Accelerated Spatial Fibrin Growth and Impaired Contraction of Blood Clots in Patients with Rheumatoid Arthritis

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease associated with thrombotic complications. To elucidate pathogenic mechanisms, hemostatic disorders in RA were correlated with other laboratory and clinical manifestations. Hemostasis was assessed using relatively new complementary tests, the spatial growth of a plasma clot (Thrombodynamics assay), and contraction of whole blood clots. Platelet functionality was assessed with flow cytometry that quantified the expression of P-selectin and the fibrinogen-binding capacity of platelets before and after activation with a thrombin receptor-activating peptide. Parameters of fibrin clot growth and the kinetics of contraction of blood clots were significantly altered in patients with RA compared to the control group. In Thrombodynamics measurements, an increase in the clot growth rate, size, and optical density of plasma clots altogether indicated chronic hypercoagulability. The rate and extent of blood clot contraction in patients with RA was significantly reduced and associated with platelet dysfunction revealed by an impaired response to activation. Changes in the parameters of clot growth and contraction correlated with the laboratory signs of systemic inflammation, including hyperfibrinogenemia. These results confirm the pathogenic role of hemostatic disorders in RA and support the validity of fibrin clot growth and the blood clot contraction assay as indicators of a (pro)thrombotic state.

Keywords: blood coagulation; contraction of blood clots; hypercoagulability; rheumatoid arthritis.

Figure 1

Results of the optical system used to quantify the spatial growth of fibrin clots (Thrombodynamics) (A) and the kinetics of contraction (retraction) of blood clots as a function of time (B). At the top are images of the cuvettes for clot growth (A) and clot contraction (B) and below are graphs of the output of these assays. For details see Materials and Methods (Section 4.3 and Section 4.4).

Figure 2

Parameters to quantify fibrin clot growth from Thrombodynamics measurements in patients with rheumatoid arthritis (RA) (n = 60) compared with the control group (n = 50). The initial growth rate of the clot (A), stationary growth rate of the clot (B), lag time (C), clot size (D), and clot density (E). The results are presented as the median and intervals between 25th and 75th, as well as between 5th and 95th percentiles (Mann-Whitney U test). n.s.—not significant.

Figure 3

Quantification of blood clot contraction in RA patients (n = 60) compared with those of the control group (n = 50). The extent of clot contraction (A), lag time of contraction (B), average velocity of contraction (C), and area under the curve (work performed by platelets in contraction) (D). The results are presented as the median and intervals between the 25th and 75th, as well as between the 5th and 95th percentiles (Mann-Whitney U test).

Figure 4

Comparative phase analysis of the averaged kinetic curves of clot contraction for RA patients (n = 63) and control group (n = 50). For the kinetic curves (A), transitions between phases 1, 2, and 3 of clot contraction were determined by finding local maxima and minima within the instantaneous first derivatives (dashed lines). The curves were fit using a piecewise function, and the rate constants of each phase were determined [33]. Rate constants of phase 1 (B), phase 2 (C), and phase 3 (D) are shown for RA patients and the control group. Data are presented as the median and interquartile ranges (25th and 75th percentiles) (Mann-Whitney U test).

Figure 5

Quantification of contraction of blood clots in patients with RA, not taking (n = 37) and taking (n = 23) pentoxifylline, compared with the control group (n = 50). The extent of clot contraction (A), lag time of contraction (B), average velocity of contraction (C), and area under the curve (work performed by platelets in contraction) (D). The results are presented as the median and intervals between 25th and 75th, as well as between 5th and 95th percentiles (Kruskal-Wallis test and Dunn’s multiple post-hoc test). n.s.—not significant.