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Platelets – From Function to Dysfunction in Essential Thrombocythaemia

Proteolytic removal of GPV by thrombin or other proteinases facilitates thrombin-induced signalling. The intracellular domain of GPIb-IX-V, comprising the cytoplasmic tails of GPIbα, GPIbβ, GPIX and GPV, is linked to structural (filamin-A, or actin-binding protein) or signalling adaptor proteins (calmodulin, 14-3-3ζ, p85), which enables extracellular ligand binding to initiate intracellular signalling pathways, resulting in platelet activation, cytoskeletal rearrangements and integrin activation. Congenital deficiency of GPIb-IX-V, Bernard–Soulier syndrome, arises from many different individual defects and is associated with giant platelets and thrombocytopaenia, as well as impaired platelet response to vWF, thrombin and other agonists.10

platelet function.5

The GPIbα–vWF-A1 interaction can be induced in vitro by applying pathological shear stress through the use of a cone-plate viscometer, by immobilising vWF on plastic or glass in flow chambers or static adhesion assays, or by using artificial vWF activators such as ristocetin (a bacterial antibiotic) or botrocetin (a snake toxin) that allow plasma vWF to bind platelets, which forms the basis of standard aggregation assays. Importantly, gain-of-function mutations within GPIbα (platelet-type von Willebrand’s disease) or vWF-A1 (type 2b von Willebrand’s disease) can enable platelet GPIbα to bind plasma vWF in vivo or in vitro.

GPVI is a platelet-specific receptor consisting of two extracellular immunoglobulin domains (see Figure 1B) and is non-covalently associated with the Fc receptor γ-chain, FcRγ, required for GPVI surface expression and transmitting signals upon cross-linking of GPVI by ligands.11,12

GPVI binds collagen and laminin.13 Non-

physiological ligands include a cross-linked collagen-related peptide and a number of snake toxins (convulxin, alborhagin and others), which can be used to assess GPVI-dependent platelet function in vitro. The cytoplasmic domain of GPVI binds the signalling molecule, Lyn, and calmodulin.11

GPVI and GPIbα are physically and functionally

co-associated on the platelet surface, providing a potent recognition– signalling complex for vWF/collagen.14

These receptors therefore play

an important role in the initiation of atherothrombosis at high shear stress and where there is exposed collagen.

Human defects of GPVI may be either an acquired deficiency, resulting from anti-GPVI autoantibodies or other causes, or a congenital deficiency where GPVI is not expressed or is expressed in a dysfunctional form with defective signalling to αIIbβ3.15

Of 13

reported cases of GPVI defects, 12 involve females and commonly there is an associated immune dysfunction. Platelets in these patients typically show defective aggregation to collagen or other GPVI ligands but aggregate in response to other platelet agonists.

On platelet activation, P-selectin and CD40 ligand are rapidly translocated to the platelet surface and are subsequently cleaved to generate soluble forms that are biologically active.16

The soluble

forms, soluble P-selectin (sP-selectin) and soluble CD40 ligand (sCD40L), promote coagulation by inducing tissue factor (TF) expression on monocytes and endothelial cells.17

Soluble CD40L also

causes platelet activation and appears to be required for thrombus formation in vivo.18

Platelets – Role in Haemostasis and Thrombosis The main role of platelets in thrombus formation is to recognise a vascular site of injury, rapidly adhere, become activated, spread over the surface and recruit additional platelets to form an aggregate or thrombus (see Figure 1C). This serves at least two purposes:


to prevent blood loss and facilitate wound healing by forming a plug and providing a pro-coagulant surface to accelerate the coagulation cascade; and to fight infection by rapid secretion of bioactive substances from granules that can activate immune cells. By providing an adhesive surface on the thrombus mass that allows direct interaction with leukocytes, these can then migrate to sites of infection or disease. In this regard, a co-ordinated response involves specific platelet surface receptors, rapid agonist-induced secretion of prothrombotic, procoagulant and proinflammatory factors, cytoskeletal changes associated with cell spreading and migration and altered membrane properties promoting coagulation, which all combine to progress from initial injury to arrest of bleeding.

The key to therapeutic management of thrombosis, therefore, is to inhibit pathological thrombosis but without inhibiting normal haemostasis.20,21

Existing antiplatelet drugs such as aspirin or

clopidogrel (one of the world’s biggest-selling medicines), which are used in the treatment or prophylaxis of thrombotic disease, can pose an unacceptable bleeding risk. The most recent approach to overcoming this problem is to consider the role of shear stress in thrombosis and haemostasis (haemorheology).22,23

In the healthy

circulation, fluid shear rates in veins or arteries/arterioles might vary from ~100s-1 to ~1,800s-1.24

However, in a stenotic blood vessel or

sclerotic large artery, turbulent shear rates can exceed 10,000s-1 (or even higher than this). This profoundly affects the capacity of platelets to form a thrombus, and a select group of platelet receptors and other clotting pathways come into play.7,25

In experimental models

of thrombosis, new ways of targeting thrombosis under pathological shear conditions, without affecting bleeding times, have raised the possibility of safer antiplatelet drugs in the future.26,27

The basic steps involved in the transition of circulating resting platelets in the bloodstream to formation of a blood clot or thrombus can be briefly summarised as follows (see Figure 1C).

Activation of platelets in response to signal transduction after engagement of GPIb-IX-V or GPVI induces shape change, cytoskeletal rearrangement, secretion of granule contents such as adenosine diphosphate (ADP) or thromboxane A2 (TXA2), surface expression of P-selectin on α-granules and ‘inside-out’ activation of the integrin αIIbβ3 (GPIIb-IIIa), and other integrins.

Aggregation is mediated by activated αIIbβ3 binding to vWF or fibrinogen, and potentiated at high shear by ADP acting at G

protein-coupled purinergic receptors, P2Y1 and P2Y12, as well as by other platelet receptors such as C-type lectin-like receptor 2 (CLEC-2), CD40L, and semaphorin-4D, which activate platelets within a developing thrombus.

Activated platelets greatly accelerate the coagulation cascade, generating active thrombin, which can further activate platelets through the G protein-coupled thrombin receptors, protease- activated receptor 1 (PAR-1) and PAR-4, which stabilises the thrombus by formation of fibrin. This is followed by αIIbβ3-mediated


The link between haemostasis and thrombosis is an important one. In the case of injury or infection, a rapid platelet response to form a thrombus is vital to prevent blood loss and promote healing. However, if the same sequence of events occurs in a diseased or damaged blood vessel, for example where an atherosclerotic plaque ruptures, then the resulting thrombosis can block blood supply to the heart or brain and cause heart attack or stroke. Similarly, platelet response to exposed collagen in an arthritic joint can promote unwanted inflammation, causing disease.19

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