The potential of Schistosoma-derived substances for use as basis for novel anti-haemostatic therapeutics: : A systematic review

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The potential of Schistosoma-derived

substances for use as basis for novel

anti-haemostatic therapeutics: A systematic review

Jonas Li

Professional degree thesis 15hp, 2020-01-04

Supervisor: Sofia Ramström

Examinator: Daniel Bergemalm

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Abstract

Background: Parasites belonging to the Schistosoma genus exert an anti-haemostatic net effect upon infection

of a definitive host, despite numerous mechanisms that should be pro-haemostatic. The anti-haemostatic mechanisms are thus highly interesting, as they may be leveraged for use as basis for novel anti-haemostatic therapeutics.

Aim: The purpose of this systematic review is to provide an overview of research, conducted between 2010 and

2020, on Schistosoma-derived anti-haemostatic substances, as well as studies on Schistosoma-caused coagulation abnormalities, to derive a multifaceted assessment on the potential of Schistosoma-derived substances for use as basis for novel anti-haemostatic therapeutics.

Method: Aggregation of publications was done through PubMed using a search string, accompanied by several

exclusion criteria. The search string used was as follows: ((Schistosoma coagulation[Title/Abstract]) or (Schistosoma haemostatic[Title/Abstract])) AND ("2010/01/01"[Date - Publication] : "2020/06/01"[Date - Publication])

Results: 10 original publications were aggregated: 3 patient studies, 1 murine study and 6 publications on

Schistosoma-derived anti-haemostatic substances. Among these, 7 Schistosoma-derived anti-haemostatic substances were found, which were all discovered to be proteases.

Conclusion: Out of the 7 proteases, 3 proteases from S. mansoni, namely SmCalp1, SmCalp2 and SmAP, were

deemed to have high potential for use as basis for novel anti-haemostatic treatment development. However, research is sparse, so more research is required for a more conclusive assessment. Furthermore, high

manufacturing costs of protease-based pharmaceuticals must be considered, by analyzing cost and competition. The other proteases, deemed less interesting, were found to have issues such as low specificity, conflicting pro-haemostatic effects or life-threatening effects if utilized systemically.

Keywords: Anti-haemostatic, anti-coagulative, pharmaceutical development, therapy development,

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1. Background

Schistosomiasis is an infectious, parasitic disease contracted by humans and other mammals upon contact by larvae of the Schistosoma genus of parasites. Taxonomically, these worms belong to the Trematoda class (flukes) under the Platyhelminthes phylum (flatworms). In adult form, they have a cylindrical body measuring approximately 1-1,5 cm in length, and are thus macroscopically visible.[1] Furthermore, they are sexually dimorph, with two separate genders that are morphologically different. [2]

1.1 Pathogenesis and transmission

Transmission of schistosomes to a new mammalian definitive host occurs through open waters and an intermediate host (see Figure 1). Matureschistosome pairs located in blood vessels surrounding the intestines within the definitive host, lay eggs that exit the body through excreta. When these eggs encounter water, the eggs hatch, and miracidia escape the shell. These miracidia have cilia, allowing free swimming in the water in search of an intermediate host to infect, in the form of specific snail species. Cercariae hatch from the snail 4-6 weeks after infection and infect water-submerged mammalian hosts by

penetrating their skin. Once in the human host, the cercariae develop into schistosomula and migrate within the bloodstream to the portal vein, where they mature into adult schistosomes.[1] After maturation, the mating process begins, involving parasitic male-female interaction within hepatic capillaries. In this competitive mating process, they bond together; a female adult schistosome will attach to a channel found on the male adult

schistosome, subsequently leading to the maturation of the couple. Only in this state will schistosomes be able to fulfill their life cycle.[2] Migration to the destination foci within the host then occurs, and the cycle

repeats.[1]

1.2 Epidemiology

Distribution and epidemiology of schistosomes largely

depends on various sources of heterogeneity, such as the snail host distribution and the socioeconomic traits of the host population.[4] Six Schistosoma species have humans as their definitive host; all with very similar morphology, with the main disease-causing species being S. haematobium, S. mansoni and S. japonicum.[5] However, the pathology differs

Figure 1. Reproductive cycle of the most common Schistosoma species. [3]

Table 1. The 6 Schistosoma species capable of infecting

humans, together with epidemiology, foci and pathology. [1]

Species Epidemiolo_gy Foci Pathology

S. japonicum China, Southeast Asia

Peri-intestinal

venula Intestinal and hepatosplenic schistosomiasis S. mansoni Africa,

Arabia, South Africa

Peri-intestinal

venula Intestinal and hepatosplenic schistosomiasis S. haematobium Africa, Arabia Perivesical

plexus Urinary schistosomiasis S. intercalatum West and

Central Africa S. mekongi Mekongi

Delta Few foci Minor importance S. malayensis Malaysia

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between these species as a result of them settling down in different locations within the host (see Table 1).

1.3 Haemostasis

The haemostatic system consists of both pro-thrombotic and anti-thrombotic mechanisms that must be finely balanced in order to retain homeostasis in haemostasis. Disrupting this balance can lead to loss of homeostasis, resulting in either shortened or prolonged blood coagulation times.

The intrinsic pathway is triggered by the release of RNA, DNA and inorganic phosphates, leading to release of prekallikrein, or contact with a negatively charged surface. (See figure 2, part A) This converts factor XII into its activated form, factor XIIa. This factor is HK (High molecular weight kininogen, HMWK) dependent, and either converts prekallikrein to kallikrein upstream, or factor XI to XIa downstream. XIa then converts factor IX Figure 2. This simplified figure shows the intrinsic pathway (A) and the extrinsic pathway (B) along with the common pathway (C)

and fibrinolysis (D). Lab tests have also been illustrated on both sides, with APTT measuring the intrinsic pathway and the common pathway (E), and PT (INR) measuring the extrinsic pathway and common pathway (F).[6-8]

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to IXa, which in turn forms a complex with factor VIIIa and factor X with co-factor assistance from Ca2+ and platelet surface (Tenase complex). This complex allows for the activation of factor X to factor Xa.

The extrinsic pathway (See figure 2, part B) is triggered by TF (Tissue Factor, Factor III) on the collagen surface. Platelet activation can be initiated by ADP, vWF or factor IIa (Thrombin) contact. TF together with Factor IXa and Factor Xa, converts VII into its active form, VIIa. VIIa forms a complex with TF (TF-VIIa complex, not shown) to activate factor X into factor Xa.

The common pathway (See figure 2, part C) is where the extrinsic and intrinsic pathways meet. Factor Xa converts factor II (Prothrombin) to its activated form, factor IIa (Thrombin), through a complex between Xa, VIIa and Va with assistance of Ca2+ and platelet surface co-factors. Factor IIa (Thrombin) then exerts several different actions, such as conversion of factor I (Fibrinogen) to Ia (Fibrin), and XIII to XIIIa, activating cross-linking of factor Ia (Fibrin). The result is cross-linked fibrin.[8]

Fibrinolysis (See figure 2, part D), the process of clot breakdown, is done by plasmin, which takes cross-linked fibrin and breaks it down into D-dimers and other breakdown products. For this to happen, plasminogen has to be converted to plasmin, which is mediated by tPA (Tissue plasminogen activator) and kallikrein. This

conversion process can be inhibited by PAI-1 (Plasminogen activator inhibitor 1).[6, 8] Measurement of fibrinolysis is done through various methods, such as measurement of D-dimer, a breakdown product of cross-linked fibrin.[7]

APTT (See figure 2, part E), activated partial thromboplastin time, is a blood coagulation test to determine coagulability of blood in vitro through an activation trigger. Typically, kaolin or a negatively charged test tube is used for this. APTT measures the intrinsic pathway together with the common pathway.

PT (INR), which stands for prothrombin time and international normalized ratio (See figure 2, part F), respectively, is also used to determine coagulative properties of blood in vitro. Activation trigger used is TF (Tissue factor, Factor III). It is utilized to measure extrinsic pathway together with the common pathway. INR is a standardization of results, taking an ISI (International Sensitivity Index) value for the specific TF batch into account for the result. This is useful as TF batches have individual variations. [8]

1.3.1 Schistosomiasis and the human host haemostatic system

During a schistosomiasis infection, the parasite thrives within the definitive host, partially by interfering with the host haemostatic system causing hypocoagulability and hyperfibrinolysis in the host. This happens despite parasite-induced alterations to endothelial function, hypercoagulability and blood flow, which are normally expected to be pro-thrombotic.

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Virchow’s triad of risk factors for venous thrombosis states that three factors contribute to an increased risk: (i) endothelial injury, (ii) hypercoagulability, and (iii) stasis.

i) Endothelial injury, and alteration of endothelial function, may occur during intravascular expulsion and extravasation of eggs originating from female schistosomes. Due to the large size of schistosome eggs, they alter the polarity of endothelial cells upon contact with their apical surface. This causes them to mobilize, retract and migrate over the eggs.[9]

ii) Hypercoagulability of blood may be caused by the superficial electronegative charge found in the tegmentum surface layer of mature schistosomes.[10] These are capable of activating platelets and the coagulation cascade, causing hypercoagulability of blood. [11]

iii) Stasis may be induced, both fully and partially through occlusion of blood vessels. Total occlusion can occur when large schistosome egg deposits trigger granuloma formation, followed by extensive fibrosis, ultimately leading to blood flow obstruction.[12] Partial occlusions may occur due to the macroscopic size of schistosome pairs; In mesenteric veins, one schistosome pair can occlude the majority of the vessel lumen.[13] Another possible source of partial occlusion are the large schistosome eggs, that can cause occlusions in smaller veins.[9] Partial occlusions may cause turbulence, which leads to decreased flow, as well as increased shear rates and shear stress, which subsequently may cause platelet activation and endothelial injury.[14]

In defiance of Virchow’s triad of risk factors, what can be seen in schistosomiasis patients are consistently prolonged blood coagulation times and decreased risk of thrombus formation. Furthermore, thrombocytopenia and hypofibrinogenemia are consistently present among patients.[15] This greatly signifies how effective Schistosoma parasites are at preventing blood clots through various different mechanisms, such as through different proteases that inhibit different components of the coagulation cascade. These proteases, together with other Schistosoma-derived anti-haemostatic substances, are highly interesting from a pharmacological

perspective, as the involved Schistosoma-derived substances may be able to be leveraged to produce anti-thrombotic pharmaceuticals for human use.

2. Aim

Recent research has identified several antigens originating from the Schistosoma family with anti-thrombotic activities. The objective of this systematic review is to identify various Schistosoma-derived antigens that have been reported to have anti-thrombotic properties between 2010 and 2020, as well as gathering information about

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Schistosoma-caused coagulation profile changes. Then, using gathered data, produce a discussion on the potential potency and plausibility of Schistosoma-derived substances for use in human anti-thrombotic pharmaceuticals.

3. Method

This study is a systematic review. The data extraction process was conducted by utilizing PubMed (www.pubmed.ncbi.nlm.nih.gov) to find internet sources for data compiling, mainly publications. To precisely gather relevant information, the constructed search string isolates the results to only return publications with the words “Schistosoma” and either “Coagulation” or “Haemostatic” in the title or abstract. Another argument was added to only gather publications published between 2010-01-01 and 2020-06-01. This was done to keep this systematic review focused on

summarizing recent Schistosoma research conducted in the past 10 years, and to give a brief idea of how much research has been focused on this topic. The following search string was used:

((Schistosoma coagulation[Title/Abstract]) or (Schistosoma haemostatic[Title/Abstract])) AND ("2010/01/01"[Date - Publication] :

"2020/06/01"[Date - Publication])

The exclusion criteria applied are as follows (see Figure 3):

1. Publications must be relevant to subject – Publications where anti-haemostatic Schistosoma substances or Schistosoma-caused coagulation profiles are not the main subject, will be excluded.

2. Publications must deliver relevant information – Schistosoma subspecies that do not infect humans will be excluded.

3. Review articles are to be excluded.

4. Studies must be credible. In order to assess this, the Protocol for Appraisal of Included Studies was utilized, published by SBU (Swedish Agency for Health Technology Assessment and Assessment of Social Service).[16] Studies included must fulfill most of the criteria in the checklist, which covers study design, setting, participant recruitment criteria, outcome assessment methods, results and discussion. This less strictly enforced assessment is due to this area of research being relatively sparse; Studies that are of less complex nature with smaller sample sizes are commonplace as a result.

Figure 3. The exclusion process utilized for aggregation of

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5. Studies must be ethically approved, according to each country’s requirement.

Information will be summarized graphically, with either a figure or a table, to categorize substances together with their gathered data, such as mechanism and lab parameters if applicable – Examples being APTT as a measurement of impact on the intrinsic coagulation pathway, and PK-INR for the extrinsic. The objective is to gather information and summarize it graphically for comprehensibility and ease of access, with a more detailed summary below.

4. Results

In summary, 10 articles were aggregated. 3 were patient studies and 1 was a murine study (see Table 2). Furthermore, 6 were functional studies of anti-haemostatic proteins.

4.1 Patient studies

Eyayu et al. (2020) conducted a cross-sectional patient study in Ethiopia to assess coagulation profile and platelet count among S. mansoni-infected adults. The study populations consisted of S. mansoni-infected adults, and non-infected adults, each population with a sample size of 100, for a total of 200 study participants.

Sociodemographic data was collected using a questionnaire survey. Blood and stool samples were collected from each study participant, and urine samples for women to exclude pregnancy. Blood samples were used for analysis of APTT, PT, INR and PLT (platelet count), whereas stool samples were used for infection intensity grading based on eggs per gram.

The study showed that the infected patient population had a higher prevalence of blood-related abnormalities, finding prolonged APTT (37%) and prolonged INR (41%), as well as thrombocytopenia (24%). This was Table 2. Patient and murine studies broken down in according to PICOS format. [17-20]

Authors Year Study design Population Intervention Control Outcome

Eyayu et al. 2020 Patient study,

cross-sectional Infected population with S. mansoni-caused schistosomiasis (n=100)

Screening of APTT, PT,

INR and PLT Healthy individuals (n=100)

Coagulation profile difference in infected population versus healthy controls

Mebius et al. 2019 Patient study,

case-control Infected population with S. haematobium-caused schistosomiasis (n=10)

Screening of vWF, OPG, TAT, ADAMTS-13 and D-Dimer Healthy individuals (n=4) Coagulation profile difference in infected population versus healthy controls

Da’dara et al. 2016 Murine study,

cohort S. mansoni-infected mice (n=?) Screening using thromboelastography Healthy mice (n=?) Coagulation and fibrinolysis profile difference between infected and healthy murine controls

Lagler et al. 2014 Patient study,

case series Infected patients with schistosomiasis (n=3) Screening of D-dimer, fibrinogen, peak thrombin generation and MP-TF activity

- Difference in coagulation profile versus widely available reference values of healthy individuals

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compared to the control group, which showed lower prevalence of prolonged APTT (7%), prolonged INR (4%), as well as thrombocytopenia being less prevalent (3%).

For comparison of coagulation profile and platelet count, the infected group was further sub-divided into light infection (n=40), moderate infection (n=24) and heavy infection (n=36).

Median INR was measured at 1.13 for the control population, whereas light, moderate and heavy populations saw 1.14, 1.21 and 1.4 respectively. Furthermore, median APTT (in seconds) was 31.2 for the control group, followed by 33.0, 34.85 and 35.7 for light, moderate and heavy infection groups respectively. Median PLT (×103_{/μL) for the control population was 268, with light, moderate and heavy infection being at 211, 202.5 and}

160, respectively. [17]

A case-control study by Mebius et al. (2019) studied the S. haematobium parasites’ ability to cause coagulation abnormalities. The study was conducted on 10 schoolchildren with a non-hepatosplenic S. haematobium

infection, as well as 4 healthy controls without infection, in Gabon.

Enzyme-linked immunosorbent assay was used to measure levels of D-dimer, vWF-antigen (vWF:ag), active vWF, ADAMTS-13 antigen, osteoprotegrin (OPG) and thrombin-antithrombin (TAT) complexes. Furthermore, ADAMTS-13 activity was measured.

The results showed statistically significant increases of vWF:ag and active vWF levels in infected children, compared to non-infected. However, these levels were found to not have any correlation with platelet count, as thrombocytopenia was absent. Changes in ADAMTS-13:ag, ADAMTS-13 activity, TAT and D-dimer were absent. It was found that S. haematobium may directly alter activation status of blood vessel walls, as OPG levels were found to be raised. [18]

Lagler et al. conducted a case series study in 2014 by observing three patients, one male and two female, in Eastern Austria, who had contracted Katayama fever (acute schistosomiasis) after a 4-week trip to the Republic of Tanzania. Schistosoma-specific antigen was found past the acute phase of infection, but no urinary or fecal Schistosoma egg excretion was detected. At initial visit, Praziquantel was administered against parasites, accompanied by corticosteroids for anti-inflammatory treatment.

Coagulation parameters were assessed in the two female patients, through D-dimer levels, Fibrinogen, Peak thrombin generation and MP-TF (microparticle-associated tissue factor) activity in vitro. Reagent used for thrombin generation was tissue factor supplied by Technoclone.

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D-dimer (Normal range <0.5 μg/ml) was recorded with an initial visit recording of 0.57 μg/ml for patient A, and 1.17 μg/ml for patient B. Normal D-dimer values were then recorded for patient A and B after 1 week and 32 weeks, respectively. Fibrinogen (Normal range 180-390 mg/dl) was recorded with initial recording values of 477 mg/dl for patient A, and 517 mg/dl for patient B, with recovery to normal values recorded at 11 weeks and 6 weeks respectively. Peak thrombin generation (Normal range 13-100 nM) was recorded with an initial value of 311 nM for patient A, and 384 nM for patient B, however none of the subjects recovered into normal range within 32 weeks. Lastly, MP-TF (microparticle-associated tissue factor) activity (pg/ml) was recorded at initial visit, followed by week 6, 11 and 32. MP-TF is a marker for thrombogenicity, which was found to be raised for patient B. Patient A showed 0.09 pg/ml at initial visit, followed by 0.05 pg/ml, 0 pg/ml and 0.85 pg/ml,

respectively. Patient B showed 1.64 pg/ml at initial visit, followed by 1.10 pg/ml, 0.12 pg/ml and 0.03 pg/ml.[19]

4.2 Murine studies

A murine blood coagulation study was made by Da’dara et al. in 2016, to determine ex-vivo effects of schistosomes on blood coagulation. S. mansoni schistosomes were used to infect mice, which had their blood collected and analyzed at the 4-week and 7-week marks post-infection. The blood was then analyzed through thromboelastography (TEG) to investigate in vivo and ex vivo effects.

Significant in vivo difference was found in blood from infected animals, compared to control animals without infection. The TEG profiles 4 weeks post-infection showed no significant differences. However, TEG profiles 7 weeks post-infection, showed significant coagulation differences, where coagulation was seen to be

significantly faster among the infected group than compared to the control group. The time points were chosen due to the life cycle of schistosomes, as the migration phase begins around 4 weeks post-infection.

The degree of fibrinolysis also differed significantly between week 4 and 7 post-infection, with ~5% clot lysis at 30 minutes and ~10% at 10 minutes for the infected blood, whereas control group blood clots remained largely intact at ~0%.

Ex vivo blood coagulation measured using TEG differed significantly, with inhibited blood coagulation in infected murine blood. However, no fibrinolysis was detected in either of the groups.[20]

4.3 Schistosoma-derived proteins of haemostatic significance

4.3.1 SmCalp1 and SmCalp2 – S. mansoni Calpain-family 1 and 2

In a 2018 study conducted by Wang et al., schistosomes’ ability to cleave HK (High molecular weight

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with their ability to generate bradykinin, a vasodilator. High molecular weight kininogen (HK) assay and kallikrein assays were conducted, together with bradykinin measurement.

The study found that S. mansoni schistosomula and/or adult male worms do cause HK cleavage using SmCalp1 and SmCalp2, however without utilizing kallikrein or kallikrein-like activity. Furthermore, the bradykinin measurement showed no detectable bradykinin, unlike the human kallikrein-mediated HK cleavage used for control that generated detectable levels.

Moreover, it was found that the serine protease inhibitor, PMSF, failed to noticeably inhibit schistosomal HK-cleaving activity. However, the cysteine protease inhibitor E64c was able to block HK cleavage, suggesting that SmCalp1 and SmCalp2 may be cysteine proteases. [21]

4.3.2 SmKI-1 – S. mansoni Kunitz Inhibitor 1

A study was conducted by Ranasinghe et al. (2015) on a gene sequence coding for SmKI-1 (Smp_147730) to identify its functional expression and its impact on haemostasis.

The results of this study showed that SmKI-1 is comprised of 146 amino acids, with a signal peptide of 20 residues and a putative molecular mass of 15.108 kDa. BLASTP analysis revealed 57% identity and 39% query cover with human tissue factor pathway inhibitor-2 (TFPI-2). Furthermore, SmKI-1 was found to be highly expressed in adult S. mansoni of both genders, but not in the earlier life cycle stages (miracidia, cercariae, schistosomula or eggs).

Immunolocalization showed presence of SmKI-1 in the tegument of adult worms and the sub-shell region of eggs in infected murine liver tissue.

Coagulation assays showed that recombinant SmKI-1 extended blood coagulation time dose-dependently, with 4 μM SmKI-1 concentration resulting in a 2.7x extension of APTT and 2.0x increase of PT. Direct thrombin inhibition was not detected, as thrombin clotting time (TCT) remained unchanged.

Protease inhibition assays identified rSmKI-1 inhibition of several proteases at difference concentrations: trypsin (35 nM), chymotrypsin (61 nM), neutrophil elastase (56 nM), FXa (142 nM) and plasma kallikrein (112 nM). Inhibition on cathepsin G or pancreatic elastase was not present.[22]

4.3.3 SmATPDase1 – S. mansoni tegumental ecto-apyrase

In this study from 2014, Da’dara et al. studied if S. mansoni could cause exogenous ATP and ADP breakdown through SmAP, SmNPP-5 and/or SmATPDase1 proteins. ADP is a potent thrombocyte activator.

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The study showed that parasites do not exhibit morphological differences compared to controls, when the three surface genes were suppressed. It was also found that SmATPDase1 alone causes exogenous ATP and ADP degradation, and that the catabolic function is only present in the full form of SmATPDase1 and not the secretory form. Its ATPase activity was found to be maximal at pH ≥ 8.5, while the ADPase activity reached maximum rates at pH ≥ 7.5.[23]

4.3.4 SmAP – S. mansoni tegumental alkaline phosphatase

Elzoheiry et al. demonstrated in a 2019 study that S. mansoni can hydrolyze polyP (polyphosphates) through its tegumental alkaline phosphatase, SmAP, into inorganic phosphate (Pi). To do this, control S. mansoni was treated with synthetic siRNA targeting the SmAP gene for gene suppression. The results showed that incubation of S. mansoni without siRNA treatment together with 2 mM polyP led to an increased release of Pi versus control, with adult male schistosomes releasing significantly more Pi, compared to females. [24]

4.3.5 SjKI-1 – S. japonicum Kunitz-type inhibitor 1

In this 2015 study, Ranasinghe et al. performed functional studies on recombinant SjKI-1 from S. japonicum. Results showed that SjKI-1 consists of 69 amino acids, with a molecular mass of 8.023 kDa and a single Kunitz domain. A signaling peptide was not identified. Gene analysis suggested that SjKI-1 may be a secretory protein. SjKI-1 gene expression was found to be the highest among adult male and female schistosomes and eggs, whereas cercariae and schistosomula showed low expression. Immunolocalization indicated SjKI-1 to only be present in eggs, specifically only those trapped in the intestinal wall.

Protease inhibitor assays showed SjKI-1 inhibition of trypsin at 0.21 nM concentration, chymotrypsin at 0.18 nM, neutrophil elastase at 122 nM, FXa at 650 nM and plasma kallikrein at 3.2 nM. No inhibitive effects were found on pancreatic elastase or cathepsin G.

Coagulation assays showed that SjKI-1 dose-dependently prolonged APTT duration compared to healthy donor blood. However, PT and TCT remained normal, indicative of no FVII or direct thrombin inhibition. SjKI-1 was also found to be a Ca2+ _{binding protein.[25]}

4.3.6 SjB10 – S. japonicum serine protease inhibitor

SjB10 was functionally characterized by Molehin et al. in a 2014 study.

Results showed that the SjB10 gene encodes a polypeptide of 403 amino acids with a predicted molecular mass of 45.7 kDa. Six N-glycosylation sites were identified on SjB10, and 8 protein kinase C phosphorylation sites. SWISS-MODEL analysis of secondary structure showed 9 α-helices and 15 β-strands. Transmembrane domains

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or signal peptides were not found. 21-65% homology with known full-length parasitic helminthic serpins was identified, with the S. haematobium serpin having the highest sequence homology.

Gene expression profile analysis detected SjB10 gene expression in adult male, schistosomula, egg and

cercariae stages, with cercariae stage showing 4-fold elevated expression compared to other stages. No evidence of SjB10 expression on female worms and miracidia was found.

Immunolocalization of SjB10 found intense prevalence in the anterior gut of the adult worm, and in the cellular layer beneath the egg shell; namely in the extra-embryonic envelopes, outer envelope and cellular inner

envelope.

Protease inhibition assays conducted to investigate potential inhibitory properties of rSjB10 showed dose-dependent inhibition of porcine pancreatic elastase, with complete inhibition at 2.5 nM concentration. Tests with trypsin, chymotrypsin and neutrophil cathepsin G showed limited to no inhibitory activity. [26]

5. Discussion

Ten articles were aggregated through the systematic search, with six on S. mansoni, two on S. japonicum, one on S. haematobium and one without specification of Schistosoma subspecies. It is evident that S. mansoni is the most studied parasite, perhaps not surprising as it is the most prevalent, being endemic in 54 countries. However, S. haematobium, being endemic in 53 countries, only had one relevant publication found. [27] This is

surprising; the assumption was made that S. haematobium would be more focused on, due to its established status as a potent, IARC Group 1, carcinogen.[28] Furthermore, S. japonicum has seen a rise in prevalence in China and South-East Asia [29], which has led to a recent rise in published research, as reflected by the two S. japonicum articles. Searching for “mansoni”, “haematobium” and “japonicum” on PubMed and observing the publications per year timeline, confirms the above observed patterns. However, much of the focus among these publications is put on anti-schistosomal treatment, not schistosomal coagulation profiles and its associated proteins. This may partially explain why the results aggregated were of limited quantity, which is reasonable as schistosomes are a major health issue in many countries. More research on Schistosoma-caused coagulation profiles and its associated anti-haemostatic proteins would have been greatly beneficial to this study.

5.1 Cost-effectiveness of protease-based pharmaceuticals

All Schistosoma-derived anti-haemostatic substances found were proteases. Protease-based pharmaceuticals remain uncommon today, as there are two main obstacles: Their labile nature and high associated production costs. Their labile nature entails the need of a strictly controlled environment during transport and storage, resulting in additional expenses. Additionally, high associated production cost is a result of the involved

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cultivation using recombinant DNA technology, in order to produce large quantities of a protease. However, there are also possibilities of great cost reductions through optimization of both manufacturing and logistical processes over time.[30]

Therefore, it is of utmost importance that protease-based pharmaceuticals make sense from a medical standpoint, but also from an economic standpoint. This can be achieved through development of a novel essential or

breakthrough therapy, development of therapy that costs less than a previous alternative, or the development of a therapy of similar effect and cost, however with a different or better side effect profile. An example of this is Alteplase, which is human tissue plasminogen activator (tPA, a serine protease) produced using recombinant DNA technology. It is a thrombolytic pharmaceutical that is currently in use mainly against acute ischemic stroke and acute myocardial infarction. Alteplase has been deemed cost-effective despite a price of $6400 per dose.[31]

5.2 Unspecified Schistosoma subspecies publications

While the 2014 Lagler et al. study did not specify which subspecies was responsible for the disease seen in the patients, the information is still valuable as it provides insight on Schistosoma-derived coagulation profiles. D-dimer, fibrinogen and peak thrombin generation were elevated, which may signify fibrin breakdown, increased fibrinogen synthesis in the liver and increased fibrinogen to fibrin conversion, respectively. These were

however in vitro tests that were stimulated using an unknown trigger, so the elevated fibrinogen to fibrin conversion reflects increased activity potential in one of the involved components. Furthermore, MP-TF was found to be elevated in one subject.[19] This set of effects may denote that blood clots form more often (Elevated peak thrombin generation), however with decreased blood clot stability (Elevated D-dimer), which results in a faster blood clot turnover rate. Increased fibrinogen is likely a result of the ongoing infection, as it is an unspecific acute phase reactant. [32] MP-TF elevations may indicate a hypercoagulable state.

5.3 S. mansoni publications

S. mansoni was found by Eyayu et al. (2020) to extend blood coagulation times, both intrinsic (APTT) and extrinsic (INR), among an infected population.[17] What this could possibly signify is either inhibition of both intrinsic and extrinsic coagulation pathways, or an inhibition of the common pathway. Seemingly contradictory, S. mansoni was found by Da’dara et al. (2016) to accelerate the formation of blood clots in murine blood in-vivo, however possibly compensated by a rapid, abnormal, fibrinolytic clot breakdown. [20] This finding is in line with the previous discussion on the study by Lagler et al.[19] Da’dara et al. hypothesized that this may be a result of reduced levels of coagulation control proteins which promotes rapid blood clotting, and as

counterbalance, rapid fibrinolytic clot breakdown may occur due to reduced blood platelet count and fibrinolysis control constituents. Ex vivo samples containing S. mansoni in the other hand, showed that

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coagulation was inhibited, with no detectable fibrinolysis. [20] A plausible explanation for this behavior is that schistosomes are anti-haemostatic in a local manner; they must be present and actively secreting their

substances for their anti-haemostatic effects to be seen.

Looking into the secreted substances of S. mansoni, the aggregated results found SmCalp1 and SmCalp2[21], SmKI-1[22], SmATPDase1[23] as well as SmAP[24] proteins. These proteins have various anti-haemostatic properties which may aid understanding of how schistosomes are anti-haemostatic, despite numerous pro-haemostatic characteristics.

•_{SmCalp1 and SmCalp2 were demonstrated to cleave high molecular weight kininogen (HK), an early} co-factor in the intrinsic coagulation pathway, without the bradykinin generation that the human kallikrein-mediated cleavage entails.[21]

As the protease actions are very specific, the two proteins may be possible subjects for use as basis for therapeutics. Since HK is a co-factor participating in FXIIa-mediated FXI activation, an early inhibition of the intrinsic pathway should occur. Moreover, this also means that FXII activity will be reduced. This should pose no issue, as deficiencies of HK and FXII have both been linked to prolonged APTT while not causing bleeding tendencies [33], making them very favorable pharmacological targets for an anti-haemostatic drug. SmCalp 1 and/or 2-based therapeutics may additionally also be used to treat HAE type III (Hereditary angioedema type 3), a disorder caused by heightened bradykinin levels as a result of elevated HK and/or FXIIa levels.[34] This may indicate that a SmCalp1 and/or SmCalp2-based therapy could have two vastly different indications, which, in essence, increases the possible market share to be acquired and thus profitability and feasibility of pharmaceutical development.

Currently, there are only expensive monoclonal antibody treatments available for reduction of bradykinin levels, such as Lanadelumab (Shire Pharma), which has an average annual cost of USD 266,994 to USD 533,988.[35] If a SmCalp1 and/or SmCalp2-based therapy could be produced and sold at a lower annual cost than Lanadelumab, there might be a competitive edge, and thus, a large market share to be gained. Lanadelumab exerts its effect by inhibiting plasma kallikrein, the endogenous protease that cleaves HK and, in the process, causes generation of bradykinin.[36] In comparison, SmCalp1 and/or SmCalp2 treatments could possibly entail fewer side effects, as the enzymes directly cleave HK without generating bradykinin.

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Thus, it could be deemed that SmCalp1 and/or SmCalp2-based pharmaceuticals may have a high potential of success. However, much of this success may be attributed to the use of the pharmaceutical for HAE type III. This stems from an assumption that it will likely not be as competitive against other anti-haemostatic drugs from an economic standpoint. Several other FXI/FXIa inhibitors are being researched, and these may involve more cost-efficient manufacturing as they’re not based on proteases.[37]

•_{SmKI-1 showed concentration-dependent inhibition of various proteases. EC}50 for the proteases

inhibited are trypsin at 35 nM, chymotrypsin at 61 nM, neutrophil elastase at 56 nM, FXa at 142 nM and plasma kallikrein at 112 nM. Many of these proteins are directly or indirectly involved in

coagulation.[22]

FXa inhibition is of interest, as SmKI-1 inhibits FXa in the nanomolar range, indicating high potency. FXa is the first protein in the common pathway, where the intrinsic and extrinsic pathways meet. Inhibition of FXa could in other words potentially lead to a vastly impeded coagulation cascade, with APTT and INR both prolonged. In this aspect, SmKI-1 could be utilized as a direct Xa inhibitor. However, direct Xa inhibitors are already on the market, at very competitive prices. For comparison, Apixaban, a direct Xa inhibitor, sold under the Eliquis brand, has an estimated annual cost of EUR 3,506 per year.[38] Thus, it is immensely difficult to compete as protease-based therapy is more expensive to manufacture.

Another considerable obstacle is that plasma kallikrein, neutrophil elastase, trypsin and chymotrypsin would be inhibited as well when FXa has been inhibited. This could cause massive side effects, such as reduced protein absorption in the jejunum, as trypsin and chymotrypsin are involved in the digestion of protein.[39] Furthermore, inhibition of plasma kallikrein results in reduced HK cleavage and

subsequently, reduced bradykinin levels. This induces a pro-thrombotic effect, and not the desired anti-thrombotic effect. Plasma kallikrein is also involved in catalyzation of plasminogen to plasmin

conversion; with inhibition by SmKI-1, plasmin levels will be reduced. As plasmin is a fibrinolytic enzyme, the effect may be slower clot breakdown. Unfortunately, this means that the net effect of plasma kallikrein inhibition is pro-coagulative, opposite of the desired anti-coagulative effect. This may weaken overall anti-haemostatic properties of SmKI-1, but it should be noted that the protein was still able to prolong APTT by 2.7x and PT by 2.0x in the 2015 study by Ranasinghe et al.[22] However, this was purely a coagulation test, which should be taken in consideration. It did not account for other factors, such as fibrinolysis potential.

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With all discussed obstacles considered; it should be deemed that SmKI-1 will not be plausible to use as basis for development of pharmaceuticals.

•_{SmATPDase1 was found to catabolize ATP, which is a potent pro-inflammatory mediator signaling cell} damage, but also ADP, a potent pro-thrombotic mediator.[23] ADP catabolization would result in an antiplatelet effect, however, ATP is also catabolized. During a Schistosomiasis infection, the effect is largely of local character and mainly anti-thrombotic and anti-inflammatory, as protease levels are not high enough to act systemically. However, having SmATPDase1 treatment applied in a systematic manner would be certainly detrimental, as all cells rely on ATP and ADP for energy. Thus,

SmATPDase1 cannot be used as basis for development of anti-haemostatic pharmaceuticals, as the side effects would be too pronounced. It could, however, be interesting for other pharmaceutical fields, such as oncology.

•_{SmAP was found by Elzoheiry et al. (2019) to cleave polyphosphates (PolyP), a relatively newly}

discovered pro-thrombotic polymer. [24] However, it should be noted that the role of polyphosphates are greatly disputed. Platelet-sized polyphosphates have been proposed to cause effects such as bradykinin release, 3000-fold enhancement of FXI th5rombin-mediated activation, acceleration of thrombin

generation, as well as clot strengthening and fibrinolysis resistance and inhibition of TFPI (Tissue Factor Pathway Inhibitor) activity. TFPI normally inhibits FXa and FVIIa, and these inhibitions are potentially lifted by polyphosphates.[40] Other publications have discussed polyphosphate-enhanced platelet binding to von Willebrand factor, as well as factor XII activation, XI activation and more.[41] If the polyphosphate findings are accurate, SmAP could be a highly interesting, potent regulator of

haemostasis. However, more research is needed to establish the roles of polyphosphates, to create an adequate assessment of SmAP utilization in anti-haemostatic pharmaceutical development.

5.4 S. japonicum publications

• SjKI-1 from S. japonicum was isolated by Ranasinghe et al. (2015), showing Ca2+_{binding properties in}

addition to the same inhibitory effects on proteases as the previously discussed SmKI-1. However, EC50

concentrations for protease inhibitions differed, with trypsin inhibited at 0.21 nM, chymotrypsin at 0.18 nM, neutrophil elastase at 122 nM, FXa at 650 nM and plasma kallikrein at 3.2 nM.[25]

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inhibitor of trypsin, chymotrypsin and plasma kallikrein, while being a weaker inhibitor of neutrophil elastase and FXa. As SjKI-1 is an even more potent inhibitor of plasma kallikrein, the potential for use as basis for novel anti-haemostatic drugs should be deemed as low.

However, SjKI-1-based pharmaceuticals could be competitive against Lanadelumab mentioned before in the discussion on SmCalp1 and SmCalp2. Both SjKI-1 and Lanadelumab are plasma kallikrein

inhibitors. Nonetheless, the same obstacle that was previously pointed out about SmKI-1 applies here as well; to reach the EC50 concentration for this effect to take place, inhibition of trypsin and chymotrypsin

would also occur. Moreover, SjKI-1 also binds Ca2+_{, meaning impact on several points of the intrinsic}

pathway as well as the common pathway, as well as systematic impact due to how widely used Ca2+

based signaling is. Further research should be conducted in order to quantify how potent the Ca2+

binding effect is, as it might pose a large side effect risk.

• SjB10, another S. japonicum protein, was studied by Molehin et al (2014). It was only shown to have dose-dependent inhibition of porcine pancreatic elastase.[26] Thus, SjB10 is not interesting for this topic.

5.5 S. haematobium publications

• S. haematobium was studied in a smaller case-control study by Mebius et al. (2019). The study did not involve blood coagulation times, and only proved direct alteration of blood vessel walls. However, this was likely due to the size of parasite eggs. Thus, the study remains inconclusive.

6. Conclusion

In summary, 3 Schistosoma-derived proteins from the past 10 years of research were determined to be of high interest: SmCalp1 and SmCalp2, together with SmAP, all derived from S. mansoni, a very well-documented Schistosoma subspecies. 4 other Schistosoma-derived proteins were also found, seemingly interesting at first. However, also discovered together with these, was the presence of issues such as low specificity or

contradictive pro-haemostatic effects, or even effects that could be life-threatening if applied systematically. The proteins of high interest may have significant potential for use as basis for novel anti-haemostatic treatment. However, further research should be conducted or awaited as many questions were left unanswered, especially concerning SmAP. This is of utmost impact, as the financial risks involved in protease-based pharmaceutical development are high. Furthermore, the high manufacturing cost of protease-based pharmaceutical must also be considered. For a protease therapy to be commercially successful, they must be cost-efficient despite high costs. Thus, a more in-depth analysis on cost and competition should be conducted.

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The potential of Schistosoma-derived substances for use as basis for novel anti-haemostatic therapeutics: : A systematic review