Rodak's Hematology: Clinical Principles and Applications, 5th Ed.

CHAPTER 43. Antithrombotic therapies and their laboratory assessment

George A. Fritsma

OUTLINE

Coumadin Therapy and the Prothrombin Time

Coumadin Is a Vitamin K Antagonist

Coumadin Prophylaxis and Therapy

Monitoring Coumadin Therapy Using the Prothrombin Time Assay

Monitoring Coumadin Therapy Using the Chromogenic Factor X Assay

Effect of Diet and Drugs on Coumadin Therapy

Effect of Polymorphisms on Coumadin Therapy

Effect of Direct Thrombin Inhibitors on the Prothrombin Time

Reversing Bleeding Caused by a Coumadin Overdose

Unfractionated Heparin Therapy and the Partial Thromboplastin Time

Heparin Is a Catalyst That Activates Antithrombin to Neutralize Serine Proteases

Unfractionated Heparin Therapy

Monitoring Unfractionated Heparin Therapy Using the Partial Thromboplastin Time

Determining the Partial Thromboplastin Time Therapeutic Range for Unfractionated Heparin Therapy

Clinical Utility of Monitoring Unfractionated Heparin Therapy Using the Partial Thromboplastin Time

Limitations of Monitoring Unfractionated Heparin Therapy Using the Partial Thromboplastin Time

Monitoring Unfractionated Heparin Therapy Using the Activated Clotting Time

Reversal of Unfractionated Heparin Overdose Using Protamine Sulfate

Low-Molecular-Weight Heparin Therapy and the Chromogenic Anti-Factor Xa Heparin Assay

Low-Molecular-Weight HeparinIs Produced from Unfractionated Heparin

Measuring Low-Molecular-Weight Heparin Therapy

Measuring Pentasaccharide Therapy Using the Chromogenic Anti-Factor Xa Heparin Assay

Measuring Oral Direct Factor Xa Inhibitors

Direct Thrombin Inhibitors

Argatroban

Bivalirudin, a Recombinant Analogue of Leech Saliva Hirudin

Dabigatran, an Oral Direct Thrombin Inhibitor

Measuring Direct Thrombin Inhibitor Therapy

Measuring Antiplatelet Therapy Using Platelet Activity Assays

Intravenous Glycoprotein IIb/IIIa Inhibitors Are Used During Cardiac Catheterization

Aspirin, Clopidogrel, Prasugrel, and Ticagrelor Reduce the Incidence of Arterial Thrombosis

Variable Aspirin and Clopidogrel Response and Laboratory Measuring of Antiplatelet Resistance

Future of Antithrombotic Therapy

Objectives

After completion of this chapter, the reader will be able to:

1. Describe the purpose of antithrombotic drug administration and distinguish between anticoagulants and antiplatelet therapy.

2. Describe the indications for, dosage of, and management of Coumadin therapy, including how to detect and manage Coumadin overdose.

3. Monitor Coumadin therapy using the prothrombin time and international normalized ratio, and compare these tests with the chromogenic factor X assay.

4. Perform and interpret prothrombin times with international normalized ratios using point-of-care instruments.

5. Describe the indications for, dosage of, and laboratory monitoring of unfractionated heparin therapy, including how to establish the unfractionated heparin partial thromboplastin time therapeutic range.

6. Perform and interpret the results of the partial thromboplastin time and activated clotting time assays for monitoring unfractionated heparin therapy, and compare these tests with the chromogenic anti-factor Xa heparin assay.

7. Describe the indications, dosage, and laboratory measurement of low-molecular-weight heparin therapy and fondaparinux therapy.

8. Perform and interpret the results of the chromogenic anti-factor Xa assay for measuring unfractionated heparin, low-molecular-weight heparin, fondaparinux, rivaroxaban, apixaban, and edoxaban therapy.

9. Measure direct thrombin inhibitor therapy, including dabigatran, using the partial thromboplastin time, ecarin clotting time, ecarin chromogenic assay, and plasma-diluted thrombin time.

10. Describe the indications for intravenous platelet glycoprotein IIb/IIIa inhibitors abciximab, eptifibatide, and tirofiban and describe how their effects are measured.

11. Describe the indications for oral platelet inhibitors aspirin, clopidogrel, prasugrel, and ticagrelor, and describe how their effects are measured.

CASE STUDY

After studying the material in this chapter, the reader should be able to respond to the following case study:

A 71-year-old woman with atrial fibrillation has been taking 5 mg of Coumadin (warfarin sodium) per day for 8 years. Her monthly prothrombin time and international normalized ratio (PT/INR) has been maintained consistently within the standard therapeutic range, 2 to 3. Last Monday, however, her INR was 6.5. For several days earlier she had noticed that her gums bled after she brushed her teeth.

1. What could cause this change in the PT/INR result?

2. What should be done about her PT/INR and symptoms?

3. What alternative test may be used to monitor Coumadin therapy?

T hrombosis, described in detail in the introduction to Chapter 39, is the pathological formation of blood clots in veins or arteries that obstruct flow and cause tissue ischemia and necrosis. Antithrombotic drugs have been employed to treat and prevent thrombosis since heparin was first developed in 1916 and then was FDA-cleared in 1936.1 Antithrombotics include anticoagulants, which suppress coagulation and reduce thrombin formation, and antiplatelet drugs, which suppress platelet activation. Fibrinolytics are also employed to disperse or reduce existing clots clogging veins and arteries. Table 43-1 provides a list of current antithrombotics with their indications.

TABLE 43-1

Current Antithrombotics, Mode of Action, Measurement, Reversal

Antithrombotic

Indication

Mode of Action

Half-Life

Measurement

Reversal

FDA Cleared

Coumadin

Prevent post-VTE rethrombosis, ischemic stroke

Oral VK antagonist

5 d

Monitor: PT/INR; CFX

Vitamin K, PCC, rFVIIa

1954

UFH

Prevent post-VTE and ACS rethrombosis; intraoperative anticoagulation

IV AT activation, anti-IIa & anti-Xa

1–2 h

Monitor: PTT, anti-Xa, ACT

PS

1936

LMWH

Prevent thrombosis after surgery, in medical conditions or in ACS; DVT/PE treatment

SC AT activation, anti-Xa

3–5 h

Anti-Xa

PS (partial)

1993

Fondaparinux

12–17 h

Anti-Xa

None

2001

Rivaroxaban

Prevent ischemic stroke, prevent thrombosis after orthopedic surgery, prevent post-VTE rethrombosis

Oral direct anti-Xa

12–15 h

PT?, anti-Xa

4-factor PCC, FEIBA, rFVIIa

2011

Apixaban

Prevent ischemic stroke

12–15 h

2012

Edoxaban

 

12 h

Clinical trials in progress

 

Argatroban

Anticoagulation in HIT

IV DTI

50 min

PTT

Discontinue

1997

Bivalirudin

IV DTI

25 min

Discontinue

2000

Dabigatran

Prevent ischemic stroke

Oral DTI

17 h

PTT, ECT, ECA, DTT

None

2010

Aspirin

Prevent acute coronary syndrome recurrence

Oral antiplatelet COX inhibitor

 

VerifyNow Aspirin, AspirinWorks, platelet aggregation

Discontinue

1900

Clopidogrel

Oral, binds platelet P2Y12

 

VerifyNow P2Y

Discontinue

2000

Prasugrel

Oral, binds platelet P2Y12

 

VerifyNow P2Y

Discontinue

2009

Ticagrelor

Oral, binds platelet P2Y12

 

VerifyNow P2Y

Discontinue

2011

Eptifibatide

Maintain vascular patency during PCI and medical therapy for acute coronary syndrome

IV, binds platelet GP IIb/IIIa

2.5 h

VerifyNow GPI

Discontinue

1998

Abciximab

GP IIb/IIIa

12–24 h

VerifyNow GPI

Discontinue

1993

Tirofiban

2.5 h

 

1998

ACS, acute coronary syndrome; ACT, activated clotting time; AT, antithrombin; CFX, chromogenic factor X activity; COX, cyclooxygenase; DTI, direct thrombin inhibitor; DTT, plasma-diluted thrombin time; ECA, ecarin chromogenic assay; ECT, ecarin clotting time; FEIBA, factor VIII inhibitor bypassing activity; GPI, glycoprotein inhibitor; INR, international normalized ratio; IV, intravenous; PCC, prothrombin complex concentrate; PT, prothrombin time; PTT, partial thromboplastin time; PS, protamine sulfate; rFVIIa, recombinant activated factor VII; SC,subcutaneous; VK, Vitamin K.

Venous thromboembolic disease (VTE, venous thromboembolism; Chapter 39) includes superficial and deep vein thrombosis (DVT) and pulmonary embolism (PE). VTE is treated using intravenous standard unfractionated heparin (UFH), subcutaneous low-molecular-weight heparin (LMWH, enoxaparin, tinzaparin), subcutaneous synthetic pentasaccharide (fondaparinux), or the oral direct factor Xa inhibitor, rivaroxaban. VTE is also treated using the oral vitamin K antagonist Coumadin (warfarin sodium). These anticoagulants are also used to prevent VTE subsequent to total hip and total knee replacement surgery, orthopedic repair surgery, and in several medical conditions.

The direct thrombin inhibitors (DTIs) argatroban and bivalirudin are intravenous anticoagulants that are substituted for UFH in patients who have developed heparin-induced thrombocytopenia with thrombosis (HIT), a devastating arterial and venous thrombotic side effect of UFH therapy (Chapters 39 and 40). Dabigatran is an oral DTI cleared in 2010 to prevent ischemic stroke, a common side effect for patients who suffer nonvalvular atrial fibrillation. The direct anti-factor Xa anticoagulants rivaroxaban and apixaban are also available to prevent ischemic stroke in atrial fibrillation.

Arterial thrombosis includes acute myocardial infarction (AMI), ischemic cerebrovascular accident (CVA, stroke), transient ischemic attack (TIA), and peripheral arterial occlusion (PAO or peripheral artery disease, PAD) and is managed with UFH, LMWH, fondaparinux, Coumadin, the intravenous DTIs, and the antiplatelet drugs aspirin, clopidogrel, prasugrel, and ticagrelor. Aspirin is taken prophylactically by many healthy people at less than 100 mg/day and is particularly effective in reducing mortality when taken within minutes of the acute onset of stroke or cardiac symptoms.2 The intravenous platelet glycoprotein IIb/IIIa inhibitor (GPI) drugs eptifibatide, abciximab, and tirofiban are used to prevent thrombosis during cardiac catheterization procedures.

Thrombolytic therapy may be used to resolve DVT, PE, PAO, AMI, and stroke, particularly when used 3 to 4 hours after the onset of symptoms. Thrombolytic therapy employs recombinant forms of tissue plasminogen activator (reteplase, alteplase, and tenecteplase). Thrombolytic therapy raises the risk of hemorrhage, particularly intracranial hemorrhage. Because thrombolytic therapy is not measured by laboratory tests (although fibrinogen levels can be checked), it is not further discussed in this chapter.

Many lives have been saved through the judicious use of antithrombotic therapy, and countless more healthy individuals have been spared thrombotic disease through long-term antithrombotic prophylaxis in moderate-risk circumstances and conditions. However, antithrombotics are dangerous because their effective dosage ranges are narrow.3 Overdose is critical and leads to emergency department visits for uncontrolled bleeding; inadequate dosages lead to secondary (repeat), often fatal, thrombotic events. Dosages and half-lives differ among the antithrombotics because of variations in formulation and metabolism.4,5

Because of these risks, laboratory monitoring or measurement of anticoagulant therapy is essential. Coagulation laboratory scientists and technicians perform countless prothrombin time (PT) assays, partial thromboplastin time (PTT, synonymous with activated partial thromboplastin time, APTT) assays, and chromogenic anti-factor Xa heparin assays to measure or monitor anticoagulant therapy; meanwhile, physicians and nurses regularly modify Coumadin and UFH dosages in response to laboratory outcomes. Although anticoagulant therapy measurement or monitoring may seem routine, vigilance is essential to provide consistently valid results in a dangerous therapeutic world.6

The antiplatelet drugs aspirin, clopidogrel, prasugrel, and ticagrelor, as well as LMWH, fondaparinux, and the direct oral anticoagulants (dabigatran, rivaroxaban, apixaban, and edoxaban), have fixed dose-response characteristics and do not require regular monitoring or laboratory-directed dose adjustment. However, though routine monitoring and dose adjustment may be unnecessary, these drugs require measurement in the conditions listed in . Box 43-1

BOX 43-1

Clinical Conditions That Require Measurement of Antiplatelet Drugs and Anticoagulants Besides Coumadin and UFH

• Renal disease: inadequate excretion, CrCl < 30 mL/min

• Detection of noncompliance and underdosing

• Detection of comedication interference

• Acute hemorrhage (usually in emergency department or surgery)

• Overdose, effects of comedication

• Detection and identification; what anticoagulant is it?

• Determine if reversal is working

• Bridging from one anticoagulant to another or discontinuing anticoagulant before surgery

• Resuming anticoagulant after surgery

• Unstable coagulation: pregnancy, liver disease, malignancy, chronic DIC

• Patients > 75 years old (excluded from clinical trials)

• Patients with marginal fluid compartment (excluded from clinical trials)

• > 150 kg: proportionally reduced fluid compartment

• < 40 kg or pediatric: proportionally increased fluid compartment

Coumadin therapy and the prothrombin time

Coumadin is a vitamin K antagonist

As detailed in Chapter 37, the coagulation factors II (prothrombin), VII, IX, and X depend on vitamin K for normal production, as do coagulation control proteins C, S, and Z. Vitamin K is responsible for the γ-carboxylation of a linear series of 12 to 18 glutamic acids near each molecule’s N-terminus (amino terminus), a posttranslational modification that enables these coagulation factors and coagulation control proteins to bind ionic calcium (Ca2+) and cell membrane phospholipids, especially phosphatidylserine (Figure 37-9). Vitamin K is concentrated in green tea, avocados, and green leafy vegetables and is produced by gut flora; its absence results in the production of nonfunctional des-γ carboxyl forms of factors II, VII, IX, and X and proteins C, S, and Z.

Coumadin (4-hydroxycoumarin, warfarin sodium) is a member of the coumarin drug family and is the formulation of coumarin most often used in North America.7 Another coumarin is dicoumarol (3,3′-methylenebis-[4-hydroxycoumarin]), the original anticoagulant extracted from moldy sweet clover, described in 1940, and used for many years as a rodenticide.8 Coumadin is a vitamin K antagonist that suppresses γ-carboxylation of glutamic acid by slowing the activity of the enzyme vitamin K epoxide reductase (Figure 37-9). During Coumadin therapy, the activities of factors II, VII, IX, and X and proteins C, S, and Z become reduced as the nonfunctional des-carboxyl proteins are produced in their place. These are sometimes called proteins induced by vitamin K antagonists (PIVKAs); they bind few calcium ions, do not assemble on phospholipid surfaces with their substrates, and therefore do not participate in coagulation. Despite several developmental efforts, until 2009, Coumadin was the only oral anticoagulant in the United States, so Coumadin therapy has often been called oral anticoagulant therapy (OAT). The new direct-acting oral anticoagulants rivaroxaban, apixaban, edoxaban, and dabigatran have broadened the meaning of “OAT.”

Coumadin prophylaxis and therapy

Physicians prescribe Coumadin prophylactically to prevent TIAs and strokes in patients with nonvalvular atrial fibrillation and to prevent VTE after trauma, orthopedic surgery, and general surgery, and in a number of chronic medical conditions. They also prescribe Coumadin therapeutically to prevent DVT or PE recurrence. Coumadin is also used therapeutically after AMI if the event is complicated by congestive heart failure or coronary insufficiency and to control clotting in patients with mechanical heart valves. Coumadin is among the 20 most commonly prescribed drugs in North America.

Whether prescribed prophylactically or therapeutically, the standard Coumadin regimen begins with a 5-mg daily oral dose. The starting dosage for people over 70 and people who are debilitated, malnourished, or have congestive heart failure is 2 mg/day. For people simultaneously taking drugs that are known to raise Coumadin sensitivity, the starting dosage is 2 mg/day, and 2 mg/day is also the dosage used for those with inherited Coumadin sensitivity. There is no loading dose, and subsequent dosing is based on patient response as measured by the PT (next section). The activity of each of the vitamin K–dependent coagulation factors begins to decline immediately but at different rates (), and it takes about 5 days for all the factors to reach therapeutic levels. Figure 43-1Table 43-2 lists the plasma half-life, plasma concentration, and minimum effective plasma percentage of normal factor activity for the coagulation factors.

Image 

FIGURE 43-1 Factor VII activity decreases to 50% of normal 6 hours after Coumadin therapy is begun, prolonging the factor VIIa–sensitive prothrombin time to near the therapeutic INR of 2 to 3. The half-lives of factors II (prothrombin), IX, and X are longer than that of VII; factor II activity requires at least 3 days to decline by 50%. The patient gains full anticoagulation effects approximately 5 days after the start of Coumadin therapy.

TABLE 43-2

Plasma Half-Life, Normal Plasma Level, and Minimum Effective Hemostatic Level as a Percentage of Normal Level for the Coagulation Factors

Factor

Half-Life

Plasma Level

Hemostatic Level

Fibrinogen

4 days

280 mg/dL

50 mg/dL

Prothrombin

60 hr

1300 μg/mL

20%

V

16 hr

680 μg/mL

25%

VII

6 hr

120 μg/mL

20%

VIII

12 hr

0.24 μg/mL

30%

IX

24 hr

5 μg/mL

30%

X

30 hr

1 mg/dL

25%

XI

2–3 days

6 μg/mL

25%

XIII

7–10 days

290 μg/mL

2%–3%

Von Willebrand factor

30 hr

6 μg/mL

50%

Control protein activities also become reduced, especially the activity of protein C, which has a 6-hour half-life, so for the first 2 or 3 days of Coumadin therapy the patient actually incurs the risk of thrombosis. For this reason, Coumadin therapy is “covered” by UFH, LMWH, or fondaparinux therapy for at least 5 days. Failure to provide anticoagulant therapy during this period may result in warfarin skin necrosis, a severe thrombotic reaction requiring débridement of dead tissue.9

Monitoring coumadin therapy using the prothrombin time assay

The PT effectively monitors Coumadin therapy because it is sensitive to reductions of factors II, VII, and X () (Figure 43-2Chapter 42). The PT reagent consists of tissue factor, phospholipid, and ionic calcium, so it triggers the coagulation pathway at the level of factor VII. Owing to the 6-hour half-life of factor VII, the PT begins to prolong within 6 to 8 hours; however, anticoagulation becomes therapeutic only when the activities of factors II and X decrease to less than 50% of normal, which takes approximately 5 days.

Image 

FIGURE 43-2 The prothrombin time (PT) reagent activates the extrinsic coagulation pathway beginning with factor VII. The PT is prolonged by deficiencies of factors VII, X, V, II (prothrombin), and fibrinogen when the fibrinogen concentration is less than 100 mg/dL. The PT is prolonged in Coumadin therapy because it responds to the reduced VII, X, and prothrombin activity. The partial thromboplastin time (PTT) reagent activates the intrinsic coagulation pathway through factor XII, in association with prekallikrein (pre-K) and high-molecular-weight kininogen (HMWK). The PTT is prolonged by deficiencies of pre-K; HMWK; factors XII, XI, IX, VIII, and X; prothrombin; and fibrinogen when the fibrinogen concentration is less than 100 mg/dL. The PTT is prolonged in unfractionated heparin (UFH) therapy because UFH activates the plasma control protein antithrombin, which neutralizes the serine proteases XIIa, XIa, Xa, IXa, and IIa (thrombin [Thr]). The PTT is also prolonged by lupus anticoagulant. TF,Tissue factor.

The first PT is collected and performed 24 hours after therapy is initiated; subsequent PTs are performed daily until at least two consecutive results are within the target therapeutic range. Monitoring continues every 4 to 12 weeks until the completion of therapy, which often lasts for 6 months following a thrombotic event.10 Coumadin therapy for stroke prevention in atrial fibrillation is indefinite, possibly lifelong. Because the therapeutic range is narrow, close monitoring is essential for successful Coumadin therapy. Under-anticoagulation signals the danger of thrombosis or secondary thrombosis (rethrombosis); overdose carries the danger of hemorrhage.

Reporting prothrombin time results and the international normalized ratio

The medical laboratory technician or scientist reports PT results to the nearest tenth of a second and provides the PT reference interval in seconds for comparison. In view of the inherent variations among thromboplastin reagents and to accomplish interlaboratory normalization, all laboratories report the international normalized ratio (INR) for patients who have reached a stable response to Coumadin therapy. Laboratory practitioners use the following formula:11

INR = (PTpatient/PTnormal)ISI

Image

where PTpatient is the PT of the patient in seconds, PTnormal is the geometric mean of the PT reference interval in seconds, and ISI is the international sensitivity index applied as an exponent.

Thromboplastin producers generate the ISI by performing an orthogonal regression analysis comparing the results of their PT reagents for 50 or more Coumadin plasma specimens and 10 or more normal specimens with the results of the international reference thromboplastin (World Health Organization human brain thromboplastin) on the same plasmas.12 Most manufacturers provide ISIs for a variety of coagulation instruments, because each coagulometer may respond differently to their thromboplastins; for instance, some coagulometers rely on photometric plasma changes, whereas others use an electromechanical system (Chapter 44). Most thromboplastin reagents have ISIs near 1.0, matching the ISI of the World Health Organization’s international reference thromboplastin. Automated coagulometers “request” the reagent ISI from the operator or obtain it electronically from a reagent vial label bar code, and compute the INR for each assay result. Although INRs are meant to be computed only for patients in whom the response to Coumadin has stabilized, they typically are reported for all patients, even those who are not taking Coumadin. During the first 5 days of Coumadin therapy, the astute physician and medical laboratory practitioner ignore the INR as unreliable and interpret the PT results in seconds, comparing it with the reference interval.

Coumadin international normalized ratio therapeutic range

The physician adjusts the Coumadin dosage to achieve the desired INR of 2 to 3, or 2.5 to 3.5 if the patient has a mechanical heart valve. INRs greater than 4 are associated with increased risk of hemorrhage and require immediate communication with the clinician who is managing the patient’s case.13 Dosage adjustments are made conservatively because the INR requires 4 to 7 days to stabilize, but an elevated INR accompanied by the symptoms of anatomic bleeding is a medical emergency.

Monitoring coumadin therapy using the chromogenic factor X assay

The chromogenic coagulation factor X assay (not to be confused with the chromogenic anti-factor Xa heparin assay) may be used as an alternative to the PT/INR system, eliminating the necessity for normalization.14 The therapeutic range is determined locally by comparison to the INR and typically is close to 20% to 40% of normal factor X activity.15 The chromogenic factor X assay is useful when the PT is compromised by lupus anticoagulant, a factor inhibitor, or a coagulation factor deficiency.16

Effect of diet and drugs on coumadin therapy

Dietary vitamin K decreases Coumadin’s effectiveness and reduces the INR. Green vegetables are an important source of vitamin K, but vitamin K also is concentrated in green tea, cauliflower, liver, avocados, parenteral nutrition formulations, multivitamins, red wine, over-the-counter nutrition drinks, and over-the-counter dietary supplements. A patient who is taking Coumadin is counseled to maintain a regular balanced diet, avoid supplements, and to follow up dietary changes or dietary supplement changes with additional PT assays and dosage adjustments, if indicated.

Coumadin is metabolized in the mitochondrial cytochrome P-450 (CYP 2C9) pathway of hepatocytes—the “disposal system” for at least 80 drugs. Theoretically, use of any drug metabolized through the CYP 2C9 pathway may unpredictably suppress or enhance the effects of Coumadin. Amiodarone, metronidazole, and cimetidine typically double or triple the INR. Any change in drug therapy, like a change in diet, must be followed up with additional PT assays and dosage adjustments.

Coumadin is contraindicated during pregnancy because it causes birth defects. When anticoagulation is desired during pregnancy—for instance, in women who possess a thrombosis risk factor—LMWH or fondaparinux is prescribed. There are no current recommendations for the direct oral anticoagulants during pregnancy.

Effect of polymorphisms on coumadin therapy

Two genetic polymorphisms generate variations in enzymes of the cytochrome P-450 pathway. These are CYP2C9*2 and CYP2C9*3, which reduce enzyme pathway activity and slow the metabolic breakdown of Coumadin. Likewise, there is a polymorphism that affects the key enzyme of vitamin K metabolism, vitamin K epoxide reductase. This polymorphism, named VKORC1, slows vitamin K reduction, which makes the patient more sensitive to Coumadin.17 In patients possessing one, two, or all three of these polymorphisms, Coumadin therapy should begin at 2 mg/day and should be adjusted and monitored daily until the INR remains consistently in the therapeutic range. The standard 5 mg/day regimen risks hemorrhage in patients who possess these polymorphisms. In 2007, the FDA required that drug manufacturers add a statement on all vials of Coumadin recommending that physicians screen patients for these common dosage-affecting polymorphisms. Although the FDA recommendation does not carry the weight of a black box warning issued by the FDA for drug use, numerous molecular diagnostics manufacturers have developed short turnaround assays for these three polymorphisms. Screening for these polymorphisms is the first and most public example of pharmacogenomic laboratory testing, although not universally endorsed.18

Conversely, Coumadin receptor insufficiency may render the patient resistant to Coumadin therapy. Some patients require dosages of 20 mg/day or higher to achieve a therapeutic INR. The search is on for polymorphisms of the vitamin K reductase pathway responsible for “Coumadin resistance.”19

Effect of direct thrombin inhibitors on the prothrombin time

The intravenously administered DTIs argatroban and bivalirudin, which are used in place of heparin as a life-saving measure for patients with HIT, and the anti-factor Xa direct oral anticoagulants rivaroxaban and apixaban, may prolong the PT (depending on the PT reagent). In switching to Coumadin therapy, the combination of a DTI or direct oral anticoagulant and Coumadin can nearly double the PT for the duration of action of the DTI or direct oral anticoagulant, which may extend 3 or 4 days.20 The chromogenic factor X assay is an effective means for monitoring Coumadin dosage during the crossover period.

Reversing bleeding caused by a coumadin overdose

provides recommendations for the reversal of a Coumadin overdose based on INR and clinical evidence of bleeding. Reversal requires oral or intravenous vitamin K and, if bleeding is severe, a means for substituting active coagulation factors such as fresh-frozen plasma, recombinant activated factor VII (NovoSeven, Novo Nordisk, Princeton, NJ), activated three-factor prothrombin complex concentrate (FEIBA FH, Baxter Healthcare Corporation, Westlake Village, CA), three-factor prothrombin complex concentrate (“non-activated” Profilnine SD; Grifols Biologicals, Inc., Los Angeles, CA), or four-factor prothrombin complex concentrate (4F-PCC, Kcentra; CSL Behring, King of Prussia, PA).Table 43-321

TABLE 43-3

Recommendations for the Reversal of Coumadin Overdose Based on International Normalized Ratio (INR) and Bleeding

Bleeding

INR

Intervention

No significant bleeding

3–5

Reduce dosage or omit one dose, monitor INR frequently

 

5–9

Omit Coumadin, monitor INR frequently, consider oral vitamin K (≤5 mg) if high risk for bleeding (surgery)

 

> 9

Stop Coumadin, give 5–10 mg oral vitamin K, monitor INR frequently

Serious bleeding

Any INR

Stop Coumadin; give 10 mg vitamin K by intravenous push, may repeat every 12 hr; give thawed fresh-frozen plasma, prothrombin complex concentrate, or recombinant factor VIIa

Life-threatening bleeding

Any INR

Same as for serious bleeding, except stronger indication for recombinant factor VIIa

Unfractionated heparin therapy and the partial thromboplastin time

Heparin is a catalyst that activates antithrombin to neutralize serine proteases

Standard UFH is a biological substance, first described in 1916. It is a mixture of sulfated glycosaminoglycans (polysaccharides) extracted from porcine mucosa. The molecular weight of UFH ranges from 3000 to 30,000 Daltons (average molecular weight 15,000 Daltons). Approximately one third of its molecules support somewhere on their length a high-affinity pentasaccharide that binds plasma antithrombin. The anticoagulant action of UFH is indirect and catalytic, relying on antithrombin. The pentasaccharide-bound antithrombin undergoes a steric change (allostery), exposing an anticoagulant site that covalently binds and inactivates the coagulation pathway serine proteases, factors IIa (thrombin), IXa, Xa, XIa, and XIIa (Chapter 37). Laboratory practitioners call activated antithrombin a serine protease inhibitor (SERPIN), and the protease-binding reaction yields, among other products, the measurable inactive plasma complex thrombin-antithrombin (TAT).

Heparin supports the thrombin-antithrombin reaction through a “bridging” mechanism (). If the heparin molecule exceeds 17 linear saccharide units, thrombin assembles on the heparin molecule near the activated antithrombin. Bridging drives the thrombin-antithrombin reaction at a rate four times that of the factor Xa-antithrombin reaction, because factor Xa becomes inactivated only through antithrombin’s steric modification, and its covalent binding is not enhanced by bridging. Figure 43-3

Image 

FIGURE 43-3 The heparin binding site of antithrombin (AT) binds a specific pentasaccharide, producing an allosteric change that activates AT. Factor IIa (thrombin) assembles on the heparin surface, provided the molecule is at least 17 saccharide units long. AT binds factor IIa, and the complex is released from unfractionated heparin (UFH) to form the soluble, measurable thrombin-antithrombin (TAT) complex. The UFH recycles. Fibrin-bound IIa does not enter the reaction.

UFH preparations vary in average molecular weight, molecule length, and efficacy. Individual patient heparin dose-responses diverge markedly, because numerous plasma and cellular proteins bind UFH at varying rates. Consequently, laboratory monitoring is essential.22

Unfractionated heparin therapy

Physicians administer UFH intravenously to treat VTE, to provide initial treatment of AMI, to prevent reocclusion after stent placement, and to maintain vascular patency during cardiac surgery using cardiopulmonary bypass (CPB) with extracorporeal circulation. Different dosing regimens are used in various settings. For VTE treatment, therapy begins with a bolus of 5000 to 10,000 units, followed by continuous infusion at approximately 1300 units/hour, adjusted to patient weight. UFH therapy is discontinued when the acute clinical state has resolved or after the procedure or surgery. If necessary the patient will be switched to a non-intravenous anticoagulant to prevent future thrombotic events. To avoid HIT (Chapters 39 and 40), LMWH or other anticoagulants are used in place of UFH where possible.

Monitoring unfractionated heparin therapy using the partial thromboplastin time

Because of its inherent pharmacologic variations and narrow therapeutic range, UFH therapy is diligently monitored using the PTT (Figure 43-2). Blood is collected and assayed before therapy is begun to ensure that the baseline PTT is normal.23 A prolonged baseline PTT may indicate the presence of a lupus anticoagulant, factor inhibitor, or a factor deficiency and confuses the therapeutic interpretation. In such cases, the laboratory practitioner switches to the chromogenic anti-factor Xa heparin assay throughout the duration of therapy (discussed later in the chapter).

A second specimen is collected at least 4 to 6 hours but not longer than 24 hours after the initial bolus and a PTT is measured on this specimen. The PTT becomes prolonged within minutes of UFH administration, which reflects the immediate anticoagulation effect of UFH. The result for this specimen should fall within the therapeutic range, which is established by the laboratory practitioner (next section) and reported with the result. The physician or nurse adjusts the infusion rate to ensure that the PTT result is within the target range. PTT measurement is subsequently repeated every 24 hours, and the dosage is continually readjusted until UFH anticoagulation is complete. The physician also monitors the platelet count daily. A 40% or greater reduction in the platelet count, even within the reference interval, is evidence for HIT (see the discussion of the “4Ts” HIT diagnosis system in Chapter 39). If HIT is suspected, UFH therapy is immediately discontinued and replaced with DTI therapy.

Determining the partial thromboplastin time therapeutic range for unfractionated heparin therapy

The hemostasis laboratory is required to establish and communicate a PTT therapeutic range to monitor and manage UFH therapy. The medical laboratory technician or scientist collects 20 to 30 plasma specimens from patients being infused with UFH at all levels of anticoagulation, ensuring that fewer than 10% of the specimens are collected from the same patient, and measures PTT for all.24 The specimens must be from patients who are not receiving simultaneous Coumadin therapy; that is, their PT results must be normal. Chromogenic anti-factor Xa heparin assays are performed on all specimens, plus at least 10 specimens from healthy normal subjects, and the paired results are displayed on a linear graph (Figure 43-4). The range in seconds of PTT results that corresponds to 0.3 to 0.7 chromogenic anti-factor Xa heparin units/mL is the therapeutic range.25 This is known as the ex vivo or Brill-Edwards method for establishing the heparin therapeutic range of the PTT, and its use is required by laboratory certification and licensing agencies. Other approaches to determining the PTT therapeutic range for UFH therapy are discouraged. For instance, experts once recommended that the PTT therapeutic range be established as 1.5 to 2.5 times the mean of the reference interval. This approach, however, must be avoided as it consistently results in under-anticoagulation, which raises the risk of a secondary thrombotic event. In addition, the practice of developing a therapeutic range by “spiking” normal plasma with measured volumes of heparin is prohibited because the curve that is generated tends to flatten at higher concentrations.26

Image 

FIGURE 43-4 Laboratory scientists establish the partial thromboplastin time (PTT) therapeutic range for unfractionated heparin (UFH) by collecting specimens from 20 to 30 patients receiving UFH at representative dosages who have normal PTs and at least 10 individuals not receiving heparin. PTT and chromogenic anti-factor Xa heparin assays are performed on all specimens, and a linear graph of paired results is prepared with PTT on the vertical scale. The PTT range in seconds is correlated with the chromogenic anti-factor Xa therapeutic range of 0.3 to 0.7 units/mL or the prophylactic range of 0.1 to 0.4 units/mL.

Clinical utility of monitoring unfractionated heparin therapy using the partial thromboplastin time

The medical laboratory practitioner reports the PTT results, the reference interval, and the UFH therapeutic range to the clinician (physician, nurse, or pharmacist) who is managing the patient’s UFH dosage. Because reagent sensitivity varies among producers and among individual producers’ reagent lots, the clinician must evaluate PTT results in relation to the institution’s current published therapeutic range and reference interval.27 No system analogous to the INR exists for normalizing PTT results, because reagents and patient responses are too variable.28 While the PTT is used most often to measure the effects of UFH therapy, LMWH (next section) selectively catalyzes the neutralization of factor Xa more avidly than the neutralization of thrombin, thus its effects cannot be measured using the PTT. However, the chromogenic anti-factor Xa heparin assay may be used to assay UFH, LMWH, and fondaparinux.

Limitations of monitoring unfractionated heparin therapy using the partial thromboplastin time

Several conditions render the patient unresponsive to heparin therapy, a circumstance called heparin resistance.29 Inflammation typically is accompanied by fibrinogen levels raised to greater than 500 mg/dL and coagulation factor VIII activities of greater than 190% above the mean of the reference interval. Both elevations render the PTT less sensitive to the effects of heparin. Further, in many patients, antithrombin activity becomes depleted as a result of prolonged therapy or an underlying deficiency secondary to chronic inflammation. In this instance, the PTT result remains below the therapeutic range, becoming only modestly prolonged despite ever-increasing heparin dosages. Inflammation may be reduced through administration of steroids and aspirin or nonsteroidal anti-inflammatory drugs, and antithrombin concentrate may be administered. In the interim, however, it is necessary to use an alternative assay such as the chromogenic anti-factor Xa heparin assay.

Platelets in anticoagulated whole-blood specimens release platelet factor 4, a heparin-neutralizing protein (Chapter 13). In specimens from patients receiving heparin therapy, the PTT begins to shorten as soon as 1 hour after collection because of in vitro platelet factor 4 release unless the specimen is centrifuged and the platelet-poor plasma is removed from the cells (Chapter 42).30 Hypofibrinogenemia, factor deficiencies, and the presence of lupus anticoagulant, fibrin degradation products, or paraproteins prolong the PTT independent of heparin levels.31

Monitoring unfractionated heparin therapy using the activated clotting time

The ACT is a 1966 modification of the time-honored but obsolete Lee-White whole blood clotting time test. The ACT is a popular point of care assay that is used in clinics, at the inpatient’s bedside, in the cardiac catheterization laboratory, or in the surgical suite, and it is particularly useful at the high UFH dosages, 1 to 2 units/mL, used in percutaneous intervention (PCI, cardiac catheterization) and in cardiac surgery using extracorporeal circulation.32

ACT assay distributors such as International Technidyne Corporation, Edison, NJ, the makers of the Hemochron Response (Chapter 44), provide evacuated blood specimen collection tubes that contain 12 mg of diatomaceous earth, a particulate clot activator. The negative pressure within the tube is calibrated to collect 2 mL of blood. As soon as the specimen is collected, the tube is placed in the instrument cuvette well, where it is rotated and continuously monitored. When a clot forms, a magnet positioned within the sample is pulled away from a sensing device, which stops the timer. The time interval to clot formation is recorded automatically. The results of the ACT assay are comparable to those of the PTT assay for UFH monitoring, provided adequate quality control steps are taken. The median of the ACT reference interval is 98 seconds. Heparin is administered to yield results of 200 to 240 seconds in PCI or 400 to 450 seconds during cardiac surgery, levels at which the PTT is ineffective.

Reversal of unfractionated heparin overdose using protamine sulfate

At completion of cardiac surgery when the extracorporeal circuit is to be terminated, heparin anticoagulation needs to be quickly reversed. In other settings, a UFH overdose, or co-administration with aspirin, fibrinolytic therapy, or a GPI, may raise the risk of bleeding. Protamine sulfate, a cationic protein extracted from salmon sperm, neutralizes UFH at a ratio of 100 units of heparin per milligram of protamine sulfate. The health care provider administers protamine sulfate slowly by intravenous push. The effect of the protamine sulfate may be detected by the shortening of the PTT or ACT. Protamine sulfate also neutralizes LMWH, although the neutralization is incompletely reflected in the results of the chromogenic anti-factor Xa heparin assay described in the following paragraphs.

Protamine sulfate has also been implicated as causing a delayed form of HIT, consequently, platelet counts for patients who have received protamine sulfate are routinely monitored.3334

Low-molecular-weight heparin therapy and the chromogenic anti–factor Xa heparin assay

Low-molecular-weight heparin is produced from unfractionated heparin

Uncertainty about UFH dose response and the ever-present threat of HIT led to the development of LMWH, which was cleared for anticoagulant prophylaxis in the United States and Canada in 1993.35 LMWH is prepared from UFH using chemical (enoxaparin, Lovenox, Sanofi-Aventis, Bridgewater, NJ) or enzymatic fractionation (tinzaparin sodium, Innohep, LEO Pharmaceutical Products, Ballerup, Denmark).36Fractionation yields a product with a mean molecular weight of 4500 to 5000 Daltons, about one third the mass of UFH. LMWH possesses the same active pentasaccharide sequence as UFH; however, the overall shorter polysaccharide chains provide little space for thrombin bridging, so the thrombin neutralization response is reduced (Figure 43-5). The factor Xa neutralization response is unchanged, however, because this reaction does not rely on factor Xa binding to heparin’s polysaccharide chain, so LMWH provides nearly the same anticoagulant efficacy as UFH, although predominantly through factor Xa inhibition.

Image 

FIGURE 43-5 The antithrombin (AT) binding site binds a specific heparin pentasaccharide, producing an allosteric change that activates AT. The low-molecular-weight heparin (LMWH) molecule is too short to support a factor IIa-AT reaction; however, the activated AT binds factor Xa independently of the bridging phenomenon, producing a soluble AT-factor Xa complex. The LMWH recycles.

LMWH is administered by subcutaneous injection once or twice a day using premeasured syringes at selected dosages—for instance, 30 mg subcutaneously every 12 hours or 40 mg subcutaneously once daily. Prophylactic applications provide coverage during or after general and orthopedic surgery and trauma, typically for 14 days from the time of the event. LMWH also is used to treat DVT, PE, and unstable angina. LMWH is indicated during pregnancy for women at risk of VTE, because Coumadin, which causes birth defects, cannot be used. When patients who are taking Coumadin require surgery it is discontinued for up to a week before the procedure and replaced with LMWH.37

The advantages of LMWH are rapid bioavailability after subcutaneous injection, making intravenous administration unnecessary; a half-life of 3 to 5 hours compared with 60 to 90 minutes for UFH; and a fixed dose response that eliminates the need for laboratory monitoring, although laboratory measurement is still required in the conditions listed in Box 43-1. The risk of HIT is reduced by 90% in people who have never received heparin before; however, LMWH may cross-react with previously formed antibodies against heparin-platelet factor 4. Consequently, LMWH is contraindicated in patients who developed HIT after UFH therapy. The risk of LMWH-induced bleeding is less than that for UFH.

Measuring low-molecular-weight heparin therapy

The kidneys alone clear LMWH, so it accumulates in renal insufficiency. Laboratory measurement of LMWH therapy is necessary when the creatinine clearance is less than 30 mL/min or the serum creatinine is greater than 4 mg/dL. During LMWH therapy, creatinine assays are performed periodically to document kidney function and avoid the risk of LMWH accumulation in plasma. LMWH therapy in children, adults under 50 kg, adults over 150 kg, and during pregnancy also requires measurement because of fluid compartment imbalances or unstable coagulation (Box 43-1).

The phlebotomist collects a blood specimen 4 hours after subcutaneous injection, and the plasma is tested using the chromogenic anti-factor Xa heparin assay. The PTT is insensitive to LMWH. The chromogenic anti-factor Xa heparin assay employs a reagent that provides a fixed concentration of factor Xa and substrate specific to factor Xa (). Some distributors add fixed concentrations of antithrombin, and others none; the latter rely on the patient’s plasma antithrombin and provide sensitivity to antithrombin depletion or deficiency. Heparin forms a complex with reagent factor Xa and antithrombin; a measured excess of factor Xa digests the substrate, yielding a colored product whose intensity is inversely proportional to heparin concentration. Figure 43-6

Image 

FIGURE 43-6 The chromogenic anti-factor Xa heparin assay. The reagent is a mixture of antithrombin (AT) and a measured excess of Xa. Most kits do not provide AT and rely solely on patient plasma AT. AT binds heparin, and this complex binds factor Xa. Excess free factor Xa digests its substrate to produce a colored end product. The color intensity of the product is inversely proportional to plasma heparin. This assay is used for unfractionated heparin, low-molecular-weight heparin, and pentasaccharide fondaparinux; a dedicated standard curve is required for fondaparinux.

To prepare a standard curve, the laboratory practitioner obtains the characteristic UFH or LMWH calibrators from distributors, then computes and prepares dilutions that “bracket” the reference and the therapeutic range. If the chromogenic anti-factor Xa heparin assay is to be used to monitor UFH and LMWH, a single hybrid standard curve may be prepared.3839 A separate curve is necessary to monitor the pentasaccharide fondaparinux (next section). The prophylactic range for LMWH is 0.2 to 0.5 unit/mL, and the therapeutic range is 0.5 to 1.2 units/mL.24

The chromogenic anti-factor Xa heparin assay is the primary assay available to measure LMWH and fondaparinux. It may also be used in place of the PTT to assay UFH with little or no modification, and it substitutes for the PTT when clinical or laboratory conditions render PTT results unreliable. The chromogenic anti-factor Xa heparin assay is “tertiary” in the sense that it measures the heparin concentration and not heparin’s anticoagulant effects; however, the assay is precise and, in contrast to the PTT, is affected by few interferences. The chromogenic anti-factor Xa heparin assay is also the reference method for establishing the PTT therapeutic range. Laboratory directors have begun to recognize the merits of the chromogenic anti-factor Xa heparin assay and substitute it for the PTT in monitoring all UFH therapy, as well as in measuring therapy levels of LMWH and fondaparinux.

Measuring pentasaccharide therapy using the chromogenic anti–factor Xa heparin assay

Fondaparinux sodium (Arixtra; GlaxoSmithKline, Research Triangle Park, NC) is a synthetic formulation of the active pentasaccharide sequence in UFH and LMWH (). Fondaparinux raises antithrombin activity 400-fold.Figure 43-740 It is equivalent in clinical efficacy to LMWH with a reduced major bleeding effect and has a reproducible dose response and a desirable half-life of 17 to 21 hours. Because of the extended half-life, fondaparinux is administered in once-a-day subcutaneous injections of 2.5 to 7.5 mg.41 Fondaparinux is FDA-cleared for prevention of VTE after orthopedic and abdominal surgery and for treatment of acute VTE events, but its use is contraindicated in patients with creatinine clearance values of less than 30 mL/min.42

Image 

FIGURE 43-7 The specific saccharide sequence in unfractionated heparin and low-molecular-weight heparin (glucosamine, glucuronic acid, glucosamine, iduronic acid, glucosamine) is synthesized to make fondaparinux, which binds and activates the heparin-binding site of antithrombin. Source: (From Turpie AGG: Pentasaccharides. Semin Hematol 39:159-171, 2002.)

The chromogenic anti-factor Xa heparin assay is used to measure fondaparinux therapy in children, adults below 50 kg or over 150 kg, patients receiving treatment for more than 7 to 8 days, and pregnant women.43-45 Blood is collected 4 hours after injection, and the target range, derived from clinical studies, though not confirmed by outcome studies, is 0.2 to 0.4 mg/mL for a 2.5 mg dose and 0.5 to 1.5 mg/mL for a 7.5 mg dose. The operator prepares a calibration curve using fondaparinux—not UFH, LMWH, or a hybrid calibrator—because concentrations are expressed in mg/mL, not units per mL. The PTT is not sensitive to the effects of fondaparinux because, although fondaparinux reacts with antithrombin and factor Xa, it does not inhibit thrombin or factors IXa, XIa, or XIIa.

In the event of bleeding associated with fondaparinux overdose, protamine sulfate is ineffective. Recombinant activated factor VII (rFVIIa, NovoSeven; Novo Nordisk, Princeton, NJ) may partially reverse the effects of fondaparinux.46

Measuring oral direct factor Xa inhibitors

Oral rivaroxaban (Xarelto; Bayer Healthcare AG, Leverkusen, Germany; Janssen Pharmaceuticals, Inc., Raritan, NJ) is an oxazolidinone derivative that directly and stoichiometrically inhibits factor Xa.4748 It inhibits free factor Xa, factor Xa that is bound by factor IXa, and clot-bound factor Xa.45 As established by results of several clinical trials, rivaroxaban has efficacy and safety characteristics equivalent to LMWH or Coumadin.

Rivaroxaban was cleared in September 2008 by both Health Canada and the European Medicines Agency and in July 2011 by the U.S. FDA for VTE prophylaxis in patients who are undergoing total knee or total hip replacement surgery. The FDA also cleared rivaroxaban in November 2011 for prevention of ischemic stroke in patients with chronic nonvalvular atrial fibrillation at 20 mg/day and for prevention of secondary thrombosis subsequent to DVT or PE at 15 mg twice a day in December 2012.4950 The European Medicines Agency cleared rivaroxaban at 2.5 mg/day for prevention of secondary thrombosis following acute myocardial infarction in March 2013, but that same month the FDA deferred clearance for the same indication having considered a dosage of 10 mg/day.4751 The distributor re-filed in August 2013. As of June 2014, rivaroxaban has not been approved for use in patients with MI.

Rivaroxaban slightly prolongs the PT, as reported in several clinical trials, but to a lesser extent than Coumadin. Attempts to correlate PT results with dosage have revealed variability among PT thromboplastin reagents, rendering the PT only partially valid as a means to measure rivaroxaban.52-54 Rivaroxaban may also be assayed using a version of the chromogenic anti-factor Xa heparin assay. The assay must be calibrated using rivaroxaban in place of UFH, LMWH, or fondaparinux.55 Using either PT or anti-factor Xa, laboratory practitioners are working to correlate laboratory results with clinical outcomes in an effort to provide a therapeutic range.56

Like rivaroxaban, oral apixaban (Eliquis; Pfizer, New York, NY; Bristol-Myers Squibb, New York, NY) is a small oxazolidinone-derived direct stoichiometric factor Xa-inhibiting anticoagulant. The results of clinical trials reveal that apixaban actually improves on the efficacy and safety of Coumadin. The FDA cleared apixaban in December 2012 for the prevention of ischemic stroke in atrial fibrillation. The dosage for this indication is 2.5 mg twice a day. Apixaban has a weaker effect on the PT than rivaroxaban and Coumadin but may be measured using the PT, provided the operator first determines the sensitivity of the reagent. The chromogenic anti-factor Xa may also be used to measure apixaban when controls and calibrators become available in the North American market.57

A third oral direct-acting anti-factor Xa anticoagulant, edoxaban (Lixiana; Daiichi-Sankyo, Tokyo, Japan), cleared in Japan in July 2011 for clot prevention in patients who have had total knee or hip replacement, is currently in phase III trials worldwide. Its characteristics mirror rivaroxaban and apixaban, and its plasma concentration may likely be measured using the same methods, but this has not yet been determined.58

The oral direct anti-factor Xa inhibitors rivaroxaban, apixaban, and edoxaban all possess half-lives of approximately 12 hours. Apixaban may have an advantage in that 70% of the active drug is cleared by the liver and only 30% by the kidney, so dosage is relatively unaffected by renal insufficiency. There is no current recommended reversal agent for hemorrhages caused by overdoses of these drugs; however, some clinicians report normalization of laboratory test results by the use of factor eight inhibitor bypassing agent (FEIBA; Baxter, Deerfield, IL), four-factor (II, VII, IX, and X) prothrombin complex concentrate (Kcentra; DSL Behring, King of Prussia, PA), or recombinant activated factor FVII (rFVIIa, NovoSeven; Novo Nordisk, Princeton, NJ). All oral direct factor Xa inhibitors are prescribed with no monitoring (contrary to Coumadin); however, the clinical conditions listed in Box 43-1 may dictate the need to measure drug levels at specific times. A drug-specific chromogenic anti-factor Xa assay, utilizing a sodium citrate plasma sample, will likely be the assay of choice in most laboratories. Although the anti-factor Xa assay has been used for several years to monitor UFH and measure LMWH and fondaparinux, it awaits FDA clearance and is currently classified as research use only when applied to measuring rivaroxaban, apixaban, and edoxaban.

Direct thrombin inhibitors

Argatroban

The intravenous use DTIs argatroban and bivalirudin reversibly bind and inactivate free and clot-bound thrombin (). DTIs are substituted for UFH or LMWH when HIT is suspected or confirmed using the “4Ts” assessment system (Figure 43-8Chapter 39). Without the use of an intravenous DTI, the risk of thrombosis is 50% for 30 days after heparin is discontinued. In HIT, Coumadin, UFH, and LMWH are contraindicated.

Image 

FIGURE 43-8 Argatroban inhibits the active site of free and clot-bound thrombin. Antithrombin is not involved in this reaction. Bivalirudin and dabigatran inhibit thrombin by interacting at the same site but with their own specific binding characteristics.

Argatroban (Novostan; GlaxoSmithKline, Research Triangle Park, NC) is a non-protein l-arginine derivative with a molecular weight of 527 Daltons. Argatroban was FDA-cleared in 1997 for thrombosis prophylaxis and treatment and for anticoagulation during cardiac catheterization for patients with HIT.5960

For patients with HIT, the physician initiates the argatroban intravenous infusion at 2 μg/kg/min or in patients with hepatic disease at 0.5 μg/kg/min. During percutaneous cardiac intervention, a bolus of 350 μg/kg is given over 3 to 5 minutes, followed by an infusion at 25 μg/kg/min. Argatroban is cleared by the liver and excreted in stool. There is a 5% general bleeding risk and no direct reversal agent; however, the half-life is 50 minutes, and argatroban clears completely in 2 to 4 hours.

Bivalirudin, a recombinant analogue of leech saliva hirudin

Bivalirudin (Angiomax; The Medicines Company, Parsippany, NJ) is a synthetic 20-amino acid (2180 Daltons molecular weight) peptide derivative of the active site of hirudin, an anticoagulant produced in trace amounts by the medicinal leech Hirudo medicinalis. Bivalirudin was cleared by the FDA in 2000 for use as an anticoagulant in patients with unstable angina at risk for HIT who are undergoing percutaneous coronary intervention.61

Bivalirudin is intended for use with concurrent aspirin therapy at a dosage of 325 mg/day and has been studied only in patients receiving aspirin.62 Physicians provide an intravenous bolus dose of 0.75 mg/kg bivalirudin, followed by an infusion of 1.75 mg/kg/hr for the duration of the percutaneous cardiac intervention. After 4 hours, an additional intravenous infusion may be given at a rate of 0.2 mg/kg/hr for 20 hours.

The rate of major hemorrhage with bivalirudin is 4%. There is no reversal agent; however, in patients with normal renal function, the half-life is 25 minutes. The dosage is decreased in patients with reduced creatinine clearance or elevated serum creatinine.6364

Dabigatran, an oral direct thrombin inhibitor

Oral dabigatran etexilate (Pradaxa; Boehringer Ingelheim, Ingelheim, Germany) is a prodrug that converts upon ingestion to active dabigatran, a reversible DTI that binds both free and clot-bound thrombin. Dabigatran’s efficacy and safety appear to match those of LMWH and Coumadin, and it has no known interaction with food. It is cleared by the kidneys, has a half-life of 12 to 17 hours, and is not metabolized by liver cytochrome enzymes; however, it does affect the P-glycoprotein transport system, which can impact drug-drug interactions.65 Dabigatran was cleared by the European Medicines Agency and Canada Health in the spring of 2008 for VTE prophylaxis following total knee or total hip replacement surgery at dosages of 220 mg/day, or 150 mg/day in the elderly or those with moderate renal impairment. In October 2010, the U.S. FDA cleared dabigatran for prevention of ischemic stroke in nonvalvular atrial fibrillation. In renal disease the half-life may be prolonged to as much as 60 hours, and in overdose-caused hemorrhage there is no known reversal agent.66-70

Measuring direct thrombin inhibitor therapy

Argatroban and bivalirudin prolong the thrombin time, PT, PTT, and ACT.71 For nonsurgical therapy, distributors recommend assaying with the PTT using the target therapeutic range of 1.5 to 3 times the mean of the laboratory reference interval (but not more than 90 seconds). Blood is collected 2 hours after the initiation of intravenous therapy for argatroban and 4 hours after therapy initiation for bivalirudin, and the dosage is adjusted to achieve a PTT within the therapeutic range. The ACT may be employed during cardiac catheterization or cardiac surgery. During these procedures, the target ACT for bivalirudin therapy is 320 to 400 seconds (median normal value, 98 seconds).

In instances in which the baseline PTT is prolonged by lupus anticoagulant, factor inhibitors, or factor deficiencies, the ecarin clotting time (ECT) is a potential alternative for assaying argatroban and bivalirudin. Ecarin (Ecarinase; Pentapharm, Basel, Switzerland) is an enzyme extracted from Echis carinatus venom that converts prothrombin to intermediate meizothrombin, which converts fibrinogen to fibrin. Argatroban and bivalirudin bind meizothrombin and generate a linear, dose-dependent prolongation of the ECT. Aside from DTIs, the ECT is prolonged only by abnormally low prothrombin or fibrinogen activity.

The oral direct thrombin inhibitor dabigatran is prescribed without routine monitoring. The clinical situations listed in Box 43-1, however, may necessitate measuring dabigatran drug levels. Dabigatran prolongs the thrombin time, PTT, and ECT.72 The standard thrombin time is exceptionally sensitive to dabigatran and is convenient for ruling out dabigatran, because a normal thrombin time indicates that no dabigatran is present. A prolonged thrombin time may indicate that dabigatran is present, but it does not indicate the plasma concentration. The PTT generates a “curvilinear” response to dabigatran and is unreliable at low levels; additionally, there is considerable variability in sensitivity to dabigatran among PTT reagents.73

The ECT and the ecarin chromogenic assay (ECA; Stago, Asnières sur Seine, France) provide a reliable, linear response to dabigatran, except at low concentrations.74 Also, a modification of the thrombin time called the plasma-diluted thrombin time (Hemoclot Direct Thrombin Inhibitor Assay; Aniara Hyphen, West Chester, OH) and a chromogenic assay that is based on the thrombin time (Biophen DTI [chromogenic] Assay; Aniara Hyphen, West Chester, OH) are available.75 ECT, ECA, plasma-diluted thrombin time, and Biophen DTI, all of which require a sodium citrate plasma sample, await FDA clearance and are currently restricted to research use only.76-78

Measuring antiplatelet therapy using platelet activity assays

Intravenous glycoprotein iib/iiia inhibitors are used during cardiac catheterization

Glycoprotein IIb (αIIb) and glycoprotein IIIa (β3) are present on the membrane of resting platelets. Upon activation by any agonist, these molecules join to form glycoprotein IIb/IIIa (αIIbβ3) heterodimers, receptors that bind fibrinogen and von Willebrand factor through their arginine–glycine–aspartic acid (RGD) sequences. Fibrinogen binding to αIIbβ3 supports the key step of in vivo platelet aggregation (Chapter 13). The intravenous glycoprotein IIb/IIIa inhibitors (GPIs) abciximab, eptifibatide, and tirofiban fill αIIbβ3 receptor sites and block fibrinogen or von Willebrand factor binding, thereby preventing platelet aggregation79(Figure 43-9). Cardiologists use intravenous GPIs to maintain vascular patency during cardiac catheterization and intracoronary stent placement.80

Image 

FIGURE 43-9 Antiplatelet drugs employ three mechanisms to inactivate platelets. Aspirin irreversibly acetylates and inactivates cyclooxygenase 1 (COX-1). Clopidogrel (irreversible), prasugrel (irreversible), and ticagrelor (reversible) bind the adenosine diphosphate (ADP) receptor, P2Y12. Intravenous abciximab, eptifibatide, and tirofiban bind the fibrinogen binding site, glycoprotein (GP) IIb/IIIa. PGG2, Prostaglandin G2PGH2, prostaglandin H2TXA synthase, thromboxane A2 synthase.

Before GPI infusion, the PT, PTT, ACT, hemoglobin, hematocrit, and platelet count are determined to detect any hemostatic or hematologic abnormality.81 During infusion, the PTT and ACT are maintained within the UFH therapeutic range as determined by the laboratory. Platelet counts are performed at 2 hours, 4 hours, and 24 hours following the initial bolus. If the platelet count drops by 25% or more, UFH and GPI are discontinued and the platelet count is monitored daily until it returns to within the reference interval. GPI efficacy may be measured using the Multiplate analyzer (DiaPharma, West Chester, OH; Roche Diagnostics Corporation, Indianapolis, IN) or VerifyNow IIb/IIIa assay (Accumetrics, San Diego, CA; International Technidyne Corporation, Edison, NJ).

Abciximab (ReoPro; Eli Lilly and Company, Indianapolis, IN) is the 47,615 Dalton Fab fragment of a mouse monoclonal antibody specific for αIIbβ3 that effectively fills the receptor site.82 The dose is 0.25 mg/kg given by intravenous bolus administered 10 to 60 minutes before the start of cardiac catheterization, followed by continuous infusion of 0.125 μg/kg/min for up to 12 hours. Abciximab is always coadministered with UFH and aspirin.

Eptifibatide (Integrilin; Schering Corporation, Kenilworth, NJ) is an 832 Dalton heptapeptide GPI. It is coadministered with aspirin and UFH. An intravenous bolus of 180 μg/kg is given as soon as possible after initial diagnosis, and a continuous intravenous drip of 2 μg/kg/min is continued for up to 96 hours following the initial bolus, including throughout cardiac catheterization.

Tirofiban hydrochloride (Aggrastat; Baxter Healthcare Corporation, Deerfield, IL) is a 495 Dalton non-protein GPI that is coadministered with aspirin and UFH. It is administered intravenously at an initial rate of 0.4 μg/kg/min for 30 minutes and then continued at 0.1 μg/kg/min throughout cardiac catheterization and for 12 to 24 hours after catheterization. Tirofiban is excreted through the kidney, so the dosage is halved when the creatinine clearance is less than 30 mL/min.

Aspirin, clopidogrel, prasugrel, and ticagrelor reduce the incidence of arterial thrombosis

The most commonly prescribed oral antiplatelet drugs are aspirin, clopidogrel (Plavix; Sanofi-Aventis, Bridgewater, NJ; Bristol-Myers Squibb, New York, NY), prasugrel (Effient; Eli Lilly and Company, Indianapolis, IN), and ticagrelor (Brilinta; AstraZeneca, Wilmington, DE). Aspirin irreversibly acetylates the platelet enzyme cyclooxygenase at the serine in position 529 (). The serine-bound acetyl group sterically hinders the access of arachidonic acid to its reactive site within the cyclooxygenase molecule. This prevents production of platelet-activating thromboxane AFigure 43-92 through the eicosanoid synthesis pathway (Chapter 13).83 Acetylation is irreversible; the eicosanoid synthesis pathway is shut down for the remainder of the life of the platelet.

In contrast, clopidogrel, prasugrel, and ticagrelor are generally considered to be members of the thienopyridine drug family, though ticagrelor is actually a purine analogue. Thienopyridines occupy the platelet membrane adenosine diphosphate (ADP) receptor P2Y12, suppressing the normal platelet aggregation and secretion response to the activating ligand (agonist) ADP. Clopidogrel and prasugrel are irreversible inhibitors, whereas ticagrelor is a reversible inhibitor.

Aspirin is often prescribed alone at 81 or 325 mg/day to prevent myocardial infarction and ischemic cerebrovascular disease in patients with stable or unstable angina,8485 AMI,86 transient cerebral ischemia,87peripheral vascular disease,88 or stroke.8990 In healthy people, aspirin prophylaxis annually prevents four thrombotic events per 1000 individuals treated, although it carries a risk of bleeding.91-93

Clopidogrel is prescribed at 75 mg/day together with aspirin at 81 or 325 mg/day. Clopidogrel is a prodrug, and patients appear to have varying responses to the fixed dose of clopidogrel, which raises the need for routine laboratory measuring using platelet function assays. Patients who possess a genetic variant of the CYP2C19 liver enzyme that activates clopidogrel may not get the full therapeutic effect. The variant polymorphism may be identified via molecular diagnostic techniques or phenotypically, as described in the next section.

Prasugrel was cleared by the FDA in July 2009.94 It is administered as an oral prodrug that is converted in the liver via several cytochrome P-450 pathways to an active metabolite whose elimination half-life is about 7 hours. Treatment begins with a single 60-mg oral loading dose and continues at 10 mg daily, or 5 mg daily for patients who weigh less than 60 kg. Prasugrel is to be taken with aspirin at 81 mg or 325 mg daily and appears to require no laboratory measuring. However, up to 14% of patients may not achieve the full effect of prasugrel due to a genetic variant of the CYP2C19 liver enzyme needed to activate the drug. Drug interactions that increase or decrease the activity of prasugrel are important to identify. Prasugrel carries a higher risk of bleeding than clopidogrel and may be associated with an increased risk of solid tumors.

Ticagrelor was cleared in July 2011 to be coadministered with aspirin. Ticagrelor is a prodrug whose main active metabolite is formed rapidly via the CYP3A4 liver enzyme. It is provided in 90-mg tablets. Therapy is begun with two tablets totaling 180 mg, taken with one 325-mg aspirin tablet, followed by 90 mg of ticagrelor twice a day and one aspirin a day, not to exceed 100 mg. Ticagrelor reaches full effectiveness in 1.5 hours and maintains steady state for at least 8 hours. Drug interactions that increase or decrease the activity of ticagrelor are important to identify.

Variable aspirin and clopidogrel response and laboratory measuring of antiplatelet resistance

Several investigations confirm that 10% to 20% of people who are taking aspirin generate an inadequate response as measured in the laboratory by light transmittance platelet aggregometry or whole-blood impedance aggregometry using arachidonic acid as the agonist. Inadequate response to aspirin has been termed aspirin resistance. Likewise, the response to clopidogrel as measured by aggregometry using ADP as the agonist varies markedly among patients, and the results of this assay may be used to adjust clopidogrel dosage.99 Mechanisms that explain aspirin resistance and clopidogrel response variation are currently under study. Unlike treatment with aspirin and clopidogrel, prasugrel and ticagrelor therapy may show less interpatient variation. Aggregometry is the reference method for determining aspirin and clopidogrel responses, but several more rapid assays are available.

• VerifyNow: The Accumetrics VerifyNow (Accumetrics, San Diego, CA; International Technidyne Corporation, Edison, NJ) system is designed for point-of-care testing and uses light transmittance aggregometry to individually test for platelet aggregation responses to aspirin, clopidogrel, prasugrel, ticagrelor, and GPIs.100 For each assay a cartridge is provided that contains the desired agonist and fibrinogen-coated beads. VerifyNow Aspirin uses arachidonic acid as its agonist. VerifyNow P2Y12 uses ADP. VerifyNow IIb/IIIa uses thrombin receptor–activating polypeptide (TRAP), which activates platelets by binding to the thrombin receptor protease-activated receptor 1 (PAR1; Chapter 13). The laboratory establishes reference interval limits and therapeutic target limits for each assay. Results that are outside the therapeutic target range indicate possible treatment failure and the need to revise dosage or change to a new antiplatelet drug. All three Accumetrics VerifyNow systems are FDA cleared.

• Multiplate: The Multiplate (DiaPharma, West Chester, OH; Roche Diagnostics Corporation, Indianapolis, IN) analyzer is automated for point-of-care testing and uses impedance aggregometry to simultaneously or individually test for platelet aggregation responses to aspirin, thienopyridines, and GPIs. The Multiplate requires 300 μL of whole blood. The aspirin resistance assay uses arachidonic acid as its agonist, the thienopyridine response assay uses ADP, and the GPI response uses TRAP. The instrument integrates three aggregometry parameters—aggregation velocity, maximum aggregation, and area under the aggregation curve—to produce measurement units. The local laboratory establishes reference interval limits and expected therapeutic target ranges. Results that are outside the therapeutic target range indicate possible treatment failure and the need to revise dosage or change to a new antiplatelet drug. The Multiplate is available in Europe and was cleared by the FDA for use in the United States in August 2012.

• Plateletworks: The Plateletworks assay from Helena Laboratories (Beaumont, TX) determines the percent platelet aggregation in whole blood. Whole blood is added to EDTA tubes coated with the agonists ADP or collagen, plus plain EDTA tubes. The practitioner performs platelet counts on the plain tube (baseline) and the agonist-treated EDTA tubes using an impedance-based electronic cell counter. The differences, expressed as percentages, indicate the degree of platelet aggregation triggered by each agonist. An expected effect of antiplatelet drug therapy would be a platelet aggregation response that is reduced 40% to 60% from a normal response.

• PFA-100 (Siemens Medical Solutions USA, Inc., Malvern, PA). The PFA-100 system uses two cartridges. The first provides an aperture impregnated with collagen and epinephrine, and the second provides an aperture impregnated with collagen and ADP. The operator pipettes 800 μL whole blood per cartridge and places the cartridge on the instrument.101 The specimen passes through the aperture until activation by the agonist causes occlusion of the aperture, generating a parameter called closure time. The PFA-100 tests only for aspirin resistance. A closure time that is shorter than the anticipated therapeutic range for aspirin indicates resistance.

• AspirinWorks (Corgenix Medical Corporation, Broomfield, CO): The AspirinWorks immunoassay measures a urine metabolite of platelet eicosanoid synthesis and thromboxane A2 activation (Chapter 13). Hepatocyte 11-hydroxythromboxane dehydrogenase acts upon stable platelet-derived plasma thromboxane B2, the end product of eicosanoid synthesis and the stable analogue of thromboxane A2, to produce water-soluble 11-dehydrothromboxane B2.99 The urine concentration of 11-dehydrothromboxane B2 is sufficient for measurement without extraction and, because platelets seem to be its primary source, proportionally reflects platelet activity within the previous 12 hours. Urine levels of 11-dehydrothromboxane B2 frequently are elevated in atherosclerosis; after stroke, transient ischemic attack, or intracerebral hemorrhage; and in atrial fibrillation. Levels of 11-dehydrothromboxane B2 typically are decreased in patients receiving aspirin therapy, even in those with atherosclerosis, myocardial infarction, and atrial fibrillation, but appear to remain normal in patients who have aspirin resistance.

Future of antithrombotic therapy

Antithrombotic therapy, unchanged for more than 50 years, is likely to see further changes between 2014 and 2020. Several oral anticoagulants currently under development or awaiting clearance are likely to replace Coumadin, the heparins, and fondaparinux. Likewise, a series of new and emerging antiplatelet drugs will augment the time-honored aspirin tablet. The work of the clinical laboratory will reflect these changes, moving from the PT and PTT to chromogenic anti-factor Xa, modifications of the thrombin time, the ecarin clotting time, chromogenic assays, and new molecular assays. Antiplatelet response measuring will grow in convenience and take advantage of flow cytometry, immunoassays, and molecular assays that are currently in development.

Summary

• Coumadin was developed in 1940 by Link and was first used in 1952. It prevents VTE, but it has a narrow therapeutic range, and an overdose causes hemorrhage. Coumadin therapy is monitored by the PT assay and reported as an INR. PT measurement is available on portable point-of-care instrumentation. Anticoagulation clinics are available to facilitate Coumadin monitoring and provide patient education and support.

• UFH is administered intravenously to provide immediate control of coagulation. Its therapeutic effect is monitored using the PTT, which requires the laboratory practitioner to develop a therapeutic range in seconds keyed to the chromogenic anti-factor Xa heparin assay. PTT results are subject to several interferences. Heparin is also used during cardiac catheterization or cardiac surgery that requires extracorporeal circulation. In these acute settings it is monitored by the ACT for its point-of-care and sensitivity to high heparin dose capabilities.

• LMWH and fondaparinux substitute for UFH and are administered subcutaneously for both prophylaxis and therapy. Both provide near-complete bioavailability, predictable dose response, and longer half-lives than UFH. LMWH and fondaparinux therapy require laboratory measurement only in patients with renal insufficiency, pregnant women, obese patients, children, and underweight adults using the chromogenic anti-factor Xa heparin assay.

• Rivaroxaban, apixaban, and edoxaban are the first to come to market of several oral direct-acting anti-factor Xa anticoagulants that require little laboratory measurement. New measurement techniques include the chromogenic anti-factor Xa assays using rivaroxaban, apixaban, and edoxaban calibrators and controls.

• The intravenous DTIs argatroban and bivalirudin directly bind thrombin without involving antithrombin and are substituted for heparin in patients with HIT. Intravenous DTI therapy is monitored using the PTT or ECT, and all affect the PT results during switchover to Coumadin therapy. At higher doses, as used during interventional procedures, these drugs are monitored by the ACT.

• Dabigatran is an oral DTI that requires minimal laboratory measurement. Dabigatran may be measured using the PTT, ECT, ECA, and plasma-diluted thrombin time.

• The antiplatelet drugs aspirin, clopidogrel, prasugrel, and ticagrelor are used after arterial thrombotic events to prevent repeat AMI, stroke, and PAO. Patient responses to aspirin and clopidogrel therapy vary. Response variation is detected using platelet aggregometry, the Accumetrics VerifyNow system, the Multiplate system, Helena’s Plateletworks, the PFA-100 or the AspirinWorks assay; the latter two measure aspirin only.

• The intravenous antiplatelet drugs abciximab, eptifibatide, and tirofiban are used during cardiac catheterization to maintain vascular patency. Because they may cause thrombocytopenia, the platelet count is monitored carefully. Their efficacy may be monitored using the Accumetrics VerifyNow system, Helena’s Plateletworks, or the Multiplate system.

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

1. What is the PT/INR therapeutic range for Coumadin therapy when a patient has a mechanical heart valve?

a. 1 to 2

b. 2 to 3

c. 2.5 to 3.5

d. Coumadin is not indicated for patients with mechanical heart valves

2. Monitoring of a patient taking Coumadin showed that her anticoagulation results remained stable over a period of about 7 months. The frequency of her visits to the laboratory began to decrease, so the period between testing averaged 6 weeks. This new testing interval is:

a. Acceptable for a patient with stable anticoagulation results after 6 months

b. Unnecessary, because monitoring for patients taking oral anticoagulants can be discontinued entirely after 4 months of stable test results

c. Too long even for a patient with previously stable test results; 4 weeks is the standard

d. Acceptable as long as the patient performs self-monitoring daily using an approved home testing instrument and reports unacceptable results promptly to her physician

3. What is the greatest advantage of point-of-care PT testing?

a. It permits self-dosing of Coumadin

b. It is inexpensive

c. It is convenient

d. It is precise

4. You collect a citrated whole-blood specimen to monitor UFH therapy. What is the longest it may stand before the plasma must be separated from the cells?

a. 1 hour

b. 4 hours

c. 24 hours

d. Indefinitely

5. What test is used to monitor high-dose UFH therapy in the cardiac catheterization lab?

a. PT

b. PTT

c. Bleeding time

d. ACT

6. What test is used most often to monitor UFH therapy in the central laboratory?

a. PT

b. PTT

c. ACT

d. Chromogenic anti-factor Xa heparin assay

7. What test is used most often to monitor LMWH therapy in the central laboratory?

a. PT

b. PTT

c. ACT

d. Chromogenic anti-factor Xa heparin assay

8. What is an advantage of LMWH therapy over UFH therapy?

a. It is cheaper

b. It causes no bleeding

c. It has a stable dose response

d. There is no risk of HIT

9. In what situation is an intravenous DTI used?

a. DVT

b. HIT

c. Any situation in which Coumadin could be used

d. Uncomplicated AMI

10. What laboratory test may be used to monitor intravenous DTI therapy when PTT results are unreliable?

a. PT

b. ECT

c. Reptilase clotting time

d. Chromogenic anti-factor Xa heparin assay

11. What is the reference method for detecting aspirin or clopidogrel resistance?

a. Platelet aggregometry

b. AspirinWorks

c. VerifyNow

d. PFA-100

12. What is the name of the measurable platelet activation metabolite used in the AspirinWorks assay to monitor aspirin resistance?

a. 11-dehydrothromboxane B2

b. Arachidonic acid

c. Thromboxane A2

d. Cyclooxygenase

13. Which of the following is an intravenous antiplatelet drug used in the cardiac catheterization laboratory?

a. Abciximab

b. Ticagrelor

c. Prasugrel

d. Clopidogrel

14. Which of the following is a newly developed oral anticoagulant?

a. Argatroban

b. Lepirudin

c. Bivalirudin

d. Rivaroxaban

15. Which of the following is not a point-of-care instrument for the measurement of PT?

a. CoaguChek XS PT

b. Gem PCL Plus

c. Cascade POC

d. Multiplate

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