Clot Structure: A Potent Mortality Risk Factor in Patients on Hemodialysis

Abstract

Patients with CKD on hemodialysis exhibit increased cardiovascular risk. Fibrin clot structure and clot lysis are crucially involved in development of cardiovascular events, but little is known about the influence of clot density on outcome in patients on hemodialysis. We determined fibrin clot structure parameters and effect on mortality in a prospective cohort of 171 patients on chronic hemodialysis (mean±SD age =59±11 years old; 54% men) using a validated turbidimetric assay. Kaplan-Meier analysis revealed that patients on hemodialysis with a denser clot structure had increased all-cause and cardiovascular mortality risks (log rank P=0.004 and P=0.003, respectively). Multivariate Cox regression models (adjusted for age, diabetes, sex, and duration of dialysis or fibrinogen, C-reactive protein, and complement C3) confirmed that denser clots are independently related to mortality risk. We also purified fibrinogen from healthy controls and patients on hemodialysis using the calcium-dependent IF-1 mAb against fibrinogen for additional investigation using mass spectrometric analysis and electron microscopy. Whereas purified fibrinogen from healthy controls displayed no post-translational modifications, fibrinogen from patients on hemodialysis was glycosylated and guanidinylated. Clots made of purified fibrinogen from patients on hemodialysis exhibited significantly thinner fibers compared with clots from fibrinogen of control individuals (mean±SD =63±2 and 77±2 nm, respectively; P<0.001). In vitro guanidinylation of fibrinogen from healthy subjects increased the formation of thinner fibers, suggesting that difference in fiber thickness might be at least partially due to post-translational modifications. Thus, in patients on hemodialysis, a denser clot structure may be a potent independent risk factor for mortality.

Keywords: chronic kidney disease; clot; coagulopathy; dialysis patients; mortality; uremia.

Figure 1.

Clot density correlates with complement C3 and CRP plasma levels. Correlation of (A) complement C3 and (B) CRP plasma levels with clot density as assessed by clot final turbidity (FT; n=171). AU, arbitrary unit.

Figure 2.

A denser clot structure is related to mortality in patients on dialysis. Kaplan–Meier analysis of (A) all-cause and (B) cardiovascular mortality in patients with denser or less dense clot structure (grouped according to median; log rank test: P=0.004 and P=0.003, respectively).

Figure 3.

Post-translational modifications of fibrinogen in uremia. (A) Characteristic mass fingerprint spectrum of tryptic-digested fibrinogen isolated from healthy controls. The arrows indicate characteristic mass signals of peptides of the tryptic-digested fibrinogen. The amino acid sequences of the peptides of interest are given. (B) Characteristic mass fingerprint spectrum of tryptic-digested fibrinogen isolated from a patient with CKD (stage 5). The arrow at 853 m/z indicates the guanidinylated peptide. The arrow at 885 m/z indicates the glycosylated peptide. AU, arbitrary unit. *Guanidinylated lysine of the corresponding amino acid sequence; **glycosylated threonine of the corresponding amino acid sequence.

Figure 4.

Thinner fibrin fibres in uremia and after in vitro guanidinylation. Scanning electron micrographs of clots prepared from pooled fibrinogen of (A) healthy control individuals and (B) patients on HD, (C) purified fibrinogen, which was not modified, and (D) in vitro guanidinylated fibrinogen. P<0.001.