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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

ACPA-mediated activation of platelets from healthy subjects. Platelets were isolated from buffy coat and incubated in the presence of plasma obtained from ACPAneg or ACPApos patients with RA (matched for age, sex and DAS44; cohort 2). To ensure we were studying active disease, only plasma samples from patients with a DAS44 > 2.4 were included. Plasma from ACPApos patients with RA induced higher platelet activation than did plasma from ACPAneg patients with RA, as indicated by increased (a) percentage and (b) MFI of CD62P expression and (d) platelet aggregation. c Histogram overlay showing representative examples of CD62P expression. e Representative pictures of aggregate formation and CD62P expression of non-aggregated and aggregated platelets. f Levels of P-selectin correlated with high ACPA titres (> 1000 AU/ml). Each symbol represents a plasma sample. ACPA anti-citrullinated protein antibodies, CD62P P-selectin, DAS44 Disease Activity Score in 44 joints, FSC forward scatter, MFI mean fluorescence intensity, RA rheumatoid arthritis, SSC side scatter
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Fig2: ACPA-mediated activation of platelets from healthy subjects. Platelets were isolated from buffy coat and incubated in the presence of plasma obtained from ACPAneg or ACPApos patients with RA (matched for age, sex and DAS44; cohort 2). To ensure we were studying active disease, only plasma samples from patients with a DAS44 > 2.4 were included. Plasma from ACPApos patients with RA induced higher platelet activation than did plasma from ACPAneg patients with RA, as indicated by increased (a) percentage and (b) MFI of CD62P expression and (d) platelet aggregation. c Histogram overlay showing representative examples of CD62P expression. e Representative pictures of aggregate formation and CD62P expression of non-aggregated and aggregated platelets. f Levels of P-selectin correlated with high ACPA titres (> 1000 AU/ml). Each symbol represents a plasma sample. ACPA anti-citrullinated protein antibodies, CD62P P-selectin, DAS44 Disease Activity Score in 44 joints, FSC forward scatter, MFI mean fluorescence intensity, RA rheumatoid arthritis, SSC side scatter

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)

ACPA-mediated activation of platelets from healthy subjects. Platelets were isolated from buffy coat and incubated in the presence of plasma obtained from ACPAneg or ACPApos patients with RA (matched for age, sex and DAS44; cohort 2). To ensure we were studying active disease, only plasma samples from patients with a DAS44 > 2.4 were included. Plasma from ACPApos patients with RA induced higher platelet activation than did plasma from ACPAneg patients with RA, as indicated by increased (a) percentage and (b) MFI of CD62P expression and (d) platelet aggregation. c Histogram overlay showing representative examples of CD62P expression. e Representative pictures of aggregate formation and CD62P expression of non-aggregated and aggregated platelets. f Levels of P-selectin correlated with high ACPA titres (> 1000 AU/ml). Each symbol represents a plasma sample. ACPA anti-citrullinated protein antibodies, CD62P P-selectin, DAS44 Disease Activity Score in 44 joints, FSC forward scatter, MFI mean fluorescence intensity, RA rheumatoid arthritis, SSC side scatter
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
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Fig2: ACPA-mediated activation of platelets from healthy subjects. Platelets were isolated from buffy coat and incubated in the presence of plasma obtained from ACPAneg or ACPApos patients with RA (matched for age, sex and DAS44; cohort 2). To ensure we were studying active disease, only plasma samples from patients with a DAS44 > 2.4 were included. Plasma from ACPApos patients with RA induced higher platelet activation than did plasma from ACPAneg patients with RA, as indicated by increased (a) percentage and (b) MFI of CD62P expression and (d) platelet aggregation. c Histogram overlay showing representative examples of CD62P expression. e Representative pictures of aggregate formation and CD62P expression of non-aggregated and aggregated platelets. f Levels of P-selectin correlated with high ACPA titres (> 1000 AU/ml). Each symbol represents a plasma sample. ACPA anti-citrullinated protein antibodies, CD62P P-selectin, DAS44 Disease Activity Score in 44 joints, FSC forward scatter, MFI mean fluorescence intensity, RA rheumatoid arthritis, SSC side scatter
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