Cancer and Metastasis Reviews 9: 381-392, 1990.

© 1990 Kluwer Academic Publishers. Printed in the Netherlands.

Proteases in the schistosome life cycle: a paradigm for tumour metastasis

M.J. DoenhofP, Rosane H.C. Curtis 2, J. Ngaiza 3 and J. Modha 4

1School of Biological Sciences, University College of North Wales, UK;
2Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, St Albans, UK;
3Department of Medicine, Cornell Medical Centre, New York, USA; 4Department of Biochemistry,
University of Leicester, Leicester, UK

Key words: schistosome, S. mansoni, protease, tumour, metastasis

Summary

Cancers and parasites have a number of properties in common, particularly those that relate to their
respective capacities to evade host defence mechanisms. This review highlights the similarities between
metastatic tumours and schistosomes in particular, and describes the role that proteases may have in the
migration, growth, survival and transmission of the different stages of the schistosome life-cycle in the
vertebrate host. An elastase-like serine protease of schistosome larvae has been particularly well character-
ized, and its substrate profile and other properties are indicative of a role in facilitating migration of the
parasite through skin tissue early after infection. The primary structures of a cathepsin B-like enzyme, and a
putative 'haemoglobinase' found in adult worms have also recently been derived, these enzymes being
responsible for degradation of haemoglobin in erythrocytes upon which the adults feed. Adult schistosome
worms reside and produce eggs intravascularly, and the processes that mediate the extravasation and
subsequent migration of the egg through host tissue are dependent on both blood platelets and the immune
response. Fibrino(geno)lytic enzymatic activity that is present in the egg could modulate the thrombogenic
potential that eggs might have as a result of their capacity to cause platelet aggregation in vitro and in vivo.
The roles of other proteases and peptidases that have been found in schistosome larvae, worms and eggs are
less clear. Some of these enzymes may modulate immunological and haemostatic defence mechanisms and
thus prolong survival of the parasite, and the consequences of the interactions between schistosomes and
host protease inhibitors could also be immune modulatory.

Introduction the body's defence mechanisms, with the corollary

that in some instances similar evasive strategies
As befits diseases of great public health impor- appear to have been adopted.
tance, cancers and parasitic infections receive con- This review, rather than ploughing the same fur-
siderable attention from medical researchers. Al- row again, will concentrate on just one parasite,
though they are seldom studied in the same in- Schistosoma mansoni, and consider in some detail
stitute, let alone in the same laboratory, parasit- the role proteases have in the life cycle of the
ologists and immunologists have in the past had infecting organism in the mammalian host. The
occasion to point out some general similarities that factors that researchers have implied are important
do exist between the disease processes of cancers in the establishment of tumour metastases have
and parasites [1-3]. Attention has been paid partic- certainly contributed to the way parasitologists
ularly to the problem they each have of surviving think about the establishment, migration, growth
382

trematodes (parasitic flat worms in which the

adults are sexually differentiated), and it is estimat-
ed that over 200 million people in the tropics are
infected, with several hundred million m o r e at risk.
There are three main species which infect man,
Schistosoma mansoni, S. japonicum and S. haema-
tobium, and they differ in their morphology and the
site of intravascular habitation of the adult worms.
The life cycle of schistosomes is illustrated dia-
gramatically in Fig. 1. One of the main features is
the requirement for two hosts. A cycle of asexual
replication occurs in the snail intermediate host,
resulting in release into water of free-swimming
cercariae. These penetrate the skin of a second
definitive vertebrate host and mature into adult
worms that produce eggs by sexual reproduction.
The eggs are produced in large numbers intravas-
cularly, and a proportion of them are swept away
by blood flow to become trapped in a capillary
0 network downstream. It is the pathological conse-
quences of this embolization of eggs in host tissues
such as the liver, and particularly the inflammatory
Fig. 1. The schistosome life cycle. Fork-tailed, free-livingcerca- immune responses generated around the eggs, that
riae which are released into water by aquatic or amphibious are responsible for most of the disease symptoms in
snails, find a suitable host such as man, and penetrate and
schistosomiasis. A proportion of eggs, however,
migrate through the skin (a). During the early stages of infection
the larvae undergo a transformation which adapts them to life in become extravasated at the site of initial deposition
their new host. After passing through the dermis the schisto- in the intestine or urino-genital tract, and these go
somula enter the vascular system and thereafter remain in the on to be excreted in the stools or urine. On contact
circulation, eventually to become trapped in the liver (b), where with water the eggs hatch into ciliated free-living
maturation of sexually differentiated adult worms commences.
miracidia which can infect further susceptible snails
Worm pairs migrate upstream in the hepatic portal vein (c), to
take up residence in the capillary beds of the intestine (S. to complete the cycle.
mansoni and S. ]aponicum ) or urino-genital tract (S. haematobi- Much of the current research being done on
urn), The adult worms lay eggs, a proportion of which become schistosomiasis is concerned with development of a
extravasated (d), and after passage through the walls of the vaccine, and recent reviews of progress in the biol-
intestine or bladder the eggs are excreted (e). On contact with
ogy and immunology of this disease are available
fresh water the eggs hatch into ciliated miracidia (f), which
infect an appropriate snail host. A cycleof asexual reproduction [4, 51.
in the snail results in production and release of cercariae.

and transmission of parasites: it is our hope that Points of similarity between schistosomes and
this description of the role of proteases in the schis- metastasizing tumours
tosome life-cycle might in return be of some in-
terest to oncologists. Table i lists on the left side a n u m b e r of steps which
m a n y reviewers agree have to occur during tumour
growth and metastasis. On the right of the Table is
The schistosome life-cycle an indication of stages of the schistosome life-cycle
which seem to have an equivalent hurdle to over-
Schistosomiasis is a disease caused by digenetic come. There follows a consideration of the role
 383

that proteases may have in facilitating the migra- nin, Type IV collagen [14], elastin [12], and the
tion and growth of schistosomes at each stage of the peptide backbone of proteoglycans [15], is consis-
life-cycle in definitive hosts. tent with it having a critical role in skin invasion.
Synthesis of the cercarial protease is limited to a
period when cercariae are still developing in the
An elastase-like enzyme involved in skin snail, and the protein is produced as a proenzyme
penetration of schistosome larvae with a structure that allows for a post-translational
modification typical of vertebrate coagulation and
Penetration and migration of schistosome larvae digestive proteases [16}. It has an amino acid se-
through the skin of their vertebrate hosts has for a quence related to that of the trypsin family of serine
long time been considered to be mediated by a proteases, and a particularly high degree of consen-
combination of secreted hydrolytic enzymes and sus with rat pancreatic elastases I and II [16]. Sub-
larval muscular activity. The penetration response strate and inhibitor profiling indicated, however,
of cercariae is chemically stimulated, particularly that aromatic amino acids were preferred at the P-1
by skin lipids, and during entry the larvae lose their site of substrates, suggesting an analogy with chy-
tails and a carbohydrate-rich glycocalyx coat. They motrypsin [12]. The enzyme has a pI of 8, a pH
undergo other morphological and physiological optimum of 9, and its activity is calcium-depend-
transformations, one of which tolerizes them to ent. It is immunogenic, with IgM and IgG antibod-
isotonic conditions [6-8]. Larvae which successful- ies found in infected patients, and its potential
ly enter the skin reach the base of the epidermis usefulness as a diagnostic aid has been considered
within 30 minutes, and they remain epidermal for [17]. A 17 kDa form of the enzyme also occurs,
40 to 48 hours before beginning to enter the dermis perhaps as a result of autoproteolysis [12].
[9, 10]. Assays for proteolytic activity indicated that re-
Early work (reviewed in [6]) indicated that pro- lease of the acetabular gland protease occurred
teolytic enzyme activity secreted from the acetab- over the first 24 hours after cercariae were trans-
ular glands of schistosome cercariae was involved formed into schistosomula [18]. The comparative
in skin penetration. The acetabular glands are con- lengths of time spent by the parasite in the epider-
nected to the apical surface of the parasite by ducts, mis compared with the dermis suggest that the
and the glands empty during the first stages of entry basement membrane may be a substantial barrier
into the skin. A serine protease(s) of Mr 25-30 kDa to progress of the larvae through skin, and electron
has been purified from cercarial extracts [11], and microscopic evidence suggests the acetabular
from skin lipid-induced cercarial secretions [12]. glands are in fact empty of contents prior to breach-
The wide range of substrates hydrolyzed by this ing of the membrane [19]. To what extent lytic
enzyme, including keratin [13], fibronectin, lami- agents other than the cercarial elastase subsequent-

Table 1. Some similarities between metastatic tumours and stages of the schistosome life cycle

Tumours Schistosomes

1. Progressive growth of tumour at primary site. i

2. Penetration of basement membrane. Schistosomula

3. Vascularization of tumour.
4. Entry of tumour ceils into the vasculature. Schistosomula
5. Migration and survival in bloodstream. Schistosomula/Worms
6. Arrest in distant capillary bed. Young Worms/Eggs
7. Exit from the vasculature. Eggs
8. Migration into tissue parenchyma. Schistosomula/Eggs
9. Progressive growth.
384

ly facilitate larval migration remains to be deter- nent of complement [29]. The inhibitory effect of
mined: serine proteases of 60 kDa and 28 kDa have glucocorticoids on schistosome survival and matu-
been identified in secretions of newly transformed ration [30] is further indirect evidence for an in-
schistosomula [20], and metalloproteinase activity flammatory response potentiating larval migra-
is also evident after exhaustion of acetabular gland tion. Cercariae are themselves capable of synthe-
proteolytic activity [18, 21]. sizing eicosanoids [31], perhaps for the same rea-
The importance of proteolytic secretions in the son. In this context it should also be mentioned that
skin invasion phase of schistosome infections larval proteases are known to react with alpha 1-
seems clear, and has been further substantiated by proteinase inhibitor [32, 33], and product(s) of the
the demonstration that a synthetic peptide inhib- interaction between proteases and serine protease
itor of the cercarial elastase, which had no effect on inhibitors are chemotactic for neutrophils [34].
cercarial mobility, prevented cercariae from invad- By virtue of its antigenicity the cercarial elastase
ing human skin explants [22]. In the context of the is a potential target for immune attack, and an IgM
anti-cancer effects of protease inhibitors [23], it monoclonal antibody raised against it inhibited
would be of interest to determine whether the com- proteolytic activity and had complement-depend-
pound niclosamide, which is being promoted as a ent cytotoxicity for cercariae [35]. Contrariwise,
prophylactic against cercarial penetration [24], has we have found that immunoglobulin G is subject to
any effect as a protease inhibitor. degradation by larval extracts. Figure 2 demon-
strates the capacity of a crude S. mansoni cercarial
extract, and a protease in a partially-purified frac-
Immune modulation by larval proteases tion therefrom, to hydrolyze radiolabelled immu-
noglobulin. The hydrolytic activity was inhibited
In addition to a direct effect of hydrolysis of the by heat, preincubation of the extract with diisopro-
extracellular matrix during migration through skin, pylfluorophosphate (DFP), and by pre-absorption
larval proteases could facilitate survival of the par- with a monospecific antiserum raised against a lar-
asite by other indirect means, including modula- val antigen with proteolytic activity. The seemingly
tion of immune effector mechanisms. The cercarial potent hydrolytic activity of this enzyme against
carbohydrate-rich glycocalyx is a strong activator IgG in fact made it difficult for us initially to dem-
of the alternative pathway of complement [25], and onstrate convincingly that serum from immunized
the hydrolytic activities of acetabular gland secre- rabbits contained antibody which could react spe-
tions [26, 27], and particularly of serine proteases cifically with the enzyme. Immunoelectro-phoretic
[20], are believed to mediate in glycocalyx loss evidence of the presence of specific antibody in
during transformation of cercariae to schistosom- such sera was seen only after the enzyme had been
ula. Loss of the glycocalyx could largely explain the pretreated with a serine protease inhibitor (Curtis
increasing resistance of schistosomula to in vitro and Doenhoff, unpublished).
and in vivo killing by complement [25, 27]. Com- IgG that is bound to the schistosomula surface
plement components appear also to be inactivated via Fc receptors is also cleaved off by proteolytic
directly as a result of their hydrolysis by secreted activity [21], and threonine-lysine-proline, a tri-
serine proteases [20]. peptide cleavage product of IgG hydrolyzed by
The vasodilation, margination of leukocytes and excreted products of schistosomula, has been
dermal inflammatory infiltrates which occur within shown to inhibit the toxic effects of macrophages
24 hours of infection [9, 28] may be generated by on schistosomula in an antibody(IgE)-dependent
anaphylactic products of schistosome-induced cytotoxicity reaction [36], and to potentiate IgE
complement degradation. That this could be im- synthesis [37].
portant in facilitating larval migration and growth
is indicated by the smaller worm burdens and short-
er worm lengths in mice deficient in the C5 compo-
 385

Fig. 2. Cleavage of human lgG by S. mansoni cercarial protease(s). Purified human IgG, 0.5 mg (Dako Corporation, California), was
labelled with 1z51by the iodogen method as described in reference 75. The radiolabelled product was separated from free isotope by
Sephadex G-50 chromatography, and preliminary analysis of the labelled preparation by sodium dodecyl sulphate- polyacrylamide gel
electrophoresis (SDS-PAGE) under reducing conditions and autoradiography indicated the presence of low molecular weight
degradation products. Intact 1251was obtained as described in reference 76 by subjecting the preparation to SDS-PAGE on 12.5% gels.
After washing the gels a 1 cm wide band containing a single radioactive band of high molecular weight, presumed to be intact IgG, was
excised and a 1 cm square slab was incubated at 37° with 100/zl phosphate buffered saline (PBS), pH 7.6, containing 16/~g (bovine serum
albumin (BSA) protein equivalent) of soluble extract of homogenized S. mansoni cercariae (prepared as in reference [71]). At
successive time intervals between 2 minutes and 10 hours samples of 10 ul were withdrawn from the incubation mixture and the enzyme
activity in these samples was terminated by boiling. The samples were analyzed in SDS-PAGE, followed by autoradiography to
determine the distribution of radiolabel, and panel A shows the results of the incubation time course, as well as, in the first lane, the
product of a control incubation containing labelled IgG without cerearial homogenate. Lane B indicates the result of incubating
radiolabelled IgG for 18 hours at 37° with a 28 kDa diisopropylfluorophosphate(DFP)-binding cercarial protease (40 p.g in 100/zl
incubate), the protease having been partially purified by preparative electrophoresis. In panel C the cercarial homogenate had been
treated with DFP at concentrations of 1.5 x 10-5 mM (left lane) and 3 x 10-5 mM (right lane), before incubation with labelled IgG. In
lane D the cercarial homogenate had been heat-inactivated prior to incubation with labelled IgG, and in lane E prior to incubation with
the labelled IgG the cercarial homogenate had been immunoabsorbed with a rabbit antiserum that had monospecific reactivity against
the 28 kDa cercarial protease.

Intravasation of larvae a proportion enter the lymphatics and can traverse

lymph nodes [9].
After entering the dermis the schistosomula take a The exact mechanisms by which schistosomula
further 20 hours or so to locate and penetrate a penetrate vascular endothelium are unknown, but
venule [10], and once inside the blood vessel they associative factors which have the potential to facil-
are probably rapidly carried away by blood flow. In itate the process, such as imflammatory infiltrates,
addition to entry into the circulation via capillaries, vasodilation, and oedema at the site of skin pene-
tration, have been noted (reviewed in [8]). An
386

immediate reaction of blood platelets might be ex- diameter from 15-25/~m when they first arrive, to a
pected upon breach of endothelium integrity, the minimum diameter of about 8 ~m. This, together
platelet activity being directed towards either dam- with muscular exertion by the parasite, is presumed
aged subendothelial matrix or the intruding para- to facilitate migration along the lumena of lung
site, or both. That platelet activity might be delete- capillaries.
rious to the parasite at this stage is indicated by the The fit is tight, however, since although the mini-
observation that mice depleted of platelets by in- mum diameter of the larval worm reduces to 8-
jections of antiplatelet serum at the time of in- 10/~m, this is still up to twice as great as the internal
fection have been found to have worm burdens bore of lung capillaries. Thus, despite the changes
increased by 30 to 45% when compared with un- which the parasites undergo in respect of extensi-
treated controls [38]. It should be noted that typ- bility, the pulmonary microvasculature would
ically only 30 to 50% of S. mansoni cercariae that seem to provide a major mechanical barrier to their
are applied percutaneously to mice mature into free movement throughout the circulation, and
adult worms resident in the hepatic portal system. hence the prolonged lung transit time of 30 hours or
The factors contributing to non-survival of parasit- more, and the retrievability of schistosomula from
es, and the sites where such losses take place are the minced lungs of infected laboratory rodents for
topics still under debate, but this attrition would up to about a week after infection. Migrating larvae
seem to occur after entry into the systemic circula- can cause endothelial damage in the lungs [40].
tion [8]. There is no apparent inflammatory activity asso-
ciated with parasites that are intravascular in the
lung, but some larvae may escape the vascular sys-
Migration and survival of larvae within the vascular tem and induce inflammatory reactions in the alve-
system oli. It is assumed that many of these ectopic parasit-
es fail to survive, but some have the capacity to
Once within the vascular system surviving schisto- re-enter the circulation, as indicated by experi-
some worms remain there for the remainder of ments showing that 12-15% of schistosomula taken
their lives in the definitive host. Work in laboratory from the lungs of donor mice and introduced into
rodents has indicated that in the few days sub- the alveoli of naive mice survived to mature in the
sequent to intravascularization there is a systemic hepatic portal vein of the recipients [41]. It remains
phase of migration of schistosomula in the direc- to be determined whether larval migration through
tion of blood flow, with an estimated mean of 30-35 lung parenchyma is protease-dependent in the
hours required for each of them to transit through same way as migration through skin.
lungs, 16 hours through non-splanchnic systemic Although some tissue damage is caused by pas-
organs and 6.5 hours through splanchnic organs sage of larvae through lungs of infected mice, con-
[8]. During this period there is reasonable agree- siderably more damage in the form of haemorrhag-
ment between total parasite numbers recorded in ic loci is evident around larvae killed in the lungs by
different systemic organs and published values for the schistosomicidal drug praziquantel [42]. That
the fractional distribution of cardiac blood output. proteases, perhaps those released from damaged
Schistosomula of S. mansoni are first found in parasites, are implicated in this is indicated by the
the lungs of laboratory rodents 3 days after in- inhibition of the haemorrhaging by administration
fection, and numbers rise to a peak on days 5 or 6 of aprotonin simultaneously with praziquantel.
[39]. Numbers of larvae recovered from the lungs
then decrease, but some can still be detected in this
tissue as late as 20 days after infection. After first The maturation of schistosome worms
arriving in the lungs the schistosomula undergo a
further physiological change which allows them Concomitant with the decline in numbers of schis-
great changes in extensibility, and a reduction in tosome larvae in the lungs of infected mice there is
 387

an increase in the number that can be recovered Protease inhibitors and schistosome w o r m survival
from the liver. The first ones are obtainable by
perfusion of the hepatic portal system on about day One feature that is remarkable about this parasite
7 after infection, and the numbers increase to reach is its capacity to survive for long periods in the
a plateau after about 3 weeks. The factors which intravascular milieu. In mice schistosomes survive
are responsible for entrapment of immature schis- for a year at least [52], and the average life-span for
tosome in the microvasculature of the liver are S. mansoni in humans is estimated at between 5 and
obscure: the proportion of parasites which survive 10 years [53], with cases of infections lasting for 3 or
into adulthood in the hepatic portal system (30- 4 decades having been recorded.
50% of the percutaneously-administered cerca- The mechanisms by which schistosomes evade
riae) could just be a stochastic reflection of the the immune response and prolong their survival
proportion of cardiac output which flows through have received a great deal of attention (see [54] for
that blood system. review). Antigenic disguise of schistosomes by ac-
At this stage of their life-cycle the worms short- quisition or mimicry of host molecules is a mecha-
en, lose motility, and begin to feed on blood and to nism for which considerable evidence existS, and
grow. As they grow they migrate upstream through the finding of alpha 2-macroglobulin (A2M) on the
the portal blood system where four to five weeks surface of adult worms was one of the earlier obser-
after infection males and females begin to pair up. vations of this genre [55], made before the pro-
Maturing schistosomes begin to feed on host eryth- tease-inhibitory function of this macroglobulin was
rocytes, and considerable effort has gone into the fully understood.
characterization of worm acidic proteinase(s) that The nature of the interaction between A2M and
were first described in 1959 [43] and which degrade the parasite surface deserves further investigation,
haemoglobin. particularly to characterize the nature of the para-
The nucleotide sequences of two molecules have site receptor for A2M, and to determine whether
recently been deduced, one having good consensus the inhibitor is taken up by the worm in native form
with human cathepsin B, and the other being that or after complexing with proteases; for example
of a putative schistosome 'haemoglobinase' that enzymes of the complement or blood coagulation
surprisingly has no obvious primary structure simi- cascades, (or does it form complexes with schisto-
larity with known protease families [44, 45]. In some proteases on the worm surface?) A2M can
vitro translation studies on the cathepsin B-like also interact with a wide variety of other molecules,
enzyme indicate that a 37kDa non-glycosylated and its immunomodulatory effects [56] in partic-
prepro-enzyme translation product can carry out ular may be of critical importance in the context of
its own processing to give a proteolytically active facilitating parasite survival.
28 kDa molecule [46]. The worm acid proteases are Contrapsin, a serine protease inhibitor (serpin)
highly immunogenic, and in purified form can in- of mice has also been found associated with adult S.
duce hypersensitivity reactions in infected patients mansoni worms [57]. It has been suggested that the
[47], and may be of use for diagnosis of schistoso- remarkably high rate of mutation in the active site
miasis [48]. regions of serpins is due to the selection pressure
Other adult worm proteases have been identified exerted by continuous exposure to proteases of
and partially characterized, including a gelatinase infectious agents [58], and further detailed study of
excreted by intact male worms [49], dipeptidylami- the nature of the interaction between the proteases
nopeptidases I and II [50], and leucine amino pepti- of the parasite and host protease inhibitors is war-
dases [51]. The specific role of these various en- ranted.
zymes in prolonging the intravascular survival of
schistosomes remains to be determined.
388

Transmission of schistosomiasis: extravasation and could serve physically to lodge the eggs in the me-
excretion of eggs senteric capillaries at sites where they are laid by
the female worms, and thus prevent their being
Egg production by female schistosome worms com- swept away by the blood stream. It has recently
mences in the mesenteric veins (S. mansoni and S. been found that platelet release products promote
japonicum) or the vesical plexus (S. haematobi- the adherence of S. mansoni eggs to cultured hu-
urn). The adult worm pairs are permanently in man umbilical vein endothelial cells in vitro (Ngai-
copula, the female being held in the gynecophoric za, Doenhoff & Jaffe, in preparation), and if this
canal, or schist, of the male, and they remain in- occurs also in vivo it may be another factor which
travascular. The eggs are laid intravascularly and it prevents dissipation of eggs away from the site of
is therefore not surprising that many are carried their initial deposition. The platelet-rich aggre-
away from the site of deposition by blood flow, to gates which form around eggs while they are still in
become lodged in the nearest capillary bed down- the mesenteric capillaries of mice may also in some
stream. A proportion are, however, extravasated way be wholly or partly responsible for actual ex-
and t~averse the intestine or bladder walls to be travasation of the egg [63, 64].
excreted with, respectively, faeces or urine. On Immunosuppressed infected mice fail to excrete
reaching water the eggs hatch into miracidia which schistosome eggs, and excretion rates can be re-
infect snails. stored by reconstitution of such animals with anti-
The excretion of viable eggs from the definitive bodies [71, 72] or immune lymphocytes [64, 65]. It
host is critical to the continuation of the schisto- is not yet clear how or where (intra- or extra-vascu-
some life-cycle. Proteolytic enzymes, together with larly, or both?) acquired immunological effectors
muscular contractions of host tissues, have tradi- mediate their effects on egg excretion. There is a
tionally been considered important mediators in numerical correlation between the number of eggs
the egg excretion process, and collagenolytic [59], excreted and the degree of immunological inflam-
thiol proteinase [60] and leucine aminopeptidolytic matory reactivity generated against tissue-bound
activities [51, 61] have been found in eggs or egg eggs in individual animals [64, 65], and Damian
extracts. The role of most of these enzymes in [66] suggested that the immune granulomas with
parasite survival has not been established, though which eggs in host tissue become surrounded are
the leucine aminopeptidase has been implicated in responsible for 'translocation' of eggs through tis-
the hatching of eggs by virtue of its activity being sue. The exact means by which endothelial integri-
inhibited by high salt concentrations [62]. ty is ruptured and the eggs are transported to the
lumen of the intestine (or of the bladder in the case
of S. haematobium) thus still need clarification.
Platelets and immune effector mechanisms in egg
excretion
Fibrino(geno)lytic schistosome egg proteases
Schistosome egg excretion has latterly been shown
to be additionally dependent on blood platelet ac- The action of one or more egg proteases may be
tivity [63], and on the immune response of the host involved in extravasation of eggs, and the protec-
[64-66]. The connection with tumour metastasis is tion they would require against inactivation by cir-
clear, since both platelets and immune effector culating protease inhibitors may be provided by
mechanisms have also from time to time been im- close aposition between host cells and the egg sur-
plicated in the extravasation of metastatic cells [67- face [73]. The potent platelet aggregating activity
70]. of S. mansoni eggs seems at variance with the ab-
S. mansoni eggs have a potent platelet aggregat- sence of overt thrombotic activity during the course
ing effect both in vitro and in vivo [63]: this reaction of schistosome infections, and one explanation for
 389

Fig. 3. Hydrolysis offibrinogen by S. raansoni eggproteases. Panel A: In an adaptation of the method described in reference 77, 10/~1of
soluble extract of S. mansoni eggs, prepared as in reference 71, and containing 120/xg of protein (BSA equivalent) was electrophoresed
under non-reducing conditions in 12.5% SDS-PAGE in which 5 mg human fibrinogen (Kabivitrum, Sweden) had been incorporated per
5 ml gel. After electrophoresis the gel was washed overnight at 37° with PBS, pH 7.6, containing 2.5% Triton X-100 in order to remove
SDS. Proteolytic activity is detectable as clear bands after staining of unhydrolyzed protein in the gel with Coomassie blue.
Panel B: Equal volumes of human fibrinogen (10 mg/ml) and soluble extract of S. mansoni eggs (12 mg/ml) were incubated at 37° in
PBS at pH 7.6. At various intervals 20 ~1 of the incubation mixture were withdrawn and the reaction in these aliquots was terminated by
heat inactivation. The samples were electrophoresed in 12.5% SDS-PAGE under reducing conditions, electrobiotted onto nitrocellu-
lose paper, and probed with a rabbit intiserum monospecific for human fibrinogen. The control lane was from a 16 hour incubate of
fibrinogen without egg homogenate.
Panel C: Fibrinogen solution (10 mg/ml) was incubated with an equal volume of human plasmin (2.5 U/ml) under the same conditions
as for incubation with egg homogenate.

this could be that the thrombogenic potential of the underwent a similar pattern of degradation (Fig. 3,
egg surface is counterbalanced by secretion of fibri- panel C). Enzymes in S. mansoni eggs act similarly
nolytic enzymes. against fibrin as against fibrinogen (Curtis, unpub-
Figure 3, panel A illustrates the presence of en- lished), but it has not yet been established that it is
zymes in S. mansoni eggs which can degrade fibri- the same enzyme which is active against both sub-
nogen in sodium dodecyl sulphate polyacrylamide strates.
gel electrophoresis (SDS-PAGE), the Mr range of The function of these proteases could be to con-
these proteases being between 22 kDa and 50 kDa. trol the level of fibrin deposition around parasite
Figure 3, panel B, shows that during an incubation eggs while they are intravascular. Comparisons be-
time course the S. mansoni egg enzymes degraded tween the respective fibrinolytic activities of S.
fibrinogen into products that, under reducing con- mansoni eggs and plasmin indicated that 1 mg of
ditions in SDS-PAGE, were initially of apparently crude soluble homogenate of freshly isolated eggs
greater size than the native protein. After incuba- contains approximately the equivalent of 20 units
tion for an hour or longer these large derivatives of plasmin fibrinolytic activity (Modha, unpublish-
disappeared and products of smaller size were gen- ed). An active fibrinolytic system such as this might
erated. Fibrinogen incubated with human plasmin
390

also be involved in egg migration through tissue, as reference to parasites and cancer. J Parasitol [66]: 705-721,
1980.
has been suggested for tumour cells [74].
2. Ashall F: Cancer cells and parasites: two of a kind. Tr
Biochem Sci [I1]: 518-520, 1986.
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Conclusions parasite relationships: a successfully transplanted concept
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Acknowledgements
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