key: cord-0736271-8bc4wb1k
authors: Kalita, Bhargab; Saviola, Anthony J.; Samuel, Stephen P.; Mukherjee, Ashis K.
title: State-of-the-art review - A review on snake venom-derived antithrombotics: Potential therapeutics for COVID-19-associated thrombosis?
date: 2021-10-14
journal: Int J Biol Macromol
DOI: 10.1016/j.ijbiomac.2021.10.015
sha: d6204d579b74fe52141227b38337c9591bf446cf
doc_id: 736271
cord_uid: 8bc4wb1k

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent responsible for the Coronavirus disease-2019 (COVID-19) pandemic, has infected over 185 million individuals across 200 countries since December 2019 resulting in 4.0 million deaths. While COVID-19 is primarily associated with respiratory illnesses, an increasing number of clinical reports indicate that severely ill patients often develop thrombotic complications that are associated with increased mortality. As a consequence, treatment strategies that target COVID-associated thrombosis are of utmost clinical importance. An array of pharmacologically active compounds from natural products exhibit effects on blood coagulation pathways, and have generated interest for their potential therapeutic applications towards thrombotic diseases. In particular, a number of snake venom compounds exhibit high specificity on different blood coagulation factors and represent excellent tools that could be utilized to treat thrombosis. The aim of this review is to provide a brief summary of the current understanding of COVID-19 associated thrombosis, and highlight several snake venom compounds that could be utilized as antithrombotic agents to target this disease.

a sterile barrier promoting pathogen recognition and prevent pathogen invasion. However, the mechanism can become maladaptive to interfere with organ perfusion [46, 69, 70] . In addition, studies on ARDS have shown both fibrin deposition (intra-and extra-vascular) and unregulated fibrinolysis during the process [64, 71] .

A mechanism for SARS-CoV-2-associated thrombus formation was proposed by McFadyen et al. [16] . Cytokine-activated endothelium upregulates von Willebrand factor, P-selectin, E-selectin, integrin αvβ3, and intercellular adhesion molecule leading to the recruitment of platelets and leukocytes and compliment activation. The inflamed endothelium releases inflammatory cytokines, including IL-8, IL-1, IL-6, RANTES, and monocyte chemoattractant protein-1. Neutrophil-derived NETs cause direct activation of the contact pathway, while the activated complement upregulates endothelial and monocyte tissue factor levels leading to activation of platelets. The hypoxic environment further upregulates endothelial tissue factors through the production of hypoxia-inducible factors. These mechanisms orchestrate an unchecked generation of thrombin, leading to thrombus formation ( Figure   1 ) [16] .

Further, phospholipase A 2 (PLA 2 ) has been implicated in SARS-CoV-2 entry and pathogenesis ( Figure 2 ). PLA 2 is involved in the early steps of the arachidonic acid (AA) pathway, and plateletactivating factor (PAF) and eicosanoids generated through AA pathway play a crucial role in the context of coagulopathy and thrombosis in COVID-19 [72] . PAF causes platelet activation and aggregation and further releases NET from neutrophils, which can activate platelets and initiate the coagulation cascade that results in thrombosis [73] [74] [75] [76] [77] [78] . Similarly, AA and the eicosanoid thromboxane A2 have platelet-activating effects [72, 79] . Indeed, studies have demonstrated NET markers, hypercoagulability, and increased platelet activity in COVID-19 patients presenting with thrombotic complications [80] [81] [82] [83] .

Because of the clinicopathologic reports showing that severe coagulopathy is associated with SARS-CoV-2 infection, the International Society on Thrombosis and Hemostasis (ISTH) has released recent interim guidance about hospital management coagulopathy in COVID-19 patients [84] . Similarly, quite a few groups of clinicians in close unanimity have developed and prepared guidelines for the diagnosis, treatment and/or prevention of venous thromboembolism (VTE) as well as risk assessment in clinically diagnosed COVID-19 patients [85, 86] . Noteworthy, several other countries may be expected to have similar thrombosis treatment guidelines soon. Table 2 shows the recommended and non-recommended antithrombotic drugs for the treatment of thrombotic complications in COVID-19 patients.

However, as mentioned below, several vital concerns are raised against the current anticoagulant treatment of COVID-19 patients with thrombotic complications.

1. There is no uniformity in treatment regime or explicit unanimity concerning the timing, dosage, and duration of anticoagulation treatment for the in-patient management of coagulopathy in patients in addition to the need for post-discharge prophylaxis [85] . [87] .

3. Should COVID-19 patients with disseminated intravascular coagulation (DIC) syndrome (a pathological condition of blood clot formation throughout the body) who do not have bleeding and are devoid of immobility be administered a prophylactic or therapeutic dose of anticoagulation [87] . 4 . Occurrence of thrombosis is frequently reported in seriously ill COVID-19 patients regardless of the prophylactic usage of low molecular weight heparin (LMWH). Consequently, there is a crucial need for randomized clinical trials, which may be used for selecting the most suitable antithrombotic drugs [88] . 5 . Some randomized clinical studies have explicitly demonstrated detrimental consequences in COVID-19 patients after treatment with oral anticoagulants [89] . Therefore, except for the use of LMWH, there is a lack of practical approaches regarding anticoagulants' therapeutic use and choice in the clinical management of COVID-19. The use of certain antithrombotic drugs to treat coagulopathy in COVID-19 patients has produced contradictory results (Table 2) . Consequently, an extensive drug discovery programme has been suggested to develop efficacious but safe antithrombotic drugs for the treatment and/or prophylaxis of hypercoagulability disorders in COVID-19 patients. In this regard, snake venoms, which contain an arsenal of relatively non-toxic but highly efficacious proteins and polypeptides, may be proposed as a choice for the treatment of thrombosis-associated complications in SARS-CoV-2 infected patients.

Snake venoms toxins often act synergistically as complexes that aid the snakes in immobilizing, killing, and digesting their prey [91] [92] [93] ; however, several of these compounds are non-toxic in their isolated forms [94, 95] . More importantly, they exhibit a wide array of pharmacological effects that can be exploited to develop life-saving drug prototypes for diverse diseases, including thrombotic disorders [96] [97] [98] . It has been observed that toxins present in the venoms of Viperid and Crotalide, and a few snakes of the Elapid family target different components of the hemostatic system, including blood coagulation factors and platelet function, that results in coagulopathy in victims upon envenomation [99, 100] . Nevertheless, it is essential to appreciate that the clinical manifestation of coagulopathy is a concerted consequence of the action of several venom toxins rather than a single protein [91] [92] [93] . Therefore, relatively non-toxic venom proteins and peptides from these families of venomous snakes have been extensively characterized for their potential use as drug prototypes for the treatment and/or prevention of thrombotic disorders. Notably, the primary advantage of these toxins over other cardiovascular drugs is the high binding affinity towards their targets. Since these toxins have naturally evolved to target the components of the hemostatic system, their specificity and selectivity are incomparable [99, 101] . Furthermore, most of these toxins are very potent and can inhibition of thrombin and factor Xa, and are additional examples of potential anticoagulant drug prototypes ( Figure 4) [94, 117] . Thrombin and factor Xa, which catalyze the penultimate steps of the blood coagulation cascade, are crucial druggable targets for the prevention and/or cure of thrombosis.

However, commercial thrombin inhibitors such as Argatroban, Dabigatran, etc., and factor Xa inhibitors including heparin, Fondaparinux are associated with life-threatening bleeding complications and other side-effects (Table 2) . Under such circumstances, thrombin and factor Xa inhibitors derived from snake venoms capable of acting at a nanomolar concentration and possessing extreme target specificity have the potential to be developed as better anticoagulant agents for the prevention of COVID-19-associated thrombosis.

Apart from inhibiting blood coagulation factors, phospholipid hydrolysis by PLA 2 enzymes also contributes to their anticoagulant action [112, 121, 122] . Phospholipids play an essential role in the blood coagulation cascade and are required during the catalytic conversion of prothrombin to thrombin [109] . Therefore, most of the PLA 2 enzymes exhibit anticoagulant action by their plasma phospholipid hydrolytic activity. Notably, concomitant non-enzymatic inhibition of other blood coagulation factors by these PLA 2 enzymes (as described above) potentiates their anticoagulant property. In addition, snake venom proteases including SVMPs (Atrase B, BpMP-I, Moojenin, etc.) and SVSPs (Bhalternin, DAnase, Russelobin) demonstrate preferential degradation of the Aα and/or Bβ chains of fibrinogen [105, [123] [124] [125] [126] [127] . Consequently, due to the unavailability of functional fibrinogen molecules in the bloodstream, fibrin clot formation is impaired, resulting in blood anticoagulation.

While platelet activation and aggregation is an integral step in thrombus formation, to date, their role in COVID-19 remains unclear. Thrombocytopenia has been reported in several COVID-19 patients [128, 129] , whereas platelet count and size were average in others [80, 130] . Increased platelet activation has also been observed in severe COVID-19 patients but not those with mild symptoms [80, 81] . Furthermore, platelets from COVID-19 patients demonstrated increased adhesion and spreading on fibrinogen [80] , a primary ligand for αIIbβ3. Notably, several snake venom toxins inhibit platelet aggregation induced by an array of agonists, including ADP, thrombin, collagen, epinephrine, and ristocetin ( Figure 3 ). While a majority of the toxins belonging to SVMP, SVSP, PLA 2 , nucleotidase, and KSPI snake venom protein families suppress platelet aggregation induced by the above agonist, disintegrins and snaclecs can specifically interact with platelet receptors (integrins) J o u r n a l P r e -p r o o f and block platelet activation and aggregation. For example, Agkisacucetin (trade name Anfibatide), a homodimeric snaclec of 30 kDa isolated from the venom of Agkistrodon acutus binds to the GPIb-IX-V platelet receptor and inhibits the association of vWF with this platelet receptor ( Figure 4 ) [131] .

Similarly, the hetero-oligomeric snaclec VP-i is an integrin α2β1 (collagen receptor) antagonist that inhibits collagen-induced platelet aggregation [132] . In addition, the disintegrins TFV-1 (purified from Trimeresurus flavoviridis venom) and Cerastategrin (purified from Cerastes cerastes venom), and disintegrin derivatives Tirofiban (a peptide mimetic from Echis carinatus venom), Eptifibatide (a peptide from the venom of Sistrurus miliarius barbouri), and Rusvikunins from Daboia russelii venom ( Figure 4 ) act as integrin αIIbβ3 antagonist and block the platelet-platelet adhesion mediated by binding of fibrinogen to the αIIbβ3 receptor [133] [134] [135] [136] [137] . Notably, while disintegrins interact with platelet receptors primarily via a conserved RGD motif [138] , the binding of Rusvikunins and their peptides (RusPep and cycRusPep) to the αIIbβ3 receptor was found to be RGD-independent [136] .

Furthermore, the Troα6 and Troα10 peptides derived from the snaclec Trowaglerix (isolated from the venom of Tropidolaemus wagleri) function as antagonists to integrin GPVI, the primary collagen receptor and thereby inhibit collagen-induced platelet aggregation [139] . These antagonistic activities of the snake venom components on different platelet receptors affect platelet activation that ultimately inhibits thrombus formation; therefore, these venom-derived toxins are potential antithrombotic agents.

The thrombolytic potential of snake venom components, especially the weak or non-hemorrhagic SVMPs, and SVSPs are well appreciated. Among them, the PI-SVMP Batroxase purified from Bothrops atrox venom deserves special attention. Under in vitro conditions, the weakly hemorrhagic PI-SVMP Batroxase directly dissolved fibrin clots without activating the endogenous fibrinolytic system (plasminogen/plasmin), whereas, in an animal model of venous thrombosis, the PI-SVMP exhibited potent antithrombotic activity [140, 141] . Other weak or non-hemorrhagic PI-SVMPs, including Colombienase-1 and 2, Barnettlysin-I, and BpirMP, have also demonstrated potent fibrinolysis under in vitro conditions [142] [143] [144] ; however, these studies lack in vivo evidence of their thrombolytic potential.

A non-hemorrhagic high molecular weight (67.8 kDa) PIII-SVMP NN-PF3 purified from the venom of Naja naja exhibited potent fibrinolytic activity under in vitro conditions and caused J o u r n a l P r e -p r o o f defibrinogentaion, prolongation of bleeding time, and reduction in the blood fibrinogen level in the Swiss albino mice model [145] . Apart from the SVMPs mentioned above, the in vivo thrombolytic potential of a few SVSPs was also demonstrated in rat and canine thrombosis models. For example, Agkihpin, a 25.5 kDa SVSP isolated from the venom of Gloydius halys exhibited fibrinolysis under in vitro conditions and reduced thrombin-induced venous thrombosis in a Sprague Dawley rat model [146] . Another SVSP GBV-PA, a 32.6 kDa plasminogen activator from the venom of Gloydius brevicaudus exhibited hydrolysis of rabbit blood clots under in vitro conditions. More importantly, intravenous injection of GBV-PA significantly reduced the thrombus weight and length in rat inferior vena cava (thrombin-induced venous thrombosis model) and ferric chloride-induced carotid artery thrombosis models. Furthermore, GBV-PA demonstrated antithrombotic potential in a canine (dog) acute ischemia-reperfusion stroke model by augmenting arterial thrombosis recanalization rates.

Notably, GBV-PA was reported with a lower hemorrhagic effect and a longer antithrombotic half-life than tissue plasminogen activator and urokinase [147] .

Considering the tremendous potential yet low abundance of this snake venom plasminogen activator, the venom toxin was cloned into a prokaryotic expression vector (pET-42a) and expressed in Escherichia coli. The recombinant molecule (rGBV-PA) was functional and it replicated the pharmacological activities exhibited by the parent toxin [148] , suggesting the prospect of utilizing recombinant DNA technology for the large-scale production of therapeutically important snake venom toxins. Moreover, considering the life-threatening bleeding complications associated with current clot bursting drugs such as Alteplase, Urokinase, and Streptokinase (Table 2) , the development of snake venom-derived thrombolytic agents against COVID-19-related cardiovascular complications seems promising.

Despite the plethora of antithrombotic drug candidates characterized from snake venom, a considerable gap exists between preliminary in vitro and pre-clinical laboratory findings and their translation for clinical applications, mainly due to cost, time, efficacy, and long term side-effects [98] .

Nevertheless, a few snake venom toxins or their derived counterparts with antithrombotic potential the venom of Crotalus durissus collilineatus in Pichia pastoris (methyltropic yeast) that resulted in the production of 56 mg/L of recombinant collinein-1 (rCollinein-1), and importantly, retained its functional integrity to hydrolyze bovine fibrinogen. A subsequent study has shown that rCollinein-1 may have therapeutic potential in preventing thrombus formation [149] . An acute and repeated dose (28 days) toxicity study of a recombinant thrombin-like defibrinogenating enzyme, batroxobin expressed in Pichia pastoris, showed no adverse effects at a dose of 2.5 NIH u/kg in rats and 1 NIH u/kg in dogs, indicating clinical potential in the treatment of hemostatic disturbances [150] . In another approach, a novel thrombin-like enzyme with fibrinogenolytic activity from Deinagkistrodon acutus venom was expressed in soluble form in E. coli. The recombinant enzyme retained its biological activity, reinforcing its large-scale industrial production for commercial exploitation as a therapeutic molecule [151] .

Several of the low molecular mass polypeptides from snake venoms (see Table 3 ) have demonstrated appreciable antithrombotic activity. With the advancement of cost-effective automated protein synthesis technology, a synthetic biology approach for the production of antithrombotic J o u r n a l P r e -p r o o f proteins and enzymes will overcome the cumbersome task of isolating and purifying protein/polypeptide from natural resources [152] [153] [154] . A similar approach may also be applied for the production of antithrombotic snake venom proteins and polypeptides.

A small peptide that has been designed to mimic a particular region of a protein or enzyme yet displays its biological function is known as a peptidomimetic. Several of such peptidomimetic inhibitors that target blood coagulation factors and platelet receptors or regulate various stages of the coagulation cascade to produce an antithrombotic effect have been projected as drug prototypes to treat thrombotic disorders [155] [156] [157] [158] . The peptidomimetics of a small stretch of snake venom toxins that target and inhibit the function of blood coagulation factors and/or platelet receptors [136] will be a unique approach for inventing improved antithrombotic drug prototypes to treat coagulation disorders in COVID-19. [159, 160] . Similarly, it has been demonstrated that site-specific mutagenesis of Fasxiator, a KSPI from Bungarus fasciatus venom, increased its potency to inhibit factor XIa both in vitro and in vivo conditions by approximately 1000 times [161] . The authors suggest that the development of Fasxiator as a novel anticoagulant candidate seems promising. A review article also highlighted the importance of recombinant and chimeric snake venom disintegrins in preclinical research [162] . Therefore, it is anticipated that there is enough scope for the improvement of therapeutic efficiency and potency of other snake venom antithrombotic molecules by in silico alanine scanning mutagenesis, followed by structural stability analysis of native protein, heterologous expression of mutated recombinant protein in a suitable host, and finally comparing the potency of the mutated recombinant protein with that of the native protein.

Such improved antithrombotic proteins will have more commercialization potential.

J o u r n a l P r e -p r o o f

In treated animals, a therapeutic combination of two antithrombotic drugs demonstrated improvement of broad preclinical efficacy against thrombotic disorders without enhancing bleeding complications [163] . A similar approach may also be adopted by combining two or more snake venom antithrombotic drugs to enhance their antithrombotic potency.

Applying a targeted nanoparticulate drug delivery system to improve the therapeutic efficacy of antithrombotic drugs has gained significant attention in recent years. Strategies such as nanoencapsulation, delivery via liposomes or inorganic nanoparticles, and PEGylation have been extensively investigated to deliver antithrombotic agents ( Figure 5 ). In a thrombus-targeting therapy approach, it has been demonstrated that activated platelet-homing liposomes containing tissue plasminogen activator (tPA) and cRGD peptides exhibit improved thrombolytic effects of tPA with low toxicity risk [164] . Mechanistically, upon incubation with activated platelets, cRGD peptides interacted with the platelet receptor integrin αIIbβ3, which resulted in the fusion of platelet membrane and liposomes and a subsequent release of tPA within one hour of incubation ( Figure 5 ) [164] . Inorganic nanoparticles such as mesoporous silica, gold, and carbon nanotubes have also been tested for cellular delivery to enhance the bioavailability and circulation time of antithrombotic drugs [165] . inhibited platelet aggregation at an IC50 value which was ~4 times lower than the parent molecule.

Furthermore, the in vivo half-life of the PEGylated form was marginally longer (~1.3 fold) than

[KGDRR]trimucrin ( Figure 5 ) [166] . Apart from increasing the circulation time of antithrombotic drugs, nanoparticle-based polymers have been employed to enhance the bioavailability of drugs via the oral route. For example, the oral bioavailability of heparin conjugated to hydroxypropyl methylcellulose phthalate (HPMCP)-modified nanoparticles was ~2.5 fold higher than in pure nanoparticles [167] .

Therefore, considering the remarkable progress in developing antithrombotic nanomedicines in recent J o u r n a l P r e -p r o o f years [165, 168, 169] , a targeted nanoparticulate drug delivery system is applied to improve the antithrombotic efficiency of snake venom toxins seems promising.

SARS-CoV-2 can result in adverse health conditions among infected individuals around the world.

Apart from the common respiratory illness, increasing reports have pointed towards cardiovascular disorders in COVID-19 patients. Although several drugs have been approved for the treatment and/or prevention of cardiovascular disorders, the clinical management of COVID-19-associated thrombosis has become challenging in terms of use, dose, and choice of anticoagulants. Under these circumstances, developing antithrombotic agents from snake venoms with high specificity and lower toxicity for the prevention and/or treatment of cardiovascular disorders in COVID-19 patients seems promising. Further, selecting an appropriate delivery system will augment the antithrombotic potential and bioavailability of such candidate snake venom molecules. No such evidence of its therapeutic use. 

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Successful use of tPA for thrombolysis in COVID related ARDS: a case series

Considerations for management of acute coronary syndromes during the SARS-CoV-2 (COVID-19) pandemic

Successful vascular interventional management of superior mesenteric vein thrombosis in a patient with COVID-19: A case report and review of literature

Pulmonary thrombosis in COVID-19 treated by thrombolysis: a small case series using streptokinase, Seminars in thrombosis and hemostasis

Stroke thrombolysis with tenecteplase to reduce emergency department spread of coronavirus disease 2019 and shortages of alteplase

Isolation and functional characterization of a new acidic PLA 2 Ba SpII RP4 of the Bothrops alternatus snake venom from Argentina

The Kv1.3 channel-inhibitory toxin BF9 also displays anticoagulant activity via inhibition of factor XIa

Purification and functional characterization of a new metalloproteinase (BleucMP) from Bothrops leucurus snake venom, Comparative biochemistry and physiology

A New Fibrinogenolytic Metalloproteinase from Bothrops moojeni Snake Venom, ISRN toxicology 2012

BmooSP, a new serine protease from Bothrops moojeni snake venom

Biochemical and enzymatic characterization of BpMP-I, a fibrinogenolytic metalloproteinase isolated from Bothropoides pauloensis snake venom, Comparative biochemistry and physiology

CCSV-MPase, a novel procoagulant metalloproteinase from Cerastes cerastes venom: purification, biochemical characterization and protein identification

Fibrinogenolytic toxin from Indian monocled cobra (Naja kaouthia) venom

LmrTX, a basic PLA 2 (D49) purified from Lachesis muta rhombeata snake venom with enzymatic-related antithrombotic and anticoagulant activity

Malabarase, a serine protease with anticoagulant activity from Trimeresurus malabaricus venom, Comparative biochemistry and physiology

Isolation of a Novel Metalloproteinase from Agkistrodon Venom and Its Antithrombotic Activity Analysis

Thrombolytic protein from cobra venom with antiadhesive properties

Characterization and structural analysis of a potent anticoagulant phospholipase A 2 from Pseudechis australis snake venom

Purification and partial upregulation of proinflammatory mediators

The pro-coagulant fibrinogenolytic serine protease isoenzymes purified from

Daboia russelii russelii venom coagulate the blood through factor V activation: role of glycosylation on enzymatic activity

Antithrombotic and anticoagulant effects of a novel protein isolated from the venom of the Deinagkistrodon acutus snake

Purification and characterization of AHPM, a novel non-hemorrhagic P-IIIc metalloproteinase with alpha-fibrinogenolytic and platelet aggregation-inhibition activities, from Agkistrodon halys pallas venom

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Purification and characterization of BmooAi: a new toxin from Bothrops moojeni snake venom that inhibits platelet aggregation

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Isolation and expression of a hypotensive and anti-J o u r n a l P r e -p r o o f Journal Pre-proof platelet acidic phospholipase A 2 from Bothrops moojeni snake venom

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Molecular cloning and pharmacological properties of an acidic PLA 2 from Bothrops pauloensis snake venom

Purification and characterization of a platelet aggregation inhibitor and anticoagulant Cc 5_NTase, CD 73-like, from Cerastes cerastes venom

Isolation and Characterization of CD39-like Phosphodiesterase (Cc-PDE) from Cerastes cerastes Venom

Molecular Inhibitory Mechanism of Antiaggregation and Anticoagulation

Purification and Characterization of a Novel Antiplatelet Peptide from Deinagkistrodon acutus Venom

A novel platelet glycoprotein Ib-binding protein with human platelet aggregation-inhibiting activity from Trimeresurus jerdonii venom

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cDNA cloning of a snake venom metalloproteinase from the eastern diamondback rattlesnake (Crotalus adamanteus), and the expression of its disintegrin domain with anti-platelet effects

First report of the characterization of a snake venom apyrase (Ruviapyrase) from Indian Russell's viper (Daboia russelii) venom

Purification and characterization of two high molecular mass snake venom metalloproteinases (P-III SVMPs), named SV-PAD-2 and HR-Ele-1, from the venom of Protobothrops elegans (Sakishima-habu)

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BjSP, a novel serine protease from Bothrops jararaca snake venom that degrades fibrinogen without forming fibrin clots

Biochemical characterization and comparative analysis of two distinct serine proteases from Bothrops pirajai snake venom

Primary structure and characterization of a non

The authors declare that there are no conflicts of interest.

All data associated with this study are present in the paper.

In vitro and in vivo [147] Jerdonibitin Trimeresurus jerdonii 25 J o u r n a l P r e -p r o o f