<figure><img src="image_1.png" /></figure>

HAL
open science

# Evidence of Toll-like receptor molecules on human platelets

Fabrice Cognasse, Hind Hamzeh, Patricia Chavarin, Sophie Acquart,

Christian Genin, Olivier Garraud

► To cite this version:

Fabrice Cognasse, Hind Hamzeh, Patricia Chavarin, Sophie Acquart, Christian Genin, et al.. Evidence of Toll-like receptor molecules on human platelets. Immunology and Cell Biology, 2005, 83 (2), pp.196-198. 10.1111/j.1440-1711.2005.01314.x. hal-04935242

HAL Id: hal-04935242

https://hal.science/hal-04935242v1

Submitted on 7 Feb 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
**Brief Communication**

# Evidence of Toll-like receptor molecules on human platelets

FABRICE COGNASSE,<sup>1,2</sup> HIND HAMZEH,<sup>2</sup> PATRICIA CHAVARIN,<sup>1</sup>
SOPHIE ACQUART,<sup>1</sup> CHRISTIAN GENIN<sup>2</sup> and OLIVIER GARRAUD<sup>1,2</sup>

¹The Auvergne-Loire Regional Blood Bank (EFS), Auvergne-Loire and ²Mucosal Immunity and Pathogen Immunity Group (GIMAP-EA3064), Faculty of Medicine, University of Saint-Etienne, Saint-Etienne, France

**Summary** Platelets are primarily involved in thrombosis and haemostasis, and they have recently been shown to have a role in innate immunity and in inflammation. We have determined the markers of innate immunity that are expressed by platelets, specifically the Toll-like receptors (TLR), originating from mixes of platelet concentrates (MPC, *n* = 5) between day zero and day five after blood collection. The surface membrane and intracellular expression of TLR were measured, both after and without permeabilization, using flow cytometry. We observed weak expression of TLR2, TLR4 and TLR9 on the surface of CD41⁺ platelets. The expression levels of TLR4 were high (59 ± 2.2%). Moreover, there was a significant expression of TLR2 (47.5 ± 4.8%), TLR4 (78.8 ± 1.3%) and TLR9 (34.2 ± 7.5%) in the cytoplasm of CD41⁺ platelets. The expression of the three receptors did not change significantly during the course of the 5 day observation period. The percentage of TLR expression is significantly modulated between activated versus non-activated platelets, both after and without permeabilization (*P* < 0.01). Study of the expression of TLR could increase our knowledge of the level of platelet participation during an immune reaction and inflammation. In the same way as the platelet ligand/receptor pair CD40L/CD40 is, the TLR are expressed by platelets, and could serve as a link between innate and adaptive immunity.

**Key words:** adaptive immunity, cytometry, innate immunity, platelet, Toll-like receptor (TLR).

**Introduction**

Platelets are non-nucleated cellular elements that have a role in the process of haemostasis (adhesion and aggregation). Platelets also have a role in innate immunity<sup>1,2</sup> and in inflammation.<sup>3</sup> We have endeavoured to determine the markers of innate immunity that are expressed by platelets and our studies have become focused on the Toll-like receptors (TLR). The TLR are thought to be of primary importance in the early processes of immunity. Eleven members of the mammalian TLR family have been reported to date (TLR1–11).<sup>4,5</sup> Each member of the TLR family recognizes distinct pathogen-associated molecular pattern (PAMP) molecules. TLR1, 2, 4, 5 and 6 are engaged in the recognition of bacterial PAMP such as LPS, peptidoglycan, lipoprotein and flagellin. TLR3, 7, 8 and 9 recognize nucleotide derivatives.<sup>6–8</sup> To the best of our knowledge, only the team of Montrucchio *et al*. (who studied the LPS ligand TLR4) have shown that platelets do not fix LPS and present neither TLR4 nor CD14 (LPS binding site) on their surface membrane.<sup>9</sup> Most of the studies concerning the expression of TLR, either on the cell surface or in the cytoplasm, for a diverse range of cell types, have been done using molecular biology techniques that are not adapted for non-nucleated cells such as platelets. The fact that very few anti-TLR monoclonal antibodies validated for flow cytometry are commercially available is probably the origin of the lack of detailed studies concerning platelets.

Correspondence: Professor Olivier Garraud, GIMAP-EA 3064, Université de Saint-Etienne, Faculté de Médecine, 15 Rue Ambroise Paré, 42023 Saint-Etienne Cedex 2, France.
Email: olivier.garraud@univ-st-etienne.fr
Received 18 June 2004; accepted 2 November 2004.

**Materials and methods**

We studied the expression of platelet TLR molecules (originating from five mixes of platelet concentrates [MPC], prepared under conditions authorized for the distribution of platelets to patients). This study was performed over a 5 day period corresponding to the period during which platelets can be delivered for blood transfusion; more precisely, from day zero to day five after blood collection. The expression of TLR was measured both at the surface membrane and intracellularly.

The preparation of MPC was performed by a double centrifugation of buffy coats, followed by leucofiltration. The Baxter OptiPure PLT DGR7012 device (Baxter, Maurepas, Belgium) allows the preparation of plate mixtures of platelet concentrates starting from buffy coats. Pockets of 450 mL of total blood were taken and centrifuged after 16–18 h of storage at 20°C. The extraction of diluted buffy coat was performed with the Baxter Optipress II device (Baxter). The buffy coat was stored for a minimum of 2 h without agitation in an enclosure regulated at 22°C. The mix was prepared for five buffy coats connected by sterile connection. After centrifugation, extraction of the platelet-rich supernatant was performed by Optipress II; the filtration was carried out during this step under pressure. The counting of the platelets was performed using the automated Abx (Abx Diagnostics, Montpellier, France) and the counting of the residual red globules was measured by flow cytometry using the leucoCOUNT kit (Becton Dickinson, Le Pont de Claix, France). The MPC were kept, under constant agitation, at 22°C for 5 days. MPC did not undergo any specific transformation. Platelets were recovered at different time points during their observation (days zero, one, two, three and five). The antibodies used to detect the expression of TRL1, 2, 4, 6, 8 and 9 were obtained from Imgenex (San Diego, CA, USA). These antibodies are validated for cytometry by their supplier. TLR2, 4, 8 and 9 were directly phycoerythretin (PE)-labelled. CD41 is expressed by platelets and stem cell precursors.<sup>10</sup> Antibodies against
**Figure 1** Toll-like receptor (TLR) expression over time by human platelets in mixes of platelet concentrates (MPC; day five). (a) TLR are expressed at the cell surface or intracellularly in platelets. Shown is the percentage of intracellular or surface expression (mean ± SD) of CD41⁺ platelets expressing the various TLR (n = 5). (b) TLR2 (panel 1), TLR4 (panel 2) and TLR9 (panel 3) was detected by antibody labelling and flow cytometry analysis after gating for CD41⁺. The histogram indicates fluorescence with isotype control (grey line); platelets after permeabilization (■) or not permeabilized (□). One representative experiment from five is shown.

<figure><img src="image_3.png" /></figure>

the platelet Ag CD41 (FITC conjugated) and against an Ag expressed by activated platelets (i.e. CD62p; APC conjugated) were purchased from BD Biosciences (Le Pont de Claix, France). Surface expression of CD62P was determined as a measure of platelet activation. Platelets were fixed in paraformaldehyde (3.6%; 10⁶ platelets/mL). Platelets were permeabilized using Triton (0.4×) or not permeabilized. An antimouse PE-coupled secondary antibody (BD Biosciences) was used to mark (indirectly) TLR1 and 6. Isotype matched mouse FITC-, allophycocyanin (APC)- and PE-conjugated antibodies of irrelevant specificity were used as negative controls (BD Biosciences). All antibody incubations were carried out in the presence of an Fc-blocking agent (BD Biosciences). This step was of particular importance because platelets are reputed to express FcγRII molecules capable of non-specific antibody binding via the Fcγ. The analysis by flow cytometry was performed by FACScalibur and CellQuest-Pro software (BD Biosciences).

## Results and discussion

We observed weak expression of TLR2 (18 ± 2.4%), TLR4 (59 ± 2.1%) and TLR9 (10.1 ± 4.2%) (independently of non-specific ligation of Ab by FcγRII) on the surface of CD41⁺ platelets (non-permeabilized). The percentage of intracellular

expression of TLR 2, 4 and 9 was more significantly augmented (after permeabilization): TLR2 (47.5 ± 4.8%, *P* < 0.001), TLR4 (78.7 ± 1.2%, *P* < 0.001) and TLR9 (34.2 ± 7.5%, *P* < 0.001) (Fig. 1). The platelet expression of TLR1, 6 and 8 was very low, whether permeabilized or not. The expression of the three receptors (TLR2, 4 and 9) did not change significantly during the course of the 5 day observation period (data not shown). We observed a significant modulation of TLR expression on activated platelets (CD41⁺/CD62p⁺) versus non-activated platelets (CD41⁺/CD62p⁻). Indeed, we showed (in Figure 2) that TLR2 (14.5 ± 4.2%, *P* < 0.01) and TLR9 (7.8 ± 2.6%, *P* < 0.01), on the platelet membrane, were increased on activated versus non-activated platelets. However, we noted a significant decrease in the expression of TLR4 (24 ± 1.4%, *P* < 0.01) on activated versus non-activated platelets. Concerning the intracellular percentage of TLR2, 4 and 9, we observed a significant increase with activated platelets (31.7 ± 5.3%, *P* < 0.01; 64.7 ± 0.9%, *P* < 0.01; and 31.9 ± 8.1%, *P* < 0.01 for TLR2, 4 and 9, respectively).

It is important to note that because platelets are known to absorb many substances from plasma, soluble TLR have not been commonly described, apart from sTLR2.¹¹ Therefore,
<figure><img src="image_4.png" /><figcaption>**Figure 2** The percentage of Toll-like receptor (TLR) expression (TLR2, 4 and 9), after gating for CD41⁺, for non-activated platelets (CD41⁺CD62⁻; ■) or activated platelets (CD41⁺CD62⁻ □). One representative of five independent experiments is shown. Inter-experiment comparisons in permeabilization versus no permeabilization (Fig. 1A) or activated versus non-activated (Fig. 2) were analysed by means of the Wilcoxon paired test (Statview, SAS Institute, Cary, NC, USA). The asterisk (*) represents statistical differences.</figcaption></figure>

the platelet TLR observed here is likely to originate from platelets. The precise role of these TLR markers has not been elucidated and furthermore, the reason that a significant percentage of the platelets (for the most part activated) do express TLR2, 4 and 9 (Fig. 2) is not clear. The present observation suggests the existence of a distinct subpopulation of platelets, considering the extreme stability of expression of TLR over the 5 day period.

The presence of TLR in the cytoplasm of nucleated cells, but not necessarily at their surface, has been described.⁸ TLR expression on the membrane or inside the cell cytoplasm, involved in the recognition of pathogens (parasite, bacterial or viral) before or after endocytosis, could participate in the antibacterial activity of platelets and the liberation of anti-microbial peptides (platelet factor 4, RANTES, connective tissue activating peptide 3, platelet basic protein, thymosin beta-4, fibrinopeptide B and fibrinopeptide A),¹² notably during the microbial colonization of endothelial lesions.

The specificity of TLR recognition for many PAMP molecules has been well documented,⁶ including (i) peptidoglycan, bacterial lipoproteins and zymosan recognition by TLR2; (ii) LPS and heat-shock protein recognition by TLR4; and (iii) CpG motifs of bacterial DNA recognition by TLR9.

The study of the expression of the TLR, important molecules of the innate immune system, could further increase our knowledge of the level of platelet participation during an immune reaction and during inflammation.¹ TLR are essential elements of host defence against pathogens, due to the activation of the innate immune system, a prerequisite for the induction of the adaptive immune system.² In the same way as the platelet ligand/receptor pair CD40L/CD40 does, the TLR, expressed by platelets, could serve as a link between innate and adaptive immunity.

## Acknowledgements

The authors gratefully acknowledge the technical help of Odile Sabido and Françoise Boussoulade. They thank deeply Mr Philip Lawrence for kindly revising the manuscript. Financial support was from a grant from the Convention Interrégionale Massif Central – Réseau Switch – MENRT 01Y0242b co-held by Professor O. Garraud and Professor M. Cogné, University of Limoges, UMR CNRS 6101.

## References

1. Danese S, de la Motte C, Reyes BM, Sans M, Levine AD, Fiocchi C. Cutting edge: T cells trigger CD40-dependent platelet activation and granular RANTES release: a novel pathway for immune response amplification. *J. Immunol.* 2004; **172**: 2011–15.

2. Elzey BD, Tian J, Jensen RJ *et al*. Platelet-mediated modulation of adaptive immunity. A communication link between innate and adaptive immune compartments. *Immunity* 2003; **19**: 9–19.

3. Klinger MH, Jelkmann W. Role of blood platelets in infection and inflammation. *J. Interferon Cytokine Res.* 2002; **22**: 913–22.

4. Chuang T, Ulevitch RJ. Identification of hTLR10: a novel human Toll-like receptor preferentially expressed in immune cells. *Biochim. Biophys. Acta* 2001; **1518**: 157–61.

5. Zhang D, Zhang G, Hayden MS *et al*. A toll-like receptor that prevents infection by uropathogenic bacteria. *Science* 2004; **303**: 1522–6.

6. Imler JL, Hoffmann JA. Toll receptors in innate immunity. *Trends Cell Biol.* 2001; **11**: 304–11.

7. Underhill DM. Toll-like receptors: networking for success. *Eur. J. Immunol.* 2003; **33**: 1767–75.

8. Takeda K, Kaisho T, Akira S. Toll-like receptors. *Annu. Rev. Immunol.* 2003; **21**: 335–76.

9. Montrucchio G, Bosco O, Del Sorbo L *et al*. Mechanisms of the priming effect of low doses of lipopoly-saccharides on leukocyte-dependent platelet aggregation in whole blood. *Thromb. Haemost.* 2003; **90**: 872–81.

10. Halle P, Rouzier C, Kanold J *et al*. Ex vivo expansion of CD34+/CD41+ late progenitors from enriched peripheral blood CD34+ cells. *Ann. Hematol.* 2000; **79**: 13–19.

11. LeBouder E, Rey-Nores JE, Rushmere NK *et al*. Soluble forms of Toll-like receptor (TLR) 2 capable of modulating TLR2 signaling are present in human plasma and breast milk. *J. Immunol.* 2003; **171**: 6680–89.

12. Tang YQ, Yeaman MR, Selsted ME. Antimicrobial peptides from human platelets. *Infect. Immun.* 2002; **70**: 6524–33.