Platelets derived citrullinated proteins and microparticles are potential autoantibodies ACPA targets in RA patients

Abstract

Citrullinated neoepitopes have emerged as key triggers of autoantibodies anti-citrullinated protein antibodies (ACPA) synthesis in rheumatoid arthritis (RA) patients. Apart from their critical role in homeostasis and thrombosis, platelets have a significant contribution to inflammation as well. Although anuclear in nature, platelets have an intricate post-translational modification machinery. Till now, citrullination in platelets and its contribution to trigger autoantibodies ACPA production in RA is an unexplored research direction. Herein, we investigated the expression of peptidylarginine deiminase (PAD) enzymes and citrullinated proteins/peptides in the human platelets and platelet derived microparticles (PDP). Both PAD4 mRNA and protein, but not the other PAD isoforms, are detectable in the human platelets. With a strict filtering criterion,108 citrullination sites present on 76 proteins were identified in the human platelets, and 55 citrullinated modifications present on 37 different proteins were detected in the PDPs. Among them, some are well-known citrullinated autoantigens associated with RA. Citrullinated forms of thrombospondin-1, β-actin, and platelet factor-4 (also known as CXCL4) are highly immunogenic and bound by autoantibodies ACPA. Furthermore, ACPA from RA sera and synovial fluids recognized citrullinated proteins from platelets and significantly activated them as evidenced by P-selectin upregulation and sCD40 L secretion. These results clearly demonstrate the presence of citrullinated autoantigens in platelets and PDPs, thus could serve as potential targets of ACPA in RA.

Keywords: anti-citrullinated protein antibodies (ACPA); citrullination; platelet derived microparticles (PDP); platelets; rheumatoid arthritis.

Figure 1

Identification of PAD4 expression in the human platelets. (A) Agarose gel electrophoresis of RT-PCR products after amplifying the mRNA of PAD isoforms in platelets and monocytes. The β-actin expression was used as an internal control. PCR without the template served as a negative control. (B) SDS-PAGE depicting the proteins from platelets. Lanes 2-5 are corresponding to platelet samples divided into 20 slices from top to bottom, followed by an in gel-digestion and LC-MS/MS analysis. Arrow indicates PAD4 expression in the gel by MS analysis. Lanes 7-10, show the paired serum samples indicating the limitation present in the carry-through of plasma proteins into platelet proteomics. (C) Detected PAD4 peptide sequence coverage. Left section: the identified tryptic peptides are indicated in green shadow. Right section: list of detected and mapped peptides.

Figure 2

Analysis of citrullinated peptides present in the human platelets by mass spectrometry. (A) Decision tree for the identification of citrullination is shown in the left panel. Representative dimer-isotopic MS2 spectrum of non-citrullinated and citrullinated 560E–575K peptide of human fibrinogen α is shown in the right panel. Purple arrow indicates the neutral loss fragment (NL, -43.0058 Da) corresponding to y3+~y7+ ions. (B) Pie-chart showing the classification of identified citrullinated proteins in the platelets, according to functional categories. (C) Protein interaction network of citrullinated proteins. Orange bobbles show the actin-myosin filaments; blue bobbles show the microtubules; yellow bobbles show the actin-myosin filaments and microtubules.

Figure 3

Measurement of citrullinated TSP-1, β-actin, and PF4 - specific antibodies in the RA sera. (A-C) Determination of citrullinated TSP-1, β-actin, and PF4-specific antibodies in the sera (1:100 dilution) of healthy controls and RA patients. (D) Distribution of citrullinated TSP-1, β-actin, and PF4-specific antibodies in the anti-CCP2⁺ and anti-CCP2^- RA sera. Broken lines indicate the cutoff values. Cit, denotes citrullination, *p < 0.05, ns = not significant.

Figure 4

Detection of platelet activation after RA serum, SF or purified ACPA stimulation. (A) Flow cytometric quantification of CD62P expression in the platelets from healthy controls and RA patients. Data are representative of six independent experiments. (B, C) Both the serum and SF from RA induced significantly a higher level of CD62P expression and sCD40L secretion in RA and healthy platelets, compared to the healthy serum or OA SF (RA serum and SF: n=11, healthy control serum: n=11, OA SF: n=8). (D) Purified ACPA from RA SF and serum samples has significantly enhanced the CD62P expression and sCD40L secretion in RA and healthy platelets. SDS-PAGE showing the purified ACPA from SF of 2 RA patients by protein G plus beads and CCP2-peptide coated resin. Lane1/lane5: SF; Lane2/lane6: flow through from protein G column; Lane3/lane7: eluted IgG from protein G column; Lane4/lane8: eluted ACPA from CCP2-coated resin. Data are presented as mean ± SD, *p < 0.05.

Figure 5

Schematic picture of platelet as a source of autoantigens and persistent inflammation in RA. Autoantibodies ACPA recognized citrullinated proteins in platelet, result in platelet activation, aggregation, and microparticles releasing. This will further lead to citrullinated proteins exposure to the immune cell, triggering of ACPA production and contribute to the persistent inflammation.