biomedicines

Review Updates on Anticoagulation and Laboratory Tools for Therapy Monitoring of , Vitamin K Antagonists and Direct Oral

Osamu Kumano 1,2,* , Kohei Akatsuchi 3 and Jean Amiral 1

1 Research Department, HYPHEN BioMed, 155 Rue d’Eragny, 95000 Neuville sur Oise, France; [email protected] 2 Protein Technology, Engineering 1, Sysmex Corporation, Kobe 651-2271, Japan 3 R&D Division, Sysmex R&D Center Americas, Inc., Mundelein, IL 60060, USA; [email protected] * Correspondence: [email protected]; Tel.: +81-78-991-2203

Abstract: drugs have been used to prevent and treat thrombosis. However, they are associated with risk of hemorrhage. Therefore, prior to their clinical use, it is important to assess the risk of bleeding and thrombosis. In case of older anticoagulant drugs like heparin and , dose adjustment is required owing to narrow therapeutic ranges. The established monitoring methods for heparin and warfarin are activated partial thromboplastin time (APTT)/anti-Xa assay and pro- time – international normalized ratio (PT-INR), respectively. Since 2008, new generation anticoagulant drugs, called direct oral anticoagulants (DOACs), have been widely prescribed to prevent and treat several thromboembolic diseases. Although the use of DOACs without routine   monitoring and frequent dose adjustment has been shown to be safe and effective, there may be clinical circumstances in specific patients when measurement of the anticoagulant effects of DOACs Citation: Kumano, O.; Akatsuchi, K.; is required. Recently, anticoagulation therapy has received attention when treating patients with Amiral, J. Updates on Anticoagulation coronavirus disease 2019 (COVID-19). In this review, we discuss the mechanisms of anticoagulant and Laboratory Tools for Therapy drugs—heparin, warfarin, and DOACs and describe the methods used for the measurement of Monitoring of Heparin, Vitamin K their effects. In addition, we discuss the latest findings on thrombosis mechanism in patients with Antagonists and Direct Oral Anticoagulants. Biomedicines 2021, 9, COVID-19 with respect to biological chemistry. 264. https://doi.org/10.3390/ biomedicines9030264 Keywords: ; activated partial thromboplastin time; heparin; warfarin; direct oral anticoagulants Academic Editor: Anand Singh

Received: 29 January 2021 Accepted: 3 March 2021 1. Introduction Published: 7 March 2021 Thrombosis disorders require prompt treatment with anticoagulant drugs at therapeu- tic doses. Although these drugs are effective and useful for the prevention and treatment for Publisher’s Note: MDPI stays neutral thrombosis, the drugs are associated with the occurrence of hemorrhage, and therefore, the with regard to jurisdictional claims in identification of patients at increased risk of bleeding is clinically important for selecting published maps and institutional affil- the optimal treatment and duration of anticoagulant therapy. Traditionally, heparin— iations. consisting of unfractionated heparin (UFH), low molecular weight heparin (LMWH), and fondaparinux—is used widely. Heparin binds to antithrombin via its pentasaccharide, catalyzing the inactivation of thrombin and other clotting factors [1]. Warfarin, a vitamin K antagonist used to hamper γ-carboxylation of vitamin K-dependent factors, Copyright: © 2021 by the authors. has also been used historically as an anticoagulant [1,2]. Drug monitoring in clinical labo- Licensee MDPI, Basel, Switzerland. ratory tests is required when using these drugs. In recent years, direct oral anticoagulants This article is an open access article (DOACs) have been developed, including direct factor IIa (i.e., dabigatran) and factor Xa distributed under the terms and inhibitors (i.e., rivaroxaban, apixaban, and edoxaban), which can overcome several of the conditions of the Creative Commons limitations of warfarin treatment, such as food interactions and the need for frequent moni- Attribution (CC BY) license (https:// toring using clinical laboratory tests. DOACs are reported to have a superior safety profile creativecommons.org/licenses/by/ 4.0/). compared with warfarin in some thrombosis disorders [3–10]. Nevertheless, even with the

Biomedicines 2021, 9, 264. https://doi.org/10.3390/biomedicines9030264 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 264 2 of 20

new generation of anticoagulant agents, the most relevant and frequent complication of anticoagulant treatment is major hemorrhage, which is associated with significant morbid- ity, mortality, and considerable costs [11–13]. Although routine monitoring of these drugs is not required, assessment of anticoagulant effect may be desirable in special situations and assessment of the individual bleeding risk might be relevant while considering the selection of the appropriate anticoagulant drug and treatment duration [14]. In addition to the introduction of new anticoagulant drugs, anticoagulation therapy has received attention with regard to treating patients suffering from coronavirus disease 2019 (COVID-19). Patients with COVID-19 associated pneumonia exhibit abnormal co- agulation and organ dysfunction, and coagulopathy based on abnormal coagulation has been associated with a higher mortality rate [15,16]. Thus, a suitable treatment for the coagulopathy and thrombosis is required. This review covers the anticoagulant mechanisms of heparin, warfarin, and DOACs as well as the measurement methods for these drugs. In addition, we attempt to provide the latest information on thrombosis mechanism in COVID-19 from the view of biological chemistry.

2. Anticoagulants 2.1. Heparin 2.1.1. The Properties of Heparin Heparin is one of the oldest biological anticoagulant drugs and its antithrombotic properties were discovered by McLean almost 100 years ago [17]. Heparin is a heterogenous mixture of branched glycosaminoglycans and acts as an indirect anticoagulant, requiring antithrombin (AT). The main anticoagulant action of heparin is mediated by the heparin/AT interaction (Figure1)[ 18]. In the heterogenous mixture of glycosaminoglycans, heparin contains a large number of sulfate groups which are negatively charged in the molecule. The negative charges bind to lysine sites on AT, producing a conformational change at the arginine reactive center, which converts AT from a slow, progressive thrombin inhibitor to a very rapid inhibitor. The arginine reactive center on the AT molecule binds covalently to the active center serine of thrombin and other coagulation enzymes, thereby irreversibly inhibiting their procoagulant activity [1,18]. Heparin has been widely used for the prevention and treatment of venous thrombosis, treatment of thromboembolism and disseminated intravascular coagulation (DIC), and prevention of clotting in artificial dialysis and extracorporeal circulation, et cetera. Two types of heparin are used clinically: UFH with a molecular weight of about 30,000–35,000 and LMWH with a molecular weight of about 4000–6000. The concept that the ability of heparin molecules to inactivate thrombin and other activated coagulation factors is chain length-dependent introduced the development of LMWH, which inhibits factor Xa mainly based on the presence of the high-affinity pentasaccharide [1,19–21].

2.1.2. Biological Properties Unfractionated Heparin UFH is heterogenous with respect to molecular size, anticoagulant activity, and phar- macokinetic properties. Only about one third of an administered dose of UFH binds to AT, and this fraction is responsible for most of its anticoagulant effect [22,23]. The remaining two thirds of the dose has minimal anticoagulant activity at therapeutic concentrations. The heparin/AT complex inactivates thrombin and factors Xa, IXa, Xia, and XIIa. Throm- bin and factor Xa are most sensitive to inhibition by heparin/AT, and thrombin is about 10-fold more sensitive to inhibition than factor Xa [1]. Although UFH binds to the coagu- lation factors and inactivates these enzymes, UFH also binds to several plasma proteins, endothelial cells, and macrophages, a property that further complicates its pharmacokinet- ics [24]. By inactivating thrombin, UFH not only prevents fibrin formation but also inhibits thrombin-induced activation of and coagulation factors [1]. Biomedicines 2021, 9, x FOR PEER REVIEW 3 of 22

Biomedicines 2021, 9, 264 3 of 20

(a) Unfractionated heparin

(b) Low molecular weight heparin

FigureFigure 1. 1. InactivationInactivation of of coagulation coagulation factors factors by by heparin. heparin. Hepari Heparinn binds binds to toantithrombin antithrombin (AT) (AT) through through a ahigh high affinity affinity pentasaccharidepentasaccharide sequence. sequence. AT AT conformational conformational structure structure is is change changedd by by binding binding to to heparin, heparin, and and it itaccelerates accelerates the the interaction interaction withwith thrombin thrombin or or factor factor Xa. Xa. (a ()a Unfractionated) Unfractionated heparin heparin (UFH) (UFH) can can inactivate inactivate both both thrombin thrombin and and factor factor Xa. Xa. High High molecular molecular weightweight heparin heparin is is required required to to bind bind to to thrombin. thrombin. (b (b) )Low Low molecular molecular weight weight heparin heparin (LMWH) (LMWH) catalyzes catalyzes the the inactivation inactivation of of factorfactor Xa Xa by by antithrombin. antithrombin. At At least least 18 18 sa saccharideccharide units units are are required required to to catalyze catalyze the the inactivation. inactivation. LMWH LMWH has has less less inhibitory inhibitory activity against thrombin than UFH. activity against thrombin than UFH.

2.1.2. BiologicalUFH is cleared, Properties with a half-life of 60–90 min, in a nonlinear fashion through the combina- Unfractionatedtion of a rapid Heparin saturable mechanism and much slower first-order mechanisms [25–27]. One of theUFH mechanisms is heterogenous reflects with the binding respect of to UFH molecular to vascular size, endothelial anticoagulant cells, activity, macrophages, and pharmacokineticand reticuloendothelial properties. cells, Only resulting about in one a rapid third andof an saturated administered elimination dose of phase. UFH UFHbinds is depolymerized through this binding to cells [28]. The other mechanism is a slow elimination Biomedicines 2021, 9, 264 4 of 20

phase equivalent to renal clearance. Many endogenous plasma proteins, including histidine- rich glycoproteins, factor 4, vitronectin, fibronectin, and bind to UFH, making it difficult to predict the pharmacokinetics of UFH [29].

Low Molecular Weight Heparin LMWH may, in principle, be prepared by fractionation of UFH or by its depolymeriza- tion. It has an average molecular weight of 5000 Da. The affinity to endothelial cells is much lower than that of UFH, and LMWH does not bind to macrophages or reticuloendothelial cells, and its plasma half-life is 2–6 h, which is 2–4 times longer than that of UFH [30]. The molecules of heparin with <18 saccharides lose their ability to bind simultaneously to thrombin and AT, and therefore, are unable to catalyze thrombin inhibition [1]. In contrast, small heparin fragments, containing the high affinity pentasaccharide sequence, catalyze the inhibition of factor Xa by AT [31,32]. The synthetic pentasaccharide fondaparinux, unlike other products containing mixtures of fragments with different molecular weight, is a homogeneous product with a longer blood half-life, 17–21 h [29,33]. LMWH and fondaparinux have a very low affinity for heparin-binding plasma proteins and are largely removed by unsaturated renal filtration. Thus, LMWH lacks the nonspecific binding affini- ties of UFH, as a result, LMWH preparations have more predictable pharmacokinetic and pharmacodynamic properties.

2.1.2.3. Monitoring of Heparin Two different strategies are commonly used to monitor the therapeutic effects of UFH: the activated partial thromboplastin time (APTT) and the anti-Xa assay. The APTT is a global assay, which reflects both the intrinsic and common pathways of the coagulation cascade, and the clotting time is prolonged in the presence of heparin because heparin in- hibits thrombin and factors Xa, IXa, XIa, XIIa, and VIIa. The anti-Xa assay is a chromogenic assay that measures inhibition of coagulation factor Xa. The assay is a kinetic method based on the inhibition of a constant amount, the reaction occurs in excess of factor Xa and the inhibition to factor Xa by heparin is assayed in the presence of AT. Hydrolysis of a chromogenic substrate specific for factor Xa by the residual factor Xa occurs, which cleaves the substrate. The amount of substrate released is inversely proportional to the concentration of heparin present. A study conducted in 1972 established that the current standard of target value was 1.5–2.5 times greater than the upper limit of the institutional reference range in APTT reagent [34]. However, heparin sensitivity in APTT varies among the reagents and it was reported that appropriate targets ranged from 1.6 to 6.2 times the control value [1]. APTT reagents consist of activators that activate contact factors and phospholipids that serve as coagulation reaction scaffold for clotting factors. Commonly, ellagic acid and silica as activators and animals/plants-derived phospholipids or synthetic phospholipids are used. It was also reported that the sensitivity was largely dependent on the composition of phospholipids and phosphatidyl-serine concentration in total phospholipids used [35]. The APTT test has not been standardized for heparin therapy and target values may need to be adjusted by the manufacturer. The APTT values can be affected by various factors, including pre-analytic (sample collection and processing), analytic (reagent and instrument), and biologic factors (level of clotting factors). Especially, it could be difficult to collect blood for APTT heparin monitoring in an intensive care unit (ICU) because of the sample , citrate anticoagulation concentration, high or low factor VIII, et cetera. Some guidelines recommend that each institution define its own therapeutic range by measuring an APTT value (corresponding to 0.3–0.7 IU/mL by factor Xa activity) used in the laboratory rather than a fixed APTT therapeutic range of 1.5–2.5 times the control value [1,36–38]. The anti-Xa assay is not affected by the above factors like APTT and has been proposed as a more accurate assay to find the concentration. The recommended anti- Xa therapeutic range is 0.3–0.7 IU/mL, and it is not necessary to establish the therapeutic Biomedicines 2021, 9, 264 5 of 20

range with each new lot of reagent because calibration curve would be created in each lot [39]. LMWH is typically administered in fixed doses, for thromboprophylaxis, or in total bodyweight-adjusted doses, for therapeutic effect [1]. Laboratory monitoring is generally not necessary. However, dose-finding trials have not been carried out in special population, such as patients with renal failure or severe obesity. It has been suggested that monitoring should be considered in such patients [40–42]. Although APTT is widely used for UFH, it is generally considered that APTT is unsuitable for LMWH monitoring because of its low sensitivity to LMWH [43]. While the anti-FXa assay is a reliable determinant of blood LMWH concentrations, it does not necessarily correlate well with the actual effect of the drug in vivo but represents the pharmacokinetics [44].

2.2. Warfarin 2.2.1. Properties of Warfarin Warfarin, an important anticoagulant used to prevent and treat thrombosis, was originally derived from dicoumarol, a coumarin that is primarily found in plants, such as sweet clover or melilot [45]. In 1939, the ingestion of sweet clover was identified as the cause of fatal hemorrhages in cattle. It was found by Campbell and Link (1944) that dicoumarol, a compound isolated from sweet clover, was the active hemorrhagic agent. Following this discovery, many synthetic anticoagulants were derived from coumarin [45–47]. Warfarin has an anticoagulant effect due to its structural resemblance to vitamin K (Figure2). Vitamin K is an essential cofactor for γ-carboxylation of glutamate residues on coagulation factors II, VII, IX, and X, which are synthesized by the liver in their biologically inactive form. Carboxylation of these factors is directly related to the oxidation of vitamin K to its epoxide form. Formation of γ-carboxyglutamate residues results in a calcium-dependent conformational change that promotes the binding of cofactors to the phospholipid surface Biomedicines 2021, 9, andx FORresults PEER REVIEW in coagulation. Warfarin can bind to vitamin K epoxide reductase, the enzyme6 of 20

that catalyzes the transformation of vitamin K epoxide back to vitamin K.

(a) warfarin (b) vitamin K

Figure 2. TheFigure chemical 2. The structure chemical structure of (a) warfarin of (a) warfarin and vitamin and vitamin (a) K.(a) K. 2.2.2. Monitoring of Warfarin Warfarin is widely used as an oral anticoagulant. However, it has been reported that it interacts with manyDespite other its therapeutic drugs and usefulness, also with the foods effect thativeness are of rich warfarin in vitamin is largely K anddependent numer- on the patient’s background and numerous other factors, and monitoring is required to ous factorskeep can interferethe therapeutic with warfarinrange for the effects, dose adju suchstment as the to age,control gender, thrombosis bodyweight, and bleeding liver and kidneyrisk. function, For a long genetic time, factors,prothrombin adherence, time (PT) andhas been others used [47 as, 48a monitoring]. Thus, the parameter. therapeutic PT range is narrow,represents and the strict most monitoring commonly isused required. coagulation test and is a single-stage screening test for extrinsic and common pathways. The reagent includes tissue factor, phospholipid, and 2.2.2. Monitoringcalcium of chloride; Warfarin and this mixture is added to plasma, then is converted to Despitefibrin. its therapeutic The test measures usefulness, the time the required effectiveness for clot formation. of warfarin The is result, largely shown dependent as the clotting time, is affected by the activity of coagulation factors II, V, VII, X, and fibrinogen, on the patient’s background and numerous other factors, and monitoring is required to and the prolongation of PT clotting time may be due to one or more coagulation factor keep the therapeuticdefects in extrinsic range and for thecommon dose pathways, adjustment or in todicate control the presence thrombosis of inhibitors and bleeding against risk. For a longthese factors, time, prothrombin including drugs. time Warfarin (PT) hasreduces been the used production as a monitoring of coagulation parameter. factor II, PT representsVII, theIX, and most X and commonly the activity used may coagulationalso be decreased, test and and thus is aPT single-stage is elevated in screeningwarfarin test for extrinsicpatients and and common used as a pathways.parameter in The monitoring reagent tests. includes tissue factor, phospholipid, and calcium chloride;Due to differing and this phospholipid mixture is compositions added to plasma, and sources then of fibrinogen tissue factor, is significant converted to fibrin. Thedifferences test measures occur in the the timeclotting required time result for from clot formation.different PT reagents The result, [49]. shownInitial efforts as the to standardize PT reagents was to establish the PT ratio (patient’s PT/mean normal PT). clotting time,However, is affected the standardization by the activity of ofPT coagulation ratio was unable factors to completely II, V, VII, resolve X, and the fibrinogen, issue of reagents’ variability because the system could not eliminate discrepancies between the reagents [50]. For further standardization, international normalized ratio (INR) was de- veloped, which reflects a mathematical calculation using PT ratio as further adjusted with a correction factor called international sensitivity index (ISI) [51].

INR = (Patient PT/Mean Normal PT) ISI

Mean normal PT is calculated as the measurement value of PT clotting time in normal samples. ISI is a specific value assigned for each reagent by manufacturers. The ISI value is assigned by the method of The World Health Organization (WHO) Expert Committee on Biological Standardization with international reference preparations (IRP) of thrombo- plastins [52,53]. In this method, manually prepared PT with IRP is used for the tests, and commercial PT reagents from manufacturers are assessed and calibrated against the WHO method in 20 healthy subjects and 60 patients with stable conditions undergoing warfarin therapy. The values obtained from the manufactured PT reagent are plotted on the X-axis and the values from the WHO method on the Y-axis. The logarithm of these values is calculated, and the ISI value is obtained from the slope of the regression line in the corre- lation graph (Figure 3) [54]. For the verification of an established PT-INR system with the specific ISI value, it is recommended to measure 20–40 samples from stable patients with warfarin and 20 normal plasma samples [55]. Warfarin is effective for the primary and secondary prevention of venous thrombo- embolism (VTE), prevention of systemic embolism in patients with prosthetic heart valves or atrial fibrillation, prevention of stroke, recurrent infarction, and acute myocardial in- farction [56–59]. However, bleeding is a major concern with warfarin therapy due to the Biomedicines 2021, 9, 264 6 of 20

and the prolongation of PT clotting time may be due to one or more coagulation factor defects in extrinsic and common pathways, or indicate the presence of inhibitors against these factors, including drugs. Warfarin reduces the production of coagulation factor II, VII, IX, and X and the activity may also be decreased, and thus PT is elevated in warfarin patients and used as a parameter in monitoring tests. Due to differing phospholipid compositions and sources of tissue factor, significant differences occur in the clotting time result from different PT reagents [49]. Initial efforts to standardize PT reagents was to establish the PT ratio (patient’s PT/mean normal PT). However, the standardization of PT ratio was unable to completely resolve the issue of reagents’ variability because the system could not eliminate discrepancies between the reagents [50]. For further standardization, international normalized ratio (INR) was developed, which reflects a mathematical calculation using PT ratio as further adjusted with a correction factor called international sensitivity index (ISI) [51].

INR = (Patient PT/Mean Normal PT) ISI (1)

Mean normal PT is calculated as the measurement value of PT clotting time in normal samples. ISI is a specific value assigned for each reagent by manufacturers. The ISI value is assigned by the method of The World Health Organization (WHO) Expert Committee on Biological Standardization with international reference preparations (IRP) of throm- boplastins [52,53]. In this method, manually prepared PT with IRP is used for the tests, and commercial PT reagents from manufacturers are assessed and calibrated against the WHO method in 20 healthy subjects and 60 patients with stable conditions undergoing warfarin therapy. The values obtained from the manufactured PT reagent are plotted on the X-axis and the values from the WHO method on the Y-axis. The logarithm of these values is calculated, and the ISI value is obtained from the slope of the regression line in the correlation graph (Figure3)[ 54]. For the verification of an established PT-INR system with the specific ISI value, it is recommended to measure 20–40 samples from stable patients with warfarin and 20 normal plasma samples [55]. Warfarin is effective for the primary and secondary prevention of venous throm- boembolism (VTE), prevention of systemic embolism in patients with prosthetic heart valves or atrial fibrillation, prevention of stroke, recurrent infarction, and acute myocardial infarction [56–59]. However, bleeding is a major concern with warfarin therapy due to the influence of environmental factors and drug interactions in the setting of a narrow therapeutic range. Treatment with warfarin increases the risk of major bleeding by 0.3–0.5% per year and the risk of intracranial hemorrhage by approximately 0.2% per year compared with the controls. The most important risk factors for hemorrhage in warfarin therapy include intensity of anticoagulant effect, time within therapeutic range, and patient charac- teristics [60,61]. For these reasons, it is necessary to maintain the INR in the therapeutic range. A significant decrease in INR may increase the risk of thrombosis, mandating higher doses of warfarin. In contrast, a significant increase in INR represents an increased risk of bleeding, indicating a reduction in the dose or temporarily discontinuing the drug. Thus, accurate reporting of PT-INR results has a direct effect on the management of patients undergoing warfarin therapy. ISI has played an important role for the INR standardization to report the same INR value in different hospitals. This standardization system provides a historically optimized way to interpret PT results independently of the PT reagent used in institutions. Biomedicines 2021, 9, x FOR PEER REVIEW 7 of 20

influence of environmental factors and drug interactions in the setting of a narrow thera- peutic range. Treatment with warfarin increases the risk of major bleeding by 0.3–0.5% per year and the risk of intracranial hemorrhage by approximately 0.2% per year com- pared with the controls. The most important risk factors for hemorrhage in warfarin ther- apy include intensity of anticoagulant effect, time within therapeutic range, and patient characteristics [60,61]. For these reasons, it is necessary to maintain the INR in the thera- peutic range. A significant decrease in INR may increase the risk of thrombosis, mandat- ing higher doses of warfarin. In contrast, a significant increase in INR represents an in- creased risk of bleeding, indicating a reduction in the dose or temporarily discontinuing the drug. Thus, accurate reporting of PT-INR results has a direct effect on the management of patients undergoing warfarin therapy. ISI has played an important role for the INR standardization to report the same INR value in different hospitals. This standardization Biomedicines 2021, 9, 264 7 of 20 system provides a historically optimized way to interpret PT results independently of the PT reagent used in institutions.

Figure 3. Example of a calibration line for determination of international sensitivity index (ISI). The Figure 3. Example of a calibration line for determination of international sensitivity index (ISI). prothrombin time (PT) clotting times of all samples including 20 normal (red) and 60 warfarin (purple) The prothrombin time (PT) clotting times of all samples including 20 normal (red) and 60 warfarin (purple)samples samples are measured are measured by the by employed the employed instrument/reagent instrument/reagent with the with reference the reference thromboplastin thrombo- and plastinthe manual and the technique manual technique (WHO method), (WHO method), and the logarithmsand the logarithms of the PT of values the PT employed values employed are plotted areagainst plotted the against logarithms the logarithms of the PT of values the PT assigned values byassigned using WHOby using method. WHO The method. regression The regres- equation is sioncalculated, equation andis calculated, the slope ofand the the line slope is established. of the line Theis established. ISI value of The the IS employedI value of instrument/reagent the employed instrument/reagentis calculated as: ISI is =calculated slope × ISI as: (ref) ISI = where slope ISI× ISI (ref) (ref) is thewhere ISI ISI of the(ref) reference is the ISI thromboplastin. of the reference thromboplastin. 2.3. Direct Oral Anticoagulant (DOAC) 2.3.2.3.1. Direct Properties Oral Anticoagulant of DOAC (DOAC) 2.3.1. PropertiesSince 2008, of dabigatran, DOAC which is an oral direct thrombin inhibitor, and the direct factor Xa inhibitors (such as rivaroxaban, apixaban, and edoxaban) have been approved by the Since 2008, dabigatran, which is an oral direct thrombin inhibitor, and the direct fac- regulatory agencies. Subsequently, betrixaban, another anti-Xa direct oral anticoagulant, tor Xa inhibitors (such as rivaroxaban, apixaban, and edoxaban) have been approved by was approved for VTE prophylaxis in the United States. These anticoagulant drugs are the regulatory agencies. Subsequently, betrixaban, another anti-Xa direct oral anticoagu- widely prescribed to prevent and treat several kinds of thromboembolic diseases. Their lant, was approved for VTE prophylaxis in the United States. These anticoagulant drugs pharmacokinetic and pharmacodynamic properties are more predictable than those of war- are widely prescribed to prevent and treat several kinds of thromboembolic diseases. farin, and thus routine monitoring of the anticoagulant effect is not required [62]. During the start of the use of these drugs, the term—novel/new oral anticoagulants (NOACs)— was employed to refer to these drugs, especially, dabigatran, rivaroxaban, apixaban, and edoxaban because of the new class of oral anticoagulants and the term NOACs has been used for several years. However, they are not so new or novel anymore and the Subcom- mittee on the Control of Anticoagulation from Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (ISTH-SSC) suggested using the term “direct oral anticoagulants” (DOACs) to reference the class of oral anticoagulants that directly inhibit a single target and have similar clinical properties [63]. DOACs directly inhibit coagulation factors, whilst warfarin inhibits the synthesis of coagulation factors (Figure4). The characteristics of dabigatran, rivaroxaban, apixaban, and edoxaban are shown in Table1. Dabigatran is generated from the oral prodrug, dabigatran etexilate, by the hydrolysis of esterases in the gut, liver, and blood [64]. Dabigatran which is the active form of dabigatran etexilate binds to the thrombin active site and inhibits free and fibrin-bond thrombin [65]. Rivaroxaban is a competitive inhibitor of FXa and it inhibits both free FXa and prothrombinase complex, and it reaches peak plasma concentration Biomedicines 2021, 9, x FOR PEER REVIEW 8 of 20

Their pharmacokinetic and pharmacodynamic properties are more predictable than those of warfarin, and thus routine monitoring of the anticoagulant effect is not required [62]. During the start of the use of these drugs, the term—novel/new oral anticoagulants (NO- ACs)—was employed to refer to these drugs, especially, dabigatran, rivaroxaban, apixa- ban, and edoxaban because of the new class of oral anticoagulants and the term NOACs has been used for several years. However, they are not so new or novel anymore and the Subcommittee on the Control of Anticoagulation from Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (ISTH-SSC) sug- gested using the term “direct oral anticoagulants” (DOACs) to reference the class of oral anticoagulants that directly inhibit a single target and have similar clinical properties [63]. DOACs directly inhibit coagulation factors, whilst warfarin inhibits the synthesis of coagulation factors (Figure 4). The characteristics of dabigatran, rivaroxaban, apixaban, and edoxaban are shown in Table 1. Dabigatran is generated from the oral prodrug, Biomedicines 2021, 9, 264 dabigatran etexilate, by the hydrolysis of esterases in the gut, liver, and blood 8[64]. of 20 Dabigatran which is the active form of dabigatran etexilate binds to the thrombin active site and inhibits free and fibrin-bond thrombin [65]. Rivaroxaban is a competitive inhibitor of FXa and it inhibits both free FXa and prothrombinase complex, and it reaches peak rapidly [66]. Apixaban is a direct and reversible inhibitor of FXa, and it inhibits free FXa, plasma concentration rapidly [66]. Apixaban is a direct and reversible inhibitor of FXa, FXa in the prothrombinase complex, and FXa bound to platelets [64]. Edoxaban is also a and it inhibits free FXa, FXa in the prothrombinase complex, and FXa bound to platelets highly selective, direct, and reversible inhibitor of FXa and it inhibits free FXa as well as [64]. Edoxaban is also a highly selective, direct, and reversible inhibitor of FXa and it in- that within the prothrombinase complex [67]. These drugs are rapidly becoming the most hibits free FXa as well as that within the prothrombinase complex [67]. These drugs are commonly prescribed oral anticoagulants for prevention of embolic stroke in patients with rapidly becoming the most commonly prescribed oral anticoagulants for prevention of non-valvular atrial fibrillation (AF) and prevention and treatment of VTE [68]. Betrixaban embolic stroke in patients with non-valvular atrial fibrillation (AF) and prevention and is a new DOAC with a different pharmacokinetic profile than other DOACs. Betrixaban treatment of VTE [68]. Betrixaban is a new DOAC with a different pharmacokinetic profile was recently approved for the indication of extended thromboprophylaxis in the United than other DOACs. Betrixaban was recently approved for the indication of extended States based on the clinical trial compared with enoxaparin in hospitalized acute medically thromboprophylaxis in the United States based on the clinical trial compared with ill patients [69]. enoxaparin in hospitalized acute medically ill patients [69].

Figure 4. The inhibition mechanisms of direct oral anticoagulants (DOACs). Dabigatran is direct thrombin inhibitor, other DOACs including rivaroxaban, apixaban, edoxaban, and betrixaban are direct factor Xa inhibitors. DOACs inhibit coagulation factors directly. On the other hand, heparin indirectly inhibits coagulation factors through AT and warfarin affects to the coagulation factor synthesis.

Table 1. The characteristics of DOACs.

Dabigatran Rivaroxaban Apixaban Edoxaban Betrixaban Target Thrombin Factor Xa Factor Xa Factor Xa Factor Xa Primary clearance Renal Renal Fecal Renal Fecal Tmax 1.5–3 h 2–3 h 3–4 h 1–2 h 3–4 h Half-life 12–14 h 5–13 h 12 h 10–14 h 19–27 h

2.3.2. Measurement of DOACs The use of DOACs without routine monitoring and frequent dose adjustment has been shown to be safe and effective in the majority of patients, thus making them more convenient anticoagulants than warfarin [70]. However, it was reported that there would be clinical circumstances in specific patients when measurements for the anticoagulant Biomedicines 2021, 9, 264 9 of 20

effects of DOAC would be required. The following cases have been discussed for the utility of the measurements [62,71]. (i) Persistent bleeding or thrombosis (ii) Decreased drug clearance resulting from impaired kidney function or liver disease (iii) Identification of subtherapeutic or supratherapeutic levels in patients taking other drugs that are known to significantly affect pharmacokinetics (iv) Extremely body weight: <40 kg or >120 kg (v) Perioperative management (vi) Reversal of anticoagulant (vii) Suspicion of overdosage (viii) Adherence to treatment protocol The measurement assays have been developed to fulfill these requirements. It was considered that the optimal laboratory assays were dependent on the cases and the tests were classified as two types as follows: qualitative (presence or absence) and quantitative (ng/mL).

Qualitative Assays for DOACs DOACs can interfere with clot-based coagulation assay because these drugs inhibit thrombin or factor Xa. Many researchers reported that the effect on clotting assays were largely dependent on principles like PT and APTT, which are the global assays for the screening of coagulation cascade. Endogenous coagulation activity changes and coagula- tion inhibitors can also affect the measurement results of PT and APTT, and these global assays lack the specificity for the measurements of DOACs anticoagulation. However, these tests have been performed as routine tests in many clinical laboratories, which indicates that the results are obtained in short turn-around-times. Timely evaluation is important because the half-lives of DOACs are short and it is critical in several scenarios, such as life-threatening bleeding or acute stroke management [72]. PT is prolonged by dabigatran and rivaroxaban in a concentration-dependent manner, and PT has higher sensitivity to rivaroxaban than dabigatran [73,74]. Specifically, the investigation for rivaroxaban has been performed by many researchers, it was reported that two-fold clotting times was 66–258 ng/mL among PT reagents in the spiked samples and it indicated that the variability among the reagents was wide. Although the relation was linear in the spiked samples, the correlation between PT and the concentration measured by mass spectrometry in clinical samples was weak (R2 = 0.3178) and the discrepancy samples were also recognized in the correlation [74,75]. The sensitivity to apixaban was lower than that of rivaroxaban. Although the sensitivity is largely dependent on PT reagents, PT may be normal with apixaban concentrations up to 200 ng/mL [76–78]. The sensitivity to edoxaban is equivalent or less than rivaroxaban, but PT is more sensitive than that of APTT, the prolongation of the PT is also concentration- and reagent-dependent [79,80]. Although INR and ISI are widely used from the view of standardization, this system had been established for warfarin monitoring based on VKA sensitivity. This system is not suitable for DOACs measurements and data interpretation [81]. It was also reported that APTT had sensitivity for dabigatran, rivaroxaban, apixaban, and edoxaban in concentration-dependent prolongation of clotting times. Particularly, the sensitivity to dabigatran was higher than those of other drugs [74,82,83] and the prolongation of APTT increased in a non-linear manner with increasing concentrations of dabigatran in clinical samples [84]. As described above, APTT is a commonly used parameter for the measurement of the degrees of anticoagulation in patients receiving anticoagulant . It has been used for the monitoring of treatment with UFH and has been discussed more recently in the clinical evaluation of direct thrombin inhibitors, such as [85–87]. Although commercial APTT reagents differ in their sensitivity, it was estimated that APTT had higher sensitivity to thrombin inhibitor than PT and the principle might be useful for the detection of dabigatran. Biomedicines 2021, 9, 264 10 of 20

Overall, APTT is more sensitive with dabigatran and PT with rivaroxaban, whilst apixaban has very little influence on both PT and APTT. The specificity was low and the sensitivity to drugs is not so high. It indicates that the ability of these tests to quantify DOAC concentrations is poor and reagent-dependent. In addition, PT and APTT are not recommended at all for monitoring DOACs. (TT) is highly sensitive to dabigatran because the reagent includes thrombin in lower concentration than fibrinogen reagent, converts fibrinogen into fibrin, and the clotting time is detected. Dabigatran concentrations in trough levels lower than 30 ng/mL lead to significant prolongation of the TT; a normal TT indicates that little or no dabigatran is present, but a prolonged TT does not necessarily equate to a high dabigatran level [88,89]. Thus, TT is only useful in the very low dabigatran concentration range to exclude its presence in plasma samples. The reagent is not affected by direct factor Xa inhibitor. In addition to these classical coagulation tests, other assays had been developed to measure DOACs in clotting principles, and one of them is diluted prothrombin time (dPT). In routine PT tests, the quantity of thromboplastin is much higher than in physiological conditions. dPT experiments were performed to increase the sensitivity of the test because the conditions of this test might be closer to the actual physiological conditions. In these dPT experiments, PT reagents were diluted with 25 mM CaCl2 solution to obtain normal clotting times of 30–40 s. The dilution ratios were different in each reagent, but these were 1/8–1/750 (one part of reagent/x parts of CaCl2 solution) [73,90]. Although the results obtained with several dPT reagents still varied, depending on the reagents used, it was confirmed that the clotting times of dPT were prolonged in rivaroxaban samples and dPT assay demonstrated weak correlation with rivaroxaban concentrations. The sensitivity to rivaroxaban in dPT was increased as the dilution ratios became higher [75,90]. Ieko et al. developed a novel dPT assay with a new formula to normalize the effect of thrombin generation. They used 500-fold diluted PT reagent for the measurements, the results were expressed as the ratio of thrombin generation in their formula, and they investigated the correlation between the formula and the drug concentration in the samples of rivaroxaban, apixaban, and edoxaban. The results showed that the formula was positively correlated with the concentrations of all drugs, and it was also recognized that the formula showed a significant decrease in cases with thrombosis and increase in those with hemorrhage [91]. Recently, we also developed a modified dPT (mdPT) assay and reported that the reagent had good correlation against the drug concentration of rivaroxaban, apixaban, and edoxa- ban, and the sensitivity of mdPT was higher than that of commercial PT reagents in these clinical samples. In addition, the discrepancy analysis in the correlation between mdPT and the drug concentrations were performed, and the results showed that the discrepancy might be related to the lower coagulation activity of factors II, V, VII, and X. Thus, it was considered that mdPT reagent reflected not only DOAC inhibition but also coagulation factor activity and this assay may predict bleeding risk in patients treated with DOAC at a lower level of coagulation activity [92,93]. Letertre et al. developed an interesting assay based on dPT. They hypothesized that the levels of anticoagulant drugs could be measured with use of the dPT and diluted factor II and X deficient plasma-PT (dFiix-PT), which is only affected by reduced levels of active factor II and X. In the dFiix-PT assays, FII- and FX- depleted plasma was mixed with the test plasma to correct for any factor deficiency other than FII or FX, and the test plasma was mixed with diluted thromboplastin, CaCl2 solution, and factor II and X depleted plasma; and the clotting time was detected. This assay showed good correlation against drug concentration in dabigatran, rivaroxaban, and apixaban. They also showed that the dFiix-PT with drug-specific calibration curves estimated the drug concentration in plasma samples from patients taking these agents and suggested that the assay may be suitable for screening for the presence of anticoagulant drug in plasma and the test might be suitable for emergency testing of anticoagulant concentrations or effects [94]. Overall, while these global assays are not specific for DOAC, they are useful for screening for anticoagulant drugs and finding the effects of drug concentration with Biomedicines 2021, 9, 264 11 of 20

coagulation factor activity. Although further studies are required from the perspective of clinical insight, it is considered that these assays are helpful to understand the status of patients taking anticoagulants.

Quantitative Assay for DOAC Measurement Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) is con- sidered the gold standard method for the quantitative assay for DOAC measurement because it has high degree of specificity, sensitivity, selectivity, and reproducibility, and this method is often used in clinical development to evaluate DOAC pharmacokinetics [95–97]. The lower limits of detection (LoD) and quantitation (LoQ) in LC-MS/MS for DOAC detection has been reported as 0.025–3 ng/mL and the reportable range of quantitation has also been reported as 5–500 ng/mL [98–103]. Although LC-MS/MS is considered the most accurate method for DOAC measurements, it is not widely used because of the limitation of availability and complexity associated with this testing. In addition, LC-MS/MS has a high intra- and inter-assay coefficient of variation and it needs to be calibrated with the purified drug, which contributes to series-to-series variations. Thus, the drug-calibrated clot-based or chromogenic methods have been developed and adapted to automated coagulation analyzers. One of the clot-based kits for dabigatran measurement is Hemoclot thrombin inhibitor (Hyphen BioMed, Neuville-sur-Oise, France). For detection, the diluted tested plasma is mixed with normal pooled plasma. Then, clotting is initiated by addition of human thrombin and the clotting time is measured in coagulation analyzers. The clotting time is directly related to the concentration of dabigatran, and the concentration is calculated from the calibration curve obtained from the dabigatran calibrator. It was shown that the clotting method and LC-MS/MS analysis correlated well, and the agreement was good [98]. Stangier et al. also reported that this kit enabled the accurate assessment of dabigatran concentrations within the therapeutic concentration range without inter- laboratory variability [104]. The correlation between the clotting method and qualitative tests as APTT and thrombin time has also been investigated. The APTT demonstrated a modest correlation with the dabigatran concentration measured by the clotting method but the correlation became less reliable at higher dabigatran levels and it was recognized that the sensitivity was different among APTT reagents. The TT was sensitive to the presence of dabigatran and showed >300 s in more than 60 ng/mL concentration. It was confirmed that the TT was too sensitive to quantify dabigatran levels [105]. The other study also demonstrated that APTT and TT could not identify the drug presence in too low or too high concentrations, respectively [106]. For dabigatran concentration detection, it is considered that the clotting method is a more suitable assay. In addition, there was an accurate correlation reported for ecarin clotting time or ecarin chromogenic assay and dabigatran concentrations [74]. Although the upper limit of the measurements is dependent on the ecarin unit and the composition, it was reported that these assays could detect dabigatran up to 940 ng/mL. Chromogenic anti-Xa assay has been used in clinical laboratories for several decades as a method to assess heparin concentration. This assay is appropriate for measuring direct factor Xa inhibitor drug concentration. The chromogenic anti-Xa assay principle is a kinetics method based on the inhibition of factor Xa at a constant and limiting concentration by the tested factor Xa inhibitor. The samples are mixed with an excess amount of factor Xa, and factor Xa inhibitor binds to factor Xa. The remaining factor Xa is then measured by its amidolytic activity on a factor Xa specific chromogenic substrate, and the residual factor Xa cleaves the substrate. The amount of cleaved substrate is inversely proportional to the concentration of factor Xa inhibitor in the tested samples. The concentration is calculated from the calibration curve obtained from rivaroxaban, apixaban, and edoxaban calibrator, respectively. It has been proved that this kit has the correlation against LC- MS/MS, and it was also shown that the inter variability was small [100,107,108]. Douxfils et al. reported that the correlation between PT and LC-MS/MS was not linear and the Biomedicines 2021, 9, 264 12 of 20

sensitivity to drugs differed among reagents [100], it could be difficult to perform the standardization to estimate drug concentration in PT reagents. Biophen DiXaI is useful to quantify drug concentration because the kit has high sensitivity to drugs and adapted to automated coagulation analyzers. DOACs have short half-lives, their concentration is time-dependent, and the concentration of peak time is quite different from that of trough time. The information when patients take the drugs is useful for the interpretation of the drug concentration data.

3. Coronavirus Disease 2019 and Thrombosis 3.1. The Thrombosis Mechanisms Anticoagulant drugs are effective for the primary and secondary prevention of VTE, prevention of systemic embolism, atrial fibrillation, et cetera [56,109]. In December 2019, a new viral infection occurred in Wuhan City, Hubei Province, China. Severe acute respi- ratory syndrome coronavirus 2 (SARS-CoV-2), a novel beta-coronavirus that can spread from human to human, is recognized as the causative virus of this infection. SARS-CoV-2 infects vascular endothelial cells through angiotensin-converting enzyme 2 (ACE2), the receptor for angiotensin II. The incubation period for SARS-CoV-2 is generally 5–6 d and once symptoms occur, the peak viremia occurs within 2–5 d of onset [110]. The virus was named COVID-19 and this infection is a mild disease in approximately 80% of patients and these patients usually present with symptoms of fever and a dry cough and it re- solves spontaneously within 10 days. However, in almost 20% of patients, viral infection progresses down the trachea to the lungs [111]. The adverse effects of COVID-19 were initially thought to affect the primarily tract by causing pneumonia and acute respiratory distress syndrome (ARDS), but it is now known that COVID-19 is associated with the thrombosis promoting state which can cause microvascular thrombosis, venous, or arterial thrombosis [112]. Serious SARS-CoV-2 infection was often associated with coagulopathy, with increased D-dimer, which was more pronounced in critically ill patients [113]. The thrombosis mechanisms in COVID-19 patients are under current investigation and the most appropriate treatment is being discussed, with guidelines being issued by scientific societies. In addition, increased C-reactive protein (CRP) and interleukin-6 (IL-6) correlated with severity, suggesting an association between innate immune response and thrombotic response [114]. Host hypercytokinemia and massive proinflammatory responses may contribute to endothelial dysfunction of COVID-19, particularly in critically ill patients. Proinflammatory cells, cytokines, and chemokines not only activate the coagulation system, but also attenuate the anticoagulant mechanism and amplify the vicious circle of vascular coagulation and thrombosis [115]. In lung autopsies from COVID-19 patients, neutrophil pulmonary infiltration and neutrophil extracellular traps (NETs) formation were revealed and NET markers were reported to be elevated in critically ill hospitalized patients [116,117]. The NETs are the extracellular web of chromatin, bactericidal proteins, and oxidative en- zymes released by neutrophils to contain infection. Thus, NET phenomenon could be related to the thrombosis in COVID-19 patients and NETs contribute to inflammation and microvascular thrombosis [118,119]. These complicated situations, including endothe- lial inflammation, complement activation, thrombin generation, and platelet/leukocyte thrombotic complications, are related to the thrombosis mechanisms in COVID-19 patients, the current proposed mechanisms of SARS-CoV-2 associated thrombosis are shown in Figure5[112]. Biomedicines 2021, 9, x FOR PEER REVIEW 13 of 20

but also attenuate the anticoagulant mechanism and amplify the vicious circle of vascular coagulation and thrombosis [115]. In lung autopsies from COVID-19 patients, neutrophil pulmonary infiltration and neutrophil extracellular traps (NETs) formation were revealed and NET markers were reported to be elevated in critically ill hospitalized patients [116,117]. The NETs are the extracellular web of chromatin, bactericidal proteins, and ox- idative enzymes released by neutrophils to contain infection. Thus, NET phenomenon could be related to the thrombosis in COVID-19 patients and NETs contribute to inflam- mation and microvascular thrombosis [118,119]. These complicated situations, including endothelial inflammation, complement activation, thrombin generation, and platelet/leu- kocyte thrombotic complications, are related to the thrombosis mechanisms in COVID-19 patients, the current proposed mechanisms of SARS-CoV-2 associated thrombosis are Biomedicines 2021, 9, 264 shown in Figure 5 [112]. 13 of 20

Figure 5. Proposed mechanisms of SARS-CoV-2 associated thrombosis. Several thrombosis mechanisms have been proposed,

the important pathogenic effect is the binding and destruction of epithelial lung cells. The phenomenon relates to tissue factorFigure release 5. Proposed into blood mechanisms circulation of and SARS-CoV-2 activate coagulation associated pathways thrombosis. at lung Several sites thrombosis and extend mechanisms it throughout have the circulatory been pro- systems.posed, the In important addition, infectedpathogenic cells effect become is the the binding trigger toand release destruction proinflammatory of epithelial cytokines lung cells. and The chemokines. phenomenon The activatedrelates to endothelialtissue factor cells release upregulate into blood P-selectin circulation and and E-selectin activate leading coagul toation recruitment pathways of at platelets lung sites and and leukocytes extend it and throughout complement the activation.circulatory systems. The infection In addition, and recruitment infected cells are become related tothe the trigger release to release of danger-associated proinflammatory molecular cytokines patterns and chemokines. (DAMPs). The activated endothelial cells upregulate P-selectin and E-selectin leading to recruitment of platelets and leukocytes and Monocytes activated through complement activation, express tissue factor, and activate coagulation cascades. Neutrophils complement activation. The infection and recruitment are related to the release of danger-associated molecular patterns release NETs which activate the contact pathway directly. Thrombin is generated through these pathways, fibrinogen is (DAMPs). Monocytes activated through complement activation, express tissue factor, and activate coagulation cascades. convertedNeutrophils to fibrin,release and NETs the which activation activate of coagulation the contact cascade pathwa isy relateddirectly. to Thrombin SARS-CoV-2 is gene associatedrated through thrombosis. these The pathways, reaction offibrinogen (i) affects is to converted cells, (ii) proteinto fibrin, expression, and the activation and (iii) theof coagulation mechanisms cascade of fibrin is clotrelated generation to SARS-CoV-2 were shown associated as red, thrombosis. yellow and greenThe reaction arrows, of respectively. i) affects to cells, ii) protein expression, and iii) the mechanisms of fibrin clot generation were shown as red, yellow and green arrows, respectively. 3.2. COVID-19 Therapy and Anticoagulants 3.2. COVID-19With the suspectedTherapy and contribution Anticoagulants of thrombotic events to morbidity and mortality in COVID-19With the patients, suspected novel contribution options for of preventing thrombotic COVID-19-related events to morbidity thrombotic and mortality diseases in COVID-19are being explored.patients, novel However, options the for data preventing is limited COVID-19-related to determination thrombotic of antithrombotic diseases therapy to improve outcomes in patients with COVID-19 for whom evidence of thrombosis has not been confirmed. An interim guideline for the management of coagulopathy in COVID-19 patients was introduced by the International Society for Thrombosis and Hemostasis (ISTH) in March 2020. The guidelines recommend prophylactic administration of LMWH in all patients with COVID-19 who require hospitalization unless there is active bleeding or a contraindication, such as a platelet count <25 × 109 /L [120]. It is also recommended by the WHO to use pharmacological prophylaxis in patients (adults and adolescents) hospitalized with COVID-19, such as low molecular weight heparin (e.g., enoxaparin) to prevent venous thromboembolism, when not contraindicated [121]. In addition, anticoagulant therapy using unfractionated heparin or low-molecular-weight heparin is recommended for reducing the depletion of coagulation substrates in patients with severe coagulation dysfunction when either fibrin/fibrinogen degradation products (FDP), D-dimer, or both, show either ≥10 µg/mL, ≥5 µg/mL, or both, respectively, in Chinese expert consensus [122]. For FDP reagents, it was reported that the reactivity to the fragments was different among the reagents and it should be considered for the data interpretation [123]. Biomedicines 2021, 9, 264 14 of 20

It has been reported that VTE frequency in subjects and a high incidence of pulmonary embolism admitted to the ICU for COVID-19 infection is increasing despite the use of standard prophylaxis for VTE with LMWH. Klok et al. reported that the occurrence of VTE was 27% and that of arterial vascular thrombosis was 3.7% despite the standard VTE prophylaxis with LMWH [124]. This was clearly higher than the 7.7% failure rate generally registered for VTE prophylaxis in ICUs [125]. White et al. demonstrated that heparin target concentrations were not reached, as measured by APTT or anti-Xa assays, in COVID-19 patients in ICU. Patient plasma spikes due to LMWH resulted in lower-than-expected anti-Xa levels in plasma. Based on these findings, the authors concluded that patients with COVID-19 were heparin resistant [126]. Decreased AT activity and increased levels of factor VIII and fibrinogen are cited as common predictors of heparin resistance [127]. Those COVID-19 patients have grossly raised fibrinogen and factor VIII levels with mild decreases in the AT [127], and these factors affect the APTT clotting times due to its global assay principle. Although there are bias risks in testing heparin in COVID-19 patients and the detailed thrombosis mechanisms are under investigation, anti-Xa assay might be more suitable for heparin monitoring of anticoagulant activity than APTT in some COVID-19 patient cases.

4. Conclusions In this review, we described anticoagulant drugs, monitoring methods, thrombosis mechanisms, and therapy in COVID-19 patients. For the older anticoagulant drugs like heparin and warfarin, the monitoring methods have been established as APTT/anti-Xa assay and PT-INR, respectively. Although the therapeutic range is narrow and strict control is required, the sensitivity to these drugs is different among APTT/PT reagents and it may be difficult to control within the therapeutic range in some cases. To proceed with treatment with a suitable anticoagulant control, the clinical laboratory data should be interpreted carefully. Although the use of DOACs without routine monitoring and frequent dose adjustment has been shown to be safe and effective, there could be clinical circumstances in specific patients when measurements for the anticoagulant effects of DOACs would be required. Several assays have been developed and it is considered that the clinical laboratory tests might be useful to understand the patient status in some causes. COVID-19 infection is related to thrombosis and coagulopathy. The data to understand the mechanisms and show the decision-making direction is limited. Further studies are desired for the better prediction and treatment.

Author Contributions: Conceptualization, Writing—Original draft preparation, visualization, O.K.; writing—part of the original draft, K.A.; Writing—Review and editing, J.A. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Acknowledgments: We gratefully acknowledge the support from the members of Sysmex corpora- tion and Hyphen BioMed. Conflicts of Interest: O. Kumano is an employee of Sysmex Corporation and K. Akatsuchi is an employee of Sysmex R&D Center Americas, Inc. J. Amiral is a consultant for Hyphen BioMed and Sysmex Corporation. The authors declare no conflict of interest.

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