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M. Rijkers et al.
platelet transfusions and repeatedly show poor incre- ments of platelet counts caused by rapid clearance of the transfused platelets.3,6 HLA-matched platelet transfusions are commonly used for treatment of HLA alloimmunized patients. However, treatment with HLA-matched platelet concentrates is challenging due to the fact that it is often difficult to find a sufficiently high number of compatible donors for refractory patients. Current transfusion approaches for HLA alloimmunized patients are exclu- sively based on binding specificity of HLA antibodies but do not take into account functional properties of circulat- ing HLA antibodies. Here, we have further characterized the pathogenic properties of different types of HLA-anti- bodies.
Previously, we showed that a subset of human mono- clonal HLA antibodies and patient sera containing HLA antibodies induce FcγRIIa-dependent platelet activation and enhanced phagocytosis by macrophages.8 However, it remains unclear to which extent this HLA antibody-medi- ated activation of platelets contributes to platelet clearance and which other mechanisms contribute to platelet clear- ance in refractory patients. In the current study we have focused on the role of complement activation by HLA antibodies.
Platelets have been shown to promote complement acti- vation via several mechanisms. It has been reported that activation of platelets, which leads to α-granule release and subsequent CD62P surface exposure, triggers deposi- tion of complement C3b. C3b can bind directly to CD62P exposed on platelet surfaces, suggesting that platelet acti- vation promotes complement deposition on platelets.9,10 In this case, the alternative pathway of the complement cas- cade is initiated, where binding of IgG and subsequent C1q deposition is bypassed. Subsequent binding of C3b facilitates further complement activation, finally leading to the formation of a membrane attack complex (MAC), also called the C5b-9 complex.9 Peerschke et al. also showed that C1q can bind to agonist-activated platelets, indicating a possible role for platelets in complement acti- vation via the classical complement pathway.11 Platelet activation can also induce complement activation in the fluid phase, where the release of chondroitin sulfate by activated platelets is the trigger.12 Also, binding of C3 to activated platelets has been suggested to stimulate forma- tion of platelet-leukocyte interactions.13 In addition, IgG- complexes can induce platelet aggregation, which is strongly enhanced by addition of C1q.14 Mouse monoclonal antibodies (mAbs) directed to beta-2 microglobulin (β2M) and a pan HLA mAb have been shown to induce C3b binding and complement dependent cytotoxicity (CDC) on platelets when added at high con- centrations.15,16
Platelet transfusion-related adverse events might be (partly) explained by complement activation in platelet products as standard storage conditions have been shown to induce complement activation with increasing C3a and C4d levels found in platelet concentrates upon prolonged storage.17
Here, we studied complement activation on platelets induced by HLA antibodies. Human HLA mAbs and sera from patients with refractory thrombocytopenia contain- ing HLA antibodies were used to study the effect of com- plement deposition, formation of a MAC, platelet activa- tion and permeabilization. Our results show that a subset of anti-HLA antibodies can induce complement activation
on platelets. We also showed that blocking pathways leading to complement deposition on platelets, prevented complement activation induced pathogenicity of HLA antibodies. Based on our findings, we propose that func- tional matching of platelet concentrates may be used to further improve treatment of refractory patients with HLA antibodies. Our results also suggest that complement- directed therapeutic interventions may be utilized to increase donor platelet survival in HLA-immunized refrac- tory patients.
Methods
Materials
Detailed information on materials used can be found in the
Online Supplementary Data.
Patient sera
Blood samples of patients refractory to platelet transfusion were used following informed consent according to the Dutch estab- lished codes of conduct for responsible use of patient material and as approved by our institute.18 HLA antibody specificities in patient sera were determined by single antigen bead assay on Luminex platform (Labscreen SA, One Lambda, Inc.). Twelve sera positive for HLA antibodies and negative for other platelet specific antibodies were used.
Platelet isolation
Platelets were isolated from citrated whole blood from healthy volunteers with known HLA type. All donors gave written informed consent and blood was drawn in accordance with Dutch regulations and after approval from the Sanquin Ethical Advisory Board in accordance with the Declaration of Helsinki. Platelets were isolated and washed as described before.19 Platelets were resuspended in platelet assay buffer (10 mM HEPES, 140 mM NaCl, 3 mM KCl, 0.5 mM MgCl2, 10 mM glucose and 0.5 mM NaHCO3, pH 7.4).
Complement deposition and platelet activation
Platelets were used at a final concentration of 0.08*108 platelets/ml and mixed with indicated inhibitors, antibodies/sera (heat inactivated for 30 min at 56°C, 25% of total sample volume) and complement source (normal human serum) (25% of total vol- ume) in platelet assay buffer. Mixtures were incubated for 30 min at 37°C while shaking (300 rpm), and then fixed by adding formaldehyde (final concentration of 1%). Platelets were washed with platelet assay buffer and stained for flow cytometry. Anti- CD42a-FITC or CD41-APC-Cy7 antibodies were used to gate for platelets. Platelets were stained with mouse anti-human CD62P- PE and mouse anti-human C3b-APC or mouse anti-human C4b- APC. For measuring the formation of a MAC, platelets were stained with rabbit anti-human C5b-9 antibody followed by the secondary antibody chicken anti-rabbit Alexa 647. Mean fluores- cent intensities and/or percentage positive platelets were meas- ured using flow cytometer FACSCanto II (Becton Dickinson, Franklin Lakes, NJ, USA).
Pore formation
Complement assay was initiated as described above. After 30 min incubation at 37°C, platelets were washed once and resus- pended in assay buffer containing live/dead marker for 30 min at room temperature (RT). Platelets were washed and stained with anti-CD42a (for gating purposes), anti-C3b and CD62P and ana- lyzed using flow cytometry.
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haematologica | 2019; 104(2)