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. 2008 Dec;84(6):1492-500.
doi: 10.1189/jlb.0508327. Epub 2008 Sep 10.

Functional expression of IgA receptor FcalphaRI on human platelets

Kun Qian  1 , Fenglong Xie, Andrew W Gibson, Jeffrey C Edberg, Robert P Kimberly, Jianming Wu
Affiliations

Functional expression of IgA receptor FcalphaRI on human platelets

Kun Qian et al. J Leukoc Biol. 2008 Dec.

Abstract

FcalphaRI (CD89) is a human IgA FcR expressed on cells of myeloid lineage such as neutrophils, monocytes, tissue macrophages, eosinophils, and subpopulations of dendritic cells. FcalphaRI mediates cell activation through Src family kinases and downstream tyrosine-based phosphorylation pathways. However, the role of IgA and the expression and role of its cognate receptor FcalphaRI (CD89) in platelet activation are undefined. In the current study, we demonstrate that human platelets express FcalphaRI mRNAs and proteins. Furthermore, we show that the platelet FcalphaRI is associated with the FcR gamma-chain, and cross-linking of FcalphaRI leads to Syk phosphorylation. Clustering of FcalphaRI induces pre-mRNA splicing and protein production of tissue factor and IL-1beta, suggesting novel roles for human platelet FcalphaRI and serum IgA in thrombosis and inflammation.

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Figures

Fig. 1.
Fig. 1.
Expression of FcαRI gene products in human platelets. (A) RT-PCR analysis of FcαRI mRNA in human platelets. Total RNA was isolated from the purified platelets from six individuals (Lanes 1, 2, and 4–7) or from the peripheral blood leukocytes of a healthy donor (Lane 3*, positive control for FcαRI mRNA). Platelet RNA was reversely transcribed, and FcαRI cDNAs were amplified by RT-PCR. The identity of FcαRI cDNA products in human platelets was confirmed further by direct sequencing. (B) Detection of FcαRI on the surface of human platelets. FcαRI was detected using anti-FcαRI mAb MIP8a (bold line) in flow cytometry analysis by comparing with the mIgG1 isotype control (dashed line). The inset shows the staining of FcαRI (bold line) compared with mIgG1 isotype control (thin line) on human neutrophils. This is representative of four independent experiments with at least four normal, healthy donors shown.
Fig. 2.
Fig. 2.
Detection of FcαRI proteins in human platelets, which were purified as described in Materials and Methods. The purified platelets (109) from three normal, healthy donors (Lanes 1–3) were lysed, and cell lysates were separated in 12% SDS-PAGE under reducing conditions. FcαRI proteins were detected on four separate blots with three anti-FcαRI mAb (A77, A59, and MIP8a) and rabbit pAb against the FcαRI cytoplasmic domain in Western blot analysis. FcαRI proteins were shown as multiple protein bands with a molecular weight (Mr) between 50 and 70 kDa. IB, Immunoblot.
Fig. 3.
Fig. 3.
hIgA binds and activates human platelets. (A) Binding of hIgA to platelets, which were preincubated with anti-FcαRI-blocking mAb MIP8a (final concentration: 50 μg/ml; thin line, MIP8a+hIgA), mIgG1 isotype control (final concentration: 50 μg/ml; dashed line), or without any treatment (bold line, hIgA). After washing, human platelets were incubated with hIgA (final concentration: 100 μg/ml) and followed by FITC-conjugated goat anti-hIgA antibody. Platelets stained with FITC-conjugated goat anti-hIgA antibody alone were used as autofluorescence control (shaded histogram). (B) Induction of platelet activation by hIgA. Human platelets were stimulated as described in Materials and Methods. Platelet activation was defined as an increase of CD62P expression. The results were representative of four independent experiments.
Fig. 4.
Fig. 4.
Induction of Syk phosphorylation by clustering of platelet FcαRI on human platelets. (A) Physical association between the FcαRI and FcR γ-chain in human platelets. Freshly isolated platelets were lysed in 1× Trition X-100 lysis buffer as described in Materials and Methods. Whole platelet cell lysates were immunoprecipitated (IP) with anti-FcαRI mAb A59 F(ab′)2-conjugated agarose beads from five donors (Lanes 1–5) or from a donor with mIgG F(ab′)2-conjugated agarose beads as control (Lane 6). Immunoprecipitated proteins were resolved on SDS-PAGE and immunoblotted with mAb MIP8a (upper panel) or anti-FcR γ-chain pAb (lower panel), followed by HRP-conjugated second antibody. The blots were developed using an ECL-detecting system. The results were representative of three independent experiments. (B) Clustering of FcαRI induced Syk phosphorylation. Purified human platelets (109) were stimulated with anti-FcαRI mAb A59 F(ab′)2 plus goat anti-mouse IgG F(ab′)2 for the various periods of time (in seconds) as indicated. Reactions were terminated by adding an equal volume of 2× lysis buffer, and the FcR γ-chain was immunoprecipitated from platelet lysates with anti-FcR γ-chain mAb 7D3 F(ab′)2-conjugated agarose beads. The immunoprecipitated proteins were resolved in SDS-PAGE and immunoblotted with anti-Syk pAb (top panel), antiphosphotyrosine antibody 4G10 (middle panel), or anti-FcR γ-chain pAb (bottom panel). The results were representative of three independent experiments.
Fig. 5.
Fig. 5.
FcαRI-mediated TF pre-mRNA splicing and protein synthesis in platelets. (A) Cross-linking of platelet FcαRI induced mature TF mRNA production. Purified human platelets (5×108) were stimulated with anti-FcαRI mAb A59 F(ab′)2 plus goat anti-mouse IgG F(ab′)2 (Anti FcαRI-marked lanes), with fibrinogen plus thrombin (Fib+Thr-marked lanes), or with hIgA plus goat anti-hIgA F(ab′)2 (hIgA-marked lanes) for various periods of time (in min). Nonstimulated platelets were labeled as Non. Total RNA was isolated from platelet pellets and used for RT-PCR. The lower panel shows RT-PCR results of GAPDH (internal controls for equal amount of total RNA) at each time-point. The results were representative of at least four independent experiments. (B) Induction of TF protein production by clustering of FcαRI. The platelets (5×108) were stimulated in 24-well tissue-culture plates coated with hIgA, anti-FcαRI mAb A59 F(ab′)2 (Anti FcαRI), and fibrinogen plus thrombin, respectively. The platelets were incubated at 37°C in 5% CO2 for various periods of time (hours). Platelets in culture media were collected by centrifugation at various points of time (0, 1, 5, and 20 h) and lysed in 1× radioimmunoprecipitation assay buffer. Proteins in platelet lysates were separated by SDS-PAGE, and the production of TF was analyzed on Western blots. The results were representative of three independent experiments.
Fig. 6.
Fig. 6.
FcαRI-mediated IL-1β pre-mRNA splicing and protein synthesis in human platelets. (A) Cross-linking of platelet FcαRI induced mature IL-1β mRNA production. Purified human platelets (5×108) were stimulated with anti-FcαRI mAb A59 F(ab′)2 plus goat anti-mouse IgG F(ab′)2 (Anti FcαR-marked lanes), with hIgA plus goat anti-hIgA F(ab′)2 (hIgA-marked lanes) or without stimulation (lane labeled as Non). Total RNA was isolated from platelets pellets and used for RT-PCR. The results were representative of at least four independent experiments. (B) Induction of IL-1β release by clustering platelet FcαRI. The platelets (2.5×108) were stimulated with plate-bound mIgG F(ab′)2, thrombin plus fibrinogen, anti-FcαRI mAb A59 F(ab′)2, and hIgA, respectively. IL-1β in culture supernatants was quantified with ELISA at 5 h and 20 h time-points. The data represent the mean ± sem of four independent experiments. *, Significant differences between platelets stimulated with plate-bound A59 F(ab′)2 or hIgA and platelets stimulated with fibrinogen plus thrombin or mIgG F(ab′)2 (P<0.0001).
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