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Anti-citrullinated protein antibodies contribute to platelet activation in rheumatoid arthritis.

Habets KL, Trouw LA, Levarht EW, Korporaal SJ, Habets PA, de Groot P, Huizinga TW, Toes RE - Arthritis Res. Ther. (2015)

Bottom Line: Furthermore, levels of P-selectin expression and sCD40L release correlated with high ACPA titres.Pre-incubation of platelets with blocking antibodies directed against low-affinity immunoglobulin G receptor (FcγRIIa) completely inhibited the ACPA-mediated activation.In addition, expression of P-selectin measured as number of platelets correlated with Disease Activity Score in 44 joints, C-reactive protein level, ACPA status and ACPA level.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology, Leiden University Medical Centre, C1-R, PO Box 9600, 2300, RC, Leiden, the Netherlands. k.l.l.habets@lumc.nl.

ABSTRACT

Introduction: Although the role of platelets in rheumatoid arthritis (RA) is relatively unexplored, recent studies point towards a contribution of platelets in arthritis. We set out to determine platelet phenotype in RA and studied whether this could be influenced by the presence of anti-citrullinated protein antibodies (ACPA).

Methods: Platelets from healthy controls were incubated in the presence of plasma of patients with RA or age- and sex-matched healthy controls and plasma from ACPA(neg) or ACPA(pos) patients or in the presence of plate-bound ACPA. Characteristics of platelets isolated from patients with RA were correlated to disease activity.

Results: Platelets isolated from healthy controls displayed markers of platelet activation in the presence of plasma derived from RA patients, as determined by P-selectin expression, formation of aggregates and secretion of soluble CD40 ligand (sCD40L). Furthermore, levels of P-selectin expression and sCD40L release correlated with high ACPA titres. In accordance with these findings, enhanced platelet activation was observed after incubation with ACPA(pos) plasma versus ACPA(neg) plasma. Pre-incubation of platelets with blocking antibodies directed against low-affinity immunoglobulin G receptor (FcγRIIa) completely inhibited the ACPA-mediated activation. In addition, expression of P-selectin measured as number of platelets correlated with Disease Activity Score in 44 joints, C-reactive protein level, ACPA status and ACPA level.

Conclusions: We show for the first time that ACPA can mediate an FcγRIIa-dependent activation of platelets. As ACPA can be detected several years before RA disease onset and activated platelets contribute to vascular permeability, these data implicate a possible role for ACPA-mediated activation of platelets in arthritis onset.

No MeSH data available.


Related in: MedlinePlus

Citrulline-specific activation of platelets is FcγRIIa-dependent. Platelets were isolated from healthy donors. ACPA plate-bound immune complexes were generated by incubating ACPAneg and ACPApos plasma on wells coated with citrulline- or arginine-containing peptides. Representative examples of citrulline-specific increase in P-selectin expression and sCD40L release are shown in a and d, respectively. b and e These effects are still present after correcting for aspecific binding by calculating the citrulline/arginine ratio. c and f Summaries of three independent experiments. Pre-incubation of platelets with a blocking antibody directed against FcγRIIa abolished the citrulline-specific induction of P-selectin (g) and sCD40L release (h). ACPA anti-citrullinated protein antibodies, CD62P P-selectin, FcγRIIa low-affinity immunoglobulin G receptor, sCD40L soluble CD40 ligand. *P < 0.05
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Fig3: Citrulline-specific activation of platelets is FcγRIIa-dependent. Platelets were isolated from healthy donors. ACPA plate-bound immune complexes were generated by incubating ACPAneg and ACPApos plasma on wells coated with citrulline- or arginine-containing peptides. Representative examples of citrulline-specific increase in P-selectin expression and sCD40L release are shown in a and d, respectively. b and e These effects are still present after correcting for aspecific binding by calculating the citrulline/arginine ratio. c and f Summaries of three independent experiments. Pre-incubation of platelets with a blocking antibody directed against FcγRIIa abolished the citrulline-specific induction of P-selectin (g) and sCD40L release (h). ACPA anti-citrullinated protein antibodies, CD62P P-selectin, FcγRIIa low-affinity immunoglobulin G receptor, sCD40L soluble CD40 ligand. *P < 0.05

Mentions: To further investigate the relationship between ACPA level and platelet activation, we incubated platelets from healthy subjects with plasma from either ACPAneg or ACPApos patients with RA (cohort 2; age-, sex- and DAS44-matched). We again observed platelet activation when plasma from ACPApos patients was used as indicated by increased platelet P-selectin expression (Fig. 2a) (ACPAneg median 19.2, IQR 17.4–23.0; ACPApos median 21.6, IQR 19.5–24.5; P<0.05), increased CD62P MFI (Fig. 2b) (ACPAneg median 38.1, IQR 34.5–43.8; ACPApos median 46.2, IQR 39.5–52.9; P<0.01) and increased number of platelet aggregates (Fig. 2d) (ACPAneg median 1.3, IQR 0.9–2.5; ACPApos median 1.97, IQR 1.3–4.5; P<0.05). Like the observations in cohort 1, a strong positive correlation was again seen between plasma samples with high ACPA level and its ability to induce P-selectin expression on platelets from healthy subjects (Pearson’s r=0.7189, P<0.05) (Fig. 2f). To address the question whether ACPA could directly activate platelets, we coated plates with arginine- or citrulline-containing peptides and incubated the coated plates with plasma from RFnegACPApos patients to generate platelet-bound ACPA–immune complexes. Plasma from RFnegACPAneg patients was used as a negative control. We observed a citrulline-dependent activation of platelets because neither the arginine control nor the use of ACPAneg plasma resulted in increased P-selectin expression or sCD40L release (Fig. 3a–f). The ACPA-mediated activation of platelets was FcγRIIa-dependent because pre-incubation of platelets with anti-CD32 inhibited upregulation of P-selectin expression and sCD40L release (Fig. 3g,h).Fig. 2


Anti-citrullinated protein antibodies contribute to platelet activation in rheumatoid arthritis.

Habets KL, Trouw LA, Levarht EW, Korporaal SJ, Habets PA, de Groot P, Huizinga TW, Toes RE - Arthritis Res. Ther. (2015)

Citrulline-specific activation of platelets is FcγRIIa-dependent. Platelets were isolated from healthy donors. ACPA plate-bound immune complexes were generated by incubating ACPAneg and ACPApos plasma on wells coated with citrulline- or arginine-containing peptides. Representative examples of citrulline-specific increase in P-selectin expression and sCD40L release are shown in a and d, respectively. b and e These effects are still present after correcting for aspecific binding by calculating the citrulline/arginine ratio. c and f Summaries of three independent experiments. Pre-incubation of platelets with a blocking antibody directed against FcγRIIa abolished the citrulline-specific induction of P-selectin (g) and sCD40L release (h). ACPA anti-citrullinated protein antibodies, CD62P P-selectin, FcγRIIa low-affinity immunoglobulin G receptor, sCD40L soluble CD40 ligand. *P < 0.05
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig3: Citrulline-specific activation of platelets is FcγRIIa-dependent. Platelets were isolated from healthy donors. ACPA plate-bound immune complexes were generated by incubating ACPAneg and ACPApos plasma on wells coated with citrulline- or arginine-containing peptides. Representative examples of citrulline-specific increase in P-selectin expression and sCD40L release are shown in a and d, respectively. b and e These effects are still present after correcting for aspecific binding by calculating the citrulline/arginine ratio. c and f Summaries of three independent experiments. Pre-incubation of platelets with a blocking antibody directed against FcγRIIa abolished the citrulline-specific induction of P-selectin (g) and sCD40L release (h). ACPA anti-citrullinated protein antibodies, CD62P P-selectin, FcγRIIa low-affinity immunoglobulin G receptor, sCD40L soluble CD40 ligand. *P < 0.05
Mentions: To further investigate the relationship between ACPA level and platelet activation, we incubated platelets from healthy subjects with plasma from either ACPAneg or ACPApos patients with RA (cohort 2; age-, sex- and DAS44-matched). We again observed platelet activation when plasma from ACPApos patients was used as indicated by increased platelet P-selectin expression (Fig. 2a) (ACPAneg median 19.2, IQR 17.4–23.0; ACPApos median 21.6, IQR 19.5–24.5; P<0.05), increased CD62P MFI (Fig. 2b) (ACPAneg median 38.1, IQR 34.5–43.8; ACPApos median 46.2, IQR 39.5–52.9; P<0.01) and increased number of platelet aggregates (Fig. 2d) (ACPAneg median 1.3, IQR 0.9–2.5; ACPApos median 1.97, IQR 1.3–4.5; P<0.05). Like the observations in cohort 1, a strong positive correlation was again seen between plasma samples with high ACPA level and its ability to induce P-selectin expression on platelets from healthy subjects (Pearson’s r=0.7189, P<0.05) (Fig. 2f). To address the question whether ACPA could directly activate platelets, we coated plates with arginine- or citrulline-containing peptides and incubated the coated plates with plasma from RFnegACPApos patients to generate platelet-bound ACPA–immune complexes. Plasma from RFnegACPAneg patients was used as a negative control. We observed a citrulline-dependent activation of platelets because neither the arginine control nor the use of ACPAneg plasma resulted in increased P-selectin expression or sCD40L release (Fig. 3a–f). The ACPA-mediated activation of platelets was FcγRIIa-dependent because pre-incubation of platelets with anti-CD32 inhibited upregulation of P-selectin expression and sCD40L release (Fig. 3g,h).Fig. 2

Bottom Line: Furthermore, levels of P-selectin expression and sCD40L release correlated with high ACPA titres.Pre-incubation of platelets with blocking antibodies directed against low-affinity immunoglobulin G receptor (FcγRIIa) completely inhibited the ACPA-mediated activation.In addition, expression of P-selectin measured as number of platelets correlated with Disease Activity Score in 44 joints, C-reactive protein level, ACPA status and ACPA level.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology, Leiden University Medical Centre, C1-R, PO Box 9600, 2300, RC, Leiden, the Netherlands. k.l.l.habets@lumc.nl.

ABSTRACT

Introduction: Although the role of platelets in rheumatoid arthritis (RA) is relatively unexplored, recent studies point towards a contribution of platelets in arthritis. We set out to determine platelet phenotype in RA and studied whether this could be influenced by the presence of anti-citrullinated protein antibodies (ACPA).

Methods: Platelets from healthy controls were incubated in the presence of plasma of patients with RA or age- and sex-matched healthy controls and plasma from ACPA(neg) or ACPA(pos) patients or in the presence of plate-bound ACPA. Characteristics of platelets isolated from patients with RA were correlated to disease activity.

Results: Platelets isolated from healthy controls displayed markers of platelet activation in the presence of plasma derived from RA patients, as determined by P-selectin expression, formation of aggregates and secretion of soluble CD40 ligand (sCD40L). Furthermore, levels of P-selectin expression and sCD40L release correlated with high ACPA titres. In accordance with these findings, enhanced platelet activation was observed after incubation with ACPA(pos) plasma versus ACPA(neg) plasma. Pre-incubation of platelets with blocking antibodies directed against low-affinity immunoglobulin G receptor (FcγRIIa) completely inhibited the ACPA-mediated activation. In addition, expression of P-selectin measured as number of platelets correlated with Disease Activity Score in 44 joints, C-reactive protein level, ACPA status and ACPA level.

Conclusions: We show for the first time that ACPA can mediate an FcγRIIa-dependent activation of platelets. As ACPA can be detected several years before RA disease onset and activated platelets contribute to vascular permeability, these data implicate a possible role for ACPA-mediated activation of platelets in arthritis onset.

No MeSH data available.


Related in: MedlinePlus