Richard C. Becker, Duke Clinical Research Institute, 2400 Pratt Street, Durham, NC 27705, USA. Tel.: 919 668 8926; fax: 919 668 7056. E-mail: email@example.com.
Summary. The evolution of platelet directed pharmacotherapy in the prevention and treatment of patients with thrombotic disorders is based soundly on a rapidly expanding knowledge of platelet biology. Traditionally viewed, throughout most of its relatively brief history in medicine, as an anucleate, passive contributor to hemostasis, a more contemporary perspective acknowledges platelets as complex, multidimensional cells that participate actively in coagulation, vascular repair, angiogenesis and thrombosis within the micro and the macro-circulatory systems. Herein, we consider platelet-directed pharmacotherapy from these fundamental, biology-based exemplars—megakaryocytes, signal transduction and the platelet—coagulation protease interface. We also highlight the emerging biopharmacology platform of oligonucleotide platelet adhesion antagonists and their complementary antidotes.
From the common hematopoietic stem cell, two types of blood cell lines are derived: lymphoid, which includes all types of white blood cells, and myeloid, which includes red blood cells and platelets. Polyploid megakaryocytes are the immediate progenitors of platelets .
Megakaryocytes regenerate in humans at the rate of 108 cells per day , and each megakaryocyte can generates up to 5000 platelets at a time. This translates to production of 5 × 1011 platelets per day. Production of such a large number of cells, each with a life-span of ∼ 9 days, offers a teleological advantage in terms of speed and flexibility in response to hemostatic challenges.
Several markers have been identified to determine the stage of progenitor cell development. At the megakaryocyte–erythroid progenitor cell stage, RNA levels are elevated, reflecting the substantial polyploidy of these cells. Histologically, there are prominent ribosomes in the endoplasmic reticulum, the alpha and dense granules begin to form, and the cell is in an early stage of what has been described as a ‘platelet demarcation system’ [2,3].
The expression of several cytokines and chemokines also has been determined. Thrombopoeitin, a heavily glycosylated, 332-amino-acid protein produced in the liver, kidney, and bone marrow stroma, stimulates megakaryocyte proliferation and differentiation (in addition to stromal-derived factor (SDF)-1 and interleukins) . Also known as the c-Mpl ligand, thrombopoeitin selectively induces maturation and release of platelets; supports the survival and expansion of all cells in the megakaryocyte line; stimulates α-granule release and augments thrombin-induced platelet aggregation . The thrombin receptor on megakaryocytes has been identified, and platelet production is reduced by nearly 85% when this receptor is inhibited .
The expression and function of the thrombopoeitin receptor is regulated in two ways. First, it is regulated by thrombopoietin itself. In addition, the receptor exists in multiple splice forms, which can be either active or inactive. Receptors can be down-regulated, by splicing the gene and truncating the protein, in response to signals indicating large numbers of circulating platelets .
The megakaryocyte undergoes a series of morphological changes during a 4- to 10-h period of platelet production—a process that follows a modified flow model (Fig. 1) . Stimulated by sliding of the microtubules, the entire megakaryocyte cytoplasm then transforms into multiple thick pseudopods in preparation for formation of ∼ 5–10 proplatelets. Organelles and granules migrate along the microtubules to the developing, elongating proplatelet ends, where new platelets will form. Proplatelets are 250–500 μm long on average and, due to extensive, actin-dependent branching along their lengths, can produce 100–200 platelets each. The proplatelets are released from the cell into the vascular sinus, often appearing paired in a dumbbell shape. The nucleus is ejected from the mass of proplatelets, and individual platelets are released or ‘budded off’ from proplatelet ends .
Megakaryocytes are found adjacent to the bone marrow stroma, and proplatelets can extend into the vascular sinus side of the marrow. Occasionally, the proplatelet spans two separate spaces, with platelet release in both the deep marrow compartment and the vascular sinus . A wide variety of important proteins are expressed during platelet development [9–12].
The expression of proteins and surface protein receptors during megakaryocyte growth and development has provided an opportunity to expand our understanding of normal biology, variations that cause platelet hemostatic disorders, and potential targets for therapeutic intervention.
As platelets are anucleate cells with a limited life span, direct molecular manipulation can neither be used as a means to investigate fundamental biological mechanisms nor serve as a reliable means for intervention. In contrast, megakaryocytes, as well as their lineage cell, are amenable to molecular manipulation . Studies of short hairpin RNA sequence transduction in hematopoeitic stem cells targeting αIIb revealed a significant reduction in integrin αIIbβ expression in megakaryocytes and platelets. RNA interference within hematopoietic cells has also been employed to knock-down talin, a major cytoskeletal protein that colocalizes with activated integrins and binds to Β cytoplasmic domains, and inhibits αIIbβ3-dependent platelet activation in vivo .
Intracellular signaling targets
Platelet G protein-coupled receptors (GPCRs) initiate and subsequently reinforce platelet activation and thrombus formation. The clinical utility of P2Y12 receptor antagonists suggests that other GPCRs and potentially their intracellular signaling pathways may represent viable targets for platelet-directed pharmacotherapy.
Beginning with the initial observations of agonist-induced platelet aggregation in 1962 , steady progress has been made in identifying cell surface receptors and accompanying intracellular signaling pathways that regulate platelet function. These discoveries have yet to be translated fully to therapeutics in patients with thrombotic disorders of the coronary arterial system, including those undergoing percutaneous coronary interventions (PCI) [11,17–32].
Vascular injury—whether caused by spontaneous rupture of atherosclerotic plaque, plaque erosion, or PCI-related trauma—exposes adhesive proteins, tissue factor, and phospholipids promoting platelet tethering, adhesion, and activation. Once bound and activated, platelets release soluble mediators to include ADP, thromboxane A2, and serotonin, thus facilitating thrombin generation. These mediators, in turn, stimulate GPCRs on the platelet surface (Fig. 2) [12–14] that initiate intracellular signaling pathways, including activation of phospholipase C (PLC), protein kinase C (PKC), and phosphoinositide (PI)-3 kinase. Both calcium and PKC contribute to activation of the small G protein, Rap1b, which, through interactions with the Rap1-GTP interacting adapter molecule (RIAM) and talin, are important for binding of fibrinogen and other multimeric ligands to integrin αIIbβ, an essential event for platelet aggregation. Members of the kindling family of focal adhesion proteins have been identified as integrin activators [15–17], potentially promoting talin–integrin interactions.
PI-3 kinases transduce signals by generating lipid secondary messengers, which then recruit signaling proteins to the plasma membrane. A principal target for PI-3K signaling is the protein kinase Akt . Platelets contain both the Akt1 and Akt2 isoforms, with Akt1 being more prevalent . In mice, both Akt1 and Akt2 are required for thrombus formation [14,18]. Mice lacking Akt2 have aggregation defects in response to low concentrations of thrombin or thromboxane A2 and corresponding defects in dense and α-granule secretion. Glycogen synthase kinase (GSK)-3β is phosphorylated by Akt in platelets and suppresses platelet function and thrombosis in mice . Akt-mediated phosphorylation of GSK-3b inhibits the enzyme’s kinase activity, attenuating platelet activation. Akt activation also stimulates nitric oxide production in platelets, which results in protein kinase G-dependent degranulation . Finally, Akt has been implicated in the activation of cAMP-dependent phosphodiesterase (PDE3A), which plays a role in reducing platelet cAMP levels after thrombin stimulation .
Rap1 members of the Ras family of small G proteins have been implicated in GPCR signaling and integrin activation. Rap1b, the most abundant Ras GTPase in platelets, is activated rapidly after GPCR stimulation and plays a key role in the activation of integrin αIIbβ3 . Stimulation of Gq-linked receptors, such as PAR-4 or PAR-1, activates PLC and with resulting increases of intracellular calcium, PKC. A final common step in integrin activation involves binding of the cytoskeletal protein talin to the integrin β-subunit cytoplasmic tail . Rap1 is required to form an activation complex with talin and the Rap effector RIAM, which redistributes to the plasma membrane and unmasks the talin binding site, resulting in integrin activation .
Platelet-coagulation protease interface
Thrombin interacts with two protease-activated receptors (PARs) on the surface of human platelets—PAR-1 and PAR-4. Signaling through the PARs is mediated by thrombin-mediated cleavage of the receptors extracellular domain and exposure of a ‘tethered ligand’ at the new end of the receptor . Signaling through either PAR-1 or PAR-4 can activate PLC and PKC and cause bioamplification through the production of thromboxane A2, the release of ADP, and generation of thrombin on the platelet surface.
PAR-1 inhibitors in clinical development
Of the existing PAR-1 antagonists, SCH 530348 and E5555 are the compounds farthest along in development and clinical testing. SCH 530348 is an oral, reversible PAR-1 antagonist derived from himbacine, a compound found from the bark of the Australian magnolia tree . SCH 530348 inhibits thrombin- and PAR-1 agonist peptide (TRAP)-induced platelet aggregation, but it has no effect on ADP, collagen, U46619, or PAR-4 agonist peptide stimulation of platelets . Single doses of SCH 530348, ranging from 5 to 40 mg, yielded > 90% inhibition of TRAP-induced platelet aggregation across healthy subjects beyond 72 h after drug dosing, and greater than 90% inhibition was achieved in 1 h following 20 or 40 mg doses. A single dose of 5 ng or less did not elicit significant platelet inhibition.
In a Phase 2 trial of SCH 530348, 1031 patients scheduled for angiography and possible stenting were randomized to receive SCH 530348 or placebo plus aspirin, clopidogrel, and antithrombin therapy (heparin or bivalirudin) . The loading doses of SCH530348 dose-dependently inhibited TRAP-mediated platelet aggregation —with a 40 mg dose producing the greatest degree of inhibition at 30 min, and nearly all patients achieving greater than 80% inhibition of TRAP-induced platelet aggregation by 120 min after dose administration. A SCH 530348 maintenance dose of either 1.0 or 2.5 mg daily produced ≥ 80% inhibition in all patients at 30- and 60-day follow-up (Fig. 3) . Major and minor bleeding did not differ substantially between the placebo and individual or combined SCH 530348 groups. Although the study was not powered to detect differences in clinical endpoints, there was a trend toward a lower incidence of major adverse cardiac events (MACE) with increasing doses of SCH 530348 vs. placebo (8.5% for 10 mg, 5.0% for 20 mg, and 4.0% for 40 mg SCH 530348 vs. 8.6% for placebo), largely due to reductions in non-fatal periprocedural MI. SCH530348 was not associated with increased TIMI major plus minor bleeding compared with placebo (odds ratio 0.85 95% CI 0.296–2.467). Two Phase 3 trials are underway to determine whether adding a PAR-1 antagonist to standard therapy can prevent ischemic events (TRA•CER; http://ClinicalTrials.gov identifier: NCT00527943 and TRA-2ºP – TIMI 50; http://ClinicalTrials.gov identifier: NCT00526474).
A second PAR-1 antagonist, E555, is a methoxy-morpholinophenyl ethanone hydrobromide that binds to the receptor in a concentration-dependent manner with an IC value of 0.019 μmol L−1. Much like SCH 530348, E5555 is a highly selective, orally active PAR-1 receptor antagonist. Using platelets from healthy volunteers, Kogushi et al.  showed that E5555 inhibited the release of SCD 40L induced by thrombin and TRAP-a biomarker associated with a high risk of events among individuals with coronary artery disease and acute coronary syndromes.
E5555 is being assessed in three randomized, double-blind, placebo-controlled, Phase 2 trials. These studies will assess safety and tolerability of E5555 vs. placebo in patients with low- and high-risk ACS (ClinicalTrials.gov identifiers: NCT00619164, NCT00548587, and NCT00312052).
Activation and signaling of PAR-1 and PAR-4 provoke a biphasic ‘spike and prolonged’ response, with PAR-1 activated at thrombin concentrations ∼ 50% lower than those required to activate PAR-4 . Thrombin may interact in tandem with PAR-1 and PAR-4, with the initial interactions involving exosite-1 and PAR-1, and subsequent docking at PAR-4 via the thrombin active site . PAR-1 and PAR-4 may form a stable heterodimer that enables thrombin to act as a bivalent functional agonist.
Pepducins, or cell-permeable peptides derived from the third intracellular loop of either PAR-1 or PAR-4, disrupt signaling between the receptors and G proteins, and inhibit thrombin-induced platelet aggregation. In mice, a PAR-4 pepducin has been shown to prolong bleeding times and attenuate platelet activation . Combining bivalirudin with a PAR-4 pepducin (P4pal-i1) inhibited aggregation of human platelets from 15 healthy volunteers, even in response to high concentrations of thrombin.
Oligonucleotide platelet adhesion antagonists
The natural history and clinical expression of atherothrombotic coronary artery disease have several distinct pathobiologic underpinnings. The most compelling is the relationship between endothelial injury, inflammation, atherogenesis and thrombogenesis . As VWF is synthesized and stored in both endothelial cells and platelets, released in response to their activation and participates in site-specific thrombus formation , it may well represent a multidimensional biomarker of underlying pathoanatomic events.
Our group has developed RNA oligonucleotides (aptamers) and their complementary antidotes as platelet adhesion antagonists. To determine whether the selected aptamers that bound to the SPIII fragment of VWF containing the A1 domain (involved primarily in platelet adhesion through the GPIbα subunit) impacted platelet performance, a PFA-100 instrument was employed. The PFA (Platelet Function Analyzer)-100 is a platelet-dependent clot instrument that uses small membranes coated with either collagen/ADP or collagen/epinephrine to determine the cessation of blood (closure time) under high shear conditions. The results are highly VWF-dependent. VWF aptamers R9.3 and R9.14 inhibited platelet-dependent clot formation completely at a concentration of 1um. In contrast, Aptamer R9.4 had no activity. The minimum effective concentration was 40 um. (Fig. 4) .
The evolution of platelet-directed pharmacotherapy is based firmly on platelet biology and physiology and on the pathogenesis of arterial thrombotic disorders. The complete platelet life-cycle, to include megakaryocyte maturation, platelet surface receptor,induced signal transduction and the fundamental adhesive events, and the platelet-coagulation protease interface collectively represents a viable target for investigation and the development of novel therapeutics.
The authors would like to thank Bruce Sullenger, PhD and Sabah Oney, PhD for their insights on oligonucleotides and platelet adhesion; Christian Gachet, PhD and Athan Kuliopulos, MD, PhD for their helpful suggestions on platelet PARs; and Neal Kleiman, MD for his work on megakaryocytes.
Disclosure of Conflict of Interests
The authors have not declared any conflicts of interest.