Thromb Haemost 2019; 119(01): 179-182
DOI: 10.1055/s-0038-1676349
Letter to the Editor
Georg Thieme Verlag KG Stuttgart · New York

Anaphylatoxin Receptor C3aR Contributes to Platelet Function, Thrombus Formation and In Vivo Haemostasis

Reinhard J. Sauter*
1  Department of Cardiology and Cardiovascular Medicine, University Clinic, Eberhard Karls University of Tübingen, Tübingen, Germany
2  Section for Cardioimmunology, Eberhard Karls University of Tübingen, Tübingen, Germany
,
Manuela Sauter*
2  Section for Cardioimmunology, Eberhard Karls University of Tübingen, Tübingen, Germany
,
Marcus Obrich
2  Section for Cardioimmunology, Eberhard Karls University of Tübingen, Tübingen, Germany
,
Frederic N. Emschermann
2  Section for Cardioimmunology, Eberhard Karls University of Tübingen, Tübingen, Germany
,
Henry Nording
1  Department of Cardiology and Cardiovascular Medicine, University Clinic, Eberhard Karls University of Tübingen, Tübingen, Germany
2  Section for Cardioimmunology, Eberhard Karls University of Tübingen, Tübingen, Germany
,
Johannes Patzelt
1  Department of Cardiology and Cardiovascular Medicine, University Clinic, Eberhard Karls University of Tübingen, Tübingen, Germany
2  Section for Cardioimmunology, Eberhard Karls University of Tübingen, Tübingen, Germany
,
Hans Peter Wendel
3  Department of Thoracic and Cardiovascular Surgery, University Clinic, Eberhard Karls University of Tübingen, Tübingen, Germany
,
Jan-Christian Reil
4  Klinik für Innere Medizin II, Kardiologie, Angiologie und Internistische Intensivmedizin, Universitätsklinikum Schleswig-Holstein, Campus Lübeck, Lübeck, Germany
,
Frank Edlich
5  Institute for Biochemistry and Molecular Biology, University of Freiburg, Freiburg, Germany
6  BIOSS, Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany
,
Harald F. Langer
1  Department of Cardiology and Cardiovascular Medicine, University Clinic, Eberhard Karls University of Tübingen, Tübingen, Germany
2  Section for Cardioimmunology, Eberhard Karls University of Tübingen, Tübingen, Germany
› Author Affiliations
Funding This work was supported by the Volkswagen Foundation (Lichtenberg program), German Heart Foundation and Wilhelm Sander Foundation (H.F.L). H.F.L. is a member of the Tuebingen platelet investigative consortium (TuePIC) funded by the Deutsche Foschungsgemeinschaft (DFG, German Research Foundation) KFO 274–Platelets – basic mechanisms and clinical implications and the SFB (Projektnummer 374031971 - TRR 240 - TP07). F.E. is supported by the Emmy Noether program of the DFG, the Else Kroener Fresenius Foundation and the German Research Council (SFB 746).
Further Information

Publication History

21 May 2018

17 October 2018

Publication Date:
31 December 2018 (online)

Complement is an essential part of our innate immune system, which is important for the host defence reaction against infectious intruders, but also participates in processes and mechanisms beyond the immune response such as synapse maturation, clearance of immune complexes, tissue remodelling after injury or angiogenesis.[1] [2] [3] During vascular and tissue injury, initiation of the complement cascade is triggered in close spatiotemporal proximity to platelet activation and thrombosis.[4] [5] [6] Beyond their classical role as thrombosis-mediating cells, platelets were recently implicated in tissue remodelling processes such as apoptosis.[7] [8] Furthermore, platelets contribute to the immediate inflammatory response after vascular injury and trigger atherosclerosis by promoting vascular inflammation.[9] At late stages of atherosclerotic diseases, for example, stroke or myocardial infarction, platelets are central for atherothrombosis and ultimately mediate formation of a vessel occluding thrombus with subsequent tissue ischaemia and organ damage.[10] [11] Whether complement receptors influence platelet activation and thrombus formation is not extensively characterized, so far. It was reported before, that C3b, which is generated after the cleavage of C3 by C3 convertases, can activate platelets and thereby may mediate thrombosis formation.[5] A role of the anaphylatoxin C3a—the other cleavage product of C3—which propagates its inflammatory reactions via its cognate receptor C3aR,[12] has not been studied sufficiently. Interestingly, normal human haematopoietic stem/progenitor cells and lineage-expanded haematopoietic precursors express functional C3aR.[13]

In line with this reported finding, we detected C3aR protein in human and murine platelets using Western blot ([Fig. 1A], [B]). Because of the C3aR expression on platelets, we sought to characterize a functional relevance of this anaphylatoxin receptor for platelet function. As dynamic platelet adhesion at endothelial wounds plays a crucial role in thrombus formation,[14] we analysed the role of the C3aR on platelets under arterial shear rates using a flow chamber approach with sub-endothelial matrix protein coating. Interestingly, the adhesion of platelets to fibrinogen was significantly increased on fibrinogen after stimulation with C3a using high shear rates (1.700 s−1) ([Supplementary Fig. S1], available in the online version). In line with those findings, murine platelets, which are deficient for C3aR, show a lower pronunciation of spreading on fibrinogen in comparison to wild-type (WT) platelets ([Fig. 1C]). Using conventional ex vivo aggregometry, we found a significant increase of platelet aggregation after co-stimulation of platelets with C3a and adenosine diphosphate (ADP) ([Supplementary Fig. S2], available in the online version). It is known that C3a receptor and C5a receptor signalling via phosphatidylinositide 3-kinase (PI3-K) results in co-stimulatory and survival signals to naive CD4+ T cells.[15] In platelets, the PI3-K/Akt signalling pathway plays a crucial role when the inflammatory chemokine CXCL16 triggers platelet activation and adhesion.[16]

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Fig. 1 (A) Protein expression of C3aR on human platelets was analysed by Western blot. Chinese hamster ovary (CHO) cells served as negative control, and human dendritic cells (DCs) served as positive control. Shown is one representative blot (n = 3). (B) Murine C3aR was detected by Western blot in cell lysates of isolated wild-type (WT) platelets. CHO cells served as negative control. Shown is one representative blot (n = 3). (C) Isolated WT and C3aR−/− platelets were stimulated with thrombin (0.01 U/mL) and adhesion to immobilized fibrinogen was analysed after 30 minutes. The samples were examined with a Cryo-FIBSEM (Auriga CrossBeamWorkstation, Oberkochen, Germany) at 3 keV with a magnification from 200,000 to 6,000 to evaluate the morphology of the cells. The analysis of the spreading of C3aR-/- platelets showed a lower pronunciation of spreading in comparison to WT platelets. Shown is one representative platelet per group. (D) Platelet aggregation was analysed in murine WT and C3aR−/− platelets. After stimulation with 4 µM adenosine diphosphate (ADP), 200 ng/mL C3a and the selective PI3-kinase β inhibitor TGX221 (25 nM), aggregation was decreased in comparison to platelets stimulated with 4 µM ADP, 200 ng/mL C3a and control (dimethyl sulfoxide [DMSO]). Shown is one representative result (n = 3). (E) Rap1 pull down assay for detection of activated Rap1 in murine platelet lysates of WT mice stimulated with C3a (200 nM) and selective PI3-K isoform-specific inhibitors or control (DMSO). Activated Rap1 in murine platelet lysates was isolated with glutathione S-transferase (GST)-tagged sepharose, subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted and detected with an activation specific Rap1 antibody. The amount of total Rap1 is equal in each sample. One representative blot of three experiments is depicted. (F) Platelet aggregation was analysed in platelet-rich plasma of C3aR−/− and WT mice using conventional aggregometry. The platelets were stimulated with 2 µM ADP, 200 nM C3a ± selective inhibition of PI3 kinase ß (TGX221). After selective inhibition of PI3 kinase ß (TGX221), there was no relevant difference between WT and C3aR−/− platelets left. Shown is one representative experiment (n = 3). (G) Tail-bleeding time was assessed in anaesthetized C3aR−/− mice and WT mice after amputation of the tail tip. C3aR−/− mice showed prolonged bleeding time compared to WT mice. n = 7; *p < 0.05. Data represent mean ± standard deviation (SD). (H) In C3aR−/− mice, tail-bleeding time was measured after intravenous injection of isolated WT or C3aR-/- platelets (1 × 108/mouse). In C3aR−/− mice transfused with WT platelets, bleeding time was significantly shorter than in those transfused with C3aR−/− platelets. Data represent mean ± SD, n = 8 (for C3aR−/− mice reconstituted with C3aR−/− platelets), n = 9 (for C3aR−/− mice reconstituted with WT platelets). *p < 0.05. Data represent mean ± SD.

As recently reported, PI3-K gets phosphorylated after treatment of platelets with C3a ([Supplementary Fig. S3], available in the online version) and this results in activation of Rap1b.[17] Here, we found that inhibition of PI3-K using Wortmannin[18] results in decreased platelet aggregation compared to vehicle control ([Fig. 1D]). To investigate this pathway in more detail, we made use of PI3-K isoform-selective inhibitors. In a Rap1 activation assay, activation of Rap1 after stimulation with C3a was inhibited by a selective PI3-K ß and γ inhibitor ([Fig. 1E]). To verify this finding, we tested one of these isoforms. Indeed, increased aggregation by C3a was no longer observed after selective inhibition of PI3-K ß ([Fig. 1F]). These in vitro and ex vivo data indicate that C3a stimulation results in platelet activation. Thus, we went on to analyse an in vivo relevance of the platelet receptor C3aR and made use of mice deficient for C3aR.[19] In line with our in vitro findings, we observed a significantly prolonged time to bleeding arrest in mice deficient for C3aR when compared with WT C57BL/6 mice using the tail-bleeding assay ([Fig. 1G]). To demonstrate cell specificity, we transfused C3aR−/− or C3aR+/+ platelets (1 × 108 platelets/mouse) into C3aR−/− mice. Before any specific analysis, we characterized this in vivo approach. For instance, we determined the percentage of platelets remaining in the recipient's circulation. In accordance with previous reports,[20] about 14% of the donor platelets were detectable in the recipient's circulation 1 hour after transfusion ([Supplementary Fig. S4], available in the online version). To verify successful platelet C3aR reconstitution, we isolated platelets 24 hours after transfusion and were able to detect C3aR+/+ platelets in C3aR−/− mice using Western blot ([Supplementary Fig. S5], available in the online version). Using this model, we were now able to scrutinize a functional relevance of the platelet C3aR for bleeding arrest after injury. Strikingly, the phenotype of a prolonged bleeding time was reversed after transfusion of C3aR+/+ platelets into C3aR−/− mice ([Fig. 1H]), demonstrating cell specificity of our observations.

Previously, it was reported that C3−/− mice and C3+/− mice show an increased bleeding time.[21] This is on the one hand explained by C3b.[5] Extending this observation, we describe here that C3a, the second cleavage product of C3, mediates platelets activation in vitro and contributes to bleeding arrest after injury in vivo. Here, we provide evidence that C3a-mediated activation of Rap1b is influenced by the PI3-K isoform ß, and may also have minor influence on the isoforms γ and δ. In line with previous findings,[22] [23] these data support the hypothesis that C3aR activation may modulate the ADP–Rap1 pathway. In patients with atherosclerosis, expression of the anaphylatoxin receptors C3aR and C5aR show a significant correlation with platelet activation markers.[24] Future investigations will have to further elucidate the relevance of the platelet C3aR in patients with atherosclerosis and atherothrombotic complications. In turn, platelets may also modulate functions of the complement system. Particularly, in diseases such as atypical haemolytic uremic syndrome and paroxysmal nocturnal haemoglobinuria, the complement system plays a crucial role for pathogenesis, progression and complications. For some time, it is known that a close association between propagation of the complement cascade and development of micro-thrombi mediating organ damage exists.[25] In recent years, a therapy with eculizumab, which inhibits the cleavage of C5 by the C5 convertase into C5a and thereby tones down clinical symptoms such as organ micro-thrombosis, has been clinically established.[26] Furthermore, new drugs targeting complement components are undergoing clinical evaluation[27] [28] [29] and should be tested for a direct impact on platelet activation and thrombosis.

In conclusion, understanding the crosstalk of platelets with the complement system is important to apprehend the exact role of this interplay for platelet and complement activation and resulting diseases featuring thromboinflammation.

Note

For materials and methods, please see [Supplementary Material] (available in the online version).


* These authors contributed equally to this work.


Supplementary Material