Platelet Function in Health and Disease: from Molecular Mechanisms,Redox Considerations to Novel Therapeutic Opportunities

1. Collagen receptors

Among the macromolecular constituents of the ECM, collagen is considered the main character in the process of platelet activation, as it is involved in platelet adhesion through direct and indirect pathways, and it directly triggers both aggregation and coagulant activity (23). Platelet adhesion and aggregation on collagen are integrated processes that involve several platelet agonists acting through a variety of surface receptors, including integrins, Ig-like receptors, and GPCRs. Indeed, platelets express several collagen receptors (64) with different tasks, as different are the collagens present in the vessel wall, of which collagens I and III are considered the most important in supporting platelet adhesion to the damaged vasculature. However, there are two receptors on the platelet surface that bind directly to collagen, the GPVI Ig-superfamily member and the integrin α2β1. An accessory receptor for collagen-dependent platelet adhesion and activation might be represented by GPV, a GP noncovalently linked to platelet GPIb–V–IX complex (292). GPVI is expressed in platelets and mature megakaryocytes, where it associates with the immunoreceptor tyrosine-based activation motif (ITAM)-containing transmembrane adaptor protein Fc receptor γ-chain (119). On the cross-linking of GPVI by its ligand, ITAM is tyrosine phosphorylated by Src family kinases Fyn and Lyn (104) bound to the cytoplasmic domain of GPVI. After binding to phosphorylated ITAM, the tyrosine kinase Syk undergoes autophosphorylation and phosphorylation by Src kinases and initiates a downstream signaling cascade, leading to the activation of a series of adapter and effector proteins (319). One of the main effector enzymes in the GPVI signaling cascade is PLCγ2, which liberates the second messengers 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃) (76). Binding of IP₃ to its receptors triggers the release of calcium from intracellular stores (Ca²⁺ mobilization), protein phosphorylation, and AA release, which, in turn, causes the aggregation and secretion of TxA₂ and ADP (Fig. 1).

The α2β1 integrin, commonly referred to as GPIa/IIa or CD49b/CD29, also plays a role in the adhesion of platelets to collagen for subsequent optimal activation. Similar to what was observed for fibrinogen receptor, integrin αIIbβ3, α2β1 integrin is expressed on resting platelets in a low-affinity state with the extracellular domains folded into a closed conformation. It has been demonstrated that the affinity of α2β1 integrin for soluble collagen increases on cellular stimulation (162), suggesting that α2β1, similar to αIIbβ3, requires an agonist-induced conformational change (via inside-out signaling) to bind to collagen (218). Moreover, recent evidence also suggests that the α2β1-triggered signaling pathway is characterized by the sequential activation of Src and Syk family tyrosine kinases (153), including Rap1b (27) and Rac1 (293), leading to the activation of PLCγ2 and the formation of lamellipodia (153). It is interesting to note that, contrarily to the ability of α2β1 to mediate full-platelet spreading, the engagement of integrin α5β1 by fibronectin (in the presence of a αIIbβ3-receptor antagonist) generates the formation of filopodia but not lamellipodia, eventually causing a stable adhesion of platelets to the ECM (268). In this regard, there is evidence indicating complex and bidirectional cross-talking between αIIbβ3 and α2β1. Indeed, it has been demonstrated that agonist-induced α2β1 activation is regulated by αIIbβ3 outside-in signaling (304) and that α2β1 promotes the activation of αIIbβ3 and induces fibrinogen binding to adherent platelets through a pathway which requires the active form of Rap1b generated downstream of PLC (27).

2. Fibrinogen receptor

A main adhesion molecule involved in platelet aggregation is the membrane protein integrin αIIbβ3. Integrin αIIbβ3 is present at a high density on platelets, both on the PM and on α-granules. After platelet activation and integrin αIIbβ3 conformational changes, receptor-bound fibrinogen acts as a bridge between two αIIbβ3 molecules on adjacent platelets (Fig. 2) (307). This is the final common pathway of platelet aggregation induced by platelet chemical agonists, and in all circulatory conditions of low shear (123), while VWF substitutes for fibrinogen as a bridge molecule between integrin αIIbβ3 for platelet aggregation induced by high shear conditions (123). Although integrin αIIbβ3 is the most widely studied mediator of bridging platelets to each other and stabilizing thrombi, other molecules have been recently proposed as mediating these responses. These include junctional adhesion molecules, signaling lymphocyte activation molecule family proteins, and CD40 ligand (CD40L) (9). Postligand binding events regulated by integrin αIIbβ3 in platelets include the stabilization of large platelet aggregates, platelet spreading, granule secretion, clot retraction, and platelet procoagulant activity (229).

One of the most intensely investigated areas of platelet biology has been defining the mechanisms by which platelet activating signals regulate the adhesive function of integrin αIIbβ3. Soluble agonist receptors coupled to Gαq stimulate PLCβ, triggering the generation of IP₃ and DAG. IP₃ plays a central role in promoting intracellular Ca²⁺mobilization, while DAG promotes the activation of specific isoforms of protein kinase C (PKC). A key step in the activation of integrin αIIbβ3 is the Ca²⁺- and DAG-dependent regulation of the guaninenucleotide exchange factor 1 (CalDAG-GEF1). CalDAG-GEF1 is a potent inducer of the small GTPase molecule Rap1b, which plays a key effector role in inducing integrin αIIbβ3 activation (72). Activated Rap1b-GTP translocates to the PM, where it binds to its effector, Rap1-GTP interacting adapter molecule (RIAM). RIAM unmasks the talin binding site, allowing talin to bind the cytoplasmic tail of integrin β3 subunits, thereby activating integrin αIIbβ3 (294). The activation of integrins by talin is enhanced by focal adhesion protein co-activators, kindlin-2 and kindlin-3. Kindlins bind to the cytoplasmic tail of β-integrin at sites distinct from those bound by talin. The C-terminal region of kindlin-3 aids the conformational activation of integrin αIIbβ3 by connecting it to the cytoskeletal protein actin (300).

The secretory phase of platelet activation amplifies the initial platelet response to ligands (Figs. 1 and 2). The exocytosis of platelet granules, namely α-granules, dense granules, and lysosomal granules, causes the release of messengers that help recruit additional circulating platelets and contributes toward altering the conformation of the αIIbβ3 receptor, finally leading to aggregation (39). In particular, platelet exocytosis of α-granules releases adhesive molecules such as fibrinogen, VWF, and P-selectin; coagulation and fibrinolitic factors; chemokines such as platelet factor 4 (PF4), RANTES (regulated on activation, normal T cell expressed and secreted), and interleukin (IL)-8; growth factors; and adhesion receptors (247). The exocytosis of dense granules releases nucleotides, including ATP and ADP, serotonin, histamine, and divalent cations. Lysosomal granules release mainly proteolytic enzymes (247).

The model of platelet activation (adhesion, shape change, secretion, and aggregation) involves an organized remodeling of the actin cytoskeleton, mediated by the activation of the small GTP-binding proteins Rho, Rac, and Cdc42. These proteins, which differentially regulate the reorganization of the actin cytoskeleton, exert different roles and lead to the formation of different cellular structures. In particular, Rho activation mainly regulates the Ca²⁺-independent cell spheration and contractility during shape change through stimulation of the Rho-kinase ROCK; Rac1 activation in platelets is Ca²⁺-dependent (286), is fundamental for the formation of lamellipodia during platelet spreading (201), and is involved in the regulation of secretion and subsequent aggregation in human platelets stimulated with thrombin (221).

3. G protein-coupled receptors for thrombin, ADP, and prostaglandins

4. Calcium signaling

An increase in [Ca²⁺]i is a major signal for platelet activation and accompanies activation by all agonists under physiological conditions (255). It occurs through Ca²⁺ release from intracellular stores and Ca²⁺ entry through the PM. Ca²⁺ is initially released from the intracellular stores of the dense tubular system (DTS) in response to the formation of IP₃ from PM phosphatidyl inositol-(4,5)-bisphosphate. This initial release from intracellular stores leads to the depletion of the cytosolic stores and triggers the influx of extracellular Ca²⁺ via store-mediated Ca²⁺ entry (SOCE) (253). The other major Ca²⁺ entry mechanism is mediated by the direct receptor-operated calcium channel, P2X1. The increase in [Ca²⁺]i is very rapid, and it is counteracted by a slower return to lower levels brought about by sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase and by plasma membrane calcium ATPase (PMCA), which pump Ca²⁺ back into the stores or through the PM out of the cell, respectively. PMCA is a highly regulated transporter whose function is to maintain low [Ca²⁺]i by catalyzing ATP-dependent Ca²⁺ efflux (46). In human platelets, where a secretion-like coupling mechanism has been demonstrated, Ca²⁺ entry is proposed to be based on the reversible trafficking of portions of the Ca²⁺ stores toward the PM to facilitate de novo coupling between the type II IP3R receptor in the store membrane and naturally expressed human canonical transient receptor potential 1 (hTRPC1) in the PM (254).

Ca²⁺ regulating mechanisms are of great importance for pathological thrombus formation, rendering Ca²⁺ entry in platelets a promising therapeutic target for both the prevention and treatment of ischemic events. However, since the description of the many proteins involved in Ca²⁺ homeostasis in platelets is beyond the focus of this review, we refer to more extensive reviews in the field (306).

1. RNOS generating systems

RNOS include superoxide anion (O₂ ⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (OH·), hypochlorous acid (HOCl), and peroxynitrite (ONOO⁻) (146). The key modulators of cellular redox regulation are NO and O₂ ⁻, which can react with each other, thus causing the formation of ONOO⁻. The sources of RNOS are a variety of cell types, including vascular smooth muscle cells (VSMCs), ECs, and mononuclear cells. Several enzyme systems contribute to the production of RNOS in vascular tissues, including xanthine oxidase, the nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases, mitochondrial sources, and NO synthase (NOS) (296).

Vascular ECs are tightly regulated by endothelium-derived NO generated via the catalytic activity of a constitutively expressed NOS, an oxido-reductase found in many cell types, also known as endothelial NO synthase (eNOS) (110). Vascular NO relaxes blood vessels, prevents platelet aggregation and adhesion, limits the oxidation of low-density lipoprotein (LDL) cholesterol, inhibits the proliferation of VSMCs, and decreases the expression of pro-inflammatory genes that advance atherogenesis (109,110). Platelet biosynthesis of NO occurs from l-arginine via eNOS (Fig. 3) (281). The first report of the NOS isoform in platelets described the presence of an l-arginine/NO pathway that is able to regulate collagen-induced aggregation (242). Later, platelet-derived NO has been shown to regulate thrombus formation in vitro and in vivo (112).

eNOS can be activated in either a Ca²⁺/calmodulin-dependent or a -independent manner, depending on the stimulus (107, 137), and several kinases (PKA, Akt, calmodulin-dependent kinase II, or AMP-activated protein kinase) appear to be involved in eNOS phosphorylation and activation. Among the kinases, Akt, a Ser/Thr kinase, appears to play a pivotal role in agonist-induced platelet activation and modulation of eNOS (325). In platelets, the β₂-adrenoceptor–mediated activation of NOS is adenylyl cyclase-dependent but is not associated with any change in [Ca²⁺]_i (241), suggesting that platelet eNOS is largely Ca²⁺-independent (107). Indeed, the rate of Ca²⁺-independent platelet eNOS activity regulation relies on the phosphorylation/dephosphorylation of Ser1177 and/or Thr495 residues. Phosphorylation of Ser1177 residue activates eNOS, while phosphorylation of Thr495 residue, being the negative regulatory site, inhibits the activity of the enzyme (243). There is evidence that persisting oxidative stress will render eNOS dysfunctional such that it no longer produces NO, but O₂ ⁻ (108).

NO bioavailability in vivo depends mainly on the inactivation induced by superoxide, with the subsequent generation of novel and more RNOS, including ONOO⁻. ONOO⁻, besides causing the initiation of lipid peroxidation, nitration of aromatic compounds, and modifications of sulfide residues, was also found to cause Tyr-residue nitration in PGI₂ synthase and Mn-superoxide dismutase (SOD) and to participate in providing the peroxide tone for COX (Fig. 3) (16). Indeed, COX-1, also known as PG endoperoxide H2 synthase-1 (PGHS-1), is converted into an active form in which a ferric heme and a tyrosine (Tyr) residue are converted to a ferryl species and a Tyr-radical, respectively. The interaction of this Tyr-radical with the substrate AA forms an arachidonyl radical, which interacts with molecular oxygen to form 15-hydroperoxy-prostaglandin-9,11,-endoperoxide (PGG₂) (270). Subsequently, PGG₂ is reduced to the 15-hydroxy derivative PGH₂, which represents the substrate for many prostanoid synthases, the most relevant of which in human platelet activation is TxA₂ synthase (130). The initiation and maintenance of the PGHS catalytic cycle by peroxides was termed “peroxide tone,” requires a steady presence of peroxides or ONOO⁻, and plays an important role in platelet-derived TxA₂ synthesis (270). In conditions of resting equilibrium, in which the uncontrolled formation of TxA₂ has to be prevented in order to avoid inappropriate platelet activation, the PGHS-2 isoform is prevalent, thus ensuring a protective release of the antiaggregatory molecule PGI₂. Conversely, under pathophysiological circumstances, associated with enhanced oxidant formation, PGI₂ formation is inhibited, while TxA₂ formation is enhanced (332).

NO bioavailability in vivo also depends on the presence of endogenous NOS inhibitors. These inhibitors take the form of methylated arginine derivatives such as asymmetric dimethylarginine (ADMA) and N^ω-monomethyl-l-arginine (L-NMMA). Their plasma concentration changes under varying physiological and pathological conditions and is regulated by the enzyme dimethylarginine dimethylaminohydrolase, which breaks down these compounds to inactive forms (303). Accordingly, there has been a recent surge of interest in the role of endogenous NOS inhibitors such as ADMA in CV diseases with debate raging over their causative roles (303).

Finally, NO insufficiency can be attributed to limited substrate/cofactor availability as well as interactions with RNOS. A balance of endothelium-derived NO and RNOS modulates endothelial function, while an imbalance between NO and RNOS, so-called oxidative stress, is involved in endothelial dysfunction through the inactivation of NO.

Platelets are capable of producing ROS, such as O₂ ⁻ and H₂O₂, during activation (86) (Fig. 3). The first evidence of the release of O₂ ⁻ and other ROS by platelets was observed by Marcus in 1977 (197).

Increased oxidative stress promotes platelet aggregation, while NO inhibits coagulation and platelet activation. The ROS derived from both platelets or other vascular sources represent modulators of platelet activity to such an extent that it has been shown that the ROS generated by platelets have a direct role in the control of their activity (120).

Agonists that induce platelet activation also activate the platelet isoform of NADPH oxidase through the activation of a gp91phox-dependent enzyme (231). The production of O₂ ⁻ by platelets that is dependent on NADPH oxidases enhances the recruitment of platelets to a growing thrombus, most likely by inactivating a platelet ectonucleotidase, thereby increasing the bioavailability of ADP. As already stated, the formation of peroxynitrate impairs the antiplatelet activity of NO. O₂ ⁻ can be converted to H₂O₂ by SOD; SOD and NO are competitors in O₂ ⁻scavenging. H₂O₂ also serves as a substrate for the production of other detrimental ROS, such as HOCl, generated by enzymatic conversion by neutrophil myeloperoxidase. In addition, H₂O₂ reacts with ferrous iron (Fe²⁺) to generate ferric iron (Fe³⁺) and the OH·. Despite playing an important role in oxidative stress and oxidative biosynthesis, evidence continues to accumulate that H₂O₂ functions as a signaling agent. Indeed, endothelial-derived H₂O₂ has proved to be a necessary component of the pathway regulating flow-mediated dilation in human coronary arterioles (192, 208).

In the presence of catalase, H₂O₂ is degraded to water and oxygen. Glutathione peroxidase (GPX) also exerts an antioxidant enzymatic function, because it catalyzes a reaction that degrades H₂O₂ by oxidizing reduced glutathione (GSH) to its disulfide form (GSSG) (for an extensive review, please refer reference 86). GSH has proved capable of potentiating platelet aggregation at concentrations (and at a ratio of GSH/GSSG) found in blood in various disease states (102). Intracellular glutathione also plays a role in platelet activation, probably by maintaining the sulfhydryl status of cytoplasmic proteins (101).

It is likely that the source of ROS, the subcellular localization of the ROS generation, and, potentially, the redox-sensitive pathway are dependent on the stimulus. Indeed, recent data indicated that thrombin-induced ROS production through PAR-1, or after membrane depolarization (177), was extracellular, while after collagen stimulation, probably mediated by GPVI, ROS production was intracellular (18). This is consistent with recent reports demonstrating that receptor-stimulated ROS generation is compartmentalized and/or targeted (295).

In patients with hypercholesterolemia, platelet-associated NAD(P)H oxidase produces a thrombogenic phenotype and mediates the arteriolar dysfunction and venular blood cell recruitment (291) [via platelet CD40 and P-selectin (290) and vessel wall CD40/CD40L interactions (105)]. The soluble form of CD40L, sCD40L, mediates the stimulation-induced platelet release of RNOS through the activation of Akt and p38 mitogen-activated protein kinase (MAPK) signaling pathways. These data suggest that sCD40L plays an important role in regulating platelet-dependent inflammatory and thrombotic responses (55). Relative to oxidative stress in platelets, redox-sensitive CD40/CD40L interactions specifically induce the activation of Akt and p38 MAPK (55).

Increased oxidative stress enhances CD40L surface expression in thrombin and GPVI-stimulated platelets, suggesting that platelet-derived CD40L surface expression is redox regulated (18, 111). Accordingly, antioxidants mediate the inhibition of agonist-induced CD40L surface expression (232).

A result of enhanced oxidative stress is the formation of isoprostanes by the peroxidation of AA esterified to membrane phospholipids and that is subsequently released, by PLA₂ activity to the blood stream (214, 216). It has been demonstrated that although many isoprostanes are present in very tiny concentrations, their biological effects may add up synergistically to biologically relevant actions (26). Isoprostanes may, thus, link lipid peroxidation seen in CV disorders to enhanced platelet activation (81). One mechanism by which isoprostanes are formed is via redox-generated protein tyrosyl radicals at Tyr385 of the COX, which may further enhance peroxidase activity of COX and thereby aggravate oxidative stress and isoprostane formation (122).

Platelet stimulation leading to various activatory responses, including aggregation and secretion, also considers the involvement of thiol groups or the rearrangement of disulfide bonds. Indeed, it has been suggested that a prerequisite for platelet responses is represented by sulfhydryl groups in platelet surface proteins (101). Sulfhydryl groups that are in proximity to each other such that they undergo reversible dithiol/disulfide conversions are considered vicinal thiols. The mechanism through which protein disulfide isomerase mediates the activation of αIIbβ3 (180) and of other platelet integrins, including α2β1 (179), might be the result of exposure or generation of vicinal thiols both in the αIIb and in the β3 subunits. Platelet redox mechanisms or low-molecular-weight thiols found in the external redox environment possibly regulate the redox-sensitive sites in both integrin subunits. Moreover, it has been suggested that a transplasma membrane oxidoreductase generates thiols from disulfides on the platelet surface (101). Modifications of protein thiols or amino groups might represent an important cause of the lipid peroxidation-mediated inactivation of different membrane-bound enzymes, including protein phosphatases (PTPs). Being the counterpart of protein tyrosine phosphorylation, PTPs are key elements of signaling pathways that regulate the physiological functions of platelets. In particular, PTP activity is essential to avoid inappropriate platelet activation or to bring platelets back to a resting state (185).

2. Antioxidant mechanisms

On the other hand, platelets also express antioxidant enzymes (248). This corroborates the role of ROS in platelet signaling, as these antioxidant enzymes likely not only prevent the cytotoxic effects of ROS but also regulate oxidation-sensitive signaling pathways in platelets (177). Antioxidant status is an important determinant of platelet function: an imbalance between the activities and the intracellular levels of these antioxidants is associated with an increase in ROS production. Antioxidants may exert an indirect inhibition of platelet function by scavenging ROS.

Enzymatic antioxidants include SOD, GPx, and catalase (302), while nonenzymatic antioxidants are represented by ascorbic acid (vitamin C), α-tocopherol (vitamin E), glutathione, carotenoids, flavonoids, and other molecules.

SOD serves as antioxidants by catalyzing the dismutation of superoxide into oxygen and H₂O₂; GPX reduces lipid hydroperoxides to their corresponding alcohols and reduces free H₂O₂ to water (108). Hydroperoxides produced by the platelets (PGG₂, 12-HpETE, and PLOOH) are metabolized by GPx. Glutathione depletion in platelets leads to attenuated GPX activity, decreased levels of α-tocopherol, and increased lipid peroxidation (43). Catalase promotes the decomposition of H₂O₂ to water and oxygen.

Vitamin C (l-ascorbate) is considered the most efficacious water-soluble antioxidant in human plasma. This molecule effectively scavenges various RNOS and regenerates α-tocopherol from its radical species (200).

Molecules with vitamin E activity scavenge lipid peroxides. However, α- tocopherol itself becomes, in turn, a radical (the tocopheroxyl radical) with potentially prooxidant activity (108). The platelet inhibitory properties of vitamin E supplementation do not appear to be entirely irrelevant, as supplementation is associated with increased hemorrhagic stroke (2). A study carried out with a mixed tocopherol preparation rich in α-tocopherol demonstrated that such a mixture caused increased NO release, endothelial constitutive NOS activation, and SOD protein content in platelets (191).

Flavonoids are polyphenols (anthocyanins) that display several biologic activities in the CV system by interfering with some of the basic mechanisms involved in the provocation of and in the protection from ischemic CV disease, such as oxidative stress and ROS production, NO formation and activity, and the expression of adhesive molecules by blood cells and the vascular wall (126).

Besides antioxidant enzymes, platelets express scavenger receptors, including CD36. Platelet CD36 is associated with nonreceptor tyrosine kinases of the Src family (151), which have previously shown to be implicated in platelet activation by oxidized LDL (oxLDL) (198). CD36-dependent signaling cascade responsible for oxLDL-dependent activation of platelets includes the Src kinases Fyn and Lyn, the upstream MAP kinase kinase 4, and the MAP kinase JNK2 (56). It has been suggested (233) that platelet CD36 might serve as a sensor of specific oxidized phospholipids generated during oxidative stress. In this review, CD36 engagement might induce an additive activating signal that, in synergism with signals from other receptors, might cause platelet activation by subthreshold concentrations of physiological agonists, thus potentiating members of the platelet activation pathway. Unlike other “co-receptor” ligands, which are primarily localized to the platelet surface during the initial phase of platelet aggregation, CD36 ligands are likely to be presented to the platelet surface before “classical” platelet agonists (233); thus, CD36 may sensitize the platelet for subsequent activation.

Another interesting scavenger receptor present in platelets is the class B scavenger receptor SR-BI, a multiligand receptor of the CD36 superfamily, which seems to be the major receptor on platelets for oxidized high-density lipoprotein (oxHDL) (301). Its major physiologic functions are selective uptake of cholesteryl esters from HDL in steroidogenic tissues and liver (176), stimulation of the bidirectional flux of free cholesterol between cells and lipoproteins, modification of membrane cholesterol distribution, and triggering of signaling events (326).

The findings in animal models have established the key role of hepatic SR-BI expression in the process of reverse cholesterol transport that has proved to be critical in maintaining adequate cholesterol homeostasis and in the prevention of CV disease. If SR-BI plays similar roles in HDL metabolism and protection against coronary artery disease (CAD) in humans as those reported in mice, then it would represent a new target for an antiatherogenic therapeutic approach (184). However, although human SR-BI has the same binding (42) and lipid trading (91) activities as rodent SR-BI, its role in human physiology requires further investigation. A fascinating hypothesis is that LDL binding and cholesteryl ester selective uptake by human SR-BI may contribute to LDL-cholesterol clearance, provided that LDL can bind to SR-BI in the presence of HDL.

To date, the major evidence that SR-BI might be relevant in controlling HDL cholesterol levels and metabolism in humans comes from recent studies of a family with a mutation in the SR-BI gene and increased levels of HDL cholesterol (309). This mutant form of SRBI results in a decreased uptake of cellular cholesteryl esters compared with wild-type SR-BI, indicating that the abnormal HDL cholesterol levels found in these patients might be due to a reduction in the functional activity of this mutant receptor (309).

1. Bleeding time

The bleeding time (BT) is the oldest in vivo test of platelet function (1) that studies the role played by the vessel wall during the interaction of platelets with endothelium/vascular cells. The BT, developed in 1910 as a clinical bedside test of global platelet function, is still, although less frequently, applied as a classical screening test for primary hemostasis. It measures the time required to stop spontaneous bleeding out of a standardized cut in the skin, on the volar surface of the forearm, using a venostatic pressure (40 mmHg) applied on the upper arm. The normal BT ranges between 2 and 10 min, but severe platelet defects including severe von Willebrand's disease (VWD) can result in a BT > 30 min (157). However, its test procedural variability renders it poorly reproducible and sensitive, and with a high inter-operator coefficient of variation and a subjective end point. Actually, an accurate bleeding history should be regarded as a more valuable screening test (134).

2. Platelet aggregometry

Light transmission aggregometry (LTA), also referred to as turbidometric, spectrophotometric, or optical aggregometry, was developed about fifty years ago and for decades was regarded as the gold standard of platelet function testing (1). LTA measures light transmittance through a platelet milieu before and after the addition of a platelet agonist. The increase in light transmittance is proportional to the platelet aggregates formed.

The principle of LTA consists of exposing platelet-rich plasma (PRP), obtained by the centrifugation of citrated blood to varying doses of platelet agonists such as collagen, ADP, thrombin, or adrenaline (either alone or in combination). On addition of the agonist, the platelets change shape from discs to a more rounded form with pseudopods, resulting in a transient small decrease in light transmission followed by a large increase on the formation of aggregates. The increase in light transmission is classically quantified using optical change technology. This is still the most widely used test for identifying platelet function defects. For instance, VWD by usage of the antibiotic ristocetin as agonist, or Glanzmann's thrombasthenia, when platelets change shape but fail to aggregate in response to all agonists, are easily diagnosed.

Major drawbacks of LTA are represented by the high amount of blood required for PRP preparation, which itself is affected by many preanalytical and analytical variables (51). Apart from the nonphysiologic no/low shear conditions during analysis, the use of PRP is responsible for the fact that LTA does not accurately simulate primary hemostasis and may result in the loss of hyper-/hypoactive platelets, extra time, and technologies experienced in preparation and cell-counting techniques (136).

Thus, whole blood aggregometery using impedance technology, which does not require further blood processing, has been recommended and is sometimes combined with luminometry to simultaneously measure dense granular ATP release. Lumiaggregometry, which measures platelet aggregation and secretion simultaneously, might be preferable to LTA for the diagnostic workup of patients with platelet function disorders (51).

Due to the many disadvantages of BT and LTA, alternative automated technologies have been developed in trying to simulate hemostasis in vitro that might potentially be utilized as point-of-care instruments for assessing bleeding risk, thrombotic risk, and monitoring antiplatelet therapy. However, since they are very poorly standardized between different laboratories, this highlights the need for new guidelines and recommendations on how to accurately perform these tests (135). Finally, large, prospective clinical trials will be required to determine whether these tests are useful for these applications (138).

Here, we briefly describe three of the most frequently used devices: multiple-electrode platelet aggregometry (MEA), PFA-100, and Verify-Now.

3. Flow cytometry

The application of flow cytometry in platelet function analysis is based on the detection of cell surface proteins with fluorescently labeled antibodies. In contrast to the analysis of platelet solutions, whole-blood cytometry allows for fewer artifacts that could occur with artificial platelet (de)activation during sample preparation, but requires access to expensive instrumentation and specialized training to be performed (207). Flow cytometry allows analyzing platelets within whole blood in their circulating state, provided both venipuncture and analysis techniques are well standardized. The most commonly used routine tests are the quantification of GP receptor density (i.e, diagnosis of deficiencies in platelet GPs, for example, Glanzmann thrombasthenia and Bernard Soulier disease), although dense, granular measurements (using mepacrine uptake and release) (313), MP formation, and exposure of anionic phospholipids (procoagulant activity) can be equally performed. Another use of flow cytometry is the detection of platelet autoantibodies in patients with idiopathic thrombocytopenic purpura and drug-induced thrombocytopenias (203).

In conclusion, platelet function testing is increasingly utilized outside of the specialized laboratory. Although this represents advancement, validation, quality control testing, and reliability of these tests will also become an increasingly important issue. Most new platelet function tests have recently and will continue to become available. However, they need to prove to be useful in addition to our existing portfolio of tests.

1. Markers of in vivo platelet activation

Since different platelet assays assess different aspects of platelet pathophysiology, the result depends on the clinical condition being investigated and the aspect of platelet activation being measured. Biomarkers of platelet activation may provide clues regarding the extent of basal platelet activation in patients at risk for CV events and may also predict the severity of the thrombotic response in patients with a previous event.

The plasma markers of platelet activation, sCD40L and sP-selectin, are promising prognostic candidates for future CV disease. Both are easily measured with a standardized enzyme-linked immunosorbent assay (ELISA). Although limited, there are some data suggesting a significant association between them and incident CV disease.

b. CD40 ligand

The immunomodulator CD40L and its receptor are also present in platelets. CD40L is a ligand for integrin αIIbβ3 that is necessary for the stability of arterial thrombi (9). When activated, platelets strongly express and release CD40L (145) (Fig. 5). CD40L also occurs in a soluble form (sCD40L) that might be biologically active and appears to mostly originate from platelets (234). The CD40/CD40L system was shown to be expressed in vascular cells, including ECs, lymphocytes, and smooth muscle cells. Platelet-derived CD40L can induce a proinflammatory and prothrombotic response in ECs, as evidenced by the generation and release of ROS, adhesion molecules, chemokines, and TF (86, 299). sCD40L significantly correlates with other markers of platelet activation, such as sP-selectin (250), and it is recognized as a useful marker of platelet activation in clinical settings. Limited data are available on the association between sCD40L levels and future CV events in subjects free of CV disease. Among healthy, middle-aged women, those who later developed CV disease had significantly higher baseline levels of sCD40L. Moreover, women with baseline levels above the 95th percentile of the normal distribution were more than thrice as likely to develop a CV adverse event (271). Elevated levels of sCD40L were observed in both stable and unstable angina (UA) (15) and in acute MI (143). Moreover, in patients with UA, the expression of CD40L on T lymphocytes surface and in vitro shedding of sCD40L from these cells were increased (15). Thus, both T lymphocytes and, particularly, platelets represent the major contributor to increased circulating sCD40L in the setting of UA. In patients with ACS, sCD40L identified a subgroup that was at an increased 6-month risk of death or nonfatal MI (263). Accordingly, it has been suggested that the combined assessment of CD40L and troponin-T levels might have a better predictive value of AMI risk. sCD40L seems to have a prognostic role not only in subjects with advanced atherosclerosis but also in the general population (12).

OPEN IN VIEWER

FIG. 5.

Role of membrane-bound and sCD40L in platelet activation. The platelet activation by agonists leads to CD40L translocation to the external membrane, followed by the release of its soluble form (sCD40L). The ligation of CD40L to constitutively expressed CD40 activates cytoskeleton rearrangements and degranulation, leading to the release of alpha and delta-granule constituents. As an alternative pathway, sCD40L may act as an agonist through the interaction with integrin αIIbβ3 and fibrinogen binding. All these phenomena might represent a self-perpetuating pathogenic loop in platelet activation and aggregation. 5HT, serotonin; bFGF, basic fibroblast growth factor; FV, Factor V; MMPs, metalloproteinases; PDGF, platelet-derived growth factor; PF-4, platelet factor-4; TGF-β, transforming growth factor-β; VEGF, vascular endothelial growth factor.

Increased plasma levels of sCD40L have been detected in both type 1 (T1) and type 2 (T2) diabetes mellitus (DM) (265, 308). Moreover, previous studies showed a significantly increased coexpression of CD40 and CD40L in platelets of diabetic patients compared with nondiabetic controls and also a correlation between sCD40L and CD40L expression in platelets (161).

The highly significant correlation between plasma CD40L levels and the urinary excretion rate of 11-dehydro-TxB₂, a noninvasive index of in vivo platelet activation, supports the evidence that CD40L is rapidly upregulated during platelet activation (145).

In addition, the positive correlation observed between enhanced sCD40L and 8-isoPGF_2α is in line with the report of increased production of endothelial ROS by CD40L (299), suggesting that in T2DM, the release of sCD40L from activated platelets may contribute to increased oxidant stress. Increased lipid peroxidation and persistent platelet activation have previously been reported in patients with T2DM (78, 80). Thus, on the basis of these findings, a possible vicious cycle may be suggested in which inflammatory stimuli involving CD40L upregulation induce increased lipid peroxidation with consequent platelet activation, resulting in further oxidant stress (145).

Although TxA₂ enzymatic metabolites have been extensively studied in relation to CV disease and in response to aspirin treatment, no data are available regarding the levels of TxA₂ metabolites prospectively used in otherwise healthy subjects as a marker of risk for CV disease (Fig. 6).

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FIG. 6.

Schematic representation of TxA₂ metabolism. TxA₂ derived from platelet COX-1, as well as COX-1 and COX-2 dependent extra-platelet sources, is rapidly hydrolyzed to TxB₂. This, in turn, is metabolized within minutes into several enzymatic metabolites that are mainly excreted in urine. Among them, 11-dehydro-TxB₂ is cleared by the kidney within 1 h and can be easily measured in the urine as an in vivo marker of platelet activation. COX, cyclooxygenase.

1. Indicators of enhanced platelet turnover (reticulated platelets and MPV)

A higher number of circulating reticulated platelets is considered an indicator of accelerated platelet turnover (97) in that circulating reticulated cells represent young, mRNA rich platelets with enhanced proaggregating and hemostatic potential. Reticulated platelets are larger and more reactive platelets, as platelet size correlates with greater platelet reactivity, measured by aggregation and total release of granular content (297).

MPV is a parameter mirroring in vivo platelet activation (22) and an independent predictor of vascular events (311). Platelets are heterogeneous in size and density. Larger platelets are metabolically and enzimatically more active and have greater prothrombotic potential. Thus, MPV, a standardized platelet parameter, commonly available in the inpatient and outpatient setting, may have prognostic value in patients with previous vascular events. Moreover, MPV is to date recognized as a new independent risk factor for MI, vascular mortality, and restenosis after coronary angioplasty. Thus, MPV is a potentially useful prognostic biomarker for CV risk stratification (60). As compared with other markers of platelet activation, MPV is less expensive, easy to interpret, and can be measured by automated cell counters.

MPV values could reflect global platelet function in that they are associated with markers of platelet activation and with adhesion molecules' expression in platelets. In fact, larger platelets are more reactive and aggregable, contain denser granules, secrete more serotonin and β-thromboglobulin, and produce more TxA₂ than smaller platelets.

These factors produce effects on inflammation and endothelial function, promoting cell adhesion and aggregation, vasoconstriction, and, ultimately, inducing thrombosis. Interestingly, large hyperactive platelets might be sequestered in the coronary circulation of CAD patients, as suggested by the finding of lower MPV values within the coronary sinus than in the arterial blood (156).

Larger and more reactive platelets can be found in association with CV risk factors such as DM, obesity, metabolic syndrome, smoking, or hypertension (60) (Fig. 9). Indeed, a higher number of reticulated platelets showing a lower response to antiplatelet therapies including aspirin and clopidogrel can be frequently found in DM (128, 129).

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FIG. 9.

Contribution of major cardiovascular risk factors to platelet turnover. Major cardiovascular risk factors are capable of acting at the megakaryocyte level, leading to higher platelet turnover and increased MPV. MPV values, in turn, could reflect global platelet function, as larger platelets are more reactive and aggregable. They contain denser granules, secrete more β-thromboglobulin, and produce more TxA₂ than smaller platelets. MPV, mean platelet volume.

The increase in MPV in diabetics does not seem to be correlated to HbA_1c, fasting blood glucose, patient age, and duration of diabetes (144), suggesting that the increase in MPV may be due to the diabetic state per se. The increase in MPV occurs at the onset of the disease and persists for its entire duration.

A recent meta-analysis (60) showed that MPV is associated with MI, mortality after MI, and restenosis after coronary angioplasty, supporting the hypothesis that platelet size may play a role in both the development and the consequences of CV disease. However, it cannot be established whether elevated MPV actually causes CV disease and ACSs, or whether it is merely associated with them. Thus, MPV represents a potentially useful prognostic biomarker in patients with CV disease, although it is likely that an increased MPV pre-dates an acute MI rather than results from it.

2. Biomarkers of platelet activation

b. Tx biosynthesis

TxA₂ is a COX product that is synthesized and released by platelets in response to a variety of stimuli, including thrombin, collagen, and ADP. Most of the vascular actions of TxA₂ result from the autocrine/paracrine activation of TP expressed on the surface of platelets, endothelial and VSMC (Fig. 10).

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FIG. 10.

Schematic representation of the major pathways initiated by thromboxane/prostanoid receptor binding on various cell types.

Eicosanoids, in general, and TxA₂, in particular, are characterized by the episodic nature of their release and the local nature of their actions. These features are ensured by a variable chemical instability (very high for TxA₂, due to nonenzymatic hydrolysis to form its stable metabolite TxB₂) coupled with a substantial metabolic instability. Thus, extensive enzymatic degradation occurs in the lungs, liver, and kidneys, and it has been suggested to account for the short-lived presence of TxA₂ in the human circulation (Fig. 6) (225). Although it has been proposed to measure circulating TxB₂ to estimate TxA₂ production, TxB₂ is not a reliable index of in vivo platelet TxA₂ production, as its concentration is readily confounded by ex vivo platelet activation, which occurs readily during sample collection and processing (222). Moreover, the chances of detecting a sudden (mostly unpredictable) and short-lasting change in TxA₂ synthesis and release, by one or two plasma sampling, is unreliable. Thus, the measurement of TxB₂ in peripheral venous plasma cannot, under any circumstances, reflect changes in TxA₂ synthesis and release occurring in vivo (86).

To circumvent this problem, it is necessary to measure a metabolite that cannot be formed in whole blood. TxB₂ undergoes enzymatic degradation that generates a series of metabolites. Among them, 11-dehydro-TxB₂ has a substantial longer plasma half life, and it is excreted at a higher rate than other metabolites (Fig. 6) (225). Experimental studies reported a linear correlation between levels of exogenously administrated TxB₂ and urinary 11-dehydro-TxB₂ excretion (61, 78). Thus, it has been hypothesized that urinary 11-dehydro-TxB₂ would provide a time-integrated index of endogenous systemic TxA₂ biosynthesis, allowing a thorough evaluation of ongoing platelet activation by multiple cell sources (48).

Urinary levels of 11-dehydro-TxB₂ have been reported as reflecting TxA₂ generation by platelets—and, possibly, other cells—in established vascular disease. Episodic increases in 11-dehydro-TxB₂ excretion have been detected in UA, or in transient cerebral ischemic attacks. Thus, TxA₂-dependent platelet activation can mediate or amplify, at least in part, the short-term occlusive consequences of acute vascular lesions (e.g., plaque rupture) in the coronary and cerebral circulation. This is likely to reflect a localized (both in time and space) stimulus to platelet activation. Moreover, increased TxA₂ biosynthesis has been associated with several clinical settings characterized by increased CV risk, including DM, visceral obesity, hypertension, hypercholesterolemia, heart failure, and PAD (21, 83, 84, 125, 264). In addition, the consistent relationship observed in vivo between the rates of formation of F₂-isoprostanes, in vivo indices of lipid peroxidation, and urinary 11-dehydro-TxB₂ suggests that a low-grade inflammatory state associated with these metabolic disorders may be the primary trigger of TxA₂-dependent platelet activation, partly mediated by enhanced lipid peroxidation (86). Furthermore, the measurement of urinary 11-dehydro-TxB₂ levels provides a useful marker that investigates the effect of various cardioprotective interventions, including lipid-lowering therapy, improved glycemic control, weight loss, and antiplatelet agents (86) on platelet activation.

Finally, it has been recently reported that urinary 11-dehydro-TxB₂ measurement predicts the future risk of MI or CV death in aspirin-treated patients (99, 100). Nevertheless, whether urinary 11-dehydro-TxB₂ levels are independent predictors of future vascular events in untreated patients still remains controversial.

1. High (on-treatment) platelet reactivity

High (on-treatment) platelet reactivity (HPR) can be observed in a significant proportion of patients treated with P2Y₁₂ inhibitors (35), the vast majority of the studies having been focused on the antiplatelet action of clopidogrel. Depending on the method used for platelet function testing and the cut-off value chosen to define HPR, the proportion of patients defined as HPR patients varies largely across commonly used assays, from <20% to >50% (13). However, rates close to or more than 50% clearly overestimate the true proportion of patients who are at risk for suffering ischemic events, considering the low rate of stent thrombosis in PCI-treated patients (280). The relation of HPR to a clinical outcome was prospectively shown in large-scale trials and meta-analysis (13, 279) that demonstrated a significant association between the occurrence of stent thrombosis and the presence of HPR. However, only recently, the “Working Group on High (On-treatment) Platelet Reactivity” provided a consensus opinion on the definition (35) of HPR to ADP, establishing specific cut-off values for the most commonly used tests in clinical routine. A crucial point is to assess the stability of the HPR phenotype over time in patients treated with clopidogrel, because platelet function testing may provide variable results measured at different time points in the same patients. Thus, only serial measurements of platelet reactivity may help in guiding tailored antiplatelet therapy (44). Interference with clopidogrel bioactivation may occur for genetic and nongenetic factors.

Apart from drug noncompliance, patients with obesity, DM, older age, or renal insufficiency show higher platelet aggregation values on clopidogrel therapy (280). In patients suffering from acute MI complicated by cardiogenic shock, the poor response to clopidogrel is due to both reduced intestinal absorption of the pro-drug and diminished bioactivation. In addition to clinical conditions, co-medication influences the efficacy of clopidogrel. Proton pump inhibitors interact with the cytochrome P450, calcium channel blockers, and statins interfere with the CYP-dependent bioactivation of clopidogrel. However, since the available data are conflicting, the relevance of this interaction in platelet response and in clinical outcome remains not clearly determined (280). Multiple genetic variants in different genes involved in clopidogrel absorption and bioactivation can be associated with high or low HPR to clopidogrel as well as to bleeding or ischemic events (282).

Several studies have involved the isoenzyme CYP2C19, and the presence of the CYP2C19*2 loss-of-function allelic variant is associated with a reduced response to clopidogrel with a consequent higher risk of stent thrombosis in PCI-treated patients (204, 282).

The CYP2C19*17 gain-of-function allelic variant results in an increased enzymatic function (280). This variant is associated with lower ADP-induced platelet aggregation and increased bleeding risk, especially for homozygous allele carriers, as demonstrated by a genetic substudy of the PLATO trial (315), which showed more bleeding events in patients with this variant treated with clopidogrel.

Other genetic variants have been associated with the clinical outcome of clopidogrel-treated patients, but the results are still conflicting (280), and further studies are needed to better characterize all the genetic variants that are able to influence clopidogrel bioavailability.

2. Variable platelet response to aspirin

Due to the multifactorial nature of atherothrombosis, patients may experience recurrent events (treatment failure) while on aspirin (Fig. 11) as with any other antithrombotic drug. This phenomenon has been inappropriately referred to as “aspirin resistance,” a definition further corroborated by the largely unreliable functional methods used to measure the response to aspirin (252). The vast majority of studies reporting the occurrence of aspirin “resistance” in different clinical settings have relied on a single measurement of platelet function ex vivo using classic LTA or bedside, whole blood assays, all exhibiting less than ideal intrasubject and intersubject variability and limited sensitivity to the effect of aspirin, not directly reflecting its mechanism of action (252). Studies of platelet function have also suggested a lower-than-expected response to aspirin as assessed by PFA-100 or LTA in response to ADP (10).

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FIG. 11.

Mechanisms contributing to aspirin efficacy. Diagram depicting the various mechanisms that may influence the action of low-dose aspirin, including reduced COX-1 acetylation due to different factors as well as enhanced platelet turnover from the MKs. These mechanisms may be responsible for a less-than-expected efficacy of chronic low-dose aspirin with a consequent increased incidence of atherothrombotic complications. MKs, megakaryocytes.

Recently, a fairly good relationship has been reported between platelet hyperreactivity, as assessed by conventional LTA, and glycemic control in T2DM patients treated with dual antiplatelet agents for coronary stenting (283). In this regard, an analysis from the aspirin-induced platelet effect (ASPECT) study challenged the issue of increasing daily aspirin dosage up to 325 mg to overcome the high platelet reactivity phenotype in diabetics with CAD (94). However, the various methods used to quantitate the anti-platelet effect of aspirin in these studies poorly reflect the biochemical path affected by aspirin, that is, platelet COX-1 activity (267) and variably reflect the aspirin-sensitive TxA₂-dependent component of platelet aggregation (86). Whether the measurement of the potential non–COX-1 effects of aspirin might also be important remains unclear (205).

In assessing the effects of a poor response to aspirin treatment, one should be aware that the different platelet function tests correlate poorly among themselves (193). In addition, whether the relative contributions of TxA₂-dependent and -independent pathways are constant for any given assay is largely unknown. This is particularly relevant to the current debate on aspirin “resistance,” because the characterization of “resistant” versus “responder” status is typically based on a single determination of platelet function, with the underlying assumption that this determination represents a stable phenotype. In addition, the clinical relevance of aspirin resistance in patients undergoing percutaneous revascularization on dual antiplatelet therapy is still uncertain. Thus, neither routine testing for aspirin resistance nor any change in antiplatelet therapy based on these tests in these patients is recommended.

In contrast, serum TxB₂ and urinary 11-dehydro-TxB₂ provide reliable information on the maximal biosynthetic capacity of circulating platelets ex vivo and on the actual rate of TxA₂ biosynthesis in vivo, respectively (252). Less-than-complete inhibition of platelet TxA₂ production and TxA₂-dependent platelet activation, as assessed by these methods, are more reliable tools that assess aspirin responsiveness. Residual TxA₂ biosynthesis despite low-dose aspirin treatment has been shown to be predictive of vascular events in high-risk patients (99). Thus, elevated urinary 11-dehydro-TxB₂ levels identify patients who are relatively resistant to aspirin and who might benefit from alternative antiplatelet therapies or treatments that more effectively block in vivo TxA₂ production or activity.

1. PAR-1 antagonists

Thrombin receptors or protease-activated receptors (PARs) are GPCRs differentially expressed by vascular cells, platelets, and other circulating cells (86). Proteolytic cleavage of the receptor by thrombin is irreversible, and is potentially coupled to different G proteins that signal through multiple pathways (11). PAR-1, -3, and -4 are activated by thrombin. Platelets express PAR-1 and -4, and are able to trigger platelet secretion and aggregation (11).

PAR-1-mediated platelet activation is mainly involved in the development of thrombosis. The inhibition of PAR-1 has been shown to reduce arterial thrombosis in a pre-clinical model by reducing thrombin-induced platelet aggregation, without prolonging BT or influencing coagulation parameters (166). PAR-1 antagonism did not inhibit ADP-induced or collagen-induced platelet aggregation, suggesting that PAR-1 antagonism does not affect other platelet signaling pathways.

Vorapaxar (SCH530348) and atopaxar (E5555) are PAR-1 antagonists, which have completed phase II testing, showing overall favorable safety profiles and encouraging clinical outcomes.

Vorapaxar is an orally active, competitive PAR-1 inhibitor. Structurally, it is a synthetic tricyclic 3-phenylpyridine analog of himbacine, a natural product that has been modified as a crystalline salt for drug development and clinical use. In animal models, it seems to completely block the thrombin receptor, inhibiting thrombin-mediated platelet activation without interfering with the cleavage of fibrinogen, which is also thrombin mediated. In addition, Vorapaxar does not affect prothrombin time or activated partial thromboplastin time, suggesting that the potential for bleeding events may not be increased (54). Accordingly, the treatment with vorapaxar either alone or in combination with aspirin plus clopidogrel does not increase BT or surgical blood loss compared with placebo (59). On the basis of these promising results, vorapaxar was advanced into clinical development.

Preclinical studies of atopaxar (E5555), the other orally active PAR-1 antagonist that has completed phase II clinical development, showed interesting antithrombotic effects in animal models (273). In fact, it inhibited neointimal hyperplasia in a rat balloon injury model (158) as well as the production of inflammatory markers: sCD40L, IL-6, and P-selectin (273). Compared with voraxapar, atopaxar has a shorter half life; thus, it may prove useful in surgery settings, in which a rapid suspension of drug effects is needed.

Although extrapolation of these results to humans is highly speculative, it seems that the inhibition of PAR-1 alone may be sufficient for the prevention or treatment of thrombotic events in humans. However, it cannot be excluded that preservation of thrombin signaling through platelet PAR isoforms other than PAR-1 can participate in the maintenance of normal hemostasis. Larger-scale phase III clinical trials will better define the efficacy and safety of PAR-1 antagonists in addition to the standard of care in patients with atherothrombotic disease manifestations.

2. New P2Y₁₂ antagonists

The pharmacopoea of thienopyridines is rapidly expanding. In addition to ticlopidine and clopidogrel, a new third generation of P2Y₁₂ antagonists is in clinical development.

The substantial advantages of prasugrel compared with clopidogrel are represented by the distinct chemical structure, which allows the conversion by esterase to an intermediate metabolite, rather than inactivation, thus resulting in higher plasma levels of the active metabolite, lower inter-individual variability, and faster onset of action (50). Several clinical studies have shown that prasugrel has a greater effectiveness in inhibiting platelet aggregation, as compared with clopidogrel. In fact, it induces 2–3 times more potent platelet inhibition in a 10 times lower dose as compared with clopidogrel or ticlopidine. Even those individuals who had responded poorly to clopidogrel seem to achieve robust platelet inhibition with prasugrel treatment (37). Prasugrel has also been shown to be more effective than clopidogrel in ACS patients undergoing PCI, but with more bleeding (323) and has been approved in the United States and Europe for this kind of patients. A greater net clinical benefit has been observed in patients with diabetes or ST-segment elevation (30).

AZD6140, known as ticagrelor, is a reversible selective P2Y₁₂ antagonist. It acts as an orally active inhibitor of ADP-induced platelet aggregation. In patients with stable atherosclerotic disease, ticagrelor has been shown to inhibit ADP-induced platelet aggregation more rapidly and effectively and with less variability than clopidogrel both after the first dose and after 28 days of therapy (DISPERSE trial) (152). Moreover, in patients with ACS, it seems to be more effective than clopidogrel in preventing MACE, including CV death, nonfatal MI, and stroke. Furthermore, no significant difference in the rates of major bleeding has been found between the patients treated with ticagrelor and those treated with clopidogrel, according to the Thrombolysis in Myocardial Infarction criteria. In particular, ticagrelor was associated with more frequent non–coronary artery bypass graft (CABG)-related bleeding that clopidogrel, but it was safer than clopidogrel in patients undergoing CABG (314). However, an unexpectedly high rate of CABG-related major bleeding has been reported in the two study groups. Regarding safety, dyspnea and bradyarrhythmias occur more frequently with ticagrelor than with clopidogrel (45).

3. TP antagonists

Aspirin-insensitive TP agonists, as well as nucleate sources of TxA₂, less affected than platelet TxA₂ production by the once-daily dosing, may limit the antiplatelet effect of aspirin (224). In fact, F₂-isoprostanes and other structurally related iso-eicosanoids, produced nonenzymatically through a process of oxygen radical-catalyzed lipid peroxidation, represent aspirin-insensitive agonists of the platelet TP. High levels of F₂-isoprostanes, such as the compound 8-iso-PGF_2α, are associated with lower aspirin effectiveness settings (86).

Thus, a selective TP antagonist could block the interaction of both aspirin-sensitive and aspirin-insensitive agonists with this platelet receptor. Several TP antagonists have been developed since the early 1980s. The most recent one, S-18886 or terutroban, is an orally active, highly specific TP antagonist. However, the PERFORM trial of terutroban versus aspirin, on nearly 19,000 patients with recent stroke, was recently halted on the basis of an interim analysis failing to support the superiority hypothesis. In fact, there was no difference between terutroban and aspirin in the vascular primary endpoint, or any of the secondary or tertiary endpoints. Furthermore, the rate of minor bleeding was slightly increased with terutroban (36). Based on this result, the clinical development of terutroban has been discontinued.

4. Therapeutic use of platelet preparations

Platelets are a source of bioactive factors in the process of wound repair. Indeed, platelets release cytokines and growth factors (e.g., transforming growth factor-β1, PDGF, vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor, and insulin-like growth factor-1) that are involved in the healing process. By virtue of the rich granule content, autologous PRP represents an advanced wound therapy used in acute and chronic wounds. The mechanism of action for PRP is thought to be the molecular and cellular induction of normal wound healing responses similar to that seen during platelet activation, implying the release of granule content. Once the bioactive factors are released at the site of injury, they recruit cells and stimulate their differentiation in order to produce the ECM necessary to replace the tissue (24).

A recent meta-analysis of the literature published over the last 10 years, reviewed randomized, controlled trials and comparative group studies using platelet preparation therapy in cutaneous wounds (47). The comparison with the treatment to standard care or other interventions demonstrated that PRP therapy was significantly favored for complete healing in chronic wounds and for the reduced incidence of infection in acute wounds.

The use of PRP and platelet-poor plasma currently extends to many areas of surgery, from the preparation of preoperative autologous blood component therapy in order to reduce the number of allogeneic blood transfusions (103), to CV and thoracic surgery, neurosurgery, plastic and reconstruction surgery, and dental surgery [see ref. (154) for a review].

In sports medicine, the effects of PRP have been tested in several tendon-related disorders, skeletal muscle traumas, ligament/fascial injuries, and in the treatment of osteoarthritis and cartilaginous injuries [for a review, see ref. (24)].

A very interesting application of PDGF in the healing process is that of MI healing. In an elegant study, Zymek and co-workers (334) demonstrated that the PDGF-mediated signaling pathway plays a crucial role in regulating postinfarction repair by promoting collagen deposition in the infarcted area, modulating fibrosis and angiogenesis, and regulating the maturation of the infarct vasculature. More recently, an intramyocardial injection of autologous platelet gel has proved capable of ameliorating cardiac dysfunction after MI in a rat model (58).