Anaphylatoxin Receptor C3aR Contributes to Platelet Function, Thrombus Formation and In Vivo HaemostasisFunding 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).
21 May 2018
17 October 2018
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.   During vascular and tissue injury, initiation of the complement cascade is triggered in close spatiotemporal proximity to platelet activation and thrombosis.   Beyond their classical role as thrombosis-mediating cells, platelets were recently implicated in tissue remodelling processes such as apoptosis.  Furthermore, platelets contribute to the immediate inflammatory response after vascular injury and trigger atherosclerosis by promoting vascular inflammation. 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.  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. A role of the anaphylatoxin C3a—the other cleavage product of C3—which propagates its inflammatory reactions via its cognate receptor C3aR, has not been studied sufficiently. Interestingly, normal human haematopoietic stem/progenitor cells and lineage-expanded haematopoietic precursors express functional C3aR.
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, 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. In platelets, the PI3-K/Akt signalling pathway plays a crucial role when the inflammatory chemokine CXCL16 triggers platelet activation and adhesion.
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. Here, we found that inhibition of PI3-K using Wortmannin 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. 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, 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. This is on the one hand explained by C3b. 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,  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. 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. 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. Furthermore, new drugs targeting complement components are undergoing clinical evaluation   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.
For materials and methods, please see [Supplementary Material] (available in the online version).
* These authors contributed equally to this work.
- 1 Ricklin D, Hajishengallis G, Yang K, Lambris JD. Complement: a key system for immune surveillance and homeostasis. Nat Immunol 2010; 11 (09) 785-797
- 2 Langer HF, Chung KJ, Orlova VV. , et al. Complement-mediated inhibition of neovascularization reveals a point of convergence between innate immunity and angiogenesis. Blood 2010; 116 (22) 4395-4403
- 3 Hertle E, van Greevenbroek MM, Arts IC. , et al. Distinct associations of complement C3a and its precursor C3 with atherosclerosis and cardiovascular disease. The CODAM study. Thromb Haemost 2014; 111 (06) 1102-1111
- 4 Amara U, Rittirsch D, Flierl M. , et al. Interaction between the coagulation and complement system. Adv Exp Med Biol 2008; 632: 71-79
- 5 Del Conde I, Crúz MA, Zhang H, López JA, Afshar-Kharghan V. Platelet activation leads to activation and propagation of the complement system. J Exp Med 2005; 201 (06) 871-879
- 6 Subramaniam S, Jurk K, Hobohm L. , et al. Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development. Blood 2017; 129 (16) 2291-2302
- 7 Schleicher RI, Reichenbach F, Kraft P. , et al. Platelets induce apoptosis via membrane-bound FasL. Blood 2015; 126 (12) 1483-1493
- 8 Kraft P, Schleicher R, Olbrich M. , et al. Platelet derived FasL contributes to apoptosis in stroke. Thromb Haemost 2016; 116 (05) 998-1000
- 9 Langer HF, Gawaz M. Platelet-vessel wall interactions in atherosclerotic disease. Thromb Haemost 2008; 99 (03) 480-486
- 10 Massberg S, Gawaz M, Grüner S. , et al. A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo. J Exp Med 2003; 197 (01) 41-49
- 11 Distelmaier K, Adlbrecht C, Jakowitsch J. , et al. Local complement activation triggers neutrophil recruitment to the site of thrombus formation in acute myocardial infarction. Thromb Haemost 2009; 102 (03) 564-572
- 12 Hajishengallis G, Reis ES, Mastellos DC, Ricklin D, Lambris JD. Novel mechanisms and functions of complement. Nat Immunol 2017; 18 (12) 1288-1298
- 13 Reca R, Mastellos D, Majka M. , et al. Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1. Blood 2003; 101 (10) 3784-3793
- 14 Ruggeri ZM. Platelets in atherothrombosis. Nat Med 2002; 8 (11) 1227-1234
- 15 Strainic MG, Liu J, Huang D. , et al. Locally produced complement fragments C5a and C3a provide both costimulatory and survival signals to naive CD4+ T cells. Immunity 2008; 28 (03) 425-435
- 16 Borst O, Münzer P, Gatidis S. , et al. The inflammatory chemokine CXC motif ligand 16 triggers platelet activation and adhesion via CXC motif receptor 6-dependent phosphatidylinositide 3-kinase/Akt signaling. Circ Res 2012; 111 (10) 1297-1307
- 17 Sauter RJ, Sauter M, Reis ES. , et al. A functional relevance of the anaphylatoxin receptor C3aR for platelet function and arterial thrombus formation marks an intersection point between innate immunity and thrombosis. Circulation 2018; 138 (16) 1720-1735
- 18 Yin H, Stojanovic A, Hay N, Du X. The role of Akt in the signaling pathway of the glycoprotein Ib-IX induced platelet activation. Blood 2008; 111 (02) 658-665
- 19 Kildsgaard J, Hollmann TJ, Matthews KW, Bian K, Murad F, Wetsel RA. Cutting edge: targeted disruption of the C3a receptor gene demonstrates a novel protective anti-inflammatory role for C3a in endotoxin-shock. J Immunol 2000; 165 (10) 5406-5409
- 20 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 (01) 9-19
- 21 Gushiken FC, Han H, Li J, Rumbaut RE, Afshar-Kharghan V. Abnormal platelet function in C3-deficient mice. J Thromb Haemost 2009; 7 (05) 865-870
- 22 Jackson SP, Schoenwaelder SM, Goncalves I. , et al. PI 3-kinase p110beta: a new target for antithrombotic therapy. Nat Med 2005; 11 (05) 507-514
- 23 Cosemans JM, Munnix IC, Wetzker R, Heller R, Jackson SP, Heemskerk JW. Continuous signaling via PI3K isoforms beta and gamma is required for platelet ADP receptor function in dynamic thrombus stabilization. Blood 2006; 108 (09) 3045-3052
- 24 Patzelt J, Verschoor A, Langer HF. Platelets and the complement cascade in atherosclerosis. Front Physiol 2015; 6: 49
- 25 Nording H, Langer HF. Complement links platelets to innate immunity. Semin Immunol 2018; 37: 43-52
- 26 Verschoor A, Langer HF. Crosstalk between platelets and the complement system in immune protection and disease. Thromb Haemost 2013; 110 (05) 910-919
- 27 Ricklin D, Mastellos DC, Reis ES, Lambris JD. The renaissance of complement therapeutics. Nat Rev Nephrol 2018; 14 (01) 26-47
- 28 Ricklin D, Lambris JD. Complement-targeted therapeutics. Nat Biotechnol 2007; 25 (11) 1265-1275
- 29 Vogel CW, Fritzinger DC, Gorsuch WB, Stahl GL. Complement depletion with humanised cobra venom factor: efficacy in preclinical models of vascular diseases. Thromb Haemost 2015; 113 (03) 548-552