FOCUS: ANTIPLATELET DRUGS AND PLATELET FUNCTION TESTING Managing Antiplatelet Therapy GEORGE A. FRITSMA LEARNING OBJECTIVES Address for Correspondence: George A. Fritsma, MS, 1. List antiplatelet drugs that are used in dual MLS, The Fritsma Factor, Your Interactive Hemostasis antiplatelet therapy. Resource, Fritsma & Fritsma LLC, 153 Redwood Drive, 2. Describe the basis for aspirin and thienopyridine Birmingham, AL 35173, [email protected] antiplatelet property. 3. Define aspirin and clopidogrel low response. Dual Antiplatelet Therapy 4. List and describe platelet function testing that is In 1955, President Dwight Eisenhower, recovering applied to detecting aspirin and clopidogrel low from an acute myocardial infarction (MI), was treated response. with warfarin (Coumadin®) to prevent a repeat MI 1 (Figures 1 and 2). Coumadin, FDA-cleared in 1954, ABBRVIATIONS: AA-arachidonic acid, ACS-acute had become an essential part of acute coronary coronary syndrome, ADP-adenosine diphosphate, ALR- syndrome treatment; it was intended to reduce the risk aspirin low responder, AMI-acute myocardial of a secondary MI, peripheral artery disease, ischemic infarction, ARU-aspirin reaction (or resistance) units, stroke, and venous thromboembolic disease subsequent ASA-acetylsalicylic acid, CABG-coronary artery bypass to an MI. Coumadin continues today as the most- graft, CLR-clopidogrel low responder, COX- prescribed anticoagulant in North America, though cyclooxygenase, CT-closure time, DAPT-dual now its most frequent indication is prophylaxis to antiplatelet therapy, CLIA-clinical laboratory reduce the risk of ischemic stroke in non-valvular atrial improvements amendment, FDA-US Food and Drug fibrillation. Because of its narrow therapeutic and safety Administration, LTA-light transmittance aggregometry, range, Coumadin requires monthly laboratory MI-myocardial infarction, NSAID-non-steroidal anti- monitoring using the prothrombin time assay, which, inflammatory drug, CYP-cytochrome oxidase pathway, beginning in 1987, was enhanced through computation 2 PCI-percutaneous intervention, PGE1-prostaglandin of the international normalized ratio (PT/INR). The E1, PGG2-prostaglandin G2, PGH2-prostaglandin H2, accepted therapeutic range worldwide is an INR of 2–3. POC-point of care, PRU-P2Y12 reaction units, An INR below 2 signals increased risk of thrombosis, 3 PT/INR-prothrombin time with international above 3, risk of hemorrhage. normalized ratio, TEG-thromboelastography, TEM- thromboelastometry, TXA2-thromboxane A2, WBA- In 1992, thienopyridine, a nucleic acid derivative whole blood aggregometry, VASP-vasodilator- distributed as ticlopidine (Ticlid®), was added to post- 4 stimulated phosphoprotein MI Coumadin therapy (Figure 3). Ticlopidine is an antiplatelet drug that reversibly occupies the platelet INDEX TERMS: Aspirin, thienopyridine, clopidogrel, membrane ADP receptor, P2Y12 (Figure 3). Ticlopidine prasugrel, ticagrelor, platelet function, platelet function competes with ADP for P2Y12 receptor sites, testing, aggregometry, thromboelastography, suppressing ADP’s platelet activation property. From thromboelastometry, antiplatelet therapy 1992 to 2000, Coumadin and Ticlid administered together were the most commonly used post-MI Clin Lab Sci 2015;28(2):132 antithrombotic regimen. This regimen was typically discontinued six months to two years after the event. George A. Fritsma, MS, MLS, The Fritsma Factor, Your Regrettably, 1 in 3–5000 ticlopidine patients developed Interactive Hemostasis Resource, Fritsma & Fritsma LLC, life-threatening thrombotic thrombocytopenic purpura Birmingham, AL or aplastic anemia, creating a negative public 5 perception. This plus the 1997 release of the VOL 28, NO 2 SPRING 2015 CLINICAL LABORATORY SCIENCE 132 FOCUS: ANTIPLATELET DRUGS AND PLATELET FUNCTION TESTING biochemically similar but seemingly safer Clopidogrel Mechanism thienopyridine clopidogrel (Plavix®) caused Ticlid to be Clopidogrel is distributed as a prodrug that is absorbed withdrawn from the North American market in 2010, within one hour of ingestion and becomes metabolized though generic ticlopidine preparations remain available through the liver cytochrome P450 C19 pathway to (Figure 3). Popular knowledge to the contrary, produce 2-oxo-clopidogrel.8 The 2-oxo-clopidogrel has thrombotic thrombocytopenic purpura continues to be a half-life of 20 minutes, during which time it reversibly associated with all forms of thienopyridine. binds the platelet membrane ADP receptor P2Y12, competing with ADP access to its receptor and thus suppressing platelet activation. 1956% Figure 1. 1956 newspaper article describing President Dwight D. Eisenhower’s Coumadin therapy. He began using the drug subsequent to an MI in 1955, just one year after it Figure 2. Vitamin K post-translational γ-carboxylation of was cleared by the FDA. coagulation factors II (prothrombin), VII, IX, and X, and control proteins C, S, and Z. Vitamin K hydroxyquinone Also around 1997, Coumadin began to be replaced with transfers a carboxyl (COO–) group to the γ-carbon of aspirin (acetylsalicylic acid, ASA) as the mainstay of glutamic acid, creating γ-carboxyglutamic acid. The post-MI therapy. Coumadin’s effect is to reduce the negatively charged pocket formed by the pair of carboxyl plasma activity of coagulation factors II, VII, IX, and X, groups attracts ionic calcium, enabling the molecule to reducing the risk of venous thromboembolic disease but bind phosphatidyl serine. Vitamin K hydroxyquinone is oxidized to vitamin K epoxide by carboxylase in the exerting little platelet suppression. Many adverse events process of transferring the carboxyl group but is that occur subsequent to MI are arterial, and arterial subsequently reduced to the hydroxyquinone form by thrombosis results mostly from platelet activation, thus epoxide reductase. Warfarin suppresses epoxide reductase, pushing post-MI therapy towards ASA. slowing the reaction, preventing γ-carboxylation. “Des- carboxy” proteins are unable to participate in coagulation. There are typically 12 to 18 γ-carboxyglutamic acid A number of clinical trials conducted between 1995 and molecules near the amino terminus of the vitamin K- 2010 have shown that dual antiplatelet therapy (DAPT) dependent factors. employing ASA and clopidogrel, provides slightly improved efficacy and safety with fewer side effects than Aspirin Mechanism Coumadin and clopidogrel, ASA alone, or clopidogrel In 1948, Lawrence Craven provided the first evidence alone.6 DAPT is now the mainstay for prevention of of ASA’s ability to reduce AMI risk.9 Doubted at the secondary adverse events such as repeat MI or stent time, his findings were confirmed by HD Lewis in 1983 thrombosis in “uncomplicated” acute coronary and subsequently confirmed in numerous large syndrome (ACS). The typical dosages are 81 mg ASA studies.10,11 For approximately 30 minutes subsequent to (one baby aspirin) and 75 mg clopidogrel per day. absorption, ASA releases its acetyl group to Coumadin is still used in ACS when complicated by cyclooxygenase (COX), an essential enzyme in the reduced ventricular ejection fraction (coronary prostaglandin (eicosanoid) synthesis pathway (Figure insufficiency).7 4).12 The acetyl group occupies COX molecule serine 133 VOL 28, NO 2 SPRING 2015 CLINICAL LABORATORY SCIENCE FOCUS: ANTIPLATELET DRUGS AND PLATELET FUNCTION TESTING 529 and blocks arachidonic acid access to the enzymatically active site at tyrosine 385 (Figure 5). Normally, arachidonic acid becomes converted to prostaglandin G2 (PGG2), then prostaglandin H2 (PGH2), and subsequently to thromboxane A2, the short-lived product that binds adenylate cyclase and activates the platelet. COX acetylation is irreversible and prevents arachidonic acid to PGG2 conversion, thereby inactivating the platelet. All platelets exposed to clopidogrel or ASA circulate inactivated through their lifespan. Figure 5. COX1 inhibited by aspirin. Arachidonic acid is released from membrane phospholipids and is converted by COX1 at the Tyr365 active site to prostaglandins G2, then H2, and finally to the platelet activating molecule, thromboxane A2. The acetyl group, released from aspirin, binds serine 529 and blocks tyrosine 385, preventing arachidonic acid access to the active site. Aspirin Resistance Between 10 and 20% of people who take 81 mg ASA Figure 3. Structures of the thienopyridines ticlopidine, clopidogrel, daily do not generate the anticipated platelet function prasugrel, and the purine analogue ticagrelor. laboratory response.13 This phenomenon is called ASA resistance and the subjects are termed “ASA low responders (ALRs).” There exist multiple models to account for ALR, one of which contrasts the cyclooxygenase variants, COX1 and COX2. COX1 is constitutive, whereas COX2, a target of non-steroidal anti-inflammatory drugs (NSAIDS, celecoxib, Celebrex®, for instance) becomes induced during inflammatory conditions like diabetes, arthritis, and AMI. COX2 becomes acetylated in the same fashion as COX1, however arachidonic acid may nonetheless gain access to the tyrosine 385 active site, as the COX2 physical configuration is broader than COX1. Consequently, as COX2 becomes induced, ASA Figure 4. The prostaglandin (eicosanoid synthesis) pathway. resistance may correlate with inflammation. A second Agonists ADP, thrombin, and collagen bind their respective model postulates that NSAIDS other than ASA may membrane receptors and activate phospholipase A2, which releases temporarily block ASA access to COX1, causing ALR arachidonic