key: cord-0987189-0szbkhiz
authors: Shi, Chen; Tingting, Wu; Li, Jin-Ping; Sullivan, Mitchell A.; Wang, Cong; Wang, Hanxiang; Deng, Bin; Zhang, Yu
title: Comprehensive Landscape of Heparin Therapy for COVID-19
date: 2020-10-22
journal: Carbohydr Polym
DOI: 10.1016/j.carbpol.2020.117232
sha: 2afda8fb35e0508fa094e9b72c1755856714d722
doc_id: 987189
cord_uid: 0szbkhiz

The pandemic coronavirus disease 2019 (COVID-19), caused by the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is rapidly spreading globally. Clinical observations found that systemic symptoms caused by SARS-CoV-2 infection are attenuated when using the anticoagulant agent heparin, indicating that heparin may play other roles in managing COVID-19, in addition to prevention of pulmonary thrombosis. Several biochemical studies show strong binding of heparin and heparin-like molecules to the Spike protein, which resulted in inhibition of viral infection to cells. The clinical observations and in vitro studies argue for a potential multiple-targeting effects of heparin. However, adverse effects of heparin administration and some of the challenges using heparin therapy for SARS-CoV-2 infection need to be considered. This review discusses the pharmacological mechanisms of heparin regarding its anticoagulant, anti-inflammatory and direct antiviral activities, providing current evidence concerning the effectiveness and safety of heparin therapy for this major public health emergency.

Since December 2019, a highly infectious coronavirus, causing coronavirus disease 2019 ,is rapidly spreadingthroughout the globe (Zhu et al., 2020) .

COVID-19 can induce a severe respiratory illness similar to that caused by severe acute respiratory syndrome coronavirus (SARS-CoV) infection.On February 11, 2020, theInternational Committee on Taxonomy of Viruses officially named the new type of coronavirus that caused COVID-19 epidemic as SARS-CoV-2 (Walls et al., 2020) .The clinical manifestations of COVID-19 includefever, dry cough and fatigue while a few patients are accompanied by nasal congestion, runny nose, sore throat, myalgia and diarrhea. Severe patients often develop dyspnea and/or hypoxemia one week after the disease's onset. For these more severe cases, symptomscan quickly progress to acute respiratory distress syndrome (ARDS), septic shock, metabolic acidosis, coagulopathy, and multiple organ failures (Guan et al., 2020) .As of September 30, 2020, the number ofSARS-CoV-2 infected patients had already risen to33 million, with an increase of more than 280,000cases in last 24 hours. By this date the cumulative number of deaths exceeded 800,000, with the estimatedmortality of COVID-19 being approximately 3.0%.This rate is much higher than influenza caused fatality (<1%) (Mehta et al., 2020) . A large number of studies have been performed to investigate the structural characteristics, pathophysiologyas well as thein vivo processes of SARS-CoV-2, which would facilitate the discovery of effective treatmentsto control the spreading of COVID-19.

The ultimate approaches to manage COVID-19 lie in the discovery of effective vaccines and antivirals. However, theprocess of developing novel therapeutics is complicated and often timeconsuming. In this regard, the use of existing, approved therapies with demonstrated safety profile is a promising strategy to meet the J o u r n a l P r e -p r o o f urgentrequirement of reducing the rising mortality.Heparin, a natural polysaccharide, is widely used as an anticoagulantagent with well-characterizedbioavailability, safety, stability and pharmacokinetic profiles (Lindahl & Li, 2020; Thachil, 2020) . In addition, heparin possesses other biological activities includingincreasing the release of hepatic lipase and lipoprotein lipase (Persson & Nilssonehle, 1990) , suppressing complement activation (Boackle, Caughman, Vesely, Medgyesi, & Fudenberg, 1983) ,inhibiting angiogenesis (Folkman, Langer, Linhardt, Haudenschild, & Taylor, 1983) , and modulating tumor progress and metastasis (Atallah, Khachfe, Berro, & Assi, 2020) .

Crucially, heparin also shows a broad-spectrum activity against a multitude of distinct viruses (Baba, Pauwels, Balzarini, Arnout, & De Clercq, 1988; Howell et al., 1996; Z. Zhang et al., 2010) . Given current insight onthe pharmacological activity and clinical efficacy of heparin, it could be anunder-exploited effectivetherapy for COVID-19 and warrants investigation.

Here we discuss the pharmacological mechanisms of the anticoagulant, anti-inflammatory and direct antiviral activities of heparin. On the basis of these critical functions of heparin, we illustrate both the preclinical and clinical evidence that heparin therapy has multiple effects for COVID-19. The therapeutic approach of using heparin to manage COVID-19 may provide a strategy to decrease SARS-CoV-2 related morbidity and mortality. Meanwhile, we alsobring up the innate adverse effects of heparin and theunderlying challenges of implementing heparin therapy for SARS-CoV-2 infection.

Heparin is structurally one of the most complex members in thefamily of polysaccharides, which isconstructed from the monosaccharide building blocks ofGlucosamine (GlcN), Iduronic Acid (IdoA) and Glucuronic Acid (GlcA) (Casu & Lindahl, 2001) . GlcNcan be N-acetylated (GlcNAc) or N-sulfated (GlcNS), both of which can be further 6-O-sulfated (GlcNAc(6S) and GlcNS(6S)).GlcNS and GlcNS(6S) can also be sulfated at carbon-3(GlcNS(3S) and GlcNS(3,6S)).GlcA and J o u r n a l P r e -p r o o f IdoA can be 2-O-sulfated (GlcA(2S) andIdoA(2S))( Figure 1A ) (Baytas & Linhardt, 2020) .Disaccharide subunits composed of the above monosaccharides form the basic sequence units of the long, linear and highly-sulfated chain of heparin. Of these, a disaccharide composed of IdoA(2S)-GlcNS(6S) accounts for the highest proportion in heparin while other combinations of monosaccharides make up the remainder (Rabenstein, 2002; T. Zhang et al., 2019) (Figure 1B) . Withthe highly-sulfated nature of the glycosaminoglycan (GAG) chain, heparin shows the highest negative charge density (∼3.3 negative charges per disaccharide) amongthe known biomolecules (Weiss, Esko, & Tor, 2017) . Moreover, the special arrangement sequence of this negative GAG chaincan createbinding sites that allow heparin to selectively and strongly interact with various proteins (Esko & Lindahl, 2001) . The most well-characterized interaction is the highly specific interactionof a pentasaccharidewith serine protease inhibitor antithrombin-III (AT-III), which confers heparin with excellent anticoagulant activity ( Figure 1C) .

Generally, the molecular weight (MW) of heparin is between 3 to 30kDa with an average MW of 15kDa. According to the length of polysaccharide chain, heparin can be classified into unfractionatedheparin (UFH) and low molecular weight heparin (LMWH).Because of the heterogeneous mixture of linear chains, UFH has variable biological activities (Bounameaux, 1998) . LMWH is generated from UFHviadifferent methods includingchemical, physical or enzymatic depolymerization. The resulting products have a mean molecular weightsof 4-5kDa, a mix of polysaccharide chain lengths and different pharmacological properties (Merli & Groce, 2010) .If there is no specific designation, heparin refers to both UFH and LMWH through the text. 

Clinical observations and laboratory evidence confirm that coagulopathy is a common phenomenon in COVID-19.Numerous SARS-CoV-2infected patients who had no other risk factors for thrombosis, suffered from various thrombotic events, such as microvascular thrombosis, venous thromboembolism,pulmonary embolism, and acute arterial thrombosis (Klok et al., 2020) .Whilethe pathogenesis of coagulopathy in COVID-19 has not been fully elucidated,several mechanisms may be involved here. For many severe infections, including pulmonary infection, the main causes for coagulation disorderincludeexcessive inflammatory cytokine production, increased levels of damage-associated molecular patterns, the stimulation of cell-death and vascular endothelial damage (Helms et al., 2020) . Similarly, J o u r n a l P r e -p r o o f SARS-CoV-2 infection could induce a cytokine storm with the activation of leukocytes, endothelium and platelets, which would promote the upregulation of tissue factor, coagulation activation, thrombin generation, and fibrin formation (Engelmann & Massberg, 2013; Sara C, 2011) .Moreover, as SARS-CoV-2 shows high affinity to angiotensin-converting enzyme 2 (ACE2) expressed on endothelial cells, endothelial cell activation may be a specific mechanism for COVID-19-inducedthrombosis(H. Zhang, Penninger, Li, Zhong, & Slutsky, 2020) .In addition, profound hypoxemia has been observed in COVID-19, which may result in Among the coagulation parameters found related to COVID-19,the most common coagulation abnormality is the remarkably elevated D-dimer concentration (up to 45% of patients) Zhou et al., 2020) .This increased D-dimer level is correlated with severity of illness. Comparedto patients with normal D-dimer levels, the patients with a concentration of 4-fold above the normal value showed approximately 5-fold higher odds of critical illness (Petrilli et al., 2020) . Elevated D-dimerlevels at the time of admission and during hospital stayare also associated with the mortality of patients. The concentration of D-dimer on admission of non-survivors (2.12 (0.77-5.27)µg/mL) is significantly higher than that of survivors (0.61 (0.35-1.29)µg/mL) (Tang, Li, Wang, & Sun, 2020) . Another typical change of coagulation parameters in COVID-19 is the dramatic increase of fibrin(ogen) degradation product (FDP), suggesting a secondary hyper-fibrinolysis following coagulation activation (Iba, Levy, Levi, & Thachil, 2020) . The prothrombin time (PT) and activated partial thromboplastin time (aPTT) are normal or slightly prolonged, which has not been shown to be related with severity of COVID-19 (Lee, Fralick, & Sholzberg, 2020) . However, a prolongation of PT > 3 or aPTT> 5 seconds are J o u r n a l P r e -p r o o f 8 independent predictors for thrombosis (Klok et al., 2020) .It was found that fibrinogen levels are elevated in the initial phase but drop late in the course of non-survivors, which may be a signal for impending death . In addition, a declining level of AT-III is found in COVID-19, which appears to be an indicator of progressive disease and is associated with death (Iba et al., 2020) .Thrombocytopenia is also observed in COVID-19 patientswitha low platelet count beingrelatedtothe elevating risk of disease severity and mortality (Lippi, Plebani, & Henry, 2020) .As coagulopathy plays an essential role in the progress and prognosis of patients, it is urgent to effectively manage the hypercoagulable conditionsof COVID-19 patients.

Discovered in 1916, heparin was introduced clinically in 1935, and became firmly established as an excellent agent to treat or prevent thromboembolism since 1960 (Conrad, 1997) . It can be used in various settings to prevent thrombotic complications, including systemic administration, catheter instillation, using an extracorporeal circuit, or by the addition of an artificial surface coating to medical devices (J. F. Lau, Barnes, & Streiff, 2018) . Mechanically, heparin cannot lyse existing thrombi as it has no intrinsic fibrinolytic activity. The major mechanism of its anticoagulant effect dependents on the presence of the active pentasaccharide sequencefor binding toAT-III (Lindahl, Backstrom, Thunberg, & Leder, 1980) .Once heparin binds with AT-III, it induces a conformational change of AT-III. It has been shown that this might involve a two-step binding mechanism (Olson, Srinivasan, Bjork, & Shore, 1981) . Firstly,the trisasaccharide at the non-reducing end of the pentasaccharide sequence forms a low-affinity complex with AT-III, which then induces the conformational change of AT-III,thereby forming a high-affinity complex.

to additional AT-III, with a recycling effect that provides a continuous anticoagulant effect ( Figure 2 ). Among thecoagulation factors, factor Xa and IIa are the most sensitive to the AT-heparin complex.Heparin's inhibition of factor Xa only requires the pentasaccharide sequence, while inhibition of factor IIa requires not only the pentasaccharide sequence but additional sugar residues adjacent to the pentasaccharide sequence (Griffith, 1982) . Therefore, heparin with a degree of polymerization of less than 18 (MW around 5.4kDa) has very low efficacy in inactivating factor IIa but a similar affinity to factor Xa (Carter, Kelton, Hirsh, & Gent, 1981) .Apart from itsanticoagulant effects, heparinhas been found to increase vessel wall permeability and suppress the proliferation ofvascular smooth muscle cells (Hirsh & Raschke, 2004) . 

The clinical application of heparin for anticoagulant therapy in COVID-19 has shown promising outcomes.It has been reported that anticoagulant therapy reduced mortality of severe COVID-19 patients. Tang et al. (Tang, Bai, et al., 2020) conducted a retrospective study that enrolled 449 patients with severe COVID-19 in Tongji hospital, Wuhan, China. In the study, 94 of the patients received LMWH (40-60 mg enoxaparin/d) while 5 of patients received UFH (10,000-15,000 U/d)for 7 days or longer. No significant difference was observed in the overall mortality between patients with or without heparin treatment(30.3% vs 29.7%, P= 0.910). However, there were significant differences in 28-day mortality in the subgroup ofpatientswith aconcentration of D-dimer (>3 μg/mL) higher than 6-fold ofthe normal upper limit (32.8% vs 52.4%, P = 0.017), or who had a sepsis-induced coagulopathy (SIC) score ≥4 (40.0% vs 64.2%, P =0.029). The results indicated that anticoagulant therapy with heparin was related with a better prognosis in severe COVID-19 patients with remarkably elevated D-dimer levels or patients whomet the criteria of SIC. In another report, the use of LMWH for anticoagulation therapy was recommended for COVID-19 patients with D-dimer concentrations 4-fold higher than the upper limit of what is considered normal (Lin, Lu, Cao, & Li, 2020) .Autopsy findings from COVID-19 patients have detected microthrombi in pulmonary microvasculature, indicating refractory hypoxemia (Tian et al., 2020; Yao et al., 2020) .In a study of 27 consecutive COVID-19 patients in SirioLibanes Hospital, São Paulo-Brazil, the researchers found that heparin therapy for COVID-19 improved oxygenation. The PaO2/FiO2 ratio, a marker for respiratory distress,was significantly increased from 254(±90) to 325(±80) (p=0.013) after anticoagulation therapy for 72 hours (Negri et al., 2020) . prophylaxistherapy with LMWH,compared with 35 (63.3%) in the non-prophylaxis group (P =0.010). Thus, using heparin for VTE prophylaxis has been included in some practice guidelinesin its efficacy regarding COVID-19 management (Bikdeli et al., 2020; Popoola et al., 2017; Qiu, Wang, Zhang, & Qian, 2020; Thachil et al., 2020) .

According to expert recommendations from the American College of Cardiology (ACC), hospitalized patients with respiratory failure or comorbidities, bedridden patients and those requiring intensive care, should receive pharmacological VTE prophylaxis in the absence of any contraindications (Bikdeli et al., 2020) . Mechanical prophylaxis should be considered in immobilized patients with a contraindication to pharmacological prophylaxis (Ho & Tan, 2013) .The International Society on Thrombosis and Haemostasis (ISTH) recommended that aprophylactic dose of LMWHshould be considered for all patients (without contraindications) who require hospital admission. The optimal dosages need to be adjusted according to the specific conditions of individual patient . For the choice of agents, the World Health Organization interim guidance statement recommends once daily dosing regimen of LMWH, or twice daily of subcutaneous UFH (Organization, 2020) .

Receiving all scheduled doses of pharmacological VTE prophylaxis has been shown to be important,with missed doseslikely resulting inthe worse outcomes (Popoola et al., 2017) . In this regard, once daily dose of LMWH may be advantageous over the twice daily UFH. However, UFH is recommended for patients with severe renal failure (creatinine clearance CrCl< 30mL/min) (Qiu et al., 2020) .

ARDS is extremely life-threatening to COVID-19 patients with a high mortality rate of 40% to 50% .Although the exact mechanism of ARDS in COVID-19 is not fully understood,initiation of a cytokine storm is considered to be J o u r n a l P r e -p r o o f one of the major contributing factors (N. Chen et al., 2020; Chousterman, Swirski, & Weber, 2017; Huang et al., 2020) . Broadly speaking, a cytokine stormis usually characterized by high release of interleukins, tumor-necrosis factors, interferons, chemokines, and other mediators (Sinha, Matthay, & Calfee, 2020) . Under ideal circumstances, the coordinated secretionof sustained inflammatory cytokines isa well-conserved consequence of appropriate innate and adaptive immune responsesfor efficient clearance of microorganisms (Pedersen & Ho, 2020) .However, in several viral infections including SARS-CoV and middle east respiratory syndrome-coronavirus (MERS-CoV), an overwhelming systematic response is triggered, leading to the excessive activation and proliferation of immune cells, withthe release of immense amounts of cytokines(S. K. P. Lau et al., 2013) . Not surprising given the phylogenetic relationship between these two coronaviruses, a similar phenomenon is also observed in patients in response to SARS-CoV-2 infection (Ragab, Salah Eldin, Taeimah, Khattab, & Salem, 2020) . However, whileSARS-CoVinfection mainly produces pro-inflammatory cytokines, SARS-CoV-2 infection produces both pro-inflammatory cytokines (e.g., interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α)) and anti-inflammatory cytokines(e.g., IL-4 and IL-10)(C. Zhang, Wu, Li, Zhao, & Wang, 2020) . According to the clinical findings, the pro-inflammatory IL-6 is the most frequently reported cytokine elevated and serves as a main contributor to the acute inflammatory responses in COVID-19 (Sinha et al., 2020) COVID-19 patients into three groups based on relevant diagnostic criteria. Among the three groups, critical cases (n = 5) had the highest IL-6 levels, while severe cases (n = 9) had higher IL-6 levels than mild cases (n = 15). In addition to the severity of illness, IL-6 is also correlated with the mortality of COVID-19 patients. In a multicenter retrospective study of 150 patients, a significantly higher concentration of IL-6 was found in fatal cases compared with discharged cases (Ruan, Yang, Wang, Jiang, & J o u r n a l P r e -p r o o f responses, especially IL-6, may ease severe symptoms and decrease the mortality of COVID-19 patients.

Heparan sulfate (HS) is a type of GAG expressed by virtually all mammalian cells (Bishop, Schuksz, & Esko, 2007) . The common form of HS found in the cell surface is as HS proteoglycans (HSPG), formedviathe covalently linking of one or more linear HS moleculeswith a core protein (Weiss et al., 2017) . HS is composed of the same saccharide building blocks as heparin. However, the primary structure of HS shows remarkable difference from heparin. The disaccharide subunits in HS present higher GlcA/IdoA and GlcNAc/GlcNSand lower SO4 content (Rabenstein, 2002) , withunsulfatedGlcA-GlcNAcbeing the most common disaccharide subunit (Malcolm Lyon, 1998) .The biological activities of HS are mainly through interaction with a wide range of proteins, including cytokines, chemokines, enzymes, enzyme inhibitors, selectins, growth factors, andextracellular matrix molecules (Sarrazin, Lamanna, & Esko, 2011) . With a highly similar structure to HS, heparin also interacts with the HS-binding proteins. By competing with HSfor protein binding, heparin will replace the original HS-mediated protein anchoring, thereby disrupting thecorresponding functional action (Figure 3) .

interact with the chemokines (e.g. CXCL-12) responsible for driving neutrophils to inflammatory area (Ma et al., 2012) . Meanwhile, heparin binds to P/L-selectin which is involvedinthe early adhesion between leukocytes and vessel walls during the process of cell rolling.Heparin also interacts with integrin adhesion molecules(e.g. (Bernfield et al., 1999) . Heparin can disrupt this process by competing with HS for IL-6 interaction. With higher levels of sulfate than HS, heparin has a much higher affinity for IL-6 (Hasan, Najjam, Gordon, Gibbs, & Rider, 1999) . Also, heparin may prevent the formation of the cell surface receptor complex of IL-6 bystronglycompeting with the binding of the rIL-6/srIL-6Ra dimer to soluble glycoprotein 130 (Mummery & Rider, 2000) . Therefore, heparin shows a dual effect in IL-6 inhibition by reducing both the release and biological activity.

Apart from the typical inflammatory factors released, some of the clinical features of cytokine storm in COVID-19 are characterized by disseminated intravascular coagulation, capillary leak syndrome and loss of blood pressure, indicating the crosstalk between hemostasis and cytokines (Mangalmurti & Hunter, attenuate thecytokine storm-induced systematicsyndrome and organ damage.

Clinical investigationson the use of heparin as an anti-inflammatory therapy have been reported (Mousavi, Moradi, Khorshidahmad, & Motamedi, 2015) . In a randomized, double-blind, crossover clinical trial for asthma, five doses of UFH (1,000 U/kg/dose) or placebo were inhaled by the patients. Compared with the placebo, heparin significantly reduced the late asthmatic response after allergen administration (P=0.005) (Diamant et al., 1996) .In a quasi-experimental (non-randomized clinicaltrial) study, nadroparin, a type of LMWH, remarkably improved the endoscopic and histological signs of inflammation in steroid refractory ulcerative colitis (Vrij et al., 2001) . Despite the clinical evidence, there are very limited applications of heparin as an anti-inflammatory therapy in COVID-19. In a retrospective cohort study，wefound thatpatients who were not treated with LMWH had a slight increase in IL-6 level after conventional treatment. However, in patients treated with LMWH, IL-6 level was significantly reduced (14.96 ± 151.09, -32.46 ± 65.97, p = 0.031) while other inflammatory factors did not have significant changes .The possible mechanism could be as follows: on one hand, LMWH could reduce the release of IL-6 by inhibiting the expression of NF-κB, which is consistent with the protective effect of LMWH;on the other hand, IL-6 can bind to HS on thecell surface to produce a high local concentration to activate signal receptors, protect them from proteolysis, and promote paracrine effects (Sarrazin et al., 2011) . This study providesdirect evidence for the favorable anti-inflammatory effect of heparin therapy in COVID-19. 

In addition to its anticoagulant and anti-inflammatory activity, heparin may possess a direct antiviral effect to SARS-CoV-2, based on the preclinical studies for other viral infections. Ito et al. (Ito et al., 1987) apoptosis.In addition to these findings, the most relevant evidence is that heparin could inhibitthe cell entry of SARS-CoVas HSPG provides a binding site for SARS-CoV invasion at the initial attachment phase (Lang et al., 2011) . Accumulated evidence indicates that heparin may be a promising candidate for antiviral therapy in COVID-19.

J o u r n a l P r e -p r o o f ACE2 is well-characterized as the major receptor for viral entry of SARS-CoV-2 (Hoffmann et al., 2020) . However, many viruses including coronaviruses, also use cellular GAG, notably HS,as co-receptors to allow the attachment of viruses to the cell surface, increasing the local concentration of viral particles for invasive infection (Tandon et al., 2020) . According to the sequence analysis of Spike glycoprotein (SGP), potential GAG binding domains and GAG binding-like motifshave been shown in SARS-CoV-2. With deeper investigation, these binding domains have been found to be distributed at site 1 (within the receptor binding domain (RBD), Y453-S459), site 2 (proteolytic cleavage site at S1/S2 junction, P681-S686) and site 3 (the S2' proteolytic cleavage site, S810-S816) (Kim et al., 2020) . Strikingly, the S1-S2 proteolytic cleavage motif is novel for SARS-CoV-2, which is not presented in the SGPs of SARS-CoV or MERS-CoV. This specific viral binding to cell surface GAG provides the possibility for the interaction of heparin with SARS-CoV-2 by competing with HS ( Figure 4) . In the direct binding assay, heparin was found to bind more tightly with both the monomeric (equilibrium dissociation constant, KD = 40 pM) and trimeric (KD=73 pM) SARS-CoV-2 spike, compared to that of SARS-CoV (KD=500 nM) and MERS-CoV SGPs (KD=1 nM) (Kim et al., 2020) . It has also been reported that a conformational change of SARS-CoV-2 S1 RBD is induced upon heparin binding.

The basic amino acidsconstitutingheparin binding domains are solvent accessible on the protein surface and can form a continuous patch for heparin binding (Mycroft-West, Su, Li, et al., 2020) . observed similar results in HS mimetic PG545, which also bind directly to SARS-CoV-2 S1 RBD and altered its conformation.It has been demonstrated that heparanase can promote viral infection and spread (Hadigal et al., 2015) . Heparin, LMWH and non-anticoagulant heparin can inhibit the enzyme activity of heparinase, thereby inhibiting viral export and spread (Nasser et al., 2006) .

Underlying the high binding affinity between heparin and SARS-CoV-2 SGP, the properties of heparin itself plays an important role. It was found that the degree and position of sulfate in heparininfluence its bindingto SARS-CoV-2. In a J o u r n a l P r e -p r o o f competitiveSGP binding study, the IC50 values of immobilized heparin, trisulfated HS (NS-2S-6S) and LWMH were 0.056 μM, 0.12 μM and 26.4 μM, respectively.

Both heparin andtrisulfated HSare at N-, 2-O-, and 6-O-sulfation. However,heparin has a higher inhibitory activity to bind with SARS-CoV-2 SGP than trisulfated HS, which could be attributed to the additional 3-O-sulfationin heparin (O'Donnell, Kovacs, Akhtar, Valyi-Nagy, & Shukla, 2010; Shukla et al., 1999) . In comparison, LMWH showed the lowest affinity for SGP binding, indicating that the chain length could be critical in this binding process (Kim et al., 2020) . Tandon et al. (Tandon et al., 2020) observed a similar result. They pseudotyped SARS-CoV-2 SGP on a third-generation lentiviral (pLV) vector and investigated the effect of heparin on transduction efficiency in HEK293T cells. The concentration-response curves displayed that the IC50 value in neutralizing pLV-S particles in UFH (599 ng/L) was significantly lower than enoxaparin, a type of LMWH (108 µg/L). Moreover, heparin's inhibitionofSARS-CoV-2 infection displays a concentration-dependent manner. When at a concentration of 100 μg/mL, heparin was capable of inhibiting 70% of invasion to Vero cells by SARS-CoV-2(Mycroft-West, Su, Pagani, et al., 2020) . 

Several adverse effects of heparin therapy are related with thewide biological activities of heparin, posing considerable challenges (Alban, 2012) . Bleeding is themajor safety concern of heparin use (Schulman, Beyth, Kearon, & Levine, 2008) .

Despite the fact thatthe bleeding incidence is hard to define, various factors could increase the risk. The elderly and patients with renal insufficiency are typical independent baseline indicators of bleeding in all patients receiving antithrombotic therapy (Bounameaux & Perrier, 2008) . In addition, recent trauma or bleeding, surgery, long hospital stay, anemia, cancer,pulmonary embolism and elevated cardiac biomarkers are also associated with heparin induced bleeding (Alban, 2012) . Apart from bleeding, heparin-induced thrombocytopenia (HIT)has been a recurrent concern.

HIT is an immunological side effect and usually occurs in the second week of heparin therapy(Theodore E. Warkentin, 2015) . The pathogenesis of HIT is caused by the binding of heparin to platelet factor 4 (PF4) and the induction of platelet-activating IgG antibodies (Chong, Pitney, & Castaldi, 1982) . As a result of the antibody-mediated platelet activation,massive thrombin generation is induced, thereby enhancingthe risk of thrombosis(Theodore E Warkentin, 2003) . HIT also induces skin lesions, which typically, occurs at heparin injection sites, while some skin necrosis also occurs at remote sites (Hirsh et al., 2001) . Systemic allergic and anaphylactic immune responses to heparin have been described more than 60 years ago (Bernstein, 1956 ). However, these immediate-type reactions are rare under current practice, with the improved pharmaceutical quality of heparinand application of LMWH.Osteoporosis is one of the most common severeside effects of long-term UFH use, with a 2.2-5% incidence of heparin-induced osteoporotic fracture (Dahlman, 1993) . The risk of LWMH for osteoporosis is lower than that of UFH, but should be J o u r n a l P r e -p r o o f noticed in pregnant women, elderly and children (Rajgopal, Bear, Butcher, & Shaughnessy, 2008; Templier & Rodger, 2008) .

In contrast to the beneficial effects of heparin in COVID-19 patients, unfortunately, HIThas been reported. Rikeret al. (Riker et al., 2020) reported three cases of thrombocytopenia with anti-PF4 antibodies among the 16 intubated COVID-19 patients with respiratory distress syndrome, where one case was confirmed to be HIT according to the results of a serotonin release assay. The authors recommended tomonitorplatelet counts during heparin therapy, facilitating the early recognition of HIT. Bleeding was also observed in COVID-19 patients who received heparin as ananticoagulation therapy.Musoke et al. (Musoke et al., 2020) reported that 20 of 80 patients had developed bleeding events and ICU patientsaccounted for 45% of these events. Thirty percent of these bleeding events were from gastrointestinal sourceswhile 30%of the events were in other sites includingpulmonary, intraabdominal and retroperitoneal. According to the ISTH guidelines , if bleeding is developed, similar principles to septic coagulopathy with respect to blood transfusions may be followed (Wada et al., 2013) . Antifibrinolytic agents could be used for patients who present a hyperfibrinolytic state such as trauma (moderate quality) or leukemia (low quality). It should be noted that thebleeding patients should keep a platelet count >50 × 10 9 /L, fibrinogen > 1.5 g/L and PT ratio <1.5 (not the same as INR). Heparin resistance, defined as the requirement of extremely high doses of heparin to achieve the target aPTT ratio, was found in COVID-19 patients under intensive care.In a cohort of 69 patients in an intensive care unit, 15 of them received either UFH or LMWH for anticoagulant therapy. 80% (8/10) of UFH-treated patients were heparin resistant while 100% (5/5) of LMWH-treated patients displayed a sub-optimal peak anti-Xa (White et al., 2020) .Robert et al. (Beun1, Kusadasi, Sikma1, Westerink, & Huisman, 2020 ) observed a similar phenomenon. All of the patients (4/75) receiving therapeutic anticoagulation necessitated a very high dose of UFH to realize the perceived adequate coagulation. Factor VIII was found to be remarkably elevated in these SARS-CoV-2 patients. As high level of factor VIII is J o u r n a l P r e -p r o o f a common reason for heparin resistance, this may be the critical cause of heparin resistance in COVID-19 patients.

The rapid spread of COVID-19poses a great threat to global healthauthoritiesas no effective antiviral drugs or vaccines are currently available.Heparin, a well-tolerated anticoagulant drug, has been used clinically for over 80years with excellent bioactivity, stability and pharmacokinetic profiles. In this review, we comprehensively discussed the potential of heparin therapy for COVID-19. With multi-functions including its anticoagulant, anti-inflammatory and antiviral activities, heparin may contribute to themanagement of COVID-19 severity. There are both preclinical evidence and clinical data to demonstrate the benefits of heparin therapy for SARS-CoV-2 infection.With anticoagulant and anti-inflammatory effects, heparin can offer supportive treatment and alleviate the systematic symptoms of COVID-19.

Notably, under the current status of lacking effective antiviral agents and vaccines, heparin shows direct antiviral efficiency by inhibitingviral entry. However, considerable safety concerns including bleeding, HIT, skin lesions and osteoporosis set a critical concern to heparin therapy.Heparin resistance was also found in COVID-19 patients. Moreover, except theanticoagulant index, heparin has not been approved as a direct anti-inflammatory or antiviral agent.

As the clinical benefits and safety concerns exist side by side, efforts should be concentrated on maximizing the therapeutic effects while minimizing the adverse effects of heparin. More detailed preclinical research should be conducted to clarify the mechanisms of heparin's function, which would provide theoretical support to develop effective solutionsfor the safety and effectivity of heparin in COVID-19 therapy. Meanwhile, more clinical trials could be performed to give greater clinical evidence and an improved understandingon the anti-inflammatory and antiviral effects of heparin.With deliberate attempts and comprehensiveinvestigation, heparin therapycan be optimized for various medical applications beyond its traditional role as J o u r n a l P r e -p r o o f an anticoagulationagent. Under the current status of the COVID-19 pandemic, the multifunctional heparin could be a promising candidate in the treatment of COVID-19.

The authors declare that they have no competing interests.

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Relationship between the antithrombotic and anticoagulant effects of low molecular weight heparin

Structure and biological interactions of heparin and heparan sulfate

Analysis of clinical features of 29 patients with 2019 novel coronavirus pneumonia. Zhonghua Jie simplex virus-1 release from cells

IL-12 Is a heparin-binding cytokine

High risk of thrombosis in patients with severe SARS-CoV-2 infection: a multicenter prospective cohort study

Heparin and low-molecular-weight heparin : the seventh ACCP conference on antithrombotic and thrombolytic therapy

Heparin and low-molecular-weight heparin mechanisms of action, pharmacokinetics, dosing, monitoring, efficacy, and safety

Stratified Meta-analysis of intermittent pneumatic compression of the lower limbs to prevent venous thromboembolism in hospitalized patients

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

Inhibition of HIV-1 infectivity by low molecular weight heparin

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Coagulopathy in COVID-19

Inhibitory effect of dextran sulfate and heparin on the replication of human immunodeficiency virus (HIV) in vitro

Glycosaminoglycan binding motif at S1/S2 proteolytic cleavage site on spike glycoprotein may facilitate novel coronavirus (SARS-CoV-2) host cell entry

Incidence of thrombotic complications in critically ill ICU patients with COVID-19

Inhibition of SARS pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans

Anticoagulation therapy. In Unfractionated Heparin and Low Molecular-Weight Heparin

Delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel Middle East respiratory syndrome coronavirus: implications for pathogenesis and treatment

Coagulopathy associated with COVID-19

Different signaling pathways involved in the anti-inflammatory effects of unfractionated heparin on lipopolysaccharide-stimulated human endothelial cells

Unfractionated heparin inhibits lipopolysaccharide-induced expression of chemokines in human endothelial cells through nuclear factor-KappaB signaling pathway

Hypothesis for potential pathogenesis of SARS-CoV-2 infection-a review of immune changes in patients with viral pneumonia

Evidence for a 3-O-sulfated D-glucosamine residue in the antithrombin-binding sequence of heparin

Heparin-an old drug with multiple potential targets in Covid-19 therapy

Thrombocytopenia is associated with severe coronavirus disease 2019 (COVID-19) infections: A meta-analysis

Modulating the interaction of CXCR4 and CXCL12 by low-molecular-weight heparin inhibits hepatic metastasis of colon cancer

Triggers, targets and treatments for thrombosis

Bio-specific sequencesand domainsin heparan sulphate and the regulation of cell growth and adhesion

Cytokine storms: understanding COVID-19

COVID-19: consider cytokine storm syndromes and immunosuppression

Pharmacological and clinical differences between low-molecular-weight heparins implications for prescribing practice and therapeutic interchange

Human coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells

Dual targeting of dengue virus virions and NS1 protein with the heparan sulfate mimic PG545

Anti-inflammatory effects of heparin and its derivatives: a systematic review

Characterization of the heparin-binding properties of IL-6

Anticoagulation and bleeding risk in patients with COVID-19

SARS-CoV-2 Spike S1 Receptor Binding Domain undergoes Conformational Change upon Interaction with Low Molecular Weight Heparins. bioRxiv

Heparin inhibits cellular invasion by SARS-CoV-2: structural dependence of the interaction of the surface protein (spike) S1 receptor binding domain with heparin

Heparanase neutralizes the anticoagulation properties of heparin and low-molecular-weight heparin

Heparin therapy improving hypoxia in COVID-19 patients -a case series

Expanding the role of 3-O sulfated heparan sulfate in herpes simplex virus type-1 entry

Binding of high affinity heparin to antithrombin III. Stopped flow kinetic studies of the binding interaction

Clinical management of severe acute respiratory infection when novel coronavirus (2019-nCoV) infection is suspected. Interim guidance 28

SARS-CoV-2: a storm is raging

Release of lipoprotein lipase and hepatic lipase activities. Effects of heparin and a low molecular weight heparin fragment

Factors associated with hospital admission and critical illness among 5279 people with coronavirus disease

Exploring the impact of route of administration on medication acceptance in hospitalized patients: Implications for venous thromboembolism prevention

Rational use and pharmaceutical care of anticoagulant drugs in patients with COVID-19

Heparin and heparan sulfate: structure and function

The COVID-19 cytokine storm; what we know so far

The effects of heparin and low molecular weight heparins on bone

Heparin-induced thrombocytopenia with thrombosis in COVID-19 adult respiratory distress syndrome

Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan

Therapeutic modulation of coagulation and fibrinolysis in acute lung injury and the acute respiratory distress syndrome. %J Current pharmaceutical biotechnology

Heparan sulfate proteoglycans

The role of heparan sulfate proteoglycans as an attachment factor for rabies virus entry and infection

Hemorrhagic complications of anticoagulant and thrombolytic treatment

The potential of low molecular weight heparin to mitigate cytokine storm in severe COVID-19 patients: a retrospective corhort study

A Novel Role for 3-O-Sulfated Heparan Sulfate in Herpes Simplex Virus 1 Entry

Is a "cytokine storm" relevant to COVID-19?

Heparan sulfate is an important mediator of Ebola virus infection in polarized epithelial cells

Genome-wide screening uncovers the significance of N-sulfation of heparan sulfate as a host cell factor for Chikungunya virus infection

Immunotherapeutic implications of IL-6 blockade for cytokine storm

Effective inhibition of SARS-CoV-2 entry by heparin and enoxaparin derivatives

Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy

Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia

Heparin-induced osteoporosis and pregnancy

The versatile heparin in COVID-19

ISTH interim guidance on recognition and management of coagulopathy in COVID-19

Pulmonary pathology of early phase 2019 novel coronavirus (COVID-19) pneumonia in two patients with lung cancer

Low molecular weight heparin treatment in steroid refractory ulcerative colitis: clinical outcome and influence on mucosal capillary thrombi

Guidance for diagnosis and treatment of DIC from harmonization of the recommendations from three guidelines

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein

Heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia

Targeting heparin and heparan sulfate protein interactions

Heparin resistance in COVID-19 patients in the intensive care unit

Risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease

Non-anticoagulant effects of low molecular weight heparins in inflammatory disorders: A review

A pathological report of three COVID-19 cases by minimal invasive autopsies

The cytokine release syndrome (CRS) of severe COVID-19 and Interleukin-6 receptor

This work was supported by the National Natural Science Foundation of China