Association of Thrombin Generation With Leukocyte Inflammatory Profile in Patients With Acute Ischemic Stroke

Abstract

Background and objectives: Thrombosis is central to the pathogenesis of acute ischemic stroke, with higher thrombin generation being associated with increased stroke risk. The immune system may contribute to thrombin generation in stroke and thus may offer novel strategies for stroke prevention. This study addresses the research question regarding the relationship of thrombin generation to leukocyte gene expression in patients with acute ischemic stroke.

Methods: We isolated RNA from whole blood and examined the relationship to thrombin generation capacity in patients with acute ischemic stroke. Due to its effects on thrombin generation, patients on anticoagulants were excluded from the study. The relationship of gene expression with peak thrombin was evaluated by analysis of covariance across peak thrombin quartiles adjusted for sex and age.

Results: In 97 patients with acute ischemic stroke, peak thrombin was variable, ranging from 252.0 to 752.4 nM. Increased peak thrombin was associated with differences in thromboinflammatory leukocyte gene expression, including a decrease in ADAM metallopeptidase with thrombospondin type 1 motif 13 and an increase in nuclear factor κB (NF-κB)-activating protein, protein disulfide isomerase family A member 5, and tissue factor pathway inhibitor 2. Pathways associated with peak thrombin included interleukin 6 signaling, thrombin signaling, and NF-κB signaling. A linear discriminant analysis model summarizing the immune activation associated with peak thrombin in a first cohort of stroke could distinguish patients with low peak thrombin from high peak thrombin in a second cohort of 112 patients with acute ischemic stroke.

Discussion: The identified genes and pathways support a role of the immune system contributing to thrombus formation in patients with stroke. These may have relevance to antithrombotic strategies for stroke prevention.

Figure 1. Hierarchical Cluster Plot of the 580 Genes Associated With Peak Thrombin

Patients are shown on the x-axis, and genes are shown on the y-axis. Patients with low peak thrombin in quartile 1 are shown in pink (median peak thrombin of 376.5 nM, range 252.0–406.4). Patients with high peak thrombin in quartile 4 are shown in blue (median peak thrombin of 565.5 nM, range 529.2–752.4). Red indicated an increased level of gene expression and green indicated a decreased level of gene expression.

Figure 2. Box Plots of Representative Genes Associated With Peak Thrombin by Quartile (p < 0.05, Partial Correlation Coefficient >|0.2|)

Quartile of peak thrombin is shown on the x-axis and fluorescence intensity log2 transformed on the y-axis. Error bars represent standard error of gene expression per quartile. From left to right: (A) NKAP, (B) ADAMTS13, (C) PDIA5, and (D) TFPI2.

Figure 3. Hierarchical Cluster Plot and Principal Component Analysis of 15-Gene Prediction Model

(A) Hierarchical cluster plot of the 15 genes (16 transcripts) used in a linear discriminant analysis model to distinguish patients with high peak thrombin (blue, quartile 4) from patients with low peak thrombin (pink, quartile 1). Genes are shown on the y-axis, and patients are shown on the x-axis. Red indicates an increased level of gene expression, and green indicates a decrease. (B) Principal component analysis of the 15 genes (16 transcripts) that distinguish patients with high peak thrombin from low peak thrombin.