Platelet integrin signaling: wherefore art thou?

In this issue of Blood, Mazzucato and colleagues demonstrate that collagen receptors GPVI and α2β1 integrin generate qualitatively distinct, independent calcium signals during platelet tethering and adhesion to collagen under flow conditions.¹ 

Rapid-spiking, short-lived, calcium signals using intracellular calcium stores are generated by α2β1 signaling in a glycoprotein (GP)VI-independent fashion. GPVI is responsible for sustained calcium mobilization dependent on extracellular calcium influx but requires the presence of α2β1 signaling. These findings add complexity to our understanding of how α2β1 and GPVI interact in response to collagen and provide additional evidence that platelet activation and platelet adhesion are not distinct and independent events.

Collagen-induced platelet adhesion and activation under flow conditions are considered primary events in the formation of the intra-arterial thrombi that cause stroke and heart attack. Studies in the past decade have identified 2 platelet collagen receptors, the immune-type receptor GPVI and the integrin α2β1, as critical for the formation of platelet thrombi on collagen under flow ex vivo and for arterial thrombus formation under some circumstances in vivo. A widely accepted model of platelet collagen responses assigns GPVI the role of generating platelet activation signals, including “inside-out” signals required to conformationally activate integrins on the platelet surface, and α2β1 the role of mediating firm adhesion to collagen in the face of powerful shear forces.^2,3  This model is supported by genetic and pharmacologic loss-of-function studies demonstrating that GPVI signaling is required for platelet activation by collagen, a prerequisite for platelet adhesion to collagen under flow.^4,5  In contrast, α2β1 is required for platelet adhesion to collagen under flow but not for platelet activation by collagen.^6,7 

This clean division of labor suggested by loss-of-function studies has been challenged by findings that have identified roles for α2β1 in the generation of intracellular signals that might initiate or prolong platelet activation. A significant report in the literature, mostly focused on αIIbβ3, supports “outside-in” signaling by integrins through the same pathway used by GPVI.^8,9  The ability of α2β1 to activate this pathway has been demonstrated and, like GPVI, uses Src-family kinases, Syk, Slp-76, and PLCg2.¹⁰  However, unmasking a role for α2β1 signaling in physiologic platelet-collagen responses has been difficult because it shares a common ligand with GPVI and requires previous activating signals to adopt a high-affinity conformation needed for collagen adhesion. Mazzucato et al make an important contribution by showing that, as platelets interact with collagen under flow, α2β1 receptors generate calcium signals that are distinct from those generated by GPVI. Surprisingly, α2β1-dependent calcium signals are even generated in the absence of GPVI, forcing us to re-examine the concept of strict dependence on previous inside-out integrin activation for α2β1 ligand binding and whether GPVI is a major source of these inside-out signals. Even more unexpected is the finding that GPVI-associated calcium signals do not take place in the absence of α2β1, suggesting a stronger synergy between α2β1 and GPVI than previously appreciated. These intriguing results are significant because they document integrin-dependent signals during the actual process of platelet interaction with collagen under flow, a response believed to faithfully reproduce the in vivo interaction between platelets and vessel wall collagen.

A major question raised by this and previous studies is the functional importance of these integrin-generated signals. Various studies have suggested that α2β1 can synergize with GPVI during platelet collagen responses,^7,11,12  but defining a direct signaling role for α2β1 has been more difficult. Mazzucato et al note that more than half of platelets successfully adhere to collagen under flow without generating such α2β1-mediated calcium signals, and GPVI-deficient platelets do not exhibit robust collagen signaling despite the presence of α2β1 integrins. Thus, one possibility is that these integrin signals are either not functionally significant or not sufficient to support platelet activation. Alternatively, as suggested by the authors, the signals generated by GPVI and α2β1, like similar signals generated by GPIb and integrin aIIbb3 during platelet rolling on von Willebrand factor,¹³  may function in an interactive and even reciprocal fashion as platelets interact with matrix ligands such as collagen that are both activating and adhesive. Such interdependent molecular interactions are particularly difficult to unravel using standard loss-of-function experiments, and a more detailed understanding of molecular mechanisms by which integrins trigger such signaling events will be needed to test their biological roles.