Overall leukocyte levels link risk factors to Von Willebrand factor and Neutrophil Extracellular Traps in stroke thrombi: a Structural Equation Modeling analysis

Abstract

Background: Von Willebrand Factor (VWF) and Neutrophil Extracellular Traps (NETs) are involved in the inflammatory response during thrombi formation and are widely found in thrombi of Acute Ischemic Stroke (AIS) patients. Inflammation may mediate the relationship between cerebrovascular risk factors (such as blood glucose) and thrombi components. This study uses overall leukocyte levels to identify potential links between risk factors and VWF, NETs in thrombi.

Methods: Thrombi samples and clinical data from 61 stroke patients treated at our hospital between 2017 and 2023 were collected. The Partial Least Squares Structural Equation Modeling (PLS-SEM) assessed direct and indirect associations, with leukocyte and its subtype counts as mediating variables, VWF and NETs as endogenous variables, and cerebrovascular risk factors as exogenous variables.

Results: Heart dysfunction and blood glucose showed a significant negative indirect effect on VWF through the overall leukocyte levels (indirect effect = -0.119 and -0.118, p < 0.05). Overall leukocyte levels acted as a mediator between vitamin B12 and VWF levels in thrombi, displaying a positive mediatory impact (indirect effect = 0.118, p < 0.05). The decrease in VWF levels within thrombi was closely associated with an increase in discharge NIHSS scores (path coefficient = -0.353, p < 0.05). Additionally, overall leukocyte levels and homocysteine (Hcy) had significant negative effects on VWF (path coefficients = -0.384 and -0.308, p < 0.05), while vitamin B12 had a negative impact on NETs (path coefficient = -0.289, p < 0.05).

Conclusion: Overall leukocyte levels mediate the influence of blood glucose levels, heart dysfunction, and vitamin B12 on content VWF in thrombi in stroke patients. VWF reduction correlates with elevated discharge NIHSS scores. These cerebrovascular factors may regulate the pathological process of AIS thrombosis through inflammatory responses, guiding further exploration of the underlying mechanisms and informing clinical strategies.

Keywords: Neutrophil Extracellular Traps (NETs); Partial Least Squares Structural Equation Modeling (PLS-SEM); Von Willebrand factor (VWF); overall leukocyte levels; stroke.

Figure 1

Immunohistochemical analysis of thrombi specimens. Representative images showing VWF and NETs staining in thrombi of different origins. These images illustrate the presence and spatial distribution of the two markers within thrombus sections. Immunohistochemically positive areas are shown in brownish-yellow. (A) Represents NETs, (B) represents VWF. 400x magnification.

Figure 2

Final PLS-SEM model incorporating the following latent constructs: NETs, VWF, cardiac dysfunction, overall leukocyte levels, blood glucose, discharge NIHSS, vitamin B12. Overall leukocyte levels are effectively represented by neutrophils, lymphocytes and eosinophils. Blood glucose is represented by fasting blood glucose and random blood glucose. Cardiac dysfunction is represented by CKMB and Myoglobin. VWF and NETs are represented by the AOD measured in five different fields of view. PLS-SEM defines the relationships between latent variables and observed variables and among latent variables in the form of a path model. The directions of the explained paths are indicated by connecting arrows. Solid lines denote statistically significant paths, whereas dashed lines denote non-significant paths. Numbers on the arrows correspond to path coefficients, indicating the strength of each association. VWF, Von Willebrand factor; NETs, neutrophil extracellular traps; Hcy, Homocysteine; NIHSS, National Institutes of Health Stroke Scale.