A subset of anti-HLA antibodies induces FcγRIIa-dependent platelet activation

Abstract

HLA antibodies are associated with refractoriness to platelet transfusion, leading to rapid platelet clearance, sometimes coinciding with clinical side effects such as fever and chills. The presence of HLA antibodies is not always manifested by clinical symptoms. It is currently unclear why refractoriness to platelet transfusion is only observed in a subset of patients. Here, we utilized the availability of a unique panel of human monoclonal antibodies to study whether these were capable of activating platelets. Three out of eight human HLA-specific monoclonal antibodies induced activation of HLA-matched platelets from healthy donors as evidenced by enhanced α-granule release, aggregation, and α_IIbb₃ activation. The propensity of HLA monoclonal antibodies to activate platelets was independent of the HLA subtype to which they were directed, but was dependent on the recognized epitope. Activation was fully inhibited either by blocking FcγRIIa, or by blocking FcγRIIa-dependent signaling with Syk inhibitor IV. Furthermore, activation required the presence of the IgG-Fc part, as F(ab')₂ fragments of HLA monoclonal antibodies were unable to induce platelet activation. Mixing experiments revealed that activation of platelets occurred in an intra-platelet dependent manner. Accordingly, a proportion of sera from refractory patients with HLA antibodies induced FcγRIIa-dependent platelet activation. Our data show that a subset of HLA antibodies is capable of crosslinking HLA and FcγRIIa thereby promoting platelet activation and enhancing these cells' phagocytosis by macrophages. Based on these findings we suggest that FcγRIIa-dependent platelet activation may contribute to the decreased platelet survival in platelet-transfusion-dependent patients with HLA antibodies.

Figure 1.

HLA monoclonal antibodies induce platelet α-granule release. (A) Platelets were matched for HLA type with the specificity of eight HLA monoclonal antibodies (mAbs) directed at different epitopes. Mean fluorescent intensity (MFI) upon staining with anti-human IgG was measured with flow cytometry for the control (buffer only, no HLA antibodies) and 2.5 mg/mL of the HLA mAbs. Right panel: representative flow cytometry plot of 10 mg/mL SN607D8 with a not matching donor and a matching donor. (B,C) CD62P surface expression of platelets incubated with 2.5 mg/mL (B) or 10 mg/mL (C) HLA mAbs compared to control (buffer only). Representative flow cytometry plots of WIM8E5, SN607D8 and SN230G6. (D) VWF release in platelet supernatant upon incubation with HLA mAbs WIM8E5, SN607D8 and SN230G6 measured by enzyme-linked immunosorbent assay. (E) Representative western blot of SPARC release in platelet supernatant upon incubation with HLA mAbs WIM8E5, SN607D8 and SN230G6. Paired t-tests (A, B and C) or paired ANOVA with the Tukey multiple comparison test (D). Each line represents a separate experiment with a separate donor (A, B and C). Mean ± SD (D). *P<0.05, **P<0.01, ***P<0.005, ****P<0.001.

Figure 2.

Integrin α_IIbb₃ activation and platelet agglutination are induced by HLA monoclonal antibodies. A) Integrin α_IIbb₃ activation, derived from PAC-1 binding, upon incubation with 10 mg/mL WIM8E5, SN607D8 or SN230G6 compared to control (buffer only, no HLA antibodies). Flow cytometry plots are representative of more than eight independent experiments with different donors. (B) Platelet agglutination upon addition of HLA monoclonal antibodies (mAbs), measured by light transmission aggregometry. Mean ± SD of percentage maximum aggregation. Paired t-tests (A) or paired ANOVA with the Tukey multiple comparison test (B). *P<0.05, **P<0.01, ***P<0.005, ****P<0.001.

Figure 3.

HLA monoclonal antibodies induce FcγRIIa-dependent platelet activation. (A) CD62P exposure upon incubation with WIM8E5 and SN607D8, inhibited by pre-incubation with Syk inhibitor IV compared to control (buffer only, no HLA antibodies). (B) VWF release, measured by enzyme-linked immunosorbent assay, induced by WIM8E5 and SN607D8, inhibited by Syk inhibitor IV. (C) SPARC release in platelet supernatant induced by WIM8E5 and SN607D8, inhibited by Syk inhibitor IV. (D) Integrin 〈IIbβ3 activation (as measured by PAC-1 binding) induced by WIM8E5 and SN607D8, inhibited by Syk inhibitor IV. (E) Agglutination induced by WIM8E5, inhibited by Syk inhibitor IV. (F) CD62P exposure induced by WIM8E5 and SN607D8, blocked by the FcγRIIa blocking antibody IV.3. (G) VWF release induced by WIM8E5 and SN607D8, inhibited by IV.3 (H) Release of SPARC in platelet supernatant inhibited by IV.3. (I) Agglutination induced by WIM8E5 and SN607D8 inhibited by pre-incubation with IV.3. (J) PAC-1 binding induced upon incubation with WIM8E5 and SN607D8 inhibited by IV.3. Data are given as mean ± SD. Paired ANOVA with the Tukey multiple comparison test. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001.

Figure 4.

HLA monoclonal antibodies activate platelets in an intra-platelet-dependent manner. (A) Schematic representation of theoretically possible inter-platelet activation and intra-platelet activation induced by the HLA monoclonal antibody WIM8E5. (B) Platelet donors were selected as either “WIM8E5 matching” or “WIM8E5 nonmatching” and their platelets were stained with calcein-green or calcein-violet, respectively. Platelets from two donors were mixed in a 1:1 ratio. Platelets from a single donor or the mixed platelets were incubated with control (buffer only, no HLA antibodies) or WIM8E5. By gating for calcein-green or calcein-violet, CD62P exposure was determined for “WIM8E5 matching” and “WIM8E5 nonmatching” platelets. (C) Platelets from a “WIM8E5 matching” donor (stained with calcein green) were mixed 1:1 with platelets from a donor with either nonmatching WIM8E5 or matching WIM8E5 (stained with calcein violet). Samples were incubated with control (buffer only, no HLA antibodies), WIM8E5 or PAR1 activating peptide (PAR1 AP). Percentage double-positive events of calcein-green/calcein-violet-stained platelets are given, representing the ability of HLA antibodies to crosslink the HLA molecule with the FcγRIIa in an inter- or intra-platelet manner. Representative flow cytometry plots are shown for a mix of WIM8E5 matching + WIM8E5 nonmatching platelets and platelets from two different donors with both matching HLA typing. Paired ANOVA with the Tukey multiple comparison test. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001.

Figure 5.

Patients’ sera with HLA antibodies induce FcγRIIa-dependent activation of platelets from a subset of donors. Thirteen sera containing HLA alloantibodies were incubated with platelets from two different donors. AB serum tested negative for HLA and other specific platelet antibodies was used as a control. Background CD62P and IgG binding are indicated by the gray background. Activation was inhibited by pre-incubation with 10 mg/mL IV.3 or 5 mM Syk inhibitor IV. (A) CD62P exposure on platelets from donor 1 (expressing HLA A1 A2 B51). (B) IgG binding to platelets from donor 1. (C) CD62P exposure on platelets from donor 2 (expressing HLA A1 A2 B35 B62). (D) IgG binding to platelets from donor 2. Antibody specificities of the sera are reported in the Online Supplementary Data.

Figure 6.

Phagocytosis of platelets opsonized by HLA monoclonal antibodies and the effect of FcγRIIa-dependent signaling. Platelets were incubated with 10 mg/mL WIM8E5, SN607D8 or SN230G6 in the presence or absence of 5 mM Syk inhibitor IV. Opsonized platelets were incubated for 1 h with monocyte-derived macrophages and internalization was analyzed through the use of imaging flow cytometry. (A–C) Representative images of imaging flow cytometry. BF: bright field; CD61: extracellular platelet staining; PKH: platelet staining; HLA-DR: macrophage staining. (A) Control without Syk inhibitor, (B) WIM8E5 without Syk inhibitor, (C) WIM8E5 with Syk inhibitor. (D) Intracellular platelet (PKH) fluorescence quantifies the amount of platelets taken up by macrophages. Data are given as mean ± SD, *P<0.05, **P<0.01. Control: buffer only, no HLA antibodies added. MF: macrophage.

Figure 7.

Proposed mechanism of platelet activation. When an activating HLA antibody (ab) binds to HLA on the platelet surface it crosslinks with FcγRIIa. This induces FcγRIIa-dependent signaling leading to the activation of Syk via the ITAM motif on FcγRIIa. Downstream signaling leads to platelet activation: α-granules are released (followed by e.g. CD62P exposure), integrin α_IIbb₃ is activated and platelets start to aggregate. This activation pathway can be inhibited by IV.3 (which blocks crosslinking with FcγRIIa) or Syk inhibitor IV (which blocks signaling via Syk). HLA antibodies that bind to an epitope on HLA preventing interaction with FcγRIIa do not induce platelet activation. FcγRIIa-dependent signaling also leads to phosphatidylserine exposure and induces enhanced phagocytosis by macrophages.