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Cardiovascular biology

Small cells, big issues

Nature volume 409, pages 145147 (11 January 2001) | Download Citation

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Identification of a long-sought ADP receptor on platelets explains the effects of two drugs used to prevent strokes and heart attacks. It also points the way to developing other such drugs.

Platelets are the smallest of the human blood cells, measuring about one-tenth the diameter of a white blood cell. They usually circulate freely, maintained in a mobile but inactive state by molecules secreted or expressed by the endothelial cells that line the walls of blood vessels. Beneath the endothelial cells is a protein matrix that includes collagen fibres. When a vessel wall is damaged by injury or disease, platelets adhere to the newly exposed collagen fibres and become active, spreading themselves along the fibres in an effort to close the gap in the layer of endothelial cells (Fig. 1). This process — together with the formation of a clot comprising the blood protein fibrinogen — is responsible for stopping bleeding from wounds. At first, platelet activation depends on the presence of collagen and a blood protein called von Willebrand factor. But as platelets accumulate at the site of injury, further activation and the growth of the platelet plug become increasingly dependent on ADP, secreted from platelets or released from damaged red blood cells.

Figure 1: Formation of a platelet plug at sites of blood-vessel injury.
Figure 1

Top, circulating platelets are maintained in an inactive state by prostacyclin and nitric oxide, released by endothelial cells. Endothelial cells also express on their surface CD39, an enzyme that converts to inactive AMP any small amounts of ADP that might otherwise activate platelets. Bottom, at sites of vascular injury, endothelial cells are damaged or removed. This exposes collagen fibrils, to which platelets adhere with help from von Willebrand factor, a blood protein synthesized by endothelial cells. Once activated in this way, platelets secrete ADP and thromboxane A2. These molecules bind to receptors on circulating platelets, causing them to change shape and become activated, and recruiting them into the growing platelet plug. At the same time, a protein mesh is formed from the plasma protein fibrinogen (not shown). These processes close the gap in the vessel wall, preventing further bleeding until healing can occur.

To accomplish all this in a timely fashion, platelets express on their surface a dense array of receptors for molecules, such as ADP, that trigger platelet activation. The molecular identity of two of the ADP receptors is well known. But pharmacological evidence indicated that there must be a third — and, on page 202 of this issue1, Hollopeter and colleagues identify this hitherto elusive receptor.

Much of the recent work on platelet biology has looked at how platelet activation begins — that is, how non-sticky cells circulating in the bloodstream are transformed into highly sticky ones that adhere to collagen fibres. This is not a trivial issue. Like many human defence mechanisms, optimal platelet responsiveness represents a balance between too much and too little. A platelet must be activated within seconds of approaching a damaged site, or bleeding will continue. On the other hand, overly responsive platelets could block normal blood flow in the smaller vessels of the heart and brain, particularly at sites where atherosclerosis — the deposition of cholesterol-laden plaques — has narrowed the vessel lumen and damaged the protective layer of endothelial cells. Hence the strong interest in identifying platelet-activating receptors that might serve as targets for drugs designed to prevent the unwanted formation of platelet plugs.

Most of these receptors are coupled to intracellular signalling pathways by molecular switches called G proteins. Human platelets express at least ten G proteins, and each type of receptor can couple to one or more types, initiating signalling through several pathways.

Pharmacological evidence indicates that human platelets express at least three different types of ADP receptor2,3,4. One type, designated P2Y1, was thought to be coupled to platelet activation through members of the Gq family of G proteins. This receptor was predicted to be largely responsible for the increase in the concentration of calcium ions inside platelets that is needed to support platelet activation. Indeed, such a role has been confirmed by inactivating the genes encoding P2Y 1 (refs 5, 6) and Gq (ref. 7) in mice. The second receptor — P2X1 — is a calcium channel, but its contribution to the activation of platelets by ADP has recently been questioned8. On the basis of circumstantial evidence, the previously unidentified third ADP receptor was predicted to be coupled to the Gi family of G proteins. It is this protein, now dubbed the P2Y12 receptor, that has been identified by Hollopeter et al.1.

The Gi family of G proteins is best known for inhibiting the formation of cyclic AMP (cAMP) by the enzyme adenylyl cyclase. But why do this? The answer lies in the balance needed to maintain platelets in an optimal state of responsiveness. Circulating platelets remain inactive in part because endothelial cells secrete prostacyclin (also known as prostaglandin I 2, PGI2). This molecule binds and activates receptors on the surface of platelets that stimulate adenylyl cyclase, increasing the formation of cAMP within the platelet. Rising cAMP levels make platelets less responsive to platelet activators. In fact, many such activators — including ADP — work in part by inhibiting adenylyl cyclase and lowering internal levels of cAMP.

The relevance of this effect is shown by the fact that, even before the third ADP receptor was identified, drugs that inhibit the ability of ADP to suppress cAMP formation in platelets were developed and found to block platelet activation. Two of these drugs, clopidogrel and ticlopidine, reduce the risk of recurrent strokes and heart attacks — catastrophic events that involve platelet activation. In the absence of safe and effective oral drugs that prevent platelets from acquiring the ability to stick to each other, ADP-receptor blockers and aspirin (which inhibits the synthesis of prostacyclin and thromboxane A2, another platelet activator) have been used widely to prevent death and disability from heart attacks and strokes.

Unfortunately, some ADP-receptor blockers have occasionally been associated with the development of a life-threatening syndrome characterized by anaemia, kidney failure and, paradoxically, the blocking of small arteries by clumps of platelets9. Until now, the development of successors to these drugs has been slow because the adenylyl cyclase-inhibiting receptor for ADP was not known. The cloning of the gene encoding the P2Y12 receptor by Hollopeter et al.1 should help considerably, because it defines a target for drug design.

As well as showing that the P2Y12 receptor is present on platelets and can mediate the inhibition of cAMP formation by ADP, Hollopeter et al. demonstrate that this protein has the expected pharmacological profile. They also speculate about how a metabolite of clopidogrel might inhibit the receptor, and show that a previously described patient whose platelets fail to respond to ADP lacks a normal form of the gene encoding P2Y12. All in all, they make a convincing case that they have indeed identified this biologically and clinically important molecule.

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Correspondence to Skip Brass.

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