Next-Generation Antithrombin Therapies

From the Duke Cardiovascular Thrombosis Center, Duke Clinical Research Institute, Durham, North Carolina. The author discloses no conflicts of interest regarding the content herein. Manuscript submitted February 26, 2009 and accepted March 3, 2009. Address for correspondence: Richard C. Becker, MD, Professor of Medicine, Director, Cardiovascular Thrombosis Center, Duke Clinical Research Institute, 2400 Pratt Street, Durham, NC 27705. _____________________________ vThe progressive and highly innovative evolution of percutaneous coronary intervention (PCI) for patients with advanced obstructive coronary artery disease (CAD) has created a concomitant need for parallel development and dedicated investigation of antithrombin pharmacotherapies. To keep pace with new technologies, operator experience and burgeoning 21st century practice, while being ever mindful of the fundamental biologic underpinnings that characterize atherosclerosis, PCI-related vascular injury and associated thrombotic phenotypes, the next generation of antithrombin agents will likely require several inherent properties including target selectivity, pathoanatomical specificity and active reversibility. The following review focuses on the activity and diversity of arterial thrombosis as a pathoanatomical substrate for clinical events and its translation to investigative platforms for antithrombin therapy in PCI. Vascular biology and coagulation, considered from the perspective of isolated biochemical reactions and normal physiology, are fundamental starting points for the development of antithrombin drugs. The complexity of atherothrombosis, viewed as a systems biology phenomenon, provides clarity to clinical investigation and, ultimately, the safe and effective treatment of patients. Biology-Based Coagulation The “waterfall” or “cascade” model of coagulation, proposed almost simultaneously by McFarlane, Davie and Ratnoff nearly 50 years ago, provided a biochemical framework for understanding coagulation reactions (and the basis for several clinical disorders of hemostasis). Its separation into intrinsic and extrinsic pathways, as well as the absence of platelets and other cellular elements from the overall working construct, however, constrained the model’s application to understanding atherothrombosis and related phenotypes.1,2 A biology-based model of coagulation3 establishes a physiological, integrated and functional view of complex biochemical events occurring on cellular (or other biological) surfaces, rather than distinct and relatively independent cascades that may be operational in static fluid systems (Figure 1). It also provides a scientific foundation for understanding the importance of platelet-coagulation protease interactions,4,5 the nonhemostatic role of coagulation proteases (which include vessel wall inflammation, cellular proliferation and apoptosis, the dynamic nature of cellular events and the interindividual variability of both platelet procoagulant activity and thrombin generation. According to the biology-based model of coagulation, initiation takes place on intact cells or cellular fragments (monocytes, macrophages, neutrophils, activated endothelial cells, smooth muscle cells, apoptotic cells, platelet microparticles, circulating vesicles) bearing the transmembrane glycoprotein tissue factor.6 Exposed tissue factor binds and fully activates coagulation factor (f) VII, which subsequently activates fIX and fX (which then activates fV), generating a small, but sufficient amount of thrombin from prothrombin (fII) for platelet activation. In the amplification phase, surface-bound thrombin activates platelets with phosphatidylserine expression as well as fV, fXI and fVIII (cleaving the latter from von Willebrand factor). fXIa generates additional fIXa whose action is accelerated by fVIIIa, whereas fVa accelerates and amplifies the action of fXa. During the propagation phase, fIXa binds to activated platelets, causing further fX activation. The complexing of fXa and fVa to membrane surfaces leads to a “burst” of thrombin generation. Thrombin’s major hemostatic roles include the conversion of soluble fibrinogen into a tridimensional network of fibrin, the activation of platelets through G protein-coupled protease-activated receptors (PARs) (3) (PAR 1 and PAR 4), and constriction of endothelium-denuded vessels. Thrombus growth in rapidly flowing blood is closely linked to the presence of soluble and surface-bound von Willebrand factor.7 This multimeric protein not only acts as a bridge for the initial tethering and translocation of platelets to subendothelial collagen (via platelet glycoprotein Ib), but also induces the surface expression of platelet glycoprotein IIb/IIIa (αIIbβ3), leading to the stable adhesion and subsequent aggregation of activated platelets to newly formed and polymerizing fibrin strands.8 The evolving paradigm of cell-based coagulation, coupled with a recognition that both circulating and vessel wall-related cellular events contribute directly and collectively to the phenotypic expression of atherothrombosis, provides a foundation for investigating novel pharmaco- and bio-therapeutics (Figure 2). The active thrombus. The traditional view of coagulation in general, and arterial thrombosis in particular, is one of a “conclusive endpoint” following vascular injury. In other words, thrombosis is a finite and predictable response. A more contemporary view acknowledges the phasic, dynamic and active nature of thrombi forming and residing within an injured vessel. This perspective may be particularly relevant in the coronary arterial vascular bed and provides a mechanistic platform for targeted pharmacotherapies in PCI. Atheromatous CAD, while often described at the time of coronary angiography as being discrete, focal and well-localized, is recognized pathologically as a diffuse process — with plaques of varying age, size and complexity. Spontaneous plaque rupture and accompanying thrombus is a common occurrence, and a majority of abrupt pathoanatomical events are, in fact, clinically silent. Histologic analyses of thrombectomy specimens obtained during PCI for acute ST-segment elevation myocardial infarction (STEMI) reveal thrombi of varying age, with more than one-third of patients having a predominance of organized (aged thrombus)9 (Figure 3). The presence of older thrombus has been identified by Kramer and Colleagues10 as an independent predictor of mortality in patients with STEMI undergoing thrombus aspiration during primary PCI. The observations surrounding thrombus-specific features and clinical outcome raise several fundamental questions with potentially translatable implications. Specifically, what are the functional characteristics of formed thrombi that might be influenced or impacted by age and degree of organization? Arterial thrombosis and organization. The histological approach to determining thrombus age provides a detailed description of its component parts and establishes a foundation for discussions regarding functionality. At the time of initiation, thrombus is attached firmly to the vessel wall. White blood cell pyknosis and monocytes with enlarged nuclei are also prevalent. After several days, endothelial cell budding and proliferative changes of the medial ring with fibroblast penetration are visible, as are hemosiderin-containing macrophages, red blood cell ghosts and fibrin architectural transformation. By 4 weeks, the free surface of the thrombus is covered by endothelium and scattered nuclear debris from fibrin-trapped leukocytes.11 The nature, history and histological organization of arterial and venous thrombi differ in several respects. First, organization occurs more slowly in arteries than in veins, with ingrowth of mesenchymal cells, macrophages, smooth muscle cells and fibroblasts seen weeks-to-months later in ligated and thrombosed arterial segments.12 In addition, loss of intimal viability occurs much more rapidly in arteries than veins — owing to differing depths of vasa vasorum penetration and dependence on luminal diffusion. Thrombus organization, from histological and molecular perspectives, provides further detail about functionality during the time course of thrombus organization. More than 1,100 genes are either upregulated or downregulated, with eight functional classes of gene expression: energy metabolism, protein synthesis (wound healing), organogenesis, cellular proliferation, extracellular signaling/inflammation, signal transduction and cytoskeletal changes.13 Platelet activity. Activated platelets release a wide variety of mediators that trigger and modulate inflammatory responses. There is evidence that platelets remain functional in vivo even after activation, and when bound to damaged endothelium, still respond to agonist stimulation hours after adhesion takes place. Disaggregation of thrombi in vitro yields platelets that maintain both basal morphology and secretory potential. Platelets encased in fibrin networks express newly synthesized proinflammatory cytokines for nearly 24 hours after clot formation, and adhesion between platelets and leukocytes remains stable, with gene expression in leukocytes increasing steadily over the subsequent 24 hours. Even platelets phagocytosed by leukocytes modulate survival markers for days, suggesting that platelets can regulate inflammatory and perhaps thrombotic events both locally and systemically. Thrombin-stimulated platelets synthesize pro-IL-1B and stimulate its subsequent processing to a biologically active protein. Circulating platelets then deliver this and other signaling proteins to target cells that amplify inflammatory responses. For example, IL-1B triggers the synthesis of E-selectin, IL-8 and ENA-78 — a protein required for leukocyte adhesion to endothelial cells. In addition, circulating platelet-leukocyte aggregates are stable and contribute to the “piggybacking” of platelets onto inflamed tissues during leukocyte transmigration. Thrombin generation. Patients with CAD, including those with acute coronary syndrome (ACS), experience heightened thrombin generation. In the acute setting, thrombin generation occurs rapidly and reaches higher levels than under more stable conditions.14 Several investigators have reported increased factor IX levels among patients with ACS.15,16 Reconstructions of patient populations (stable CAD and ACS) employed to reproduce thrombin generation computational models have suggested that collective alterations in factor VIII, factor II and antithrombin III represent the driving prothombotic force.14 The maintenance of a blood clot for hemostatic purposes requires a series of integrated events with a structural goal of a stable fibrin matrix. The prothrombinase complex (factor V, factor Xa, calcium, and phospholipid membrane) accumulated during tissue factor-initiated coagulation is the primary catalyst for prothrombin activation and thrombin generation; however, factors VIIIa and IXa are major “re-supply substrates” for clot maintenance.17 Thus, a rapid renewal of declining prothrombinase would be impaired with defects (or pharmacological inhibitors) of intrinsic factor Xase proteins — factor VIII and/or factor IX. Clot-bound thrombin is present, but its contribution to the process of renewed prothrombin activation during resupply may be modest. Antithrombins in Clinical Development: Focus on Factor IXa Biology and biochemistry of factor IX. Factor IX (fIX) is synthesized in the liver where it undergoes vitamin K-dependent carboxylation. Following release, fIX circulates in the plasma compartment as a 57kDa zymogen with a half-life of 18–24 hours.18 Clearance of fIX from the circulation is mediated by low-density lipoprotein-related protein (LRP), an endocytic receptor on hepatocytes.19 Factor IX has a total of six structural domains, each with functional significance. The amino-terminal Gla domain, containing 12γ-carboxyglutamic acid residues is essential for calcium and cell surface binding through the molecule’s hydrophobic domain. The two epidermal growth factor (EGF) domains are critical for catalytic domain orientation, as well as tissue factor-fVII and fVIIIa binding. The activation peptide domain is cleaved by fXIa or TF-VIIa, yielding a 17 kDa light chain and 30 kDa heavy chain protease containing the active site. The catalytic domain itself, in addition to its role in enzymatic reactions governing fX activation, is also critical for maintaining molecular integrity and fVIIIa binding. The importance of fIXa-fVIIIa complex formation is reflected in the modest enzymatic potential of either substrate in isolation.20–23 The relative contribution of coagulation proteases assembled on phospholipids toward thrombin generation has been investigated by several highly experienced groups.24 As the concentration of fIX increases from 0% to 10% (of normal in vivo plasma levels), the rate of thrombin generation increases on average by 4-fold, with fIX concentrations ranging from 0% to 200%. The most dramatic increase occurs between levels of 0% and 3%. The area under the curve for thrombin generation, reflecting total thrombin production, does not vary with fIX concentration. In contrast, maximal rate, peak and total thrombin generation are achieved with fX at concentrations between 0% and 3% (Figures 4 A–D and 5 A–D). This very efficient mechanism for thrombin generation is particularly evident in experiments performed with platelets, suggesting a pivotal role of platelet fV. Considered collectively, observations generated through experiments employing an ex vivo cell-based model suggest that inhibition of fIXa represents an effective means to attenuate thrombin generation on cell surfaces. Pharmacotherapies designed to target factor IXa have been developed and include: active site-blocked competitive antagonists (IXai), monoclonal antibodies, small-molecule inihbitiors and RNA aptamers.25 RNA aptamers will be the focus of discussion. Aptamers are short oligonucleotides that fold into defined three-dimensional (3-D) conformations enabling them to bind with high specificity to a chosen small molecule or protein target. In our laboratory, oligonucleotides are identified using an in vitro screening method known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment).26 Target protein recognition by an aptamer is achieved by several variations of nucleic acid-protein interactions to include hydrogen bonding, salt bridges and Vanderwall forces.27 A distinguishing structural-functional feature of oligonucleotide aptamers is their ability to form stable secondary conformations that maintain proper spatial arrangement of the target recognition elements. In fact, aptamers do not maintain a stable conformation in the free state, but acquire one after binding. Usually, one or two structural motifs comprise the basis of an aptamer — hairpins and pseudo knots.28,29 Complementary RNA antidotes targeting one or more sites for binding through conventional Watson-Crick base paring alters the 3-D fold of the aptamer, attenuating or completely “turning off” the oligonucleotides functional activity. (Figure 6)30 Factor IX aptamer-antidote clinical development program. Regado 1a. Regado 1a was a phase 1, first-in-human study of an aptamer-based inhibitor to factor IXa (RB006) and its complementary antidote (RB007)31 A total of 85 healthy subjects participated in a dose escalation, placebo-controlled study and received a bolus of RB006 or placebo followed 3 hours later by a bolus of RB007 or placebo. The doses of RB006 — 15 mg, 30 mg, 60 mg and 90 mg — were chosen to provide a large margin of safety to the study subjects and to parallel the pharmacodynamic effects, based on the activated partial thromboplastin time (APTT), according to an in vitro dose-response curve. The fixed dose of RB007 was twice the RB006 dose chosen (antidote-to-drug ratio of 2:1), representing a 4-fold increase above the minimal amount required to fully neutralize RB006 (and return factor IX activity to its baseline level). In subjects receiving RB006, the APTT increased rapidly in a dose-dependent manner, with a stable pharmacodynamic effect over the initial 3 hours. The duration of effect was also dose-dependent, with return to baseline APTT values at 3 hours, 20–24 hours and 30 hours for the 15, 30 and 60 mg doses, respectively. To evaluate the relationship between drug dose, APTT values (15 minutes after drug administration) and factor IX inhibition, we compared the relative increase in APTT (fold-increase compared to baseline) with the factor IX activity calibration curve. The analysis revealed that a 1.1-, 1.3-, 2.1- and 2.9-fold increase in APTT corresponded to a 35–40%, 80%, 98% and > 99% loss of factor IX activity, respectively. Activated clotting time (ACT) values followed a similar response pattern, with relative increases of 1.1-, 1.3-, 1.4- and 1.5-fold, respectively. Administration of RB007 prompted a rapid, sustained return of the APTT to baseline values — on average within 1–5 minutes of administration.32 Regado phase 1b. The Regado phase 1b study randomized 50 subjects between the ages of 50 and 75 years with stable CAD receiving aspirin and/or clopidogrel to a bolus of RB006 (15, 30 50 or 75mg) and RB007, given 3 hours later, at respective doses of 30, 60, 100 and 150 mg. As in Regado phase 1a, RB006 and RB007 were well tolerated, with no major bleeding, serological evidence of complement activation or other serious adverse events during the 7-day follow-up period. There were 5 subjects with minor bleeding at peripheral intravenous line sites.33 There was a dose-dependent increase of APTT and ACT values 10 minutes following RB006 administration. Complete neutralization of the anticoagulant effect with RB007 was rapid, within 1–2 minutes of administration, predictable, consistent and durable. Pharmacokinetics and pharmacodynamic measures, to include RB006 plasma concentrations, factor IXa activity and APTT values derived from the phase 1a and 1b studies, provided the required information to elucidate RB006 dose-response relationships according to subject body weight.34 (This information played a pivotal role in subsequent dose selection in the phase 2 pilot study (described in greater detail below.) Regado 1c. Regado 1c was a double-blind, single-center study that included 32 healthy subjects randomized sequentially to either fixed doses of RB006 (0.75 mg kg-1), followed by varying, de-escalating doses of RB007, or to double placebo. On treatment days 1, 3 and 5, subjects received RB006 (repeat drug dosing) and RB007 (repeat antidote dosing) 60 minutes later (1.5–0.094 mg kg-1) or placebo. Blood samples for APTT, PT and complement Bβ were collected daily for 6 days and again on a follow-up visit at days 10–14. In addition, whole blood ACT and APTT measurements were made using point-of-care coagulation monitors. Highly reproducible APTT measurements were observed with each of the three drug-antidote cycles. RB007 restored APTT levels to baseline (determined prior to RB006 administration) with all consecutive drug-antidote cycles.35 Descending doses or RB007 (Concluding Thoughts The next generation of antithrombin therapies for use in PCI will require several inherent properties, including target selectivity, pathoanatomical specificity and controlled, active reversibility. Atherothrombotic substrate variability and dynamic conditions determined by thrombus age and associated activity collectively form a translatable platform for innovation and clinical investigation, with a uniform objective of optimal contemporary practice.

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