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Platelet activation by HLA antibodies
sure on the platelet surface. At low concentrations (2.5 mg/mL), a subset of HLA monoclonal antibodies induced α-granule release, as shown by significantly increased CD62P membrane exposure (Figure 1B). Statistically sig- nificant enhanced CD62P exposure was observed only for the broadly reactive WIM8E5 and HLA-A2/A28-specific SN607D8, whereas GV5D1 (anti-HLA A1/A23/A24) showed a trend of enhanced CD62P exposure, but this was not statistically significant. SN230G6, with a speci- ficity similar to that of SN607D8 and GV2D5, binding to HLA-A1 like GV5D1, did not induce platelet activation. VTM1F11, HDG8D9 and BRO11F9 also did not induce enhanced CD62P membrane exposure on platelets. Increased CD62P exposure was observed upon incubation with higher concentrations of WIM8E5, SN607D8 and GV5D1 (Figure 1C). Addition of 10 mg/mL of the other HLA monoclonal antibodies included in this study did not induce significantly increased CD62P membrane exposure (Figure 1C). Additionally, incubation of platelets with two human monoclonal anti HPA-1a antibodies did not induce CD62P exposure despite their efficient binding to platelets (Online Supplementary Figure S1A).To further confirm the release of α-granules from platelets, the release of von Willebrand factor (VWF) and SPARC (secreted protein acidic and rich in cysteine), both proteins residing in α- granules,26 was measured in the supernatant of platelets incubated with HLA monoclonal antibodies. VWF (Figure 1D) and SPARC (Figure 1E, Online Supplementary Figure S2A) were released by platelets upon incubation with either WIM8E5 or SN607D8. Incubation with SN230G6 resulted in significant, but low levels of released VWF and SPARC. Together these results indicate that a subset of HLA monoclonal antibodies can induce α-granule release in platelets.
HLA monoclonal antibodies induce integrin α b IIb 3
activation and platelet agglutination
We subsequently studied the activation of GPIIb/IIIa (integrin αIIbb3) using the PAC-1 antibody, which recog- nizes the active configuration of this integrin.27 PAC-1 binding was increased significantly upon incubation with WIM8E5 and SN607D8, while SN230G6, an antibody not potent in inducing α-granule release, did not lead to acti- vation of integrin αIIbb3 (Figure 2A). We then performed light aggregometry to study whether platelets aggregate upon incubation with HLA monoclonal antibodies. In agreement with the results for α-granule release and inte- grin αIIbb3 activation, WIM8E5 and SN607D8 induced
Table 1. Human HLA-specific monoclonal antibodies used in this study.
dose-dependent platelet aggregation. No platelet aggrega- tion was observed upon incubation with SN230G6 (Figure 2B). A combination of suboptimal concentrations of PAR1 activating peptide and HLA monoclonal antibodies did not induce enhanced CD62P exposure. In combination with low suboptimal concentration of PAR1 activating peptide or collagen, both activating (WIM8E5 and SN607D8) and non-activating HLA monoclonal antibodies (SN230G6) significantly enhanced platelet aggregation, which was most pronounced for WIM8E5 (Online Supplementary Figure S1B,C).
WIM8E5 also induced exposure of phosphatidylserine, as measured by annexin V binding. No significantly increased levels of annexin V binding were observed for SN607D8 and SN230G6, suggesting that only strongly activating HLA antibodies can induce phosphatidylserine exposure on platelets (Online Supplementary Figure S2B). These results indicate that the subset of HLA antibodies which induces α-granule release also stimulates integrin αIIbb3 activation, platelet aggregation and phospatidylser- ine exposure.
Activation of platelets by HLA monoclonal antibodies is FcγRIIa dependent
Next, we tested whether the platelet-Fc receptor, FcγRIIa,28,29 is involved in anti-HLA mediated platelet acti- vation. The pathway of FcγRIIa-dependent platelet activa- tion by IgG is well described in, for instance, heparin- induced thrombocytopenia.17 FcγRIIa-mediated platelet activation via its ITAM motif has been shown to be dependent on the tyrosine kinase Syk.30 We tested whether HLA antibody-mediated activation of platelets required Syk,31 employing the extensively characterized Syk inhibitor IV.30 This inhibitor has no effect on activa- tion via PAR1 receptor (Online Supplementary Figure S2C), confirming its specificity. CD62P exposure induced by WIM8E5 or SN607D8 was inhibited in a dose-dependent manner by Syk inhibitor IV (Figure 3A). Involvement of Syk was further supported by blocking experiments employing Syk inhibitor I and II (Online Supplementary Figure S2D). Similarly, VWF release (Figure 3B) and SPARC release (Figure 3C) were blocked by Syk inhibitor IV. Activation of integrin αIIbb3 (Figure 3D) and platelet aggre- gation (Figure 3E) were completely abrogated following the addition of Syk inhibitor IV. These results indicate that HLA monoclonal antibody-induced platelet activation is dependent on Syk, which acts downstream of FcγRIIa. To further substantiate the involvement of FcγRIIa, blocking monoclonal anti-FcγRIIa-antibody IV.3 was used to pre- vent binding of the Fc tail of the HLA monoclonal antibod- ies to FcγRIIa.29 Analysis of CD62P surface exposure and of VWF and SPARC secretion revealed that α-granule release was completely blocked upon pre-incubation with IV.3 (Figure 3F-H). In addition, IV.3 completely blocked WIM8E5- and SN607D8-induced platelet aggregation (Figure 3I), integrin αIIbb3 activation (Figure 3J) and annexin V binding (Online Supplementary Figure S2B). The R131H polymorphism in FcγRIIa did not affect platelet activation by the HLA monoclonal antibodies used in this study (Online Supplementary Figure S3). Together these results indicate that platelet α-granule release, activation and aggregation induced by HLA monoclonal antibodies are FcγRIIa dependent.
To confirm the involvement of the Fc-tail of the HLA monoclonal antibodies in platelet activation, F(ab’)2 frag-
Antibody name
WIM8E5
SN607D8*
SN230G6*
GV5D1$
GV2D5$
VTM1F11
HDG8D9
BRO11F6
HLA specificity
A1/A10(A25/A26/A34/A43/A66)/A11/A9(A23/A24)/A29/ A30/A31/A33/A28(A68/A69)
A2/A28(A68/A69)
A2/B57/B58
A1/A23/A24 {not A*2403; A80 weak}
A1
B27/B7/B60
B51/B35
A3/A11/A24
* SN607D8 and SN230G6 originate from the same patient. $GV5D1 and GV2D5 origi- nate from the same patient.
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