Characterization of Procoagulant COAT Platelets in …

 

 

Article
Characterization of Procoagulant COAT Platelets in
Patients with Glanzmann Thrombasthenia

Alessandro Aliotta
Francisco J. Gomez, Ana P. Batista Mesquita Sauvage and Lorenzo Alberio *

, Debora Bertaggia Calderara

, Manuel Krüsi

†

†

, Maxime G. Zermatten,

Division of Hematology and Central Hematology Laboratory, Lausanne University Hospital (CHUV)
and University of Lausanne (UNIL), Rue du Bugnon 46, CH-1011 Lausanne, Switzerland;
Alessandro.Aliotta@chuv.ch (A.A.); manuel.krusi@unil.ch (M.K.); Debora.Bertaggia-Calderara@chuv.ch (D.B.C.);
Maxime.Zermatten@chuv.ch (M.G.Z.); Francisco-Javier.Gomez@chuv.ch (F.J.G.);
Ana-Patricia.Batista-Mesquita@chuv.ch (A.P.B.M.S.)
* Correspondence: Lorenzo.Alberio@chuv.ch
† These authors contributed equally to this work.

Received: 15 November 2020; Accepted: 10 December 2020; Published: 14 December 2020

Abstract: Patients affected by the rare Glanzmann thrombasthenia (GT) suffer from defective
or low levels of the platelet-associated glycoprotein (GP) IIb/IIIa, which acts as a fibrinogen
receptor, and have therefore an impaired ability to aggregate platelets. Because the procoagulant
activity is a dichotomous facet of platelet activation, diverging from the aggregation endpoint,
we were interested in characterizing the ability to generate procoagulant platelets in GT patients.
Therefore, we investigated, by flow cytometry analysis, platelet functions in three GT patients as well
as their ability to generate procoagulant collagen-and-thrombin (COAT) platelets upon combined
activation with convulxin-plus-thrombin. In addition, we further characterized intracellular ion
fluxes during the procoagulant response, using specific probes to monitor by flow cytometry kinetics
of cytosolic calcium, sodium, and potassium ion fluxes. GT patients generated higher percentages of
procoagulant COAT platelets compared to healthy donors. Moreover, they were able to mobilize
higher levels of cytosolic calcium following convulxin-plus-thrombin activation, which is congruent
with the greater procoagulant activity. Further investigations will dissect the role of GPIIb/IIIa
outside-in signalling possibly implicated in the regulation of platelet procoagulant activity.

Keywords: procoagulant platelets; Glanzmann thrombasthenia; procoagulant activity; ion fluxes

1. Introduction

In order to maintain physical integrity during episodes of injury and disease, primary and
secondary hemostasis are important processes to limit life-threatening bleeding [1]. The first process
encompasses mainly vessel wall reactivity and aggregation of platelets. The second consists of soluble
clotting factors mainly synthesized in the liver parenchyma [2]. Activation of these clotting factors
leads to a common endpoint: the transformation of fibrinogen in fibrin. The latter will engulf and
stabilize a preformed primary clot, mainly made up of aggregated platelets, and stop the bleed.

Aggregation of platelets is mediated by the surface receptor glycoprotein (GP) IIb/IIIa (integrin
αIIbβ3). In the first steps, agonists activate GPIIb/IIIa (called inside-out signaling). Activation of
GPIIb/IIIa is characterized by a conformational change to increase its affinity principally for fibrinogen
but also for other adhesive molecules such as fibronectin or von Willebrand factor [3]. The binding
of fibrinogen to activated GPIIb/IIIa receptors allows platelet bridging and also mediates outside-in
signaling to culminate with a stable and irreversible aggregation of platelets [2–4].

Int. J. Mol. Sci. 2020, 21, 9515; doi:10.3390/ijms21249515

www.mdpi.com/journal/ijms

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Glanzmann thrombasthenia (GT) is a rare autosomal inherited disorder, characterized by a
quantitative or qualitative defect in GPIIb/IIIa, thus impairing platelet aggregation and normal primary
hemostasis [2,4]. Pathogenic mutations in the genes ITGA2B and ITGB3 prevent the normal function
of GPIIb/IIIa receptor, weakening platelet aggregation and leading to unstable clot formation and
thus to bleeding phenotype [5–7]. Affected patients can range in their symptoms from being nearly
asymptomatic to bleeding episodes that can vary in intensity and frequency and are characterized by
easy mucocutaneous bleeding and bruising [8].

In recent decades, it became increasingly clear, that platelets do not represent a homogenous
population of cells but rather a heterogeneous assortment of subpopulations that differ upon activation
in their structural features as well as their functional properties [9–11].

Platelets show great variability in their agonist-induced response patterns and the propensity
to expose phosphatidylserine (PS) on their surface, which in turn is one of the hallmarks of the
procoagulant platelet population [12,13].

Platelet procoagulant activity, as an additional activation endpoint to traditional secretion and
aggregation, is generated upon strong platelet activation [14,15]. In particular, the combination of
potent agonists such as thrombin (THR) and collagen [or convulxin (CVX), a selective agonist of the
GPVI collagen receptor] induce the formation of procoagulant platelets that become highly efficient in
sustaining thrombin generation [16]. Procoagulant collagen-and-thrombin (COAT) activated platelets
are characterized by high and sustained intracellular free calcium levels, loss of the mitochondrial
potential, the coating of their surface by pro-hemostatic α-granule proteins, downregulation of activated
GPIIb/IIIa (losing their aggregatory property), and the expression of PS to support the tenase and
prothrombinase complexes for the coagulation process [14–18]. Investigations of the ability to generate
procoagulant COAT platelets is of high clinical relevance as increased levels of procoagulant COAT
platelets have been correlated with thrombotic events [19–21] while low levels were associated with a
bleeding diathesis and its severity [22–25].

Moreover, because the procoagulant activity is a dichotomous facet of platelet activation,
complementary and diverging from the aggregation endpoint [18], we were interested to
characterize the functionality of procoagulant platelets in GT patients lacking platelet aggregation.
Therefore, we systematically characterized platelet functions in GT patients as well as their ability
to generate procoagulant COAT platelets, and we further analyzed intracellular ion fluxes upon the
procoagulant response.

2. Results

2.1. Characterization of Platelet Function by Flow Cytometry

In addition to a complete medical history and traditional laboratory workup, including platelet
aggregation studies, we characterized three GT patients with a comprehensive platelet phenotypic and
functional analysis by flow cytometry (summarized in Table 1). The flow cytometry analysis (FCA)
confirmed an absence of both components of the fibrinogen receptor, namely GPIIb (CD41) and GPIIIa
(CD61), and a markedly impaired ability to bind PAC-1 following activation with increasing doses of
ADP, THR or CVX. In addition, we observed a conserved platelet size and granularity, slightly in the
higher range for the patient with GT number 3 (PAT_GT3), and a conserved surface density of the
receptors for the von Willebrand factor (GPIb and GPIX) and the collagen receptor GPVI. The second
collagen receptor GPIa (CD49b), which mainly supports platelet adhesion, was decreased in the three
patients. The higher surface density measured for PAT_GT3 is corrected when data are normalized
according to the platelet size (data not shown).

Int. J. Mol. Sci. 2020, 21, 9515

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Table 1. Patient Characteristics Revealed by Flow Cytometric Analysis and In-House Reference Ranges.

Characterization

Indicator

PAT_GT1 PAT_GT2 PAT_GT3

In-House Ranges
(n = 73; 2.5–97.5 Percentiles)
(cid:48)

(cid:48)

Unit

MFI
MFI

MFI
MFI
MFI
MFI
MFI
MFI

Size
Granularity

Surface GP Markers

FSC
SSC

GPIIb
GPIIIa
GPIb
GPIX
GPVI
GPIa

anti-CD41 mAb
anti-CD61 mAb
anti-CD42b mAb
anti-CD42a mAb
anti-GPVI mAb
anti-CD49b mAb

Dense granules

Content
After CVX (500 ng/mL)

CVX-induced secretion

After THR (0.5 U/mL)

THR-induced secretion

Content
After CVX (500 ng/mL)

CVX-induced secretion

After THR (0.5 U/mL)

THR-induced secretion

Mepacrine 0.17 µM MFI
MFI
%§
MFI
%§
MFI
MFI
%§
MFI
%§

Mepacrine 1.7 µM

anti-CD62P mAb

Alpha granules

Baseline

ADP 0.5 µM
ADP 5 µm
ADP 50 µm
THR 0.005 U/mL
THR 0.05 U/mL
THR 0.5 U/mL
CVX 5 ng/mL
CVX 50 ng/mL
CVX 500 ng/mL
GPIIb/IIIa Activation

Baseline
ADP 0.5 µM
ADP 5 µm
ADP 50 µm
THR 0.005 U/mL
THR 0.05 U/mL
THR 0.5 U/mL
CVX 5 ng/mL
CVX 50 ng/mL
CVX 500 ng/mL

anti-CD41/CD61 (PAC−1) mAb

(cid:48)

74
(cid:48)
6

739
322 *

(cid:48)

93
(cid:48)
7

372
588

(cid:48)

125
(cid:48)
9

674
610

184 *
229 *
(cid:48)
23
539.0
(cid:48)
033
27
(cid:48)
738
4
(cid:48)
244 *
1

445 *
640 *
(cid:48)

20
22
(cid:48)
5
(cid:48)
1

565.0
(cid:48)
486
491
291 *

225 *
328*
(cid:48)
31
(cid:48)
32
(cid:48)
7
(cid:48)
1

463 *
646 *
851 *
463 *

374
322
−13.9 *
170
−55
618
563
−9 *
199
−68

0.2
182
265 *
524 *
529 *
376
(cid:48)
4
304 *
145 *
5
(cid:48)
682
1
(cid:48)
614 *
3
(cid:48)
132 *
4

(cid:48)

150 *
147 *
155 *
151 *
151 *
158 *
160 *
152 *
150 *
144 *

4.1
99
57 *
(cid:48)

404
304
−25
176
−56
632
516
−18
200
−68

0.8
194
527
(cid:48)
374
1
(cid:48)
322
1
(cid:48)
979
1
(cid:48)
067
6
(cid:48)
585 *
6
(cid:48)
362
4
(cid:48)
918
5
(cid:48)
058
6

376*
583 *
821 *
776 *
(cid:48)
128 *
1
(cid:48)
1
886 *
(cid:48)
621 *
1
656 *
1
544 *
1
574 *
1

(cid:48)

(cid:48)

(cid:48)

5.9
99
64 *
(cid:48)

398
350
−12 *
180
−55
701
651
−7 *
216
−69

1.1
183
314
704
731
(cid:48)
510
1
221
6
(cid:48)
719 *
6
(cid:48)
908
1
781
5
132
6

(cid:48)

(cid:48)

(cid:48)

153 *
151 *
144 *
145 *
141 *
105 *
103 *
128 *
101 *
96 *

1.6
99
50
(cid:48)
741

74
(cid:48)
7

002
073

(cid:48)

(cid:48)

14
25
18
19
(cid:48)
4
(cid:48)
1

(cid:48)

(cid:48)

392
111
647
407
584
485

278
215
−14
162
−37
441
294
−16
181
−58

0.2
168
297
620
766
250
(cid:48)
062
5
(cid:48)
6
757
789
(cid:48)
643
4
609
5

(cid:48)

469
(cid:48)
564
1
(cid:48)
356
4
(cid:48)
6
323
980
(cid:48)
060
9
(cid:48)
017
15
(cid:48)
026
2
(cid:48)
605
5
(cid:48)
293
6

0.8
96
25
(cid:48)
645

13

–
–

–
–
–
–
–
–

–
–
–
–
–
–
–
–
–
–

–
–
–
–
–
–
–
–
–
–
–

–
–
–
–
–
–
–
–
–
–

–
–
–
–

126
(cid:48)
10

189
808

(cid:48)

(cid:48)

21
39
28
27
(cid:48)
7
4

(cid:48)

(cid:48)

(cid:48)

923
696
661
401
518
227

502
377
−41
188
−64
817
565
−53
229
−74

4.9
285
958
(cid:48)
564
2
(cid:48)
081
3
(cid:48)
735
2
(cid:48)
957
9
(cid:48)
321
11
(cid:48)
312
9
(cid:48)
030
10
891
10

(cid:48)

(cid:48)
1
(cid:48)
5
12
16
(cid:48)
5
22
28
12
14
14

(cid:48)

145
049
(cid:48)
182
678
116
(cid:48)
867
176
135
931
789

(cid:48)

(cid:48)

(cid:48)

(cid:48)

4.0
100
55
(cid:48)

116

434

Absolute %
MFI
MFI
MFI
MFI
MFI
MFI
MFI
MFI
MFI
MFI

MFI
MFI
MFI
MFI
MFI
MFI
MFI
MFI
MFI
MFI

Absolute %
Absolute %
Absolute %
MFI

Procoagulant Activity

Annexin V

Baseline
Ionophore
COAT platelets (CVX + THR)

Abbreviations: ADP, adenosine diphosphate; CVX, convulxin; FSC, forward scatter; mAb, monoclonal antibody; MFI,
median fluorescence intensity; SSC, side scatter; THR, thrombin; PAT_GT#, Patient with Glanzmann thrombasthenia
number #. § Percentage decrease in fluorescence in stimulated platelets relative to the basal fluorescence (mepacrine
uptake). * values out of the reference range.

23

561

23

403

31

Moreover, we observed a conserved content of dense granule, assessed by mepacrine staining,
with normal secretion following THR activation but slightly reduced after CVX for PAT_GT1 and
PAT_GT3. Alpha-granule secretion following increasing doses of ADP, THR (except the highest dose),
and CVX was conserved for two patients; only one patient (PAT_GT1) showed a reduced surface
expression of P-selectin following stimulation with increasing concentrations of the agonists.

In our FCA, we also systematically investigated the ability to generate procoagulant COAT
platelets following the combined activation with CVX-plus-THR. Interestingly, all three GT patients
were revealed to generate a higher percentage of procoagulant COAT platelets or at least in the high
range (for PAT_GT3) compared to normal subjects.

Int. J. Mol. Sci. 2020, 21, 9515

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2.2. Procoagulant COAT Platelet Formation in GT Patients

Because of the consistency of our observation, we decided to investigate further the high percentage
of procoagulant COAT platelets measured in the three GT patients. First, this aspect of platelet function
was replicated (n = 2–7) among these three GT patients, and we confirmed that their individual mean
value is in the high range or above our in-house reference range (Figure 1). The group of healthy donors
(HD) reach a median value of 39%, with the 25th-percentile (lower bar) at 33% and the 75th-percentile
(upper bar) at 44%. Taken together, the percentage of procoagulant COAT platelets generated by the
three GT patients is statistically different compared to the group of HD, with PAT_GT1 reaching a value
of 54% ± 2% (mean ± standard deviation). PAT_GT2 attained a value of 57% ± 10.3%, and PAT_GT3
with a value of 56% ± 5%.

Figure 1. Procoagulant collagen-and-thrombin (COAT) platelet formation in patients with Glanzmann
thrombasthenia and healthy donors. Absolute percentage of the Annexin-V-positive platelets following
activation with convulxin-plus-thrombin in healthy donors (HD) and means of at least two replicates
for patients with Glanzmann thrombasthenia (GT). The grey zone (25–55%) indicates the in-house
reference ranges (2.5–97.5 percentiles). Statistical significance was determined by Mann–Whitney test,
*** p < 0.001. 2.3. Intracellular Ion Fluxes Following CVX-Plus-THR Platelet Activation Second, we visualized the effects of combined activation CVX-plus-THR on ion kinetics in GT patients, especially on calcium, which is an important player of the procoagulant response (Figure 2). To assess ion mobilization, platelets were loaded with the respective ion probes (Fluo-3 for calcium, ING-2 for sodium and or IPG-2 for potassium) and fluorescence was acquired over time [26]. Int. J. Mol. Sci. 2020, 21, 9515 5 of 15 Figure 2. Intracellular ion kinetics following platelet activation with convulxin and thrombin. Platelets from healthy donors (HD) or patients with Glanzmann thrombasthenia (GT) were loaded with ion fluorescent indicator. Fluorescence from ion probes was monitored in FL1 (488:530/30) and median fluorescence intensity (MFI) fold change (relative to the baseline [BSL]) for the whole platelet population was plotted over time. (A) Calcium monitoring with Fluo-3. (B) Sodium monitoring with ION NaTRIUM Green-2 (ING-2). (C) Potassium monitoring with ION Potassium Green-2 (IPG-2). Data from HD represented as mean ± SEM, from experiments with platelets from different donors (n = 6). Data for each GT patient (named PAT_GT1, PAT_GT2, and PAT_GT3) are a mean of individual replicates, n = 3 for PAT_GT1, n = 1 for PAT_GT2, and n = 2 PAT_GT3. As illustrated in Figure 2A, the combined platelet activation by CVX-plus-THR resulted in a rapid increase in calcium mobilization. We observed a rapid and sustained calcium mobilization in platelets from HD as well as in the three GT patients. Interestingly, the initial peak mobilization and the time-dependent mobilization are constantly higher in the GT patients than in the control population. Intracellular sodium (Figure 2B) was also characterized by a rapid mobilization after activation followed by a gradual efflux (progressive fluorescence decline) over time. This behavior was observed in HD as well as in GT patients. GT patients seemed to have a slightly higher sodium mobilization than the donors, in particular PAT_GT1 and PAT_GT2. Cytosolic potassium (Figure 2C) showed a progressive decrease in efflux after activation, with the rate of decrease seemingly attenuating over time. In this situation, GT patients did not greatly seem to differ in their kinetics with the HD group, with the sole exception of PAT_GT3 who reached rapidly lower potassium levels following the strong CVX-plus-THR activation. 2.4. Intracellular Calcium during the First Three Minutes after Activation It has been described that procoagulant COAT platelets are generated in the order of 1–2 min after combined CVX-plus-THR activation [18,27]. Since GT patients revealed the ability to generate a higher Int. J. Mol. Sci. 2020, 21, 9515 6 of 15 proportion of procoagulant COAT platelets with a global higher calcium mobilization, we decided to better dissect the calcium kinetics in each GT patient individually with a special focus on this early time frame. Moreover, we co-stained platelets with Annexin V to discriminate the procoagulant COAT platelets, identified as Annexin-V-positive. Therefore, for each GT patient, we individually evaluated the first 3 min post-activation (Figure 3) in comparison with kinetics from HD. In addition to the whole platelet kinetics, we are also able to appreciate the calcium mobilization in Annexin-V-positive platelet as soon as they are generated. Figure 3. Intracellular calcium within first 3 min after activation. Each of the three patients with Glanzmann thrombasthenia (GT) had their first 3 min kinetics after activation plotted with respect to their Fluo-3 median fluorescence intensity (MFI) fold change compared to healthy donors (HD). Co-staining with Cy5 Annexin V allowed differentiating procoagulant COAT platelets (Annexin V positive) from the whole platelet population. (A) PAT_GT1; (B) PAT_GT2; (C) PAT_GT3. HD data are mean ± SEM from experiments with platelets from different donors (n = 6). Data for each GT patient are a mean of individual replicates, n = 3 for PAT_GT1, n = 1 for PAT_GT2, and n = 2 PAT_GT3. Baseline, BSL. As already shown in the previous experiments (Figure 2A), CVX-plus-THR activation leads to a significant increase in calcium mobilization. Within the expected time frame, procoagulant COAT platelets began to appear in the HD kinetics as well as in each GT patient. Subsequently, as indicated in Figure 3A, PAT_GT1 showed a higher calcium mobilization in generated procoagulant COAT platelets than the ones in the HD pool. Figure 3B and 3C confirmed this observation for PAT_GT2 and PAT_GT3, respectively. In this context, it was remarkable that all three GT patients showed a higher initial calcium mobilization following activation in their procoagulant COAT platelet population compared to the HD group (Figure 3A–C). Int. J. Mol. Sci. 2020, 21, 9515 7 of 15 2.5. GPIIb/IIIa Antagonists and Their Effect on COAT Platelet Formation With a higher capacity for the formation of procoagulant COAT platelets in GT patients, the GPIIb/IIIa receptor—missing in this patient population—could be a critical regulator of intracellular calcium mobilization and procoagulant COAT platelet formation. GPIIb/IIIa antagonists, in our case eptifibatide and tirofiban, seemed an enticing and simple approach to block the GPIIb/IIIa receptor and thus mimic the effect of GT pathophysiology. This approach would potentially allow the evaluation of GPIIb/IIIa blockage on procoagulant COAT platelet formation in an in vitro model. Platelets from HD were pretreated with increasing concentrations of eptifibatide or tirofiban and were activated with CVX-plus-THR. Then, we evaluated the percentage of Annexin-V-positive platelets, namely procoagulant COAT platelets, at each drug concentration point. Figure 4 displays the relative change in Annexin-V-positive events with respect to the antagonist vehicle (H2O or DMSO). The data did not show any remarkable and meaningful difference between basal conditions and increasing concentrations of either eptifibatide (Figure 4A) or tirofiban (Figure 4B). GPIIb/IIIa-antagonists effect on the formation of procoagulant COAT platelets. Figure 4. Platelets were pretreated with increasing doses of (A) eptifibatide or (B) tirofiban, and activated with convulxin-plus-thrombin. Relative change of the fraction of Annexin-V-positive platelets compared to control condition (vehicle H2O or DMSO, respectively) is represented as mean ± SEM from experiments with platelets from different donors (n = 3–5). 3. Discussion Blood platelets are crucial and active players of primary and secondary hemostasis. They contribute to forming a stable hemostatic plug in order to stop bleeding and prevent further blood loss. In order to achieve an efficient hemostatic response, activation of the fibrinogen receptor GPIIb/IIIa and to Int. J. Mol. Sci. 2020, 21, 9515 8 of 15 another extent surface expression of negatively charged amino-phospholipids are both essential for platelet bridging and aggregation, and clot stabilization by fibrin, respectively [12]. GT is a rare autosomal recessive inherited platelet dysfunction. The underlying deficiency is defined by the dysfunctional receptor, low expression or complete absence thereof [8]. Due to the absence or dysfunction of the GPIIb/IIIa receptor, activated platelets fail to aggregate with each other by means of fibrinogen binding [3,8]. At our institution, we systematically apply FCA as an extension to our diagnostic workup for patients with a bleeding diathesis in whom laboratory analysis could not identify a cause ([25] and Adler et al., manuscript in preparation) or in selected instances. In the case of these three GT patients, after a complete clinical and laboratory workup, including platelet aggregation tests, FCA confirmed the absence of GPIIb/IIIa at the platelet surface in all three GT patients and the impaired binding of PAC-1 following activation with increasing doses of ADP, THR, and CVX. Moreover, GT patients were revealed to have conserved platelet size and morphology, and the surface density for other GP was normal, as already documented [28–30]. A single exception was for GPIa. GPIa is a subunit of the adhesion collagen receptor GPIa/IIa (integrin α2β1), which plays an essential role to mediate platelet adhesion to collagen. It is known that the single nucleotide polymorphism C807T in the integrin alpha 2 gene (ITGA2) correlates with platelet GPIa/IIa density [31]. This gene polymorphism—with 807T allele expressing the highest receptor density, 807C the lowest and heterozygous individual expressing intermediate levels—was documented to be present in GT population, and GT patients with homozygous 807C allele were correlated with clinical bleeding severity [7,8]. The lower alpha-granule secretion observed in PAT_GT1 is congruent with its slightly lower granularity according to SSC properties. Common to two of our GT patients is the lower dense-granule secretion following CVX activation. Interestingly, the initial phase of GPIIb/IIIa outside-in signaling resembles that induced by the collagen receptor GPVI, with activation of Src-family kinases and Syk [3,32]. Therefore, the lacking outside-in signaling in our GT patients would impair the part of the activation pathway needed for appropriate granule secretion. The original contribution of our work is the investigation of the procoagulant platelet activity in GT patients. Not much is known about the characteristics of procoagulant COAT platelets in the context of dysfunctional hemostasis. By confirming the presence of procoagulant COAT platelets in our GT patients and comparing them to a pool of HD, we could indeed establish a higher potency of procoagulant platelet formation in our GT patients (Figure 1). To our knowledge, the capacity of procoagulant COAT platelet formation in GT patients has been poorly evaluated, in particular, it has not been comprehensively compared to reference ranges established in healthy subjects [25,33]. In the few publications present in the literature, the number of procoagulant platelets in GT patients differ and they have lower values than the ones we obtained [34–38]. However, the sample preparation in these studies (gel-filtration or washed platelets) differs from our protocol (diluted PRP). Of note, it was reported that, at least in GT patients, different platelet reactivity was observed in gel-filtered platelets compared to diluted-PRP [39] as well as according to the experimental setting (such as the use of stirring or not) [40]. Therefore, we can barely compare our results with the sparse data present in the literature and we rely on the comparison with our in-house ranges established with the same technique in HD. The investigation of procoagulant COAT platelets in the context of GT is also of great interest in order to study and explore the role of GPIIb/IIIa in the formation of procoagulant COAT platelets. Indeed, COAT platelets gradually inactivate the GPIIb/IIIa fibrinogen receptor to become procoagulant, through surface exposure of PS. We were therefore interested to explore if we could take advantage of this natural model in order to investigate and better understand the role of GPIIb/IIIa, increasing the knowledge already generated by several studies in the past [14,17,18]. To this end, we used a flow-cytometry based approach to monitor ion fluxes (notably calcium, sodium, and potassium) in our three patients affected by GT. Moreover, flow cytometry allowed for