Background/Purpose: The establishment of longitudinal pre-clinical RA cohorts is beginning to provide important insights into the mechanisms that precede the onset of clinically detectable disease. In an attempt to identify candidate biomarkers that may suggest the activation of specific biological pathways associated with RA onset, we used aptamer based proteomic technologies to mine the serum proteome of a cohort of individuals who were followed longitudinally into disease onset.

Methods: We have prospectively followed a large cohort of first-degree relatives of North American Native RA patients, a subset of whom developed clinically detectable synovitis after an extended period of follow up. Representative serum samples were selected from 10 such individuals and analyzed using the SOMAmer (slow off-rate modified aptamer) platform (SOMAlogic Inc., Boulder, Co) to generate simultaneous quantitative levels on 1132 serum proteins. For each individual, a sample was obtained as close as possible to the RA onset date, while a matched sample had been obtained a minimum of 2 years prior to the onset. Fold changes for each individual protein at the two time points were derived from the data. The significance of the fold change was calculated by paired analysis using Stanford Analysis of Microarrays (SAM) software set at a false discovery rate <1%. For selected proteins, levels were confirmed using ELISA at multiple time points.

Results: Of the 10 patients who converted from pre-RA to clinical RA, 6 were female and all were active smokers. The average age at conversion was 34.8 (+/- 13.6) years, while the average number of months to conversion was 51.8 (+/- 30.6). Anti-CCP antibodies were detectable in the pre-RA sample in 4 patients. All 10 patients were anti-CCP positive at the time of clinical disease onset. Quantification using SOMAlogic platform indicated that 18 proteins had a mean fold increase of 3.2 (range=1.2-12.8), while 28 proteins had a mean fold decrease of 0.7 (range=0.3-0.8). Ingenuity pathway analysis revealed activation of angiogenesis, and both mucosal and cellular immunity. Of note, there was a consistent proteomic signature suggesting activation of the intrinsic coagulation cascade at the time of RA onset: Factor X increased by a mean fold increase=1.6, Factor IX increased by a mean fold increase=3.4, and Factor V increased by a mean fold increase=1.3, compared to the pre-RA sample. This pattern was detectable in 9 of 10 patients studied.

Conclusion: Based on broad-based quantitative analysis of more than 1000 serum proteins in longitudinally followed individuals who ultimately developed RA, we present evidence indicating activation of the intrinsic coagulation cascade at the time of clinical development of synovitis. Aligning with a growing body of evidence, we hypothesize that these hemostatic cofactors contribute to the pathophysiology of RA, possibly through cytokine signalling as well as synovial fibrin deposition. Our approach also demonstrates the utility of a novel assay for biomarker discovery, one that provided a rich dataset that will help shape the direction of future studies.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/broad-based-interrogation-of-the-serum-proteome-suggests-that-ra-onset-is-associated-with-activation-of-the-intrinsic-coagulation-cascade/