling events downstream of platelet GPIb

Several recently published studies in Blood have attempted to unravel the signaling events operating downstream of GPIb.^1-3  One of these studies by Garcia et al¹  examined the role of Erk in promoting VWF/GPIb-dependent activation of integrin α_IIbβ₃ in platelets. These investigators analyzed changes in Erk phosphorylation in VWF/ristocetin-stimulated platelets and examined the effects of a range of pharmacological signaling inhibitors on Erk activation and integrin α_IIbβ₃-dependent platelet aggregation. Based on these studies, the authors conclude that there is an important role for Erk in GPIb signaling and propose a model in which GPIb initiates a linear signaling cascade involving Src kinases → PLC → MEK → Erk → PLA2 that stimulates integrin α_IIbβ₃ and platelet aggregation through an indirect mechanism dependent on the generation of TXA2. Although the results presented are consistent with such a model, we have some concerns with the definitive nature of these conclusions.

The main concern is fundamental and relates to the individual contributions of GPIb and integrin α_IIbβ₃ to VWF-induced signaling. It is generally accepted that the VWF-GPIb interaction induces weak signals to initiate integrin α_IIbβ₃ activation, and the subsequent VWF binding to activated integrin α_IIbβ₃ in concert with released ADP and TXA2 triggers global platelet activation. Thus, many of the commonly used suspension-based functional assays to investigate signals downstream of soluble agonist receptors (ie, classical platelet aggregation, secretion, or ligand binding to activated integrin α_IIbβ₃) are not ideal for analysis of signals derived exclusively downstream of GPIb. In particular, when VWF/ristocetin or VWF/botrocetin induces biphasic platelet aggregation the authors need to consider that output signals are derived from both GPIb and integrin α_IIbβ₃, not solely from GPIb. These factors compound the analysis of the findings presented by Garcia et al¹  and also those by Liu et al²  investigating a role for Btk in GPIb signaling and Rathore et al³  examining PECAM-1 regulation of GPIb signals.

It should be acknowledged that dissecting signaling events downstream of GPIb is difficult, mainly because GPIb-induced signals per se are weak regardless of the experimental approaches used.^4-6  Elucidating GPIb-specific signaling events independent of integrin α_IIbβ₃, ADP, or TXA2 preferably uses methodology with high sensitivity and rapid temporal resolution. This has been most clearly demonstrated from analysis of GPIb-dependent calcium flux.^4,5  Furthermore, GPIb-derived signals in an adherent platelet population are not coincident, and at any given time point detectable signals may be identified only in a relatively small subpopulation of platelets, further complicating analysis. Experimental approaches have been devised by a number of laboratories to investigate GPIb signals independent of other activating stimuli; however, a number of these assays are technically demanding and not widely available. Ideally, findings with respect to GPIb-dependent integrin α_IIbβ₃ activation in suspension-based aggregation assays should be confirmed in adhesion-based assays using direct reporters of integrin α_IIbβ₃ activation, such as those described by Kasirer-Friede et al.⁷ 

With these considerations in mind, we believe some caution should be exercised in the interpretations by Garcia et al,¹  Liu et al,²  and Rathore et al³  that ERK, Btk, and PECAM-1 are major players in GPIb signaling.