Role of Von Willebrand factor level as a biomarker in acute ischemic stroke

Background Von Willebrand factor (VWF) is a large, multimeric glycoprotein that plays a role in thrombus forma‑ tion; it is also an important mediator of inflammation. Our study aims to determine the association of VWF plasma level and acute ischemic stroke and determine plasma level of VWF in different subtypes of acute ischemic stroke. This case–control study was conducted on 90 subjects: 30 acute ischemic atherosclerotic stroke patients, 30 acute cardioembolic stroke patients and 30 healthy age and sex‑matched control subjects. Stroke patients were recruited within the first week of stroke onset with an age range from 18 to 75 years. All subjects underwent complete neuro‑ logical examination, duplex ultrasonography (U/S), CT brain, routine laboratory work‑up and serum level of VWF. Results VWF serum levels were significantly elevated in patients of acute ischemic stroke, compared to control sub‑ jects. Higher plasma levels of VWF were observed in patients with acute ischemic atherosclerotic stroke. Conclusion Serum level of VWF can be used as a marker for acute ischemic stroke, especially the atherosclerotic subtype.


Background
Von Willebrand factor (VWF) is a large multimeric plasma glycoprotein that plays an important role in primary hemostasis.It is synthesized in vascular endothelium and megakaryocytes, then stored in Weibel-Palade bodies of endotheliocytes and granules of platelets or released into the circulation.Upon vascular wall damage, plasma VWF binds to collagen in the exposed subendothelial matrix, and platelet glycoprotein Iba (GPIba) triggers platelet aggregation and thrombus formation [1].
Levels of VWF were significantly elevated in patients with transient ischemic attack (TIA) or ischemic stroke attributed to large artery atherosclerosis, compared with the other stroke subtypes.VWF serves as a useful marker of atherosclerosis and subsequently, a risk marker of stroke [2].
The aim of this work is to evaluate the association of VWF plasma level in subtypes of acute ischemic stroke and correlate its level with the severity of the stroke measured by National Institute of Health Stroke Scale (NIHSS) [3], and functional outcome measured by modified Rankin Scale (mRS) [4].

Methods
This is a case-control study, 90 subjects were recruited from Kasr Al Ainy Hospital ED and were admitted to the Stroke Unit.Patients were subsequently divided into 3 groups: group I, 30 acute ischemic atherosclerotic stroke patients; group II, 30 acute cardioembolic stroke patients; and group III, 30 age and sex-matched normal healthy control subjects.All ischemic stroke patients were recruited within the first week of stroke onset.The study was conducted between January 2020 and January 2021.
The G*Power 3.1.9.7 program (Heinrich Heine Universität Düsseldorf ) was used to estimate the sample size for our study.For an effect size 0.8, error 0.05 and at a Power of 0.8 a minimum of 27 for each group was determined.In this study 30 subjects were recruited in each group.
Inclusion criteria were as follows: age range 18-75 years, both sexes, acute ischemic stroke patients, within 7 days of symptoms onset, patients with a good temporal window.Exclusion criteria: patients < 18 years and > 75 years, patients with subarachnoid, subdural, extradural, or intracerebral hemorrhage, malignancy, and CNS infection.
Patients' demographics and vascular risk factors including diabetes, hypertension, smoking, and hyperlipidemia were documented.General and neurological examinations including NIHSS [3] were used for assessment of stroke severity and mRS was [4] used for assessment of functional outcome.
Applying extracranial and transcranial neuro-sonological assessments using Philips IU22 duplex machine software, version 2.0.13,USA 2012, carotid artery stenosis was defined according to the criteria set by society of radiologists in ultrasound 2003 [5].Diagnosis of intracranial stenosis was interpreted according to the internationally published criteria [6].Intima medial thickness (IMT) above 1 mm at any age is associated with a significantly increased risk of myocardial infarction and/or cerebrovascular disease [7].
Serum level of VWF, was measured by enzyme linked immunosorbent assay (ELISA) based on the biotin double antibody sandwich technology using stat fax-2100 device.The reference interval and normal range of VWF was calculated and was determined to be 6-24 ng/dl.The reference range was established in the lab according to CLSI EP28-A3C guideline to define and establish reference intervals in clinical laboratories.
Statistical methods: Data were coded and entered using the statistical package SPSS (Statistical Package for the Social Sciences) version 25.Data were summarized using median and interquartile range in quantitative data and using frequency (count) and relative frequency (percentage) for categorical data.Comparisons between quantitative variables were done using the non-parametric Kruskal-Wallis and Mann-Whitney tests [8].Correlations between quantitative variables were done using spearman correlation coefficient [9].P-values less than 0.05 were considered as statistically significant.
The ethical review board of Cairo University revised and approved the study protocol.All the study participants were treated according to the Helsinki declaration of Biomedical ethics.Written informed consent was obtained from the patients or their caregivers.

Results
Sixty stroke patients and 30 age and sex-matched control subjects were recruited in this study.Patients were subsequently divided into 3 groups: group I, 30 acute ischemic atherosclerotic stroke patients; group II, 30 acute cardioembolic stroke patients; and group III, normal healthy control subjects.
The age of the selected patients ranged from 31-74 years, and age of control subjects ranged from 34-60 years.In patients' groups: there were 39 males (65%) and 21 females (35%), while in control subjects there were 20 males (66.7%) and 10 females (33.3%).In group I there were 20 males and 10 females, while in group II there were 19 males and 11 females.
Tables 1 and 2 show the comorbidities and vascular risk factors in patients and control groups.Thirtyfive out of 60 patients had a cardiac problem which includes: valvular heart disease, cardiomyopathy (ischemic or non-ischemic) (58.3%), 27 had abnormal ECG (45%) in the form of atrial fibrillation.Thirtytwo patients had abnormal echo (53.3%) in the form of dilated left atrium, cardiomyopathy, impaired contractility, regional wall motion abnormalities and valvular dysfunction.
Patients' NIHSS ranged from 2 to 19 with a mean of 10.28 ± 4.56 SD.Patients' mRS score ranged from (1 to 5) with mean of 3 ± 0.99 as in Tables 3 and 4.
Forty-eight patients (80%) had anterior circulation stroke, while 12 patients (20%) had posterior circulation stroke.In group I: 23 patients had anterior circulation stroke, 7 patients had posterior circulation stroke, while in group II: 25 patients had anterior circulation stroke, 5 patients had posterior circulation stroke as shown in Table 5.
Among 60 patients, 36 had increased intima media thickness (IMT) on both sides (60%) and 24 patients with normal (IMT) on both sides (40%), compared to 30 control subjects who had normal IMT on both sides, as described in Table 6.
Forty patients had abnormal duplex finding either intracranial or extracranial (66.7%), 20 patients had normal duplex (33.3%).Duplex findings are shown in Tables 7 and 8.It was noticed that higher levels of VWF were present in acute ischemic patients than in control subjects.Such a difference was statistically highly significant (P value < 0.001***) as in Table 9 and Fig. 1.
Higher plasma level of VWF was noticed in patients with acute ischemic atherosclerotic stroke (group I) than in patients with acute cardioembolic stroke (group II) with statistical significance.P value = 0.025* as in Table 10 and Fig. 2.
We found increased level of VWF in patients with increased intima media thickness (IMT) (49.68 ± 28) compared to patients with normal intima media thickness (IMT) (40.35 ± 21.82 ng/dl), however such difference was not statistically significant, P value = 0.386 as described in Table 11 and Fig. 3.
No statistical significance was detected while comparing VWF plasma levels with degree of overall stenosis in patients, P value = 0.112 as in Table 12.
Also, no statistical significance was found while comparing VWF plasma levels with degree of stenosis of extracranial or intracranial arteries in patients p value = 0.090, P value = 0.316 as in Table 13.
There was no statistically significant comparative between VWF plasma levels and renal function, hepatic function, lipid function, uric acid and HbA1C.
No statistically significant difference in VWF level between males and females with P value = 0.768.
No statistically significant relation was observed between VWF plasma level and NIHSS score, mRS score and age in patients' group (P value 0.787, 0.411, 0.625), respectively.

Discussion
In the current study, it was observed that VWF plasma levels were significantly higher in patients with acute ischemic stroke within the first week of stroke onset than in serum of control subjects.It can be explained by the role of VWF in platelet adhesion and thrombus formation.VWF is a large, multimeric glycoprotein present in blood plasma endothelium and binds to other proteins, particularly factor VIII, preventing its rapid degradation.It is mediating initial platelet adhesion at sites of vascular injury.Although this is a prerequisite for normal hemostasis, adhesion of platelets is also the first step in thrombosis and an important mediator of inflammation [10].Similar to our results, Menih et al. [11], found that elevated level of von Willebrand Factor (VWF) is associated with increased risk for coronary heart disease and ischemic stroke.Barakzie et al. [12], also found that VWF is associated with risk of ischemic stroke, stroke severity, and clinical outcome.Hanson et al. [13] found that VWF levels are increased in patients with ischemic stroke   compared to control subjects, it was explained by role of VWF in acute phase of ischemic stroke and prothrombotic effects of VWF that play a role in atherosclerosis/ inflammation or endothelial damage [13,14].VWF is predominantly released from endothelial cells.Damage to the endothelium in atherosclerosis increases the release of VWF, which can lead to a sudden thrombus formation in the arteries [11].The VWF is a major factor for the accumulation and activation of platelets in stenotic arteries, which can lead to acute thrombotic occlusion, and it has a dual role, participating in normal hemostasis as well as in the pathological process of thrombus formation in arteries [11].
In our study higher plasma levels of VWF were more significantly associated with acute ischemic atherosclerotic stroke patients, than in patients with acute cardioembolic stroke (P value = 0.025).Our results were similar to the studies performed by Sonneveld et al., and Buchtele et al., where VWF plasma levels were significantly raised in patients of acute ischemic stroke.A strong positive association was detected between the extent of atherosclerosis, and VWF levels [15,16].Kraft et al. [17] noticed that subjects with carotid plaques have elevated VWF plasma levels.
It could be explained by association of VWF level in the acute phase after ischemic stroke and its association with inflammation and thrombo-inflammation.The term ''thrombo-inflammation'' describes the interaction of thrombotic (for example, coagulation factors, platelets) and inflammatory (for example, immune cells) circuits that occurs at the ischemic neurovascular unit and has been identified as key mechanism of stroke occurrence and propagation.
On the other hand, Maida et al. [18], observed higher levels of VWF in patients with cardioembolic acute ischemic stroke compared to other stroke subtypes.Hanson et al. [13] found that levels of VWF in the acute phase of both cardioembolic and atherosclerotic stroke were significantly higher compared to the small vessel disease subtype of stroke.
In our study we found no significant association between VWF plasma levels and the degree of stenosis of intracranial or extracranial arteries in patients with acute ischemic stroke.On the contrary Murphy et al. [19], found that VWF antigen expression is enhanced in symptomatic versus asymptomatic carotid stenosis patients.Also, Kinsella et al. [20], found that endothelial activation is enhanced in symptomatic versus asymptomatic carotid stenosis patients.VWF levels increase in early symptomatic versus asymptomatic carotid stenosis [21,22].
In the current study, NIHSS and mRS scores were not associated with higher plasma levels of VWF, there was no statistical significance for this.However, it may be related to a limited number of patients.Meanwhile, Menih et al. [11], found that patients with high NIHSS score on admission had significantly higher levels of VWF as a predictor for the severity of stroke.Also, patients

Fig. 3
Fig.3Comparative between VWF levels (ng/dl) and IMT in patients using independent-samples Mann-Whitney U test

Table 1
Comorbidities and risk factors in patients and control groups HTN: hypertension, DM: diabetes mellitus

Table 2
Risk factor in subgroups of stroke patients and control groups

Table 3
NIHSS score in subgroups of patients

Table 4
mRS score in patients' subgroups

Table 5
Site of infarction in patients' subgroups

Table 6
IMT in patients' subgroups

Table 7
Transcranial and intracranial duplex findings in patients' groups

Table 8
Duplex state in group I and II

Table 9
Comparing VWF plasma levels between patients and control groups

Table 12
Comparative between VWF levels and overall degree of stenosis in duplex VWF: Von Willebrand factorNumber of patients (

Table 13
Comparative between VWF levels and degree of stenosis of extracranial and intracranial arteries in patientsVWF: Von Willebrand factor