Platelet integrin activation surfs the calcium waves

Glycoprotein (GP) IIbIIIa is the most abundant integrin on the platelet surface and plays a crucial role in hemostasis. With over 80,000 copies on the platelet surface, together with an additional pool that can be trafficked to the membrane during platelet activation, GPIIbIIIa is complexed from the calcium-dependent association of the α and β integrin subunits that require inside-out signaling events to modulate their function in a temporal and spatial manner.^1,2

Calcium (Ca²⁺), originally coined as coagulation factor IV, is well-known to orchestrate a plethora of extracellular and intracellular pathways essential for hemostasis. Platelet inside-out signaling is triggered by platelet agonists including adenosine diphosphate (ADP), thrombin, or extracellular matrix proteins ranging from major to minor constituents including collagen, laminin, and nidogen, respectively.^3,4 These agonists drive a cytosolic Ca²⁺ flux resulting in a conformational change of GPIIbIIIa from a low affinity state at rest to a high affinity state upon activation requisite for ligation of adhesive proteins including fibrinogen and fibronectin.⁵ Upon ligation, GPIIbIIIa then returns the favor by inciting outside-in signaling events to maintain or even further promote platelet activation.

In the propagation phase of platelet plug formation, Ca²⁺ mobilization and accompanied secretion of ADP and thromboxane A2 form a coalition mediated by P2Y₁, P2Y₁₂, and thromboxane prostanoid receptors to recruit, activate, and form aggregates with other platelets. The ensemble of information from the last 20 years of platelet research has improved our mechanistic understanding of the pathways by which G-protein coupled receptors and tyrosine kinase-linked receptors combine to facilitate and regulate GPIIbIIIa inside-out signaling. Studies by Sun et al were among the first to suggest a link between ADP-mediated signaling downstream of P2Y receptors, the PI3 kinase (PI3K) pathway, and oscillations in calcium transients associated with GPIIbIIIa signaling and activation.⁶ Cosemans et al refined this to show that GPIIbIIIa activation and its role in maintenance of platelet aggregates relies on continuous outside-in signaling mediated in part by ADP via P2Y₁₂ and downstream of PI3Kβ and PI3Kγ.⁷ In 2007, Schoenwaelder et al was the first to report an in vivo role for PI3K-mediated signaling in regulating GPIIbIIIa activation and function using a p110γ^−/− mouse model.⁸ Although one cannot deny the fundamental role of the phospholipase C (PLC) signaling pathway downstream of P2Y₁ in regulating the activation state of GPIIbIIIa, signaling functions of PI3K isoforms downstream of P2Y₁₂ are indispensable and complementary to PLC-mediated signaling in driving the cytosolic Ca²⁺ flux crucial for maintaining GPIIbIIIa activation.

When assessed in purified systems on an individual basis, select platelet agonists have been observed to activate integrins with varying degrees of efficiency. Consistent with the non-uniform and complex spatiotemporal nature of Ca²⁺ waves observed in model cell systems, there likely exists a nonlinear relationship between the pattern of intraplatelet Ca²⁺ flux and the combinations of platelet agonists and antagonists present in the milieu of the blood microenvironment in the setting of hemostasis, thrombosis, or inflammation. As an example, the procoagulant activity of platelets is dependent on the pattern, duration, and amplitude of Ca²⁺ spikes.^9–11 The field awaits the use of system biology approaches to fully develop to understand of the spatiotemporal regulation of inside-out signaling driven by Ca²⁺ dynamics.

In the recent issue of Science Signaling, Bye and colleagues describe and visualize signaling events by which the ADP receptors, P2Y₁ and P2Y₁₂, synergize to generate the P2Y₁-dependent Ca²⁺ transients and P2Y₁₂-dependent activation of the PI3K intracellular signal transduction pathway to facilitate polarized GPIIbIIIa activation and subsequent fibrinogen binding. Importantly, this study shows that the spatiotemporal pattern of GPIIbIIIa activation is correlated to the direction of the transient Ca²⁺ waves.¹² Albeit, the relevance of this mechanism in regulating platelet biology is based on the assumption that pathways are conserved between platelets and their precursor cell, the megakaryocyte (MK). Prior studies including the work by Tolhurst et al have demonstrated the synergy between P2Y₁ and P2Y₁₂ receptors in orchestrating the activation of GPIIbIIIa in murine MKs, leading to the suggestion that the rat MK may serve as a functional model of select P2Y-based receptor signaling pathways observed in platelets.¹³ By taking advantage of freshly isolated rat MKs, a primary cell type that displays reproducible, measurable Ca²⁺ waves and robust inside-out activation of GPIIbIIIa, together with fluorescent imaging techniques and quantification of cellular polarity, Bye et al provide insight into the timing, directionality, and synergy of signaling events mediated by Ca²⁺ flux within MKs. This study confirms and complements the long-standing observations on the crucial role of PI3K and PLC signaling axis in mediating Ca²⁺ transients and sustained GPIIbIIIa activation.

Using this model system, Bye and colleagues demonstrated that stimulation of MKs with ADPβS, a hydrolysis-resistant agonist of P2Y₁ and P2Y₁₂, leads to series of transient increases in cytosolic Ca²⁺ waves. The amplitude and frequency of Ca²⁺ waves dictate the degree of GPIIbIIIa activation and correspondingly fibrinogen binding. A noteworthy observation made in this study was a consistent time delay between integrin activation resultant from P2Y₁-dependent cytosolic Ca²⁺ flux and GPIIbIIIa-mediated fibrinogen binding. Moreover, an even more pronounced delay in fibrinogen binding was observed when ionomycin rather than ADPβS was used to induce the release of calcium stores in rat MKs, despite the fact that ionomycin induced far greater calcium peaks than what was observed for ADPβS. The study employed pharmacological inhibitors of P2Y₁, P2Y₁₂, and inhibitors of downstream signaling components such as PLC, protein kinase C (PKC), PI3K, and adenylyl cyclase, to tease apart the interdependencies between the P2Y₁ and P2Y₁₂ signaling cascades. They show that P2Y₁-dependent Ca²⁺ flux relies on P2Y₁₂-dependent PI3K activation, which in synergy with PLC-mediated signaling downstream of G_q evokes GPIIbIIIa activation and fibrinogen binding.

The Bye et al study concludes by validating the concept that spatial reorganization of activated GPIIbIIIa correlates with localized fibrinogen binding in primary MKs. Such polarity in integrin distribution depends on its association with the actin cytoskeleton, a finding in accord with well-established phenomena observed in other model cell systems. While often the platelet apple doesn’t fall far from the MK tree, it is yet to be seen whether this observed role for polarity in MKs will translate to platelet physiology.

In summary, Bye et al have elegantly showcased the intricate spatiotemporal regulation of GPIIbIIIa activation, affinity, and avidity by P2Y receptor-induced calcium transients. This work highlights the key roles of PLC and PI3K signaling in regulating megakaryocyte biology. It remains to be seen whether platelets recapitulate such phenomena, like mother like daughter, or whether platelets adopt a distinct phenotype during their journey of discovery in the circulation.

Figure 1. Synergy between P2Y₁ and P2Y₁₂ are required for integrin activation and fibrinogen binding.

P2Y₁-mediated calcium transients downstream of PLC and P2Y₁₂-mediated PI3K activation synergize to facilitate GPIIbIIIa activation and subsequent polarized fibrinogen binding in a manner correlating to the amplitude and frequency of Ca²⁺ waves during platelet inside-out signaling.