Clinical Relevance of Preclinical and Clinical Studies of Four-Factor Prothrombin Complex Concentrate for Treatment of Bleeding Related to Direct Oral Anticoagulants.

Direct oral anticoagulants (DOACs) are widely used for the prevention and treatment of venous thromboembolism and stroke. When emergency reversal of DOAC-related anticoagulation is required, specific DOAC reversal agents are recommended, including idarucizumab for dabigatran reversal and andexanet alfa for apixaban and rivaroxaban reversal. However, specific reversal agents are not always available, andexanet alfa has not been approved for urgent surgery, and clinicians need to know the patient's anticoagulant medication before administering these treatments. Four-factor prothrombin complex concentrates (4F-PCCs) are recognized as nonspecific, alternative hemostatic agents for treatment of DOAC-related bleeding. Evidence from preclinical and clinical studies shows that they may reduce the anticoagulant effects of DOACs and may help control DOAC-related bleeding. However, randomized controlled trials are lacking, and most data are from retrospective or single-arm prospective studies in bleeding associated with activated factor X inhibitors. There are no clinical data showing the efficacy of 4F-PCC for the treatment of bleeding in dabigatran-treated patients. This review focuses on the current evidence of 4F-PCC use in controlling bleeding associated with DOACs and provides an expert opinion on the relevance of these data for clinical practice. The current treatment landscape, unmet needs, and future directions are also discussed.

Red blood cell microvesicles activate the contact system, leading to factor IX activation via 2 independent pathways.

Storage lesion-induced, red cell-derived microvesicles (RBC-MVs) propagate coagulation by supporting the assembly of the prothrombinase complex. It has also been reported that RBC-MVs initiate coagulation via the intrinsic pathway. To elucidate the mechanism(s) of RBC-MV-induced coagulation activation, the ability of storage lesion-induced RBC-MVs to activate each zymogen of the intrinsic pathway was assessed in a buffer system. Simultaneously, the thrombin generation (TG) assay was used to assess their ability to initiate coagulation in plasma. RBC-MVs directly activated factor XII (FXII) or prekallikrein, but not FXI or FIX. RBC-MVs initiated TG in normal pooled plasma and in FXII- or FXI-deficient plasma, but not in FIX-deficient plasma, suggesting an alternate pathway that bypasses both FXII and FXI. Interestingly, RBC-MVs generated FIXa in a prekallikrein-dependent manner. Similarly, purified kallikrein activated FIX in buffer and initiated TG in normal pooled plasma, as well as FXII- or FXI-deficient plasma, but not FIX-deficient plasma. Dual inhibition of FXIIa by corn trypsin inhibitor and kallikrein by soybean trypsin inhibitor was necessary for abolishing RBC-MV-induced TG in normal pooled plasma, whereas kallikrein inhibition alone was sufficient to abolish TG in FXII- or FXI-deficient plasma. Heating RBC-MVs at 60°C for 15 minutes or pretreatment with trypsin abolished TG, suggesting the presence of MV-associated proteins that are essential for contact activation. In summary, RBC-MVs activate both FXII and prekallikrein, leading to FIX activation by 2 independent pathways: the classic FXIIa-FXI-FIX pathway and direct kallikrein activation of FIX. These data suggest novel mechanisms by which RBC transfusion mediates inflammatory and/or thrombotic outcomes.

Preclinical Development of a vWF Aptamer to Limit Thrombosis and Engender Arterial Recanalization of Occluded Vessels.

Endothelial surface and circulating glycoprotein von Willebrand factor (vWF) regulates platelet adhesion and is associated with thrombotic diseases, including ischemic stroke, myocardial infarction, and peripheral vascular disease. Thrombosis, as manifested in these diseases, is the leading cause of disability and death in the western world. Current parenteral antithrombotic and thrombolytic agents used to treat these conditions are limited by a short therapeutic window, irreversibility, and major risk of hemorrhage. To overcome these limitations, we developed a novel anti-vWF aptamer, called DTRI-031, that selectively binds and inhibits vWF-mediated platelet adhesion and arterial thrombosis while enabling rapid reversal of this antiplatelet activity by an antidote oligonucleotide (AO). Aptamer DTRI-031 exerts dose-dependent inhibition of platelet aggregation and thrombosis in whole blood and mice, respectively. Moreover, DTRI-031 can achieve potent vascular recanalization of platelet-rich thrombotic occlusions in murine and canine carotid arteries. Finally, DTRI-031 activity is rapidly (<5 min) and completely reversed by AO administration in a murine saphenous vein hemorrhage model, and murine toxicology studies indicate the aptamer is well tolerated. These findings suggest that targeting vWF with an antidote-controllable aptamer potentially represents an effective and safer treatment for thrombosis patients having platelet-rich arterial occlusions in the brain, heart, or periphery.

Abnormal joint and bone wound healing in hemophilia mice is improved by extending factor IX activity after hemarthrosis.

Wound healing requires interactions between coagulation, inflammation, angiogenesis, cellular migration, and proliferation. Healing in dermal wounds of hemophilia B mice is delayed when compared with hemostatically normal wild-type (WT) mice, with abnormal persistence of iron deposition, inflammation, and neovascularity. We observed healing following induced joint hemorrhage in WT and factor IX (FIX) knockout (FIX-/-) mice, examining also parameters previously studied in an excisional skin wound model. Hemostatically normal mice tolerated this joint bleeding challenge, cleared blood from the joint, and healed with minimal pathology, even if additional autologous blood was injected intra-articularly at the time of wounding. Following hemarthrosis, joint wound healing in hemophilia B mice was impaired and demonstrated similar abnormal histologic features as previously described in hemophilic dermal wounds. Therefore, studies of pathophysiology and therapy of hemophilic joint bleeding performed in hemostatically normal animals are not likely to accurately reflect the healing defect of hemophilia. We additionally explored the hypothesis that the use of a FIX replacement protein with extended circulating FIX activity could improve synovial and osteochondral wound healing in hemophilic mice, when compared with treatment with unmodified recombinant FIX (rFIX) in the established joint bleeding model. Significantly improved synovial wound healing and preservation of normal osteochondral architecture are achieved by extending FIX activity after hemarthrosis using glycoPEGylated FIX when compared with an equivalent dose of rFIX. These results suggest that treating joint bleeding only until hemostasis is achieved may not result in optimal joint healing, which is improved by extending factor activity.

The Tissue Factor Pathway and Wound Healing.

The role of tissue factor (TF) as the major initiator of hemostatic blood coagulation is well recognized. The ability to form an adequate hemostatic clot is essential to the normal healing of an injury by staunching bleeding, stabilizing the injured tissue, and serving as a scaffold for repair processes. Also, some molecules produced during hemostasis, particularly thrombin, have cytokine and growth factor-like activities that contribute to inflammation and repair. However, TF itself has activities as a regulator of cellular processes via direct signaling, as well as by facilitating activation of proteolytically activated receptors by activated factors VII and X. The importance of hemostasis in the host response to injury makes it very difficult to separate the hemostatic from nonhemostatic effects of TF on wound healing. The literature in this area remains sparse but suggests that TF influences the course and tempo of healing by cell signaling events that impact inflammation, epithelialization, and angiogenesis.

Impact of Non-Vitamin K Antagonist Oral Anticoagulants From a Basic Science Perspective.

The biochemical properties of the non-vitamin K antagonist oral anticoagulants (NOACs) and their differences from the mechanism of action of vitamin K antagonists contribute to their properties as anticoagulants. These properties include as follows: (1) Inhibiting a single protease is much less effective at inhibiting coagulation than is inhibiting at multiple steps. Thus, the dose-response relationship between NOAC level and intensity of anticoagulation is shallower and more linear than that of vitamin K antagonists. This partially accounts for the greater safety of NOACs than vitamin K antagonists reported in some studies. (2) Because they are small molecules, NOACs can reach their target proteases in locations that plasma protease inhibitors, such as antithrombin, cannot. (3) NOACs compete with substrates for binding at the active site of the target protease and that binding is reversible. When the drug level falls, the drug dissociates from its target, and protease activity is restored. Thus, there is the possibility of a rebound in procoagulant activity if the drug is abruptly terminated. (4) The effects of a NOAC can be overcome by increasing the amount of substrate available for the target protease or the amount of protease produced. This property may contribute to the safety of NOACs and their potential reversibility by coagulation factor concentrates. The biochemical properties of NOACs contribute to their suitability for use in conditions that require a predictable moderate degree of anticoagulation when administered orally at a consistent dose. Their effects can be overcome by a sufficiently strong procoagulant stimulus. This characteristic likely contributes to their generally reduced risk of serious bleeding. However, they are not well suited for use in settings that require a profound degree of anticoagulation.

Differentiating between cold agglutinins and rouleaux: a case series of seven patients.

We present a case series of seven patients with suspected cold agglutinin antibodies, discovered after initiation of bypass. Laboratory analysis of blood samples intraoperatively determined the cause of the aggregation to be rouleaux formation in three of the patients and cold agglutinins in the other four.

No association between donor age and recipient outcomes: transfusion of plasma in patients undergoing coronary artery bypass grafting surgery.

BACKGROUND: Recent animal studies suggest that transfusion of plasma from young donors reverses age-related neurologic and cardiac changes in older recipients. Associations between age of blood product donors and corresponding outcomes in recipients have not been studied in humans. Therefore, our primary objective was to examine this relationship between donor age and recipient outcomes among patients that received plasma during and after coronary artery bypass grafting (CABG) surgery. STUDY DESIGN AND METHODS: This retrospective cohort included patients undergoing CABG surgery who received plasma during or after surgery. All plasma units transfused were evenly divided into tertiles based on the plasma donor age (17-37, 38-50, and 51-86 years), and CABG patients receiving all perioperative plasma within a single donor tertile were studied. Patient demographics and outcomes including mortality, length of stay (LOS), and acute kidney injury (AKI) were measured. RESULTS: Overall, 1306 patients (24% of 5339) received American Red Cross plasma perioperatively, with a median dose of 2 units. In a multivariate model of 1-year mortality, transfusion of a greater number of plasma units (p = 0.0007) and EuroSCORE (p < 0.0001) were significantly associated with patient mortality while donor age was not. There was no difference in mortality between patients receiving plasma from donors in the youngest, middle, or oldest age tertile (10.2 and 8.1% vs. 7.8%, respectively, p = 0.76). Other outcomes, including rates of AKI or LOS, were also independent of plasma donor age. CONCLUSIONS: We did not observe an association between donor age and recipient outcomes among patients who received plasma perioperatively while undergoing CABG surgery.

Novel oral anticoagulants and reversal agents: Considerations for clinical development.

This white paper provides a summary of presentations and discussions that were held at an Anticoagulant-Induced Bleeding and Reversal Agents Think Tank co-sponsored by the Cardiac Safety Research Consortium and the US Food and Drug Administration (FDA) at the FDA's White Oak Headquarters on April 22, 2014. Attention focused on a development pathway for reversal agents for the novel oral anticoagulants (NOACs). This is important because anticoagulation is still widely underused for stroke prevention in patients with atrial fibrillation. Undertreatment persists, although NOACs, in general, overcome some of the difficulties associated with anticoagulation provided by vitamin K antagonists. One reason for the lack of a wider uptake is the absence of NOAC reversal agents. As there are neither widely accepted academic and industry standards nor a definitive regulatory policy on the development of such reversal agents, this meeting provided a forum for leaders in the fields of cardiovascular clinical trials and cardiovascular safety to discuss the issues and develop recommendations. Attendees included representatives from pharmaceutical companies; regulatory agencies; end point adjudication specialist groups; contract research organizations; and active, academically based physicians. There was wide and solid consensus that NOACs overall offer improvements in convenience, efficacy, and safety compared with warfarin, even without reversal agents. Still, it was broadly accepted that it would be helpful to have reversal agents available for clinicians to use. Because it is not feasible to do definitive outcomes studies demonstrating a reversal agent's clinical benefits, it was felt that these agents could be approved for use in life-threatening bleeding situations if the molecules were well characterized preclinically, their pharmacodynamic and pharmacokinetic profiles were well understood, and showed no harmful adverse events in early human testing. There was also consensus that after such approval, efforts should be made to augment the available clinical information until such time as there is a body of evidence to demonstrate real-world clinical outcomes with the reversal agents. No recommendations were made for more generalized use of these agents in the setting of non-life-threatening situations. This article reflects the views of the authors and should not be construed to represent FDA's views or policies.

Coagulation in Liver Disease.

The liver plays a key role in hemostasis as the site of synthesis of many of the proteins involved in the coagulation, antithrombotic and fibrinolytic systems that interact to both establish hemostasis, and preventing thrombosis. The common laboratory tests, prothrombin time (PT) and activated partial thromboplastin time (aPTT), evolved from studies of plasma clotting in test tubes. Such studies laid the basis for the coagulation cascade model of hemostasis. However, thought has evolved to place a greater emphasis on the active roles of cells in localizing and regulating hemostasis. The PT and aPTT do not reflect the roles of cellular elements in hemostasis, nor do they reflect the crucial roles of antithrombotic and fibrinolytic systems. Thus, though the PT may indeed reflect the synthetic capacity of the liver, it does not accurately reflect the risk of bleeding or thrombosis in patients with liver failure.

Reversal of dabigatran effects in models of thrombin generation and hemostasis by factor VIIa and prothrombin complex concentrate.

BACKGROUND: The oral thrombin inhibitor dabigatran has the drawbacks that it does not have a validated antidote. Data from animal studies and plasma coagulation assays suggest that prothrombin complex concentrate (PCC) or recombinant factor VIIa (FVIIa) might reverse dabigatran anticoagulation. METHODS: Cellular elements make a significant contribution to hemostasis. Our goals were to (1) test the hypothesis that both FVIIa and a 4-factor PCC improve parameters of thrombin generation in the presence of dabigatran in a cell-based model; and (2) determine whether results in a cell-based model correlate with hemostasis in vivo. RESULTS: PCC reversed dabigatran effects on the rate, peak, and total amount of thrombin but did not shorten the lag (n = 6 experiments in triplicate). By contrast, FVIIa shortened the lag, increased the rate and peak, but did not improve total thrombin (n = 6). Effects of PCC were seen at both therapeutic and markedly supratherapeutic dabigatran levels, whereas beneficial effects of FVIIa decreased as the dabigatran level increased. The PCC effect was reproduced by adding prothrombin, factor X, and factor IX. At therapeutic dabigatran levels, both PCC and FVIIa normalized hemostasis time in a mouse saphenous vein bleeding model. CONCLUSIONS: A cell-based model reflects the effects on thrombin generation of clinically relevant levels of FVIIa and PCC in the presence of dabigatran. Enhancing the rate of thrombin generation and peak thrombin level appear to correlate best with hemostasis in vivo. The ineffectiveness of FVIIa at supratherapeutic dabigatran levels may explain conflicting reports of its efficacy in dabigatran reversal.

Superactivated platelets: thrombus regulators, thrombin generators, and potential clinical targets.

Platelets contribute to hemostasis by forming the platelet plug and then contributing to coagulation by providing a catalytic surface where thrombin generation occurs efficiently. This catalytic activity, known as the platelet procoagulant response, is being recognized as a nuanced response. This review examines platelets' response to strong stimuli, which results in the formation of a platelet subpopulation (superactivated platelets) with several unique properties, including enhanced procoagulant activity. These platelets contribute uniquely to thrombus architecture and seem to have thrombus regulatory activity. Superactivated platelets' role in diseases of thrombosis and hemostasis, as either potentiating or mitigating factors, is not currently known, but may be an important pharmacological target.

Emicizumab promotes factor Xa generation on endothelial cells.

BACKGROUND: Until recently, the treatment of hemophilia A relied on factor (F)VIII replacement. However, up to one-third of patients with severe hemophilia A develop neutralizing alloantibodies that render replacement therapies ineffective. The development of emicizumab, a bispecific antibody that partially mimics FVIIIa, has revolutionized the treatment of these patients. However, the use of an activated prothrombin complex concentrate [FEIBA (Takeda)] to treat breakthrough bleeding in patients on emicizumab has been associated with thrombotic complications including a unique microangiopathy. OBJECTIVES: We hypothesized that the thrombotic complications observed with the combination of emicizumab and FEIBA might be due to excessive expression of procoagulant activity on the surface of endothelial cells. METHODS: We examined the ability of emicizumab to promote FX activation on endothelial cells using 2 cell culture models. RESULTS: We found that endothelial cells readily support emicizumab-mediated activation of FX by FIXa. The level of FXa generation depends on the concentration of available FIXa. The addition of FEIBA to emicizumab increased FXa generation in a dose-dependent manner on endothelial cells in both models. The rate of FXa generation was further enhanced by endothelial cell activation. However, unlike emicizumab, we found limited FXa generation in the presence of FVIII(a), which followed a significant lag time and was not dependent on FIXa concentration under these conditions. CONCLUSION: Emicizumab promotes FXa generation on the surface of endothelial cells, which is markedly enhanced in the presence of FEIBA. These findings demonstrate a potential mechanism for the thrombotic complications seen with the combined use of emicizumab and FEIBA.

Ultrasoft platelet-like particles stop bleeding in rodent and porcine models of trauma.

Uncontrolled bleeding after trauma represents a substantial clinical problem. The current standard of care to treat bleeding after trauma is transfusion of blood products including platelets; however, donated platelets have a short shelf life, are in limited supply, and carry immunogenicity and contamination risks. Consequently, there is a critical need to develop hemostatic platelet alternatives. To this end, we developed synthetic platelet-like particles (PLPs), formulated by functionalizing highly deformable microgel particles composed of ultralow cross-linked poly (N-isopropylacrylamide) with fibrin-binding ligands. The fibrin-binding ligand was designed to target to wound sites, and the cross-linking of fibrin polymers was designed to enhance clot formation. The ultralow cross-linking of the microgels allows the particles to undergo large shape changes that mimic platelet shape change after activation; when coupled to fibrin-binding ligands, this shape change facilitates clot retraction, which in turn can enhance clot stability and contribute to healing. Given these features, we hypothesized that synthetic PLPs could enhance clotting in trauma models and promote healing after clotting. We first assessed PLP activity in vitro and found that PLPs selectively bound fibrin and enhanced clot formation. In murine and porcine models of traumatic injury, PLPs reduced bleeding and facilitated healing of injured tissue in both prophylactic and immediate treatment settings. We determined through biodistribution experiments that PLPs were renally cleared, possibly enabled by ultrasoft particle properties. The performance of synthetic PLPs in the preclinical studies shown here supports future translational investigation of these hemostatic therapeutics in a trauma setting.

Thrombin generation and implications for hemophilia therapies: A narrative review.

Thrombin plays an essential role in achieving and maintaining effective hemostasis and stable clot formation. In people with hemophilia, deficiency of procoagulant factor (F)VIII or FIX results in insufficient thrombin generation, leading to reduced clot stability and various bleeding manifestations. A correlation has been found between the bleeding phenotype of people with hemophilia and the extent of thrombin generation, with individuals with increased thrombin generation being protected from bleeding and those with lower thrombin generation having increased bleeding tendency. The amount, location, and timing of thrombin generation have been found to affect the formation and stability of the resulting clot. The goal of all therapies for hemophilia is to enhance the generation of thrombin with the aim of restoring effective hemostasis and preventing or controlling bleeding; current treatment approaches rely on either replacing or mimicking the missing procoagulant (ie, FVIII or FIX) or rebalancing hemostasis through lowering natural anticoagulants, such as antithrombin. Global coagulation assays, such as the thrombin generation assay, may help guide the overall management of hemostasis by measuring and monitoring the hemostatic potential of patients and, thus, assessing the efficacy of treatment in people with hemophilia. Nevertheless, standardization of the thrombin generation assay is needed before it can be adopted in routine clinical practice.

Analytical validation and quality control/quality assurance practices for improved rigor and reproducibility of biochemical assays in orthopaedic research.

Orthopaedic research, and biomedical research in general, has made enormous strides to develop treatments for conditions long thought to be inevitable or untreatable; however, there is growing concern about the quality of published research. Considerable efforts have been made to improve overall research quality, integrity, and rigor, including meaningful proposals focused on transparency of reporting, appropriate use of statistics, and reporting of negative results. However, we believe that there is another key component to rigor and reproducibility that is not discussed sufficiently-analytical validation and quality control (QC). In this commentary, we discuss QC and method validation principles and practices that are systematically applied in the clinical laboratory setting to verify and monitor the analytical performance of quantitative assays, and the utility of applying similar practices to biochemical assays in the orthopaedic research setting. This commentary includes (1) recommendations for validation and QC practices, including examples of assay performance limitations uncovered by validation experiments performed in our laboratory, and (2) a description of an ongoing QC program developed to monitor the ongoing performance of commonly used assays in our lab. We hope that this commentary and the examples presented here will be thought-provoking and inspire further discussion and adaptation of analytical validation and QC procedures to advance our shared pursuit of high-quality, rigorous, and reproducible orthopaedic research.

Inhalation of an RNA aptamer that selectively binds extracellular histones protects from acute lung injury.

Acute lung injury (ALI) is a syndrome of acute inflammation, barrier disruption, and hypoxemic respiratory failure associated with high morbidity and mortality. Diverse conditions lead to ALI, including inhalation of toxic substances, aspiration of gastric contents, infection, and trauma. A shared mechanism of acute lung injury is cellular toxicity from damage-associated molecular patterns (DAMPs), including extracellular histones. We recently described the selection and efficacy of a histone-binding RNA aptamer (HBA7). The current study aimed to identify the effects of extracellular histones in the lung and determine if HBA7 protected mice from ALI. Histone proteins decreased metabolic activity, induced apoptosis, promoted proinflammatory cytokine production, and caused endothelial dysfunction and platelet activation in vitro. HBA7 prevented these effects. The oropharyngeal aspiration of histone proteins increased neutrophil and albumin levels in bronchoalveolar lavage fluid (BALF) and precipitated neutrophil infiltration, interstitial edema, and barrier disruption in alveoli in mice. Similarly, inhaling wood smoke particulate matter, as a clinically relevant model, increased lung inflammation and alveolar permeability. Treatment by HBA7 alleviated lung injury in both models of ALI. These findings demonstrate the pulmonary delivery of HBA7 as a nucleic acid-based therapeutic for ALI.

The clotting system - a major player in wound healing.

Wound healing involves a complex series of interactions between coagulation, inflammation, angiogenesis, and cellular migration and proliferation. Our laboratory has developed an excisional dermal wound model in mice in order to study some of these processes and to determine how coagulation defects affect wound healing. In contrast to wild type mice, haemophilia B mice typically show delayed healing, signs of bleeding into the wound, and significant wound expansion. The difference in wound size may result from limited fibrin deposition in haemophilic animals and the subsequent inability to anchor the platelet plug to the surrounding tissues, thus allowing wound expansion through oedema. Haemophilic mice also demonstrate impaired wound healing times. However, while pre-treatment with factor IX or human activated factor VII improves some wound characteristics in haemophilia B animals, the time to wound healing is still delayed and signs of ongoing bleeding are evident. Haemophilic mice also show a deficient initial inflammatory response and increased angiogenesis, which, in turn, leads to increased bleeding: in the absence of robust haemostasis, these fragile, newly sprouted vessels have a tendency to bleed. Taken together, these observations suggest that ongoing haemostasis is necessary for normal wound healing. If this is correct, then optimal wound healing in haemophilia would require therapy until at least the point that vessel formation is stabilized. The goal of such treatment would be to avoid a feedback cycle in which bleeding tends to lead to further bleeding. Once initiated, this cycle may be difficult to control.

Low intensity laser therapy speeds wound healing in hemophilia by enhancing platelet procoagulant activity.

Our group has previously shown that cutaneous wound healing is delayed and histologically abnormal in a mouse model of hemophilia. Hemostasis is not only required to stop bleeding at the time of wounding, but also produces bioactive substances that promote appropriate inflammatory and proliferative responses during healing. Low intensity laser therapy (LILT) has been reported to enhance impaired wound healing in a variety of animal and human studies. The current studies were conducted to test the hypothesis that LILT can improve healing in a hemophilia B mouse model. Three daily treatments with 12 J/sq cm of 650 nm laser illumination reduced the time to closure of a 3-mm cutaneous punch biopsy wound in the hemophilic mice. All wounds were closed at 13 days in the sham-treated hemophilic mice, compared with 10 days in the LILT-treated hemophilic mice, and 9 days in wild-type mice. While LILT can speed healing by enhancing proliferation of cutaneous cells, we found that an additional mechanism likely contributes to the efficacy of LILT in the hemophilic mice. LILT enhanced the mechanical rigidity and platelet activity of clots formed from human platelet-rich plasma. Illumination of isolated platelets increased the mitochondrial membrane potential and enhanced binding of coagulation factors to the surface of activated platelets. Thus, while LILT can directly promote proliferative responses during healing, it also appears to enhance hemostasis in an animal model with impaired coagulation. These data suggest that trials of LILT as an adjunct to the usual hemostatic therapies in hemophilia are warranted.