The Kunitz-Like Modulatory Protein Haemangin Is Vital
for Hard Tick Blood-Feeding Success
M. Khyrul Islam1, Naotoshi Tsuji1*, Takeharu Miyoshi1, M. Abdul Alim1, Xiaohong Huang1, Takeshi
Hatta1, Kozo Fujisaki2
1Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural and Food Research Organization, Tsukuba, Ibaraki, Japan, 2Department of
Emerging Infectious Diseases, School of Veterinary Medicine, University of Kagoshima, Kagoshima, Japan
Ticks are serious haematophagus arthropod pests and are only second to mosquitoes as vectors of diseases of humans and
animals. The salivary glands of the slower feeding hard ticks such as Haemaphysalis longicornis are a rich source of bioactive
molecules and are critical to their biologic success, yet distinct molecules that help prolong parasitism on robust
mammalian hosts and achieve blood-meals remain unidentified. Here, we report on the molecular and biochemical features
and precise functions of a novel Kunitz inhibitor from H. longicornis salivary glands, termed Haemangin, in the modulation
of angiogenesis and in persistent blood-feeding. Haemangin was shown to disrupt angiogenesis and wound healing via
inhibition of vascular endothelial cell proliferation and induction of apoptosis. Further, this compound potently inactivated
trypsin, chymotrypsin, and plasmin, indicating its antiproteolytic potential on angiogenic cascades. Analysis of Haemangin-
specific gene expression kinetics at different blood-feeding stages of adult ticks revealed a dramatic up-regulation prior to
complete feeding, which appears to be functionally linked to the acquisition of blood-meals. Notably, disruption of
Haemangin-specific mRNA by a reverse genetic tool significantly diminished engorgement of adult H. longicornis, while the
knock-down ticks failed to impair angiogenesis in vivo. To our knowledge, we have provided the first insights into
transcriptional responses of human microvascular endothelial cells to Haemangin. DNA microarray data revealed that
Haemangin altered the expression of 3,267 genes, including those of angiogenic significance, further substantiating the
antiangiogenic function of Haemangin. We establish the vital roles of Haemangin in the hard tick blood-feeding process.
Moreover, our results provide novel insights into the blood-feeding strategies that enable hard ticks to persistently feed and
ensure full blood-meals through the modulation of angiogenesis and wound healing processes.
Citation: Islam MK, Tsuji N, Miyoshi T, Alim MA, Huang X, et al. (2009) The Kunitz-Like Modulatory Protein Haemangin Is Vital for Hard Tick Blood-Feeding
Success. PLoS Pathog 5(7): e1000497. doi:10.1371/journal.ppat.1000497
Editor: David S. Schneider, Stanford University, United States of America
Received March 13, 2009; Accepted June 3, 2009; Published July 10, 2009
Copyright: ? 2009 Islam et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Grant-in-aids from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and also by a grant from
the Program for Promotion of Basic Research Activities for Innovative Biosciences. The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Angiogenesis or neovascularization, the formation of new blood
vessels from pre-existing ones, is involved in a variety of
physiological processes, such as corpus luteum formation,
embryonic development, and wound healing [1,2]. Also, angio-
genesis plays a vital role in the development and progression of
various pathological conditions, including rheumatoid arthritis,
diabetic retinopathy, and tumor metastasis . The process of
angiogenesis involves activation and release of angiogenic factors,
release of proteolytic enzymes to degrade extracellular matrix
protein (ECM), migration and proliferation of endothelial cells,
and microvessel formation . Pericellular proteinases, comprised
of membrane-type matrix metalloproteinases (MT-MMPs), serine
proteinases (SPs), and membrane-bound aminopeptidases, are
known to play key roles in angiogenesis .
The proteinase inhibitors of SPs belonging to the Kunitz, Kazal,
a-macroglobulin, and serpin families play critical roles in
physiological and pathophysiological states such as coagulation,
intravascular fibrinolysis, wound healing, angiogenesis, and tumor
metastasis [6,7]. The best studied serine proteinase inhibitors
(SPIs) to date in angiogenesis, tumor invasion, and metastasis are
the plasminogen activator inhibitor-1 (PAI-1), PAI-2 , and
maspin . Kunitz-type SPIs such as tissue factor pathway
inhibitor (TFPI) inhibit the proliferation of basic fibroblast growth
factor (bFGF)-induced endothelial cells (ECs) in addition to their
inhibitory activity against tissue factor–mediated blood coagula-
tion cascade . Snake venoms contain many unique proteins,
including Kunitz-type SPIs . These Kunitz proteins are potent
inhibitors of trypsin, chymotrypsin, kallikrein, and plasmin;
however, little is known about their physiologic roles in
angiogenesis and angiogenesis-dependent diseases such as cancer.
Hard ticks are notorious ectoparasites that feed on blood for
quite a long period (e.g., 10 days or more) in contrast to fast-feeder
soft ticks, which usually feed for an hour . Tick feeding is a
complex process and involves severe tissue damage caused by the
probing actions of barbed mouthparts and release of salivary
secretions to the feeding lesions, leading to host’s haemostatic,
inflammatory, and immune responses. However, despite the host’s
armoury of rejection mechanisms, the ticks manage to remain
attached and achieve engorgement [12,13]. Increasing evidence
suggests that success in blood-feeding relies on a pharmacy of
PLoS Pathogens | www.plospathogens.org1July 2009 | Volume 5 | Issue 7 | e1000497
chemicals located in the salivary glands (SGs) . A recent study
has reported that the saliva of the hard tick Ixodes scapularis is a
negative modulator of angiogenesis and wound healing ,
though the specific molecular component(s) is yet to be identified.
Moreover, the implications of hard-tick-modulated angiogenesis
and wound healing in persistent blood-feeding remain unknown.
To this end, we have looked for a novel salivary-specific
molecule(s) of interest in the hard tick Haemaphysalis longicornis.
We have selected a full-length cDNA that shows moderate
sequence homologies with Kunitz-type proteinase inhibitors from
the SG-specific expressed sequence tag (EST) library of adult
female H. longicornis  for its functional analysis. Here, we show
that an Escherichia coli–expressed recombinant protein, herein
called Haemangin, inhibits trypsin, chymotrypsin, and plasmin.
Also, we provide novel evidence that this Haemangin-like
modulatory protein is the key to the modulation of angiogenesis
and angiogenesis-dependent wound healing during persistent
blood-feeding and is vital for hard ticks in achieving host blood-
Molecular Characterization of Haemangin
The composite full-length Haemangin cDNA sequence was 583
nucleotides long with a single open reading frame (ORF) of 363
bases. The ORF coded for a protein of 120 amino acids, including
a signal peptide of 19 residues (data not shown). The putative
mature protein has a molecular mass of 14,157 Da and an
isoelectric point (pI) of 10.65. The deduced protein has three
potential N-glycosylation sites. The unique primary structure of
the predicted protein consists of 10 cysteines forming 5 disulfide
bonds, a lysine- and histidine-rich carboxy terminus, and a single
Kunitz-like inhibitory domain that belongs to bovine pancreatic
trypsin inhibitor (BPTI)/Kunitz family of SPI, as detected with
Scan-Prosite program. A BLASTX analysis of the translated
product deduced from the ORF revealed that Haemangin shares
the greatest sequence identity (56%) with kalicludine 1, a K+
channel inhibitor that belongs to the BPTI/Kunitz family of SPI,
obtained from the toxin of Anemonia sulcata (accession number
AAB35413). Haemangin also shares 51% sequence identity with
venom trypsin inhibitor isolated from the venom of Naja naja
(P20229), 49% with isoinhibitor K, a trypsin-kallikrein inhibitor K
from Helix promatica (P00994), 48% with TFPI from Homo sapiens
(AA089075), and 47% with venom basic protease inhibitor I of
Vipera ammodytes ammodytes (P00992). A comparison of the deduced
amino acid sequence of Haemangin with those of venom basic
proteinase inhibitors belonging to the BPTI/Kunitz family of SPIs
is shown (Figure 1A). Alignment data revealed that out of 5
disulfide bonds, 3 were conserved. The predicted signal peptides,
however, were not conserved. Phylogenetic data revealed that
Haemangin within the BPTI/Kunitz family of SPIs forms a
separate single cluster and is distantly related to Kunitz-type
inhibitors of snake-venom origin (data not shown).
Haemangin-specific transcript profiling at different blood-
feeding stages was analyzed by quantitative RT-PCR. Data
revealed that the target transcript was up-regulated as blood-
feeding progressed. A dramatic increase of Haemangin expression
was observed at 96 h of blood-feeding prior to acquisition of a full
blood-meal, which then sharply declined to a minimal level (data
not shown), indicating that Haemangin plays a role in the
acquisition of blood-meals.
The endogenous expression of Haemangin in SGs of a partial-
fed adult H. longicornis was shown to be localized in the salivary
acini by immunofluorescence staining (Figure 1B, left panel), but
was not detected in the SGs treated with pre-immune mouse sera
(Figure 1B, right panel).
Inhibition of Vascular Sprouting in Chick Aorta
A chick aortic ring assay was employed to examine whether
Haemangin inhibited angiogenesis in vitro. The absence of
Haemangin (control) allowed the formation of dense vascular
sprouts in aortic ring explants, while its presence (,500 nM)
potently inhibited vascular sprouting in a dose-dependent manner
(Figure 2A). HlSGE (,500 mg/ml) also strongly inhibited vascular
sprouting in a dose-response manner. The IC50for Haemangin
and HlSGE was 100.81 nM and 129.63 mg/ml, respectively. A
dose-dependent inhibition curve of vascular sprouting is shown
(Figure 2B). We also observed that transfected E. coli lysate
(,100 ml/well) but not non-transfected E. coli lysate (,100 ml/
well) exerted an inhibitory activity (data not shown). Addition of
rTpx (500 nM) did not show any inhibitory effect on vascular
sprouting (Figure 2A). OmSGE (,500 mg/ml) completely failed to
inhibit vascular sprouting (Figure 2A).
Inhibition of Capillary Tube Formation
To further evaluate the potential of Haemangin in inhibiting
angiogenesis, we used human umbilical vein endothelial cells
(HUVECs) in a 96-well plate to examine Matrigel-induced
morphogenic differentiation of ECs into capillary-like tubes. Data
revealed that in the absence of Haemangin, HUVECs were
rapidly organized, forming a number of well-defined capillary-like
structures in Matrigel (Figure 3A). The presence of Haemangin
(,250 nM) strikingly inhibited tube formation. HlSGE (,200 mg/
ml) also inhibited tube formation in a dose-dependent manner.
Tube formation was also inhibited by transfected E. coli lysate
(,100 ml/well), but not by non-transfected E. coli lysate (,100 ml/
well) (data not shown) or by rTpx (250 nM). Of interest, OmSGE
(,200 mg/ml) did not inhibit tube formation. Dose-dependent
inhibition curves of tube formation are shown in Figure 3B. The
IC50for Haemangin and HlSGE was 55.82 nM and 20.13 mg/ml,
Ticks are notorious ectoparasites that exclusively feed on a
host’s blood for a period of 10 days or longer. Upon blood-
feeding, an adult female tick gains 100–200 times its body
weight compared to its pre-feeding stage. Despite the
host’s armoury of rejection mechanisms, ticks manage to
remain attached until a full blood-meal is ensured. The
molecular machineries that make the tick a success with its
feeding, however, remain unknown. We demonstrate that
the Kunitz-like protein Haemangin, identified from the
salivary glands of the tick Haemaphysalis longicornis, plays
vital roles in blood-feeding success. Using both cell- and
chick embryo–based bioassays, we have shown that
Haemangin efficiently disrupted angiogenesis and wound
healing processes, enabling ticks to remain attached and
allowing persistent feeding. Additionally, in a rabbit model,
we reveal that an elevated expression of Haemangin is
associated with the acquisition of full blood-meals.
Importantly, Haemangin-knockdown ticks fail to prevent
angiogenesis in the host’s tissues and consequently
achieve only a poor blood-meal as compared to normal
ticks. We conclude that Haemangin is vital for ticks’
survival and can be a novel therapeutic target against ticks
and tick-borne diseases, including tumor angiogenesis.
Haemangin Assists in Tick Feeding
PLoS Pathogens | www.plospathogens.org2July 2009 | Volume 5 | Issue 7 | e1000497
were subjected to SDS-PAGE. Plasmin degradation was examined
by Coomassie staining of the proteins. To further examine the
effect of Haemangin on fibrinolytic activity of plasmin, plasmin-
dependent fibrinolysis inhibition assays were performed at 25uC to
monitor changes in turbidity at 450 nm . Fibrin polymer was
prepared in a volume of 400 ml buffer and 1.5 mM plasmin (Sigma)
without or with Haemangin, or HlSGE was added to initiate
fibrinolysis. After the sample was incubated at 25uC for 12 h,
plasmin-dependent fibrinolysis was observed and turbidity as
function of plasmin was measured at an absorbance of 450 nm.
RNAi studies were carried out using dsRNA . The coding
sequenceofHaemanginwascloned intopBluescriptIISK+ plasmid
(Toyobo). The dsRNA complementary to the E. coli malE gene was
used as a negative control . cDNA corresponding to malE
mRNA was synthesized and was cloned into pBluescript II SK+
plasmid using the oligonucleotides 59-CCGCTCGAGCGGTTAT-
GAAAATAAAAACAGGTGCA-39 and 59-GAATTCGCTTG-
TCCTGGAACGCTTTGTC-39 as forward and reverse primers,
respectively. The inserted sequences of Haemangin and malE were
amplified by PCR using the oligonucleotide T7 (59-GTAATAC-
GACTCACTATAGGGC-39) and CM0422 primers (59-GCG-
to attach T7 promoter recognition sites at both ends. The PCR
products were purified using a gel extraction kit (Qiagen). dsRNA
complementary to the DNA insert was synthesized by in vitro
transcription using T7 RNA polymerase (Promega). One micro-
gram each of Haemangin and malE dsRNA in 0.5 ml of PBS
separately was injected into each unfed adult tick. Ticks were
allowed to rest for 24 h at 25uC prior to placement on the host .
Rabbit tissues were also processed for H&E and silver nitrate
RNA Extraction and RT-PCR Analysis
The total RNA from tick SGs was isolated using an RNAeasy
Mini Kit (Qiagen) and was submitted to reverse transcription (RT)
before PCR. cDNA was synthesized and was employed to perform
PCRs using either Haemangin-specific oligonucleotides (59-
CAGCAGCTATG-39) or oligonucleotides specific for b-actin.
Quantitative RT-PCR was done using LightCycler FastStart DNA
Master SYBR Green I (Roche) in a LightCycler 1.5 instrument
RNA Extraction and Microarray Analysis
To perform oligonucleotide microarray analysis, HUVECs were
cultured in the absence (control) or presence of Haemangin
(100 nM) for 48 h as described above. Approximately 26106cells
were harvested and the total RNA was extracted as described
above. The mRNAs were then prepared and analyzed by
hybridization to microarrays (Filgen Array Human35k, oligo
Data are reported as means6standard errors, where appropri-
ate. The statistical significance (p,0.05) was determined by
Student’s t test/Mann-Whitney’s U test.
Nucleotide Sequence Accession Number
The nucleotide sequence data reported in this paper will appear
in the DDBJ/EMBL/GENBANK nucleotide sequence databases
with the accession number AB434485.
was performed using total RNA extracted from HUVECs treated
with Haemangin for 48 h. The total numbers of genes filtered to
include only those with "2.0 fold up-regulated compared with
untreated control are categorized according to their molecular
functions and are indicated with their relative numbers.
Found at: doi:10.1371/journal.ppat.1000497.s001 (0.05 MB PDF)
Cluster of up-regulated genes. Microarray analysis
extracted from HUVECs treated with Haemangin for 48 h and
was subjected to microarray analysis. The "2.0 fold down-
regulated genes compared with untreated control are categorized
according to their molecular functions and are indicated with their
Found at: doi:10.1371/journal.ppat.1000497.s002 (0.03 MB PDF)
Cluster of down-regulated genes. Total RNA was
different biological processes. Transcripts were filtered to include
only those with "2.0 fold changes compared with untreated
Found at: doi:10.1371/journal.ppat.1000497.s003 (0.10 MB PDF)
Haemangin-induced up-regulated genes ordered into
into different biological processes. Transcripts were filtered to
include only those with "2.0 fold changes compared with
Found at: doi:10.1371/journal.ppat.1000497.s004 (0.08 MB PDF)
Haemangin-induced down-regulated genes ordered
We thank H. Shimada and M. Kobayashi for assistance with the
Conceived and designed the experiments: MKI NT KF. Performed the
experiments: MKI NT TM MAA XH TH. Analyzed the data: MKI NT
KF. Wrote the paper: MKI NT KF.
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