Tissue plasminogen activator modified thromboelastography identifies fibrinolysis resistance in dogs with immune-mediated hemolytic anemia

Abstract

Introduction: Immune-mediated hemolytic anemia (IMHA) is an important immunologic disorder in dogs that is associated with high mortality rates, frequently due to thromboembolism. Multiple factors contribute to the pathophysiology of thrombosis in IMHA including intravascular tissue factor expression, platelet activation, and neutrophil extracellular trap (NET) formation. It was hypothesized that dogs with IMHA have impaired fibrinolysis that can be detected using a modified viscoelastic assay and that biomarkers of NET formation are associated with this hypofibrinolysis.

Methods: Twenty dogs with non-associative IMHA were enrolled and paired thromboelastography (TEG) assays with and without additional tissue plasminogen activator (tPA) performed. A panel of hemostasis tests including measurement of plasma thrombin-activatable fibrinolysis inhibitor (TAFI) activity, active plasminogen activator inhibitor-1 (PAI-1), and concentrations of cell-free DNA (cfDNA) and nucleosomes were also performed.

Results: Dogs with IMHA had hypercoagulable TEG tracings, increased TAFI activity and frequently displayed fibrinolysis resistance defined as minimal lysis in tPA augmented TEG assays. Increased concentrations of cfDNA, nucleosomes and active PAI-1 in dogs with IMHA compared to healthy controls were identified.

Discussion: These observations support the hypothesis that hypofibrinolysis is a common feature of IMHA in dogs. Increased plasma active PAI-1 concentrations and TAFI activities might contribute to the observed hypofibrinolysis. The combined hypercoagulability and hypofibrinolysis observed supports recent recommendations to provide thromboprophylaxis to all dogs with IMHA. These findings also suggest that NETosis might contribute to the common prothrombotic imbalance of IMHA in dogs.

Keywords: cell-free DNA; immunothrombosis; neutrophil extracellular traps; nucleosomes; plasminogen activator inhibitor-1; thrombin-activatable fibrinolysis inhibitor.

Figure 1

Kaolin and tissue factor activated thromboelastography assays with and without additional tissue plasminogen activator indicate that dogs with sepsis are hypofibrinolytic and display fibrinolysis resistance. (A,B) Kaolin LY30 +/− tPA. (C,D) Kaolin LY60 +/− tPA. (E,F) TF 1:3,600 LY30 +/− tPA. (G,H) TF 1:3,600 LY60 +/− tPA. Comparisons were performed using Wilcoxon signed rank tests. (A,C,E,G) Central horizontal line represents the median, the boxes represent the interquartile range, and the whiskers represent the minimum and maximum values. (B,D,F,H) Paired values are connected by lines to indicate the direction of change in the lysis values without and with additional tPA as indicated.

Figure 2

Kaolin and tissue factor (TF) activated thromboelastography assays with and without additional tissue plasminogen activator (tPA) indicate that dogs with sepsis are hypofibrinolytic and display fibrinolysis resistance. Note, healthy control data for the kaolin assay are derived from Spodsberg et al. (22), and healthy control data for the TF assay are derived from Fletcher et al. (49). All panels compare values in healthy controls with those in dogs with IMHA. (A) Kaolin LY30. (B) Kaolin LY60. (C) TF 1:3,600 LY30. (D) TF 1:3,600. For all panels, symbols represent the mean value, whiskers represent the standard deviation. Comparisons were performed using unpaired t-tests with Welch’s correction.

Figure 3

Representative thromboelastography (TEG) tracings of a dog with expected tPA-induced fibrinolysis. (A) Tissue factor-activated TEG tracings from a dog with IMHA where hypofibrinolysis was not apparent. The black line is the tracing with only the activator, while the green line is the tracing with both the activator and the addition of tPA. In these tracings, the addition of tPA caused clot lysis to occur relative to the paired tracing without the addition of tPA. Although the degree of fibrinolysis is diminished compared to healthy dogs, these tracings indicate that this dog did not have fibrinolytic shutdown. (B,C) Kaolin activated and tissue factor activated TEG tracings from dogs with IMHA displaying fibrinolysis resistance (also termed fibrinolytic shutdown). The black lines are the tracings with only the activator, while the green lines are the tracings with both the activator and the addition of tPA. In dogs with hypofibrinolysis (also termed fibrinolysis resistance or fibrinolytic shutdown) the addition of tPA does not cause clot lysis to occur.

Figure 4

Box and whisker plots of (A) plasma concentrations of active plasminogen activator inhibitor-1 (PAI-1) and (B) plasma activity of thrombin-activatable fibrinolysis inhibitor (TAFI). Dogs with IMHA had significantly higher PAI-1 concentrations and TAFI activities than healthy controls. Comparisons were performed using the Mann–Whitney U test. The central horizontal line represents the median, the boxes represent the interquartile range, and the whiskers represent the minimum and maximum values.

Figure 5

Scatterplots depicting the correlations between select coagulation variables in dogs with IMHA. (A) Plasma concentration of active plasminogen activator inhibitor-1 (PAI-1) against plasma activity of thrombin-activatable fibrinolysis inhibitor (TAFI). (B) Plasma concentration of active plasminogen activator inhibitor-1 (PAI-1) against plasma D-dimer concentration. (C) Plasma activity of thrombin-activatable fibrinolysis inhibitor (TAFI) against activated partial thromboplastin time (aPTT). (D) Plasma activity of thrombin-activatable fibrinolysis inhibitor (TAFI) against plasma D-dimer concentration. On each panel, the Spearman’s rank correlation coefficient (r_s) and associated p-value are displayed.