Aspirin

Aspirin produces dose-dependent inhibition of platelet COX activity after a single oral dose. A single dose of 100 mg effectively suppresses biosynthesis of TxA₂ within several minutes of administration via acetylation of platelet COX in the presystemic circulation.¹³ Owing to aspirin’s irreversible inhibition of COX and the inability of platelets to synthesize new proteins, aspirin’s effect is maintained for the lifespan of the platelet (7-10 days). Cyclooxygenase activity returns only as new platelets are generated.

Aspirin reduces acute coronary and cerebrovascular events such as unstable angina, myocardial infarction (MI), sudden cardiac death, and stroke.¹³ Its utility is enhanced by its modest cost and nominal side effects. Although aspirin effectively reduces platelet secretion and aggregation, it is a relatively weak platelet inhibitor. The inhibitory effects of aspirin are pronounced when using relatively weak platelet agonists, but less so against stronger agonists like thrombin that can induce platelet activation in the absence of TxA₂. Importantly, the majority of platelet responses remain unaffected by aspirin treatment. Aspirin does not inhibit shear stress–induced platelet activation and platelet adhesion. In addition to its antiplatelet properties, aspirin also exerts antiinflammatory effects.¹⁴ In a meta-analysis of antiplatelet therapy studies in patients with acute coronary syndrome (ACS), administration of several doses of aspirin (from 75 mg/day up to 150 mg/day) significantly decreased the overall risk of nonfatal MI, nonfatal stroke, and death rates.¹⁵ Several studies, however, have demonstrated that aspirin monotherapy is inadequate because of high intraindividual variability to aspirin response, as well as increased aspirin resistance, especially observed in patients with diabetes mellitus.¹⁶

Nonsteroidal antiinflammatory drugs

There are several nonsteroidal antiinflammatory drugs (NSAIDs) that act as competitive reversible inhibitors of PGH-synthase. Sulfinpyrazone, indobufen, and triflusal are several of the drugs in this class evaluated for their antithrombotic activity in randomized clinical trials. The active sulfide metabolite of sulfinpyrazone administered in the highest dose allowable (200 mg four times a day) inhibits only 60% of platelet COX activity, with results suggesting no significant clinical efficacy.¹⁷ The clinical, biochemical, and functional effects of the more effective inhibitor, indobufen, are similar to those of aspirin. An oral dose of indobufen 200 mg twice daily inhibits 95% of platelet TxA₂ synthesis.¹⁸ Triflusal, a derivative of salicylic acid, is also able to inhibit platelet COX, but only after conversion to a longer-lasting metabolite.¹⁹ None of the three are currently approved for use as antiplatelet drugs in the United States.

Thienopyridine platelet P2Y12 receptor antagonists

The thienopyridine class of antiplatelets (ticlopidine, clopidogrel, and prasugrel) selectively and irreversibly inhibit the P2Y₁₂ purinoreceptor throughout the life of the platelet. Clopidogrel is the dominant member within the class and provides for modest platelet inhibition, delayed onset of action, and significant interpatient variability, including nonresponsiveness to the drug, which necessitated the search for more potent and stable alternatives.²³ Ticlopidine has been eclipsed because of its adverse hematological side effects, including neutropenia and thrombotic thrombocytopenic purpura.²⁴ The opposite, however, is true of third-generation thienopyridines, namely prasugrel.

Oral thienopyridines are prodrugs that require conversion into their active metabolites by hepatic cytochrome P450 (CYP) enzymes (CYP3A4 isozyme). Whereas clopidogrel requires esterase inactivation and a two-step CYP-dependent activation, prasugrel requires only one reaction to yield its active metabolite.²⁵ This difference in metabolism translates into different patient responses and drug interactions. Genetic variations of P450 (CYP) enzymes affect clopidogrel’s active metabolite formation, resulting in lower platelet inhibition and, most importantly, a higher rate of major adverse cardiovascular events. Prasugrel’s pharmacology, however, is not affected by CYP polymorphisms and provides a stable platform for antiplatelet therapy. Prasugrel has a faster onset of action and a tenfold higher potency than clopidogrel.²⁶

Clopidogrel

Clopidogrel is metabolized by CYP P450. Its active metabolite irreversibly binds to the platelet P2Y₁₂ receptor, thus inhibiting the effect of ADP on platelets. As a result, GPIIb/IIIa receptors have decreased activation, thereby resulting in reduced platelet function. The Clopidogrel versus Aspirin in Patients at Risk of Ischemic Events (CAPRIE) study, a randomized trial that included patients with ischemic stroke, MI, or symptomatic atherosclerotic peripheral artery disease, showed that clopidogrel-treated patients had an 8.7% relative risk reduction for acute MI, stroke, or vascular death, compared to those patients treated with aspirin.²⁷ The Clopidogrel in Unstable Angina to Prevent Recurrent Events (CURE) trial was the first study establishing the significance of dual antiplatelet therapy (acetylsalicylic acid [ASA] plus clopidogrel) in patients with ACS²⁸ and involved a population of 12,562 patients presenting with non-ST-elevation ACS who were randomized to receive either a combination of ASA (75-325 mg/day) and clopidogrel (300 mg loading dose, followed by 75 mg/day) or ASA and placebo for 3 to 12 months. At 12 months, a lower incidence of MI, stroke, or cardiovascular death was observed in the clopidogrel plus ASA group compared to the placebo group; however, the risk of major bleeding was increased among patients treated with clopidogrel plus ASA.

Similar results were reported by the COMMIT study.²⁹ Patients received either a combination of clopidogrel (75 mg/day) and ASA (162 mg/day) or placebo and ASA for 28 days or until hospital discharge. There was a 9% relative risk reduction in the composite endpoint of stroke, vascular reinfarction, or death and a 7% relative risk reduction for death in the group receiving dual antiplatelet therapy. In the CREDO study, which was the first to evaluate the significance of dual antiplatelet therapy pre- and post-percutaneous coronary intervention (PCI), patients undergoing PCI received either a 300-mg loading dose of clopidogrel 3 to 24 hours before the procedure and then 75 mg/day for 12 months, or 75 mg/day for 30 days after the procedure without a loading dose.³⁰ All patients also received a 325 mg/day dose of ASA during the 12-month follow-up period. A 27% relative risk reduction for death, MI, or stroke was observed in the group receiving long-term dual antiplatelet therapy. Given the established antiplatelet effect of clopidogrel, the Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization Management and Avoidance (CHARISMA) study investigated the potential benefit from 28-month dual antiplatelet therapy (75 mg/day clopidogrel and 75-162 mg/day ASA) in patients 45 years of age or older who experienced one of the following conditions: multiple atherothrombotic risk factors, documented coronary disease, documented cerebrovascular disease, or documented symptomatic peripheral artery disease.³¹ The control group received ASA and placebo. No benefit was observed in the primary endpoint of MI, stroke, or cardiovascular death in the dual antiplatelet therapy group. By contrast, asymptomatic patients of the group with risk factors but without clinical evidence of CAD had a higher risk of bleeding.³² A further analysis in the subgroup of patients with documented atherothrombotic cardiovascular disease showed a significant risk reduction for new MI, stroke, or cardiovascular death in those patients compared with controls.³³

Prasugrel

Prasugrel is a P2Y₁₂ inhibitor acting similarly to clopidogrel. Prasugrel is rapidly converted to its active metabolite by the P450 cytochrome and has higher bioavailability than clopidogrel.²⁶ Recently, a 60-mg loading dose of prasugrel achieved high platelet inhibition both in healthy subjects and in patients scheduled for PCI, whereas healthy clopidogrel poor-responders achieved satisfactory platelet inhibition of up to 80% after prasugrel administration.²⁶ The beneficial effect of prasugrel was also established in the TRITON-TIMI 38 study.³⁴ Patients scheduled for PCI were randomized to receive either clopidogrel (300-mg loading dose and 75 mg/day afterwards) or prasugrel (60-mg loading dose and 10 mg/day afterwards). All patients also received ASA, and about half the patients from each group were treated with a GPIIb/IIIa inhibitor as well. The prasugrel group demonstrated a significant risk reduction for the composite primary endpoint of death, nonfatal MI, and nonfatal stroke. In addition, risk was significantly lower in the diabetes mellitus patient subgroup³⁴; however, incidence of major hemorrhagic events was more frequent in the prasugrel group, but even when this parameter was added to the study’s primary endpoints, the net clinical benefit findings still favored prasugrel compared to clopidogrel. Subgroup analysis demonstrated a higher rate of major bleeding in those with body weight of less than 60 kg, history of stroke or transient ischemic attack, and age older than 75 years.³⁴

Non-thienopyridine platelet P2Y12 receptor antagonists

Novel non-thienopyridine platelet P2Y₁₂ receptor antagonists include ticagrelor, cangrelor, and elinogrel, which are direct and reversible P2Y₁₂ antagonists with rapid onsets and short durations of action. Ticagrelor is highly selective and very specific for the P2Y₁₂ receptor, and it exhibits a greater, more consistent inhibition of platelet aggregation than clopidogrel.³⁵ Ticagrelor is administered orally and does not require metabolic activation, providing a rapid onset peaking within 2 to 4 hours of dosing. The metabolism of ticagrelor yields an active molecule (AR-C124910XX) that has similar P2Y₁₂-blocking activity as its parent molecule. Ticagrelor’s plasma half-life is approximately 12 hours, which corresponds to twice-daily dosing and a 1- to 2-day restoration of normal platelet-mediated hemostasis upon discontinuation. These pharmacokinetics are in contrast to clopidogrel and prasugrel, which require discontinuation approximately 5 days before restoration of normal platelet-mediated hemostasis is achieved. Ticagrelor’s potential advantage in plasma half-life also carries the risk of increased thrombotic events if patients miss a dose.³⁶

Multiple trials have demonstrated the benefits of ticagrelor in clinical practice. The Dose Confirmation Study Assessing Antiplatelet Effects of AZD6140 vs. Clopidogrel in Non-ST-Segment Elevation Myocardial Infarction (DISPERSE-2) trial showed no difference in major bleeding or MI, with an increase in minor bleeding at higher doses of ticagrelor in 990 patients with non-ST-segment elevation ACS.³⁷ In the PLATO (the Study of Platelet Inhibition and Patient Outcomes) trial comparing ticagrelor and clopidogrel with respect to their efficacy in preventing cardiovascular events and safety showed ticagrelor significantly reduced the rate of death from vascular causes, MI, or stroke, without an increase in the rate of overall major bleeding compared to clopidogrel in 18,624 patients with ACS.³⁸ Ticagrelor is the first investigational antiplatelet drug to demonstrate a reduction in cardiovascular death when compared to clopidogrel in patients with ACS.

Finally, research has shown ticagrelor to produce platelet inhibition regardless of genotypic variations in the three genes that had been associated with variability to clopidogrel in platelet inhibition.³⁹ All these trials underline the potential for ticagrelor to achieve a rapid and sustained antiplatelet effect that could be reversed and could overcome nonresponsiveness and interpatient variability to clopidogrel, thus addressing the main limitations of clopidogrel therapy.²⁴ Nonetheless, its adverse effects (e.g., dyspnea, bradycardia) and weight-based dosing require further investigation before ticagrelor may advance toward routine use in antiplatelet therapy.³⁸

Abciximab

Abciximab is a large chimeric monoclonal antibody Fab fragment with high affinity for the GPIIb/IIIa receptor.⁴⁰ Abciximab is the largest agent with binding sites located on the β-chain of the GPIIb/IIIa receptor which, because of its large size, causes a steric hindrance to ligand access.

Abciximab has the strongest affinity for the GPIIb/IIIa receptor and dissociates at a slower rate than other GPIIb/IIIa antagonists. Unbound abciximab is rapidly cleared from plasma by proteolytic degradation, resulting in a very short plasma half-life (several minutes), whereas the biological half-life ranges from 8 to 24 hours. As such, some GPIIb/IIIa receptors may still be occupied by drug up to 2 weeks after discontinuation of drug infusion. In the event of bleeding, the antithrombotic effects of abciximab cannot be rapidly reversed by discontinuing therapy, but rather are diminished by exogenous platelet transfusion.

The efficacy and safety of abciximab in patients undergoing PCI has been evaluated in several trials, including EPIC, EPILOG, and EPISTENT.^41–43 The Intracoronary Stenting and Antithrombotic Regimen–Rapid Early Action for Coronary Treatment (ISAR-REACT) trial demonstrated that the rate of death, MI, and urgent revascularization at 30 days was low and comparable between patients undergoing elective PCI after pretreatment with clopidogrel 600 mg and allocated to abciximab vs. placebo.⁴⁴ Rates of major bleeding were similar between groups, although abciximab was associated with a significantly higher rate of thrombocytopenia. ISAR-REACT-2 evaluated the same abciximab and clopidogrel treatment regimens in high-risk patients with non-ST-segment elevation ACS undergoing PCI.⁴⁵ Death, MI, and urgent revascularization at 30 days occurred significantly less frequently with abciximab compared to placebo; however, the treatment benefit of abciximab was confined to patients with elevated troponin levels. Rates of major bleeding were similar between groups. Overall, the findings suggest that in the modern era of interventional cardiology using high clopidogrel dosing regimens, GPIIb/IIIa inhibition should be reserved only for high-risk ACS patients with positive cardiac markers.

Eptifibatide

Eptifibatide is a competitive antagonist of the GPIIb/IIIa receptor. It is a synthetic small-molecule inhibitor that fits directly into the Arg-Gly-Asp binding pocket of the GPIIb/IIIa receptor, directly competing with the binding of ligands such as fibrinogen and vWF.⁴⁶ Eptifibatide rapidly dissociates from its receptor, is cleared by the kidney largely as active drug, and has a plasma half-life of approximately 1.5 to 2.5 hours. The return of hemostatic platelet function is largely dependent on clearance of the drug from plasma. Cessation of drug infusion restores platelet function and, in patients with normal renal function, normal hemostasis returns within 15 to 30 minutes after drug discontinuation. Unlike abciximab, however, the platelet inhibitory effect of eptifibatide is not significantly influenced by platelet transfusion.⁴⁶

Eptifibatide has demonstrated efficacy and safety in patients with non-ST-segment elevation ACS or undergoing PCI in a number of randomized clinical trials. Most recently, the Early Glycoprotein IIb/IIIa Inhibition in Non-ST-Segment Elevation Acute Coronary Syndrome (EARLY ACS) trial demonstrated that early administration of eptifibatide vs. provisional eptifibatide after angiography (delayed eptifibatide) resulted in similar 30-day rates of death, MI, urgent revascularization, or thrombotic complications during PCI in patients with non-ST-segment elevation ACS undergoing invasive management.⁴⁷ Major and minor bleeding rates were significantly higher with early eptifibatide vs. delayed eptifibatide. Overall, these findings do not support the use of upstream compared with ad hoc GPIIb/IIIa inhibition in ACS patients undergoing PCI.

Tirofiban

Tirofiban is a tyrosine-derived nonpeptide inhibitor associated with rapid onset and short duration of action, with a plasma half-life of approximately 2 hours. Tirofiban, like eptifibatide, is a competitive inhibitor of the GPIIb/IIIa receptor that has high specificity but relatively low affinity.⁴⁶ Tirofiban is excreted by the kidney, predominantly as unchanged drug; it rapidly dissociates from the receptor and has a biological half-life of 1.5 to 2.5 hours. Restoration of hemostasis is best achieved by discontinuing the drug infusion. Efficacy and safety of tirofiban in PCI patients has been investigated in several trials.

Von willebrand factor–GPIP/IX inhibitors

A number of vWF-GPIb/IX inhibitors, including aurin tricarboxylic acid, peptide fragments from vWF A1 domain, and a soluble GPIb-immunoglobulin (Ig)G, have been examined for inhibiting vWF and platelet interactions.⁵⁰ Glycoprotein-290 is a recombinant protein consisting of a 290 amino acids sequence of the N-terminal of human GPIba, with two gain-of-function mutations that increase the affinity for the vWF A1 domain. The in vivo antithrombotic and antihemostatic effects have been evaluated with good GPIba/vWF inhibition, which could be reversed with a currently approved (DDAVP) treatment regimen. A lower clopidogrel dose combined with GPIba/vWF inhibition could theoretically have improved efficacy with less bleeding risk.

AJvW-2 is a murine monoclonal antibody to human vWF A1 domain that blocks the GPIba/vWF interaction and has demonstrated antithrombotic activity in animal models.⁵¹ In order to reduce immunological response, AJvW-2 was humanized and converted from an IgG1 to an IgG4.⁵² This humanized antibody (AJW200) exhibited similar inhibition of in vitro vWF-mediated platelet activation to AJvW-2. In human volunteer studies, AJW200 demonstrated no clinically significant adverse events or immunogenicity. Ristocetin cofactor (Ri:CoF) assays showed a significant reduction at 1 hour post infusion compared with baseline that lasted for up to 12 hours. The template bleeding time was not significantly prolonged at any time or dose of AJW200. Platelet function as measured by the PFA-100 was reduced by up to 3 to 6 hours at the lower dose and 12 hours at the highest dose administered.

ARC1779 is an aptamer that blocks the GPIba/vWF A1 domain interaction. Aptamers are nucleic acid molecules with high affinity and specificity for a selected target molecule, discovered through in vitro selection on the basis of their ability to fold into unique three-dimensional structures that promote binding to that target.⁵³ ARC1779 is a modified deoxyribonucleic acid/ribonucleic acid (DNA/RNA) oligonucleotide composed of hybrid terminal ends to minimize endonuclease and exonuclease digestion, with nucleotide segments designed to enhance affinity for vWF. ARC1779 has demonstrated efficacy comparable or superior to that of previously published dosing regimens of abciximab with respect to protection from thrombus formation and average time to occlusion. ARC1779 was evaluated in a randomized double-blind placebo-controlled study that demonstrated it was well tolerated, and no bleeding was observed.⁵⁴ An S-nitroso derivative of a mutated fragment of the A1 domain (S-nitroso-AR545C) was shown to inhibit effectively arterial thrombosis in the carotid artery.⁵⁵ In unpublished observations, it has also been reported that targeting the vWF A1 domain with the recombinant nanobody ALX-0081, a novel class of antibody therapeutics, resulted in inhibition of vWF-mediated platelet activation, providing novel options for future therapies.

Collagen-GPVI inhibitors

Glycoprotein VI is also expressed on the surface of platelets. Signaling by GPVI is via an immunoreceptor tyrosine activation motif (ITAM) promoting phosphorylation and initiating the syk signaling cascade. Syk activation results in activation of integrin-induced platelet aggregation, release of ADP and thromboxane, and procoagulant activity.⁵⁶ Rat monoclonal antibody (JAQ1) to mouse GPVI results in inhibition of collagen-induced aggregation.⁵⁷ In animal models, JAQ1 caused mild thrombocytopenia and resulted in a 34% decrease in platelet counts within 24 hours of treatment, which returned to normal levels within 3 days of treatment. Platelets showed no indication of activation or change in surface protein expression. A single dose of JAQ1 resulted in 14 days of inhibition of ex vivo collagen-induced aggregation. Further evaluation of in vivo response determined that binding of antibody to the platelet GPVI resulted in depletion of platelet GPVI. Collagen-induced adhesion was significantly reduced in these GPVI-depleted platelets. Bleeding times of JAQ1-treated mice were significantly elevated over control mice (330 seconds vs. 158 seconds), but less than that seen following inhibition of GPIIb/IIIa (330 seconds vs. > 600 seconds).

Heparin

Heparin is a sulfated polysaccharide and is isolated from mammalian tissues rich in mast cells, most notably porcine intestinal mucosa. Heparin acts as an anticoagulant by activating antithrombin (antithrombin III), thereby accelerating the rate at which antithrombin inhibits clotting enzymes, particularly thrombin and factor Xa. Heparin activates antithrombin by binding to a unique pentasaccharide sequence to induce a conformational change within antithrombin, rendering it more readily accessible to its target proteases (Fig. 7-4). This conformational change enhances the rate at which antithrombin inhibits factor Xa but has little effect on the rate of thrombin inhibition. To catalyze thrombin inhibition, heparin serves as a template that simultaneously binds antithrombin and thrombin. Formation of this ternary structure allows close apposition, promoting formation of a stable covalent thrombin-antithrombin complex. Heparin additionally causes release of TFPI from the endothelium, a tissue factor–bound factor VIIa inhibitor, further enhancing its anticoagulant effects.

Figure 7-4 Comparative mechanisms of action of selected anticoagulants.

Heparin binds to the endothelium and to plasma proteins, explaining its dose-dependent clearance. At low doses, the half-life of heparin is short because it binds rapidly to the endothelium. Clearance is mainly extrarenal, as heparin binds to macrophages, which internalize and depolymerize the long heparin chains and secrete shorter chains back into the circulation. Because of its dose-dependent clearance mechanism, the plasma half-life of heparin ranges from 30 to 60 minutes.

Heparins binding to plasma proteins, especially acute-phase reactants whose levels are elevated in ill patients, may make the anticoagulant response unpredictable to fixed or weight-adjusted doses. Consequently, coagulation monitoring is essential to ensure that a therapeutic response is obtained. This is particularly important when heparin is administered for treatment of established thrombosis because a subtherapeutic anticoagulant response may render patients at risk for recurrent thrombosis, whereas excessive anticoagulation increases bleeding risk. Heparin therapy can be monitored using the APTT or anti–factor Xa level. Anti–factor Xa levels also can be used to monitor heparin therapy. Although gaining in popularity, anti–factor Xa assays have yet to be standardized, and results can vary widely between laboratories.

The most common side effect of heparin is bleeding. Other complications include thrombocytopenia, osteoporosis, and elevated levels of transaminases. The risk of heparin-induced bleeding increases with higher heparin doses. Concomitant administration of drugs that affect hemostasis, such as antiplatelet or fibrinolytic agents, increases bleeding risk, as does recent surgery or trauma. Heparin-treated patients with serious bleeding can be given protamine sulfate to neutralize the heparin. Protamine sulfate binds heparin with high affinity, and the resultant protamine-heparin complexes are then cleared. Typically, 1 mg of protamine sulfate neutralizes 100 units of heparin. Anaphylactoid reactions to protamine sulfate can occur, and drug administration by slow IV infusion is recommended to reduce the risk.

Heparin can cause a drop in platelet count in the form of heparin-induced thrombocytopenia (HIT). Heparin-induced thrombocytopenia is an antibody-mediated process triggered by antibodies directed against neoantigens on platelet factor 4 (PF4). These antibodies (IgG isotype) bind simultaneously to the heparin-PF4 complex and to platelet Fc receptors. Such binding activates the platelets and generates platelet microparticles. Circulating microparticles are prothrombotic and can bind clotting factors and promote thrombin generation. Typically, HIT occurs 5 to 14 days after initiation of heparin therapy, but it can manifest earlier. It is rare for the platelet count to fall below 100,000/mL in patients with HIT, and even a 50% decrease in platelet count from the pretreatment value should raise the suspicion of HIT in those receiving heparin. The diagnosis of HIT is established using enzyme-linked assays to detect antibodies against heparin-PF4 complexes or with platelet activation assays. Enzyme-linked assays are sensitive but can be positive in the absence of any clinical evidence of HIT. Another diagnostic test is the serotonin release assay. This test is performed by quantifying serotonin release when washed platelets loaded with labeled serotonin are exposed to patient serum in the absence or presence of varying concentrations of heparin. If the patient serum contains the HIT antibody, heparin addition induces platelet activation and serotonin release.

Heparin is stopped in patients with suspected or documented HIT, and an alternative anticoagulant should be administered to prevent or treat thrombosis. The agents most often used for this indication are parenteral DTIs (e.g., lepirudin, argatroban, bivalirudin) or factor Xa inhibitors (e.g., fondaparinux, danaparoid). Patients with HIT, particularly those with associated thrombosis, often have evidence of increased thrombin generation that can lead to consumption of protein C; if given warfarin without a concomitant parenteral anticoagulant, this can trigger skin necrosis.

Low-molecular-weight heparin

Low-molecular-weight heparin consists of smaller fragments of heparin and is prepared from unfractionated heparin (UFH) by controlled enzymatic depolymerization. Low-molecular-weight heparin provides advantages over heparin in that it has better bioavailability and longer half-life, simplified dosing, predictable anticoagulant response, lower risk of HIT, and lower risk of osteoporosis.

Like heparin, LMWH exerts its anticoagulant activity by activating antithrombin. Even though LMWH consists of shorter pentasaccharide-containing chains, they retain greater capacity to accelerate factor Xa inhibition by antithrombin (see Fig. 7-4). Consequently, many forms of LMWH catalyze factor Xa inhibition by antithrombin more than thrombin inhibition.

Since LMWH contains shorter chains, they bind less avidly to ECs, macrophages, and heparin-binding plasma proteins. Reduced binding to ECs and macrophages eliminates the rapid dose-dependent and saturable mechanism of clearance that is a characteristic of UFH. Instead, clearance of LMWH is dose independent, and its plasma half-life is longer at approximately 4 hours. Low-molecular-weight heparin is cleared almost exclusively by the kidney, and the drug can accumulate in patients with renal insufficiency.

Because Low-molecular-weight heparin binds less avidly to heparin-binding proteins in plasma than heparin, LMWH produces a more predictable dose response, and resistance is rare. With a longer half-life and more predictable anticoagulant response, LMWH can be given subcutaneously once or twice daily without coagulation monitoring, even when the drug is given in treatment doses. In the majority of patients, LMWH does not require coagulation monitoring. If monitoring is necessary, anti–factor Xa levels must be measured because most LMWH preparations have little effect on APTT.

Indications for LMWH monitoring include renal insufficiency and obesity. Low-molecular-weight heparin monitoring in patients with a creatinine clearance of less than 50 mL/min is advisable to ensure there is no drug accumulation. It may also be advisable to monitor the anticoagulant activity of LMWH during pregnancy, because dose requirements can change, particularly in the third trimester. Monitoring should also be considered in high-risk settings, such as in patients with mechanical heart valves who are given LMWH for prevention of valve thrombosis, and when LMWH is used in treatment doses in infants or children.

The major complication of LMWH is bleeding. Meta-analyses suggest that the risk of major bleeding is lower with LMWH than with UFH. Heparin-induced thrombocytopenia and osteoporosis are less common with LMWH than with UFH. As with heparin, bleeding with LMWH is more common in patients receiving concomitant therapy with antiplatelet or fibrinolytic drugs. Recent surgery, trauma, or underlying hemostatic defects also increase the risk of bleeding with LMWH. Although protamine sulfate can be used as an antidote for LMWH, it incompletely neutralizes the anticoagulant activity of LMWH because it only binds the longer chains of LMWH and is only partially reversed.

The risk of HIT is about fivefold lower with LMWH than with heparin. Low-molecular-weight heparin binds less avidly to platelets and causes less PF4 release. Furthermore, with lower affinity for PF4 than heparin, LMWH is less likely to induce the conformational changes in PF4 that trigger the formation of HIT antibodies. Low-molecular-weight heparin should not be used to treat HIT patients because most HIT antibodies exhibit cross-reactivity with LMWH. This in vitro cross-reactivity is not simply a laboratory phenomenon; there are case reports of thrombosis when HIT patients are treated with LMWH.

Fondaparinux

Fondaparinux is a synthetic analog of the antithrombin-binding pentasaccharide sequence that differs from LMWH in several ways. As a synthetic analog of the antithrombin-binding pentasaccharide sequence found in heparin and LMWH, fondaparinux binds only to antithrombin and is too short to bridge thrombin to antithrombin (see Fig. 7-4). Consequently, fondaparinux catalyzes factor Xa inhibition by antithrombin and does not enhance the rate of thrombin inhibition. Fondaparinux is licensed for thromboprophylaxis in general surgical and high-risk orthopedic patients and as an alternative to heparin or LMWH for initial treatment of patients with established venous thromboembolism (VTE).

Fondaparinux does not bind to ECs or plasma proteins. Clearance of fondaparinux is dose independent, and its plasma half-life is 17 hours. Because fondaparinux is cleared unchanged via the kidney, it is contraindicated in patients with a creatinine clearance below 30 mL/min and should be used with caution in those with a creatinine clearance below 50 mL/min. Fondaparinux produces a predictable anticoagulant response after administration in fixed doses because it does not bind to plasma proteins.

Fondaparinux does not cause HIT because it does not bind to PF4. In contrast to LMWH, there is no cross-reactivity of fondaparinux with HIT antibodies. Consequently, fondaparinux appears to be effective for treatment of HIT patients, although large clinical trials supporting its use are lacking.

The major side effect of fondaparinux is bleeding. There is no antidote for this drug. Protamine sulfate has no effect on the anticoagulant activity of fondaparinux because it fails to bind to the drug. Recombinant activated factor VII reverses the anticoagulant effects of fondaparinux in volunteers, but it is unknown whether this agent will control fondaparinux-induced bleeding.

Hirudins

Hirudin consists of a single polypeptide chain of 65 amino acids with increased affinity for thrombin. Various recombinant hirudins have since been developed (lepirudin and desirudin). They are bivalent DTIs and bind thrombin with high affinity, forming noncovalent irreversible complexes. Their plasma half-life is approximately 1 to 2 hours, and they distribute widely in the extravascular space. Clearance is primarily through the kidney, so dose adjustment is required in the setting of impaired renal function.⁵⁸ Monitoring hirudin is challenging, since plasma levels must be determined using enzyme-linked immunosorbent assays (ELISA), which are costly and limited in availability. Hirudins are associated with bleeding, thus requiring monitoring to avoid excessive anticoagulation. Since hirudin prolongs APTT, it is widely used to monitor therapy. Lepirudin is licensed for use in patients with HIT with thrombosis. Major bleeding occurs in 18% to 20% of patients receiving lepirudin for treatment of HIT. Clinical use of commercially available hirudins is complicated by the development of antibodies. Studies have shown that over 40% of lepirudin- treated patients develop IgG antibodies against lepirudin.⁵⁹ Antilepirudin antibodies typically form 1 to 4 weeks after initiation of treatment and in most cases are not associated with any adverse clinical outcomes. In 10% of lepirudin-treated patients, however, formation of antibodies delay clearance of lepirudin, enhancing its anticoagulant effect.⁵⁹ Anaphylaxis, which can be fatal, has also been reported in patients receiving intravenous bolus lepirudin for treatment of HIT.⁶⁰ Lepirudin use in patients with HIT is based on the findings of three prospective, historically controlled cohort studies, HAT-1, HAT-2, and HAT-3. These studies found that compared with historical controls, lepirudin reduced the frequency of the composite endpoint of all-cause mortality, new thrombosis, or limb amputation in patients with HIT.⁶¹ Lepirudin was also associated with significantly reduced new thrombosis. Lepirudin additionally has been studied in the setting of non-ST-segment ACS. When compared with heparin, 10,141 patients with ACS lepirudin significantly reduced the incidence of cardiovascular death, refractory angina, or new MI at 7 days from 6.7% to 5.6%, but also significantly increased the rate of major bleeding from 0.7% to 1.2%.⁶² Lepirudin has not been approved for use in ACS, but such results support the hypothesis that hirudins are superior to heparin in preventing recurrent ischemia; however, they do so within a narrow therapeutic window.

Desirudin is a hirudin used for thromboprophylaxis in patients undergoing elective hip or knee surgery. Desirudin is used for 9 to 12 days, or until the patient is fully ambulatory. Unlike lepirudin, desirudin monitoring is unnecessary. Like lepirudin, bleeding is the most common side effect. When receiving desirudin for thromboprophylaxis, approximately 10% of patients also develop antibodies of the IgG class against hirudin. These antibodies have not been associated with altered plasma concentrations of desirudin, deep vein thrombosis (DVT), pulmonary embolism (PE), allergic reactions, or hemorrhage.⁶³ Desirudin is currently approved in Europe for thromboprophylaxis in patients undergoing elective hip or knee surgery. Approval is based on findings of two multicenter randomized double-blind trials. In the first study, desirudin 15 mg subcutaneously twice daily was compared to UHF, 5000 international units, three times daily in patients undergoing primary elective total hip replacement (THR). Desirudin was associated with a significantly lower rate of all DVT and proximal DVT.⁶⁴ In the second study, desirudin 15 mg subcutaneously twice daily was compared to enoxaparin 40 mg subcutaneously once daily in patients undergoing elective THR. Duration of treatment was 8 to 12 days, and DVT during the treatment period was verified by mandatory bilateral venography. Desirudin was associated with a significantly lower rate of proximal DVT and overall DVT. There was no significant difference in bleeding, transfusion requirements, or thrombocytopenia between the groups.⁶⁵

Bivalirudin

Bivalirudin is a hirudin analog with high-affinity binding to thrombin. Once bound, however, thrombin cleaves the Arg-Pro in the N-terminus of bivalirudin, allowing for recovery of thrombin activity and subsequent competing of fibrinogen with the bivalirudin remnant for thrombin.⁶⁶ Unlike hirudin, only 20% of bivalirudin is excreted via the kidney; the remainder is eliminated by proteolytic enzymatic degradation. Both APTT and activated clotting time (ACT) have been used to monitor bivalirudin. Bivalirudin has been licensed as an alternative to heparin in patients undergoing PCI and for patients with HIT who require PCI. A prospective trial comparing bivalirudin with high-dose heparin in 1261 patients demonstrated a lower rate of death, MI, or repeat revascularization.⁶⁷ The REPLACE-1 trial compared bivalirudin to heparin in 1056 patients undergoing coronary stenting with GPIIb/IIIa inhibitors. There was a trend toward a reduction in the combined endpoint of death, MI, or revascularization with bivalirudin at 48 hours; there was no difference in major bleeding.⁶⁸ In REPLACE-2, bivalirudin was as effective as heparin plus GPIIb/IIIa inhibition in reducing death, MI, and revascularization. Bivalirudin was also associated with significant reduction in the incidence of bleeding and thrombocytopenia.⁶⁹ In the Acute Catheterization and Urgent Intervention Triage Strategy (ACUITY) trial, patients with moderate- to high-risk unstable angina or non-ST-segment elevation myocardial infarction (NSTEMI) undergoing early invasive management⁷⁰ demonstrated that bivalirudin alone was noninferior to heparin (either UFH or enoxaparin) plus GPIIb/IIIa inhibitors in terms of mortality and the primary endpoint. It was also associated with significantly less bleeding. At 1 year, bivalirudin was still noninferior to heparin in terms of the primary endpoint and mortality rates; however, bivalirudin, either alone or with GPIIb/IIIa inhibitors, continued to be associated with significantly less bleeding. Results were similar in patients triaged to medical management, PCI, or coronary artery bypass surgery. The ACUITY trial showed a strong association between major bleeding in the first 30 days and risk of death over 1 year.⁷⁰

Argatroban

Argatroban is a potent agent that differs from parenteral DTIs in that it binds reversibly to the active site of both free and clot-bound thrombin.⁷¹ Argatroban is metabolized by the liver, and its clearance is reduced in patients with moderate hepatic impairment (Child-Pugh > 6). Thus, significant reductions in argatroban dose are required for individuals with moderate hepatic impairment, and the drug is contraindicated in patients with severe hepatic dysfunction. No dose adjustment is needed in the setting of renal impairment.⁷² Argatroban increases APTT, PT/INR, thrombin time, ecarin clotting time, and ACT in a dose-dependent fashion.

The major side effect of argatroban is bleeding. Because there is no specific antidote, excessive bleeding can only be managed by stopping the argatroban infusion and providing supportive therapy. In patients with normal hepatic function, the anticoagulant effect of argatroban disappears 2 to 4 hours after stopping the infusion. However, the anticoagulant effects of argatroban may persist for up to 24 hours in patients with hepatic impairment. A major challenge of argatroban is its effect on PT/INR. When overlapped with warfarin, PT/INR is prolonged beyond what would be expected with warfarin alone, making dose adjustment of either drug difficult.⁷³

Two multicenter phase III prospective trials of argatroban in HIT have been completed. When compared with historical controls, patients on argatroban had reduced rates of thrombosis and death due to thrombosis, without an increase in bleeding.⁷⁴ On the basis of these findings, argatroban has been approved for treatment of thrombosis and for thromboprophylaxis in patients with HIT, including those undergoing PCI with HIT.⁷⁵ Increasing data supporting the use of argatroban in patients without HIT undergoing PCI has been emerging. A 2007 multicenter prospective pilot study evaluated efficacy and safety of argatroban in combination with the GPIIb/IIIa inhibitors abciximab or eptifibatide in 152 patients. The primary efficacy endpoint (a composite of death, MI, or urgent revascularization at 30 days) occurred in 2.6% of patients, and major bleeding occurred in 1.3% of patients.⁷⁶ This study also showed that argatroban in combination with GPIIb/IIIa inhibition was an adequate anticoagulant with an acceptable bleeding risk.

Ximelagatran

Because of the significant limitations of warfarin, alternative oral DTIs have undergone significant development and clinical study. Ximelagatran, the orally available prodrug of the univalent DTI melagatran, was the first drug in this class to generate widespread interest.⁷⁷ Although ximelagatran was shown to be as effective as warfarin in preventing stroke or systemic embolism in the setting of atrial fibrillation,⁷⁸ there were questions about its safety profile. The open-label SPORTIF III trial compared ximelagatran with warfarin for prevention of stroke and systemic embolism, and although ximelagatran was shown to be noninferior to warfarin in preventing stroke and systemic embolism, serum alanine aminotransferase levels rose to greater than three times the upper limit of normal in 6% of individuals in the ximelagatran group and greater than five times the upper limit of normal in 3.4% of individuals in the ximelagatran group.⁷⁹ Significant safety concerns regarding increased liver toxicity without a significant offsetting advantage in major bleeding led the U.S. Food and Drug Administration (FDA) to reject the sponsor’s application for ximelagatran in 2004.

Dabigatran etexilate

Like ximelagatran, dabigatran etexilate is a prodrug. Once absorbed, the drug is rapidly converted by esterases to dabigatran, whose levels peak in approximately 1 to 2 hours. Dabigatran is a small-molecule reversible inhibitor that binds to the active site of thrombin. The half-life of dabigatran is approximately 12 hours, and it is primarily eliminated via the kidney.⁸⁰ The half-life is prolonged in the elderly, reflecting their impaired renal function.⁸¹ The pharmacokinetics and pharmacodynamics of dabigatran are not influenced by CYP P450 enzymes and other hepatic oxidoreductases and thus do not interfere with drugs that are metabolized by the P450 enzyme system.⁸⁰ Dabigatran produces a predictable anticoagulant response. Therefore, routine coagulation monitoring is not necessary. Dabigatran prolongs ecarin clotting time, APTT, and PT/INR in a dose-dependent fashion.⁸¹ Because these widely available tests have not been used in the clinical setting for monitoring, target levels are unknown.

The major side effect of dabigatran is hemorrhage. No specific antidote is available. Consequently, bleeding complications must be managed symptomatically. Although not well studied, dialysis or hemoperfusion likely removes this compound from the circulation, and administration of activated coagulation factor complexes such as FEIBA, Autoplex, or rFVIIa may overcome its anticoagulant effect.⁸² Several clinical trials have demonstrated dabigatran’s antithrombotic effect. The phase II Boehringer Ingelheim Study in Thrombosis II (BISTRO II) trial compared oral dabigatran with enoxaparin as thromboprophylaxis after total hip or total knee replacement.⁸³ Dabigatran was administered at doses of 50 mg, 150 mg, or 225 mg twice daily, or 300 mg once daily for 6 to 10 days. A significant dose-dependent decrease in VTE occurred with increasing doses of dabigatran etexilate. Overall VTE rates were 28.5% in patients receiving 50 mg of dabigatran twice daily, 17.4% in patients receiving 150 mg of dabigatran twice daily, 13.1% in patients receiving 225 mg of dabigatran twice daily, 16.6% in patients receiving 300 mg of dabigatran once daily, and 24% in patients receiving enoxaparin. The risk of serious bleeding with dabigatran increased in a dose-dependent manner as well, but did not reach statistical significance at any dose. Serious bleeding occurred in 0.3% of patients receiving 50 mg of dabigatran twice daily, 4.1% of patients receiving 150 mg of dabigatran twice daily, 4.8% of patients receiving 225 mg of dabigatran twice daily, and 4.7% of patients receiving 300 mg of dabigatran once daily. Serious bleeding occurred in 2.0% of patients receiving enoxaparin.

The RE-NOVATE study demonstrated that dabigatran etexilate was as effective as enoxaparin for preventing VTE after THR, with a similar safety profile.⁸⁴ This double-blind, noninferiority trial randomized 3494 patients to treatment for 28 to 35 days with dabigatran etexilate 220 mg or 150 mg once daily, or subcutaneous enoxaparin 40 mg once daily. The primary efficacy outcome was the composite of total VTE and all-cause mortality. Both doses of dabigatran etexilate were noninferior to enoxaparin, with the primary efficacy outcome occurring in 6.7% of patients in the enoxaparin group vs. 6.0% of patients in the dabigatran etexilate 220-mg group, and 8.6% of patients in the 150-mg group. There was no significant difference in major bleeding rates with either dose of dabigatran etexilate compared to enoxaparin. There was no difference in the frequency of liver enzyme elevation.

The subsequent REMODEL trial reproduced these results in 2076 patients undergoing total knee replacement (TKR).⁸⁵ The primary efficacy outcome occurred in 37.7% of the enoxaparin group vs. 36.4% of the dabigatran etexilate 220-mg group and 40.5% of the 150-mg group. Both doses of dabigatran etexilate were thus noninferior to enoxaparin. Incidence of major bleeding did not differ significantly between the three groups (1.3% vs. 1.5% and 1.3%, respectively), and there were no significant differences in liver enzyme elevation.

The RE-MOBILIZE trial was similar in design to the RE-MODEL and, in contrast to REMODEL, failed to show equivalence for a composite endpoint of proximal DVT, distal DVT, PE, and all-cause mortality.⁸⁴ Warfarin is often used for thromboprophylaxis after knee arthroplasty in centers in North America, but it has not been directly compared to dabigatran in a clinical trial. Dabigatran has undergone study for initial and long-term treatment of patients with established VTE. The Randomised Evaluation of Long-Term Anticoagulant Therapy (RE-LY) trial demonstrated that in 18,133 patients with atrial fibrillation, primary outcome of stroke or embolism was lower in patients on dabigatran as compared to warfarin, as was bleeding.⁸⁶ Dabigatran has been approved for use in the United States and is in use in Europe.

Rivaroxaban

Rivaroxaban is in development for prevention and treatment of thromboembolic disorders, including VTE prevention following orthopedic surgery, treatment of DVT and PE, ACS, and stroke prevention in patients with atrial fibrillation. Rivaroxaban is a potent inhibitor of factor Xa and does not inhibit thrombin-induced platelet aggregation, but it attenuates tissue factor–induced platelet aggregation indirectly through inhibition of thrombin generation.⁸⁷ The antithrombotic efficacy of rivaroxaban has been demonstrated in various animal models of arterial or venous thrombosis across doses that do not prolong bleeding times.⁸⁷ When combined with ASA or clopidogrel, the antithrombotic potency of rivaroxaban is enhanced.⁸⁸

In healthy subjects and patients undergoing orthopedic surgery, rivaroxaban displays predictable pharmacokinetics and pharmacodynamics.⁸⁹ The half-life is between 7.6 and 9.1 hours. Plasma levels of rivaroxaban correlate well with both inhibition of factor Xa activity and prolongation of PT, as assessed in healthy subjects who received multiple doses of rivaroxaban across a wide dose range.⁹⁰ The pharmacodynamic effects of rivaroxaban (as measured by endogenous thrombin potential) are sustained for 24 hours after single oral doses, thus supporting once-daily dosing.⁹¹ Age, gender, and body weight have not been shown to exert clinically significant effects on the pharmacokinetic or pharmacodynamic profiles of rivaroxaban. Rivaroxaban has a dual mode of elimination: one third is excreted unchanged via the kidneys and the remaining two thirds of the drug is metabolized by the liver; there are no major or active circulating metabolites.⁹² It does not interact with or mobilize PF4 on platelets, and is therefore unlikely to induce the conformational changes in PF4 necessary for cross-reaction with HIT antibodies.

Four dose-finding clinical studies (one phase IIa and three phase IIb) have assessed the potential efficacy and safety of rivaroxaban for thromboprophylaxis in patients undergoing major orthopedic surgery.^84,93–95 All four studies assessed rivaroxaban relative to conventional anticoagulants and measured the composite of the incidence of any DVT or objectively confirmed nonfatal PE or all-cause mortality as the primary endpoint and major bleeding as the primary safety endpoint. Results from these studies support the feasibility of daily dosing. The 10 mg daily dose provided the optimal balance between efficacy and safety in the phase II trials and was therefore selected for further study in phase III trials. There has been no evidence of liver toxicity. The RECORD program (Regulation of Coagulation in Major Orthopedic Surgery Reducing the Risk of DVT and PE) was initiated in December 2005 and enrolled more than 12,500 patients worldwide to participate in four multicenter randomized, active-controlled, double-blind studies of rivaroxaban prophylaxis in patients undergoing THR (RECORD1 and RECORD2) and TKR (RECORD3 and RECORD4).^96–99 Rivaroxaban was significantly more effective in prevention of VTE after TKR and THR. These three phase III studies demonstrate that a fixed daily unmonitored dose of rivaroxaban provides a safe and effective option for short-term and extended thromboprophylaxis after major orthopedic surgery.

Rivaroxaban was also assessed for treatment of VTE in two randomized phase IIb double-blind, dose-ranging studies of rivaroxaban administered for 12 weeks in patients with acute symptomatic proximal DVT (without PE) vs. parenteral UFH or LMWH and a vitamin K antagonist.¹⁰⁰ These two studies of more than 1150 patients suggested that efficacy of rivaroxaban for treatment of proximal DVT was similar to that achieved with standard anticoagulation therapy, with no significant dose-response relationship for the primary efficacy endpoints and low rates of VTE recurrence and bleeding events. Following on from the promising findings in the VTE treatment studies, phase III studies of long-term rivaroxaban for stroke prevention in patients with atrial fibrillation are underway. ROCKET-AF is a randomized double-blind study designed to assess efficacy and safety of rivaroxaban (20 mg daily) relative to dose-adjusted warfarin for stroke prevention in approximately 14,000 patients with atrial fibrillation. A large dose-finding randomized, double-blind, placebo-controlled phase II study is also underway to investigate the efficacy and safety of rivaroxaban alone or in combination with ASA or ASA and thienopyridine, for secondary prevention of fatal and nonfatal cardiovascular events in patients with recent ACS.

Apixaban

Apixaban is in clinical development for prevention of VTE in patients undergoing THR and TKR, in patients with advanced metastatic cancer and in medically ill patients, secondary prevention in patients with ACS, prevention of stroke in nonvalvular atrial fibrillation, and treatment of VTE. Apixaban is a follow-up to razaxaban. Clinical development of razaxaban was stopped following a phase II trial in patients undergoing TKR, in which the three higher dosages of razaxaban caused major bleeding. Apixaban has an improved pharmacological profile relative to razaxaban.

Apixaban is a highly potent and selective direct inhibitor of factor Xa. Apixaban is eliminated via multiple pathways; 70% of the compound is eliminated in feces, and 25% is eliminated via the renal pathway. In two double-blind randomized, placebo-controlled, dose-escalation studies in healthy males, oral apixaban demonstrated predictable pharmacokinetics with single doses, with maximum plasma concentrations achieved 1.5 to 3.5 hours after oral administration of the drug.

Efficacy and safety of apixaban for prevention of VTE in patients undergoing TKR was evaluated in a phase IIb trial (APROPOS).¹⁰¹ In this study, 1217 patients were randomized to receive one of six doses of apixaban (5 mg, 10 mg, or 20 mg, administered once or twice daily), open-label enoxaparin 30 mg twice daily, or warfarin for 10 to 14 days. Apixaban and enoxaparin were initiated 12 to 24 hours after surgery, whereas warfarin was started in the evening of the day of surgery. Rates of VTE and all-cause mortality, the primary efficacy endpoint, were significantly lower in the combined apixaban groups (8.6%) than in either the enoxaparin or warfarin groups (15.6% [P < 0.02] and 26.6% [P < 0.001], respectively). The primary safety endpoint of major bleeding was similar between treatment groups.

A recent double-blind randomized dose-finding trial investigated the efficacy and safety of apixaban in patients with confirmed DVT (proximal DVT or extensive calf DVT). Frequency of the primary efficacy endpoint (composite of symptomatic recurrent VTE and deterioration of the thrombotic burden, as assessed by repeat bilateral compression ultrasound and perfusion lung scan) was similar in the apixaban 5 mg twice daily and 10 mg twice daily groups (6.0% and 5.6%, respectively) and lower in the apixaban 20 mg daily group (2.6%). Early evaluation of results from phase III study of apixaban for prevention of VTE in patients undergoing TKR, however, indicate that the primary endpoint was not met.¹⁰²

A placebo-controlled phase II pilot study is in progress to investigate apixaban for prevention of thromboembolic events in patients undergoing treatment for advanced cancer (ADVOCATE). Efficacy and safety of a 30-day regimen of apixaban compared with enoxaparin for prevention of VTE in acutely medically ill patients has been initiated in a phase III study. Two phase III studies of apixaban for stroke prevention in atrial fibrillation are in progress. One is designed to evaluate efficacy and safety of apixaban vs. warfarin in preventing stroke and systemic embolism in 15,000 patients with nonvalvular atrial fibrillation and at least one additional risk factor for stroke (ARISTOTLE). The second trial is assessing whether apixaban is superior to ASA in preventing stroke or systemic embolism in 5600 patients with atrial fibrillation and at least one additional risk factor for stroke who refuse or are unsuitable for treatment with a vitamin K antagonist (AVERROES). A large phase II placebo-controlled study is examining the efficacy and safety of apixaban in patients with recent ACS (APPRAISE-2).

DU-176b

DU-176b is an oral direct factor Xa inhibitor in early clinical development for prophylaxis and treatment of thrombotic disorders. It is a potent inhibitor of factor Xa, with a 10,000-fold higher selectivity for factor Xa than for thrombin. DU-176b dose-dependently prolongs clotting times and decreases thrombin generation and platelet aggregation. A phase I study in 12 healthy adults demonstrated that DU-176b was able to reduce thrombus formation ex vivo in a Badimon chamber. The antithrombotic effects of DU-176b were sustained for up to 5 hours, with maximum inhibition of factor Xa activity occurring 1.5 hours after administration.¹⁰³ A recent phase II study evaluating the efficacy and safety of DU-176b for prevention of VTE in patients undergoing total knee arthroplasty demonstrated significant dose-dependent reductions in VTE in patients undergoing total knee arthroplasty, with a bleeding incidence similar to placebo.¹⁰⁴ In a phase II study of prevention of stroke in atrial fibrillation, patients will receive DU-176b or warfarin for 3 months.

LY517717

LY517717 is an indol-6-yl-carbonyl derivative in development for treatment and prophylaxis of thromboembolic disorders. It is a factor Xa inhibitor with 1000-fold higher selectivity for factor Xa than other serine proteases, and high oral availability.¹⁰⁵ In humans, anticoagulant activity of LY517717 peaked within 0.5 to 4 hours of administration, and a terminal half-life of approximately 27 hours was observed, with the gastrointestinal tract as the main elimination route. A phase II double-blind parallel-group, dose-ranging study of LY517717 was undertaken in 511 patients undergoing THR or TKR. LY517717 was investigated relative to enoxaparin.¹⁰⁶ The primary efficacy endpoint was a composite of DVT, and for the higher doses of LY517717, incidences of VTE were 19% (100 mg), 19% (125 mg), and 16% (150 mg), compared to 21% for enoxaparin, indicating that LY517717 at these doses was noninferior to enoxaparin according to prespecified criteria. Further development of LY517717 is planned, with phase III trials for prevention of VTE.

Betrixaban

Betrixaban is a potent inhibitor of factor Xa, with a half-life of 19 hours. The antithrombotic activity of betrixaban, demonstrated in different animal models of arterial and venous thrombosis, has been shown to occur at doses that inhibit thrombin generation in human blood. A phase I dose-escalation study in 64 subjects revealed that betrixaban had minimal interactions with food and predictable pharmacokinetics and pharmacodynamics.¹⁰⁷ Furthermore, betrixaban undergoes minimal renal excretion because it is predominantly eliminated unchanged in bile. In a phase IIa proof-of-concept study (EXPERT), betrixaban was investigated relative to enoxaparin administered for 10 to 14 days.¹⁰⁸ The primary efficacy endpoint was the incidence of VTE (symptomatic DVT or PE or asymptomatic DVT on a mandatory venogram) on days 10 to 14. Rates of VTE were 20% and 15%, respectively, in patients receiving betrixaban, and 10% in patients receiving enoxaparin. Further clinical studies for prevention and treatment of VTE, stroke prevention in atrial fibrillation, and secondary prevention of stroke and MI are planned.

YM150

YM150 is in development for prevention of VTE. YM150 has a major active metabolite, YM-222741, against factor Xa. A randomized open-label, phase IIa dose-escalation trial in 178 patients undergoing THR assessed YM150 for 7 to 10 days after surgery, relative to enoxaparin.¹⁰⁹ The primary endpoint was major and/or clinically relevant nonmajor bleeding, and the main efficacy endpoint was the composite of DVT detected by mandatory bilateral venography, confirmed symptomatic DVT, PE, and all-cause mortality. There were no major bleeding events during the study and three clinically relevant nonmajor bleeding events. Venous thromboembolism incidence was dose dependent, ranging from 52% for 3-mg dosing to 19% at 60 mg; incidence of VTE in the enoxaparin group was 39%.¹¹⁰ A phase IIb study of YM150 (5-120 mg daily) for prevention of VTE after THR has been completed recently.¹¹⁰ Incidence of the primary efficacy endpoint (composite of DVT, symptomatic VTE, PE, and death up to day 7 to 10 of treatment) ranged from 31.7% to 13.3% and decreased significantly with increasing doses of YM150 (P < 0.0002). A further phase II study will assess the pharmacokinetics, pharmacodynamics, safety, and tolerability of YM150 in an atrial fibrillation patient population.

Active-site competitive antagonists

The earliest investigation of FIXa inhibitors was based on an active-site competitive antagonist, IXai, a protein without functional anticoagulant activity.¹¹⁴ Intravenous infusion of IXai inhibited thrombosis in animal models of coronary thrombosis and stroke in a dose-dependent fashion and produced less bleeding than UFH.^114–118 To date, clinical trials of factor IXai have not been conducted.

Monoclonal antibodies as anticoagulants

Monoclonal antibodies are currently used to treat cancer, autoimmune disease, and allergy. Several antibodies directed against the Gla domain of factor IX have been developed. The 10 C12 clone was an effective anticoagulant, prolonging APTT as well as inhibiting platelet-mediated clotting in vitro.¹¹⁹ It effectively inhibited arterial thrombosis in a rabbit model of carotid artery injury, without increasing blood loss from a standardized cutaneous incision.¹²⁰ A humanized monoclonal antibody, SB 249417, is a chimeric molecule directed against the human FIX Gla domain. In a rat arterial thrombosis model, the antibody produced significant reductions in thrombus formation, with modest APTT prolongation. In a murine stroke model, SB 249417 reduced infarct volume and was associated with reduced neurological deficits compared to tPA.¹²¹ Suppression of FIX activity and APTT prolongation were rapid and dose dependent. A phase I clinical trial with SB 249417 has been completed. Designed as a single-blind randomized placebo-controlled, single intravenous infusion dose-escalating trial, the study was undertaken to establish pharmacokinetic and pharmacodynamic properties. The antibody displayed a dose-dependent effect on clotting times, with a maximal effect at completion of a 50-minute continuous infusion.¹²² There were no major safety concerns.

RIBONUCLEIC ACID Aptamers as anticoagulants

Aptamers are short oligonucleotides (< 100 bases) selected for their ability to bind a chosen target, typically a protein or small molecule.¹²³ A complex between RNA and the selected target protein (or small molecule) involves a three-dimensional folding of the RNA such that it is complementary with the surface of the target protein. Molecular recognition of a target protein by an aptamer can involve several types of RNA protein interactions, including hydrogen bonding, salt bridges, van der Waals forces, and stacking with aromatic amino acids.¹²⁴

Aptamer 9.3 t, specific for factor IXa, showed that the aptamer bound factors IX and IXa with high affinity but exhibited minimal affinity for the structurally related proteins, factors VII, X, or XI, or protein C.¹²⁵ Since factor VIIa binds FIX via the Gla and EGF domains, the aptamer may interact with the EGF domain.¹²⁶ An RNA antidote (5.2) to the FIXa aptamer has been made and can reverse 9.3 t-induced anticoagulation in human plasma.¹²⁷ Other advantages of the aptamer/antidote pair include reduced generation of thrombin and inflammatory mediators (interleukin [IL]-1b, IL-6), reduced postoperative hemorrhage, and improved cardiac output.¹²⁸ The anti-IX aptamer/antidote pair 9.3 t and its antidote 5-2 were subsequently optimized for in vivo stability and manufacturability to generate the REG-1 anticoagulation system. Regado-1A was a subject-blinded dose-escalation placebo-controlled study that randomized 85 healthy volunteers to receive a bolus of drug (FIX aptamer RB006) or placebo, followed 3 hours later by a bolus of antidote (RB007) or placebo.¹²⁹ Among subjects treated with RB006, APTT and ACT increased rapidly in a dose-dependent fashion, and the observed pharmacodynamic effect was stable over a 3-hour time period. The Regado-1 C study randomized 39 healthy human subjects in a double-blind fashion to either three consecutive weight-adjusted drug/antidote treatment cycles or double placebo. Each treatment cycle consisted of an intravenous bolus of RB006, followed an hour later by an ascending dose of RB007. There was a graded response to varying doses of antidote, showing an ability to titrate anticoagulant response and reversibility. There were no major bleeding or other serious adverse events.¹³⁰ Potential clinical applications for this injectable factor IXa–specific drug antidote system include percutaneous and surgical coronary revascularization procedures, bridging therapy for elective noncardiac surgery in patients on Coumadin therapy, prophylaxis and treatment of venous and arterial thromboembolic disorders, and maintenance of hemodialysis circuit patency. A subcutaneous formulation that is currently being studied may extend the pharmacodynamic half-life of the drug, minimizing the number of daily injections and enabling home use. A key concern will be the potential for equipment-related thrombosis, a phenomenon that has hindered clinical development of other specific coagulation protease inhibitors.¹³¹

TTP889

TTP889 is an orally available small-molecule selective partial antagonist of factor IX/IXa. The FIXIT study group conducted a phase II clinical trial to determine the safety and antithrombotic efficacy of TTP889 in patients at risk for VTE. This multicenter placebo-controlled trial enrolled 261 hip fracture surgery patients,¹³² and there was no significant difference between treatment groups in the composite primary outcome of venographic or symptomatic DVT or PE at the end of the study period. However, TTP889 had no effect on markers of thrombin generation and fibrin degradation (D-dimer) compared with placebo, despite the use of TTP889 dose levels considered sevenfold higher than that required to prevent venous thrombosis in animal models. This apparent lack of pharmacodynamic effect raises concerns about the appropriateness of the dose of TTP889 selected.

Factor IX-binding proteins

It is known that natural anticoagulants occur from snake venom, including a family of homologous proteins that complex with factor IX (IX-binding proteins [bp]), factor X (X-bp), or both (IX/X-bp). The family includes habu IX-bp and habu IX/X-bp of Trimeresurus flavoviridis, echis IX/X-bp of Echis carinatus leucogaster, and acutus X-bp of Deinagkistrodon acutus.¹³³ The venom of Agkistrodon acutus contains agkisacutacin, a homologous protein that binds both platelet GPIb and coagulation factors IX and X.¹³⁴ These proteins have structures similar to disulfide-linked heterodimers of C-type lectin-like subunits. In vitro studies with IX-bp from T. flavoviridis showed anticoagulant activity, with prolongation of APTT and interference of FIXa binding to phosphatidyl serine on the plasma membrane.