Direct factor Xa inhibitors, such as rivaroxaban, apixaban, edoxaban (factor Xa inhibitors), and direct thrombin inhibitors such as dabigatran are direct oral anticoagulants (DOACs) or non-vitamin K antagonist oral anticoagulants (NOACs) used for secondary prophylaxes in atrial fibrillation and treatment of deep venous thrombosis (DVT) and venous thromboembolism (VTE). One case-report report addressed the uneventful use of rivaroxaban for 10 days in a COVID patient on V-V ECLS with suspected HITT, with no other intravenous anticoagulation alternatives. In this case, anti-Xa assays were used to monitor the rivaroxaban levels [ 49 ]. So far, no further evidence for the use of direct factor Xa inhibitors in ECLS as anticoagulation is available [ 50 ].
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Desirudin is another recombinant-DNA form of hirudin with an irreversible inhibition action to thrombin. It has been proven to be more effective than UFH or LMWH in reducing the risk of deep venous thrombosis [ 47 ] and to have a similar effect compared to argatroban in the treatment of HITT [ 48 ]. However, there are no case reports or case series discussing the use of desirudin during ECLS.
Lepirudin is a recombinant form of hirudin. It is a bivalent direct thrombin inhibitor, binding to the catalytic site and exosite-1 of thrombin. It is approved by the Food and Drug Administration (FDA) as an alternative drug for heparin in the occurrence of HITT. The half-life of lepirudin is 1&#;2 h and administration by bolus can increase aPTT to a maximum within 10 min. Due to the renal elimination route, dosages must be adjusted in acute kidney injury [ 43 ]. This agent has been used in patients undergoing ECLS with contra-indications for UFH. The literature reports two pediatric cases of lepirudin use in patients diagnosed with HITT and suffering from biventricular heart failure requiring ECLS [ 44 ]. Another two cases reported on lepirudin use in adults with similar conditions [ 45 , 46 ]. In both cases, aPTT and ACT were used to titrate dosages, and, in one case, a lower dose was required based on acute kidney injury. In all described patients, no bleedings or thromboses occurred. Since , lepirudin is no longer available on the market [ 24 ].
Hirudin has been reported as a possible alternative for UFH. It is a naturally occurring anticoagulant in the salivary glands of leeches, and different recombinant (and synthetic) forms are available as anticoagulants but none of them is paired to an antidote.
Low molecular weight heparin (LMWH) has been described as anticoagulation during ECLS with promising results in clinical trials, even if its use is uncommon. The standard test for monitoring LMWH is an anti-Xa essay [ 38 ]. Thromboelastography is an assay to measure the stages of clot development and has also been described as a monitoring assay for LMWH. However, it has not been proven superior to anti-Xa assays. ROTEM does not fully detect the effects of LMWH [ 38 , 39 ]. Since LMWH selectively targets factor Xa through antithrombin, it has more predictable pharmacokinetics and therefore does not need routine monitoring [ 40 ]. The risk for HITT is also lower with LMWH [ 7 ]. Krueger et al. reported a rate of 18% relevant bleeding complications in 61 patients undergoing V-V ECLS support for 7 days with only LMWH as anticoagulation. In 4 (6.5%) patients severe thrombotic events occurred, but all after more than 5 days of ECLS [ 41 ]. In lung transplantation patients, similar outcomes were found. Of 102 patients with perioperative ECLS during lung transplant 80 patients received LMWH, and the remaining 22 received UFH as anticoagulation. No significant differences in bleeding complications were found between both groups, but thromboembolic events occurred more often in the UFH group [ 40 ]. LMWH seems promising, but it is difficult to predict the ending of its effect in the case of need and it cannot be considered as an alternative to UFH in the case of HITT due to the potential remaining risk of HITT antibody formation [ 42 ].
Argatroban is a small molecule direct thrombin inhibitor and can also be an alternative for UFH in patients with a contraindication for UFH and renal failure. Differently from bivalirudin, argatroban binds to the active site of thrombin (univalent), whereas bivalirudin binds to the active site and an additional exosite-1 on thrombin (bivalent) [ 22 ]. The onset of action is within 30 min and the half-life of this agent is around 45 min, with no antidote available [ 24 ]. Argatroban is eliminated by hepatic metabolism, and liver dysfunction requires dosage change [ 22 , 32 , 33 ]. No randomized controlled trials are available on argatroban, and its clinical use is justified based on case series and case reports [ 24 ]. A preclinical study showed lower fibrinolytic levels and higher platelet count in animals treated with argatroban compared to heparin and supported with CPB, using circuit components with or without heparin coating [ 34 ]. Another study tested three sham ECLS circuits with blood priming and demonstrated that thrombin formation was lower in the argatroban anticoagulated circuits compared to heparin, despite a less prolonged aPTT [ 35 ]. Even in ARDS patients requiring ECLS, argatroban administration was found feasible and safe, and comparable to heparin. Outcomes of bleeding complications, requiring transfusion, thrombotic complications, and replacement of ECLS components did not differ between heparin or argatroban anticoagulated patients [ 36 ]. The use of argatroban has been reported in patients simultaneously receiving continuous renal replacement therapy (CRRT) and veno-venous (V-V) ECLS. In these patients, a dosage of 2 µg/kg/min resulted in bleeding complications, and lowering the dose to 0.2 µg/kg/min showed promising effects [ 33 ]. The use of argatroban is associated with higher aPTT values and requires more frequent measurements to titrate the drug to an optimal therapeutic level [ 37 ].
Bivalirudin , a synthetic hirudin, is a direct thrombin inhibitor peptide often used as anticoagulation in HITT patients or patients with heparin resistance [ 6 ]. There is no antidote available, however the half-time of bivalirudin is 25 min and the onset of action is within 4 min [ 23 ]. It is mostly cleared by the kidneys and dosages should be adjusted in renal dysfunction [ 22 , 24 ]. It can be monitored by aPTT but also with ROTEM [ 25 ]. Bivalirudin has been used as an off-label anticoagulation therapy in ECLS with no significant increased risk of bleeding or thrombosis [ 24 ]. In post-cardiotomy ECLS patients, bivalirudin-based anticoagulation, compared to conventional heparin, has been associated with less bleeding and transfusion rates [ 26 ]. Similar outcomes were found in a mixed ECLS adult cohort, where bivalirudin showed less bleeding complications and a lower rate of thrombosis compared to heparin. In the same study, heparin was associated with higher aPTT variations compared to bivalirudin [ 27 ]. Indeed, it has been demonstrated that time within the therapeutic range is better with bivalirudin, especially in high-intensity anticoagulation protocols [ 28 ]. On the other hand, other studies failed to show the significant superiority of bivalirudin in terms of mortality and adverse events. For example, Kaseer et al. were not able to demonstrate any differences in 30-day and in-hospital mortality, major bleedings, renal and hepatic impairment, and thrombotic events between heparin and bivalirudin [ 29 ]. Again, bivalirudin showed more consistency than heparin in ACT and aPTT levels without higher risk for bleeding in patients with normal hepatic function [ 29 , 30 ]. However, dose adjustment is required in patients with hepatic impairment due to possible false and unpredictable aPTT prolongation and changes [ 31 ]. Different dosages of bivalirudin have been reported in studies with ACT and aPTT as monitoring tools to test the effect of medication [ 30 ]. Indeed, the optimal bivalirudin dosage still needs to be defined.
Direct thrombin inhibitors are known alternatives for heparin in HITT patients. These agents bind directly to thrombin and inhibit the actions of thrombin, including feed back-activation of factors V, VIII, and XI, and conversion of fibrinogen to fibrin, and the stimulation of platelets [ 22 ].
A study comparing NM to UFH in dogs on ECLS revealed decreased hemoglobin levels after 1 hour of ECLS in all animals. However, the NM group experienced no cannulation site bleeding as opposed to the UFH group. Thrombo-elastography and aPTT results were comparable between groups, but pro-inflammatory cytokine levels were lower with NM [ 18 ]. A retrospective study of patients on ECLS showed a longer duration of oxygenators, less transfusion of red blood cells, fresh frozen plasma, and cryoprecipitate when NM was used as an anticoagulation agent compared to UFH. In addition, the rate of bleeding, thrombosis, and mortality was higher in the heparin group [ 19 ]. Similarly, Han et al. observed more bleedings with UFH, but 3 cases of intracerebral hemorrhage with NM. Survival was higher in the NM group (38.2% vs. 13.6%) and heparin was found to be the only independent predictor of bleeding complications [ 20 ]. Conflicting results were presented in another retrospective study based on propensity-matched data. In this case, bleeding events occurred more in the NM group, probably because of the lack of an antidote for NM [ 21 ]. In conclusion, evidence on NM is still controversial and it is mainly used as an alternative anticoagulation agent, especially in patients with a high bleeding risk on hemodialysis.
A possible alternative for UFH is nafamostat mesilate (NM). NM is a synthetic serine protease inhibitor, often used as an anticoagulant for patients with a high bleeding risk on hemodialysis. It inhibits thrombin, factor Xa, and XIIa, the kallikrein-kinin system, complement system, and lipopolysaccharide-induced nitric oxide production. There is no antidote available, but NM has a short half-life of 8&#;10 min [ 17 ].
Regardless, in the case of HITT, alternative anticoagulants should be administered, and all sources of heparin should be removed, including heparin-coated components [ 12 , 15 ]. In addition, protamine sulfate can be administered to reverse the effects of UFH. To summarize, UFH is still the most used anticoagulation agent used in ECLS patients but its monitoring uncertainty and the risk of HITT prompt exploring of new anticoagulant agents [ 16 ].
The most commonly used anticoagulation during ECLS is unfractionated heparin (UFH). It has an inhibitory effect by binding the enzyme inhibitor antithrombin and increasing its inhibitory potential toward coagulation enzymes factor Xa and thrombin [ 5 , 6 ]. UFH is administered continuously and usually titrated based on activated clotting time (ACT), anti-factor Xa activity levels, or activated partial thromboplastin time (aPTT) [ 5 ]. Though, these measurements do not always correlate correctly with the heparin dose and effect, leading to some uncertainty in the monitoring of patients&#; anticoagulation status [ 7 ]. Anti-Xa does correlate superiorly on heparin concentrations compared to ACT and aPTT, on the other hand, it does not represent the overall hemostatic state of the patient [ 8 ]. Thromboelastography (TEG) and thromboelastometry (ROTEM) have been studied in ECLS populations, where ROTEM showed moderate correlation with standard coagulation test and [ 9 ] ROTEM has been found to be a good indicator of anticoagulation status in pediatric patients undergoing ECLS as well [ 10 ]. Furthermore, UFH might stimulate the development of antibodies against heparin-platelet factor 4 complexes, which induce heparin-thrombocytopenia and thrombosis (HITT) [ 11 ]. The incidence of HITT varies between 0.36% [ 12 ] and 3.1% [ 13 ], and 50% of ECLS patients diagnosed with HITT develop clinically significant thrombotic events if no alternative anticoagulant is given [ 12 ]. While circulating UFH is surely related to HITT, it is unclear if heparin-coated circuits may induce HITT [ 14 ].
To minimize the risk of thrombosis or clotting in the circuit, and subsequently the failure of the ECLS system, patients receive systemic anticoagulation. An optimal anticoagulation agent should be easy to administer and monitor and have a moderate risk for bleeding complications while maintaining the anti-thrombotic effects. Moreover, it should have an antidote or short half-life to ensure possible counteraction or fast extinguishing effect. Currently, multiple anticoagulant drugs are available, but each of them has specific advantages and disadvantages, implying the fact that the perfect agent still needs to be found ( Table 1 ).
The complex interaction between inflammation and coagulation significantly affects a patients&#; safety, but it has also important consequences on the ECLS devices as well, especially in terms of durability. Despite the routine patient&#;s systemic anticoagulation, deposition of blood proteins onto the artificial ECLS surfaces may still occur, leading to inefficient membrane functioning, insufficient gas transfer, and finally, device failure [51]. This is a major limitation for the long-term use of ECLS systems and a major obstacle toward the development of totally implantable durable devices [52,53]. The main limiting factors are related to platelet and coagulation activation leading to clot formation within the system, and protein adsorption which gradually impairs gas exchange in the oxygenator [52]. For these reasons, research efforts are aiming to improve hemocompatibility of foreign surfaces, optimize gas and blood flows, miniaturize ECLS systems, and decrease the imbalance of coagulation and inflammation [52].
From an engineering point of view, the new ECLS circuits should aim to mimic the physiologic conditions in order to avoid hemolysis and reduce the shear stress and/or the stasis zones [54,55,56,57]. The artificial surface area of the ECLS systems should be minimized by simplifying the circuit, reducing shear stress and stasis, while maintaining or increasing usability [58]. On the other hand, the ultimate goal is to mimic healthy endothelial tissue in circuits´ surfaces such as oxygenators´ membranes and housing parts, pumps, cannula, and tubing to eliminate both the systemic inflammatory and the coagulation pathway responses.
Normally, anticoagulant regulation of procoagulant processes is regulated by the endothelium which is absent at the artificial surfaces of the ECLS circuit. The artificial surfaces not only activate platelets and factor XII, but also adsorb plasma proteins like fibrinogen, immunoglobulins, hemoglobin, fibronectin, and van Willebrand factor, in varying amounts depending on the material, but especially on hydrophobic surfaces [59]. This protein adhesion is thought to be the initiating factor of the procoagulant response [60]. As a consequence, to improve the hemocompatibility of these artificial ECLS surfaces, a replication of the anti-thrombotic and anti-inflammatory properties of the endothelium would be ideal. According to Ontaneda and Annich, surface modifications addressing this goal can be classified into three major groups [61]: bioactive surfaces (also called biomimetic surfaces); biopassive surfaces; and endothelialization of blood-contacting surfaces.
An overview of the commercially available hemocompatibility improving coatings for extracorporeal circulation systems is available in Table 2.
Overview of the commercially used coatings in extracorporeal life support circuit components.
Main Coating Compount(s) Commercial Name of Coating Company Bioactive Heparin Cortiva Bioactive surface Medtronic Heparin Rheoparin Xenios/Fresenius Albumin + Heparin Bioline Maquet/Getinge Albumin + Heparin X.ellence Xenios/Fresenius Biopassive Albumin Rheopak Chalice Medical Albumin Recombinant Albumin Coating Hemovent Albumin Safeline (discontinued) Maquet/Getinge Albumin X.eed Xenios/Fresenius Phosphorylcholine PC phosphorylcholine Eurosets Phosphorylcholine PH.I.S.I.O Coating Liva Nova poly(2-methoxyethylacrylate) (PMEA) Xcoating Terumo Sulphate and sulphonate groups and polyethylene oxide (PEO) Balance Biosurface Medtronic Sulphonate groups, polyethylene oxide (PEO) and heparin Trillium Biosurface Medtronic Amphyphilic polymer Softline Maquet/Getinge Open in a new tabHeparin-coated systems for ECLS were developed to reduce the hemorrhagic risk by lowering the systemic heparinization [62,63,64,65]. The first heparin coating to become commercially available was developed by the company Carmeda in [66,67]. From that time on, several new coatings with different bonding techniques have been developed and became available in the market. The local release of heparin can minimize the negative effects of foreign materials coming in contact with blood [68]. In an early study, Videm et al. found that heparin coatings have the ability to reduce complement activation by 45% [69]. Wendel and Ziemer analyzed several studies and assumed that oxygenators coated with heparin can reduce the following effects in comparison to uncoated devices: activation of contact activation of coagulation, complement system activation, alteration of granulocytes, inflammation, and pulmonary complications, activation of platelets, disturbance of homeostasis, loss of blood, and cerebral damage [70]. However, the utility of heparin-coated materials has been questioned. Covalently- and ionic-bonded heparin coating on oxygenators reduced some effects of the inflammatory response, thrombi formation, but other complications remained the same when compared to uncoated oxygenators [60]. In general, these studies need to be interpreted with some caution as most were performed either in 6 h in vitro tests or in short-term use in CPB. Thus, their relevance for long-term ECLS is limited, but no evident contraindications are reported so far [71].
Nitric Oxide (NO) is also known as an endothelium-derived relaxing factor and is released by endothelial cells to induce vasodilatation. NO activates an increase in cyclic guanosine monophosphate (GMP) in platelets and vascular smooth muscle cells [61]. Indeed, coatings with NO-catalytic bioactivity can inhibit collagen-induced platelet activation and adhesion, proliferation, and migration of arterial smooth muscle cells through the cGMP signaling pathways. Studies showed good anti-thrombogenic properties in extracorporeal circuits [61,72]. Moreover, stents implanted in rabbits with this coating showed improved endothelial mimetic microenvironment, stronger recovery to the endothelium, and had less restenosis and thrombosis after 4 weeks [73]. A significant reduction in platelet consumption and activation was also observed in animal studies. The latest generation of NO coating is characterized by a lipophilic NO donor complex embedded into plasticized PVC to prevent uncontrolled NO release in the circulatory system. This technology showed not only platelet inhibition but also less fibrinogen consumption. The main disadvantage with NO is the fact that its storage cannot exceed 4 weeks. This can be a problem in long-term ECLS runs [61,72]. So far, NO-coatings have not been used commercially. However, NO was clinically used as a fraction of the sweep gas (20 ppm) of the oxygenator in 31 pediatric ECLS runs in order to use its anti-thrombotic properties by diffusion through the gas exchanger membrane [74].
To further improve hemocompatibility, a novel covalent C1-esterase inhibitor (C1-INH) coating has been introduced by Gering et al. [53]. Besides complement inhibition, C1-INH also prevents factor XII (a) activation, an early event of contact phase activation at the crossroads of coagulation and inflammation [53]. This coating is still under development and thus not commercially available.
Albumin has been used as coating material since and it is often indicated in case of contraindications from heparin [75]. Albumin coating is used as a base layer with a hydrophilic surface, which reduces the biological response to hydrophobic surfaces [23]. Albumin lacks binding sequences for platelets, leukocytes, and coagulation enzymes and therefore slows down the platelet activation when used as a coating. Nevertheless, albumin coatings do not last long due to displacement by procoagulant proteins [75]. Some manufacturers use albumin as part of a multi-layer, bioactive coating in alternating layers with heparin (Table 2: Bioline and X.ellence coatings).
Phosphorylcholine (PC) is anti-thrombogenic, protein resistant, antibacterial, and has anti-fouling properties [67]. Coatings with phosphorylcholine (PC) have been developed as an alternative to heparin-bound systems. PC is a hydrophilic polar headgroup of phospholipids. It contains a negatively charged phosphate bonded to a positively charged choline. Phospholipids containing PC are non-thrombogenic. PC coatings in extracorporeal circuits have been found to induce plateau formation of thromboxane B2 and thromboglobulin and even reduce thrombin formation [76]. However, other studies did not find PC favorable over heparin-coated circuits [61]. A study by Thiara et al. compared heparin-albumin coating with PC coating in elective cardiac surgery patients. The PC group showed significantly higher lactate dehydrogenase, thus hemolysis, but this was allocated to the fact that the group had significantly longer aortic clamping time and CPB duration. Further, hemoglobin, platelet counts, numbers of leukocytes and cytokines, levels of complement activation, and endothelial shedding molecule syndecan-1 were not significantly different between the two coating groups [77].
Poly(2-methoxy-ethyl-acrylate) (PMEA) is a blood-compatible polymer composed of a hydrophobic polyethylene chain and a mild hydrophilic tail. This combined hydrophobic and hydrophilic polymer allows the polymer to adhere to the hydrophobic site to different materials and create a hydrophilic surface for the blood to contact with the other side. Proteins and platelets will not denature or adhere to the hydrophilic surface [59]. Animal studies involving CPB revealed suppression of the complement system activation [61]. Compared to non-coated systems in patients undergoing coronary artery bypass grafting, PMEA coating was superior in reduction of platelet adhesion, aggregation, and protein adsorption [78]. However, other studies found a higher risk of postoperative leukopenia and systemic inflammatory response syndrome (SIRS) without a decrease in platelet aggregation [79]. Finally, there is no consensus on whether or not PMEA is superior to heparin-bound systems.
Polyethylene oxide (PEO), commercially used in combination with negatively charged sulphonate groups and sulphate, is used as a biopassive coating, which has been proposed as an alternative to the heparin-loaded coatings. In an ex vivo study with human blood (n = 40), Teliguia et al. found no differences in coagulation activation (factor IIa, prothrombin fragment 1 + 2 were assessed) when compared to a heparin coating. All groups demonstrated similar adhesion scores following ultrastructural oxygenator assessment by scanning electron microscopy and no difference in the pressure gradients of the oxygenators was observed [80].
Poly(MPC-co-BMA-co-TSMA) (PMBT), a zwitterionic copolymer, is also a polymer with both positive and negative charged components [81]. PMBT coating was shown to be stable on polypropylene hollow fiber membranes, tested by Wang et al. by elution with ethanol and washing and sterilizing solutions of peracidin. In the same study in animal models, almost no change in fibrinogen and platelets in the blood after blood circulation through PMBT copolymer circuits was observed. In the uncoated circuits, fibrinogen and platelets were significantly reduced due to absorption and consumption. Thrombus formation was significantly lower in the PMBT circuits. PMBT&#;s influence on gas exchange was not tested in the study [82]. The mimetic surface seems promising and might be applicable in artificial lung systems, however, it is not commercially available yet.
In an in vitro study by Preston et al., different coatings were tested in ECLS circuits with bovine blood. Coatings were tested regarding the adsorption of morphine and fentanyl. Safeline® coating&#;a synthetic albumin (Maquet), Softline® coating&#;a heparin free polymer (Maquet), Bioline® coating&#;recombinant albumin and heparin (Maquet), Xcoating®&#;poly2methoxylacetylate (Terumo), Carmeda® coating&#;covalently bonded heparin (Metronic), and Trillium®&#;covalently bonded heparin (Metronic) were compared to one another. All circuit coatings were associated with the loss of drugs. The Carmeda® and Xcoating® had significantly more morphine adsorption than Safeline®, Softline®, Bioline® and Trillium®. Fentanyl was adsorbed more in Safeline®, Softline®, Bioline®, and Trillium® compared to Carmeda® and Xcoating®, but was not statistically significant [83].
Surface endothelialization is a technique where an endothelial layer is created onto circuit surface areas by seeding cells onto the surface to achieve complete hemocompatibility between blood and materials. Creating a surface with endothelial cells would achieve higher hemocompatibility than replicating specific thrombo-regulatory aspects of the endothelium. Few studies have investigated the feasibility of establishing an endothelial monolayer on the gas exchange ECLS membranes [51], although it is known that endothelial cells do not adhere easily to hydrophobic surfaces [75]. To provide an endothelial monolayer, the base of the material must enable endothelial attachment and bonding while preserving the viability of the endothelial cells. Heparin/albumin-coated PMP membrane fibers were found to be a good base for a viable and confluent endothelial monolayer of endothelial cells. Moreover, the heparin/albumin coating avoids thrombogenic events in areas not covered with cells [84]. Pflaum et al. demonstrated the effectiveness of a stable titanium dioxide (TiO2) coating achieved by pulsed vacuum cathodic arc plasma deposition (PVCAPD) technique on hydrophobic poly(4-methyl-1-pentene (PMP) membranes, with a functional monolayer of endothelial cells as a result. Although the use of the TiO2 coating resulted in a reduction in the oxygen transfer rate (OTR) of the membrane by 22%, it successfully mediated EC attachment. The endothelial layer was resistant to shear stress and able to repair itself when monolayer disruption appeared. [51]. A study experimented with endothelial cell seeding from cells derived from juvenile sheep carotid arteries and searched for the best protein coating for endothelial cell attachment. Seeding endothelial cells to uncoated oxygenator membranes was ineffective, and using gelatin, fibrinogen, and collagen IV did not enhance the cell seeding process. Cornellissen et al. considered fibronectin to be a good base for cell attachment on flat sheet membranes, however, they did not perform gas exchange performance tests [85]. However, current research on how to establish a single layer of endothelial tissue on the gas exchange of ECLS equipment is not advanced [23]. In addition, the shelf life of an endothelialized oxygenator can, under hypothermic conditions, be stretched up to two weeks [86] compared to the shelf life of an otherwise coated oxygenator being typically 2 years. This would result in complex resource planning and management for both manufacturers and ECLS centers. The use of immune-silenced cells might at least help in quicker response times as production for a particular patient would not depend on the availability of autologous cells. Indeed, Wiegmann et al. showed that the rejection of allogeneic endothelial cells could be prevented by silencing HLA-class I expression [87]. However, many questions in relation to costs, timely production, quality assurance, and approval of endothelialized oxygenators remain open, leaving a wide field of potential research.
Heparin, also known as unfractionated heparin (UFH), is a medication and naturally occurring glycosaminoglycan.[3][4] Heparin is a blood anticoagulant that increases the activity of antithrombin.[5] It is used in the treatment of heart attacks and unstable angina.[3] It can be given intravenously or by injection under the skin.[3] Its anticoagulant properties make it useful to prevent blood clotting in blood specimen test tubes and kidney dialysis machines.[4][6]
Common side effects include bleeding, pain at the injection site, and low blood platelets.[3] Serious side effects include heparin-induced thrombocytopenia.[3] Greater care is needed in those with poor kidney function.[3]
Heparin is contraindicated for suspected cases of vaccine-induced pro-thrombotic immune thrombocytopenia (VIPIT) secondary to SARS-CoV-2 vaccination, as heparin may further increase the risk of bleeding in an anti-PF4/heparin complex autoimmune manner, in favor of alternative anticoagulant medications (such as argatroban or danaparoid).[7][8][9]
Heparin appears to be relatively safe for use during pregnancy and breastfeeding.[10] Heparin is produced by basophils and mast cells in all mammals.[11]
The discovery of heparin was announced in .[12] It is on the World Health Organization's List of Essential Medicines.[13] A fractionated version of heparin, known as low molecular weight heparin, is also available.[14]
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Heparin was discovered by Jay McLean and William Henry Howell in , although it did not enter clinical trials until .[15] It was originally isolated from dog liver cells, hence its name (&#;παρ hēpar is Greek for 'liver'; hepar + -in).
McLean was a second-year medical student at Johns Hopkins University, and was working under the guidance of Howell investigating pro-coagulant preparations, when he isolated a fat-soluble phosphatide anticoagulant in canine liver tissue.[16] In , Howell coined the term 'heparin' for this type of fat-soluble anticoagulant. In the early s, Howell isolated a water-soluble polysaccharide anticoagulant, which he also termed 'heparin', although it was different from the previously discovered phosphatide preparations.[17][18] McLean's work as a surgeon probably changed the focus of the Howell group to look for anticoagulants, which eventually led to the polysaccharide discovery.
It had at first been accepted that it was Howell who discovered heparin. However in the s, Jay McLean became unhappy that he had not received appropriate recognition for what he saw as his own discovery. Though relatively discreet about his claim and not wanting to upset his former chief, he gave lectures and wrote letters claiming that the discovery was his. This gradually became accepted as fact, and indeed after his death in , his obituary credited him as being the true discoverer of heparin. This was elegantly restated in in a plaque unveiled in Johns Hopkins to commemorate the major contribution (of McLean) to the discovery of heparin in in collaboration with Professor William Henry Howell.[19]
In the s, several researchers were investigating heparin. Erik Jorpes at Karolinska Institutet published his research on the structure of heparin in ,[20] which made it possible for the Swedish company Vitrum AB to launch the first heparin product for intravenous use in . Between and , Connaught Medical Research Laboratories, then a part of the University of Toronto, perfected a technique for producing safe, nontoxic heparin that could be administered to patients, in a saline solution. The first human trials of heparin began in May , and, by , it was clear that Connaught's heparin was safe, easily available, and effective as a blood anticoagulant. Prior to , heparin was available in small amounts, was extremely expensive and toxic, and, as a consequence, of no medical value.[21]
Heparin production experienced a break in the s. Until then, heparin was mainly obtained from cattle tissue, which was a by-product of the meat industry, especially in North America. With the rapid spread of BSE, more and more manufacturers abandoned this source of supply. As a result, global heparin production became increasingly concentrated in China, where the substance was now procured from the expanding industry of breeding and slaughtering hog. The dependence of medical care on the meat industry assumed threatening proportions in the wake of the COVID-19 pandemic. In , several studies demonstrated the efficacy of heparin in mitigating severe disease progression, as its anticoagulant effect counteracted the formation of immunothrombosis. However, the availability of heparin on the world market was decreased, because concurrently a renewed swine flu epidemic had reduced significant portions of the Chinese hog population. The situation was further exacerbated by the fact that mass slaughterhouses around the world became corona hotspots themselves and were forced to close temporarily. In less affluent countries, the resulting heparin shortage also led to worsened health care beyond the treatment of covid, for example through the cancellation of cardiac surgeries.[22]
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A vial of heparin sodium for injectionHeparin acts as an anticoagulant, preventing the formation of clots and extension of existing clots within the blood. Heparin itself does not break down clots that have already formed, instead it prevents clot formation by inhibiting thrombin and other procoagulant serine proteases. Heparin is generally used for anticoagulation for the following conditions:[23]
Heparin and its low-molecular-weight derivatives (e.g., enoxaparin, dalteparin, tinzaparin) are effective in preventing deep vein thromboses and pulmonary emboli in people at risk,[24][25] but no evidence indicates any one is more effective than the other in preventing mortality.[26]
In angiography, 2 to 5 units/mL of unfractionated heparin saline flush is used as a locking solution to prevent the clotting of blood in guidewires, sheaths, and catheters, thus preventing thrombus from dislodging from these devices into the circulatory system .[27][28]
Unfractionated heparin is used in hemodialysis. Comparing to low-molecular-weight heparin, unfractionated heparin does not have prolonged anticoagulation action after dialysis, and low cost. However, the short duration of action for heparin would require it to maintain continuous infusion to maintain its action. Meanwhile, unfractionated heparin has higher risk of heparin-induced thrombocytopenia.[29]
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A serious side-effect of heparin is heparin-induced thrombocytopenia (HIT), caused by an immunological reaction that makes platelets a target of immunological response, resulting in the degradation of platelets, which causes thrombocytopenia.[30] This condition is usually reversed on discontinuation, and in general can be avoided with the use of synthetic heparins. Not all patients with heparin antibodies will develop thrombocytopenia. Also, a benign form of thrombocytopenia is associated with early heparin use, which resolves without stopping heparin. Approximately one-third of patients with diagnosed heparin-induced thrombocytopenia will ultimately develop thrombotic complications.[31]
Two non-hemorrhagic side-effects of heparin treatment are known. The first is elevation of serum aminotransferase levels, which has been reported in as many as 80% of patients receiving heparin. This abnormality is not associated with liver dysfunction, and it disappears after the drug is discontinued. The other complication is hyperkalemia, which occurs in 5 to 10% of patients receiving heparin, and is the result of heparin-induced aldosterone suppression. The hyperkalemia can appear within a few days after the onset of heparin therapy. More rarely, the side-effects alopecia and osteoporosis can occur with chronic use.[23]
As with many drugs, overdoses of heparin can be fatal. In September , heparin received worldwide publicity when three prematurely born infants died after they were mistakenly given overdoses of heparin at an Indianapolis hospital.[32]
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Heparin is contraindicated in those with risk of bleeding (especially in people with uncontrolled blood pressure, liver disease, and stroke), severe liver disease, or severe hypertension.[33]
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Protamine sulfate has been given to counteract the anticoagulant effect of heparin (1 mg per 100 units of heparin that had been given over the past 6 hours).[34] It may be used in those who overdose on heparin or to reverse heparin's effect when it is no longer needed.[35]
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Heparin's normal role in the body is unclear. Heparin is usually stored within the secretory granules of mast cells and released only into the vasculature at sites of tissue injury. It has been proposed that, rather than anticoagulation, the main purpose of heparin is defense at such sites against invading bacteria and other foreign materials.[36] In addition, it is observed across a number of widely different species, including some invertebrates that do not have a similar blood coagulation system. It is a highly sulfated glycosaminoglycan. It has the highest negative charge density of any known biological molecule.[37]
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In addition to the bovine and porcine tissue from which pharmaceutical-grade heparin is commonly extracted, it has also been extracted and characterized from:
The biological activity of heparin within species 6&#;11 is unclear and further supports the idea that the main physiological role of heparin is not anticoagulation. These species do not possess any blood coagulation system similar to that present within the species listed 1&#;5. The above list also demonstrates how heparin has been highly evolutionarily conserved, with molecules of a similar structure being produced by a broad range of organisms belonging to many different phyla.[citation needed]
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In nature, heparin is a polymer of varying chain size. Unfractionated heparin (UFH) as a pharmaceutical is heparin that has not been fractionated to sequester the fraction of molecules with low molecular weight. In contrast, low-molecular-weight heparin (LMWH) has undergone fractionation for the purpose of making its pharmacodynamics more predictable. Often either UFH or LMWH can be used; in some situations one or the other is preferable.[51]
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Heparin binds to the enzyme inhibitor antithrombin III (AT), causing a conformational change that results in its activation through an increase in the flexibility of its reactive site loop.[52] The activated AT then inactivates thrombin, factor Xa and other proteases. The rate of inactivation of these proteases by AT can increase by up to -fold due to the binding of heparin.[53] Heparin binds to AT via a specific pentasaccharide sulfation sequence contained within the heparin polymer:
The conformational change in AT on heparin-binding mediates its inhibition of factor Xa. For thrombin inhibition, however, thrombin must also bind to the heparin polymer at a site proximal to the pentasaccharide. The highly negative charge density of heparin contributes to its very strong electrostatic interaction with thrombin.[37] The formation of a ternary complex between AT, thrombin, and heparin results in the inactivation of thrombin. For this reason, heparin's activity against thrombin is size-dependent, with the ternary complex requiring at least 18 saccharide units for efficient formation.[54] In contrast, antifactor Xa activity via AT requires only the pentasaccharide-binding site.
This size difference has led to the development of low-molecular-weight heparins (LMWHs) and fondaparinux as anticoagulants. Fondaparinux targets anti-factor Xa activity rather than inhibiting thrombin activity, with the aim of facilitating a more subtle regulation of coagulation and an improved therapeutic index. It is a synthetic pentasaccharide, whose chemical structure is almost identical to the AT binding pentasaccharide sequence that can be found within polymeric heparin and heparan sulfate.
With LMWH and fondaparinux, the risk of osteoporosis and heparin-induced thrombocytopenia (HIT) is reduced. Monitoring of the activated partial thromboplastin time is also not required and does not reflect the anticoagulant effect, as APTT is insensitive to alterations in factor Xa.
Danaparoid, a mixture of heparan sulfate, dermatan sulfate, and chondroitin sulfate can be used as an anticoagulant in patients having developed HIT. Because danaparoid does not contain heparin or heparin fragments, cross-reactivity of danaparoid with heparin-induced antibodies is reported as less than 10%.[55]
The effects of heparin are measured in the lab by the partial thromboplastin time (aPTT), one of the measures of the time it takes the blood plasma to clot. Partial thromboplastin time should not be confused with prothrombin time, or PT, which measures blood clotting time through a different pathway of the coagulation cascade.
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Heparin vial for subcutaneous injectionHeparin is given parenterally because it is not absorbed from the gut, due to its high negative charge and large size. It can be injected intravenously or subcutaneously (under the skin); intramuscular injections (into muscle) are avoided because of the potential for forming hematomas. Because of its short biologic half-life of about one hour, heparin must be given frequently or as a continuous infusion. Unfractionated heparin has a half-life of about one to two hours after infusion,[56] whereas LMWH has a half-life of four to five hours.[57] The use of LMWH has allowed once-daily dosing, thus not requiring a continuous infusion of the drug. If long-term anticoagulation is required, heparin is often used only to commence anticoagulation therapy until an oral anticoagulant e.g. warfarin takes effect.
The American College of Chest Physicians publishes clinical guidelines on heparin dosing.[58]
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Unfractionated heparin has a half-life of about one to two hours after infusion,[56] whereas low-molecular-weight heparin's half-life is about four times longer. Lower doses of heparin have a much shorter half-life than larger ones. Heparin binding to macrophage cells is internalized and depolymerized by the macrophages. It also rapidly binds to endothelial cells, which precludes the binding to antithrombin that results in anticoagulant action. For higher doses of heparin, endothelial cell binding will be saturated, such that clearance of heparin from the bloodstream by the kidneys will be a slower process.[59]
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Native heparin is a polymer with a molecular weight ranging from 3 to 30 kDa, although the average molecular weight of most commercial heparin preparations is in the range of 12 to 15 kDa.[60] Heparin is a member of the glycosaminoglycan family of carbohydrates (which includes the closely related molecule heparan sulfate) and consists of a variably sulfated repeating disaccharide unit.[61] The main disaccharide units that occur in heparin are shown below. The most common disaccharide unit* (see below) is composed of a 2-O-sulfated iduronic acid and 6-O-sulfated, N-sulfated glucosamine, IdoA(2S)-GlcNS(6S). For example, this makes up 85% of heparins from beef lung and about 75% of those from porcine intestinal mucosa.[62]
Not shown below are the rare disaccharides containing a 3-O-sulfated glucosamine (GlcNS(3S,6S)) or a free amine group (GlcNH3+). Under physiological conditions, the ester and amide sulfate groups are deprotonated and attract positively charged counterions to form a heparin salt. Heparin is usually administered in this form as an anticoagulant.
GlcA = β-D-glucuronic acid, IdoA = α-L-iduronic acid, IdoA(2S) = 2-O-sulfo-α-L-iduronic acid, GlcNAc = 2-deoxy-2-acetamido-α-D-glucopyranosyl, GlcNS = 2-deoxy-2-sulfamido-α-D-glucopyranosyl, GlcNS(6S) = 2-deoxy-2-sulfamido-α-D-glucopyranosyl-6-O-sulfate
One unit of heparin (the "Howell unit") is an amount approximately equivalent to 0.002 mg of pure heparin, which is the quantity required to keep 1 ml of cat's blood fluid for 24 hours at 0 °C.[63]
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The three-dimensional structure of heparin is complicated because iduronic acid may be present in either of two low-energy conformations when internally positioned within an oligosaccharide. The conformational equilibrium is influenced by sulfation state of adjacent glucosamine sugars.[64] Nevertheless, the solution structure of a heparin dodecasaccharide composed solely of six GlcNS(6S)-IdoA(2S) repeat units has been determined using a combination of NMR spectroscopy and molecular modeling techniques.[65] Two models were constructed, one in which all IdoA(2S) were in the 2S0 conformation (A and B below), and one in which they are in the 1C4 conformation (C and D below). However, no evidence suggests that changes between these conformations occur in a concerted fashion. These models correspond to the protein data bank code 1HPN.[66]
Two different structures of heparin
In the image above:
In these models, heparin adopts a helical conformation, the rotation of which places clusters of sulfate groups at regular intervals of about 17 angstroms (1.7 nm) on either side of the helical axis.
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Either chemical or enzymatic depolymerization techniques or a combination of the two underlie the vast majority of analyses carried out on the structure and function of heparin and heparan sulfate (HS).
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The enzymes traditionally used to digest heparin or HS are naturally produced by the soil bacterium Pedobacter heparinus (formerly named Flavobacterium heparinum).[67] This bacterium is capable of using either heparin or HS as its sole carbon and nitrogen source. To do so, it produces a range of enzymes such as lyases, glucuronidases, sulfoesterases, and sulfamidases.[68] The lyases have mainly been used in heparin/HS studies. The bacterium produces three lyases, heparinases I (EC 4.2.2.7), II (no EC number assigned) and III (EC 4.2.2.8) and each has distinct substrate specificities as detailed below.[69][70]
Heparinase enzyme Substrate specificity Heparinase I GlcNS(±6S)-IdoA(2S) Heparinase II GlcNS/Ac(±6S)-IdoA(±2S)The lyases cleave heparin/HS by a beta elimination mechanism. This action generates an unsaturated double bond between C4 and C5 of the uronate residue.[71][72] The C4-C5 unsaturated uronate is termed ΔUA or UA. It is a sensitive UV chromophore (max absorption at 232 nm) and allows the rate of an enzyme digest to be followed, as well as providing a convenient method for detecting the fragments produced by enzyme digestion.
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Nitrous acid can be used to chemically depolymerize heparin/HS. Nitrous acid can be used at pH 1.5 or at a higher pH of 4. Under both conditions, nitrous acid effects deaminative cleavage of the chain.[73]
IdoA(2S)-aMan: The anhydromannose can be reduced to an anhydromannitolAt both 'high' (4) and 'low' (1.5) pH, deaminative cleavage occurs between GlcNS-GlcA and GlcNS-IdoA, albeit at a slower rate at the higher pH. The deamination reaction, and therefore chain cleavage, is regardless of O-sulfation carried by either monosaccharide unit.
At low pH, deaminative cleavage results in the release of inorganic SO4, and the conversion of GlcNS into anhydromannose (aMan). Low-pH nitrous acid treatment is an excellent method to distinguish N-sulfated polysaccharides such as heparin and HS from non N-sulfated polysaccharides such as chondroitin sulfate and dermatan sulfate, chondroitin sulfate and dermatan sulfate not being susceptible to nitrous acid cleavage.
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Current clinical laboratory assays for heparin rely on an indirect measurement of the effect of the drug, rather than on a direct measure of its chemical presence. These include activated partial thromboplastin time (APTT) and antifactor Xa activity. The specimen of choice is usually fresh, nonhemolyzed plasma from blood that has been anticoagulated with citrate, fluoride, or oxalate.[74][75]
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Considering the animal source of pharmaceutical heparin, the numbers of potential impurities are relatively large compared with a wholly synthetic therapeutic agent. The range of possible biological contaminants includes viruses, bacterial endotoxins, transmissible spongiform encephalopathy (TSE) agents, lipids, proteins, and DNA. During the preparation of pharmaceutical-grade heparin from animal tissues, impurities such as solvents, heavy metals, and extraneous cations can be introduced. However, the methods employed to minimize the occurrence and to identify and/or eliminate these contaminants are well established and listed in guidelines and pharmacopoeias. The major challenge in the analysis of heparin impurities is the detection and identification of structurally related impurities. The most prevalent impurity in heparin is dermatan sulfate (DS), also known as chondroitin sulfate B. The building-block of DS is a disaccharide composed of 1,3-linked N-acetyl galactosamine (GalN) and a uronic acid residue, connected via 1,4 linkages to form the polymer. DS is composed of three possible uronic acid (GlcA, IdoA or IdoA2S) and four possible hexosamine (GalNAc, Gal- NAc4S, GalNAc6S, or GalNAc4S6S) building-blocks. The presence of iduronic acid in DS distinguishes it from chrondroitin sulfate A and C and likens it to heparin and HS. DS has a lower negative charge density overall compared to heparin. A common natural contaminant, DS is present at levels of 1&#;7% in heparin API, but has no proven biological activity that influences the anticoagulation effect of heparin.[87]
In December , the US Food and Drug Administration (FDA) recalled a shipment of heparin because of bacterial growth (Serratia marcescens) in several unopened syringes of this product. S. marcescens can lead to life-threatening injuries and/or death.[88]
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In March , major recalls of heparin were announced by the FDA due to contamination of the raw heparin stock imported from China.[89][90] According to the FDA, the adulterated heparin killed nearly 80 people in the United States.[91] The adulterant was identified as an "over-sulphated" derivative of chondroitin sulfate, a popular shellfish-derived supplement often used for arthritis, which was intended to substitute for actual heparin in potency tests.[92]
According to the New York Times: "Problems with heparin reported to the agency include difficulty breathing, nausea, vomiting, excessive sweating and rapidly falling blood pressure that in some cases led to life-threatening shock".
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In , Petr Zelenka, a nurse in the Czech Republic, deliberately administered large doses to patients, killing seven, and attempting to kill ten others.[93]
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In , a nurse at Cedars-Sinai Medical Center mistakenly gave the 12-day-old twins of actor Dennis Quaid a dose of heparin that was 1,000 times the recommended dose for infants.[94] The overdose allegedly arose because the labeling and design of the adult and infant versions of the product were similar. The Quaid family subsequently sued the manufacturer, Baxter Healthcare Corp.,[95][96] and settled with the hospital for $750,000.[97] Prior to the Quaid accident, six newborn babies at Methodist Hospital in Indianapolis, Indiana, were given an overdose. Three of the babies died after the mistake.[98]
In July , another set of twins born at Christus Spohn Hospital South, in Corpus Christi, Texas, died after an accidentally administered overdose of the drug. The overdose was due to a mixing error at the hospital pharmacy and was unrelated to the product's packaging or labeling.[99] As of July , the exact cause of the twins' death was under investigation.[100][101]
In March , a two-year-old transplant patient from Texas was given a lethal dose of heparin at the University of Nebraska Medical Center. The exact circumstances surrounding her death are still under investigation.[102]
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Pharmaceutical-grade heparin is derived from mucosal tissues of slaughtered meat animals such as porcine (pig) intestines or bovine (cattle) lungs.[103] Advances to produce heparin synthetically have been made in and .[104] In , a chemoenzymatic process of synthesizing low molecular weight heparins from simple disaccharides was reported.[105]
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As detailed in the table below, the potential is great for the development of heparin-like structures as drugs to treat a wide range of diseases, in addition to their current use as anticoagulants.[106][107]
As a result of heparin's effect on such a wide variety of disease states, a number of drugs are indeed in development whose molecular structures are identical or similar to those found within parts of the polymeric heparin chain.[106]
Drug molecule Effect of new drug compared to heparin Biological activities Heparin tetrasaccharide Nonanticoagulant, nonimmunogenic, orally active Antiallergic Pentosan polysulfate Plant derived, little anticoagulant activity, anti-inflammatory, orally active Anti-inflammatory, antiadhesive, antimetastatic Phosphomannopentanose sulfate Potent inhibitor of heparanase activity Antimetastatic, antiangiogenic, anti-inflammatory Selectively chemically O-desulphated heparin Lacks anticoagulant activity Anti-inflammatory, antiallergic, antiadhesive[
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