Anticoagulants: BPS 338 Lecture Notes PDF

BPS 338 Inhibitors of Blood (fibrin) clot formation: Anticoagulants (prevent fibrin) Learning the structural features of the coagulation cascade proteins helps to understand their action and the action of the anticoagulant drugs Dr. King...

BPS 338 Inhibitors of Blood (fibrin) clot formation: Anticoagulants (prevent fibrin) Learning the structural features of the coagulation cascade proteins helps to understand their action and the action of the anticoagulant drugs Dr. King 1  Themes – protein activation 1. Protein conformation (and activity) change caused by binding of another large or small molecule a. Coagulation cofactors (V, VIII, tissue factor) increase activity of serine proteases (prothrombin/thrombin, VII, IX, X, XI, XIII) b. Glycosaminoglycan binding interactions (heparin + antithrombin + thrombin) c. Carboxylation of coagulation factors 2. Protease Enzyme activators: an enzyme hydrolyzes a peptide bond in another enzyme, and converts the second enzyme to an active (or more active) form via conformational change a. coagulation cascade factors b. plasminogen (thrombolytics) In coagulation, activation of the Serine Protease (serpin) family of enzymes is critical!! 2 How does the coagulation factor activation occur? (co-factor) and/ 3  Drug classes Inhibitors of clot formation I. Anticoagulants (inhibit fibrin formation) (inhibit clot formation) A. Direct Factor Xa inhibitors: apixaban, rivaroxaban, edoxaban B. Direct Thrombin inhibitors: dabigatran C. Antithrombin enhancers: Unfractionated Heparin; Low MW heparins LMWH: -parin; fondaparinux (Arixtra) D. γ-Glutamate carboxylation inhibitor: Warfarin II. Antiplatelets (inhibit platelet aggregation) (inhibit clot formation) A. Thromboxane A2 synthesis inhibitor: Aspirin B. Glycoprotein IIb/IIIa inhibitors: abciximab, tirofiban, eptifibatide C. Purinergic receptor inhibitors (ADP-receptor, P2Y12) (and more) -clopidogrel, prasugrel, ticagrelor, cangrelor Stimulators of clot degradation III. Thrombolytics (degrade existing clots) Plasmin activators (Alteplase, reteplase, anistreplase, streptokinase) 4  These slides focus on the Fibrin Formation Formation of blood clots… Occurs by two fundamental processes: 1. Platelet activation/aggregation 2. Fibrin formation 2. 1. Arteriosclerosis, Thrombosis, and Vascular Biology. 2019;39:7–12  Clots are formed by two fundamental processes Simplified pathways 1. Aggregation of platelets 2. Formation of fibrin Boxes and open arrows note the drugs that inhibit clot formation. 6 (Med Res Reviews, 2004, 24:151-181)  Extrinsic pathway Intrinsic pathway wound The coagulation cascade (formation of fibrin). Initiated by factor VIIa/tissue factor complex (extrinsic). Propagated (intrinsic) by (also called factor Xa and cofactor Va, prothrombinase) factor IXa and cofactor VIIIa. Factor Xa converts inactive Prothrombin (II) to active Apixaban, Thrombin (IIa), Rivaroxaban Edoxaban Thrombin converts soluble Apixaban, fibrinogen into insoluble fibrin, (II) (IIa) Rivaroxaban Edoxaban and leads to more activation of *Note: Heparin, LMWH, *Heparin V, VIII, XI, and XIII. and fondaparinux act indirectly via antithrombin Fibrin is the major matrix protein of a clot. NOTE: “a” designation means activated conformation. 7  The coagulation cascade is an enzyme amplification network The factors were named before we knew what they were—just given numerals. – Roman numerals for “inactive or less active” form (X), addition of “a” for “active or more active” form (Xa) sometimes called Serpins Later found that some of the factors are serine protease enzymes (VII, IX, X, XI, thrombin, XIII) – Active forms are designated VIIa, IXa, Xa, XIa, XIIIa – Each one is activated by proteolytic cleavage (one factor cleaves the next in the cascade). VII can slowly cleave other VII’s to form VIIa. – (prothrombin is II; thrombin is IIa) Some (V, VIII) are NOT proteases (now usually called co-factors), but bind with activated proteases to increase activity ~100-fold. – Active form of the cofactor requires binding to a membrane and active factor (causes conformational change in increase serpin activity) – (Note: the Cofactors are proteins, but they are not enzymes) 8  Structure of the factors Learning the structural features of the factors helps to understand their action and the action of the anticoagulant drugs Visualize the 3-D interactions, understand what interacts with what (and how) Remember, The coagulation factors and cofactors are all folded proteins 9  Tissue Factor (TF) Extrinsic pathway (wound) (is a co-factor) TF is shaped like this……………... Initiates coagulation cascade by binding factor VII or VIIa Tissue Factor Only comes into contact with blood components when endothelium is damaged Cell membrane (endothelium of vasculature) Image from: Protein Databank, “Tissue Factor” 10 March 2006 Molecule of the Month by David S. Goodsell. www.rcsb.org Tissue Factor activates factor VII a Factor VII auto-activates to VIIa Factor VIIa binds to tissue factor, ‘embraces’ it, contacting entire TF molecule Tissue Factor Results in 1000x increase in serpin activity (VII vs. VIIa/TF) Extrinsic pathway What serpin activity? 3 actions, See Image from: next slide… Protein Databank, , “Tissue Factor” 11 March 2006 Molecule of the Month by David S. Goodsell. www.rcsb.org  What does TF/VIIa complex do? 3 serpin actions… wound Hydrolyze: Extrinsic pathway Factor IX to IXa, Factor X to Xa, Factor VII to VIIa 12  Factor VIIa/TF hydrolyses Factor X (and IX and VII) a Factor VIIa/TF binds to Factor X They actually lay right up to one another, not separate as in this figure (see the movies). Factor VIIa serine protease activity hydrolyzes a peptide bond within factor X, converts it to Xa (see green region) Xa is more active than X because its protease site is exposed (see green region) – Conformational change in protein – Remember slide 3, Asp-His-Ser active conformation Image from: Protein Databank, , “Tissue Factor” 13 March 2006 Molecule of the Month by David S. Goodsell. www.rcsb.org  Movie of interactions Tissue Factor (green) binds to VIIa 14 Movie made in BPS 455  Movie of interactions TF-VIIa binds to Xa 15 Movie made in BPS 455  Cascade – feedback stimulates it Tissue factor activates a few molecules of Factor VII to VIIa/TF. – & Feedback to activate more VII to VIIa VIIa/TF activates Factor X to Xa, binds to Va. – Feedback to activate more VII to VIIa VIIa/TF also activates IX to IXa, binds to VIIIa – Activates more X to Xa Now have multiple amounts of Prothrombin (II) Thrombin (IIa) thrombin (IIa) 16 Summary: Structures of protease enzyme coagulation factors (VII, IX, X: the ones that utilize cofactors) Head 3 structural domains in each protein a – Serine protease (hydrolysis) domain (head) – Co-factor binding domain Tissue Factor – GLA-domain with 9 Tail modified glutamic acids (γ-carboxylated glutamic acid-rich domain, red) to bind calcium (yellow) Image from: Protein Databank, “Tissue Factor” March 2006 Molecule of the Month 17 by David S. Goodsell. www.rcsb.org  Function of each domain (VII, IX, X: the ones that utilize cofactors) Head The protease domain (head) binds the protease substrate (other enzyme which gets hydrolyzed) and catalyzes the hydrolysis reaction. The co-factor binding domains bind the cofactor Tail and change conformation around the protease active site, making it more active. Cofactors bind to this ‘tail’ region. Carboxylation of the glutamic acids in the Gla domain puts negative charges (red) on the protein to bind (yellow) Ca2+ ions. Calcium ions bind to the membrane surface and help the factor and co-factors “find” one another. The Gla domain is at the end of the “tail” region. – More about GLA domain later (warfarin acts there) 18  Factors VII, IX, X, XI: Summary All need amino acid cut (hydrolyzed) to convert from inactive/less active to active form (example, X → Xa) All are serine protease enzymes (serpins) which hydrolyze other coagulation factors (see cascade figure to see which hydrolyze which) All except XI/XIa need a membrane-associated cofactor protein bound for highest protease activity Co-factors V, VIII and factor XI are hydrolyzed by thrombin (providing feedback activation) VII/TF is the only one that autoactivates (hydrolyzes other VII’s) (Later topic) Thrombin, Xa, IXa, XIa, XIIa are naturally inhibited by antithrombin (Later topic) Heparin decreases the activity of coagulation factors by enhancing their natural inhibition by antithrombin. 19 Anticoagulant drug class: – NEWEST Direct Factor Xa inhibitors Name hint: Direct Xa – Rivaroxaban (Xarelto) inhibitors FDA approved July 1, 2011 have suffix Xa ban – Apixaban (Eliquis) FDA approved December 2012 – Edoxaban (Savaysa) FDA approved January 2015 ORALLY ADMINISTERED – Not injection, advantage!!! – DOAC (direct oral anticoagulants) – SAFE !! 20  Why Factor Xa inhibitors? Because 1 molecule of Xa produces 1000 (or more) molecules of thrombin (thrombin burst) – More effective to inhibit Xa and prevent thrombin (IIa) formation versus inhibiting later thrombin action – See the cascade figure Another more recently recognized advantage of direct Xa inhibitors versus direct thrombin (IIa) inhibitors is… – that thrombin has been discovered to have many other non- coagulation related activities. – Potentially undesirable to interrupt these other activities. – These direct thrombin inhibitors (DTI) are in later slides but have very limited therapeutic use. 21 Zoom in on the protease domain (“head”) of factor X (Xa) Xa, “head” protease domain only 22 3D Figure prepared by Joe Christian, URI, for BPS 455, Fall 2015  Fills both the “cleft” Rivaroxaban (Xarelto) and the “deep hole” These drugs were designed to bind very specifically to the Xa binding pocket. -used the crystal structure to design -“Structure-based drug design” Zoom in on binding pocket 23 3D Figures prepared by Joe Christian, URI, for BPS 455, Fall 2015  Apixaban (Eliquis) Fills both the “cleft” and the “deep hole” Zoom in on binding pocket 24 Figures prepared by Joe Christian, URI, for BPS 455, Fall 2015  Edoxaban (Savaysa) FDA approved January 2015 Fills both the “cleft” and the “deep hole” 25  Another class of anticoagulant drugs: Direct Thrombin inhibitors Another class: Direct Thrombin inhibitors (DTIs) – Thrombin distinguishes them from the newer Xa inhibitors. – “Direct” distinguishes them from thombin inhibitors that act indirectly through antithrombin (heparin, LMWH, fondaparinux) (later slides) First, we need to know more about thrombin and how it works… Thrombin has at least 5 roles in the cascade – very important – and its inhibition is rational drug target But we already saw that Factor X is an even better target because it forms thrombin 26 Thrombin: what is, how work? Another serine protease enzyme Hydrolyzes co-factors V & VIII, and factor XI to multiply cascade effect (feedback activation) Cleaves (soluble) fibrinogen to form (insoluble) fibrin for the clot Cleaves XIII to XIIIa to increase crosslinked fibrin. Will come to this Prothrombin later…Antithrombin (AT) inhibits thrombin activity, this inhibition is enhanced by glycosaminoglycans Factor XIIIa Factor XIII (like heparin) 27 Crosslinked fibrin  What is thrombin: Conversion of Prothrombin (II) to thrombin (IIa) – Prothrombin 3 amino acids Cleaved by Xa/Va complex (prothrombinase) Thrombin – only the yellow/orange part left! Thrombin catalytic site is circled PNAS | May 27, 2014 | 28 vol. 111 | no. 21  Structure of Thrombin Protease (head) portion only, does not have cofactor binding ‘tail’ !!! Serine Protease site is in a deep canyon which restricts substrate and inhibitor access and determines substrate/inhibitor Active site specificity. Red = oxygen of key serine residue Blue = two nitrogens of histidine which activates the serine (Hidden) = aspartate which assists in serine activation Remember this from slide 3? 29  Drugs in Direct Thrombin Inhibitor class: Historical: Large molecule (peptide) direct thrombin inhibitors, block both active site and another site called exosite I Development history of direct thrombin inhibitors Hirudin is a 65-amino acid peptide isolated from the medicinal leech. – Very tight binding makes it essentially irreversible and very potent (Kd = 10-14 M = femtomolar) Lepirudin (recombinant form) was approved by FDA in 1998. (Refludan), But no longer manufactured (2013) Hirudin is in white sticks Bivalirudin (Angiomax) is a smaller, 20-amino acid analogue, FDA approved Dec 2000 – available but minor use. (Green color indicates hydrophobic regions in thrombin protein, but this is not relevant for this class) TIPS, 2003, 24:589-595. 30  The direct thrombin inhibitors block the active site: small molecules Melagatran Argatroban Argatroban (Acova™) was FDA-approved for injection in 2000. Small molecule (about 500 MW) Non peptide!!!! Still injection!!! Orally available prodrug form of melagatran (Ximelagatran, Exanta™) was approved in Europe (withdrawn Feb 2006), but failed approval in USA (Oct. 2004). WAS first orally available thrombin inhibitor. 31 TIPS, 2003, 24:589-595.  Newest one: Orally available direct thrombin inhibitor Orally administered - Direct Thrombin inhibitor (blocks active site) – Dabigatran (Pradaxa), Approved by FDA in 2010 Prodrug form is administered: etexilate, mesylate A dabigatran antidote (idarucizumab) approved by the FDA in 2015 (an antibody that binds excess dabigatran) Pro-Drug form: Dabigatran etexilate mesylate Active Drug form, hydrolyze esters at both ends 33  Next group of anticoagulants… Direct inhibitors of coagulation 1. Factor Xa inhibitors – rivaroxaban, apixaban, edoxaban Prevent formation of thrombin 2. Thrombin inhibitors- bivalirudin, argatroban, dabigatran … Prevent serine protease catalytic action of thrombin Indirect inhibitors of coagulation – Antithrombin enhancers (enhance protease inhibitory activity of the natural anticoagulant antithrombin) Heparin (can enhance thrombin-AT and Xa-AT), LMWHs, fondaparinux (can only enhance Xa-AT) – Prevent biosynthesis of active forms of procoagulant proteases Coumarins (warfarin) By reducing the carboxylation of the glutamic acid residues in 34 the Gla domain  Antithrombin enhancers First, what is antithrombin? How does it act as a natural anticoagulant? – antithrombin’s normal mechanism of action – What are antithrombin's normal binding partners? 35  What is Antithrombin (AT)? How does it work? Physiological inhibitor of thrombin and factor Xa, controls normal hemostasis Antithrombin present at high concentration in whole blood, ~2.3 mM When thrombin or Xa are bound to antithrombin, their normal protease action is prevented – (remember antithrombin acts as anticoagulant) Specific glycosaminoglycan binding enhances binding of antithrombin to either thrombin or Xa. (up to 10,000 fold) – Heparin, low molecular weight heparins, fondaparinux (Arixtra®) – Different binding causes somewhat different clinical effect 36  First, what is Heparin? Heparin is a naturally occurring complex carbohydrate known as a glycosaminoglycan or proteoglycan. Molecular weight ranges from 3,000-30,000 MW (average 15,000 = 45 sugar residues). It has a protein core with branches of carbohydrate extending out. Heparin’s structure is too complex to be synthesized. For drug use, it is isolated from porcine intestinal mucosa or bovine lung. Heparin is naturally present, medicinal heparin just increases its concentration. carbohydrate branches protein core 37 Minimum structure of glycosaminoglycan to bind antithrombin The glycosaminoglycan binding site on antithrombin needs a minimum sequence of 5 specific sugar residues (pentasaccharide) Heparin naturally contains this pentasaccharide sequence (about 1 of every 3 side-chains) The activity of heparin, LMWH heparin and synthetic preparations depend on presence of this specific carbohydrate sequence. O3SO RO O3SO CH2 COO CH2 COO CH2 O O O O O OH OH OSO3 OH OH O O O O O O NHR' OH HN OSO3 HN SO3 SO3 R = H or SO3 R' = SO3 or COCH3 Pentasaccharide structure needed for binding to antithrombin. The sulfates (OSO3-) are important for binding to (+) lysine & arginine on antithrombin. 38  Cartoon: how pentasaccharide enhances binding of antithrombin to factor Xa 1. 2. 3. 1. nAT = native antithrombin – Factor Xa binding sites too far apart – Single molecule of Xa can only interact with 1 site at a time, weak interaction 2. Heparin (glycosaminoglycan) – Pentasaccharide portion of heparin binding causes conformational change, – conformational change to aAT – aAT = – more active (enhanced) antithrombin When thrombin or Xa are bound to antithrombin, their normal protease action is prevented 3. After conformational change– enhanced (tighter) binding of Xa and Specific glycosaminoglycan binding enhances binding of antithrombin to thrombin to aAT either Xa by this conformational change from nAT to aAT. 39 Johnson et al (2006) The EMBO Journal, 25: 2029-2037. Figure 6.  Structure of antithrombin Remember: antithrombin binds to and inhibits thrombin and factor Xa. This binding is assisted by carbohydrates (pentasaccharide, glycosaminoglycan) Two binding regions – Factor Xa and thrombin bind here. – Pentasaccharide binds here to blueish portion e.g. heparin, LMWH, or fondaparinux (Arixtra®) Native Antithrombin structure 40 Li et al, Nature Structural and Molecular Biology (2004) 11:857-862  Activation of antithrombin shown with movement Li et al, Nature Structural and Molecular Biology (2004) 11:857-862 This is what happens with the pentasaccharide binds to antithrombin, converts it from nAT to aAT  Glycosaminoglycan assisted binding of antithrombin to factor Xa Factor Xa + heparin +AT Factor Xa + penta + AT First, even a pentasaccharide can initiate Xa binding to antithrombin (e.g., LMWH, fondaparinux) 500 fold enhancement If full length heparin is present, then Small glycosaminoglycan, Only 5 sugar units needed Xa/antithrombin binding is even tighter. 10000 fold enhancement Needs about 16-30 sugar residues for full enhancement Remember: When factor Xa is bound to antithrombin, it cannot stimulate coagulation. 42 TIPS, 2003, 24:589-595.  Glycosaminoglycan assisted binding of antithrombin to thrombin Fondaparinux (and shorter LMWH) ONLY act via Factor Xa! (not thrombin) thrombin Heparin (and longer LMWH) act via Factor Xa and Thrombin The Pentasaccharide is NOT long enough to enhance thrombin- antithrombin binding! No heparin enhancement Thrombin needs a longer bridge to assist in antithrombin binding antithrombin – Heparin is long enough (minimum ~16 residues), – But fondaparinux and most LMWH are too short to form the bridge to thrombin 43 Li et al, Nature Structural and Molecular Biology (2004) 11:857-862 Needs to be AT LEAST 16 sugar resudes to “reach” thombin SOME LMWH mixtures are long enough to bind BOTH Xa and Thrombin, but others are too short and only act through Xa Factor Xa thrombin heparin antithrombin 44 Li et al, Nature Structural and Molecular Biology (2004) 11:857-862 Sequence needed for bridging thrombin to antithrombin Polysaccharide units, minimum 16 residues Li et al, Nature Structural and Molecular Biology (2004) 11:857-862 45  Antithrombin enhancers Now let’s talk about the drugs… They are all variations of the glycosaminoglycan 46  Commercial heparin Large molecule, but a minimum of 5 specific sugar residues are required for activity to enhance AT binding to factor Xa. Minimum of ~16 sugar residues required for enhanced AT binding to thrombin. The commercial product (Unfractionated Heparin, UFH) is always of variable composition, but each preparation is standardized according to its blood clotting activity and administered as Units of clotting activity. Problem: Heparin interacts with several other proteins in the circulation – Reduces bioavailability, poorly predicted dose-response – Can lead to heparin-induced thrombocytopenia (HIT, serious problem!) Must be administered by injection – Immediate onset of action (because i.v. and its mechanism) Dose must be individually titrated to appropriate effect 47  Low Molecular Weight Heparins (LMWH) The natural heparin also may be digested into smaller pieces, partially purified, and administered as Low Molecular Weight (LMW) Heparins (range 2,000–10,000 MW, 6-30 sugar units). Advantage: LMW heparins have highly predictable dose- response, can be administered subcutaneous, and have lower incidence of thrombocytopenia (HIT) (because better selectivity). Many companies make LMWH, but they are NOT interchangeable. – Length determines whether enhance both thrombin and Xa, or only Xa – Enoxaparin, dalteparin, tinzaparin, Ardeparin, nadroparin, reviparin Most LMWH are too small to act as bridge for thrombin mechanism. Thus, LMWH typically only stimulate antithrombin inhibition of factor Xa (not thrombin). 48  What is Fondaparinux (Arixtra®)? Synthetic version of specific pentasaccharide sequence corresponding to antithrombin binding site of heparin (glycosaminoglycan binding site of antithrombin) Arixtra (approved December 2001), fondaparinux sodium Injectable, subcutaneous Same advantages and activity as LMWH Additional advantages: 100% subQ bioavailability, predictable PK – Selectivity for antithrombin NaO3S NaO3S NaO3S HO O HO O NaOOC OH HO NH OCH3 NH O OH O HO O O O O NH O O NaOOC O O NaO3S O O NaO3S NaO3S NaO3S NaO3S 5 sugar residues, 1700 MW – Some disadvantage: renal excretion limits use in renal impairment 49  Still in development “Full-length” heparin mimics – Same benefits as LMWH, fondaparinux – But long enough to bridge thrombin – Smallest one active: 16 sugar residues 5 sugars—specific for antithrombin binding at glycosaminoglycan site, sulfated 7 sugars—spacer, uncharged (no sulfates) 4 sugars—thrombin-specific binding – None brought to market yet 50  Last group of anticoagulants… next I. Anticoagulants (inhibit fibrin formation) (inhibit clot formation) Direct inhibitors of coagulation A. Factor Xa inhibitors – rivaroxiban, apixaban B. Thrombin inhibitors- argatroban, dabigatran … C. Antithrombin enhancers: Unfractionated Heparin, Low MW heparin, fondaparinux D. γ-Glutamate carboxylation inhibitors: Warfarin, other coumarins These agents prevent biosynthesis of functional forms of procoagulant proteases (VII, IX, X) by reducing the carboxylation of the glutamic acid residues in the Gla domain They act INDIRECTLY– because their molecular binding target is several steps removed from the anticoagulation activity Their DIRECT molecular binding target is Vitamin K oxido-reductase enzyme, type C1 (VKORC1) See figure on next slide… 51  Mechanism of action Warfarin (VERY Indirect….) CYP1A1 CYP1A2 CYP2C9 CYP3A4 Vitamin K Oxido- Reductase VKORC1 Oxidized Vitamin K Reduced Vitamin K CO2 O2 Calumenin (non-Carboxylated) (Carboxylated) Hypofunctional γ-glutamyl Functional Factors VII, IX, X carboxylase Factors VII, IX, X Protein C, S, Z Proteins C, S, Z 52 Remember the Gla-domain of factors VII, IX, X? It is a region containing many glutamic acid residues Carboxylation of the gamma carbon increases number of negative charges (-COO¯) at physiological pH -COO¯ Binds calcium ions (Ca2+), for better affinity to cofactors and membrane O O O H2 H2 H2 N N N N N N H2 H2 H2 O O O O O O O- O- O- glutamic acid gamma-carboxy glutamic acid GLA-domain with 9 modified glutamic acids (dark red) (γ-carboxylated glutamic acid-rich 53 domain) to bind calcium (yellow)  What is responsible for carboxylating the coagulation factors? (also see the figure) Very indirect pathway from warfarin to factors… Gamma-glutamylcarboxylase (Vitamin K-dependent) – Carboxylates prothrombin, factor VII, IX, X (also protein S, C, & Z) – Needs reduced vitamin K to assist in the carboxylation – Oxidative regeneration of vitamin K is inhibited by the coumarins (warfarin) Regeneration is catalyzed by vitamin K oxidoreductase— (VKORC1) —this is the molecular target of warfarin!!!!!!!!!! Delayed action—no effect on factors already carboxylated! – Few weeks after first dose before warfarin is effective as anticoagulant – And it remains effective few weeks after last dose. 54  Additional difficulties with use of warfarin… Very narrow therapeutic window (low therapeutic index) Dose must be individually titrated for each patient! Drug-drug interactions (mostly CYP2C9) Genetic differences (sensitive and resistant) Diet differences (vitamin K intake) See next few slides for details… 55  Warfarin–mechanisms for drug interactions The S-warfarin (5-6 times more potent than R) is cleared by CYP 2C9 CYP2C9 inhibitors–coadministration requires lower warfarin dose – Amiodarone (antiarrhythmic-class III) – Sulfinpyrazone (uricosuric-gout, antiplatelet) – Miconazole (antifungal) – Fluconazole (antifungal) 56 Individuals sensitive to warfarin (need lower doses) Some patients have hereditary warfarin sensitivity due to a genetic polymorphism in CYP2C9. These patients express variant alleles of CYP2C9 which are much less-active than the wild-type allele— 2C9 poor metabolizers. Since CYP2C9 is the major enzyme responsible for conversion of S-warfarin to inactive metabolites, patients with the variant CYP2C9 metabolize warfarin very slowly (causing lower clearance rate). The low activity variant alleles are present in 10-20% of Caucasians,