Current Anticoagulation Therapy: Practice and Monitoring

## **Chapter 2**

## Pharmacological Review of Anticoagulants

*Hobart Owen Ng Tsai*

## **Abstract**

The art and science of anticoagulation have never gotten more complicated than it has now. Newer anticoagulants have entered the market and have provided more options to the patients and healthcare professionals. This chapter will review the basic physiology of hemostasis, pharmacology of the anticoagulants, and how these medications are used in the clinical setting. The mechanism of action, pharmacokinetics and pharmacodynamics, clinical evidence of use and clinical pearls, laboratory monitoring in clinical practice, and adverse effects will be examined individually for each drug considered. This chapter will serve as a review for the practicing clinician and a thorough introduction for the beginning reader.

**Keywords:** anticoagulants, warfarin, heparin, low molecular weight heparin, enoxaparin, dabigatran, rivaroxaban, apixaban, edoxaban

## **1. Introduction**

Anticoagulants are the mainstay of treatment for stroke and systemic embolism prevention in patients with atrial fibrillation (AF) or flutter. They can be used as well for prevention and treatment of venous thromboembolism (VTE) and treatment of thrombus formation in other places. This class of medications must be used carefully because using them incorrectly can lead to either ineffective prevention of clot formation or bleeding. It is vital for the clinician who uses any of these anticoagulants to have a basic understanding of their pharmacology and evidence of use.

## **2. The coagulation cascade**

The coagulation system is composed of two separate pathways that convene on a single pathway. The two pathways are extrinsic pathway and intrinsic pathway. Injury to the endothelial system exposes *tissue factor* out in the bloodstream. The extrinsic pathway begins with factor VII. Circulating factor VII in the bloodstream will then get activated to factor VIIa when they come into contact with tissue factor. Factor VIIa then converts factors X and IX to factor Xa and IXa, respectively. The presence of factor IXa, together with factor VIIIa, work to produce more factor Xa. Factor Xa and factor Va then activate factor II (prothrombin) to factor IIa (thrombin). Factor IIa then converts fibrinogen to fibrin [1, 2].

#### **Figure 1.**

*Coagulation cascade showing the intrinsic, extrinsic, and common pathway. Reprint with permission from [30].*

The result of this cascade is the production of fibrin molecules that bind to GPIIb/ IIIa receptors on platelets and hold them together to form a platelet plug. The extrinsic pathway is what protects humans when bleeding occurs involving trauma to the vasculature or when the blood comes in contact with extravascular tissues [1, 2].

The intrinsic pathway gets activated upon trauma to the blood or when the blood gets exposed to collagen found on damaged blood vessels. At the beginning of the intrinsic pathway activation, exposure of factor XII to collagen, for example, stimulates a configurational change in factor XII to become factor XIIa. Together the help of high molecular weight kininogen and prekallikrein, factor XIIa enzymatically activates factor XI to XIa. Factor XIa in turn activates factor IX to IXa. Factor IXa then works with factor VIIIa to convert factor X to Xa. Factor Xa then converts factor II to factor IIa, which in turn activates fibrinogen to fibrin [1, 2].

The extrinsic pathway and intrinsic pathway converge as the common pathway when factor X gets converted to factor Xa [1, 2] (see **Figure 1**).

#### **3. Basic and clinical pharmacology of the anticoagulants**

#### **3.1 Unfractionated heparin (UFH)**

#### *3.1.1 Mechanism of action*

Unfractionated heparin is a long string of glycosaminoglycan molecules that can range from 3000 to 30,000 Daltons. UFH with its specific pentasaccharide sequence binds to antithrombin III and catalyzes its efficiency in inhibiting factor Xa and IIa in a ratio of 1: 1. However, not all heparin molecules given are active; only about a third of the heparin molecules in a solution contain the required pentasaccharide sequence [1–3].

#### *3.1.2 ADME*

Heparin is not absorbed orally and is given either subcutaneously (SQ ) or intravenously (IV). Heparin is highly protein bound and is cleared in the bloodstream by endothelial cells and macrophages. The half-life of heparin increases as the dose increases; it can vary from 1 hour at a dose of 100 units/kg to 2.5 h for 400 units/kg to 5 h for 800 units/kg. In general, the clinical effect of IV UFH dissipates 4–6 hours after stopping the infusion [1–3].

#### *3.1.3 Clinical use*

UFH can be either given SQ or IV as mentioned above. However, SQ administration of UFH has erratic bioavailability. Hence, SQ is not a preferred route if a patient requires treatment dose of UFH. Clinical studies have also shown a higher rate of treatment failure rate with SQ compared to IV heparin [3, 4]. For VTE prophylaxis, SQ administration of UFH would suffice. The usual dose is 5000 units q8-12h [4].

Initial dosing of IV UFH depends on the indication. Besides VTE treatment, IV UFH can also be used for patients with acute coronary syndrome, as a bridging agent for patients with atrial fibrillation, mechanical valves, etc. The dose of IV UFH could be either a fixed dose or a weight-based dose. A study by Raschke et al. [3] compared fixed dose vs. weight-based dose of IV heparin in patients with venous and arterial thromboembolism. In that study, significantly more patients (97%) in the weight-based dosing group achieved an Activated Partial Thromboplastin Time (aPTT) > 1.5x the baseline within 24 hours vs. the fixed dose group (77%), leading the authors to conclude that weight-based dosing is superior to fixed dose IV heparin [3, 4]. The 2016 Guidance for the practical management of the heparin anticoagulants in the treatment of venous thromboembolism and the 2012 American College of Chest Physicians (ACCP) Guidelines on Antithrombotic Therapy and Prevention of Thrombosis Supplement on Parenteral Anticoagulants list both fixed dose and weight-based dose for IV heparin [3].

#### *3.1.4 Monitoring*

There are two ways of monitoring the heparin activity in the body. These are the aPTT and Activated Clotting Time (ACT). The usual aPTT target for a therapeutic effect of heparin is 1.5–2x the baseline, which is equivalent to an anti-factor Xa activity of 0.3–0.7 units/ml [1, 3, 4]. The target aPTT varies based on what reagent is used to measure it. Thus, the clinician should check with the institution's laboratory for the target aPTT for patients receiving IV heparin. When using IV UFH for treating heparin or for bridging purposes, the aPTT is used. Another lab test used to monitor heparin is the ACT. ACT is available as a point-of-care test and is used when patients are receiving high doses of heparin. ACT can be seen being used in instances such as during cardiopulmonary bypass surgery and percutaneous coronary intervention. The target ACT varies based on the indication. ACT is reported in seconds and denotes how long it takes for the blood to clot.

#### *3.1.5 Adverse effects*

Patients on heparin also need close monitoring of platelet counts. Thrombocytopenia could be a consequence of heparin infusion and severe reaction called heparin-induced thrombocytopenia (HIT) may occur [1–5]. There are two kinds of heparin-induced thrombocytopenia: HIT type 1 and HIT type 2. HIT Type 1, which may also be called heparin-associated thrombocytopenia, is a benign, transient drop in platelets counts usually within the first 2–4 days after initiation of heparin infusion. Platelet counts rarely go below 100,000 [3–6]. The mechanism behind HIT type 1 is unknown but may involve dilutional effect or decreased platelet production associated with the acute illness [6].

The other more serious reaction, which is HIT type 2, involves antibody formation against heparin-platelet factor 4 complex. Heparin may bind to platelet factor-4 (PF 4), which is a cationic protein product of platelets that binds heparin and prevents heparin from binding with antithrombin. The heparin-PF4 complex is highly antigenic and induces the formation of IgG molecules against it. The IgG molecule-heparin-PF 4 complex binds to platelets and activates it, further releasing more PF4. The activated platelets with bound IgG-heparin-PF 4 complex also produce prothrombotic molecules that may cause thrombosis [3–6]. HIT type 2 can cause both arterial and venous thromboses, although venous thrombosis is more common. The activated platelets with bound IgG-heparin-PF 4 get removed from the body quickly, hence causing thrombocytopenia [6].

For the treatment of HIT, the American Society of Hematology 2018 guidelines for the management of venous thromboembolism: heparin-induced thrombocytopenia suggests use of non-heparin options such as argatroban (a direct thrombin inhibitor), fondaparinux (an ant-factor Xa inhibitor), or DOAC (specifically rivaroxaban due to most experience at a dose of 15 mg twice a day for 3 weeks if thrombosis is present, or 15 mg twice a day until the platelet counts have recovered to ≥150 × 109 /L, then followed by 20 mg daily if there is an indication for continued anticoagulation) [7].

#### *3.1.6 Reversibility*

In the event of bleeding, heparin can be reversed with protamine sulfate. Protamine is a cationic protein from fish sperm that can bind to heparin (which is anionic) and neutralize heparin immediately [1]. 1 mg of protamine reverses approximately 100 units of heparin, with a maximum dose of 50 mg at a time. For patients who are receiving continuous IV heparin infusion, only the last 2–3 hours dose of heparin given needs to be taken into when calculating the dose for protamine. For patients who received SQ heparin, protamine has to be given as a prolonged infusion. aPTT can be used to monitor the efficacy of protamine. One needs to be careful when giving protamine as protamine itself is prothrombotic [3, 5].

#### **3.2 Low molecular weight heparin (LMWH)**

There are various preparations of LMWH available in the market. Some examples are enoxaparin, dalteparin, tinzaparin, etc. The rest of the discussion in this section will focus on enoxaparin.

#### *3.2.1 Mechanism of action*

LMWH's are shorter molecular version of UFH. They are only a third of the size of UFH. Same as UFH, LMWH bind to antithrombin and catalyzes its efficiency. But unlike UFH, the combination LMWH-antithrombin is only capable of deactivating factor Xa and very little factor IIa [1, 3, 5].

#### *3.2.2 ADME*

Enoxaparin can be either given SQ or IV depending on the indication. It is predominantly cleared renally hence dose adjustment is needed in patients with renal impairment (when Cockcroft-Gault calculated creatinine clearance is <30 ml/ min). It has a half-life of 3–6 hours and reaches peak concentration 3–5 hours after SQ injection [1, 3–5].

#### *3.2.3 Clinical use*

Enoxaparin can be given as a once a day or twice daily dosing. Once daily dosing is not advisable in certain populations such as obese patients because the effect of enoxaparin would not last for 24 hours [4]. The dose varies based on the indication. It can be used for VTE treatment and prophylaxis, arterial thromboses, bridging agent for patients with atrial fibrillation, mechanical valves, etc. It is easier to use in practice than IV heparin if full treatment dose is needed as there is no laboratory monitoring required.

## *3.2.4 Monitoring*

The activity of LMWH is more predictable than UFH. Hence, monitoring is not needed when LMWH is given. However, its anticoagulation effects may be determined by checking the anti-factor Xa activity. Several guidelines including the 2016 Guidelines for the practical management of the heparin anticoagulants in the treatment of venous thromboembolism and the 2018 American Society of Hematology guidelines for the management of venous thromboembolism: optimal management of anticoagulation therapy do not suggest routing monitoring of anti-factor Xa activity due to uncertainties regarding its clinical utility and its cost-effectiveness [4, 8]. Additionally, there is currently no standardized method of adjusting the dose of enoxaparin based on anti-factor Xa activity [8], except for pediatric patients.

## *3.2.5 Adverse effects*

As with any anticoagulants, bleeding is a major concern for patients receiving LMWH. Hematoma surrounding the injection site may also appear if patients rub on the injection site. In terms of major side effects, LMWH has a lower incidence of HIT type 2 compared to UFH. However, patients who have a history of HIT type 2 should best avoid LMWH if antibodies are still present.

## *3.2.6 Reversibility*

In cases of bleeding, enoxaparin may be partially reversed with protamine if it was given within 8 hours. Protamine can only reverse 65–70% of enoxaparin at most [5]. Protamine neutralizes anti factor IIa bound to the LMWH-antithrombin complex completely but only variably to factor Xa bound to the LMWHantithrombin complex [3, 5].

## **3.3 Warfarin**

## *3.3.1 Mechanism of action*

Warfarin has been used around since the early 1930s but it was not used clinically until the 1950s. Warfarin is the oldest oral anticoagulant around. Warfarin inhibits vitamin K epoxide reductase (VKOR) that leads to the decrease in production of factors II, VII, IX, and X. These factors depend on vitamin K for carboxylation in order to become active. In addition to these four factors, vitamin K also decrease the production of protein C and S, which also depend on carboxylation to become active [5, 9, 10] (See **Figure 2**).

## *3.3.2 ADME*

Warfarin is present as a racemic mixture of two enantiomers, S-warfarin and R-warfarin. S warfarin is about three times more potent than R-warfarin. The S-warfarin is metabolized by CYP 2C9, whereas R-warfarin is metabolized by CYP 1A1, 1A2, and 3A4. Hence, any metabolic interactions involving these enzymes, especially 2C9, would affect the clinical efficacy and safety of warfarin significantly [9, 10].

#### **Figure 2.**

*Vitamin K cycle showing where warfarin acts and the enzymes that metabolize each enantiomer. Reprint with permission from [16].*

Warfarin is readily absorbed and is almost 100% bioavailable. It has similar volume of distribution as albumin (0.11–0.18 L/kg) and is metabolized by the liver. It is a highly protein-bound drug (>98%) and has a half-life of 36–42 hours (R-warfarin 45 hours, S-warfarin 29 hours). Advances in genetics have elucidated that polymorphisms in the gene that encode VKOR (VKORC1) and CYP2C9 enzyme dramatically affect the dose requirement of a patient [9, 10]. Dose calculators based on the genetic polymorphisms of these two enzymes exist and some of them are available online. How accurate they are is still a question. It should be noted that each patient's warfarin dose requirement does not rely only on genes. Genetics can only account for 30–50% of each patient's dose requirement. Diet, medical condition, and drugs (including supplements) have a role to play as well in determining how much warfarin one needs.

#### *3.3.3 Clinical use*

Clinically, warfarin is used for various conditions such as prevention of stroke and systemic embolism in patients with atrial fibrillation or atrial flutter, treatment and prevention of venous thromboembolism, cerebral venous thrombosis, etc. Clinicians have the most experience with warfarin and warfarin has a wider range of indications than the direct-acting oral anticoagulants (DOACs).

There are several published sample algorithms on initiation and dosing of warfarin. Several institutions also have in-house protocols and dose adjustment guidelines for patients on warfarin. However, due to the multiple factors that can affect warfarin, the protocols may not be necessarily apt to follow. Picking the correct warfarin dose to start patients on and adjusting of warfarin doses subsequently is usually not as simple clinically. Choosing what dose to start patients on require a thorough review of that patient's medical condition, weighing thrombotic risk against bleeding risk, and having considerable experience in managing patients on warfarin.

## *3.3.4 Monitoring*

Upon initiation, it takes about 2–3 days usually to see an increase on the international normalized ratio (INR) and about 5–7 days to see the full effect of warfarin on INR (corresponding approximately to the amount of time it takes for factor IIa to be depleted). A usual starting dose is 5 mg. Smaller doses such as 2 or 3 mg may be given in patients who are elderly or who are expected to have lesser dose requirements (e.g., CKD patients, patients with lighter body weight, presence of drugs that could raise the INR). Due to the delayed effect of warfarin, it may be overlapped with some faster-acting anticoagulants such as heparin, enoxaparin, or DOACs if immediate anticoagulation is needed.

INR is affected mainly by three factors, namely, drugs including natural supplements, patient's medical condition, and diet and lifestyle. These factors need to be considered when dosing a patient and when an explanation for a subtherapeutic or supratherapeutic INR is being sought.

## *3.3.5 Adverse effects*

The most important adverse effect of warfarin is bleeding. Bleeding risk increases when the INR is >4. The risk of bleed is generally <3% annually if INR is kept between 2 and 3 [1]. If bleeding occurs and warfarin has to be reversed, patients should be given IV Vitamin K and Prothrombin Complex Concentrate (PCC). Fresh frozen plasma (FFP) may also be used as an alternative to PCC but it carries some disadvantages such as delayed administration due to thawing, and infusion of large volumes of fluid [9, 10].

Other notable adverse effects of warfarin include skin necrosis, which occurs 3–8 days after initiation of warfarin [5]. Skin lesions appear due to thrombi in the capillaries and veins. They are usually found in areas rich with fatty tissue such as the breast, abdomen, and extremities. Skin lesion may occur in two types of patients. First is in patients treated with warfarin who have active HIT. Second is in patients who have protein S and/or C deficiency. The exact pathophysiology is not known but may be due to the abrupt drop in protein S and/or C before factors IIa, VIIa, IXa, and Xa drops sufficiently that cause the scale to tip over to the prothrombotic side. In such events, warfarin may be started slowly with concurrent heparin. Stop heparin when INR has reached the therapeutic range [10, 11].

Another adverse effect is purple toe syndrome that occurs 3–10 weeks after initiation of warfarin. The exact pathophysiology is not known but may be due to cholesterol deposits embolizing from the arterioles when the patient develops microbleeds in the atherosclerotic arterioles. The cholesterol emboli could occlude the arteries downstream. It can be reversed when it is discovered early and when warfarin is discontinued. Otherwise, it may lead to gangrene necessitating amputation [5, 11–13].

Other less serious adverse effects include osteoporosis, alopecia, etc. [5, 10]. Warfarin has also been associated with acute kidney injury termed warfarin-related nephropathy.

#### **3.4 Dabigatran**

#### *3.4.1 Mechanism of action*

Dabigatran is a competitive direct thrombin (factor IIa) inhibitor. It binds to both free and clot-bound thrombin [1, 5, 10, 14]. This is in contrast to heparin which can only bind to free thrombin. As a result, fibrinogen cannot be converted to fibrin. Parenteral direct factor IIa inhibitors have been available in the market before the introduction of dabigatran. Some examples are argatroban, bivalirudin, etc.

#### *3.4.2 ADME*

Dabigatran has low oral bioavailability of only 3–7% and is a substrate of P-glycoprotein (P-gp). It comes as a prodrug called dabigatran etexilate that gets hydrolyzed to dabigatran. It reaches its peak concentration about 2 hours after ingestion. Dabigatran is 35% bound to protein and is highly (80%) renally excreted. Dabigatran is 50–60% dialyzable, an important point to take note of especially in cases of toxicity. It has a half-life of 12–17 hours and is dosed twice a day [15, 16] (see **Table 1**).

### *3.4.3 Clinical use*

Dabigatran was the first medication under the new class of medications called novel oral anticoagulants (NOAC) or Direct-acting Oral Anticoagulant (DOAC). Dabigatran is approved for stroke and systemic embolism (SSE) prevention for patients with non-valvular atrial fibrillation, treatment of VTE, and VTE prophylaxis post-hip replacement.

Dabigatran is available in three doses: 75, 110, and 150 mg. Some countries, in the United States, for example, only have 75 and 150 mg. As with the other NOACs, dabigatran can be given without the need for any monitoring of anticoagulation intensity. However, renal function must be monitored carefully as dabigatran is highly renally cleared (80% of the drug).

In terms of efficacy as compared to warfarin for SSE prevention, dabigatran 110 mg has similar efficacy to warfarin titrated to an INR of 2–3. Dabigatran


#### **Table 1.**

*Pharmacokinetic and pharmacodynamic properties of the different oral anticoagulants. Reprint with permission from [16].*

#### *Pharmacological Review of Anticoagulants DOI: http://dx.doi.org/10.5772/intechopen.88407*

150 mg, on the other hand, is superior to warfarin titrated to an INR of 2–3. The superiority is mainly driven by a decrease in stroke events among patients taking dabigatran 150 mg. This is based on the RE-LY (Dabigatran versus Warfarin in Patients with Atrial Fibrillation) trial that led to the approval of dabigatran. In terms of bleeding risk, dabigatran 110 mg has lower overall major bleeding risk, similar gastrointestinal (GI) bleeding risk, and lower intracranial hemorrhage (ICH) compared to warfarin. With regards to dabigatran 150 mg vs. warfarin, dabigatran 150 mg has a similar overall bleeding risk, higher rates of GI bleed, and similar ICH risk compared to warfarin. These have to be taken into consideration when choosing which DOAC is more suitable for a patient [17] (see **Table 2**).

In terms of VTE treatment, dabigatran is not inferior to warfarin in terms of efficacy and safety based on the RE-COVER and RE-COVER II studies [17] (see **Table 3**). One caveat in its use for VTE treatment is that it requires a parenteral lead-in of at least 5 days with UFH or LMWH based on how it was carried out in the studies [19].

### *3.4.4 Monitoring*

No pharmacodynamic monitoring is needed for any of the DOACs including dabigatran. Dabigatran has little effect on prothrombin time (PT) and INR. With regards to the activated partial thromboplastin time (aPTT), a normal aPTT does not exclude the presence of dabigatran. More sensitive tests for dabigatran are Thrombin Time (TT), Ecarin Clotting Time (ECT) and Diluted Thrombin Time (dTT) [20, 21] (see **Table 1**).

## *3.4.5 Adverse effects*

Dabigatran, being a blood thinner, has bleeding as its main side effect. Careful consideration of the bleeding risk factors of the patients needs to be done prior to prescribing dabigatran 150 mg twice daily. History of prior GI bleeding, peptic ulcer disease, colonic angiodysplasia, and other pathologies that predispose a patient to GI bleeding are some practical issues that the prescriber needs to be mindful of. Dyspepsia, abdominal pain, and abdominal discomfort are other side effects that occurred more frequently compared to warfarin [5, 16]. The RE-LY trial has raised concerns about increased myocardial infarction (MI) risk among patients taking dabigatran but the US Food and Drug Administration (FDA) has not found an increase MI risk among patients taking dabigatran in its observational study [22]. Because dabigatran is highly renally cleared, the serum creatinine and creatinine clearance has to be regularly monitored.

### **3.5 Rivaroxaban**

## *3.5.1 Mechanism of action*

Rivaroxaban is the first direct factor Xa inhibitor [16]. It therefore prevents the formation of factor II to factor IIa. It acts one step higher on the coagulation cascade compared to dabigatran. Rivaroxaban is able to bind to both free and clot-bound factor Xa due to its small size (436 g/mol) [10].

## *3.5.2 ADME*

Rivaroxaban has good bioavailability of 66% when taken without food. The bioavailability dramatically increases when it is taken with food to 80–100%.



#### **Table 2.**

*Characteristics, efficacy, and safety data of warfarin vs. DOACs for stroke and systemic embolism prevention in patients with non-valvular atrial fibrillation. Adapted from [31] under Creative Commons (CC BY) Attribution 4.0 International License.*

Rivaroxaban reaches its peak concentration about 2–4 hours after ingestion. Rivaroxaban is highly protein bound at 95%. Two-thirds of the drug is degraded by the liver via CYP3A4 and CYP3A5, and CYP2J2 to a lesser extent, half of which is then excreted renally and the other half is excreted by the hepatobiliary route into the feces. The remaining one-third of the drug is excreted renally. Rivaroxaban is a P-gp substrate both at the gut and at the kidney, hence

#### *Pharmacological Review of Anticoagulants DOI: http://dx.doi.org/10.5772/intechopen.88407*


#### **Table 3.**

*Efficacy and safety data of DOACs venous thromboembolism treatment. Adapted from [18] under Creative Commons (CC BY) Attribution 4.0 International License.*

drug–drug interaction between rivaroxaban and P-gp substrates, inhibitors, or inducers must be taken note of. The half-life of rivaroxaban is about 5–9 hours for the younger patients. It has a longer half-life of 11–13 hours among the elderly [15, 16] (see **Table 1**).

#### *3.5.3 Clinical use*

Rivaroxaban comes in various strengths of 2.5, 10, 15, and 20 mg tablets. It is approved for stroke and systemic embolism prevention in patients with nonvalvular AF, VTE treatment and prophylaxis including patients who had Total Knee Replacement (TKR) and Total Hip Replacement (THR), and for cardiovascular risk reduction among patients with Coronary Artery Disease (CAD) or Peripheral Artery Disease (PAD) in the United States.

For SSE prevention in patients with non-valvular AF, rivaroxaban has demonstrated non-inferiority both in efficacy and safety compared to warfarin. Careful examination of the data from the Rivaroxaban versus Warfarin in Nonvalvular Atrial Fibrillation (ROCKET AF) trial shows that rivaroxaban has higher GI bleeding rates but lower ICH rates compared to warfarin [23] (see **Table 2**).

For VTE treatment, rivaroxaban is non-inferior as well compared to warfarin (see **Table 3**). However, the initial treatment dose is 15 mg twice daily × 3 weeks followed by 20 mg daily [18]. The reason for the twice daily dosing is likely due to the need for a higher concentration for clot resolution and prevention of further propagation, knowing that the risk of VTE occurrence is highest during the first 3–4 weeks [24].

At a dose of 2.5 mg twice daily in combination with aspirin and either clopidogrel or ticlopidine, rivaroxaban has shown small 16% relative risk reduction, or 1.6% absolute risk reduction (NNT = 63) for death due to cardiovascular causes, MI, or stroke. This is at the backdrop of higher bleeding events (NNH = 83 for TIMI major bleeding events not associated with CABG) [25]. Patients subjected to rivaroxaban 2.5 mg twice daily need to be carefully selected.

#### *3.5.4 Monitoring*

No routine laboratory monitoring is needed for rivaroxaban for its pharmacodynamic effect. Rivaroxaban may affect PT/INR depending on the reagent used; elevated PT/INR may signal the presence of the drug but rivaroxaban cannot be excluded even if the PT/INR is normal [15, 20, 21]. Rivaroxaban may affect aPTT but less so compared to PT/INR. The best test to measure for the effect of the drug is via anti-factor Xa assay [20, 21], which is not routinely available in most hospital laboratories. Patient's serum creatinine and creatinine clearance should be periodically monitored to see if dose adjustment might be needed.

#### *3.5.5 Adverse effects*

Bleeding is the adverse effect of utmost concern among patients started on rivaroxaban. Patients with history of GI bleeding should select other oral anticoagulants besides rivaroxaban due to its higher GI bleed risk when studied against warfarin.

#### **3.6 Apixaban**

#### *3.6.1 Mechanism of action*

Similar to rivaroxaban, apixaban is also a direct factor Xa inhibitor [16, 26]. It thus inhibits one step higher on the coagulation cascade compared to dabigatran. It binds to both free and clot-bound factor Xa.

#### *3.6.2 ADME*

Apixaban has a bioavailability of 50% and is an active drug itself. It is also a substrate of P-gp. It reaches peak concentration 2–4 hours after ingestion. It is

#### *Pharmacological Review of Anticoagulants DOI: http://dx.doi.org/10.5772/intechopen.88407*

highly protein bound as well at 87%. Apixaban is cleared both via renal and nonrenal pathway. Fifty percent of the drug is excreted into the feces unchanged, 27% is also excreted in the urine unchanged. The remaining (~25%) undergoes metabolism by CYP3A4 and by other minor CYP enzymes such as CYP1A2, 2J2, 2C8, 2C9, and 2C19. It has a half-life of 9–14 hours [15, 16, 19, 26] (see **Table 1**).

## *3.6.3 Clinical use*

Apixaban is approved in the US for SSE in patients with non-valvular AF, VTE treatment and prophylaxis, including patients' post-TKR and post-THR, and for longterm recurrent VTE prophylaxis. It comes in two strengths of 2.5 and 5 mg tablets.

In the Apixaban versus Warfarin in Patients with Atrial Fibrillation (ARISTOTLE) trial, apixaban demonstrated superiority over warfarin titrated to an INR of 2–3 with relative risk reduction of 21%, absolute risk reduction of 0.33% giving an NNT of 303. This statistically significant result is driven mainly by a decrease in hemorrhagic stroke rates. In terms of bleeding events, apixaban demonstrated 31% less bleeding compared to warfarin, mainly driven by decrease in ICH and bleeding other than GI bleeding rates. Of note, apixaban has similar GI bleeding rates versus warfarin. Apixaban is used at a dose of 5 mg twice daily for SSE prevention (see **Table 2**). The dose is reduced to 2.5 mg twice daily if two out of the three following criteria are met: weight of ≤60 kg, serum creatinine of >1.5 mg/dl, or age of ≥80 years old according to the drug label. It should be noted that patients with CrCl of <25 ml/min are not included in the trial and hence other countries do not allow use of apixaban in patients with CrCl of <25 ml/min [27]. However, in the US, apixaban can be used at a dose of 2.5 mg twice daily even in patients with end-stage renal disease, including patients on dialysis, based on pharmacokinetic modeling.

For the treatment of VTE, apixaban is non-inferior to warfarin but has lower bleeding episodes (see **Table 3**). Apixaban is started at a dose of 10 mg twice daily for 7 days followed by 5 mg twice daily [19, 26]. Similarly, apixaban should be avoided in patients with CrCl of <25 ml/min as these patients were excluded in the trial.

### *3.6.4 Monitoring*

Apixaban does not require any laboratory monitoring as well. PT/INR is even less sensitive to apixaban compared to rivaroxaban. Anti-factor Xa assay specifically calibrated to apixaban is a sensitive test that could detect the presence and could give the quantity of the drug present in the sample. However, anti-factor Xa assay for DOACs are not readily available in most hospital laboratories [20, 21, 26].

### *3.6.5 Adverse effects*

Patients on apixaban need to watch for bleeding, though apixaban has a better safety profile than warfarin.

### **3.7 Edoxaban**

#### *3.7.1 Mechanism of action*

Edoxaban is also a direct factor Xa inhibitor, like rivaroxaban and apixaban [16].

## *3.7.2 ADME*

Edoxaban is 62% bioavailable and is a P-gp substrate as well. Peak concentration occurs 1–2 hours after ingestion of edoxaban. Edoxaban is cleared both renally and

non-renally in a 50-50 manner. Fifty percent of the drug is metabolized hepatically via hydrolysis. Only 4% of the drug is metabolized by CYP3A4. The remaining 50% of the drug is excreted renally. It has a half-life of 10–14 hours [15, 19, 28] (see **Table 1**).

#### *3.7.3 Clinical use*

Edoxaban is approved both for SSE prevention in patients with non-valvular AF and VTE treatment. In the Edoxaban versus Warfarin in Patients with Atrial Fibrillation (ENGAGE AF-TIMI 48) trial, edoxaban has shown to be non-inferior to warfarin for SSE prevention [28] (see **Table 2**). However, a separate analysis that was published shows that at CrCl of >95 ml/min, edoxaban is not as protective as warfarin against SSE. Patients with CrCl >95 ml/min have lower edoxaban concentration likely due to higher clearance of the drug among those with CrCl of 95 ml/ min [29]. Hence, edoxaban is not approved by the US FDA for use among patients with CrCl of >95 ml/min. In terms of bleeding episodes, edoxaban has lesser bleeding risk than warfarin. Edoxaban comes in 30 and 60 mg doses. The 30 mg dose is used if patients have CrCL of 30–50 ml/min, weighs ≤60 kg, or is on verapamil, quinidine, or dronedarone (medication that are strong P-gp inhibitors) [28].

For the treatment of VTE, edoxaban is non-inferior to warfarin in terms of efficacy and has a lesser bleeding occurrence (see **Table 3**). There is no FDA recommendation whether it should be avoided in patients with CrCl > 95 ml/min due to lack of studies. However, it would seem prudent to also do the same for patients with VTE [18].

#### *3.7.4 Monitoring*

Edoxaban does not require monitoring like other DOACs. Similar to rivaroxaban and apixaban, PT/INR has low sensitivity towards edoxaban's pharmacodynamic effect and is therefore not a good laboratory test to check on the presence of the drug. Anti-factor Xa assay calibrated to edoxaban remains to be the most sensitive test for edoxaban so far [20, 21, 28].

#### *3.7.5 Adverse effects*

Edoxaban has demonstrated lesser bleeding risk compared to warfarin in the clinical phase III studies.

### **4. Conclusion**

The use of anticoagulants requires holistic evaluation of the patient and careful balancing of the thrombotic and bleeding risks of the patient. Understanding the pharmacology, pharmacodynamics, pharmacokinetics, and clinical evidence behind the use of these drugs will help the clinician in selecting the best therapy for the patient.

#### **Conflict of interest**

The author has no conflict of interest to declare.

#### **Notes/thanks/other declarations**

None.

*Pharmacological Review of Anticoagulants DOI: http://dx.doi.org/10.5772/intechopen.88407*

## **Author details**

Hobart Owen Ng Tsai Khoo Teck Puat Hospital, Singapore

\*Address all correspondence to: hngtsai@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## **Chapter 3**

## Update on Mechanisms, Pathogenicity, Heterogeneity of Presentation, and Laboratory Diagnosis of Heparin-Induced Thrombocytopenia

*Jean Amiral and Anne Marie Vissac*

## **Abstract**

Heparin-induced thrombocytopenia (HIT) is the most life-threatening adverse effect of heparin therapy and is provoked by the development of drug-dependent antibodies. It occurs more frequently in patients with cardiac or orthopedic surgery or severe circulatory diseases, and the risk depends on the patient pathological status. As heparin is an anticoagulant used for treating thrombotic events or their risk, this iatrogenic complication has a paradoxal effect as it can induce thromboembolic diseases, frequently associated to severe morbidity or fatal outcomes. Diagnosis involves clinical evaluation of disease probability and laboratory tools for testing the presence of heparin-dependent antibodies with immunoassays or their capability to activate platelets with functional assays. Antibodies developed when stoichiometric complexes of platelet factor 4 (PF4) with heparin are formed during therapy. In few cases non-platelet factor 4 antigens can be involved. Antibodies can remain asymptomatic, but pathogenicity occurs in the presence of high concentrations of IgG isotype antibodies, with high avidity: they target and activate platelets or endothelial cells exposing heparin-PF4 (HPF4) complexes and produce thrombocytopenia and sometimes thrombosis. Risk factors which favor the development of antibodies and their pathological effect are discussed. The present understanding of mechanisms underlying disease development and diagnostic strategies of this heparin adverse effect is presented.

**Keywords:** platelet factor 4, heparin, antibodies, pathogenicity, thrombosis, thrombocytopenia, diagnosis

## **1. Introduction**

The major adverse effect of heparin therapy concerns probably the development of thrombocytopenia and thromboembolic complications, which are directly caused by the drug itself [1–6]. This heparin paradox is associated with a characteristic platelet fall and thrombosis in some heparin-treated patients, especially when unfractionated heparin (UFH) is used, but it can also occur with low molecular weight heparin (LMWH) therapy. The patient's clinical context can favor the

development of this iatrogenic complication, called heparin-induced thrombocytopenia (HIT) without or with associated thrombosis (HITT). When this complication occurs, it requires an immediate management with the withdrawal of heparin and use of an alternative anticoagulant [7–11]. If incorrectly managed, it can rapidly cause severe burden and become life-threatening. This complication is reported to occur in about 1–5% of patients treated with UFH and 0.2–0.5% of those treated with LMWH, but the incidence highly depends on the clinical context [1, 3, 5, 6]. Cardiology or orthopedic surgery, trauma, circulatory diseases, and the presence of tumors are increased risk factors for that disease. A recent meta-analysis in the USA reported a different HIT/HITT incidence and clinical association than that usually accepted but shows that it remains a critical clinical issue in hospitals [5]. The first alert signal for HIT is a platelet count drop by more than 30–50% between two successive measures, occurring between 5 and 15 days following the onset of heparin therapy, in the absence of any other thrombocytopenia cause (**Table 1**). However, platelet fall can develop earlier if patients have been exposed to heparin during the 3 months preceding the treatment. The mechanisms producing HIT involve the generation of a heparin-dependent antibody, usually of the IgG isotype (but IgA and IgM isotypes can also be present). This antibody has been demonstrated to be targeted to complexes of heparin and platelet factor 4 (PF4) in most of the cases [12, 13], but non-PF4 antigens can be present in some atypical patients [14–17]. Frequently, heparin-dependent antibodies, including IgG isotypes, are asymptomatic [18, 19]. They are symptomatic and harmful only in a few subsets of affected patients. What renders the antibodies pathogenic in those patients is not totally understood, but some evidence becomes available. IgG isotypes present at high concentration, and with high avidity, provoke frequently the development of disease [19, 20]. Clinical diagnosis and laboratory diagnosis are of high importance to rapidly identify the patients with an active disease and treat them [6, 8, 21–24]. It includes a multiple strategy approach:


It is of essence to duly characterize patients with HIT: if this complication is excluded, it is not necessary to deprive them from heparin, as it is the most effective anticoagulant in many acute conditions. Conversely, if HIT/HITT is confirmed, it is


*Update on Mechanisms, Pathogenicity, Heterogeneity of Presentation, and Laboratory Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.86475*

mandatory to not reintroduce any heparin treatment and to switch to an alternative anticoagulation. In this book chapter, we will present and discuss the following: (a) the present understandings of conditions which can favor development of heparindependent antibodies in heparin-treated patients; (b) why antibodies generate HIT or HITT only in a few subset of patients; (c) the mechanisms of action of these antibodies, as they are presently understood; (d) the available laboratory tools and their indications; (e) the diagnostic strategy for rapidly characterizing patients at risk; (f) the occurrence of atypical presentations of HIT in patients with pre-existing antibodies to PF4 or to interleukin 8 (IL8) or with antibodies to protamine sulfate (PrS); and (g) cross-reactivity of the various polysaccharide anticoagulants in immunoassays and functional methods. This chapter mainly focuses on antibodies generated to heparin-PF4 (HPF4) complexes, which concern most of the patients with HIT/HITT, but other non-PF4 antigens can be involved in few cases and will be rapidly discussed.

### **2. Development of heparin-induced thrombocytopenia**

Heparin-dependent antibodies develop in many patients treated with UFH or LMWH. Their incidence is higher in patients undergoing extracorporeal circulation (ECC) for cardiopulmonary bypass (CPB) or extracorporeal membrane oxygenation (ECMO) [5, 26, 27]. Antibodies' development is not rare during heparin therapy, but they are often of the IgM isotype with a rapid reversal without any clinical incidence [18, 19]. They can also be of the IgA or IgG isotypes, and the three isotypes are associated in many patients and are frequently asymptomatic. IgGs have been demonstrated to be those which can become pathogenic, especially when present at high concentration, with high affinity for their target heparin-dependent antigen [19, 25]. A subset of the IgG isotype heparin-dependent antibodies can then activate platelets [20], which induce thrombocytopenia, platelet aggregation, and sometimes thrombosis. HIT was first characterized for the white thrombus formed in arteries (platelets and white blood cells), when patients with this complication were first identified, but there is evidence now that arterial (about 30% of cases) or venous (about 70% of cases) thrombosis can occur [28]. Skin necrosis at the injection site or elsewhere, or thrombosis, frequently at limb extremities, is often observed, but thrombosis can occur at many different sites.

In addition to platelet activation, heparin-dependent antibodies can induce activation of endothelial cells (ECs) and of monocytes, and they can release tissue factor (TF) from these cells, which contribute to thrombosis [29–31]. Plateletleukocyte aggregates are also formed and contribute to thrombogenicity. When this multiple blood activation process is initiated, it is enhanced at pathological sites where platelet and white blood cells can be chemo-attracted and accumulate with a high density. If blood activation and prothrombotic process are strong enough to overwhelm the antithrombotic body's defenses, thrombus formation occurs. The first clinical warning for HIT is the occurrence of thrombocytopenia, with a characteristic time kinetics from the onset of therapy, when other causes of decreased platelet counts are excluded [6, 25, 28]. Platelet fall typically occurs between 5 and 15 days following the initiation of treatment, as shown in **Figure 1**, except if patients already received heparin within the 100 preceding days or in the rare cases with pre-existing anti-PF4 antibodies. Thrombocytopenia can then develop earlier and possibly just at the onset of heparin therapy. In HIT/HITT thrombocytopenia is usually moderate, between 20 and 100 giga platelets per liter (G/L), and it is rarely very severe (<10 G/L). When it develops, the clinical probability for HIT/HITT

#### **Figure 1.**

*Typical platelet count kinetics in heparin-treated patients who develop heparin-dependent antibodies responsible for heparin-induced thrombocytopenia.*


#### **Table 2.**

*The pretest probability for HIT based on the 4Ts score.*

must be evaluated. It is an important criterion for estimating the risk to develop this complication in heparin-treated patients, and various pretest methods for estimating disease risk are available. The most frequently used is the 4Ts score [28], which considers four major criteria: the presence of thrombocytopenia, the timing of platelet count fall, the occurrence of new thrombosis or sequelae, and the investigation of other causes of thrombocytopenia. For each criterion, a score from 0 to 2 is given, as shown in **Table 2**. It allows to classify patients from 0 to 8 (risk is low for 0–3, intermediate for 4–5, and high for ≥6, indicating an elevated disease probability). In cardiology patients with ECC, thrombocytopenia is frequently observed, and HIT can be identified when a biphasic platelet count kinetics is present: in the absence of HIT, thrombocytopenia is progressively corrected, but, if present, platelet count starts to increase and falls again when symptomatic antibodies develop [27].

*Update on Mechanisms, Pathogenicity, Heterogeneity of Presentation, and Laboratory Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.86475*

Other HIT clinical evaluation approaches have been proposed (such as the expert score), but the 4Ts score remains the most widely used. When HIT is suspected, heparin treatment must be stopped and replaced by another anticoagulant. The possible drugs which can be used include argatroban, direct oral anticoagulants (DOACs), danaparoid sodium, fondaparinux, and bivalirudin [7, 9–11, 32, 33]. Nevertheless, if HIT is excluded, heparin can be reintroduced, as it can be of full benefit for the patient, especially in cardiac surgery and circulatory diseases. Establishing rapidly a safe and reliable diagnosis of HIT is then of essence for the right management of patients [6, 25, 28].

## **3. Heparin-dependent antibodies in clinical settings**

HIT/HITT occurs in some of the patients who develop heparin-dependent antibodies, a major risk factor for the disease occurrence. In most of the cases, they are targeted to stoichiometric complexes of heparin and PF4 (HPF4) and are of the IgG isotype but are the only ones present in few patients with atypical HIT/HITT antibodies to IL8 or to PrS [12, 14, 15, 17]. In rare cases, the antibody specificity remains non-identified, although patients present the suggestive clinical complication of heparin therapy. What causes the heparin-dependent antibodies' generation is not yet fully understood, but drug immunogenicity tends to develop when heparin forms complexes with its high-affinity binding blood protein, PF4, a chemokine from the CXC family [34–36], and eventually IL8 [37, 38]. In healthy individuals, PF4 is normally present at very low concentrations in blood circulation (<10 ng/ml). It is released from platelets' α-granules upon activation or aggregation, as a complex of eight PF4 tetramers with a platelet proteoglycan dimer, with a molecular weight (MW) of about 350 kDa. This complex is rapidly cleared from circulation as PF4 is captured by endothelial cells' glycosaminoglycans (GAGs) and

#### **Figure 2.**

*At the onset of heparin therapy, TFPI and PF4 "storage pool" are displaced from endothelial cells and released into blood circulation. Heparin complexes with PF4, and this can stimulate the immune system (especially if heparin and PF4 stoichiometric concentrations are met), and antibodies to these complexes are generated.*

remains in this endothelial storage pool. In patients with inflammation or blood activation, PF4 concentrations can be much higher, either in blood circulation or on the endothelial storage pool. In addition, at pathological sites, platelets and white blood cells can be chemo-attracted and stimulated. Much higher PF4 concentrations can be present at these sites. At the onset of heparin therapy, PF4, which has a higher affinity for this drug than for GAGs or physiological proteoglycans, forms complexes with it, as presented in **Figure 2**. In some circumstances these complexes can activate the immune system and induce the generation of antibodies. The immune response can be innate, mediated via the toll-like receptors, and adaptive with a T cell-mediated response, followed by the generation of antibodies. The three isotypes (IgG, IgA, or IgM) can be present [13, 19], but IgGs are formed very rapidly, which is unusual in the early stage of the immune response, and IgGs can become rapidly pathogenic [19]. In rare cases, only IgA (especially in patients with cancer) or IgM isotypes are identified [39]. Following heparin treatment cessation, antibodies disappear from blood circulation within about 3 months. The respective concentrations of PF4 and heparin in blood circulation or at pathological sites are key factors for inducing immunogenicity [40, 41]. The clinical context is then a risk factor for heparin-dependent antibodies' development. Another initial cause which can favor generation of antibodies has been described and concerns a previous exposure of patients to bacterial infections [42]. PF4 can complex with bacterial polysaccharides and then becomes immunogenic. The immune response induces generation of antibodies to this chemokine. When patients with this former stimulation receive heparin, PF4 released from endothelium forms HPF4 complexes which reactivate the immune system (**Figure 1**), and IgG isotypes are rapidly generated. In addition to PF4, heparin treatment (and more especially LMWH) can also release tissue factor pathway inhibitor (TFPI) bound to ECs into blood circulation. No immune reaction to TFPI has been observed until now, but its increased concentration contributes to elevate the anticoagulant activity of heparin at the beginning of treatment.

#### **4. Heparin-dependent antigens in HIT**

The major heparin-dependent antigen involved in HIT/HITT is PF4, a CXC chemokine present in platelet α-granules and released upon platelet activation and aggregation. PF4 is a 70 amino acid (AA) protein with a MW of 7800 kDa, released in blood circulation as a tetramer with a MW of about 30 kDa [34–36, 43]. This chemokine has a structure involving one α-helix and three β-sheets organized in an antiparallel manner; it is highly electropositive, with many lysine and arginine residues, and has two disulfide bridges per monomer. The tetramer is organized in such a way that it exposes an external ring of positive charges, as shown in **Figure 3**. The formation of HPF4 complexes depends on the respective concentrations of heparin and PF4 [13, 24, 44]. Stoichiometric complexes are formed at a concentration of about 150 μg of heparin (i.e., about 27 IU UFH) per mg of PF4 (**Figure 4**). High- and low-affinity heparin molecules have the same reactivity with PF4, as well as LMWH, and the sulfation grade is of essence for these interactions. Patients who develop antibodies are those with the highest extracellular concentrations of PF4 in blood circulation, or at pathological sites, and with heparin concentrations permitting the formation of stoichiometric HPF4 complexes.

If heparin treatment is given through continuous infusion, heparin concentration remains constant in blood circulation, and the risk to form stoichiometric HPF4 reactive complexes is reduced. When heparin is given through the subcutaneous route, blood concentrations present high variations, from <0.1 IU/ml at trough to >0.7 IU/ml at peak. For current curative UFH treatments (2–3 injections/day),

*Update on Mechanisms, Pathogenicity, Heterogeneity of Presentation, and Laboratory Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.86475*

#### **Figure 3.**

*Reaction of heparin with PF4 tetramers at stoichiometric concentrations. There is an intimate interaction between the ring of positive charges on the PF4 tetramer and the negative charges of the sulfated polysaccharide, heparin. This strong interaction induces an alteration of PF4 structure, rendering it immunogenic. Heparin (UFH or LMWH) molecules with at least 12 monosaccharides are required for this interaction.*

#### **Figure 4.**

*PF4 is released from platelets as a complex with a proteoglycan dimer and is displaced by heparin for which it has a higher affinity. Complexes of heparin and PF4 depend on their respective ratios. When heparin and PF4 are at a stoichiometric concentration, large multimolecular complexes are formed and can be exposed on platelets or other blood cells. They can bind heparin-dependent antibodies and focus the deleterious immune reaction onto these cells. This can induce HIT or HITT in some patients.*

the PF4 concentrations needed for forming stoichiometric complexes must be of about 4 μg/ml for heparin concentrations ≤0.1 IU/ml (trough) or of ≥28 μg/ml for heparin concentrations ≥0.7 IU/ml (peak). Required PF4 concentrations are high comparatively to expected heparin concentrations in blood circulation, even in

disease states, but these high concentrations could be present at pathological sites. In ECC, blood heparin concentrations are high (about 4–5 IU/ml) and constant: formation of stoichiometric complexes can only occur with 25–30 μg/ml of PF4, which is unlikely. For information, the total amount of PF4 releasable from platelets, when they are totally activated and aggregated, is of about 5 μg per ml of blood (depending on platelet count and PF4 content; it is of ±12.5 ng/106 platelets).

But PF4 can accumulate and be at higher concentrations at pathological sites. Immunogenic stimulation occurs when body detects a non-self-component, which can be heparin used as anticoagulant. When bound to PF4, it forms large complexes, which can activate the immune response, which is targeted to these complexes and possibly extended to PF4 itself, through epitope spreading. Generated antibodies can be considered as alloantibodies. In few cases, PF4 antibodies can be pre-existing chronically or generated transitory as a side response to an infectious disease [14, 42, 45]. Anti-PF4 autoantibodies can bind to HPF4 complexes formed during heparin therapy and are then targeted to platelets or other blood cells which expose HPF4 complexes, focusing the deleterious immune response [22, 29, 30, 46]. In few cases, non-PF4 antigens can be involved [14, 15, 17, 46, 47]. HIT/HITT presentation and disease kinetics are then frequently atypical, although a moderate or characteristic thrombocytopenia develops during heparin therapy. IL8 has been reported in some patients as another heparin-dependent antigen in HIT/HITT. Anti-IL8 antibodies are pre-existing in many patients with chronic inflammation and are generated as a regulatory response to control this pathological context. Pathogenicity can occur because IL8 can bind heparin, and these complexes are fixed onto platelets and other blood cells through IL8 receptors (IL8-RA and IL8-RB) or through direct heparin binding [37, 38]. Interestingly, heparin binding to platelets increases with their activation grade. Anti-IL8 antibodies then focus the immune response deleterious effects to blood cells exposing heparin IL8 complexes which are then activated or destroyed. Neutrophilactivating peptide 2 (NAP-2), the β-thromboglobulin precursor, is another platelet CXC chemokine reported as a possible heparin-dependent antigen in rare HIT cases [14]. Lastly, in patients undergoing ECC [16, 26, 27], heparin is used as anticoagulant and is neutralized with a defined concentration of PrS at the end of the process. Anti-PrS or anti-heparin-PrS antibodies have been the only ones identified in few patients treated with heparin and presenting with a HIT-/HITT-like syndrome [17], with a possible fatal outcome. These antibodies can activate platelets in the presence of heparin [15, 46, 47]. Recent investigations have shown that anti-PrS antibodies are rather frequent in patients receiving this drug for heparin neutralization, but only very few of them develop severe clinical complications. Recurrent ECC in the same patient, with various exposures to heparin and PrS over time, can be an increased risk for development of antibodies and associated pathogenicity, with a HIT-/HITT-like syndrome.

## **5. Pathogenicity and mode of action of heparin-dependent antibodies**

Heparin-dependent antibodies, and especially those to HPF4 complexes, induce thrombocytopenia and thrombosis in some clinical circumstances [46]. Particularly IgG isotypes can activate platelets, ECs, or other white blood cells such as monocytes, when they bind to their target antigenic structure, present at the surface of these cells [28, 29, 44, 46, 47]. There is now evidence that heparin and HPF4 complexes bind to platelets' surface, and this binding increases with their activation grade. HPF4 complexes fix antibodies and target the immune response, provoking platelet activation, aggregation, and interaction with other blood cells. During the process, IgGs react with platelet CD32, which is the FcɣRIIa receptor [44, 46]. This contributes to amplify platelet activation and aggregation. The CD32 surface density is an

*Update on Mechanisms, Pathogenicity, Heterogeneity of Presentation, and Laboratory Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.86475*

important factor for the amplitude of platelet activation induced by antibodies. In patients with platelets presenting a CD32 polymorphism (131 Arg-His), activation is enhanced: the 131-Arg-His heterozygous or 131-His-His homozygous CD32 phenotypes are more reactive than the 131-Arg-Arg one. The patient propensity to develop HIT or HITT can depend on platelet activation grade and density or polymorphism of CD32. Antibodies to HPF4 can activate ECs and monocytes, favoring the release of TF, a potent procoagulant starter [29, 30]. In patients with HIT/HITT, neutrophils are activated and form aggregates with platelets, which can be detected in blood circulation. Therefore, the presence of anti-HPF4 IgG antibodies initiates multiple abnormal activities in blood circulation, which induce platelet activation and destruction and a concomitant prothrombotic risk (**Figure 5**). Blood activation can be out of control from body's antithrombotic defenses, which are overwhelmed, and thrombosis occurs. Interestingly, thrombosis tends to occur at pre-existing pathological sites, where blood activation and inflammation are already activated, and the risk is greatly amplified by anti-HPF4 antibodies, as summarized in **Figure 4**. We have the experience that an additional factor is very important for the initiation and amplification of the pathological process. This concerns the antibody avidity for HPF4 complexes [20]. In three patients with HIT or HITT, we succeeded to separate anti-HPF4 IgGs into two groups: the most important (>90%) one had a low affinity for HPF4 and no or only a weak platelet activation capacity, while the minor one (≤10%) activated highly platelets, as evidenced with the C14-serotonin release assay. In few cases, only IgA isotypes specific for HPF4 complexes were identified in patients with HITT and malignant diseases. Although rare, IgAs can be pathogenic in some autoimmune disorders [49, 50], and this is not unexpected to note their effect in HIT. More rarely, IgM can be present at high concentration in patients with HIT, without IgGs. The mechanisms involved are not totally understood, but recently it was demonstrated that anti-HPF4 IgM antibodies can activate complement and induce platelet destruction [39]. Altogether, the different activities described here above help to understand why HPF4 antibodies, including IgG isotypes, can remain

#### **Figure 5.**

*Scheme showing how heparin-dependent antibodies, targeted to HPF4 complexes, bind to platelets and endothelial cells but also to monocytes and induce platelet and EC activation, monocyte stimulation, release of TF, and formation of aggregates, all contributing to thrombocytopenia and thrombosis.*

asymptomatic in many patients and produce (especially IgG isotypes with high HPF4 affinity) HIT or HITT only in a few of them. The pathogenic process is multifactorial and involves activation and interaction of various blood cells, with the prothrombotic activity of TF. Patients' pathophysiological history and clinical status provide additional risk factors for the occurrence of disease [5, 6].

Nevertheless, there is still a fortuity context for the occurrence of the HIT/HITT complication, which relies on the formation of the immunoreactive HPF4 complexes, requiring defined concentrations of PF4 and heparin, exposed on blood cells [48]. This is a pre-requisite condition for permitting the binding of antibodies and starting the pathogenic process. This explains why this disease develops so rapidly when the critical conditions are met.

#### **6. Diagnosis of heparin-induced thrombocytopenia**

Many different assays are available for the diagnosis of heparin-dependent antibodies and for testing their capability to activate platelets. They are classified into two groups: immunoassays [23, 25, 51, 52], developed following the discovery of PF4 as the major target heparin-dependent antigen, and functional assays, performed with a low and a high heparin concentration, which were already used before [53]. A murine monoclonal antibody (KKO) has been developed and mimics HIT-associated antibodies, with platelet activation capability [54]. Here below we discuss the laboratory methods, and their combination, for the diagnosis of HIT/HITT. Diagnosis combines the clinical probability pretest with laboratory investigations [25]. For laboratory testing, the specimen used is plasma or serum for immunoassays and citrated plasma or heat-inactivated serum for functional assays. These techniques provide a laboratory support to establish, confirm, or exclude the diagnosis of HIT/HITT and must always be used in association with the pretest clinical probability. When HIT is suspected with a characteristic thrombocytopenia, heparin must be discontinued and replaced with another anticoagulant.

#### **6.1 Immunoassays for heparin-dependent antibodies**

With the discovery of the major target antigen for heparin-dependent antibodies, i.e., HPF4 complexes, immunoassays were developed, optimized, and standardized [23, 24, 52]. The first immunoassay introduced was a two-site enzyme-linked immunosorbent assay (ELISA), for measuring antibodies to HPF4 [12]. The antigen, HPF4, is coated on the plate, which is then saturated and stabilized. A well-defined stoichiometric concentration of PF4 tetramer and heparin (about 150 μg heparin per mg PF4) must be used for presenting epitopes reactive with antibodies. Heparindependent antibodies can be caught from the diluted tested plasma or serum (usually a 1:100 dilution is used), during the first incubation step. Following a washing step, the immunoconjugate, specific for human immunoglobulins or their isotypes, is introduced, and a second incubation step is performed. The immunoconjugate is often a rabbit or goat antibody, specific for human whole immunoglobulins (IgGAM) or for only an isotype (IgG, IgA, or IgM), and labeled with peroxidase. In current practice, this tag reagent is an antihuman IgG-peroxidase conjugate. Following a new washing step, the substrate is introduced, and a color develops. Tetramethylbenzidine (TMB) with hydrogen peroxide (H2O2) is now the most often used substrate, producing a blue color, which turns yellow when the reaction is stopped with sulfuric acid. Absorbance is measured using a microplate reader at 450 nm. Different variant methods have been introduced. Heparin can be replaced with another sulfated polymer (electronegative) such as polyvinyl sulfonate. However, using heparin matches better

#### *Update on Mechanisms, Pathogenicity, Heterogeneity of Presentation, and Laboratory Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.86475*

with the context of antibody generation and in vivo pathogenicity. Magnetic latex particles can be used in place of the solid phase capture micro-ELISA. Different tag antibody labels can be used instead of peroxidase, such as alkaline phosphatase (with its appropriate substrate). The "enzyme-substrate" detection system with chemiluminescence or fluorescence can also be used (direct measurement). Combining latex magnetic particles and chemiluminescence or fluorescence allows immunoassay automation. Lastly, performing immunoassays in the presence of an excess of heparin allows confirming antibody specificity [25, 55].

**Figure 6** shows the general immunoassay principle for detecting heparin-dependent antibodies. For testing the non-PF4 antigen-dependent antibodies, similar immunoassays can be designed by replacing PF4 with the concerned protein (e.g., IL8 or PrS). We developed an original patented approach, where heparin in excess is coated in the presence of PrS and remains biologically available. The tested patient's sample is then incubated in the presence of a concentrated platelet lysate (containing all the platelet releasable proteins, but not plasma factors). If antibodies are present, a ternary complex is formed between tetrameric PF4 (or eventually another platelet protein), immobilized heparin, and antibodies. Caught antibodies are then detected as previously described [24]. This method offers a kinetic model for testing antibodies and mimics their binding to heparin-protein complexes bound onto platelet or blood cell surfaces. This assay reflects better the mechanisms occurring in pathology and offers improved and optimized sensitivity and specificity.

#### **6.2 Platelet activation methods for disease confirmation**

Functional assays rely on testing the capability of heparin-dependent antibodies to activate platelets at a low (0.1–1.0 IU/ml) and a high (10–100 IU/ml) heparin concentration. In HIT/HITT, platelets are only activated at the low heparin

#### **Figure 6.**

*General principle of immunoassays used for heparin-dependent antibodies, either globally or for specific isotypes. Enzyme tag with substrate is used in ELISA. Chemiluminescent immunoassays, using magnetic latex particles, can be automated on immunological analyzers. Using heparin in excess in sample diluent allows confirming antibody specificity.*

concentration. Functional assays need to use normal donor platelets, freshly prepared. They must be duly selected for the right reactivity. This is the constraint which limits the use of this technique. Platelets are used as platelet-rich plasma (PRP) or as washed platelets. What induces donor to donor responsiveness in platelet activation assays used for HIT antibodies is not totally understood. The CD32 platelet density or His polymorphism could favor reactivity. In practice, platelets need to be qualified with a known positive sample for their appropriateness. Frequently, platelets from four normal donors are used, and the assay is positive if at least two out of the four donors give a positive platelet activation test. Other factors can regulate platelet activity, and interestingly washed platelets are usually more reactive than PRP. This can be explained by some platelet activation induced by the washing process, and a higher amount of PF4 is present on platelet surface. Functional assays concern PAT, SRA, heparin-induced platelet activation (HIPA), and flow cytometric assays (FCA); but other assays have been reported and elegantly reviewed in 2017 [53]. PAT is a simple aggregation assay performed with PRP and the tested patient citrated plasma. SRA is performed with washed platelets labeled with C14, incubated with tested patient's plasma, and released C14-serotonin is measured. HIPA is also performed with washed platelets, incubated with the tested sample, and platelet activation/aggregation is visually evaluated. FCA is a technique that requires to mix PRP (washed platelets are possible) with patient's citrated plasma and to measure platelet activation through the expression

#### **Figure 7.**

*Scheme showing the algorithm for the diagnosis of HIT/HITT: when suspected (thrombocytopenia and/or thrombosis), the disease diagnosis involves the clinical probability estimation and laboratory testing, first with immunoassay, which allows ruling out disease when not present, and then with a confirmatory functional assay.*

*Update on Mechanisms, Pathogenicity, Heterogeneity of Presentation, and Laboratory Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.86475*

of P-selectin [24]. FCA can also be used for the measurement of antibody-induced release of platelet microparticles. SRA is considered as the reference and most sensitive method. PAT has a poor sensitivity. HIPA needs trained laboratory operators and is mainly used in Germany and some neighboring countries. FCA is now a more standardized approach and looks promising but needs to be confirmed through practical experience in clinical laboratories. This method can be available in many centers for testing in emergency, provided a flow cytometer and fresh platelets are available.

#### **6.3 Diagnostic approach for HIT/HITT**

The diagnosis of HIT/HITT must be done accurately and reliably for a safe management of concerned patients [25]. The first alert signal is thrombocytopenia occurring 5–15 days following the onset of heparin therapy or earlier if the patient had a previous exposure to that drug within the 3 preceding months. HIT, or HITT if thrombosis is present, is then suspected and must rapidly be confirmed. If this complication is excluded, patients can continue to receive heparin, the most effective anticoagulant in many critical clinical situations. If the disease is confirmed or cannot be excluded, or if HIT is suspected but the diagnosis cannot be conducted, heparin must be replaced by another anticoagulant, according to the clinical context and practitioners' experience. **Figure 7** shows an algorithm for establishing or excluding the diagnosis of HIT. When HIT is suspected, the pretest clinical probability must be evaluated with the 4Ts method or another one in use in the clinical setting [28]. The 4Ts score is simple and relatively well-standardized. When HIT/ HITT is suspected, heparin is immediately stopped, and another anticoagulant is used to avoid any risk of severe complication. Nevertheless, the diagnosis must be established and confirmed, as the patient can need heparin later. The first laboratory investigation involves immunological testing for antibodies. If the test is negative, and the clinical probability is low or moderate, HIT can be excluded. But if clinical probability is high, HIT cannot be excluded and remains possible with non-PF4 heparin-dependent antigens involved. If positive, antibodies are present. HIT develops mainly when IgGs are generated and present at high concentration. Many authors consider that HIT occurs when the optical density (OD) in ELISA is >1.00 (the cutoff value for the positive range being at ≥0.5). When the IgG immunoassay is positive, a functional assay must be performed for confirming the diagnosis, as many heparin-dependent antibodies are asymptomatic. This functional assay must be as sensitive and specific as possible. In any case if clinical probability is high, the possibility of HIT complication remains present, whether the laboratory testing is. Testing must be repeated [56], and other antigens than HPF4 can be investigated.

### **7. Cross-reactivity of the various heparins, heparin-like compounds, or danaparoid sodium**

HIT/HITT is associated with UFH or LMWH therapy, both drugs being sulfated polysaccharides. Other heparin-like anti-FXa anticoagulants, such as fondaparinux or danaparoid sodium, do not generate drug-specific antibodies [7, 10]. However, cross-reactivity of these drugs with antibodies present in patients with characterized HIT/HITT can be observed in laboratory assays [57]. This cross-reactivity has been reported for danaparoid sodium when it is tested in the immunoassay at a high concentration in the presence of PF4 (about 3.00 mg danaparoid sodium per mg of PF4). This can be due to the high-affinity heparan sulfate component present in this drug, which represents about 4% of the total. Cross-reactivity has also

been reported in functional assays. However, there is no evidence that danaparoid sodium can generate drug-induced antibodies, and cross-reactivity is opposed by the other non-affinity components (about 80% low-affinity heparan sulfate, 12% dermatan sulfate, and 4% chondroitin sulfate), present in large excess, which disrupt the possible complexes formed, as do other low-sulfated polysaccharides [9, 58]. Therefore, there is no evidence that danaparoid sodium can provoke HIT/ HITT, and the reported results and long-term clinical experience in many countries suggest that cross-reactivity is totally inhibited by the major non-affinity fractions. Furthermore, danaparoid sodium at therapeutic concentrations can inhibit the heparin-induced platelet aggregation. Conversely, pentosan polysulfate was found to be as effective as heparin and to form complexes with PF4 at similar ratios than UFH, for binding all heparin-dependent antibodies [8, 14]. Lastly, fondaparinux is not expected to induce drug-dependent antibodies or to cross-react with existing antibodies [32].

#### **8. Conclusions and perspectives**

In this chapter we have reviewed the present understanding of the generation of heparin-dependent antibodies in UFH- or LMWH-treated patients, which are the primary cause for HIT/HITT and a major adverse effect of heparin therapy. This risk is much higher when UFH is used, and disease develops more frequently in some clinical situations including cardiac or orthopedic surgery, traumatology, or malignancy. The occurrence of HIT/HITT tends to decrease thanks to a better control of therapy with UFH, shorter treatment times, and the use of LMWH when possible. When heparin therapy needs to be stopped, a large panel of alternative anticoagulants is available, although in some applications heparin remains the most effective one.

The mechanisms, which can induce generation of heparin-dependent antibodies, and pathogenicity for some of them have been extensively described and discussed in literature [14, 19, 20, 22, 42, 46]. Immunization develops when defined concentrations of heparin and PF4, forming stoichiometric multimolecular complexes, are present. In vivo, immunogenic complexes with PF4 can also be formed with other polyanions such as polyphosphates [59]. Various isotypes can be generated, IgM, IgA, or IgG, but almost all clinical complications of this iatrogenic disease are reported with IgGs present at high concentration and with high affinity. For HIT/HITT pathological development, various patient-associated and fortuity factors are required. Stoichiometric HPF4 complexes must be present for stimulating the immune system and developing antibodies but also for expressing pathogenicity. Heparin-dependent antibodies are harmful only if they bind to target antigenic structures (mainly HPF4 stoichiometric complexes), present on platelets, ECs, or other blood cells, focusing the immunological response. The immune system is then deviated from its protective role and destroys the patient's own cells [60].

HIT/HITT diagnosis is of essence for confirming or excluding this disease, and heparin treatment can be continued if the risk is ruled out. The first step when thrombocytopenia and/or thrombosis occur is to suspect this heparin adverse effect and to evaluate the pretest clinical probability. The 4Ts score is frequently used and allows risk classification from 0 to 8, 6–8 being the highest risk. Concomitantly, performing an immunoassay allows to detect and to measure IgG heparin-dependent antibodies targeted to HPF4. If the assay is negative and if clinical probability is low or moderate (score ≤ 5), HIT can be excluded, but patients need to be monitored closely, especially if thrombocytopenia is not corrected. If positive, IgG heparin-dependent antibodies are present, and HIT probability is higher if ELISA OD ≥ 1.00. Finally, the use of functional assays allows differentiating asymptomatic antibodies from those

*Update on Mechanisms, Pathogenicity, Heterogeneity of Presentation, and Laboratory Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.86475*

which can activate platelets and provoke disease. The presence of HIT or HITT is confirmed when the functional assay is positive [60]. However, even when negative, if clinical probability is high (6–8), HIT/HITT remains possible, and patients must be managed accordingly. Heparin cannot be continued in any patient with a possible or probable HIT diagnosis, and an alternative anticoagulant must be used.

## **Acknowledgements**

The authors would like to thank the research groups with whom they had the opportunity and honor to collaborate and especially Profs. D. Meyer (Paris, France), A. Greinacher (Greifswald, Germany), T. Bakchoul (Tübingen, Germany), S. Panzer (Vienna, Austria), M. Poncz and G. Arepally (Philadelphia, USA), J. Fareed and J. Walenga (Chicago, USA), Y. Gruel and C. Pouplard (France), I. Elalamy (France), I. Gouin and P. Guéret (France), and all those who gave us the opportunity to investigate HIT.

## **Author details**

Jean Amiral1 \* and Anne Marie Vissac2

1 Scientific-Hemostasis, Andresy, France

2 Hyphen-BioMed, Neuville sur Oise, France

\*Address all correspondence to: jean.amiral@scientific-hemostasis.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[52] Greinacher A, Potzsch B, Amiral J, Dummel V, Eichner A, Mueller-Eckhardt C. Heparin-associated thrombocytopenia: Isolation of the antibody and characterization of a multimolecular PF4-heparin complex as the major antigen. Thrombosis and Haemostasis. 1994;**71**(2):247-251

[53] Minet V, Dogné JM, Mullier F. Functional assays in the diagnosis of heparin-induced thrombocytopenia: A review. Molecules. 2017;**22**:617

[54] Arepally GM, Kamei S, Park KS, Kamei K, Li ZQ, Liu W, et al. Characterization of a murine monoclonal antibody that mimics heparin-induced thrombocytopenia antibodies. Blood. 2000;**95**:1533-1540

[55] Zheng G, Streiff MB, Takemoto CM, Bynum J, Gelwan E, Jani J, et al. The clinical utility of the heparin neutralization assay in the diagnosis of heparin-induced thrombocytopenia. Clinical and Applied Thrombosis/ Hemostasis. 2018;**24**(5):749-754

[56] Omer T, Mullaguri N, George P, Newey CR. False-negative platelet factor 4 antibodies and serotonin release assay and the utility of repeat testing in the diagnosis of heparininduced thrombocytopenia and

thrombosis, Hindawi case reports. Hematology. 2019;**2019**:1-4. DOI: 10.1155/2019/1585014

[57] Pouplard C, Amiral J, Borg JY, Vissac AM, Delahousse B, Gruel Y. Differences in specificity of heparindependent antibodies developed in heparin-induced thrombocytopenia and consequences on cross-reactivity with danaparoid sodium. British Journal of Haematology. 1997;**99**:273-280

[58] Joglekar MA, Quintana Diez PM, Marcus S, Qi R, Espinasse B, Wiesner MR, et al. Disruption of PF4/H multimolecular complex formation with a minimally anticoagulant heparin (ODSH). Thrombosis and Haemostasis. 2012;**107**:717-725

[59] Cines DB, Yarovoi SV, Zaitsev SV, Lebedeva T, Rauova L, Poncz M, et al. Polyphosphate/platelet factor 4 complexes can mediate heparinindependent platelet activation in heparin-induced thrombocytopenia. Blood Advances. 2016;**1**(1):62-74

[60] Greinacher A. Heparin induced thrombocytopenia. The New England Journal of Medicine. 2015;**373**:252-261

## **Chapter 4**

## Examination of Laboratory for Monitoring Heparin Anticoagulant Therapy

*Yetti Hernaningsih and Ersa Bayung Maulidan*

## **Abstract**

Heparin-derivative anticoagulants include unfractionated heparin (UFH), low molecular weight heparin (LMWH), pentasaccharide (fondaparinux), and ultralow molecular weight heparin (ULMWH). Heparin contains an active pentasaccharide sequence that binds to antithrombin (AT). This bond produces conformational changes that accelerate its binding with AT and inactivation of coagulation factors XIIa, XIa, Xa, and IXa and thrombin (IIa). Thrombin and factor Xa are the most sensitive to inhibition by the heparin-AT complex, and the strength of inhibiting thrombin is ten times more sensitive than factor Xa. The UFH anticoagulant response is monitored using activated partial thromboplastin time (APTT), a measurement that is sensitive to inhibition of thrombin and factor Xa. Protamine titration examination is the standard for measuring UFH concentrations in plasma. Recommendations from the American College of Chest Physicians (ACCP) suggest that the APTT target range for the UFH therapy is equivalent to 0.2–0.4 IU/mL with protamine titration or 0.35–0.7 IU/mL with an anti-Xa examination. A new examination is thrombodynamics (TD), measuring the level of development of clots. This method is considered most able to mimic the coagulation process that occurs in vivo compared to other examinations.

**Keywords:** heparin, anticoagulant, PPT, APTT, anti-Xa, protamine, thrombodynamics

## **1. Introduction**

At present the use of anticoagulants is very wide; about 0.7% of the population in the west receives anticoagulant treatment. Basic anticoagulant therapy is a vitamin K antagonist; a derivative of warfarin, which is most commonly used, is coumadin (warfarin). This drug has been used for more than 50 years and is consistently able to eliminate recurrent venous thrombosis at adequate doses. However, warfarin has disadvantages, namely, the interaction with other drugs and with food, slow onset and excessive effects, and a narrow therapeutic range. Drug responses and pharmacodynamics are varied and unpredictable, so routine monitoring is needed. For most patients who take drugs in the long term, this is quite troublesome [1].

The current world medical need is to find anticoagulants that are more effective and safer than warfarin for both doctors and patients in long-term use. Responding to this need, a new drug is needed that can change molecules that are difficult to absorb to be easily absorbed through the digestive tract; this is used as the basis for making oral preparations of unfractionated heparin (UFH). In theory the use of the oral form of heparin or low molecular weight heparin (LMWH) is given at fixed doses, two or three times a day, and does not require overly frequent coagulation monitoring checks or dosage adjustments which are too tight, and the potential for interactions between drugs and medications is also low, making this drug an anticoagulant needed for long-term use [1].

Coagulation monitoring for patients receiving heparin therapy is very important. This is intended to obtain a range of heparin therapy that is effective in reducing the incidence of thrombus and bleeding. The effective use of heparin anticoagulant therapy must increase the activated partial thromboplastin time (APTT) value from 1.5 to 2.5 times. This value is equivalent to levels of heparin 0.2–0.4 U/mL based on protamine titration and is equivalent to anti-Xa levels 0.3–0.7 U/mL [2]. This chapter will discuss laboratory tests that are used to monitor patients receiving heparin therapy.

### **2. Development of heparin**

Heparin is the oldest anticoagulant used in medicine. Heparin was discovered by McLean in 1916 while trying to isolate thromboplastic agents. Heparin is a polysaccharide from the class of glycosaminoglycans (GAG) which naturally appears on all mast cells. Further research in 1935 resulted in clinical use of heparin. Since then, heparin has been widely studied for various applications and modifications [3].

Unfractionated heparin (UFH) is a product of GAG purified from animal tissue, most often from pig intestines. Heparin provides indirect anticoagulant properties by binding to antithrombin III (ATIII) and facilitating the inhibitory effects possessed by AT on thrombin and activated X factor (factor Xa). It is known that only UFH contains at least 18 saccharide sequences that can affect AT activity and thrombin, whereas UFH with a series of certain pentasaccharides can inhibit the activity of factor Xa [3] (**Figure 1**).

The heterogeneity of the structure of the UFH causes extensive bioactivity and physiological activity. Some heparin chains bind to other plasma proteins and have an effect on bone metabolism resulting in osteoporosis or heparin-induced thrombocytopenia (HIT) and other unpredictable effects that require continuous monitoring. Further research and discoveries resulted in low molecular weight heparins (LMWH) in the late 1970s to early 1980s; this was to find anticoagulants which were more predictable in their activities [4]. LMWH, such as enoxaparin, dalteparin, and tinzaparin, is made by chemical control or enzymatic cutting of UFH in a depolymerization reaction.

This controlled process produces fragments with lower molecular weight and more predictable action than UFH. As a result, side effects are lighter than UFH, monitoring needs are decreased, and bioavailability increases, making LMWH potentially used for outpatients. This makes LMWH the standard of care replacing UFH except in certain cases such as kidney failure and acute coronary syndrome where UFH is still preferred because the liver clearance is lighter and better reversible with protamine sulfate [5].

Ultralow molecular weight heparin (ULMWH) was discovered in early 2000 through a process of chemical synthesis. The reason is to get agents with lighter side effects but have the same or better anticoagulant effect which causes a higher antifactor Xa ratio to antithrombin activity [6].

*Examination of Laboratory for Monitoring Heparin Anticoagulant Therapy DOI: http://dx.doi.org/10.5772/intechopen.88401*

**Figure 1.**

*Mechanism of heparin in the coagulation cascade. Box A: AT (red) binds to a heparin fragment (green) with any series length provided that certain pentasaccharide bonds can inhibit factor Xa. Box B: AT (red) binds to heparin (green) with a chain length > 17 U of disaccharide that can inhibit thrombin (factor IIa) [3].*

#### **3. Structure and biosynthesis of heparin**

Heparin is a polydisperse and highly sulfated GAG with a molecular weight between 5 and 40 kDa. The structure of the complex contains repetitive disaccharide units that contain uronic acid residues (l-iduronic (IdoA) or d-glucuronic acid (GlcA)) and N-acetyl-d-glucosamine. The biosynthesis process of heparin starts in the endoplasmic reticulum and the Golgi apparatus of mast cells. The tetrasaccharide link attaches to the residue of serine in the core protein, serglycin, and then adds a unit of d-glucuronic acid (1→4) N-acetyl-d-glucosamine disaccharide. Disaccharide sulfonation and epimerization of glucoronate to iduronate are carried out by various enzymes in the biosynthetic pathway. In total there are 12 enzymes involved in this pathway, which act together to form the desired molecule. These involved enzymes have many isoforms, which cause heterogeneity of heparin and allow these enzymes to directly biosynthesize associated GAG, heparin sulfate. The degree of sulfation and sulfate residue allocation depends on the spectrum of activity of the product. In mast cell degradation, peptidoglycan heparin changes to GAG heparin through protease and β-endo glucuronidase activity [3].

The first glycosaminoglycan-protein bonding region is formed due to glycotransferase activity. Repeated disaccharide units undergo elongation by GlcA and GLcNAc transferase. Modified chains include N-deacetylation and N-sulfonation, O-sulfonation, and epimerization, which then occur due to specific enzyme activity. The monosaccharide symbol in this figure follows the symbol nomenclature for glycan (SFNG) system [3] (**Figure 2**).

*Heparin biosynthesis.*

### **4. Mechanism of heparin as an anticoagulant**

Heparin has anticoagulant effects through interactions with coagulation factors and inhibitors. Coagulation is a complex process involving proteins, platelets, and cellular components such as endothelium and monocytes. The balance of hemostasis is maintained if the activity of procoagulant can be balanced with an inhibitor. The main inhibitor of plasma coagulation factor is AT, which acts on active coagulation factors such as FXIIa, FXIa, FXa, FIXa, FVIIa, and FIIa. These coagulation factors are serine proteases, so AT is a serine protease inhibitor (serpin).

Heparin, acting as a catalyst, provides anticoagulation activity by potentiating other AT and serpin inhibitor activities (**Figure 3**). The interaction of heparin with AT requires a certain sequence of pentasaccharides, but not so with other serpins.

#### **Figure 3.**

*Interaction of the coagulation factor with serpin. The blue line shows the place of inhibition of each serpin [11]. TFPI, tissue factor pathway inhibitor; AT, antithrombin; HCII, heparin cofactor II.*

#### *Examination of Laboratory for Monitoring Heparin Anticoagulant Therapy DOI: http://dx.doi.org/10.5772/intechopen.88401*

Another important heparin-binding inhibitor on the extrinsic pathway is tissue factor pathway inhibitors (TFPI) [7–9].

Antithrombin (AT) under conditions when it does not bind to heparin is a slowacting serpin, because the reactive center loop is partially folded to the center of the b-sheet structure. When binding to heparin, AT inhibition activity will increase significantly, which is a characteristic of serpin. The high-affinity pentasaccharides in heparin bind to antithrombin through two stages: first, the initial binding process involves three monosaccharide units and initiates conformational changes to AT, and then after the interaction is complete, AT conformation after activation by heparin is stabilized. This conformational change is transmitted to the AT structure, causing the opening of the reactive center loop and an increase in exosite exposure from AT which binds directly to FXa [10].

### **4.1 Inhibition of antithrombin in factors IIa and Xa**

Active AT conformation caused by pentasaccharides is sufficient to increase inhibitory activity in FXa, a protease that converts prothrombin to thrombin (FIIa). Factor Xa interacts directly with AT in specific exosite exposed when binding to heparin. In addition, in the presence of calcium, a single heparin chain can bind directly to AT and FXa, increasing AT-FXa interactions, but this is not absolutely necessary. This calcium-dependent effect can account for reports of calcium-induced-specific molecules during the FXa inhibition process by AT [12]. Heparin-mediated FXa binds to AT, indicating the longer heparin chain will increase its affinity with the presence of Ca2+ and will strengthen inhibition [13].

Unlike FXa, the potentiation of thrombin inhibition by AT requires an additional 13 saccharide chains attached to the nonreductive end of the pentasaccharide sequence, so thrombin and AT are bound to the same heparin molecule. Thrombin interacts with heparin in exosite II, which is basically a different method with AT; no specific heparin is needed for this interaction [14].

#### **4.2 Inhibition of antithrombin in other coagulation factors**

Antithrombin also inhibits several other protease coagulant factors. The way antithrombin blocks FIXa is similar to FXa, which binds to the same exosite on AT. The high-resolution crystal structure of the pentasaccharide-FIXa-AT complex shows that one pentasaccharide binds to AT and the second binds to exosite from FXa, which allows a relationship between the two proteins with one molecule of heparin [13].

The ability of heparin to increase AT inhibition in FXI and kallikrein in the intrinsic coagulation pathway is relatively limited compared to FXa and thrombin. It should be noted that FXI activity in AT mutants that bind to the heparin site is still slightly potentiated by heparin, suggesting that there is a direct interaction between heparin and FXI involved in the binding sites of potential heparin found in the FXI catalytic domain [11].

Antithrombin inhibits the FVIIa complex in the extrinsic pathway, and this effect is reinforced by fondaparinux, LMWH, and UFH. Direct and calcium-dependent interactions are found between FVIIa and heparin [11].

#### **4.3 Other serpins activated by heparin**

Heparin cofactor II (HCII), another serpin potentiated by heparin, is a coagulation inhibitor that only inhibits thrombin. HCII is also reported to inhibit chymotrypsin and neutrophil cathepsin G. Heparin cofactor II is found in plasma at the same level as AT, but HCII cannot replace AT if there is a deficiency. HCII deficiency has no effect on the coagulation system but results in increased formation of an occlusive arterial thrombus after endothelial damage. In vivo HCII is potentiated by dermatan sulfate, which is found in the walls of blood vessels. HCII activated by dermatan sulfate may play a role in preventing excessive thrombosis of injured blood vessels [15].

Heparin and dermatan sulfate both potentiate inhibition of thrombin through HCII in several stages. Unlike AT, HCII does not require a certain series of heparin to interact. Other polyanions can also bind to HCII. The HCII bond with heparin causes conformational changes similar to AT while also releasing thrombin-binding N-terminal tail. The combination of reactive center loop expulsion initiated by GAG by exposure to exosite protease-binding is controlled by AT and HCII in the opposite way [16, 17].

Protein C inhibitors (PCI) regulate the activity of activated protein C (APC), which is the active form of zymogen protein C. Protein C is converted to APC by thrombin and in its active form acts as an anticoagulant by inactivating FVa and FVIIIa, in the presence of protein cofactors S. PCI regulates coagulation inhibitors, in this case acting more as a supporter of coagulation than inhibiting coagulation. The bond of heparin to PCI strengthens the inhibition of APC and FXa in the presence of calcium. A long chain of heparin is needed to strengthen APC inhibition, which suggests both PCI and protease require simultaneous bonding with heparin. The basic structure of the PCI complex with heparin and thrombin if separated, binding sites for heparin will appear involving the H helix, which is located close to the reactive loop [18].

The importance of protease nexin (PN)-1 in the last biology and hemostasis is known. In vitro serpin is known to have an effect of approximately 100 times faster than AT, and heparin increases about 3 times. In vivo PN-1 does not contribute to the activity of heparin as an anticoagulant because its concentration in plasma is very low; on the contrary, PN-1 is found to bind to the cell surface in several organs and tissues, including blood vessel walls. Protease nexin (PN)-1 is detected in platelet granules on the platelet surface and secreted during platelet activation. In this context, the contribution of PN-1 to antithrombotic activity from heparin in vivo is ignored [19].

The crystal structure of PN-1 is obtained from the breakdown of complexes with heparin and thrombin. This protein has a typical serpin fold with the heparin-binding site in helix D. Contrary to AT, heparin-binding site PN-1 and thrombin are not parallel when the reactive center loop of PN-1 productively interacts with the active part of thrombin. The initial formation of the ternary complex between PN-1 and thrombin and heparin can be said to be the initial phase of two-phase interaction, with loss of heparin-thrombin interactions when covalent complex PN-1 thrombin is formed. Heparin is not released when forming the PN-1-thrombin complex; this shows that the PN-1-thrombin complex is still bound to HS on the cell surface [11, 20].

Protein Z-dependent proteinase inhibitors are known to inhibit both FXa and FXIa. Heparin speeds up this reaction 20–100×. The heparin-binding site on protein Z-dependent protease inhibitors involves basic residues in helix D (such as AT) and helix C (unlike AT), and the presence of unstructured N-terminal ends can indicate similarities with HCII [21].

Other serpins, c1inh, inhibit both the complement cascade and the intrinsic pathway, where the coagulation system and innate immune system interact. Clinical deficiency leads to congenital angioedema through excessive contact system activity. The effects of c1inh are not limited to complement and contact systems. Heparin potentiates the activity of the c1inh, and the crystal structure of the c1inh indicates

*Examination of Laboratory for Monitoring Heparin Anticoagulant Therapy DOI: http://dx.doi.org/10.5772/intechopen.88401*

a different model of activity against different protease and heparin-binding sites against AT [22].

#### **4.4 Non-serpin inhibitors: tissue factor pathway inhibitors**

Tissue factor pathway inhibitors (TFPI) are structurally serpin, but not serine protease inhibitors. Tissue factor pathway inhibitors (TFPI) are major inhibitors in the extrinsic pathway. The heparin-binding TFPIα isoform contains an acidic N-terminal region, three Kunitz domain pairs, and a basic C-terminal end. Kunitz domain is involved in anticoagulant activity, with the first domain inhibiting the FVIIa-tissue factor complex and the second domain inhibiting FXa. The C-terminal circuit in TFPI has a high affinity for heparin. Heparin injection releases TFPI bound to the endothelium to the circulation. Heparin bound to TFPI potentiates inhibitory activity in both free FXa and FXa in the FVIIa-TF-FXa complex [11].

## **5. The drug derivate heparin**

#### **5.1 Unfractionated heparin**

Unfractionated heparin (UFH) is one of the most commonly used parenteral anticoagulants to treat or prevent thromboembolism and has been used for almost a century. This drug is used in various methods, such as systemic use, through a catheter, extracorporeal, or on the surface of a medical device to prevent thrombotic complications. Heparin depends on the presence of antithrombin (AT) to inhibit clotting factors, so heparin is called an anticoagulant which acts indirectly. Heparin does not have fibrinolytic activity and will not lyse the thrombus [23].

Heparin contains an active pentasaccharide sequence that binds AT. The active pentasaccharide sequence responsible for catalyzing AT is found in one third and one tenth of the UFH and LMWH chains. After heparin binds and activates AT, heparin can release AT and bind other ATs, thus providing a continuous anticoagulant effect. This bond produces conformational changes that accelerate binding of AT and inactivation of coagulation factors XIIa, XIa, Xa, and IXa and thrombin (IIa). Thrombin and factor Xa are the most sensitive to inhibition by the heparin/AT, and tenfold thrombin complex is more sensitive to inhibition than factor Xa [23, 24].

The inhibition of UFH on thrombin requires binding of coagulation enzymes and AT through high-affinity pentasaccharides, whereas inhibition of factor Xa requires only heparin binding to AT. By deactivating thrombin, heparin not only prevents fibrin formation but also inhibits platelet activation induced by thrombin and coagulation factors V and VIII. In addition to its anticoagulation effects, heparin increases the permeability of blood vessel walls, suppresses smooth muscle proliferation, suppresses osteoblast formation, and activates osteoclasts [23, 24].

#### *5.1.1 Pharmacokinetics and pharmacodynamics*

Intravenous (IV) or subcutaneous (SC) injection is the route available for UFH administration, and IV is the most frequently used route. When given by SC injection for therapeutic anticoagulation, the dose must be large enough (30,000 U/day) to compensate for the low bioavailability of UFH, as can be seen in **Table 1**. UFH is already bound to plasma proteins, which results in variations in anticoagulant responses [25].


#### **Table 1.**

*Pharmacological properties of UFH and LMWH [26].*

UFH clearance depends on the dose and occurs through two independent mechanisms. The initial phase is a fast and saturated bond in endothelial cells, macrophages, and local proteins where UFH is depolymerized. The second phase is slower and unsaturated clearance through the kidneys. At therapeutic doses, UFH is cleared mainly through depolymerization, where higher molecular weight chains are cleared faster than those with lower weight. When clearance tends to the kidneys, an increase or extension of the dose of UFH provides a disproportionate increase in both the intensity and duration of the anticoagulant effect. Anticoagulant responses to UFH administration are usually monitored using activated partial thromboplastin time (APTT). APTT must be measured every 6 hours with IV administration and the dose adjusted until the patient has reached a stable level of therapy. After a stable condition is reached, the frequency of monitoring can be extended [24, 26].

#### *5.1.2 Monitoring*

The UFH anticoagulant response is monitored using APTT, a measurement that is sensitive to inhibition of thrombin and FXa. APTT examinations have a large variety of reagents (even the same reagents have different lots) so that they have varying sensitivity to the anticoagulant effect of UFH. Each laboratory must ensure that their therapeutic range of heparin and APTT is based on levels of heparin measured by anti-Xa (target range 0.3–0.7 U/mL) or protamine titration (0.2–0.4 U/mL). APTT must be measured every 6 h based on UFH half-life and the dose adjusted until the patient reaches the therapeutic level based on the APTT target range. When APTT values are obtained in the treatment range twice in a row, monitoring can be extended to one or two times a day depending on the clinical scenario. Weight-based dose nomograms, consisting of bolus doses and infusion droplet speeds with regular monitoring using APTT, are recommended for the treatment of thromboembolic disease [25].

The UFH dose nomogram differs in each hospital due to differences in thromboplastin reagents, calibration, and interlaboratory standards in APTT measurements. This causes the need for alternative monitoring methods. Functional heparin, also known as anti-Xa, has been promoted as a more reliable measure of UFH because it is not sensitive to factors other than UFH, such as concomitant use of warfarin, sodium citrate in the sample tube, impaired lupus anticoagulant (LA), increased factor activity VIII, and liver disease [25].

Acquired inhibitors, such as LA, cause an extension of APTT, which results in not being able to accurately measure the level of anticoagulation due to UFH.

#### *Examination of Laboratory for Monitoring Heparin Anticoagulant Therapy DOI: http://dx.doi.org/10.5772/intechopen.88401*

In this case, if APTT is maintained within the usual therapeutic range, it may result in underdose of UFH and cause development or recurrence of thrombosis. Simultaneous testing of APTT and anti-Xa levels is needed to estimate the APTT value of therapy in patients receiving heparin [27].

### **5.2 Low molecular weight heparin (LMWH)**

LMWH is a polysaccharide derived from the pig's intestine containing an active pentasaccharide sequence which is needed for anticoagulant activity as in UFH. LMWH is produced from UFH through chemical or enzymatic degradation. Each LMWH product is prepared by a different method. Clinical development of LMWH driven by certain observations includes the reduction of thrombin activity in relation to anti-factor Xa activity, the ratio of benefits or risks that are more favorable in animal studies, and good pharmacokinetic properties. The molecular weight of the LMWH is approximately one third of the molecular weight of UFH (4000–5000 Da). Because of their smaller size, LMWH has a lower affinity for thrombin because they cannot bind AT and thrombin together. However, LMWH has the same affinity as UFH for FXa [23].

Factor Xa does not require heparin to stabilize its interaction with AT, so smaller molecules such as LMWH deactivate factor Xa equivalent to larger molecules such as UFH. The length of the polysaccharide chain of at least 18 saccharides, including the order of active pentasaccharides, is needed to bridge between AT and thrombin. About 25–50% of LMWH molecules are above the length of this chain. All LMWH chains contain active pentasaccharide sequences, so 100% can inactivate factor Xa [23].

#### *5.2.1 Pharmacodynamics and pharmacokinetics*

There are several biological consequences of the small size of LMWH compared to UFH, a decrease in binding of LMWH to other plasma proteins, macrophages, and endothelial cells. This resulted in a more predictable dose-response relationship and a longer plasma half-life for LMWH. In contrast to UFH, routine plasma monitoring is not needed which makes it easier for outpatient management. The lower incidence of HIT has also been investigated because of the reduced bond to PF4 and platelets. LMWH has also reduced bonds in osteoblasts resulting in decreased incidence of osteoclast activation and lower bone destruction [23].

All LMWH products have half-lives ranging from 3 to 7 h and bioavailability 87–90%. Anti-Xa peak activity occurs 3–5 h after SC injection with predictable dose-based responses. All agents are metabolized through desulfation or depolymerization, and all agents metabolized are excreted through the kidneys [25].

#### **5.3 Pentasaccharides (fondaparinux)**

Fondaparinux is a chemically synthesized anticoagulant that is specifically developed as a selective indirect inhibitor for FXa. Factor Xa is an important target for anticoagulant therapy given its position at the meeting of the intrinsic and extrinsic coagulation pathways. Its inhibition significantly decreases thrombin formation [28] (**Figure 4**).

Factor Xa has one function in the coagulation cascade, as a gatekeeper to the path along the coagulation cascade. Conversely, thrombin (FIIa) has many roles in the coagulation process, including activation and mediation of endogenous anticoagulation by binding to thrombomodulin and activation of protein C. Factor Xa may be a purer target than thrombin [29].

#### **Figure 4.**

*Formation of antithrombin complexes with FIIa and FXa. (1) Antithrombin (AT), active thrombin (FIIa), active X factor (FXa). (2) UFH (chain shape) forms a complex with AT both in FIIa and FXa. (3) Shorter polysaccharides of 18 U form AT complex with FXa but not with FIIa. (4) The sequence of pentasaccharides forms a bond with FXa only [30].*

#### *5.3.1 Pharmacodynamics and pharmacokinetics*

Fondaparinux binds non-covalently and reversibly to AT, increasing AT anticoagulant activity by up to 300 times. The AT-fondaparinux complex then binds and neutralizes FXa, which reduces prothrombin (FII) conversion to thrombin (FIIa), thereby inhibiting clot formation. Fondaparinux is then released and can catalyze other AT molecules. When AT plasma becomes saturated, excess unbound circulating fondaparinux (which has no anticoagulant activity) is excreted through the kidneys.

Because it does not affect pre-existing thrombin circulation, fondaparinux may in theory have some residual hemostatics, if needed, at the site of injury. Fondaparinux has no effect on the examination of fibrinogen platelet function, thrombin time, or antithrombin tests. Fondaparinux can affect PT and APTT and can interfere with factor VIII testing. Although not routinely recommended, if measurements of fondaparinux are needed (e.g., changes in kidney function, weight, or extreme age), the most accurate plasma concentration is measured using the anti-factor Xa test. The results of this examination are in IU/mL, which is directly proportional to the plasma concentration of fondaparinux. The results were extrapolated to the mcg/mL plasma concentration using a standard curve calibrated by fondaparinux. The test must be calibrated specifically for fondaparinux, because the use of calibration for UFH or LMWH will produce inaccurate results.

#### *Examination of Laboratory for Monitoring Heparin Anticoagulant Therapy DOI: http://dx.doi.org/10.5772/intechopen.88401*

The use of fondaparinux and drugs that affect concomitant coagulation (e.g., antiplatelet, NSAIDs) results in pharmacodynamic drug interactions that can increase the risk of bleeding and should be avoided as much as possible. After stopping fondaparinux, the anticoagulant effect will last up to 4 days and even longer in patients with low clearance [31].

Fondaparinux is not absorbed through the gastrointestinal mucosa, so it must be given parenterally. Subcutaneous administration showed rapid absorption and complete absorption with 100% bioavailability. The peak plasma concentration is reached about 2–3 h after subcutaneous administration. A stable state is achieved after 3–4 doses of administering fondaparinux once a day [25].

Fondaparinux is highly protein bound and cannot be distributed to tissues without binding to proteins. The volume of distribution is 7–11 L, which is close to blood volume. Fondaparinux does not undergo metabolism in the liver and is not susceptible to the pharmacokinetics of drug interactions with the cytochrome P450 isoenzyme system substrate [25].

Reduced bonding with macrophages and endothelial cells increases the half-life of plasma fondaparinux compared to UFH and LMWH. Elimination of fondaparinux is influenced by several patient parameters, including kidney function, age, and low body weight. These factors must be evaluated regularly, because they can block the use of fondaparinux or require increased monitoring for signs and symptoms of drug accumulation [25].

#### **5.4 Ultralow molecular heparin**

Therapy using LMWH provides clear pharmacokinetic advantages over UFH, so LMWH has become the standard of care for prevention and treatment of venous thromboembolism (VTE) in patients with and without cancer. The development of the ULMWH drug is based on the theory that, because of the much higher ratio of anti-Xa and anti-IIa activity, ULMWH will be associated with similar or better antithrombotic efficacy from the efficacy achieved by LMWH products but with lower bleeding and HIT risks. ULMWH has a molecular weight of <4000 Da and an increase in anti-factor Xa activity compared to LMWH. There is only one ULMWH marketed outside the United States (bemiparin), and another, RO-14, is currently in clinical development [32].

#### *5.4.1 Pharmacokinetics and pharmacodynamics*

Bemiparin was approved for once-daily use of subcutaneous VTE primary prophylaxis in medical patients and patients undergoing general or orthopedic surgery and for prophylaxis in patients with deep vein thrombosis (DVT). Bemiparin originates from alkaline depolymerization and UFH fractionation from pig intestinal mucosa. Pharmacokinetic studies in healthy volunteers given bemiparin showed an increase in anti-factor Xa activity depending on the dose given [32].

#### **6. Heparin therapy monitoring**

Treatment using UFH requires routine monitoring to see its functional activity due to the high variation in plasma concentrations and functional activities after fixed doses in each patient. This is due to several reasons, including AT levels in plasma that are different in each patient, UFH elimination with two mechanisms that also differ in each individual, and neutralization and bonding of heparin with various activated plasma proteins and platelets. It is estimated that 10% of people

are not sensitive to heparin. The nonlinear pharmacodynamics of UFH make it difficult to predict. Unfractionated heparin (UFH) is usually measured by APTT or activated coagulation time (for high levels of heparin), but this examination has a low sensitivity and is not standardized; besides, this examination is also not sensitive to the use of low-dose UFH as prophylaxis. The results also depend on the reagent and the instrument used. The reference value for this examination must be determined by each laboratory, so it is difficult to compare the results obtained from each laboratory [33].

Monitoring the therapy of coagulation UFH activity should be measured 6 h after bolus and 6 h after dose change. It takes 6 h for the heparin to reach a stable phase, so monitoring heparin therapy less than 6 h after administration gives the wrong results for determining the dose. Protamine titration is the gold standard for heparin monitoring, but this examination is not widely available and expensive, so it is only used for research [34].

Unlike UFH, LMWH has a more stable pharmacokinetics, and its bioavailability reaches 100% at any dose with subcutaneous administration. The maximum LMWH concentration in plasma is directly proportional to the LMWH dose, and many experts believe that routine coagulation monitoring during therapy is not necessary because the clinical dose of LMWH can be corrected based solely on the patient's body weight. In a healthy population after a fixed dose of LMWH, it turns out that heparin concentration varies and only partially correlates with the patient's body weight. More precisely correction needs to be done in some cases (e.g., low or high BMI; critical condition; kidney failure (creatinine clearance <30 ml/min); when changing anticoagulants; age (children or elderly >75 years); and pregnancy) [35].

Anti-Xa activity measurement is the most widely used examination for LMWH therapy management. This examination has a high sensitivity (lower limit of determination using chromogenic substrate is <0.03 anti-Xa IU/mL). This method is not a method of global coagulation examination because it only measures the concentration of one factor (Xa) but does not react with AT deficiency or changes in concentration from other factors that influence patient hemostasis. This method is difficult to predict thrombosis or bleeding. Until now there is still no universal and reliable method for monitoring heparin properly [30].

#### **6.1 Activated partial thromboplastin time (APTT)**

The APTT examination is currently used by most laboratories for monitoring UFH therapy. This examination uses plasma citrate from the patient and is measured based on clot formation. Phospholipids and activators are added to platelet-poor plasma (PPP) patients and then incubated. Calcium is added and then the clotting time is measured. This examination has several advantages, fast, inexpensive, and widely available, but this does not directly measure the level of heparin. Many APTT reagents are available, and each reagent has a varied response to heparin therapy, and besides that several physiological factors can influence the results of this examination [36].

The clinical condition of patients can also affect the results of APTT, but it does not correlate with the presence of bleeding or thrombosis. The most frequent cases are patients receiving vitamin K antagonist therapy. Patients with international normalized ratio (INR) of more than 1.3 because warfarin therapy can also affect APTT results in heparin monitoring. Other cases that also affected were patients with antiphospholipid antibodies, which could affect clotting tests. Coagulation factor deficiencies, such as in patients with liver disease, or consumptive coagulopathy in disseminated intravascular coagulopathy (DIC) will affect APTT results. These conditions cause heparin anticoagulation activity not to be measured properly [36].

The conditions mentioned above can prolong the APTT results and result in underdose anticoagulant doses. Factor VIII and fibrinogen are the most frequent causes and can significantly extend or shorten the APTT baseline. Patients with acute disease are also known to have a deficiency in antithrombin. This condition can lead to excessive anticoagulation when using APTT for monitoring [36].

Preanalytic variables also play a role in the APTT response in monitoring heparin. The wrong APTT results can be due to improper sampling, and less samples also make too much citrate concentration in the tube. Underdose therapy causes a high risk of thrombosis [37].

Basu et al. [38] conducted a study on patients with venous thromboembolism who received heparin therapy and found the risk of recurrent thromboembolism associated with the APTT ratio which did not reach 1.5–2.5 times the normal value. This ratio is used as a standard for therapeutic ranges. Based on this study, the authors mention that the ratio is equivalent to levels of heparin 0.2–0.4 U/ mL based on protamine titration and is equivalent to anti-Xa levels of 0.3–0.7 U/ mL. The range of anti-Xa levels is higher because of heparin clearance. Smaller heparin molecules are cleared more slowly, so LMWH has a stronger inhibitory effect on factor Xa than thrombin. Examinations that measure anti-factor Xa activity, such as anti-Xa examination, will detect higher levels than examinations that measure antithrombin activity, such as protamine titration, which utilizes thrombin time [30].

Other studies evaluated the upper limit of anti-Xa examinations related to the incidence of bleeding. This study produced anti-Xa levels of more than 0.74–0.88 U/mL related to the incidence of bleeding complications [30]. The use of the APTT standard ratio for the range of heparin therapy is difficult to apply because the response of each APTT reagent to heparin is different. Researchers from the joint study found that APTT corresponds to a concentration of heparin 0.2–0.4 U/mL with protamine titration, based on the APTT reagent used. APTT reagents from other laboratories showed different sensitivity to heparin, and a ratio of 1.5–2.5 times the normal value did not correlate with the concentration of heparin in the therapeutic range. Various types of laboratories and reagents can produce an APTT ratio of 1.6–3.7 times the normal value, which is equivalent to a level of heparin 0.3 U/mL to a ratio of 2.4–6.2 times the normal value equivalent to the level of heparin 0.7 U/mL with anti-Xa [39].

#### **6.2 Anti-Xa activity assay**

Variations in results were also obtained for each anti-Xa examination if compared with protamine titration as a reference but far smaller compared to APTT. The therapeutic range for this examination will remain to be 0.3–0.7 U/mL, although with different machines and reagents. In contrast to APTT, anti-Xa results are not affected by poor sampling, also are not affected by factor VIII or fibrinogen, and are not affected by deficiency factors in patients with liver disease and consumptive coagulopathy [30].

Anti-Xa assay is not a test of factor X activity or factor X level antigen. This examination is also called an anti-factor Xa test or a functional test of heparin. The principle of this examination is to monitor indirect factor Xa inhibitors, such as LMWH and fondaparinux, or direct factor Xa inhibitor drugs such as rivaroxaban. These anticoagulants require monitoring in certain patient populations and in certain clinical settings. Each anticoagulant requires its own anti-X curve, and this must be done by each laboratory [37].

Anti-Xa activity assay uses the chromogenic method. The known factor Xa is added to platelet-poor plasma, wherein there is heparin. Heparin strengthens the inhibition of antithrombin to factor Xa, and the uninhibited factor Xa chromogenic substrate is added. This process produces color detected by a spectrophotometer and directly proportional to the level of factor Xa. The amount of color correlates with the level of heparin in the plasma with the correct heparin curve [36].

Like other tests, the anti-Xa assay is imperfect, with the chromogenic method; this examination will be affected by the conditions of the hemolysis, jaundice, and lipemic samples, which will affect the ability of the machine to measure and distinguish chromogenic reactions. Anti-Xa reagents which are not added with antithrombin will make false low heparin concentrations in the condition of patients with severe antithrombin deficiency, but there can be a misdiagnosis of antithrombin deficiency if antithrombin is added. Viewed from the laboratory side, anti-Xa assay is considered expensive and needs special attention in the process [36].

#### **6.3 Protamine titration**

Protamine is a small protein, rich in arginine, and positively charged, has a similarity to histones, and is involved in folding and stabilizing DNA in sperm heads. Protamine can neutralize the effects of heparin through electrostatic bonds between cation arginine groups from protamine and heparin anions with a ratio of 1:1. This results in a neutral protamine-heparin aggregate which can be seen in the form of a white suspense formed in a few seconds. Binding of heparin by protamine will release the AT-heparin complex, resulting in AT activity returning to its original state [40].

Protamine titration is the gold standard for measuring UFH concentrations in plasma. The results of this examination are quite promising, but it is still not considered a good examination in terms of clinical management of UFH because it is still not automatic. UFH clinical trials determined that heparin concentrations of 0.2–0.4 U/mL with protamine titration were equivalent to APTT lengthening 1.5–2.5 times the normal values, providing safe UFH levels and good patient outcomes [41].

The principle of this examination is to measure thrombin clotting time (TCT), which is the time required for clot formation after the addition of thrombin to plasma. The presence of UFH in plasma will result in TCT prolongation depending on the dose used. Protamine competed in binding to UFH, and the results of protamine titration measurements showed the amount of protamine needed to restore TCT to baseline [42].

This method is neither expensive nor difficult, but because it is done manually, the workload is quite heavy. The results given by this examination are very dependent on two important factors, the operator that works and the origin of thrombin. Protamine titration measurements depend on the operator when identifying clot formation in the test tube, so efforts to standardize this are difficult. The reproducibility of results can be achieved by limiting operators who work on checks and equating perceptions between operators about the process of forming clots and when the test is certain to be completed.

First time this method was found, thrombin used came from rabbits or young cattle. Its concentration is determined by identifying the amount of thrombin which results in TCT 18–20 s. At present the thrombin used is from humans, so it can increase the specificity of this test. Thrombin originating from humans has different interactions with plasma patients than thrombin from rabbits and young cattle. Thrombin originating from humans also needs to be determined by *Examination of Laboratory for Monitoring Heparin Anticoagulant Therapy DOI: http://dx.doi.org/10.5772/intechopen.88401*

calculating the amount of thrombin needed to achieve TCT 18–20 s. Each laboratory must determine the concentration of thrombin that is needed based on thrombin available in each lab [41].

UFH therapy is optimal if the levels are 0.2–0.4 IU/mL based on protamine titration. Recommendations from the American College of Chest Physicians (ACCP) suggest that the APTT target for the UFH therapy range is equivalent to 0.2–0.4 IU/mL with protamine titration of 0.35–0.7 IU/mL with anti-Xa assay [23].

This method is carried out at room temperature, plasma samples and protamine solutions that have been titrated at each concentration, and thrombin stored in ice until it is transferred to the test tube. First take a normal plasma, and leave it at room temperature for 30 s. Then the titration solution is added, and thrombin is then calculated when the clot is formed. The time obtained will be used as TCT baseline. Assay was repeated using a sample of patients with different protamine concentrations starting from the highest concentration first (0.9 IU/mL). After finding the smallest concentration that still provides TCT baseline, this result illustrates the level of heparin in the blood [41].

#### **6.4 Thrombodynamics**

A new examination for the coagulation function, thrombodynamics (TD), is examined under different circumstances. This TD measures the level of clot formation. Coagulation is triggered on the surface of an activator which is bound to a space and extends to the plasma layer. The scattered beam increases in the area formed by the clot, so clot formation and time needed can be measured. This method is considered most able to mimic the coagulation process that occurs in vivo compared to other examinations. This examination is very sensitive to the conditions of either hypocoagulation or hypercoagulation. **Figure 5** shows the

#### **Figure 5.**

*Examination of thrombodynamics simulates the in vivo coagulation process. (a) Plastic cuvette schemes and activators that have been added for TD measurements. (b) Photos of the sequence of clot growth from spontaneous activator in vitro and clot in a portion of the sample. (c) Schematic image of clot growth from damage to blood vessel walls in vivo [44].*

test tube scheme used in TD examination and the formation of clots and compared with the process of clot in vivo [43].

Clot formation curve depends on several parameters obtained: Tlag (time of formation of clot); Vi (initial speed of formation of clots) and V (speed of formation of clots) in clot formation (slope of clot formation curve versus time at 2–6 min and 15–25 min from the initial formation of clots for Vi and V); and CS (clot size 30 min after the coagulation process is activated). Other important parameters that are also measured are the maximum optical density of the formed clot (D) which shows the quality of the clot and the time required for the appearance of spontaneous clot (Tsp). The last two parameters have important clinical values because spontaneous clot (which grows not from activators on the surface) is only found in serious hypercoagulability states [44].

Heparin anticoagulants form complexes with ATIII and inhibit factors II, X, and IX. Research by Sinauridze et al. measure Tlag, Vi, and V parameters in post hip surgery patients who get heparin. The result is only Vi and V have significant different results, whereas Tlag has results that are not much different before and after heparin is given [44].

## **7. Conclusion**

Heparin monitoring is needed to achieve an adequate dose. Laboratory tests for heparin monitoring include APTT examinations especially for UFH, anti-FXa activity assays especially for LMWH, protamine titration especially for UFH, and thrombodynamic tests that better reflect in vivo conditions.

#### **Abbreviations**


*Examination of Laboratory for Monitoring Heparin Anticoagulant Therapy DOI: http://dx.doi.org/10.5772/intechopen.88401*


## **Nomenclature**


## **Author details**

Yetti Hernaningsih\* and Ersa Bayung Maulidan Department of Clinical Pathology, Faculty of Medicine, Dr. Soetomo General Academic Hospital, Universitas Airlangga, Surabaya, Indonesia

\*Address all correspondence to: yettihernaningsih@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## Section 3
