The concentration of clottable fibrinogen in plasma decreases rapidly after severe traumatic injury and resuscitation, a reduction associated with both greater transfusion requirements and mortality. Fibrinogen is very sensitive to oxidative modification by oxidants generated during ischemia and inflammation, including hypochlorous acid and peroxynitrite, which both can oxidize methionine to methionine sulfoxide. Here, we investigated the potential of this mechanism to contribute to the coagulopathy of trauma.
We obtained plasma, clinical data, and outcomes from a biorepository of trauma patients presenting to the Emergency Department of a local Level I trauma center. Subjects with plasma INR ≥1.3 were defined as being coagulopathic while those with INR<1.3 were used as non-coagulpathic controls. We precipitated fibrinogen from plasma with glycine and tested it for the presence of methionine sulfoxide using tandem mass spectrometry. We also tested for defects in fibrin polymerization using thrombin time, reptilase time, and fibrinogen activity/antigenic ratio and correlated these changes with the presence of methionine sulfoxide in fibrinogen. The effects of selectively oxidizing certain methionine residues on fibrinogen structure and polymerization were then simulated using molecular dynamics.
Trauma patients who were coagulopathic (N=31) required more blood products in the first 24 hours of care compared to non-coagulopathic subjects (N=30) (mean PRBC: 3.9 vs. 0.5 units, p < 0.001; mean plasma transfused: 3.2 vs. 0.2 units, p < 0.001; and mean platelets transfused: 0.8 vs. 0.2 units, p=0.008) and were more likely to die (29% vs. 10% mortality, ChiSqr Likelihood Ratio p=0.056). Methionine sulfoxide was selectively increased in coagulopathic trauma patients at position 476 in the alpha C domain compared to controls [mean(SEM) 1.8(0.1)% vs. 2.5(0.1)%, p=0.003] and this low-level of oxidation was confirmed to be associated with an increasing reptilase time in vitro. Mean thrombin time (16 vs. 17.8 sec, p = 0.01) and reptilase times (18.2 vs. 23.7 sec, p<0.001) were prolonged in coagulopathic subjects and the fibrinogen activity/antigen ratio, standardized to albumin content to adjust for hemodilution, tended to be higher in coagulopathic patients (1.3 vs. 1.6, p = 0.06). Percent M476alpha oxidation was associated with increased INR (R=0.5, p=0.016), lower clottable fibrinogen concentration (R=-0.546, p=0.009), increased thrombin time (R=0.4, p=0.04), and increased reptilase time (R=0.5, p=0.01). Using multivariate regression, M476alpha oxidation remained significantly associated with reptilase time (M476alpha p=0.035) after adjusting for D-dimer concentration, fibrinogen concentration, and volume of plasma transfused at the time of measurement (whole model R2=0.77, p=0.003). When M476alpha oxidation was introduced into a human fibrinogen alpha C domain homology model, thermodynamic fluctuations were reduced, favoring a more-open configuration for beta sheet-hairpins that are hypothesized to play a role in fibrin monomer lateral aggregation by their dimerization and oligomerization via beta-hairpin swapping.
The level of oxidation of methionine residues in fibrinogen was increased in coagulopathic trauma patients presenting to the Emergency Department. Even at low levels, this modification was associated with altered fibrin polymerization suggesting that it may contribute mechanistically to coagulopathy by altering fibrin alpha C domain dimerization during the lateral aggregation of fibrin monomer.
White:Stasys Medical Corp: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Vidacare Corp: Honoraria; NIH: Research Funding; Coulter Foundation: Research Funding; Washington State Life Sciences Discovery Fund: Research Funding.
Asterisk with author names denotes non-ASH members.