Platelets

ISSN: 0953-7104 (Print) 1369-1635 (Online) Journal homepage: https://www.tandfonline.com/loi/iplt20

Inhibition of agonist-induced platelet aggregation

by magnesium sulfate warrants its use
as an alternative in vitro anticoagulant in
pseudothrombocytopenia

Steffen Mannuß, Peter Schuff-Werner, Katrin Dreißiger & Christine Burstein

To cite this article: Steffen Mannuß, Peter Schuff-Werner, Katrin Dreißiger & Christine Burstein
(2019): Inhibition of agonist-induced platelet aggregation by magnesium sulfate warrants
its use as an alternative in vitro anticoagulant in pseudothrombocytopenia, Platelets, DOI:
10.1080/09537104.2019.1663804

To link to this article: https://doi.org/10.1080/09537104.2019.1663804

Published online: 11 Sep 2019.

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ISSN: 0953-7104 (print), 1369-1635 (electronic)

Platelets, Early Online: 1–5

© 2019 Taylor & Francis Group, LLC. DOI: https://doi.org/10.1080/09537104.2019.1663804

Inhibition of agonist-induced platelet aggregation by magnesium sulfate

warrants its use as an alternative in vitro anticoagulant in
pseudothrombocytopenia
Steffen Mannuß1,2, Peter Schuff-Werner1,2, Katrin Dreißiger1, & Christine Burstein1
1
Institute of Clinical Chemistry and Laboratory Medicine, Rostock University Medical Center, Rostock, Germany , 2Medizinisch-Diagnostische Institute
(MVZ), Berlin, Germany

Abstract Keywords
MgSO4 is effective in preventing spontaneous in vitro platelet agglutination in anticoagulant- Impedance aggregometry, light transmission
induced pseudothrombocytopenia (PTCP). In order to learn more about its potential as an aggregometry (LTA), magnesium sulfate
in vitro anticoagulant, platelets from MgSO4-anticoagulated blood were stimulated by several (MgSO4), platelet function
differentially-acting agonists (ADP, ARA, TRAP, epinephrine, collagen and ristocetin). Platelet
aggregation in blood samples from 11 and 17 volunteers was measured by light-transmission History
aggregometry (LTA) according to Born and impedance aggregometry (MultiplateTM), respec-
Received 26 January 2019
tively. Agonist-induced platelet aggregation was markedly lower in MgSO4-anticoagulated
Revised 21 June 2019
samples when compared with citrate-anticoagulated samples (decrease of 95.75% (ristocetin),
Accepted 8 August 2019
69.02% (collagen) and 75.73% (epinephrine)) or hirudin-anticoagulated samples (decrease of
Published online 14 September 2019
85.99% (ADP), 80.98% (ARA), 77.24% (ristocetin), 54.37% (collagen) and 50.14% (TRAP)). The
anti-aggregatory effect of MgSO4 is dose-dependent and readily detectable at a concentration
of 7.5 mmol/l. Analysis of the agonist signaling pathways suggest that MgSO4 interferes with
the final step of platelet aggregation, namely the intracellular mobilization of Ca2+.

Introduction In 1986, Nakamoto used MgSO4-anticoagulated blood for

platelet counting in individuals with PTCP [12], but due to the
With the introduction of automated blood cell counting, EDTA was
fact that these data were published in Japanese they were not
recommended as the standard in vitro anticoagulant [1].
noticed by the scientific community. In 2013, MgSO4 was again
Nevertheless, the search for alternative in vitro anticoagulants sui-
shown to be an effective in vitro anticoagulant for estimating
table for automated platelet counting remained a subject of interest.
platelet counts in anticoagulant-induced PTCP [13]. Further
The reason for this is that a disadvantage of EDTA is the rare
investigations into the use of MgSO4 as in vitro anticoagulant
possibility of induction of spontaneous time- and temperature-
[14,15] focused primarily on technical issues of automated plate-
dependent in vitro platelet aggregation, subsequently followed by
let counting and volume determination rather than on platelet
spurious low platelet counts (“pseudothrombocytopenia”) [2,3].
function.
Other in vitro anticoagulants such as citrate and heparin are
Platelet function is ideally tested using hirudin-anticoagulated
significantly less associated with in vitro platelet aggregation,
blood samples. As a pure thrombin inhibitor, hirudin does not affect
although this is not completely excluded from occurring [4–6].
platelet physiology and therefore is commonly utilized as an antic-
The use of MgSO4 as an anticoagulant dates back to the 19th
oagulant for impedance aggregometry measurements [16]. Citrate is
century when Bizzozero investigated different chemicals for their
the standard anticoagulant used for measuring platelet function by
ability to avoid agglutination of peripheral blood used for micro-
LTA according to Born [17]. Therefore, we used these recommended
scopic analysis. As a result of his search, he discovered that
anticoagulants as a reference for their respective aggregation mea-
MgSO4 (“sulfuric acid bitter earth”) was the most suitable antic-
surements in comparison to MgSO4-anticoagulated samples.
oagulant for this purpose [7].
The intention of our study was to test the degree of agonist-
In the second half of the last century, MgSO4 was displaced by
induced platelet aggregation in MgSO4-anticoagulated whole
EDTA and fell into disuse as in vitro anticoagulant. On the other
blood in comparison to aggregation in samples anticoagulated
hand, scientific interest in the anti-aggregatory ability of MgSO4
by hirudin or citrate.
remained [8]. For example, orally administered magnesium was
investigated for use in adjuvant antithrombotic treatment and for
its impact on platelet activation [9–11]. Material and Methods
Blood Sampling from Volunteer Probands
Healthy volunteers (11 males, 17 females; age range: 23 to 65 years)
Correspondence: Peter Schuff-Werner, Institute of Clinical Chemistry and
Laboratory Medicine, Rostock University Medical Center, Ernst- were informed about the purpose and aim of the study, and asked for
Heydemann-Straße 8, D-18057 Rostock, Germany. E-mail: written consent for donation of differently anticoagulated blood
pschuffw@web.de samples (citrate: S-MonovetteTM Coagulation 2.9 ml, hirudin:
2 S. Mannuß et al. Platelets, Early Online: 1–5

S-MonovetteTM Hirudin 2.7 ml and MgSO4: ThromboExact 2.7 ml,
final MgSO4-concentration: 33.8 µmol/ml, all from Sarstedt AG,
Nümbrecht, Germany).
To avoid advance activation of the platelets, blood was care-
fully drawn after release of the tourniquet, gently and carefully
mixed with the anticoagulant and immediately brought to the
laboratory for further processing.
The study was approved by the ethics committee of the medical
faculty of the Rostock University (registration number: A 2011/44).
LTA according to Born
Measurements were performed with the PAP-4 aggregation
profilerTM (möLab ltd, Langenfeld, Germany). In the first step,
platelet-rich (PRP) and platelet-poor plasma (PPP) from citrate-
and MgSO4- anticoagulated samples (n = 11) was prepared
according to our standard laboratory protocol. In the second
step, the analyzer zeroed aliquots of PRP against the respective
PPP, and aggregation was measured as the increase of light
transmittance over time after stimulation with different agonists
at appropriate concentrations (ADP: 85.44 µg/ml, collagen:
1.9 mg/ml, ARA: 5 mg/ml, epinephrine: 183.2 µg/ml and risto-
cetin: 1.5 mg/ml) [18].
Impedance Aggregometry
Immediately after sampling, agonist-induced platelet aggregation
was measured in hirudin- and MgSO4-anticoagulated blood sam-
ples (n = 14) using the MultiplateTM analyzer (Roche
Diagnostics, Mannheim, Germany). This device measures the
time-dependent increase in electrical impedance caused by the
adhesion of platelets onto the sensor electrodes during platelet
aggregation. The changes in impedance are recorded over 6 min-
utes, and results are presented dimensionless as “area under the
curve” (AUC) [16,19].
Platelet aggregation was elicited by different reagents at the
concentration specified by the manufacturer test instructions:
ADP (0.2 mM), ARA (15 mM), ristocetin (10 mg/ml), collagen
(100 µg/ml) and TRAP (1 mM).
3.7 mg/ml (15 mmol/l), 5.55 mg/ml (22.5 mmol/l) and
7.41 mg/ml (30 mmol/l). The spiked samples and controls were
measured by impedance aggregometry as described above.
Statistical Analysis
Statistical analysis (Wilcoxon signed-rank test) was carried out
using the software SigmaPlot version 13 (Systat Software, San
Jose, CA), and graphical presentation of data was performed
using Excel 2013 (Microsoft Corporation, Redmond, WA).
Results
Agonist-induced Aggregation of MgSO4-anticoagulated
Platelets
LTA according to Born
The aggregation of MgSO4-anticoagulated platelets elicited by
different agonists and expressed as a percentage is depicted in
Table I. MgSO4-anticoagulated platelets did not aggregate upon
stimulation with ADP or ARA, marginally aggregated with risto-
cetin and moderately aggregated with collagen and epinephrine.
This is in contrast to the aggregation of citrate-anticoagulated
platelets, which ranged from 81.91% to 92.45% for the various
agonists. The difference in induced aggregation between citrate-
and MgSO4-anticoagulated samples was highly significant
(p < .001) for each agonist.
Impedance Aggregometry
Impedance aggregometry is another approach for measuring pla-
telet aggregability. The agonist-induced platelet aggregation in
MgSO4-anticoagulated compared to hirudin-anticoagulated
whole blood samples is depicted in Figure 1.
Table I. Agonist-induced platelet aggregation (LTA) in differently antic-
oagulated blood samples (n = 11).

Dose-dependency of MgSO4-induced Suppression of Platelet Agonist Citrate MgSO4

Aggregation ADP 81.91 ± 2.78% No aggregation
In parallel experiments, hirudin-anticoagulated sample tubes ARA 80.82 ± 5.77% No aggregation
spiked with MgSO4 were used to determine the dose- Collagen 92.45 ± 5.77% 28.64 ± 9.94%
Epinephrine 89.55 ± 2.15% 21.73 ± 5.86%
dependency of MgSO4 anticoagulation (n = 3). The Ristocetin 85.64 ± 1.72% 3.64 ± 0.64%
final MgSO4 concentrations were: 1.85 mg/ml (7.5 mmol/l),

Figure 1. Comparison of agonist-induced plate- 160

let aggregation in MgSO4 (dashed columns) and
hirudin (black columns) anticoagulated samples 140
as measured by impedance aggregometry. The
Area under the curve (AUC)

results are shown as mean ± SD of the respec- 120

tive AUCs (n = 14).
100

80

60

40

20

0
ADP ARA Ristocetin Collagen TRAP
DOI: https://doi.org/10.1080/09537104.2019.1663804 Platelet aggregation in MgSO4-anticoagulated blood 3

The mean AUC of MgSO4-anticoagulated blood samples was
10.63 ± 4.07 when stimulated by ADP and 17.75 ± 6.18 when
stimulated by ARA, whereas the mean AUCs in the respective
hirudin-anticoagulated blood samples were 75.89 ± 9.79 and
93.33 ± 22.54. The response to ristocetin was similarly poor for
MgSO4-anticoagulated blood (2.57 ± 2.31) and hirudin- antic-
oagulated blood (11.29 ± 6.92). The agonists collagen and TRAP
in MgSO4-anticoagulated blood induced a mean AUC of
34.29 ± 12.88 and 64.64 ± 17.43, respectively, while the respec-
tive mean AUCs in hirudin-anticoagulated blood were
75.14 ± 13.65 and 129.64 ± 15.31. The difference in induced
aggregation between MgSO4- and hirudin-anticoagulated samples
was highly significant (p < .001) for each agonist.
Dose-dependent Anti-aggregatory Efficacy of MgSO4
In order to investigate the effect of increasing MgSO4 concentra-
tions on agonist-induced platelet aggregation, hirudin samples
from 3 volunteers were spiked with different concentrations of
MgSO4 and stimulated by ADP, ARA and TRAP. Finally, the
measured AUCs were compared to the AUCs measured in the
unspiked hirudin anticoagulated sample.
The results of the spiking experiments are summarized in Table II.
The AUCs of the spiked samples after TRAP stimulation decreased in
a dose-dependent manner to a maximum decrease between 39.3% and
46.6% when compared with unspiked hirudin samples. The dose-
dependent effect of MgSO4 spiking was also shown after stimulation
with ADP and ARA. Even at the lowest concentration of MgSO4
(1.85 mg/ml), the AUC is reduced to 26.3% and 73.3% of the respec-
tive AUC of unspiked samples for ADP and ARA. The addition of 3-
to 4-fold higher MgSO4 concentrations leads to a maximal suppression
of aggregation that amounts to values between 9.8% and 26.6% of the
unspiked samples.
Discussion
We have previously shown that in vitro anticoagulation with
MgSO4 effectively averts anticoagulant-induced platelet aggrega-
tion [13]. To better understand the significance and possible uses
of MgSO4-anticoagulation, we were interested in exploring its
anti-aggregatory potential in conventional tests of agonist-
induced platelet aggregation, such as Born aggregometry and
impedance aggregometry. Therefore, we compared the degree of
aggregation elicited in MgSO4-anticoagulated whole blood with
that of citrate- and hirudin-anticoagulated control samples.
Citrate acts by effectively binding Ca2+ and the anticoagulant
properties of hirudin are attributed to direct inhibition of thrombin
[20,21], whereas the anti-aggregatory effect of MgSO4 is not entirely
clear.
Platelet activation and subsequent aggregation is regulated by
a distinct intracellular signaling network that facilitates an elevation
of cytosolic Ca2+-levels and conformational changes of the fibrino-
gen receptor GpIIb/IIIa [22]. The subsequent activation of the GpIIb/
IIIa fibrinogen receptor is a prerequisite for the formation of stable
platelet aggregates via platelet recruiting through fibrinogen bind-
ing. Gawaz and colleagues showed that Mg2+ inhibits fibrinogen-
mediated platelet aggregation, regardless of the choice of agonist,
and reduces the adhesion of platelets to immobilized fibrinogen [23].
However, their approach focussed on the intravenous application
of Mg2+ as an in vivo therapeutic rather than on a usage as in vitro
anticoagulant for diagnostic purposes.
The data of our study shows that agonist-induced platelet
aggregation in MgSO4-anticoagulated blood is suppressed regard-
less of the agonist used. The fact that the pro-aggregatory proper-
ties of these agonists comprise different intracellular signaling
pathways let us assume that MgSO4 inhibits a common final step.
Platelet aggregation-eliciting ARA is metabolized to thrombox-
ane A2 (TXA2) via cyclooxygenase and TXA2-synthase. TXA2
acts via the TXA2-receptor, inducing an increase of cytoplasmic
Ca2+ levels [24–26]. Since the investigations of Hwang and collea-
gues revealed that a Mg2+ concentration of 4 mmol/l inhibits the
synthesis of TXA2 from ARA by 50% [27], it is possible that
higher Mg2+ concentrations suppress ARA-induced Ca2+-influx,
thus inhibiting subsequent triggered platelet aggregation.
Platelet stimulation with collagen leads to an activation of phos-
pholipase Cγ2 (PLCγ2). PLCγ2 metabolizes PIP2 (phosphatidyli-
nositol-4,5-bisphosphate) into inositol trisphosphate (IP3) and
diacylglycerol (DAG). These second messengers induce intracellular
Ca2+ mobilization and activation of protein kinase C (PKC), respec-
tively, subsequently initiating intra-platelet TXA2 formation with
consecutive platelet aggregation [28]. Other agonists follow
a similar process. The agonist ADP binds to the purinergic receptor
P2Y1, also leading to the synthesis of IP3 and DAG [29], while
TRAP induces the activation of phospholipase Cß (PLCß), which
promotes the conversion of PIP2 to IP3 and DAG via G protein-
coupled receptors [30].
Thus, the agonists collagen, ADP and TRAP all finally lead to
intracellular Ca2+ mobilization, eliciting platelet aggregation. The
addition of MgSO4 obviously interferes with this common last
step. Ristocetin as a cofactor of von Willebrand factor (vWF)
induces platelet aggregation by mediating the binding of vWF to
the platelet glycoprotein receptor Gp Ib-IX-V, likewise leading to
the subsequent activation of PLC [31]. Volpe and colleagues
demonstrated that Mg2+ is a non-competitive inhibitor of IP3

Table II. AUCs in MgSO4-spiked hirudin samples compared with the AUC of the unspiked hirudin sample (%) using three different agonists (n = 3).

MgSO4 concentration in the hirudin sample (mg/ml)

0 1.85 3.70 5.55 7.41

Proband Agonist AUC % AUC % AUC % AUC % AUC %

A TRAP 148 100 121 81.8 121 81.8 75 50.7 69 46.6

ARA 109 100 70 64.2 33 30.3 21 19.3 20 18.3
ADP 76 100 20 26.3 15 19.7 13 17.1 17 22.4
B TRAP 107 100 99 92.5 86 80.4 55 51.4 42 39.3
ARA 86 100 63 73.3 40 46.5 23 26.7 15 17.4
ADP 60 100 34 56.7 35 58.3 15 25.0 14 23.3
C TRAP 131 100 111 84.7 84 64.1 60 45.8 57 43.5
ARA 79 100 24 30.4 42 53.2 26 32.9 21 26.6
ADP 61 100 25 41.0 9 14.8 7 11.5 6 9.8
4 S. Mannuß et al.
mediated Ca2+ release [32], thus indicating that Mg2+ might
inhibit this final step of aggregation. Beyond this, MgSO4 also
inhibits PKC activation which is another common step of agonist-
induced aggregation [27,33,34].
Epinephrine acts as aggregation agonist via Gi protein-coupled
α2-adrenoceptors and induces platelet aggregation by inhibiting
adenylate cyclase and the subsequent formation of cAMP [35,36],
which is an inhibitor of PLC and TXA2. This is a common
pathway with that of ADP-induced platelet aggregation via
P2Y12, thus explaining the requirement of ADP for epinephrine-
induced platelet activation [37]. Hardy and colleagues demon-
strated that the presence of MgSO4 also inhibits the ADP-induced
decrease in cAMP [38].
The observation of Ravn and Hwang that Mg2+ causes a dose-
dependent inhibition of platelet aggregation [8,27] was confirmed
by our spiking experiments with hirudin-anticoagulated blood
samples containing increasing amounts of MgSO4. As shown in
Table II, platelet aggregation is already impaired at a MgSO4
concentration of 7.5 mmol/l, indicating that the MgSO4 concen-
tration of 33.8 mmol/l in the commercially available sampling
device [13] is markedly above the necessary inhibitory concentra-
tion to effectively antagonize platelet aggregation, even when
stimulated by highly effective agonists.
Taken together, the findings of our study reveal that MgSO4 at
higher concentrations might be a reliable alternative as in vitro
anticoagulant for blood cell counting due to the fact that even
high agonist concentrations cannot induce platelet aggregation
in vitro in the presence of MgSO4. This was impressively
shown by anticoagulant-induced in vitro platelet aggregation
(PTCP) that occurred in five differently anticoagulated blood
samples, but not in those anticoagulated with MgSO4 [39]. We
therefore believe that MgSO4 is suitable as in vitro anticoagulant
in the hematological laboratory.
Acknowledgements
The authors thank Prof. Günther Kundt (Institute for Biostatistics and
Informatics in Medicine and Aging Research, Rostock University
Medical Center) for his statistical advice.
Declaration of interest
The authors report no conflict of interest.
Contribution of each author
SM defined the study design and performed the experiments, PSW wrote
parts of the manuscript, KD was responsible for data acquisition and CB
contributed to the discussion and corrected the final manuscript.
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