CHAPTER 43: Antithrombotic Therapies and Their Laboratory Assessment

INTRODUCTION and Thrombocytosis

• Absence of Vitamin K results in: nonfunctional des-g
Thrombosis Overview carboxyl forms of factors II, VII, IX, and X and proteins C, S,
• Thrombosis - formation of blood clots in veins or arteries and Z.
that cause tissue ischemia and necrosis • Coumadin(vitamin K antagonist): suppresses g-
• Antithrombotic drugs have been used since heparin was carboxylation of glutamic acid by slowing the activity of
first developed in 1916 and FDA-cleared in 1936. vitamin K epioxide reductase
• Antithrombotics include anticoagulants and antiplatelet • During Coumadin therapy: factors II, VII, IX, and X and
drugs. proteins C, S, and Z become reduced as nonfunctional des-
carboxyl proteins are produced.
Venous Thromboembolic Disease (VTE) • Until 2009: Coumadin was the only oral anticoagulant in
• VTE includes superficial and deep vein thrombosis (DVT) the United States, hence "oral anticoagulant therapy."
and pulmonary embolism (PE). • New direct-acting oral anticoagulants have broadened
• Treatment includes: the meaning of "OAT."
o intravenous standard unfractionated heparin (UFH)
o subcutaneous low-molecular-weight heparin Coumadin Prophylaxis and Therapy
(LMWH) Clinical Conditions That Require Measurement of
o subcutaneous synthetic pentasaccharide Antiplatelet Drugs and Anticoagulants Besides Coumadin
(fondaparinux) and UFH
o oral direct factor Xa inhibitor, rivaroxaban. • Renal disease: inadequate excretion, CrCl ,30
• VTE is also treated with the oral vitamin K antagonist mL/min
Coumadin. • Detection of noncompliance and underdosing
• Detection of comedication interference
Arterial Thrombosis • Acute hemorrhage (usually in emergency
• Arterial thrombosis includes: department or surgery)
o acute myocardial infarction (AMI) o Overdose, effects of comedication
o ischemic cerebrovascular accident (CVA) o Detection and identification; what
o transient ischemic attack (TIA) anticoagulant is it?
o peripheral arterial occlusion (PAO) o Determine if reversal is working
• Treatment: includes UFH, LMWH, fondaparinux, Coumadin, • Bridging from one anticoagulant to another or
the intravenous DTIs, and the antiplatelet drugs aspirin, discontinuing
clopidogrel, prasugrel, and ticagrelor. anticoagulant before surgery
• Resuming anticoagulant after surgery
Thrombolytic Therapy • Unstable coagulation: pregnancy, liver disease,
• Thrombolytic therapy may resolve DVT, PE, PAO, AMI, and malignancy,
stroke, particularly when used 3 to 4 hours after the onset of chronic DIC
symptoms. • Patients .75 years old (excluded from clinical trials)
• Thrombolytic therapy raises the risk of hemorrhage, • Patients with marginal fluid compartment (excluded
particularly intracranial hemorrhage. from clinical
trials)
Risks and Importance of Laboratory Monitoring o >150 kg: proportionally reduced fluid
• Antithrombotics - dangerous due to their narrow effective compartment
dosage ranges. o <40 kg or pediatric: proportionally increased
• Overdose - critical and inadequate dosages lead to fluid compartment
secondary, often fatal, thrombotic events.
• Laboratory monitoring or measurement of anticoagulant Factor Half-Life Plasma Level Hemostatic Level
therapy is essential due to these risks.

COUMADIN THERAPY AND THE PROTHROMBIN TIME

Coumadin Is a Vitamin K Antagonist
• Coagulation factors II, VII, IX, and X rely on Vitamin K for
normal production.
• Vitamin K:
o responsible for g-carboxylation of 12 to 18 glutamic
acids, enabling these factors and control proteins
to bind ionic calcium and cell membrane
phospholipids
o concentrated in green tea, avocados, and green
leafy vegetables and produced by gut flora

TRANSCRIBED BY: JOLAN HERCE

Monitoring Coumadin Therapy Using the Prothrombin Time • Coumadin therapy should start at 2 mg/day in patients
Assay with these polymorphisms, adjusted daily until therapeutic
• The PT monitors Coumadin therapy due to its sensitivity to INR remains within therapeutic range.
reductions of factors II, VII, and X. • Coumadin receptor insufficiency may render patients
PT reagent triggers the coagulation pathway at factor VII resistant to Coumadin therapy, requiring 20 mg/day or
level. higher doses.
• Anticoagulation becomes therapeutic when factors II and • Search for polymorphisms of the vitamin K reductase
X decrease to less than 50% of normal, which takes pathway responsible for "Coumadin resistance."
approximately 5 days.
• The first PT is performed 24 hours after therapy is initiated, Effect of Direct Thrombin Inhibitors on the Prothrombin Time
followed by daily monitoring until at least two • DTIs argatroban and bivalirudin used as life-saving
consecutive results are within the target therapeutic measures.
range. • Anti-factor Xa direct oral anticoagulants rivaroxaban and
• Monitoring continues every 4 to 12 weeks until therapy apixaban may prolong PT.
completion, which often lasts for 6 months following a • Switching to Coumadin therapy can nearly double PT,
thrombotic event. potentially extending 3-4 days.
• Close monitoring is essential for successful Coumadin • Chromogenic factor X assay: used for monitoring
therapy due to the narrow therapeutic range. Coumadin dosage during crossover period.

Reversing Bleeding Caused by a Coumadin Overdose

• Determines therapeutic range near 20%-40% of normal
PTpatient: is the PT of the patient in seconds factor X activity
PTnormal: is thegeometric mean of the PT reference intervaL Bleeding INR Intervention
in seconds
ISI: is the international sensitivity index applied as an
exponent

Coumadin International Normalized Ratio Therapeutic

Range
• Physician adjusts Coumadin dosage to achieve desired
INR of 2 to 3.
• INRs greater than 4 increase hemorrhage risk.
•Dosage adjustments are conservative due to INR
stabilization time of 4 to 7 days.
•Elevated INR accompanied by anatomic bleeding
symptoms is a medical emergency.

Monitoring Coumadin Therapy Using the Chromogenic

Factor X Assay UNFRACTIONATED HEPARIN THERAPY
• Determines therapeutic range near 20%-40% of normal AND THE PARTIAL THROMBOPLASTIN TIME
factor X activity. Heparin Is a Catalyst That Activates Antithrombin to
Neutralize Serine Proteases
Effect of Diet and Drugs on Coumadin Therapy • Extracted from porcine mucosa.
• Coumadin's effectiveness decreases with dietary vitamin • Molecular weight: 3000 to 30,000 Daltons.
K. • Anticoagulant action: indirect, catalytic, relies on plasma
• Patients advised to maintain balanced diet and avoid antithrombin.
supplements. • Heparin supports thrombin-antithrombin reaction through
• Coumadin metabolized in CYP 2C9 pathway. "bridging" mechanism.
• Changes in drug therapy need additional PT assays and • Preparations vary in molecular weight, molecule length,
dosage adjustments. and efficacy.
• Coumadin contraindicated during pregnancy due to birth • Individual patient dose responses diverge due to varying
defects. plasma and cellular protein binding.

Effect of Polymorphisms on Coumadin Therapy Unfractionated Heparin Therapy

• CYP2C9*2 and CYP2C9*3 polymorphisms in cytochrome P • Treats VTE, AMI, prevents reocclusion, maintains vascular
450 pathway reduce enzyme activity, slowing Coumadin patency.
metabolism. • Dosing regimens vary, starting with bolus and infusion.
• VKORC1 polymorphism affects vitamin K epoxide • Therapy discontinued post-acute clinical state or surgery.
reductase enzyme, slowing vitamin K reduction, increasing • LMWH or anticoagulants used to avoid thrombotic
Coumadin sensitivity. events.

TRANSCRIBED BY: JOLAN HERCE

Monitoring Unfractionated Heparin Therapy Using the Partial Monitoring Unfractionated Heparin Therapy Using the
Thromboplastin Time Activated Clotting Time
• 1966: modification of Lee-White whole blood clotting time
test.
• Used in clinics, inpatient bedsides, cardiac catheterization
labs, and surgical suites.
• Especially useful at high UHF dosages in percutaneous
intervention and cardiac surgery.
• Distributors like International Technidyne Corporation
provide evacuated blood specimen collection tubes.
• Results comparable to PTT assay for UFH monitoring.

Reversal of Unfractionated Heparin Overdose Using

Protamine Sulfate
• Protamine sulfate: a cationic protein from salmon sperm,
• Uses PTT for monitoring due to pharmacologic variations neutralizes UFH at a 100 units of heparin per milligram.
and narrow therapeutic range. • It is administered slowly by intravenous push, affecting the
• Blood collected and assayed before therapy to ensure PTT or ACT.
normal baseline PTT. • It also neutralizes LMWH, though its effect is incompletely
• If prolonged baseline PTT detected, switch to reflected in the chromogenic anti-factor Xa heparin assay.
chromogenic anti-factor Xa heparin assay. • Protamine sulfate has been linked to a delayed form of
• Second specimen collected and PTT measured within Heparin Intensity Test (HIT), hence routine monitoring of
minutes of UFH administration. platelet counts.
• Adjustment of infusion rate and daily monitoring of platelet
count. LOW-MOLECULAR-WEIGHT HEPARIN THERAPY AND THE
• If HIT suspected, UFH therapy discontinued and replaced CHROMOGENIC ANTI–FACTOR Xa HEPARIN ASSAY
with DTI therapy. Low-Molecular-Weight Heparin Is Produced from
Unfractionated Heparin
Determining the Partial Thromboplastin Time Therapeutic • LMWH reduces risk of thromboembolic events (HIT) due to
Range for Unfractionated Heparin Therapy uncertainty about UFH dose response.
• Collect 20-30 plasma specimens from patients not • Approved for anticoagulant prophylaxis in the US and
receiving Coumadin therapy. Canada in 1993.
• Perform Chromogenic anti-factor Xa heparin assays on all • Prepared from UFH using chemical or enzymatic
specimens. fractionation.
• Determine therapeutic range using ex vivo or Brill-Edwards • Administered by subcutaneous injection, providing
method. coverage during or after surgery and trauma.
• Avoid approaches like 1.5-2.5 times the mean of the • Treats DVT, PE, and unstable angina.
reference interval due to risk of under-anticoagulation and • Offers rapid bioavailability, 3 to 5 hour half-life, and fixed
flattening curves. dose response.
• Contraindicated in patients developing HIT after UFH
Clinical Utility of Monitoring Unfractionated Heparin Therapy therapy.the conversion of fibrinogen to fibrin polymer
Using the Partial Thromboplastin Time
• Reports PTT results, reference interval, and UFH therapeutic
range to clinician.
• Evaluates results against institution's current therapeutic
range and reference interval.
• No system for normalizing PTT results.

Limitations of Monitoring Unfractionated Heparin Therapy

Using the Partial Thromboplastin Time
• Patient unresponsive to heparin therapy due to
inflammation, fibrinogen levels, and antithrombin depletion.
• PTT remains below therapeutic range despite increased
heparin dosages.
• Inflammation reduced with steroids, aspirin, or anti
inflammatory drugs.
• Platelets release platelet factor 4, shortening PTT.
• Hypofibrinogenemia, factor deficiencies prolong PTT.

TRANSCRIBED BY: JOLAN HERCE

Measuring Low-Molecular-Weight Heparin Therapy Dabigatran, an Oral Direct Thrombin Inhibitor
• Kidneys clear LMWH in renal insufficiency. • Reversible DTI binds thrombin.
• Laboratory measurement: crucial for creatinine clearance • Similar efficacy and safety to LMWH and Coumadin.
and plasma levels. • No known food interaction.
• Chromogenic anti-factor Xa heparin assay: measures • Kidney-cleared.
LMWH and fondaparinux. • Cleared for VTE prophylaxis and ischemic stroke
• Precise, insensitive to heparin concentration, can replace prevention.
PTT. • May prolong half-life in renal disease.
• Used for monitoring all UFH therapy and LMWH and • No reversal agent in overdose-caused hemorrhage.
fondaparinux therapy levels.
Measuring Direct Thrombin Inhibitor Therapy
MEASURING PENTASACCHARIDE THERAPY USING THE • Argatroban and bivalirudin: prolong thrombin time, PT, PTT,
CHROMOGENIC ANTI–FACTOR Xa HEPARIN ASSAY and ACT.
• Blood collected 2 hours post-therapy initiation for
argatroban and 4 hours for bivalirudin.
• Dosage adjusted to achieve therapeutic PTT.
• ACT: used during cardiac catheterization or surgery.
• Ecarin clotting time (ECT): used for assaying argatroban
and bivalirudin.
• Dabigatran: an oral thrombin inhibitor, also prolongs
thrombin time, PTT, and ECT.
• Increases antithrombin activity 400-fold.
• ECT, ECA, plasma-diluted thrombin time, and Biophen DTI
• FDA-cleared for VTE prevention and treatment.
require FDA clearance.
• Contraindicated in patients with creatinine clearance
below 30 mL/min.
MEASURING ANTIPLATELET THERAPY USING PLATELET
• Used in subcutaneous injections.
ACTIVITY ASSAYS
Intravenous Glycoprotein IIb/IIIa Inhibitors Are Used During
MEASURING ORAL DIRECT FACTOR Xa INHIBITORS
Cardiac Catheterization
• Rivaroxaban: an oxazolidinone derivative that directly
inhibits factor Xa.
• Equivalent to LMWH or Coumadin in efficacy and safety.
• Cleared for VTE prophylaxis in total knee or hip
replacement surgery.
• Temporarily prolongs thromboplastin time, but to a lesser
extent than Coumadin.
• Improves efficacy and safety of Coumadin.
• All oral direct anti-factor Xa inhibitors have approximately
12 hour half-lives.

DIRECT THROMBIN INHIBITORS

Argatroban

• Glycoprotein IIb and IIIa form heterodimers on resting

platelets, bind fibrinogen and von Willebrand factor.
• Intravenous GPIs
o Abciximab
o Eptifibatide
o tirofiban
prevent platelet aggregation.
• GPIs: used to maintain vascular patency during cardiac
catheterization and stent placement.
• Patients' PT, PTT, ACT, hemoglobin, hematocrit, and
• Argatroban and bivalirudin bind and inactivate thrombin. platelet count are checked before GPI infusion.
• Without DTIs, 50% thrombosis risk for 30 days. • Administered with aspirin and UFH, dosage halved for
• FDA-cleared for thrombosis prophylaxis and treatment. creatinine clearance less than 30 mL/min.

TRANSCRIBED BY: JOLAN HERCE

Aspirin, Clopidogrel, Prasugrel, and Ticagrelor Reduce the FUTURE OF ANTITHROMBOTIC THERAPY
Incidence of Arterial Thrombosis • New oral anticoagulants and antiplatelet drugs replace
• Aspirin: Reversely acetylates platelet enzyme o Coumadin
cyclooxygenase, preventing platelet-activating o Heparins
thromboxane A2 production. o fondaparinux
• Clopidogrel: Prescribed at 75 mg/day with aspirin at 81 or • Clinical laboratories adapt with chromogenic anti-factor
325 mg/day. Xa, thrombin time, ecarin clotting time, and molecular
• Prasugrel: Administered as an oral prodrug with aspirin at assays.
81 mg or 325 mg daily.
• Ticagrelor: Provided in 90-mg tablets, whose main active
metabolite forms rapidly via the CYP3A4 liver enzyme.

Variable Aspirin and Clopidogrel Response and Laboratory

Measuring of Antiplatelet Resistance
• Aspirin resistance: condition where 10%-20% of people
take aspirin, causing an inadequate response.
• This resistance is also observed in clopidogrel, with
significant variation among patients.
• Current research is focused on the mechanisms underlying
aspirin resistance and clopidogrel response variation.

VerifyNow:
• Designed for point-of-care testing.
• Uses light transmittance aggregometry to test platelet
aggregation responses to various drugs.
• Each assay includes a cartridge with desired agonist and
fibrinogencoated beads.
• Aspirin uses arachidonic acid as agonist.
• VerifyNow P2Y12 uses ADP.
• VerifyNow IIb/IIIa uses TRAP.
• Laboratory sets reference interval and therapeutic target
limits.
• Results outside target range indicate treatment failure.
• All systems are FDA cleared.

Multiplate:
• Automated point-of-care tool for platelet aggregation.
• Tests responses to aspirin, thienopyridines, GPIs.
• Requires 300 mL of whole blood.
• Results outside target range indicate treatment failure.

Plateletworks:
• Measures platelet aggregation in whole blood.
• ADP or collagen agonists affect aggregation.
• Antiplatelet drug therapy reduces aggregation response
by 40%-60%.

PFA-100 (Siemens Medical Solutions USA, Inc., Malvern,

PA):
• Uses two cartridges.
• Operator pipettes 800mL whole blood per cartridge.
• Shorter closure time indicates aspirin resistance.

AspirinWorks (Corgenix Medical Corporation, Broomfield,

CO):
• Measures Urine 11-dehydrothromboxane B2
• Elevated in patients with atherosclerosis, stroke, ischemic
attacks, atrial fibrillation.
• Aspirin therapy decreases levels, even in atherosclerosis,
myocardial infarction, and atrial fibrillation.

TRANSCRIBED BY: JOLAN HERCE