British Journal of Anaesthesia 1997; 78: 724730

Heterogeneous effects of protamine on human mast cells and basophils

V. PATELLA, A. CICCARELLI, B. LAMPARTER-SCHUMMERT, A. DE PAULIS, M. ADT AND
G. MARONE

Summary
To investigate the mechanisms of anaphylactoid
reactions to protamine, we have examined the
in vitro effects of increasing concentrations of
protamine (10963
1094 mol litre91) on the release
of preformed (histamine and tryptase) and de novo
synthesized (peptide leukotriene C4 (LTC4) or
prostaglandin D2 (PGD2)) mediators from human
basophils and mast cells isolated from lung
parenchyma, heart, skin and synovial tissues.
Protamine 10963
1094 mol litre91 induced
release of histamine, but not de novo synthesis of
LTC4 from basophils. At concentrations from 1095
to 3
1094 mol litre91 it induced histamine release
from human heart (mean 6.5 (SEM 1.5) %), skin (17.7
(4.1) %) and to a lesser extent from synovial mast
cells, but not from lung mast cells. Protamine also
caused the release of tryptase from heart mast
cells (12.8 (3.2) g/107 cells), but did not induce de
novo synthesis of LTC4 and PGD2 from lung and
skin mast cells. In these experiments cross-linking
of IgE by anti-IgE caused release of LTC4 or PGD2
from human basophils or mast cells. These results
demonstrate that protamine acted as an incomplete secretagogue, causing the release of preformed mediators from human basophils and mast
cells. (Br. J. Anaesth. 1997; 78: 724730).
Key words
Allergy. Histamine. Pharmacology, protamine.

hypersensitivity reactions.2 6 It has also been suggested that protamine induces or potentiates histamine release from human basophils and mast
cells.1618
Techniques are now available to isolate large
numbers of mast cells from human lung
parenchyma,19 heart,20 skin21 and synovial tissues.22
These techniques have led to the demonstration that
human basophils and mast cells isolated from different anatomical sites vary markedly in their morphological, biochemical and functional responses.1925
These cells differ with respect to the releasing
activity of various stimuli and release of different
preformed (histamine and tryptase) and de novo
synthesized mediators (peptide leukotriene C4
(LTC4) and prostaglandin D2 (PGE2)).26
Preliminary evidence suggests that human
basophils and mast cells may also vary in their
histamine releasing capacity when challenged with
general anaesthetics. For example, thiopentone
activates skin mast cells24 but not human basophils.
It should be emphasized that previous studies have
focused on the in vitro histamine releasing capacity
of protamine1618 but have not evaluated its possible
effects on de novo synthesis of chemical mediators.
To investigate the mechanisms of anaphylactoid
reactions to protamine, we have tested in vitro to see
if protamine induces the release of preformed and
de novo synthesized mediators from human basophils
and mast cells isolated from human heart, lung
parenchyma, skin and synovial tissues.

Materials and methods

Protamine sulphate is a basic protein extracted from
fish sperm heads.1 The use of i.v. protamine to
reverse heparin anticoagulation has increased in the
past decade with the advent of cardiopulmonary
bypass technology, cardiac catheterization, haemodialysis and leukapheresis.26 However, protamine
may be a major cause of morbidity and mortality in
these procedures.2 4 710 During the past decade,
reports of adverse reactions associated with i.v.
protamine (rash, urticaria, bronchospasm, hypotension and increased pulmonary artery pressure) have
also increased.26
The mechanism(s) by which protamine produces
adverse reactions is still not completely understood.
It has been suggested that they are caused by complement activation,5 11 12 inhibition of serum carboxypeptidase,13 thromboxane production14 15 and
anti-protamine IgE antibody via type I immediate

REAGENTS

The following were purchased: 60% HClO4 (Baker

Chemical Co., Deventer, The Netherlands);
piperazine-N,N-bis
(2-ethanesulphonic
acid),
hyaluronidase, collagenase type II, chymopapain,
elastase type I, synthetic PGD2, (Sigma Chemical
Co., St Louis, MO, USA); Hanks balanced salt
solution (HBSS) and fetal calf serum (FCS) (Gibco,
VINCENZO PATELLA, MD, ANNA CICCARELLI, MD, AMATO DE
PAULIS, MD, GIANNI MARONE, MD, Division of Clinical
Immunology and Allergy, University of Naples Federico II,
School of Medicine, Via S. Pansini 5, 80131 Napoli, Italy.
BRBEL LAMPARTER-SCHUMMERT, MD, MONIKA ADT, MD,
Department of Anaesthesiology, German Heart Institute Berlin,
Berlin, Germany. Accepted for publication: February 27, 1997.
Correspondence to G. M.

Protamine induces histamine release

Grand Island, NY, USA); deoxyribonuclease I and
pronase (Calbiochem, La Jolla, CA, USA); RPMI
1640 with Hepes buffer 25 mmol litre91, Eagles
minimum essential medium (Flow Laboratories,
Irvine, Scotland); Percoll (Pharmacia Fine
Chemicals, Uppsala, Sweden); protamine chlorhydrate (Roche SpA, Milan, Italy); (3H)-LTC4 39.3 Ci
mmol91 and (3H)-PGD2 210 Ci mmol91 (New
England Nuclear, Boston, MA, USA). Rabbit antihuman-Fc antibody was kindly donated by Drs
Teruko and Kimishige Ishizaka (La Jolla Institute for
Allergy and Immunology, La Jolla, CA, USA). The
rabbit anti-PGD2 antibody was a generous gift from
Dr L. M. Lichtenstein (The Johns Hopkins
University, Baltimore, MD, USA). The rabbit antiLTC4 was kindly provided by Dr E. J. Kusner
(Zeneca, Wilmington, DE, USA).27 The tryptase
radioimmunoassay kit (Pharmacia Tryptase RIACT
50, Pharmacia Diagnostics AB, Uppsala, Sweden)
was kindly donated by Kabi Pharmacia SpA (Milan,
Italy).
BUFFERS

The Pipes buffer used in these experiments was a

mixture of Pipes 25 mmol litre91, NaCl 110 mmol
litre91, KCl 5 mmol litre91, pH 7.37, referred to as
P. P2CG contains, in addition to P, CaCl2 2
mmol litre91 and glucose 1 g litre91 28; pH was
titrated to 7.4 with sodium bicarbonate. PGMD is:
MgCl2i6H2O 0.25 g litre91, DNase 10 mg litre91
and gelatine 1 g litre91 in addition to P, pH 7.37.
HISTAMINE RELEASE FROM HUMAN BASOPHILS

Informed consent was obtained from all subjects and

approximately 50 ml of blood were obtained and
diluted into a final concentration of EDTA 0.008
mol litre91 and 1.1% Dextran 70.26 The procedures
for isolation of peripheral blood basophils and in
vitro mediator release have been described in detail
elsewhere.29 The cell-free supernatant was assayed
for histamine with an automated fluorometric technique.29 Net percentage release was calculated by
subtracting histamine released spontaneously from
unstimulated samples (mean 3.6 (SEM 0.9) %) from
total histamine released from cell samples lysed with
2% perchloric acid.29 The difference between
replicate histamine measurements was less than
10%.
ISOLATION AND PARTIAL PURIFICATION OF HUMAN
HEART MAST CELLS (HHMC)

The heart tissue used in this study was obtained from

patients (3665 yr) undergoing heart transplantation
at the Deutsches Herzzentrum, Berlin, mostly for
cardiomyopathy. The explanted heart was immersed
immediately in cold (4C) cardioplegic solution,
shipped to Naples by air (4C) and processed within
518 h of removal. Fat tissue, large vessels and pericardium were removed from the heart (100400 g).
The tissue, was minced finely into 25-mm fragments, suspended in P buffer (10 ml/g of wet tissue)
and washed three times by centrifugation (the first

725
time 150
g, 4C, 8 min and twice at 150
g, 22C,
8 min). The heart fragments were filtered through a
150-m pore Nytex cloth (Tetko, Inc., Elmsford,
NY, USA) and incubated for 15 min at 37C
under constant stirring in P buffer containing 10 mg
collagenase/g of wet tissue.20 28 Three additional
cycles of enzymatic digestion were performed, with a
new preparation of collagenase each time. After the
last enzymatic digestion, the cell suspension was
centrifuged (150
g, 22C, 8 min) and filtered first
through a 150-m pore Nytex cloth and then
through a 60-m pore Nytex cloth to remove large
particles and large cells (mostly myocytes). Lastly,
cells were washed twice in PGMD (P 25 mmol
litre91, NaCl 110 mmol litre91, KCl 5 mmol litre91,
Mg 1 mmol litre91, gelatin 1 g litre91, DNase 20 mg
litre91; pH 7.37) by centrifugation at 150
g, 22C
for 8 min. Cell pellets were resuspended in 250 ml of
P buffer containing 2% BSA and centrifuged (25
g,
22C, 2 min) to remove sedimented myocytes.18
Myocytes (100 m long) were pelleted and discarded; supernatants containing endothelial cells,
fibroblasts and mast cells were then collected and
centrifuged (150
g, 22C, 8 min). At this stage of
purification, Alcian blue-positive cells amounted to
1% of the total cells. HHMC were partially purified
by flotation through a discontinuous Percoll gradient
prepared by mixing 9 parts Percoll and 1 part 10
P
solution. This mixture was then diluted with isotonic
P to give Percoll concentrations of 40, 50, 60, 70, 80
and 90%. The cell suspension was overlaid on the
Percoll gradient in 50-ml polypropylene tubes and
the mixture centrifuged at 350
g, 22C, for 20 min.
The cells found at the interface between the 6070%
and 7080% fractions were removed and washed
twice with P. Mast cells in these populations ranged
from 0.1 to 16%, with an average of 14.3 (2.4) %.
The enzymatic dispersion of tissue yields ranged
between 2 and 6
104 mast cells per gram of heart
tissue.
PURIFICATION OF HUMAN LUNG MAST CELLS
(HLMC)

Human lung tissue (2080 g) was obtained from

patients undergoing thoracotomy and lung resection, mostly for lung cancer. General anaesthesia
was induced using the following drugs: droperidol
with fentanyl and atropine (premedication);
droperidol with fentanyl, thiopentone, suxamethonium and pancuronium (anaesthesia). Lung
parenchymal mast cells were isolated as described
previously.19 This technique yielded between 3 and
8
105 mast cells/g of lung tissue, and purities
ranged between 1% and 8%.19
PURIFICATION OF HUMAN SKIN MAST CELLS
(HSMC)

Skin (1565 g) was obtained from either mastectomies for breast cancer or elective cosmetic surgery
procedures. General anaesthesia was induced using
the following drugs: thiopentone, pancuronium,
atropine, fentanyl, ethrane or fluorane. Tissue was
placed immediately in Eagles MEM at 4C and

726

British Journal of Anaesthesia

used within 1 h. HSMC were isolated as described

previously.21 24 This technique yielded between 1
and 7
105 mast cells/g of wet tissue, and purities
ranged between 1% and 4%.
PURIFICATION OF HUMAN SYNOVIAL MAST CELLS
(HSYMC)

Synovial tissue (2235 g) used in this study was

obtained from patients (2766 yr) with osteoarthritis
or rheumatoid arthritis, undergoing synovectomy at
the Division of Orthopaedic Surgery. Extradural
anaesthesia was induced with mepivacaine i.v. and
atropine, fentanyl and benperidol were given systemically. Resected joint tissue was placed immediately
in Eagles MEM at 4C and processed within 2 h of
removal using a technique described recently.22 Fat,
cartilage and fibrous tissue were removed, and the
tissue was minced finely with scissors into 25-mm
fragments, suspended in P buffer (10 ml/g of wet
tissue) and washed twice by centrifugation at 150
g
for 8 min at 4C and then at 22C. The minced
synovium was incubated for 45 min at 37 C in a
shaking water bath with chymopapain 1 mg ml91
and pronase 0.5 mg ml91 in 1 ml of Tyrodes
buffer/g synovial tissue. Fragments of remaining
tissue were digested for another 45 min at 37C with
collagenase 1 mg ml91. The resulting cell suspensions were pooled, filtered twice through 200-m
pore Nytex cloth (Tetko, Elmsford, NY, USA),
centrifuged at 200
g, 22C for 8 min, and washed
twice with P buffer. Cells were washed three times in
P and resuspended in P2CG. This technique yielded
between 1 and 9
105 mast cells/g of wet tissue, and
purities ranged between 1% and 8%.
MEDIATOR RELEASE FROM HUMAN MAST CELLS

Mast cells (approximately 3
104 cells per tube)

were resuspended in P2CG, and 0.3 ml of the cell
suspension was placed in 12
75 mm polyethylene
tubes and warmed to 37C; 0.2 ml of each prewarmed releasing stimulus was added, and incubation was continued at 37C for 30 min.26 29 At the
end of this step, the reaction was stopped by centrifugation at 1000
g 22C for 2 min, and the
cell-free supernatants were stored at 970C for

subsequent assay of histamine, tryptase, LTC4 and

PGD2 content. The cell-free supernatants were
assayed for histamine with an automated fluorometric technique.29 To calculate histamine release
as a percentage of total cellular histamine,
spontaneous release from mast cells (212% of the
total cellular histamine) was subtracted from both
numerator and denominator. The total histamine
content in mast cells was obtained by cell lysis with
8% HClO4.2426 All values are based on the means of
duplicate or triplicate determinations. Replicates
differed from each other by less than 10% in
histamine content.
RIA OF TRYPTASE, LTC4 AND PGD2

Total tryptase content was assessed by lysis induced

by incubating cells with 0.1% of Triton X-100.
Tryptase was analysed by a solid-phase radioimmunoassay (Pharmacia Tryptase RIACT 50).20 26
PGD2 was analysed on 100-l fractions obtained
from the supernatant fluids. The samples were
stored at 970 C. LTC4 and PGD2 were analysed by
RIA within 18 h of the experiment to minimize
degradation of the compounds.23
STATISTICAL ANALYSIS

Results are mean (SEM). Statistical analysis was performed by two-way non-parametric analysis of variance (Friedmans chi-square test). The extended
Tukey test was used for multiple comparisons. The
level of statistical significance was P0.05.30

Results
In the first series of experiments we evaluated the
effects of increasing concentrations (109693
1094
mol litre91) of protamine on histamine release from
human basophils. Protamine caused concentrationdependent histamine release from basophils from 11
normal donors (table 1). In one experiment, protamine released less then 5% of histamine content but
in all other experiments release varied from 5% to
26%. The release caused by protamine reached a
plateau between 1095 and 3
1095, with less release
above these concentrations. Anti-IgE, used as a

Table 1 Effect of increasing concentrations of protamine on histamine release from human basophils
Histamine release (%)
Protamine (mol litre91)

Experiment
No.

1096

3
1096

1095

3
1095

1094

3
1094

1
2
3
4
5
6
7
8
9
10
11
Mean (SEM)

0
0
5
4
0
0
0
0
0
3
1
1.2 (0.5)

1
0
8
1
0
2
2
3
2
3
1
2.1 (0.6)

3
1
14
9
4
14
12
26
16
5
3
9.7 (2.3)

8
4
3
14
9
14
16
18
17
3
2
9.8 (1.9)

15
5
0
8
4
6
9
9
12
3
1
6.5 (1.4)

16
4
0
3
4
5
6
8
8
5
1
5.5 (1.3)

Protamine induces histamine release

727

Figure 1 Effect of increasing concentrations of protamine on

release of histamine from HHMC and HLMC. Results are mean
(SEM) of 11 (HHMC) or nine (HLMC) different donors. Error
bars are not shown when graphically too small. **P0.01
compared with spontaneous release; P0.01 compared with
histamine release induced by protamine from HLMC.

positive control in these experiments, induced 28.9

(5.4) % of histamine release. There was no correlation between the maximum release caused by protamine and anti-IgE (rs:0.01; ns). Cell viability in the
presence of protamine was always 95% as detected
by trypan blue exclusion.28
In the second series of experiments we compared
the effect of protamine on histamine release from
HHMC and HLMC. Protamine 10953
1094
mmol litre91 induced histamine release from
HHMC in a concentration-dependent manner (fig.
1) with maximum release between 3% and 17% (8.8
(1.4) %) (P0.01 compared with spontaneous
release). In none of nine HLMC preparations did
protamine induce the release of more than 5% of
histamine content (fig. 1).
In view of the functional heterogeneity of human

Figure 2 Effect of increasing concentrations of protamine on

histamine and tryptase release from HHMC and HLMC.
Results are mean (SEM) of four (HHMC) or five (HLMC)
different donors. Error bars are not shown when graphically too
small.

mast cells isolated from different anatomical sites,20

2226 28
we evaluated the effects of increasing concentrations (10953
1094 mmol litre91) of protamine
on histamine release from mast cells from skin and
synovial tissues. These concentrations of protamine
induced histamine release from HSMC (table 2) and
to a lesser extent from HSyMC (table 3). The maximum release varied between 7% and 25% (HSMC)
and 12% and 13% (HSyMC).
Tryptase is a neutral protease that is a selective
marker for mast cells.31 The secretory granules of all
mature human mast cells contain large amounts
(approximately 24.2 (4.3) g/106 HHMC of
tryptase).20 22 We investigated if activation of
HHMC with protamine caused release of tryptase in
addition to histamine. Tryptase was released in
parallel with histamine from HHMC whereas protamine did not induce release of histamine and
tryptase (5%) from HLMC in three experiments
(fig. 2).

Table 2 Effect of increasing concentrations of protamine on histamine release from HSMC

Histamine release (%)
Experiment
No.
1
2
3
4
Mean (SEM)

Protamine (mol litre91)

1096

3
1096

1095

3
1095

1094

3
1094

0
0
0
4
1.0 (1.0)

1
1
2
7
2.8 (1.4)

18
4
8
7
9.2 (3.0)

23
9
8
7
11.8 (3.8)

23
16
6
7
13.0 (4.0)

25
23
16
7
17.7 (4.1)

Table 3 Effect of increasing concentrations of protamine on histamine release from HSyMC

Histamine release (%)
Experiment
No.
1
2
3
Mean (SEM)

Protamine (mol litre91)

1096

3
1096

1095

3
1095

1094

3
1094

0
0
0
0(0)

0
0
0
0(0)

0
0
0
0(0)

0
0
0
0(0)

0
4
2
2 (1.1)

13
10
12
11.6 (0.8)

728

Figure 3 Effect of increasing concentrations of protamine and

anti-IgE on release of histamine and LTC4 from human
basophils. Results are mean (SEM) of three experiments. Error
bars are not shown when graphically too small. **P0.01
compared with spontaneous release.

Different experimental models have shown that

some of the adverse effects of protamine are caused
by generation of eicosanoids.32 33 We therefore compared production of the newly formed lipid mediators from human basophils and mast cells activated
by protamine 10953
1094 mmol litre91 and antiIgE 10923
1091 g ml91. In this group of experiments protamine induced concentration-dependent
release of histamine from basophils, but did not
stimulate de novo synthesis of LTC4 by these cells
(fig. 3). Anti-IgE stimulated release of large amounts
of LTC4. Protamine also did not induce de novo
synthesis of PGD2 or LTC4 from HHMC and
HSMC, whereas anti-IgE stimulated the release of
these eicosanoids (data not shown).

Discussion
Our results indicate that protamine is an incomplete
secretagogue inducing only release of preformed
mediators (histamine and tryptase) from human
basophils and mast cells without stimulating de novo
synthesis of eicosanoids. Protamine activated not
only peripheral blood basophils, but also mast cells
isolated from human heart, skin and synovial tissues,
but not from lung parenchyma.
Intravascular administration of high concentrations of protamine is used increasingly to reverse
heparin anticoagulation during cardiac catheterization,4 cardiothoracic and vascular surgical procedures34 35 and after dialysis36 and leukapheresis.37 I.v.
protamine can cause acute reactions such as rash,
urticaria, bronchospasm, hypotension, cardiovascular collapse, pulmonary hypertension and even
death.2 4 79 These adverse reactions are thought to be
caused by multiple mechanisms, including
anaphylactic26 and anaphylactoid reactions.1114 1618
Intravascular administration of protamine causes

British Journal of Anaesthesia

rapid and massive exposure of basophils to this agent.
It is therefore likely that the release of histamine from
peripheral blood basophils might at least partly
explain some of the adverse effects of protamine in
some patients.
Intravascularly administered compounds rapidly
expose circulating basophils and tissue mast cells to
very high concentrations that tend to decline according to their pharmacokinetics. Thus we investigated
the effects of a wide range of concentrations of protamine which must include concentrations reached
during intravascular administration of the compound. The percentage of histamine release from
basophils caused by protamine varied greatly from
donor to donor, presumably because of the influence
on basophil releasibility.19 23 38 This may partly
explain the wide variability of protamine-induced
adverse effects in patients given this drug intravascularly.6 At first sight, the bell shape of the
doseresponse curve of protamine on release of
histamine from basophils might be surprising.
However, other stimuli (anti-IgE, antigens, etc.) can
produce a similar doseresponse curve.20
Although it has been demonstrated in different
experimental models that the adverse reactions to
protamine are to some extent caused by synthesis of
eicosanoids and other lipid mediators,32 33 protamine
did not induce release of LTC4 and PGD2 from
human basophils and mast cells in vitro. Possibly in
vivo protamine induces the release of lipid mediators
from other cells.
Radiocontrast media, which can cause anaphylactoid reactions, also act as incomplete secretagogues
on human basophils and mast cells26 and pretreatment with histamine H1 and H2 receptor antagonists
reduces the incidence and severity of adverse reactions to these agents.39 In the light of our results, it
might be appropriate to investigate if these antagonists also reduce the incidence of anaphylactoid
reactions to protamine.
Protamine caused a concentration-dependent
release of tryptase from HHMC, HSMC and
HSyMC. This neutral protease, present in the cytoplasmic granules of all human mast cells,20 22 31 can
activate complement leading to the formation of
anaphylatoxins (C3a and C5a).40 Complement
activation and C3a formation can occur during in
vivo administration of protamine1114 and tryptase
release from mast cells might therefore contribute, as
an amplification factor, to the pathogenesis of the
generalized reactions to protamine, through activation of the complement system. Tryptase has a
longer half-life than histamine in plasma.31
Therefore, it may be useful to measure plasma concentrations of tryptase in order to identify adverse
reactions caused by protamine in humans.
Our results showed that protamine can induce
release of vasoactive and proinflammatory mediators
from HHMC in vitro. Coronary artery spasm,
hypotension and shock can occur during intravascular administration of protamine.8 9 Mast cells
have been demonstrated clearly within human heart
tissue,20 28 around coronary vessels and in the
shoulder region of atherosclerotic human coronary
vessels.41 During intravascular administration of

Protamine induces histamine release

protamine, cardiac mast cells are likely to be exposed
to high concentrations and our findings suggest that
in vivo they can thus be activated to release histamine, tryptase and possibly other mediators.
Histamine exerts significant cardiovascular effects in
humans42 and vasoactive mediators released locally
from coronary and cardiac mast cells might therefore
contribute to the pathogenesis of severe adverse
reactions to protamine.
Unlike HHMC and HSMC, HLMC were not
activated by protamine in vitro. However, transient
increases in pulmonary artery pressure are frequent
when protamine is administered i.v.7 14 It is therefore
possible that the pulmonary reactions to protamine
involve alternative mechanisms such as direct activation of basophils, tryptase-mediated formation of
anaphylatoxins that in turn activate C5a receptors
on HSMC18 23 and HHMC,20 or other unknown
mechanisms. Our results were obtained with preparations of mast cells isolated from different tissues of
patients receiving several drugs before and during
surgical procedures. Consequently, we cannot
exclude the possibility that some of the heterogeneous effects of protamine on the release of mediators from mast cells isolated from different tissues
might be influenced by the in vivo treatment of the
cell donors.
In conclusion, these findings suggest that protamine selectively activated different types of human
mast cells and basophils, acting as an incomplete
secretagogue. These results are of interest for several
reasons. They contribute to a better understanding
of the pathogenesis of immediate generalized
reactions caused by rapid injection of protamine and
they also suggest that anti-H1 and anti-H2 blockers
might be useful in attenuating anaphylactoid
reactions to this substance. Finally, they indicate
that measurements of plasma tryptase might be
useful to identify adverse reactions.

Acknowledgements
This work was supported in part by grants from the C.N.R.
(Targeted Project F.A.T.M.A.; Prevention and Control of
Disease Factors; SP2 No. 94.00607.PF41 and 95.00856.PF.41)
and the M.U.R.S.T. (Rome, Italy). This work was performed
within the frame of the EU ENDA Programme BIOMED 1, CT
931661. We thank Drs G. Aliperta and F. Tatangelo for
supplying lung and skin tissue specimens.

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