Donna S. Woulfe

Defects in secretion, aggregation, and thrombus formation in platelets from mice lacking Akt2

Prior studies have shown that PI3Ks play a necessary but incompletely defined role in platelet activation. One potential effector for PI3K is the serine/threonine kinase, Akt, whose contribution to platelet activation was explored here. Two isoforms of Akt were detected in mouse platelets, with expression of Akt2 being greater than Akt1. Deletion of the gene encoding Akt2 impaired platelet aggregation, fibrinogen binding, and granule secretion, especially in response to low concentrations of agonists that activate the G(q)-coupled receptors for thrombin and thromboxane A(2). Loss of Akt2 also impaired arterial thrombus formation and stability in vivo, despite having little effect on platelet responses to collagen and ADP. In contrast, reducing Akt1 expression had no effect except when Akt2 was also deleted. Activation of Akt by thrombin was abolished by deletion of Galpha(q) but was relatively unaffected by deletion of Galpha(i2), which abolished Akt activation by ADP. From these results we conclude that Akt2 is a necessary component of PI3K-dependent signaling downstream of G(q)-coupled receptors, promoting thrombus growth and stability in part by supporting secretion. The contribution of Akt1 is less evident except in the setting in which Akt2 is absent.

Activation of Rap1B by Gi Family Members in Platelets

It has become increasingly appreciated that receptors coupled to Gαi family members can stimulate platelet aggregation, but the mechanism for this has remained unclear. One possible mediator is the small GTPase, Rap1, which has been shown to contribute to integrin activation in several cell lines and to be activated by a calcium-dependent mechanism in platelets. Here, we demonstrate that Rap1 is also activated by Gαi family members in platelets. First, we show that platelets from mice lacking the Gαi family member Gαz (which couples to the α2A adrenergic receptor) are deficient in epinephrine-stimulated Rap1 activation. We also show that platelets from mice lacking Gαi2, which couples to the ADP receptor, P2Y12, exhibit reduced Rap1 activation in response to ADP. In contrast, platelets from mice that lack Gαq show no decrease in the ability to activate Rap1 in response to epinephrine but show a partial reduction in ADP-stimulated Rap1 activation. This result, combined with studies of human platelets treated with ADP receptor-selective inhibitors, indicates that ADP-stimulated Rap1 activation in human platelets is dependent on both the Gαi-coupled P2Y12 receptor and the Gαq-coupled P2Y1 receptor. Gαi-dependent activation of Rap1 in platelets does not appear to be mediated by enhanced intracellular calcium release because no increase in intracellular calcium concentration was detected in response to epinephrine and because the calcium response to ADP was not diminished in platelets from the Gαi2−/− mouse. Finally, using human platelets treated with selective inhibitors of phosphatidylinositol 3-kinase (PI3K) and mouse platelets selectively lacking the Gβγ-activated form of his enzyme (PI3Kγ), we show that Gi-mediated Rap1 activation is PI3K-dependent. In summary, activation of Rap1 can be stimulated by Gαi- and PI3K-dependent mechanisms in platelets and by Gq- and Ca2+-dependent mechanisms, both of which may play a role in promoting platelet activation.

Interactions between Eph kinases and ephrins provide a mechanism to support platelet aggregation once cell-to-cell contact has occurred

Eph kinases are receptor tyrosine kinases whose ligands, the ephrins, are also expressed on the surface of cells. Interactions between Eph kinases and ephrins on adjacent cells play a central role in neuronal patterning and vasculogenesis. Here we examine the expression of ephrins and Eph kinases on human blood platelets and explore their role in the formation of the hemostatic plug. The results show that human platelets express EphA4 and EphB1, and the ligand, ephrinB1. Forced clustering of EphA4 or ephrinB1 led to cytoskeletal reorganization, adhesion to fibrinogen, and α-granule secretion. Clustering of ephrinB1 also caused activation of the Ras family member, Rap1B. In platelets that had been activated by ADP and allowed to aggregate, EphA4 formed complexes with two tyrosine kinases, Fyn and Lyn, and the cell adhesion molecule, L1. Blockade of Eph/ephrin interactions prevented the formation of these complexes and caused platelet aggregation at low ADP concentrations to become more readily reversible. We propose that when sustained contacts between platelets have occurred in response to agonists such as collagen, ADP, and thrombin, the binding of ephrins to Eph kinases on adjacent platelets provides a mechanism to perpetuate signaling and promote stable platelet aggregation.

G-Protein–Coupled Receptors as Signaling Targets for Antiplatelet Therapy

Platelet G protein–coupled receptors (GPCRs) initiate and reinforce platelet activation and thrombus formation. The clinical utility of antagonists of the P2Y12 receptor for ADP suggests that other GPCRs and their intracellular signaling pathways may represent viable targets for novel antiplatelet agents. For example, thrombin stimulation of platelets is mediated by 2 protease-activated receptors (PARs), PAR-1 and PAR-4. Signaling downstream of PAR-1 or PAR-4 activates phospholipase C and protein kinase C and causes autoamplification by production of thromboxane A2, release of ADP, and generation of more thrombin. In addition to ADP receptors, thrombin and thromboxane A2 receptors and their downstream effectors—including phosphoinositol-3 kinase, Rap1b, talin, and kindlin—are promising targets for new antiplatelet agents. The mechanistic rationale and available clinical data for drugs targeting disruption of these signaling pathways are discussed. The identification and development of new agents directed against specific platelet signaling pathways may offer an advantage in preventing thrombotic events while minimizing bleeding risk.

ADP and platelets: the end of the beginning

Along with thrombin and collagen, ADP has long been recognized for its contribution to the propagation of platelet activation at sites of vascular injury. However, recent studies have brought a new appreciation of the extent of that contribution and identified the two G protein–coupled receptors that appear to be largely responsible for platelet responses to ADP: P2Y1, which activates phospholipase C (1), and P2Y12, which suppresses cAMP formation so that activation can occur (Figure ​(Figure1)1) (2, 3). P2Y12 (also known as P2T, P2Yac, and SP1999) was identified last year by two teams of investigators using distinctly different strategies (2, 3). Now, in this issue of the JCI, Carolyn Foster and her colleagues at Schering-Plough Research Institute describe the consequences of deleting the gene encoding P2Y12 in mice (4). Their results help to validate parts of the current model for platelet activation by ADP. They also bring to a conclusion a phase of research in which platelet responses to ADP were initially characterized, the contribution of heterotrimeric G proteins was recognized, and the existence of more than one class of ADP receptor was established. Given the growing use of antiplatelet agents that target P2Y12 in patients with cardiovascular disease, this is a good opportunity to review the role of ADP in platelet activation and consider some of the questions that have yet to be answered. Figure 1 ADP and platelet activation. Platelet activation by potent agonists such as thrombin or collagen causes the release of secondary agonists such as thromboxane A2 (TxA2) and the secretion of ADP from platelet dense granules. Platelet responses to ADP require ...

GSK3β is a negative regulator of platelet function and thrombosis

Glycogen synthase kinase (GSK)3beta is a ser-thr kinase that is phosphorylated by the kinase Akt. Although Akt has been shown to regulate platelet function and arterial thrombosis, its effectors in platelets remain unknown. We show here that agonist-dependent phosphorylation of GSK3beta in platelets is Akt dependent. To determine whether GSK3beta regulates platelet function, platelets from mice lacking a single allele of GSK3beta were compared with those of wild-type (WT) controls. GSK3beta+/- platelets demonstrated enhanced agonist-dependent aggregation, dense granule secretion, and fibrinogen binding, compared with WT platelets. Treatment of human platelets with GSK3 inhibitors renders them more sensitive to agonist-induced aggregation, suggesting that GSK3 suppresses platelet function in vitro. Finally, the effect of GSK3beta on platelet function in vivo was evaluated using 2 thrombosis models in mice. In the first, 80% of GSK3beta+/- mice (n=10) formed stable occlusive thrombi after ferric chloride carotid artery injury, whereas the majority of wild-type mice (67%) formed no thrombi (n=15). In a disseminated thrombosis model, deletion of a single allele of GSK3beta in mice conferred enhanced sensitivity to thrombotic insult. Taken together, these results suggest that GSK3beta acts as a negative regulator of platelet function in vitro and in vivo.

The kinetics of αIIbβ3 activation determines the size and stability of thrombi in mice: implications for antiplatelet therapy

Two major pathways contribute to Ras-proximate-1-mediated integrin activation in stimulated platelets. Calcium and diacyglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI, RasGRP2) mediates the rapid but reversible activation of integrin αIIbβ3, while the adenosine diphosphate receptor P2Y12, the target for antiplatelet drugs like clopidogrel, facilitates delayed but sustained integrin activation. To establish CalDAG-GEFI as a target for antiplatelet therapy, we compared how each pathway contributes to thrombosis and hemostasis in mice. Ex vivo, thrombus formation at arterial or venous shear rates was markedly reduced in CalDAG-GEFI(-/-) blood, even in the presence of exogenous adenosine diphosphate and thromboxane A(2). In vivo, thrombosis was virtually abolished in arterioles and arteries of CalDAG-GEFI(-/-) mice, while small, hemostatically active thrombi formed in venules. Specific deletion of the C1-like domain of CalDAG-GEFI in circulating platelets also led to protection from thrombus formation at arterial flow conditions, while it only marginally increased blood loss in mice. In comparison, thrombi in the micro- and macrovasculature of clopidogrel-treated wild-type mice grew rapidly and frequently embolized but were hemostatically inactive. Together, these data suggest that inhibition of the catalytic or the C1 regulatory domain in CalDAG-GEFI will provide strong protection from athero-thrombotic complications while maintaining a better safety profile than P2Y12 inhibitors like clopidogrel.

Akt signaling in platelets and thrombosis

Akt is a Ser–Thr kinase with pleiotropic effects on cell survival, growth and metabolism. Recent evidence from gene-deletion studies in mice, and analysis of human platelets treated with Akt inhibitors, suggest that Akt regulates platelet activation, with potential consequences for thrombosis. Akt activation is regulated by the level of phosphoinositide 3-phosphates, and proteins that regulate concentrations of this lipid also regulate Akt activation and platelet function. Although the effectors through which Akt contributes to platelet activation are not definitively known, several candidates are discussed, including endothelial nitric oxide synthase, glycogen synthase kinase 3β, phosphodiesterase 3A and the integrin β3 tail. Selective inhibitors of Akt isoforms or of proteins that contribute to its activation, such as individual PI3K isoforms, may make attractive targets for antithrombotic therapy. This review summarizes the current literature describing Akt activity and its regulation in platelets, including speculation regarding the future of Akt or its regulatory pathways as targets for the development of antithrombotic therapies.

Somatostatin receptor activation of cellular effector systems.

Somatostatin (SRIF) induces its multiple biological actions by interacting with a family of receptors, referred to as SSTR1-SSTR5. These receptors are capable of associating with particular guanine nucleotide binding proteins to couple the receptors to distinct cellular effector systems. Therefore, G proteins have an important role in directing SRIF signalling and may provide the molecular basis for the diverse cellular actions of SRIF.

Contact-dependent signaling during the late events of platelet activation.

Summary.  Signaling events downstream from collagen receptors and G protein‐coupled receptors are responsible for the initiation and extension of platelet plug formation. This creates the platelet plug and hopefully results in the cessation of bleeding. It is not, however, all that is required for hemostasis, and growing evidence is emerging that the perpetuation of a stable hemostatic plug requires additional intracellular signaling. At least part of this process is made possible by the persistent close contacts between platelets that can only occur after the onset of aggregation. This review discusses several examples of such signaling mechanisms that help to perpetuate the platelet plug in a contact‐dependent manner, including outside‐in signaling through integrins, signaling though Eph kinases and ephrins, and the role of CD40L.