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Crosstalk Between Activated Platelets and the Complement System
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology. (Complement And Biomaterial)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Several studies have shown that complement and thrombotic events co-exist. Platelets have been suspected to act as the bridge between the two cascade systems.

To study the platelet-induced complement activation we developed a system in which platelets were activated by thrombin receptor activating peptide (TRAP) in platelet rich plasma (PRP) or whole blood anti-coagulated using the specific thrombin inhibitor, lepirudin.

TRAP-activated platelets induced a fluid-phase complement activation measured as generation of C3a and sC5b-9, triggered by released chondroitin sulphate-A (CS-A) which interacted with C1q and activated the complement system through the classical pathway.

Complement components C1q, C3, C4 and C9 were also shown to bind to TRAP-activated platelets but this binding did not seem to be due to a complement activation since blocking of complement activation at the C1q or C3 levels did not affect the binding of the complement proteins. The C3 which bound to activated platelets consisted of C3(H2O), indicating that bound C3 was not proteolytically activated. Binding of C1q was partially dependent on CS-A exposure on activated platelets. The abolished complement activation on the surface of activated platelets was suggested to be dependent on the involvement of several complement inhibitors. We confirmed the binding of C1INH and factor H to activated platelets. To this list we have added another potent complement inhibitor, C4BP. The binding of factor H and C4BP was shown to be dependent on exposure of CS-A on activated platelets.

The physiological relevance of these reactions was reflected in an elevated expression of CD11b on leukocytes, and increased generation of platelet-leukocyte complexes. The platelets were involved in these events by at least two different mechanisms; generation of C5a which activated leukocytes and binding of C3(H2O)/iC3(H2O), a ligand to the intergrin CD11b/CD18 on their surface.

These mechanisms add further to the understanding of how platelets interact with the complement system and will help us to understand the role of the complement system in cardiovascular disease and thrombotic conditions.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2010. , p. 82
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 567
Keywords [en]
platelets, activated platelets, TRAP, chondroitin sulfate, C1q, factor H, C4BP, C3, Compstatin, complement, platelet-leukocyte complexes, platelet microparticles
National Category
Immunology in the medical area
Research subject
Clinical Immunology
Identifiers
URN: urn:nbn:se:uu:diva-123681ISBN: 978-91-554-7822-3 (print)OAI: oai:DiVA.org:uu-123681DiVA, id: diva2:315297
Public defence
2010-06-01, Rudbecksalen, Rudbeck Laboratory, Dag Hammarskjölds väg 20, Uppsala, 09:15 (Swedish)
Opponent
Supervisors
Projects
Platelet Mediated Complement ActivationAvailable from: 2010-05-11 Created: 2010-04-28 Last updated: 2018-01-12Bibliographically approved
List of papers
1. Complement activation triggered by chondroitin sulfate released by thrombin receptor-activated platelets
Open this publication in new window or tab >>Complement activation triggered by chondroitin sulfate released by thrombin receptor-activated platelets
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2008 (English)In: Journal of Thrombosis and Haemostasis, ISSN 1538-7933, E-ISSN 1538-7836, Vol. 6, no 8, p. 1413-1421Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Chondroitin sulfate (CS) is a glycosaminoglycan released by activated platelets. OBJECTIVE: Here we test the hypothesis that CS released by activated platelets can trigger complement activation in the fluid phase. METHODS AND RESULTS: Thrombin receptor-activating peptide (TRAP)-6 was used to activate platelets in platelet-rich plasma and blood, anticoagulated with the thrombin inhibitor lepirudin. TRAP activation induced fluid-phase complement activation, as reflected by the generation of C3a and sC5b-9, which could be attenuated by the C3 inhibitor compstatin. Chondroitinase ABC treatment of supernatants from activated platelets totally inhibited the activation, indicating that platelet-derived CS had initiated the complement activation. Furthermore, addition of purified CS to plasma strongly triggered complement activation. C1q was identified as the recognition molecule, as it bound directly to CS, and CS-triggered complement activation could be restored in C1q-depleted serum by adding purified C1q. TRAP activation of whole blood increased the expression of CD11b on leukocytes and generation of leukocyte-platelet complexes. It was demonstrated that these leukocyte functions were dependent on C3 activation and signaling via C5a, as this expression could be inhibited by compstatin and by a C5aR antagonist. CONCLUSIONS: We conclude that platelets trigger complement activation in the fluid phase by releasing CS, which leads to inflammatory signals mediated by C5a.

Keywords
chondroitin sulfate, coagulation, complement, platelets, thrombin receptor-activating peptide (TRAP)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-16841 (URN)10.1111/j.1538-7836.2008.03034.x (DOI)000257757000024 ()18503629 (PubMedID)
Projects
Platelet mediated complement activation
Available from: 2008-06-05 Created: 2008-06-05 Last updated: 2019-12-14Bibliographically approved
2. Non-proteolytically activated C3 promotes binding of activated platelets and platelet-derived microparticles to leukocytes via CD11b/CD18
Open this publication in new window or tab >>Non-proteolytically activated C3 promotes binding of activated platelets and platelet-derived microparticles to leukocytes via CD11b/CD18
2012 (English)In: Immunobiology, ISSN 0171-2985, E-ISSN 1878-3279, Vol. 217, no 11, p. 1191-1191Article in journal, Meeting abstract (Refereed) Published
Abstract [en]

Background:

We have previously demonstrated that complement component C3 binds to the surface of activated platelets, independent of proteolytic activation. The resulting form of C3, termed C3(H2O), was shown to be a ligand for recombinant CD35 (complement receptor 1, CR1). Previous studies by others have indicated that platelet-leukocyte complex (PLC) formation is dependent on the interaction between platelet exposed P-selectin (CD62P) and its ligand, PSGL-1, on leukocytes. In addition, CD11b/CD18 (Mac-1 or CR3) has been shown to participate in this reaction, but its ligand has not yet been identified.

Objective:

To test the hypothesis that C3 bound to activated platelets and platelet-derived microparticles (PMPs) can act as a ligand for CD11b/CD18 (CR3) and contribute to PLC formation.

Methods and results:

Blood cells were depleted of plasma proteins. After extensive washing, C3 was added, and the leukocytes were activated with C5a and the platelets with thrombin receptor-activating peptide (TRAP). PLC formation was detected by flow cytometry (monocytes: CD14+/CD42a+, granulocytes: CD16+/CD42a+). For both granulocytes and monocytes, the addition of C3 significantly enhanced PLC formation. Formation of PLC was inhibited by both anti-P-selectin and anti-CD11b monoclonal antibodies. In addition, PMPs isolated from serum, were found to expose C3(H2O) and bind to leukocytes in a fashion similar to activated platelets.

Conclusion:

We have identified proteolytically non-activated C3 as a ligand for CD11b in the formation of PLC and possibly the binding of PMPs to leukocytes. This observation most likely has pathophysiological implications for the recently reported links between thrombotic disease and the complement system.

Keywords
platelet activation, complement, platelet-leukocyte complexes, PMP, CD11b/CD18, complement component 3
National Category
Immunology in the medical area
Research subject
Clinical Immunology
Identifiers
urn:nbn:se:uu:diva-123624 (URN)10.1016/j.imbio.2012.08.178 (DOI)000311187800190 ()
Conference
Aegean Conferences, XXIV International Complement Workshop, 10-15 October, 2012, Chania, Crete, GREECE
Projects
platelet mediated complement activation
Available from: 2010-04-28 Created: 2010-04-28 Last updated: 2018-01-12Bibliographically approved
3. Contribution of chondroitin sulfate A to the binding of complement proteins to activated platelets
Open this publication in new window or tab >>Contribution of chondroitin sulfate A to the binding of complement proteins to activated platelets
2010 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 5, no 9, p. e12889-Article in journal (Refereed) Published
Abstract [en]

Exposure of chondroitin sulfate A (CS-A) on the surface of activated platelets is well established.  The aim of the present study was to investigate to what extent CS-A contributes to the binding of C1q and the complement regulators C1 inhibitor (C1INH), C4b-binding protein (C4BP), and factor H to platelets. Human serum was passed over Sepharose conjugated with CS-A, and bound proteins were identified by Western blotting, and mass spectrometric analysis. C1q was identified as the main protein that specifically bound to CS-A, but C4BP and factor H were also shown to interact. Binding of C1INH was dependent of the presence of C1q and not bound to CS-A from C1q-depleted serum. The specific interactions observed of these proteins with CS-A were subsequently confirmed by surface plasmon resonance analysis using purified proteins. Importantly, C1q, C4BP, and factor H were shown to bind also to activated platelets and this interaction was inhibited by a CS-A-specific monoclonal antibody, thereby linking the binding of C1q, C4BP, and factor H to exposure of CS-A on platelets. CS-A-bound C1q was also shown to amplify the binding of model immune complexes to both microtiter plate-bound CS-A and to activated platelets. In conclusion, this study supports the concept that CS-A contributes to the binding of C1q, C4BP, and factor H to platelets, thereby adding CS-A to the previously reported binding sites for these proteins on the platelet surface. CS-A-bound C1q seems to amplify the binding of immune complexes to activated platelets, suggesting a role for this molecule in immune complex diseases.

Keywords
chondroitin sulfate, activated platelets, complement proteins, complement inhibitors, TRAP, C1q
National Category
Immunology in the medical area
Research subject
Clinical Immunology
Identifiers
urn:nbn:se:uu:diva-123614 (URN)10.1371/journal.pone.0012889 (DOI)000282091100006 ()
Projects
platelet mediated complement activation
Available from: 2010-04-28 Created: 2010-04-28 Last updated: 2018-01-12Bibliographically approved
4. Complement component C3 binds to activated normal platelets without preceding proteolytic activation and promotes binding to complement receptor 1
Open this publication in new window or tab >>Complement component C3 binds to activated normal platelets without preceding proteolytic activation and promotes binding to complement receptor 1
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2010 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 184, no 5, p. 2686-2692Article in journal (Refereed) Published
Abstract [en]

It has been reported that complement is activated on the surface of activated platelets, despite the presence of multiple regulators of complement activation. To reinvestigate the mechanisms by which activated platelets bind to complement components, the presence of complement proteins on the surfaces of nonactivated and thrombin receptor-activating peptide-activated platelets was analyzed by flow cytometry and Western blot analyses. C1q, C4, C3, and C9 were found to bind to thrombin receptor-activating peptide-activated platelets in lepirudin-anticoagulated platelet-rich plasma (PRP) and whole blood. However, inhibiting complement activation at the C1q or C3 level did not block the binding of C3 to activated platelets. Diluting PRP and chelating divalent cations also had no effect, further indicating that the deposition of complement components was independent of complement activation. Furthermore, washed, activated platelets bound added C1q and C3 to the same extent as platelets in PRP. The use of mAbs against different forms of C3 demonstrated that the bound C3 consisted of C3(H(2)O). Furthermore, exogenously added soluble complement receptor 1 was shown to bind to this form of platelet-bound C3. These observations indicate that there is no complement activation on the surface of platelets under physiological conditions. This situation is in direct contrast to a number of pathological conditions in which regulators of complement activation are lacking and thrombocytopenia and thrombotic disease are the ultimate result. However, the generation of C3(H(2)O) represents nonproteolytic activation of C3 and after factor I cleavage may act as a ligand for receptor binding.

Keywords
complement, platelet activation, TRAP, complement component 3, chondroitin sulfate
National Category
Immunology in the medical area
Research subject
Clinical Immunology
Identifiers
urn:nbn:se:uu:diva-123613 (URN)10.4049/jimmunol.0902810 (DOI)000274768900054 ()20139276 (PubMedID)
Projects
Platelet Mediated Complement Activation
Available from: 2010-04-28 Created: 2010-04-28 Last updated: 2018-01-12Bibliographically approved

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