Blood pressure modulation following activation of mast cells by cationic cell penetrating peptides.
ABSTRACT Short cell penetrating peptides (CPP) are widely used in vitro to transduce agents into cells. But their systemic effect has not been yet studied in detail. We studied the systemic effect of the cell penetrating peptides, penetratin, transportan and pro-rich, on rat hemodynamic functions. Intra-arterial monitoring of blood pressure showed that injection of the positively charged penetratin and transportan in a wide range of concentrations (2.5-320 μg/kg) caused highly significant transient decrease in the systolic and diastolic blood pressure in a dose dependent manner (p<0.01). Pretreatment with histamine receptors blockers or with cromolyn, a mast cell stabilizing agent, significantly attenuated this effect. Furthermore, in vitro incubation of these both peptides with mast cells line, LAD2, caused a massive mast cell degranulation. In vitro studies showed that these CPP in a wide range of concentrations were not cytotoxic without any effect on the survival of LAD2 mast cell line. In contrast, the less positively charged and proline-rich CPP, pro-rich, had no systemic effects with no effect on mast cell degranulation. Our results indicate that intravenously administrated positively charged CPP may have deleterious consequences due to their induced BP drop, mediated by mast cell activation. Therefore, the major effect of mast cell activation on BP should be considered in developing possible future drug therapies based on the injection of membrane-permeable and positively charged CPP. Nevertheless, lower levels of such CPP may be considered as a treatment of systemic high BP through moderate systemic mast cell activation.
- SourceAvailable from: Raphael Gorodetsky[Show abstract] [Hide abstract]
ABSTRACT: We tested the ability of purified, ultraviolet C virally inactivated components of human fibrin sealant (FS) to modulate the chemotaxis, adherence, and proliferation of cultured cells. A fibrin clot formed on a near-confluent layer of human fibroblasts (HFs) recruited cells from the surrounding area. Thrombin (Thr) enhanced HF proliferation by a factor of 1.5 to 1.8, whereas fibrinogen (Fib) exerted only a minimal proliferative effect. We developed a new cell haptotactic/attachment assay by using Thr and Fib covalently bound to Sepharose beads (SBs). The kinetics of cell binding were approximately equivalent for beads coated with either protein. Uncoated SBs or fibrinogen-bound SBs (Fib-SB) pretreated with plasmin did not attract HFs. AlphaThr-SB induced a positive migratory response that was not affected by blocking its proteolytic site, whereas gammaThr-SB elicited no response. X irradiation of HFs at a dose of 6 Gy showed that the migratory response of HF is independent of proliferation, as confirmed by a bromodeoxyuridine uptake assay. Several types of cultured cells (murine fibroblasts, smooth muscle cells, aortic endothelial cells, and murine mammary carcinoma cells) also attached to Fib-SB. By contrast, human keratinocytes, human ovarian carcinoma cells, murine macrophage-like cells, leukemic cells, and murine mast cells did not attach. Our results provide some mechanistic insights into the haptotactic and proliferative effects of Fib and Thr on different cells.Journal of Laboratory and Clinical Medicine 04/1998; 131(3):269-80. · 2.62 Impact Factor
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ABSTRACT: Fibrinogen molecules are comprised of two sets of disulfide-bridged Aalpha-, Bbeta-, and gamma-chains. Each molecule contains two outer D domains connected to a central E domain by a coiled-coil segment. Fibrin is formed after thrombin cleavage of fibrinopeptide A (FPA) from fibrinogen Aalpha-chains, thus initiating fibrin polymerization. Double-stranded fibrils form through end-to-middle domain (D:E) associations, and concomitant lateral fibril associations and branching create a clot network. Fibrin assembly facilitates intermolecular antiparallel C-terminal alignment of gamma-chain pairs, which are then covalently 'cross-linked' by factor XIII ('plasma protransglutaminase') or XIIIa to form 'gamma-dimers'. In addition to its primary role of providing scaffolding for the intravascular thrombus and also accounting for important clot viscoelastic properties, fibrin(ogen) participates in other biologic functions involving unique binding sites, some of which become exposed as a consequence of fibrin formation. This review provides details about fibrinogen and fibrin structure, and correlates this information with biological functions that include: (i) suppression of plasma factor XIII-mediated cross-linking activity in blood by binding the factor XIII A2B2 complex. (ii) Non-substrate thrombin binding to fibrin, termed antithrombin I (AT-I), which down-regulates thrombin generation in clotting blood. (iii) Tissue-type plasminogen activator (tPA)-stimulated plasminogen activation by fibrin that results from formation of a ternary tPA-plasminogen-fibrin complex. Binding of inhibitors such as alpha2-antiplasmin, plasminogen activator inhibitor-2, lipoprotein(a), or histidine-rich glycoprotein, impairs plasminogen activation. (iv) Enhanced interactions with the extracellular matrix by binding of fibronectin to fibrin(ogen). (v) Molecular and cellular interactions of fibrin beta15-42. This sequence binds to heparin and mediates platelet and endothelial cell spreading, fibroblast proliferation, and capillary tube formation. Interactions between beta15-42 and vascular endothelial (VE)-cadherin, an endothelial cell receptor, also promote capillary tube formation and angiogenesis. These activities are enhanced by binding of growth factors like fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF), and cytokines like interleukin (IL)-1. (vi) Fibrinogen binding to the platelet alpha(IIb)beta3 receptor, which is important for incorporating platelets into a developing thrombus. (vii) Leukocyte binding to fibrin(ogen) via integrin alpha(M)beta2 (Mac-1), which is a high affinity receptor on stimulated monocytes and neutrophils.Journal of Thrombosis and Haemostasis 09/2005; 3(8):1894-904. · 5.55 Impact Factor
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ABSTRACT: Haptides are 19-21mer cell-binding peptides equivalent to sequences on the C-termini of fibrinogen beta chain (Cbeta), gamma chain (preCgamma) and the extended alphaE chain of fibrinogen (CalphaE). In solution, Haptides accumulated in cells by non-saturable kinetics [Exp. Cell Res. 287 (2003) 116]. This study describes Haptide interactions with liposomes and Haptide-mediated liposome uptake by cells. Haptides became incorporated into negatively charged liposomes, changing their zeta potential. Atomic force microscopy and particle sizing by light scattering showed that the liposomes dissolved Haptide nanoparticles and absorbed them from solution. Pre-mixing fluorescent rhodamine-containing liposomes or "stealth" doxorubicin (DOX)-containing liposomes (Doxil) with Cbeta, preCgamma or to a lesser degree CalphaE, significantly enhanced their uptake by fibroblasts and endothelial cells. Confocal microscopy showed Haptide-induced liposome uptake saturated above approximately 40 microM Haptide. Cytotoxicity tests with lower concentrations of Doxil liposomes indicated that premixing with approximately 40 microM Cbeta or preCgamma increased their toxicity by one order of magnitude. It was evident that the liposomes complexed with an amphiphilic Haptide are transduced through cell membranes, probably by a non-receptor-mediated process. These results suggest that Cbeta or pre-Cgamma could be employed to augment the cellular uptake of drugs in liposomal formulations.Journal of Controlled Release 04/2004; 95(3):477-88. · 7.26 Impact Factor
Mast cell activation by fibrinogen-related homologous
c-terminal peptides (haptides) modulates systemic blood
Maamoun Basheer, MSc,aHerzl Schwalb, PhD,bMaoz Nesher, MSc,cDan Gilon, MD,dIrit Shefler, PhD,f
Yoseph A. Mekori, MD,fOz M. Shapira, MD,b,eand Raphael Gorodetsky, PhDa
Jerusalem and Kfar Saba, Israel
Background: Haptides are a family of short peptides
homologous to C-termini sequences of fibrinogen chains b and g
(haptides Cb and preCg, respectively) which were previously
shown to penetrate and bind cells.
Objectives: This work investigates the systemic effect of the
haptides with possible clinical implications.
Methods: Intra-arterial monitoring in rats recorded the
haptides’ effects on systemic blood pressure. In parallel, their
effect was also tested in vitro on isolated rat peritoneal mast cells
and on human mast cells.
Results: Intra-arterial monitoring in rats showed that
intravenous administration of low haptides concentrations
(35-560 mg/kg rat) caused a shocklike behavior with transient
decrease in the systolic and diastolic blood pressure by up to
55% (P < .05) in a dose-dependent manner and a minor
increase in their heart rate. Randomly scrambled sequences of
the haptides had no such effect, suggesting a specific
interaction with receptors. Intravenous administration of
blockers to histamine receptors H1 and H2 before haptides
administration attenuated this effect. Furthermore, in vitro
incubation of human LAD2 mast cell line or isolated rat
peritoneal mast cells with the haptides caused degranulation of
the mast cells. We found that the haptides Cb and preCg
activated mast cells causing histamine release, resulting in a
steep decrease in blood pressure, comparable to anaphylactic
Conclusion: In treating vascular occlusive diseases, massive
fibrinolysis is induced, and haptide-containing sequences are
released. We suggest that treatment with histamine receptor
blockers or with mast cell stabilizing agents in such pathological
conditions may overcome this effect. (J Allergy Clin Immunol
Key words: Haptides, fibrin, mast cells, hypotension, histamine,
Fibrinogen molecules and fibrin gels were found to be highly
cell-binding.1Part of the mediation of cell binding was recently
attributed to small conserved family of short homologous
(;20mer) sequences on the c-termini of b chain and just preced-
ing the c-termini of the g chain (Cb and preCg, respectively).2
Another partially homologous sequence is present on the ex-
tended a-chain aE, which is seen only in small fraction of fibrin-
ogen molecules in adult mammals because of posttranslational
modification.2-4We termed this family of homologous peptides
haptides because of their haptotactic (cell-binding) properties.
Haptides also showed high penetration properties into cultured
cells,2,5probably mediated by their structure and their relatively
It is of major interest that all the haptides in a wide range of con-
centrations tested had no apparent cytotoxic effect invitro on dif-
ferent cell types, including normal fibroblasts and endothelial
cells.2When bound to collagen matrix implants in rats, haptides
increased cell binding with no adverse effects.6Haptides’ effect
was also tested ex vivo on isolated perfused beating hearts, in
which high levels of haptides caused significant vasoconstriction
of the coronaries, resulting in impaired heart functions.7
Nevertheless, the systemic effect of the haptides has not been
The possible effect of haptides administered into the circula-
tion is highly relevant to clinical practice. Fibrinolysis is a
prominent treatment for salvaging ischemic organs caused by
obstructive thrombotic events, resulting in a release of peptides
containing haptidic sequences.8,9The effectiveness in reducing
morbidity in acute myocardial infarction is dependent on early
thrombolytic therapy.10-12Streptokinase is widely used to initiate
this cascade by inducing the activation of the fibrinolytic enzyme
plasmin, which degrades fibrin clots at specific sites, yielding
identifiable soluble fragments that are released to the circula-
circulating fragments, was observed in numerous pathological
conditions14-17and could serve as a marker of many vascular
Low-molecular-weight fibrinogen degradation products derived
fibrin degradation products mediate platelet activation,19,20fibro-
blast proliferation,21endothelial cells spreading, proliferation and
capillary tube formation,20and promotion of angiogenesis.22,23
The current study examines whether haptides can contribute to
the systemic side effects that are observed after massive fibrinol-
ysis. Our findings in the current report suggest that haptides
exhibit hemodynamic effects with significant induction of
Cardiothoracic Surgery, and the Departments ofcPhysiology,dCardiology, and
eCardiothoracic Surgery, Hadassah-Hebrew University Medical Center, Jerusa-
Center, Kfar Saba.
ited P/S, New Orleans, La (Joseph Lunenfeld Cardiac Surgery Center).
Disclosure of potential conflict of interest: The authors have declared that they have no
conflict of interest.
Received for publication December 14, 2009; revised July 18, 2010; accepted for publi-
cation July 21, 2010.
Available online September 15, 2010.
Reprint requests: Raphael Gorodetsky, PhD, Head, Biotechnology and Radiobiology
Laboratory, Sharett Institute of Oncology Hadassah-Hebrew University Medical
Center, POB 12000, Jerusalem 91120, Israel. E-mail: firstname.lastname@example.org.
? 2010 American Academy of Allergy, Asthma & Immunology
athe Laboratory of Biotechnology and Radiobiology, Sharett Institute of
bthe Cardiothoracic and Thoracic Research Center, Department of
fthe Laboratory of Allergy and Clinical Immunology, Meir Medical
BP: Blood pressure
Cb: Haptides, homologous to sequences on the
C-termini of fibrinogen beta chains
DBP: Diastolic blood pressure
HR: Heart rate
LAD2: Human mast cell line
MAP: Mean arterial pressure
preCg: Haptides, homologous to sequences on the
C-termini of gamma chains
SBP: Systolic blood pressure
hypotension. Furthermore, we propose that the mechanism of
such effect may be associated with histamine and serotonin
secretion as a result of systemic activation of mast cells.
(KTRWYSMKKTTMKIIPFNRL) correspond to addresses 471-491 of the C-
the g chain, respectively. The control peptide Ca (SEADHEGTHSTKR-
GHAKSRP) corresponds to a sequence close to the C-terminal of fibrinogen
a chain. The arbitrary scrambled sequences of Cb and preCg were
ISSWYKFPMQGQKRPRMSFKM and FIKLMNPWSTRYIKMRTKTK, re-
spectively. The peptides were synthesized by GeneScript (Piscataway, NJ).
The experiments used peptides that were >90% pure as determined by HPLC
and mass spectrometry.
Human fibrinogen as a component of a tissue sealant was obtained
nadine, famotidine, pyrilamine, cimetidine, cromolyn A, and p-nitrophenyl-
N-acetyl-b-D-glucosaminide were supplied by Sigma (Rehovot, Israel).
Male Sprague-Dawley rats (275-300 g; Harlan Laboratories, Rehovot,
Israel) were grown in specific pathogen-free conditions. All animals received
Animals (http://www.nap.edu/catalog/5140.html). The experiments were ap-
proved by the Institutional Animal Care and Utilization Committee.
Blood pressure monitoring
Male Sprague-Dawley rats (250-300 g; Harlan Laboratories) were kept in
specific pathogen-free conditions in accordance with the Guide for the Care
and Use of Laboratory Animals (NIH pub no. 86-23, revised 1996). Experi-
ments were approved by the Institutional Animal Care and Utilization Com-
mittee (Application MD-103.05-1).
on a heated pad. Rectal temperature was adjusted to 37.5 to 388C by a rectal
probe. After tracheotomy and insertion of a PE260 catheter, the right femoral
artery and vein were cannulated with a PE50 heparinized tube. Blood pressure
ducer (MEMSCAP, Skoppum, Norway) coupled to an amplifier (Laboratory
in parallel. Mean arterial blood pressure (MAP) was calculated as the sum of
two thirds diastolic blood pressure and one third systolic pressure (mmHg).
After 20 minutes of equilibration, peptides dissolved in saline were
administered by a bolus injection through the femoral vein cannula in a final
minutes, a higher dose of the peptide was injected, and the hemodynamic
parameters were measured for additional 15 minutes. The maximal effect of
the haptides was recorded after 3 minutes.
In the histaminergic blockade experiment, rats were intravenously admin-
istrated with the histamine antagonists cimetidine (15 mg/kg) and pyrilamine
haptide injection. Cromolyn A (intravenous, 1 mg/kg/min) was injected for 5
minutes before haptide injection. Administration of the saline and antagonist
H1 and H2 alone had no significant effect on the BP.
Isolation and purification of rat peritoneal mast
Rat peritoneal mast cells were obtained from Sprague-Dawley rats by a
peritoneal lavage, and the cells were purified as previously described.24
Briefly, a suspension of washed peritoneal cells was layered over a cushion
of 30% Ficoll 400 (Pharmacia Biotech, Uppsala, Sweden) in buffered saline
supplemented with 0.1% BSA and centrifuged at 1500g for 15 minutes. The
by toluidine blue staining.
Purified mast cells (5 3 105cells/mL in a final volume of 0.5 mL) were in-
cubated in Tyrode buffer (137 mM NaCl, 2.7 mM KCl, 20 mM HEPES, pH
7.4, 1 mM CaCl2, 5.6 mM glucose, 1 mg/mL BSA) and were stimulated by
the activating agent for 20 minutes. Reactions were terminated by placing
the tubes on ice, followed by a brief centrifugation by microfuge (12,000
rpm for 20 seconds) at 48C. Aliquots of 50 mL were collected from the super-
natant for determination of histamine levels.
The amount of histamine released was determined by using the O-phthal-
dialdehyde fluorometric method, as previously described.24,25The results are
presented as final concentration of histamine above the baseline values.
Histamine released after haptides administration
Blood samples were collected after intravenous administration of haptides
(preCg or Cb) or the control peptide Ca at a concentration of 280 mg/kg
animal. The blood samples were centrifuged for 10 minutes at 3000 rpm.
Aliquots of 50 mL plasma were used for the determination of histamine, as
previously described.24,25Results are presented as net concentration of hista-
mine released. The baseline histamine and negligible readings of haptides
alone in the system were deducted from the relevant readings.
Human-derived LAD2 mast cells line
The LAD2 human mast cell line was maintained and used as previously
described.26The LAD2 human mast cells (106/mL) were incubated with the
Cb and preCg haptides. A dose response for both haptides activity was per-
formed for 2 minutes incubation for doses of 0.1 to 100 mg/mL. Incubation
with the peptides Cb or preCg as well as with the control Ca at concentration
of 50 or 100 mg/mL. Mast cell degranulation was measured by b-hexosamin-
idase (b-hex) release as previously described.27,28Briefly, 20 mL sample
supernatants were mixed with 50 mL substrate solution (8 mmol/L p-nitro-
phenyl-N-acetyl-b-D-glucosaminide in 48 mmol/L citric acid and 56 mmol/
L Na2HPO4,pH 4.5), and incubated for 90 minutes at 378C. The reaction
was stopped by addition of 150 mL ice-cold glycine (0.2 mol/L, pH 10.7),
and the optical density measurement was immediately performed by using a
spectrophotometer (OD 405 nm). Results were expressed as percentage of to-
tal b-hex activity present in the cells as previously described.26,29Total activ-
ity was the one measured in cell lysates plus supernatant. Haptides alone had
no effect on the readings. For IgE-mediated activation, cells were sensitized
overnight with 100 ng/mL human myeloma IgE-biotin (Calbiochem, Merck
KGaA, Darmstadt, Germany), then stimulated with 100 ng/mL streptavidin
(Jackson ImmunoResearch Laboratories, West Grove, Pa) as previously
All values are expressed as means 6 SEs. One-way ANOVA and a 2-tailed
Student t test were used for comparing between relevant groups. Statistical
differences of P <.05 were considered significant.
J ALLERGY CLIN IMMUNOL
1042 BASHEER ET AL
Effect of haptides on the rat hemodynamic
Intravenous injection of a 1.5 mg/kg bolus of both fibrin-related
the animals with a shocklike behavior. In sedated rats, intravenous
administration through the femoral vein resulted in significant
reduction of blood pressure as monitored by intra-arterial monitor
(Fig 1). The maximal effect was observed within 3 minutes after
pulse administration of the haptides. Full recovery was recorded
effect on arterial blood pressure, made up of an initial intense but
that persisted until the full recovery in about 15 minutes (Fig 1, A).
The administration of haptides (preCg and Cb) within the
concentration range of 35 to 560 mg/kg produced a dose-
dependent decrease of the systolic BP (SBP), diastolic BP
(DBP), and MAP (Fig 1, B-D). Maximal MAP decrease of as
much as 50% (P < .01) was recorded at a higher dose of
280 mg/kg (Fig 1, D). In addition, the HR minimally increased
in correlation with doses of haptides given, reaching a significant
elevation at 560 mg/mL (P <.05; Fig 1, E). However, the control
peptide Ca at the same concentration did not affect both BP and
HR (Fig 1, B-E).
Effect of scrambled haptides sequences and whole
fibrinogen on rat blood pressure
To study the specific effect of haptides sequences that
modulate BP, scrambled sequences of haptides were tested. In
parallel, the effect of the whole intact fibrinogen molecule was
also studied. Administration of 280 mg/kg scrambled sequences
of the haptides, preCg and Cb, or fibrinogen did not affect the
FIG 1. Effect of escalating doses of intravenous (IV) administration of haptides on blood-pressure and heart
rate in the rat. A, An arterial pressure records at baseline and 6 minutes after intravenous injection of 1.5 mg/
kg Cb showing a triphasic response effect on blood pressure (lines 1-3). Haptides effect (35-560 mg/kg) on
diastolic, systolic, and mean arterial BP as well as heart rate were tested (n 5 6) (B-E, respectively). Results
were compared with the control peptide Ca (*P < .05; **P < .01).
FIG 2. Effect of haptides versus scrambled haptides sequences on DBP,
SBP, and MAP.Rats were injected with 280 mg/kg of the haptides Cb, preCg
or their scrambled versions as well as with whole human fibrinogen (n 5 6).
The results (mean 6 SE) are presented as percentages of the baseline
values. The effects are compared with the baseline values (P < .01).
J ALLERGY CLIN IMMUNOL
VOLUME 126, NUMBER 5
BASHEER ET AL 1043
SBP, DBP, MAP, or HR (Fig 2). In contrast, haptides preCg and
Cb at a concentration of 280 mg/kg decreased SBP, DBP, and
MAP by 50% of baseline blood pressure values (P < .01).
Effect of the mast cell secretagogue compound 48/
80 and serotonin on BP
The reduction of BP by haptides is similar to the effect
observed after repeated injections of the mast cell secretagogue
compound 48/80 (Fig 3, A). This suggests that haptides-mediated
hypotension could be generated by mast cell activation and hista-
Intravenous bolus injection of compound 48/80 (35-280 mg/
kg) in rats resulted in a dose-dependent decrease in SBP, DBP,
and MAP (Fig 3, B). Serotonin injection in a range of 3 to
30 mg/kg also resulted in a dose-dependent decrease in the
BP (Fig 3, C).
Histaminergic blockade attenuates haptide
To verify that haptides-induced hypotension is associated with
mast cell activation, the rats were pretreated with histamine
rats with cimetidine, a histamine receptor type 2 antagonist, and
pyrilamine, a histamine receptor type 1 antagonist (Fig 4, A),
resulted in a significant attenuation of haptides-induced decrease
of the BP (P < .05). Infusion of the mast cell stabilizing agent
cromolyn A 5 minutes before haptide injection hindered the
haptide-mediated hypotension effect (Fig 4, A). The effect was
most pronounced for preCg (P <.05). Cromolyn alone did not af-
fect any of the parameters tested. An example of a chart of the re-
corded BP drop caused by intravenous administration of haptide
and modulation with pretreatment with cromolyn is presented in
Fig 4, B. The red arrows indicate the amplitude and time interval
FIG 3. Compound 48/80–mast cell activator and serotonin decreases rat BP in the same manner as haptides.
A, Trace record of arterial pressure at baseline and following intravenous (IV) injection in rat of 2 mg/kg
compound 48/80. Hemodynamic parameters in response to escalating doses of compound 48/80 and sero-
tonin are summarized in B and C, respectively (n 5 6). Results presented as percentages of the baseline
(mean 6 SE). The effects are compared with the baseline values (*P < .05; **P < .01).
FIG 4. Histaminergic blockade attenuates haptides’ effect on rat blood pressure. A, Effect of haptides (280
mg/kg) on hemodynamic parameters in rats pretreated with cromolyn or with pyrilamine and cimetidine. B,
A chart of blood pressure drop caused by intravenous haptide administration and pretreatment with cromo-
lyn. C, Effects of haptides on BP in rats pretreated with fexofenadine or famotidine. The results (mean 6 SE)
are presented as percentages of the baseline (n 5 6; *P < .05).
J ALLERGY CLIN IMMUNOL
1044 BASHEER ET AL
The samearrowsaresuperimposedontheresponserecorded with
of the reduction of the haptide effect. The newly introduced
antihistamine agent fexofenadine showed a significant inhibition
(P <.05) of haptides-induced decrease of BP (Fig 4, C). These re-
sults clearly demonstrate that mast cell stabilizers and histamine
antagonists significantly attenuated the haptides-induced hypo-
tensive response. This further indicates that the systemic effect
of the haptides, as reflected by a drop of BP, seems to be associ-
ated with regulation of histamine secretion by mast cells.
Haptides activate rat peritoneal mast cells
mast cells was tested. Thus, peritoneal mast cells were incubated
in the absence or presence of haptides (100 mg/mL), and
histamine content was measured in the supernatant. Trypan blue
exclusion assay showed that the haptides had no effect on cell
viability, which was found to be greater than 98% in all
Incubation of rat peritoneal mast cells with haptides, preCg,
and Cb for 30 minutes resulted in a significant histamine release
(Fig 5, A) relative to the group that was treated with the Ca inac-
tive control peptide (P <.05). Furthermore, intravenous haptides
administration induced a significant increase in plasma histamine
level (Fig 5, B) relative to the control group (P <.05).
Haptides activate cultured human mast cells
tested by b-hex release. A dose response of the haptides’ effect
after 2 minutes of exposure is shown in Fig 6, A. The effect was
tested after incubation for 2 or 30 minutes in the presence of 2
doses of the haptides (50 and 100 mg/mL), as seen in Fig 6, B
and C. In both concentrations, the haptides induced a highly sig-
nificant b-hex release (Fig 6, B and C; P <.05) not dependent on
the time of incubation. In the same conditions and experimental
system, activation of the LAD2 human mast cells for 2 and 30
minutes via the FceRI (biotinylated IgE plus streptavidin)
resulted in only 5% and 15% b-hex release, respectively (data
not shown). The effect of the haptides on b-hex release was
also evident within the very short time of 2 minutes after the ad-
dition of the haptides, which coincides with the time scale of the
systemic effect by in vivo administration. The control unrelated
peptide Ca had no such effect (Fig 6, B and C).
Effect of serotonin receptor antagonist on the
vasopressor phase mediated by haptides
Both the injection of serotonin and high-dose haptides induced
a triphasic effect on arterial BP made up of an initial intense but
brief vasodepressor response followed by a moderatevasopressor
response (Fig 1, A). Finally, a longer-lasting vasodepressor re-
sponsewas recorded. Thevasopressor phasevaried quantitatively
depending on the species and the experimental conditions.31-34
by ketanserin, a serotonin receptor type 2 (5-HT2) antagonist
(Fig 7, A-D).
We have previously identified and characterized the haptides, a
family of cell-binding and penetrating fibrin-related peptides.2,5
The importance of the current study is in demonstrating that the
venous administration of relatively low doses (35-560 mg/kg) of
haptides Cb and preCg induced significant hypotension (Fig 1).
injection of the haptides. Higher doses of haptides (1.5 mg/rat)
resulted in triphasic effect on the arterial BP,made up of an initial
intense brief vasodepressor response followed by a moderate
vasopressor response. Later, a longer-lasting vasodepressor re-
occlusive arterial diseases, where vast amounts of fragments of
fibrin withhaptides sequences arereleased intothe circulation.8,9
result of the activation of mast cells, followed by histamine and
serotonin release, as observed during anaphylaxis.31This is fur-
ther supported by the direct evidence whereby intravenous hapti-
des injection indeed caused a significant histamine release (Fig 5,
A). Asimilar effect on BP was also observed, with similar timing,
after the injections of compound 48/80 (Fig 3), a known mast cell
secretagogue, or serotonin, an active mediator and histamine an-
alog in rats that is stored in mast cell granules.32-34In parallel, the
effect of haptides on blood pressure was suppressed by histamine
receptor type 1 and 2 antagonists or cromolyn A, a mast cell–
significantly suppressed the effect of haptides on blood pressure
more than famotidine. Although famotidine is a histamine
H2-receptor antagonist that inhibits H-pump and is used in the
treatment of peptic ulcer disease and gastroesophageal reflux,35
it is only marginally involved in regulating blood pressure
compared with histamine H1 receptors that are blocked more ef-
ficiently by fexofenadine.36Therefore, it is not surprising that fa-
motidine did not mask the effect of haptides as much as recorded
The time interval of haptides-induced in vivo maximal effect
was short, within ;3 minutes after their administration. To
FIG 5. Haptides activate rat mast cells. Effect of haptides on histamine
release in isolated rat peritoneal mast cells. The mast cells (2 3105cells/mL)
were preincubated for 30 minutes with 100 mg/mL haptides (A). Plasma his-
tamine level in rats pretreated with 280 mg/kg preCg, Cb, or the control (Ca;
n 5 5; B). Results are presented as means 6 SEs. The effects are compared
with the control peptide Ca values (*P < .01).
J ALLERGY CLIN IMMUNOL
VOLUME 126, NUMBER 5
BASHEER ET AL 1045
investigate whether this timing is sufficient for an induced effect
caused by mast cells activation, we tested the timing for a direct
effect of haptides on cultured mast cells. Both haptides preCg
and Cb induced significant degranulation of the LAD2 cells
(Fig 6) within a short time of 2 minutes. This indeed conforms
to the timing of the onset of the effect observed in the whole an-
imal (Fig 1).
The vasopressor response to serotonin can be blocked by
5-HT2 receptor antagonists such as ketanserin.37,38Accordingly,
are generally mediated by 5-HT2 receptors.33,37-39We found that
the vasopressor response to haptides was blocked by 5-HT2
receptor antagonist ketanserin (Fig 7), indicating that haptides
affect on cardiovascular function is associated with activation
of the serotonin receptors.
It is expected that in treating certain pathological conditions
such as occlusive blood vessels disease, massive degradation of
fibrin and fibrinogen is induced. In such conditions, peptides
FIG 7. Effect of intravenous administration of ketanserin, 5-HT2 receptor antagonists, on BP variability in
rats. Trace records of arterial BP at baseline and 6 minutes after injection into the femoral veins of 2 mg/kg
haptides Cb (A) or preCg (C). Tracings of arterial BP records where both haptides were injected 10 minutes
after a singledose of intravenous ketanserin (1mg/kg) administrationare presented in B and D, respectively.
FIG 6. Effect of haptides on degranulation of cultured LAD2 cells. Haptides effects on LAD2 cells activation
as monitored by b-hex secretion. A, Dose response of 2-minute incubation with haptides. The cells were
preincubated for 2 minutes (B) or 30minutes (C) with 50 or 100mg/mLwith haptides preCg,Cb, or the control
Ca (n 5 4). Values are percentages of total b-hex release. The haptides effects are presented relative to Ca
(**P < .01).
J ALLERGY CLIN IMMUNOL
1046 BASHEER ET AL
containing haptides sequences are released into the circulation.
Each fibrinogen molecule contains 4 haptidic sequences.8,9In
cases with massive fibrinolysis, fibrin degradation products with
haptide containing sequences are equivalent to haptides levels
of ;1 to 5 mg/mL.40,41In our rat model, the effective levels of
haptides in the circulation that influenced BP were in the range
of 0.5 to 7 mg/mL. In the in vitro system with LAD2 cells
(Fig 6, A), the potent concentrations tested (<10 mg/mL) were
equivalent to those used in vivo (;0.5-7 mg/mL blood or
perimental rat model seem to be equivalent to the physiological
situation in cases associated with massive fibrinolysis.
It was previously demonstrated that low-molecular-weight
fibrinogen degradationproducts causeddose-dependentsecretion
of collagenase and histamine from mast cells.42Our study iden-
tifies the haptides as potential fibrin-related sequences that could
trigger such effects after fibrinolysis.
The damage to blood vessels caused by clot formation induced
endothelial-mediated mast cell attraction. The activated mast
cells were previously shown to release b-tryptase, a specific
enzyme of mast cells.43Tryptase, a very potent proteolytic en-
zyme, was shown further to enhance full degradation of fibrin,
as well as fibrinogen, to produce fibrinopeptides of smaller size
containing haptides sequences.44This positive feedback cycle
may increase the haptides release, which may further activate
the mast cells that in turn secrete more tryptase.
Therapeutic use of plasminogen activators remains controver-
sial because of the sometimes inefficient restoration of blood
Streptokinase-based treatment was found to be associated with a
risk of developing hypotension.12,45The mechanism responsible
for this streptokinase effect remains unknown.18,46,47We suggest
results in the release of smaller fragments containing the C-ter-
mini of fibrin with haptide sequences,8,9which, as shown here,
may trigger hypotension.
It is of interest whether the specific haptides effect may be
mediated by a receptor. The control peptide Ca, which is not re-
lated to the haptides, did not affect the BP (Fig 1). Even arbitrary
scrambled sequences of the haptides that have similar mean
hydrophobicity and positive charge had no such effect (Fig 2).
Furthermore, we found that haptides caused major mast cell acti-
vation in a system without IgE and added complement (Fig 6),
whereas IgE and the specific antigen caused merely 15% activa-
tion at 30 minutes.29These findings suggest that the haptides ef-
fect is probably not equivalent to classical IgE mediated
activation. Their major effect may be mediated through a specific
receptor on the target cells that has not been identified yet.
Nevertheless, a direct effect, such as the one recorded by basic
secretagogues that were previously proposed as effectors of ana-
phylaxis, still cannot be ruled out.29,48
Integrin aIIbb3 is highly expressed in mouse, rat, and human
mast cells. In the presence of stem cell factor and FceRI, it was
reported to bind to fibrinogen and activate the mast cells.49We
found that the haptides alone, but not the whole fibrinogen, with
no addition of other factors, induced mast cells activation. There-
fore, other candidate targets may also be considered.
To conclude, haptides were shown to induce release of hista-
mine and serotonin from mast cells, which can explain their me-
diation of hypotension. This response can be abrogated by
histamine receptor antagonists and other anti-inflammatory
drugs. These findings may be relevant to the possible mode of ac-
tion of circulating fibrinogen fragments containing haptides as a
result of induced fibrinolysis in the management of vascular oc-
We thank Prof Levi-Schaffer (Department of Pharmacology and Experi-
mental Therapeutics, School of Pharmacy, Faculty of Medicine, Hebrew
University of Jerusalem) for her assistance in assessing rat-derived mast cells
and Esther Houminer (Cardiac Surgery Research Center, Hadassah-Hebrew
Levdansky and Elena Gaberman for their general laboratory help on this
Clinical implications: Haptides, short peptides homologous to
sequences on the C-termini of fibrinogen, induce hypotension
by mast cell activation; thus, they are presented as possible
key mediators of hypotension after streptokinase-induced
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