Decreased clot burden is associated with factor XIII Val34Leu polymorphism and better functional outcomes in acute ischemic stroke patients treated with intravenous thrombolysis

István Szegedi; Rita Orbán-Kálmándi; Attila Nagy; Ferenc Sarkady; Nikolett Vasas; Máté Sik; Levente István Lánczi; Ervin Berényi; László Oláh; Alexandra Crișan; László Csiba; Zsuzsa Bagoly

doi:10.1371/journal.pone.0254253

. 2021 Jul 7;16(7):e0254253. doi: 10.1371/journal.pone.0254253

Decreased clot burden is associated with factor XIII Val34Leu polymorphism and better functional outcomes in acute ischemic stroke patients treated with intravenous thrombolysis

István Szegedi ¹, Rita Orbán-Kálmándi ², Attila Nagy ³, Ferenc Sarkady ², Nikolett Vasas ⁴, Máté Sik ⁴, Levente István Lánczi ⁴, Ervin Berényi ⁴, László Oláh ¹, Alexandra Crișan ⁵, László Csiba ^1,⁶, Zsuzsa Bagoly ^2,^6,^*

Editor: Arijit Biswas⁷

PMCID: PMC8263307 PMID: 34234378

Abstract

Background

Intravenous thrombolysis using recombinant tissue plasminogen activator remains the mainstay treatment of acute ischemic stroke (AIS), although endovascular treatment is becoming standard of care in case of large vessel occlusions (LVO). To quantify the thrombus burden in LVO, a semiquantitative CT angiography (CTA) grading system, the clot burden score (CBS) can be used. Here we aimed to study the association between CBS and various hemostasis parameters, and to evaluate which parameters are major determinants of thrombolysis outcome.

Methods

In this single-centered prospective observational case-control study, 200 anterior circulation AIS patients receiving intravenous thrombolysis treatment without thrombectomy were enrolled: 100 AIS patients with LVO (CBS 0–9) and 100 age- and sex-matched AIS patients without LVO (CBS 10). Fibrinogen, α2-plasmin inhibitor, plasminogen, factor XIII and D-dimer were assessed from blood samples taken before and 24 h after thrombolysis, and FXIII-A Val34Leu was genotyped. CBS was calculated using admission CTA. Short-term outcomes were defined based on the change in NIHSS by day 7, long-term outcomes were assessed according to the modified Rankin scale at 3 months post-event.

Results

Poor outcomes were significantly more frequent in the CBS 0–9 group. Plasminogen activity on admission was significantly higher in the CBS 0–9 group. In a univariate analysis, significant protective effect of the Leu34 allele against developing larger clots (CBS 0–9) could be demonstrated (OR:0.519; 95%CI:0.298–0.922, p = 0.0227). Multivariate regression analysis revealed that CBS is an independent predictor of short- and long-term functional outcomes, while such effect of the studied hemostasis parameters could not be demonstrated.

Conclusions

CBS was found to be a significant independent predictor of thrombolysis outcomes. FXIII-A Leu34 carrier status was associated with smaller thrombus burden, which is consistent with the in vitro described whole blood clot mass reducing effects of the allele, but the polymorphism had no effect on thrombolysis outcomes.

Introduction

Acute ischemic stroke (AIS) is a potentially severe vascular disease that leads to disability or death without proper treatment [1]. The patient’s only chance of recovery depends on the successful opening of the occluded vessel. As of today, two evidence-based therapies are approved. The endovascular therapy known as mechanical thrombectomy is used more and more frequently in case of large vessel occlusions worldwide, nevertheless, intravenous thrombolysis using recombinant tissue plasminogen activator (rt-PA) is still the mainstay therapy for AIS [2]. Unfortunately, the 4.5-hour-long therapeutic time window is narrow and thus only a fraction of patients are eligible for this therapy. Moreover, thrombolysis is only effective in approximately 30–40% of patients, while in a smaller subset of cases (6–8%) life-threatening side-effects, e.g., intracerebral hemorrhage occurs. These complications cannot be foreseen at the initiation of therapy and their occurrence remains unexplained [3].

The outcome of thrombolysis can be affected by several factors: higher systolic blood pressure on admission, higher NIHSS score on admission, history of coronary artery disease and/or atrial fibrillation all lead to worse functional outcome [4]. Another important prognostic factor is the presence of large-vessel occlusion (LVO) [5]. Approximately 24–46% of acute ischemic strokes are caused by the occlusion of a large vessel [6]. These patients have more severe neurological symptoms on admission and their prognosis is also worse compared to patients without LVO [6].

In order to quantify the thrombus burden in LVO, a semiquantitative computed tomography angiography (CTA) grading system, the so-called “clot burden score” (CBS) has been developed [7]. The CBS is a 10-point system, evaluating the anterior circulation based on the presence of contrast opacification on CTA. Two points each are subtracted for the absence of contrast opacification in the complete cross-section of any part of the proximal M1 segment, distal M1 segment or supraclinoid ICA, and 1 point each for M2 branches, A1 segment and infraclinoid ICA.

Although it is logical to assume that thrombus size affects the outcome of AIS thrombolysis treatment, little is known about the mechanisms controlling thrombus burden. We can surmize that levels of certain hemostasis factors and genetic polymorphisms are likely to be associated with thrombus size and the prognosis of AIS [8–10]. An important regulator of thrombus burden might be coagulation factor XIII (FXIII), a plasma protransglutaminase, a key regulator of fibrinolysis and clot structure. Its active form (FXIIIa) is responsible for the cross-linking of fibrin chains and the cross-linking of α2-antiplasmin to fibrin, providing mechanical stability to the fibrin clot and protecting it from premature fibrinolysis [11]. FXIII-A has a common genetic alteration: a G->T in codon 103 of the gene, which leads to a change from valine to leucine in position 34 (FXIII-A Val34Leu) [12]. FXIII-A Leu34 allele influences the rate of FXIII activation by thrombin and as a result it affects clot structure. A protective effect of the polymorphism against myocardial infarction and venous thromboembolism has been described by a number of studies and meta-analyses [13–16]. Most recently, by in vitro experiments we have identified that the protective effect of the FXIII-A Leu34 allele might be attributed to its decreasing effect of whole blood clot mass in the presence of the elevated fibrinogen levels [17]. On the other hand, in vivo data on potential associations between FXIII-A Val34Leu polymorphism and clot burden is still lacking.

Here we aimed to study the association between CBS and various hemostasis parameters, and to evaluate which parameters are major determinants of the outcome of intravenous thrombolysis.

Materials and methods

Patients

In this single-centered prospective observational case-control study, anterior circulation AIS patients eligible for intravenous thrombolysis treatment were enrolled at the Stroke Center of the Department of Neurology, University of Debrecen, Hungary. The 2008 ESO guideline was used for the inclusion and exclusion of patients for rt-PA administration [18]. In this case-control design, each AIS patient with LVO (CBS 0–9) was matched with an age- and sex-matched AIS patient without LVO (CBS 10). Patients were selected from 519 consecutive AIS patients admitted to the stroke center over a period of 59 months. All patients received intravenous thrombolysis within the 4.5 h therapeutic time window using rt-PA according to standard protocols [18]. None of the patients underwent mechanical thrombectomy: thrombectomy was either unavailable or the patient was unsuitable to perform the procedure. The diagnosis of AIS was made based on clinical symptoms, brain imaging using non-contrast computed tomography (NCCT) scan and CT angiography (CTA). A follow-up NCCT was performed for every patient 24 h after the thrombolysis. CBS was calculated using admission CTA imaging data, while Alberta Stroke Program Early CT Score (ASPECTS) was calculated at both NCCT examinations by 4 independent radiologists [19]. Baseline clinical and demographic characteristics were recorded for every patient (age, sex, BMI, previous medications, history of cerebrovascular and cardiovascular diseases, cerebrovascular risk factors). Severity of stroke was determined using NIHSS (National Institutes of Health Stroke Scale) on admission and on day 7 as short-term outcome [20]. In order to identify the etiology of stroke, Trial of ORG 10172 in Acute Stroke Treatment (TOAST) criteria was used [21]. The European Cooperative Acute Stroke Study (ECASS) II criteria was applied to grade haemorrhagic transformation as symptomatic (SICH) or asymptomatic intracranial haemorrhage (SICH) [22]. The following outcomes were investigated: 1/ Short-term functional outcome (day 7): poor short-term outcome was defined as a less than 4 points decrease or any increase of NIHSS by day 7 post-event or death by day 7, while a decrease in NIHSS score by at least 4 points or to 0 was defined as favorable outcome [23, 24]. 2/ Long-term functional outcome (90 days post-event): modified Rankin Scale (mRS) at 90 days post-event was used to assess long-term outcomes [24]. Poor long-term outcome was defined by mRS 3–6 [25].

Blood sampling and laboratory measurements

Peripheral venous blood samples were collected before the administration of intravenous thrombolysis and 24 hours after the event. Routine laboratory examinations (electrolytes, glucose, renal and liver function, high-sensitivity C-reactive protein (hsCRP), complete blood count) were performed before thrombolysis using standard methods (Roche Diagnostics, Mannheim, Germany and Sysmex Europe GmbH, Hamburg, Germany). For the examination of hemostasis tests, blood samples were collected in tubes containing 0.109 M sodium citrate (Becton Dickinson, Franklin Lane, NJ). Samples were processed immediately and were centrifuged twice at 1500 g, room temperature for 15 min. Screening tests of coagulation (prothrombin time, activated partial thromboplastin time and thrombin time) were performed from freshly separated plasma samples using routine methods on a BCS coagulometer (Siemens Healthcare Diagnostic Products, Marburg, Germany). Fibrinogen concentration was determined using the Clauss assay. For specific hemostasis measurements, plasma aliquots were labelled with a unique code and stored at −80°C until further assaying in batches. Standard methods were used for measurement of plasminogen and α2-plasmin inhibitor (α2-PI) activity using a BCS coagulometer (Siemens Healthcare Diagnostic Products, Marburg, Germany). D-dimer levels were measured by an immuno-turbidimetric assay (Innovance D-dimer). Plasma levels of FXIII activity were determined by ammonia release assay using a commercially available reagent kit (Technochrome FXIII, Technochlone, Austria). The isolation of DNA was performed from buffy coats of blood samples by QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). FXIII-A Val34Leu polymorphism (c.103 G > T; rs5985) was determined by real-time PCR using fluorescence resonance energy transfer detection and melting curve analysis on a LightCycler® 480 instrument (Roche Diagnostics GmbH, Mannheim, Germany) [26]. All primers are available from the authors upon request. Investigators performing laboratory measurements were blinded to patient identification and clinical data.

Statistical analysis

Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, Release 22.0, Chicago, IL), GraphPad Prism 8.0 (GraphPad Prism Inc., La Jolla, CA) and Stata 12 (Stata Corp, College Station, TX). Shapiro-Wilk test was used to evaluate the normality of the data. For two-group analyses Student’s t test (parametric data) or Mann–Whitney U test (non-parametric data) were performed. Kruskal–Wallis analysis with Dunn–Bonferroni post hoc test was used for multiple comparisons. χ2 test or Fisher’s exact were used for the evaluation of differences between categorical variables. Binary backward logistic regression model was used to determine the independent predictors of unfavourable short- and long-term outcome after thrombolysis. Results of the logistic regression analysis were expressed as odds ratio (OR) and 95% confidence interval (CI). A P-value of <0.05 was considered statistically significant.

Informed consent

The study was approved by the Ethics Committee of the University of Debrecen, Hungary and the Ethics Committee of the National Medical Research Council. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki. All patients or their relatives provided written informed consent.

Results

In this single-centered prospective observational case-control study, 200 patients with anterior circulation AIS undergoing intravenous thrombolysis by rt-PA were enrolled: 100 patients with LVO (CBS 0–9) and 100 age- and sex-matched patients without LVO (CBS 10) (Table 1). Smoking was significantly more frequent and NIHSS was significantly higher in the CBS 0–9 group. There were significant differences in both radiological and clinical outcomes between the two groups. Worse ASPECTS at 24 h post-event and poor short-term and long-term outcomes were significantly more frequent in the CBS 0–9 group. Of the measured routine laboratory parameters, hsCRP was significantly increased in the CBS 0–9 group. Plasminogen activity on admission was significantly higher in the CBS 0–9 group, while other parameters were not significantly different between the two groups. Genotype frequencies of FXIII-A Val34Leu polymorphism were consistent with Hardy-Weinberg equilibrium in the total cohort (FXIII-A Val34Val: n = 112 (56%), FXIII-A Val34Leu: n = 78 (39%) and FXIII-A Leu34Leu: n = 10 (5%). Allele frequencies of the FXIII-A Val34Leu polymorphism in this cohort did not significantly differ from a large cohort of population control group tested earlier [27]. Factor XIII-A Leu34 allele was significantly more frequent in the CBS 10 group, which is consistent with the in vitro described whole blood clot mass reducing effects of the allele. In a univariate analysis, a significant protective effect of the Leu34 allele against developing larger clots (CBS 0–9) could be demonstrated (OR: 0.519; 95%CI: 0.298–0.922, p = 0.0227). Given the modifying effects of fibrinogen concentration on FXIII-A Val34Leu genotype dependent clot structure and thrombus burden, we further looked at the specific relationships between admission fibrinogen concentration (below or above 3.5 g/L) and the CBS for each FXIII-A genotype, but no significant difference was found between groups (S1 Table). In the CBS 0–9 group, 11 patients (11%) suffered post-lysis ICH, while in the CBS 10 group this complication occurred in 7 patients (7%). Differences between CBS groups were not significant when ICH was considered according to ECASS II (aSICH or SICH), suggesting that clot burden was not associated with post-lysis bleeding complications in the studied cohort of patients.

Table 1. Baseline characteristics of patients according to their CBS.

	CBS 0–9	CBS 10	p
Number of patients	100	100
Age (years), median (IQR)	71 (62–79)	69 (62–76)	0.156
Male, n (%)	51 (51)	51 (51)	0.671
Cerebrovascular risk factors, n (%)
Arterial hypertension	85 (85)	86 (86)	0.841
Atrial fibrillation	26 (26)	16 (16)	0.083
Previous stroke	20 (20)	28 (28)	0.185
Hyperlipidaemia	56 (56)	66 (66)	0.147
Diabetes mellitus	25 (25)	28 (28)	0.631
Smoking, n (%)
Non-smoker	59 (59)	71 (71)	0.002
Previous smoker	16 (16)	2 (2)
Current smoker	19 (19)	26 (26)
Undetermined	6 (6)	1 (1)
BMI, mean (SD)	28.1 (5.2)	27.9 (6.5)	0.766
Duration of thrombolysis (min), median (IQR)	60 (60–62)	60 (60–60)	0.234
Time-to-treatment (min), mean (SD)	143.5 (115–176.3)	145 (109.3–182.8)	0.523
rt-PA dose (mg), median (IQR)	67 (56.75–81.25)	68 (58–80)	0.988
NIHSS, median (IQR)	11 (8–16)	7 (4–10)	<0.001
Medication at enrollment, n (%)
Antihypertensive therapy	76 (76)	62 (62)	0.032
Antiplatelet drug	40 (40)	37 (37)	0.663
Anticoagulant drug	5 (5)	6 (6)	0.756
Lipid lowering therapy	24 (24)	24 (24)	>0.999
Antidiabetic therapy	15 (15)	15 (15)	>0.999
Laboratory measurements, median (IQR)
INR	0.96 (0.94–1.04)	0.97 (0.93–1.03)	0.613
APTT (sec)	27.70 (25.90–31.20)	27.65 (25.68–30.02)	0.583
WBC (G/L)	7.84 (6.44–9.40)	8.02 (6.52–9.54)	0.530
RBC (T/L), mean (SD)	4.4 (0.5)	4.6 (0.6)	0.035
Hemoglobin (g/L), mean (SD)	133.6 (16.0)	139.2 (16.2)	0.016
Platelets (G/L)	215.00 (177.00–266.50)	212.50 (179.00–254.50)	0.438
Serum glucose (mmol/l)	6.70 (5.80–8.08)	6.40 (5.63–7.88)	0.439
hsCRP (mg/L)	4.30 (2.14–8.58)	2.63 (1.37–6.01)	0.017
Creatinine (umol/L)	78.00 (66.25–95.75)	78.50 (68.00–90.00)	0.870
Plasminogen activity on admission (%	111.50 (92.00–132.80)	97.00 (88.00–118.00)	0.032
Plasminogen activity at 24 h (%)	91.00 (78.00–105.00)	89.00 (81.00–101.00)	0.728
α2-plasmin inhibitor activity on admission (%)	101.00 (94.00–109.80)	101.00 (88.75–109.00)	0.494
α2-plasmin inhibitor activity at 24 h (%)	74.00 (67.00–86.00)	76.00 (67.00–86.00)	0.305
D-dimer level on admission (mg/L)	0.82 (0.53–1.46)	0.70 (0.50–1.60)	0.431
D-dimer level at 24 h (mg/L)	2.69 (1.37–4.92)	1.85 (1.03–5.17)	0.101
Fibrinogen level on admission (g/L)	3.88 (3.47–4.62)	3.91 (3.20–4.55)	0.468
Fibrinogen at 24 h (g/L)	3.73 (2.99–4.31)	3.76 (3.18–4.31)	0.848
Factor XIII activity on admission (%) mean (SD)	119.50 (33.77)	128.70 (51.53)	0.137
Factor XIII activity at 24 h (%)	114.10 (86.3–132.5)	104.30 (80.61–130.3)	0.168
Stroke etiology (TOAST), n (%)
Large-artery atherosclerosis	72 (72)	22 (22)	<0.001
Small-vessel occlusion	0 (0)	18 (18)
Cardioembolic	16 (16)	16 (16)
Other/undetermined	12 (12)	44 (44)
Imaging data, n (%)
ASPECTS on admission
0–7	4 (4)	0 (0)	0.059
8–10	93 (96)	98 (100)	0.059
ASPECTS at 24h after thrombolysis
0–7	51 (53)	8 (8)	<0.001
8–10	46 (47)	90 (92)	<0.001
Outcomes, n (%)
Short-term outcome (ΔNIHSS by day 7)
Good outcome (at least -4 points or NIHSS = 0)	39 (39)	53 (53)	0.001
Unchanged status (± 3 points)	33 (33)	35 (35)
Poor outcome (+4 points or more)	28 (28)	8 (8)
Undetermined	0 (0)	4 (4)
Long-term outcome (90 days)
mRS 0–2	34 (34)	67 (67)	<0.001
mRS 3–6	61 (61)	31 (31)
Undetermined	5 (5)	2 (2)
Intracranial hemorrhage (ECASS II)
No hemorrhage	89 (89)	93 (93)	0.226
aSICH	4 (4)	5 (5)
SICH	7 (7)	2 (2)
Factor XIII-A Leu34 carrier n (%)	36 (36)	52 (52)	0.023

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APTT, activated partial thromboplastin time; aSICH, asymptomatic intracerebral hemorrhage; ASPECTS, The Alberta Stroke Program early CT score; CBS, clot burden score; ECASS II, European Co-operative Acute Stroke Study-II; hsCRP high sensitive CRP; INR, international normalized ratio; IQR, interquartile range, mRS, modified Rankin Scale; NIHSS, National Institutes of Health Stroke Scale; RBC, red blood cell count; rt-PA, recombinant tissue plasminogen activator; SD, standard deviation; SICH, symptomatic intracerebral hemorrhage; TOAST, Trial of ORG 10172 in Acute Stroke Treatment; WBC white blood cell count

In order to learn more on the potential associations between clot burden and key factors/markers of fibrinolysis, the patient group with CBS 0–9 was further divided to subgroups according to the degree of clot burden (S2 Table). None of the investigated hemostasis parameters showed significant associations with the extent of clot burden at admission or at 24 h post-event, which rules out the possibility of excessive consumption in case of larger AIS thrombi. D-dimer levels 24 h post-event did not show significant differences between subgroups of different CBS. Taken together, this suggests that the degree of clot burden is not associated with the investigated fibrinolysis factors/markers and these measurements would not be useful in the clinical practice to estimate thrombus size. To find out which clinical or laboratory parameters are associated with thrombolysis outcomes in the study cohort, a binary backward multiple logistic regression analysis was performed. Univariate analysis revealed that low CBS, higher NIHSS at admission, history of previous stroke, and higher plasminogen at admission were significantly associated with poor short-term outcome (Table 2). Although the FXIII 34Leu allele was significantly more frequent in the patient group with CBS 10, the polymorphism showed no association with short-term outcomes. Based on the logistic regression model including age, sex, previous stroke, NIHSS on admission, plasminogen activity on admission, and CBS, only age, NIHSS on admission and CBS were identified as independent predictors of poor short-term outcome (Table 3).

Table 2. Baseline parameters and specific hemostasis markers according to short-term functional outcomes in the studied cohort.

	Favorable outcome	Unfavorable outcome	p
Number of patients	92	104
Age (years), median (IQR)	68 (62–76)	70 (63–79)	0.108
Male, n (%)	47 (51)	57 (55)	0.602
Cerebrovascular risk factors, n (%)
Arterial hypertension	80 (87)	87 (84)	0.516
Atrial fibrillation	17 (18)	25 (24)	0.344
Previous stroke	28 (30)	19 (18)	0.047
Hyperlipidaemia	56 (61)	64 (62)	0.924
Diabetes mellitus	18 (20)	31 (30)	0.098
Smoking, n (%)
Non-smoker	60 (65)	66 (63)	0.092
Previous smoker	4 (5)	14 (13)
Current smoker	23 (25)	22 (21)
Undetermined	5 (5)	2 (2)
BMI, median (IQR)	27.2 (23.8–31.5)	27.6 (24.6–30.9)	0.794
Duration of thrombolysis (min), median (IQR)	60 (60–60)	60 (60–60)	0.603
Time-to-treatment (min), mean (SD)	139 (104–173)	145 (115–181)	0.362
rt-PA dose (mg), median (IQR)	67 (58–80)	69.5 (57–81)	0.522
Clot burden score, n (%)
CBS 0–9	39 (42)	61 (59)	0.023
CBS 10	53 (58)	43 (41)	0.023
NIHSS, median (IQR)	10 (7–13)	7 (4–13)	0.042
Medication at enrollment, n (%)
Antihypertensive therapy	69 (75)	66 (63)	0.082
Antiplatelet drug	36 (39)	41 (45)	0.967
Anticoagulant drug	4 (4)	7 (7)	0.469
Lipid lowering therapy	25 (27)	22 (21)	0.325
Antidiabetic therapy	10 (11)	18 (17)	0.199
Laboratory measurements, median (IQR)
INR	0.98 (0.94–1.05)	0.98 (0.93–1.02)	0.242
APTT (sec)	27.2 (25.2–31.1)	27.9 (26.2–30.8)	0.464
WBC (G/L)	7.7 (6.3–8.7)	8.0 (6.5–9.9)	0.153
RBC (T/L)	4.5 (4.1–4.8)	4.6 (4.2–4.9)	0. 268
Hemoglobin (g/L)	136.0 (124.0–146.5)	139.0 (124.0–152.0)	0.114
Platelets (G/L)	214 (175–257)	215 (180–265)	0.967
Serum glucose (mmol/l)	6.4 (5.7–7.5)	6.6 (5.7–8.3)	0.251
hsCRP (mg/L)	3.0 (1.8–6.9)	3.9 (1.7–7.6)	0.675
Creatinine (umol/L)	78.5 (67.0–91.0)	77.5 (67.0–91.0)	0.938
Plasminogen activity on admission (%	99.5 (85.3–122.3)	107.0 (93.0–132.0)	0.031
Plasminogen activity at 24 h (%)	89.5 (76.3–104.5)	90.0 (80.0–105.3)	0.302
α2-plasmin inhibitor activity on admission (%)	99.0 (89.5–108.5)	101.00 (93.3–110.8)	0.347
α2-plasmin inhibitor activity at 24 h (%)	75.0 (67.3–87.8)	76.5 (66.0–85.3)	0.737
D-dimer level on admission (mg/L)	0.7 (0.5–1.3)	0.7 (0.5–1.4)	0.841
D-dimer level at 24 h (mg/L)	2.3 (1.1–3.8)	1.8 (1.1–3.9)	0.879
Fibrinogen level on admission (g/L)	3.9 (3.3–4.5)	3.9 (3.4–4.6)	0.668
Fibrinogen at 24 h (g/L)	3.7 (2.9–4.4)	3.8 (3.2–4.3)	0.642
Factor XIII activity on admission (%) mean (SD)	121.4±34.9	126.1±37.0	0.371
Factor XIII activity at 24 h (%)	106.2±31.6	108.7±39.3	0.624
Stroke etiology (TOAST), n (%)
Large-artery atherosclerosis	7 (8)	11 (11)	0.326
Small-vessel occlusion	40 (43)	53 (51)
Cardioembolic	14 (15)	17 (16)
Other/undetermined	31 (34)	23 (22)
Imaging data, n (%)
ASPECTS on admission
0–7	0 (0)	4 (4)	0.123
8–10	92 (100)	97 (93)	0.123
ASPECTS at 24h after thrombolysis
0–7	13 (14)	45 (43)	<0.001
8–10	79 (86)	55 (53)	<0.001
Factor XIII-A Leu34 carrier n (%)	47 (51)	41 (39)	0.101

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APTT, activated partial thromboplastin time; aSICH, asymptomatic intracerebral hemorrhage; ASPECTS, The Alberta Stroke Program early CT score; CBS, clot burden score; ECASS II, European Co-operative Acute Stroke Study-II; HGB, hemoglobin; hsCRP high sensitive CRP; INR, international normalized ratio; IQR, interquartile range, mRS, modified Rankin Scale; NIHSS, National Institutes of Health Stroke Scale; RBC, red blood cell; rt-PA, recombinant tissue plasminogen activator; SD, standard deviation; SICH, symptomatic intracerebral hemorrhage; TOAST, Trial of ORG 10172 in Acute Stroke Treatment; WBC white blood cell

Table 3. Independent predictors of poor short-term outcome^a in the studied cohort.

	OR	95%CI	p
Age	1.026	1.000 to 1.053	0.049
NIHSS on admission	0.916	0.861 to 0.974	0.005
Clot burden score (0–9 vs. 10)	2.777	1.439 to 5.361	0.002

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Last step of backward multiple regression analysis is provided.

^aPoor short-term outcome is defined as a less than 4 points decrease or any increase of NIHSS or death by day 7 post-event. Backward multiple regression model included age, sex, previous stroke, NIHSS on admission, plasminogen activity on admission, clot burden score (CBS 0–9 vs CBS 10). 95%CI, 95% confidence interval; NIHSS, National Institutes of Health Stroke Scale; OR, odds ratio.

As for long-term outcomes, univariate analysis revealed that older age, diabetes mellitus, lower CBS, higher NIHSS at admission, elevated serum glucose, elevated CRP, elevated creatinine, higher admission D-dimer value and higher admission fibrinogen levels and lower FXIII activity 24 h post-lysis were significantly associated with poor-long term outcomes (Table 4). Similarly to what was observed in case of short-term outcomes, FXIII-Val34Leu polymorphism showed no association with long-term outcomes in this patient cohort. In a binary backward multiple regression model including all variables that showed significant differences between long-term outcomes, CBS proved to be an independent predictor of long-term functional outcomes (CBS 0–9 vs. 10: OR: 2.501; 95%CI: 1.179–5.306, p = 0.017), among with age, NIHSS on admission and creatinine levels at admission (Table 5).

Table 4. Baseline parameters and specific hemostasis markers according to long-term functional outcomes in the studied cohort.

	mRS 0–2	mRS 3–6	p
Number of patients	101	92
Age (years), median (IQR)	67 (60–75)	73 (66–80)	<0.01
Male, n (%)	56 (55)	46 (50)	0.449
Cerebrovascular risk factors, n (%)
Arterial hypertension	85 (84)	81 (88)	0.437
Atrial fibrillation	19 (19)	21 (23)	0.492
Previous stroke	22 (22)	26 (28)	0.281
Hyperlipidaemia	62 (61)	55 (60)	0.820
Diabetes mellitus	17 (17)	33 (36)	0.003
Smoking, n (%)
Non-smoker	62 (61)	64 (70)	0.416
Previous smoker	8 (8)	9 (10)
Current smoker	26 (26)	17 (18)
Undetermined	5 (5)	2 (2)
BMI, median (IQR)	27.2 (24.3–30.5)	27.7 (24.3–32.1)	0.374
Duration of thrombolysis (min), median (IQR)	60 (60–60)	60 (60–60)	0.770
Time-to-treatment (min), mean (SD)	143 (102–180)	145 (116–180)	0.488
rt-PA dose (mg), median (IQR)	69 (57–80)	68 (57–82)	0.847
Clot burden score, n (%)
CBS 0–9	34 (33)	61 (66)	<0.001
CBS 10	67 (67)	31 (34)	<0.001
NIHSS, median (IQR)	7 (4–10)	11.5 (8–15)	<0.001
Medication at enrollment, n (%)
Antihypertensive therapy	64 (64)	70 (76)	0.055
Antiplatelet drug	34 (34)	41 (45)	0.121
Anticoagulant drug	7 (7)	4 (4)	0.440
Lipid lowering therapy	20 (220)	27 (29)	0.123
Antidiabetic therapy	11 (11)	18 (20)	0.092
Laboratory measurements, median (IQR)
INR	0.97 (0.93–1.02)	0.99 (0.93–1.04)	0.369
APTT (sec)	27.7 (25.5–31.1)	27.7 (25.9–30.5)	0.930
WBC (G/L)	8.1 (6.5–9.0)	7.8 (6.4–10.0)	0.962
RBC (T/L)	4.6 (4.2–4.9)	4.5 (4.1–4.9)	0.197
Hemoglobin (g/L),	138.5 (127.0–150.0)	135.0 (120.0–145.0)	0.066
Platelets (G/L)	210 (178–255)	217 (177–269)	0.490
Serum glucose (mmol/l)	6.3 (5.4–7.2)	6.8 (5.9–8.6)	0.003
hsCRP (mg/L)	2.8 (1.7–5.4)	4.8 (1.8–11.7)	0.016
Creatinine (umol/L)	75.0 (66.0–86.5)	85.5 (69.3–100.0)	0.011
Plasminogen activity on admission (%	102.5 (89.3–127.0)	107.0 (90.1–127.8)	0.586
Plasminogen activity at 24 h (%)	90.0 (81.5–107.5)	89.0 (76.8–102.3)	0.107
α2-plasmin inhibitor activity on admission (%)	101.0 (90.5–109.0)	100.5 (92.5–110.3)	0.850
α2-plasmin inhibitor activity at 24 h (%)	76.0 (68.5–86.5)	73.5 (65.8–83.3)	0.238
D-dimer level on admission (mg/L)	0.66 (0.49–0.99)	0.85 (0.53–1.63)	0.012
D-dimer level at 24 h (mg/L)	1.74 (1.1–3.1)	2.5 (1.2–4.6)	0.060
Fibrinogen level on admission (g/L)	3.7 (3.3–4.3)	4.1 (3.5–4.8)	0.020
Fibrinogen at 24 h (g/L)	3.8 (3.2–4.4)	3.7 (2.94.3)	0.374
Factor XIII activity on admission (%) mean (SD)	124.7±36.1	121.4±35.7	0.542
Factor XIII activity at 24 h (%)	117.2 (88.9–132.6)	99.1 (81.5–122.4)	0.025
Stroke etiology (TOAST), n (%)
Large-artery atherosclerosis	12 (11)	4 (4)	0.035
Small-vessel occlusion	38 (38)	52 (57)
Cardioembolic	17 (17)	14 (15)
Other/undetermined	34 (34)	22 (24)
Imaging data, n (%)
ASPECTS on admission
0–7	0 (0)	3 (3)	0.101
8–10	100 (99)	85 (92)	0.101
ASPECTS at 24h after thrombolysis
0–7	14 (13)	42 (46)	<0.001
8–10	87 (87)	44 (48)	<0.001
Factor XIII-A Leu34 carrier n (%)	42 (42)	43 (47)	0.471

Open in a new tab

APTT, activated partial thromboplastin time; aSICH, asymptomatic intracerebral hemorrhage; ASPECTS, The Alberta Stroke Program early CT score; CBS, clot burden score; ECASS II, European Co-operative Acute Stroke Study-II; HGB, hemoglobin; hsCRP high sensitive CRP; INR, international normalized ratio; IQR, interquartile range, mRS, modified Rankin Scale; NIHSS, National Institutes of Health Stroke Scale; RBC, red blood cell; rt-PA, recombinant tissue plasminogen activator; SD, standard deviation; SICH, symptomatic intracerebral hemorrhage; TOAST, Trial of ORG 10172 in Acute Stroke Treatment; WBC white blood cell

Table 5. Independent predictors of unfavorable long-term outcome^a (mRS) in the total cohort.

	OR	95%CI	p
Creatinine level on admission	1.019	1.001 to 1.038	0.043
NIHSS on admission	1.119	1.039 to 1.205	0.003
Age	1.033	1.000 to 1.066	0.048
Clot burden score (0–9 vs. 10)	2.501	1.179 to 5.306	0.017

Open in a new tab

Last step of backward multiple regression analysis is provided.

^aUnfavorable long-term outcome is defined as mRS 3–6 by day 90 post-event. Backward multiple regression model included age, sex, diabetes mellitus, NIHSS on admission, clot burden score (CBS 0–9 vs CBS 10), admission glucose level, hsCRP, creatinine level on admission, D-dimer level on admission, fibrinogen level on admission, FXIII activity at 24 hours post-event, TOAST values. 95%CI, 95% confidence interval; hsCRP high sensitive CRP, mRS, modified Rankin Scale; NIHSS, National Institutes of Health Stroke Scale; OR, odds ratio, TOAST, Trial of ORG 10172 in Acute Stroke Treatment.

Discussion

Understanding the mechanisms driving thrombus burden are critical for improving acute stroke care. The CBS grades thrombus size and length accurately and has been proposed to be useful in identifying poor responders to intravenous thrombolysis treatment [7, 28]. Here we confirm that a higher CBS is associated with better short-term and long-term outcomes and CBS might be used as a prognostic marker of AIS thrombolysis treatment. The results of our study suggest that recanalization and thrombolysis outcome depend greatly on the size of the thrombus. Although a number of studies have proposed CBS to be useful in predicting stroke outcomes, our study is among the first to measure key hemostasis parameters that could be associated with thrombus size in AIS patients. It is plausible to think that thrombus size directly relates to major coagulation or fibrinolysis proteins regulating clot structure and lysis. Interestingly, here we show that with the exception of plasminogen levels at admission, there is no direct relation between thrombus size and the investigated fibrinolysis parameters at admission or 24 h later. In a previous study by our group involving 131 AIS patients, we found that PAI-activity and antigen levels before thrombolysis, 1 hour and 24 hours after thrombolysis showed no association with short‐term or long‐term functional outcomes [29], thus levels of this marker were not measured in this cohort. In this patient cohort, plasminogen levels were significantly lower in case of higher CBS (smaller thrombi) that might be a result of significantly higher incorporation of plasminogen to thrombi and as a consequence, the consumption of the protein. Lower plasminogen levels were associated not only with smaller clot burden but with better short-term functional outcomes in an univariate model. On the other hand, in a multivariate model, the independent effect of admission plasminogen levels on short-term outcomes was not proved. In the literature only one study can be found where the relation of hemostasis proteins and CBS was assessed [30]. In this report only fibrinogen was measured in a smaller subset of AIS patients, and higher fibrinogen levels were found to be associated with smaller clot burden. In our study of much larger sample size, admission and 24 h post-lysis fibrinogen levels were practically identical in patient groups of different CBS, therefore, this association could not be confirmed. Apart from the fibrin and fibrinolytic network, thrombus size might be influenced by red blood cells and platelets that are important components of thrombi. Here we show that slightly, but significantly decreased red blood cell count and hemoglobin concentration was found in patients with higher thrombus burden (CBS<10). Without further experiments, it is difficult to interpret whether this difference might be the result of consumption, but overall, red blood cell count was not associated with outcomes in this cohort. Platelet count did not show an association with thrombus burden or outcomes.

Here we show for the first time that FXIII-A Val34Leu polymorphism has a significant effect on clot burden in vivo. FXIII has been implicated in limiting thrombus size by various mechanisms, including the regulation of the retention of red blood cells in thrombi or by down-regulating the accumulation of platelets on fibrin, among others [16, 17]. Of the major FXIII polymorphisms, the FXIII-A Val34Leu polymorphism has been investigated most extensively in relation with the risk of thrombotic diseases. It has been confirmed by meta-analyses that the Leu34 allele provides a moderate protection against coronary artery disease and venous thromboembolism, but evidence is still lacking in case of ischemic stroke [16]. The effect of FXIII-A Val34Leu polymorphism has been known to be influenced by complex gene-environmental interactions [31]. A number of reports have shown that the protective effect of the Leu34 prevails at high fibrinogen concentrations [27, 32]. Experimental reports demonstrated that in the presence of the Leu34 allele the formed fibrin is more susceptible to fibrinolysis than fibrin formed in the presence of Val34 [16]. Still, explaining the biochemical link between earlier FXIII activation provided by the Leu34 allele and protection against thrombotic events remains a challenge. In a most recent report, we have shown for the first time that in in vitro experiments, reconstituted whole blood clot mass is greatly determined by the FXIII-A Val34 Leu polymorphism [17]. Using plasma samples with high fibrinogen levels (>3.5 g/L), clot mass was higher for clots with Val34 as compared with clots with homozygous Leu34. In the current cohort of AIS patients, median fibrinogen level on admission was above 3.5 g/L in both groups of patients (3.88 g/L vs. 3.91 g/L in CBS 0–9 vs. CBS 10), therefore the effect of Leu34 allele could potentially prevail. Further statistical analysis of the FXIII-A Leu34 allele’s effect on clot burden with respect to fibrinogen levels did not reveal statistically significant differences among groups, which might be explained by the influence of the acute event on fibrinogen levels. Here we show that the presence of the Leu34 allele provided a significant protective effect against developing larger (CBS 0–9) clots (OR: 0.519; 95%CI: 0.298–0.922, p = 0.0227). Interestingly, in multivariate analysis, the polymorphism did not prove to be an independent predictor of functional outcomes. As thrombolysis outcomes are influenced by several factors, most importantly by the severity and localization of stroke, it is plausible that the FXIII-A Val34Leu polymorphism is not a significant contributor of the overall functional outcome of patients. This is in line with our previous report where FXIII-A Val34Leu had no effect on outcomes in a smaller AIS patient cohort undergoing thrombolysis treatment [33]. In that report, other major FXIII polymorphisms, FXIII-A p.Tyr204Phe (c.614 A > T; rs3024477), FXIII-B p.His95Arg (c.344 G > A; rs6003) and FXIII-B Intron K (IVS11 c.1952 + 144 C > G; rs12134960) were also investigated and they showed no association with stroke severity, unfavorable outcomes of therapy, therapy-associated symptomatic intracranial hemorrhage and mortality.

Based on multivariate models, CBS was found to be a significant independent predictor of short-term and long-term outcomes in the studied cohort of AIS patients treated by intravenous thrombolysis (CBS 0–9 vs. 10: OR: 2.777; 95%CI: 1.439–5.361, p = 0.002 and OR: 2.501; 95%CI: 1.179–5.306, p = 0.017, respectively). It has been proposed that patients with lower CBS (larger thrombi) are more likely to benefit from endovascular treatment as opposed to intravenous thrombolysis alone [34, 35]. On the other hand, several lines of evidence suggest that besides thrombus length, clot characteristics also play an important role in the likelihood of recanalization with intravenous thrombolysis and/or thrombectomy [36, 37]. Here we show that thrombus size is indeed among the most important factors for the outcome of intravenous thrombolysis, and potential differences between cellular composition or the levels of key fibrinolysis factors in larger or smaller thrombi cannot be detected using peripheral blood samples of patients on admission and 24 h post-lysis. In univariate models, the association of higher admission D-dimer levels, higher admission fibrinogen levels and lower FXIII activity 24 h post-lysis were significantly associated with poor-long term outcomes. However, in a multiple logistic regression model including CBS, the association of these hemostasis proteins or fibrinolysis markers with long-term outcomes could not be revealed.

Limitations of the study

Results of the present study should be interpreted in the context of its limitations and strengths. Due to the single-centered study design, the sample size is limited, however, as compared to other published studies measuring hemostasis or fibrinolysis biomarkers in acute ischemic stroke patients from pre-thrombolysis and post-thrombolysis samples, it is among the largest studies as yet [38]. Being single-centered, our study had the advantages of uniform sample handling and uniform patient care, moreover, the proportion of patients that were lost to follow-up was remarkably low (2% and 3.5% for short-term and long-term follow up, respectively) as compared to that observed in other studies involving post-stroke patients [38]. It must be noted, however, that the relatively low sample size together with this drop-out rate at follow-up might have influenced the results to a certain extent and thus larger clinical studies are needed to confirm and to validate our data.

As the in vitro described whole blood clot mass reducing effects of the FXIII-A Leu34 allele has not been proven in conditions resembling acute thromboembolism, it must be acknowledged as a limitation that conclusions of this study based on previously reported in vitro data may be speculative to a certain extent.

Conclusions

To conclude, CBS was found to be a significant independent predictor of short-term and long-term outcomes in the studied cohort of AIS patients treated by intravenous thrombolysis. FXIII-A 34Leu carrier status was found to be associated with smaller thrombus burden, which is consistent with the in vitro described whole blood clot mass reducing effects of the allele, but the polymorphism had no effect on thrombolysis outcomes.

Supporting information

S1 Table. Effects of fibrinogen and FXIII-A Val34 or Leu34 alleles on clot burden score.

(DOCX)

Click here for additional data file.^{(14.9KB, docx)}

S2 Table. Levels of different hemostasis markers according to CBS, median (IQR).

(DOCX)

Click here for additional data file.^{(16.1KB, docx)}

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The research was founded by grants from the National Research, Development and Innovation Office (NKFI) (2019-2.1.11-TÉT-2019-00065 to I.S., FK128582 to Z.B. and K120042 to L.C.), by the GINOP-2.3.2-15-2016-00048 project co-financed by the European Union and the European Regional Development Fund to L.C., and the Hungarian Academy of Sciences (ELKH-DE Cerebrovascular and Neurodegenerative Research Group) to L.C..

References

1.Donkor ES. Stroke in the 21(st) Century: A Snapshot of the Burden, Epidemiology, and Quality of Life. Stroke research and treatment. 2018;2018:3238165. doi: 10.1155/2018/3238165 ; PubMed Central PMCID: PMC6288566. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Powers WJ, Rabinstein AA, Ackerson T, Adeoye OM, Bambakidis NC, Becker K, et al. 2018 Guidelines for the Early Management of Patients With Acute Ischemic Stroke: A Guideline for Healthcare Professionals From the American Heart Association/American Stroke Association. Stroke. 2018;49(3):e46–e110. doi: 10.1161/STR.0000000000000158 . [DOI] [PubMed] [Google Scholar]
3.Lees KR, Emberson J, Blackwell L, Bluhmki E, Davis SM, Donnan GA, et al. Effects of Alteplase for Acute Stroke on the Distribution of Functional Outcomes: A Pooled Analysis of 9 Trials. Stroke. 2016;47(9):2373–9. doi: 10.1161/STROKEAHA.116.013644 ; PubMed Central PMCID: PMC5024752. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Mehrpour M, Afrakhte M, Shojaei SF, Sohrabi A, Ashayeri R, Esmaeili S, et al. Factors predicting the outcome of intravenous thrombolysis in stroke patients before rt-PA administration. Caspian journal of internal medicine. 2019;10(4):424–30. doi: 10.22088/cjim.10.4.424 ; PubMed Central PMCID: PMC6856912. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Smith WS, Lev MH, English JD, Camargo EC, Chou M, Johnston SC, et al. Significance of large vessel intracranial occlusion causing acute ischemic stroke and TIA. Stroke. 2009;40(12):3834–40. doi: 10.1161/STROKEAHA.109.561787 ; PubMed Central PMCID: PMC2796543. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Rennert RC, Wali AR, Steinberg JA, Santiago-Dieppa DR, Olson SE, Pannell JS, et al. Epidemiology, Natural History, and Clinical Presentation of Large Vessel Ischemic Stroke. Neurosurgery. 2019;85(suppl_1):S4–S8. doi: 10.1093/neuros/nyz042 ; PubMed Central PMCID: PMC6584910. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Puetz V, Dzialowski I, Hill MD, Subramaniam S, Sylaja PN, Krol A, et al. Intracranial thrombus extent predicts clinical outcome, final infarct size and hemorrhagic transformation in ischemic stroke: the clot burden score. International journal of stroke: official journal of the International Stroke Society. 2008;3(4):230–6. doi: 10.1111/j.1747-4949.2008.00221.x . [DOI] [PubMed] [Google Scholar]
8.Bagoly Z, Szegedi I, Kalmandi R, Toth NK, Csiba L. Markers of Coagulation and Fibrinolysis Predicting the Outcome of Acute Ischemic Stroke Thrombolysis Treatment: A Review of the Literature. Front Neurol. 2019;10:513. Epub 2019/07/19. doi: 10.3389/fneur.2019.00513 ; PubMed Central PMCID: PMC6611415. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Hudak R, Szekely EG, Kovacs KR, Nagy A, Hofgart G, Berenyi E, et al. Low thrombin generation predicts poor prognosis in ischemic stroke patients after thrombolysis. PLoS One. 2017;12(7):e0180477. Epub 2017/07/12. doi: 10.1371/journal.pone.0180477 ; PubMed Central PMCID: PMC5503253. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Toth NK, Szekely EG, Czuriga-Kovacs KR, Sarkady F, Nagy O, Lanczi LI, et al. Elevated Factor VIII and von Willebrand Factor Levels Predict Unfavorable Outcome in Stroke Patients Treated with Intravenous Thrombolysis. Front Neurol. 2017;8:721. Epub 2018/02/08. doi: 10.3389/fneur.2017.00721 ; PubMed Central PMCID: PMC5787073. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Muszbek L, Yee VC, Hevessy Z. Blood coagulation factor XIII: structure and function. Thrombosis research. 1999;94(5):271–305. doi: 10.1016/s0049-3848(99)00023-7 . [DOI] [PubMed] [Google Scholar]
12.Mikkola H, Syrjala M, Rasi V, Vahtera E, Hamalainen E, Peltonen L, et al. Deficiency in the A-subunit of coagulation factor XIII: two novel point mutations demonstrate different effects on transcript levels. Blood. 1994;84(2):517–25. . [PubMed] [Google Scholar]
13.Wartiovaara U, Mikkola H, Szoke G, Haramura G, Karpati L, Balogh I, et al. Effect of Val34Leu polymorphism on the activation of the coagulation factor XIII-A. Thrombosis and haemostasis. 2000;84(4):595–600. . [PubMed] [Google Scholar]
14.Ariens RA, Lai TS, Weisel JW, Greenberg CS, Grant PJ. Role of factor XIII in fibrin clot formation and effects of genetic polymorphisms. Blood. 2002;100(3):743–54. doi: 10.1182/blood.v100.3.743 . [DOI] [PubMed] [Google Scholar]
15.Voko Z, Bereczky Z, Katona E, Adany R, Muszbek L. Factor XIII Val34Leu variant protects against coronary artery disease. A meta-analysis. Thrombosis and haemostasis. 2007;97(3):458–63. . [PubMed] [Google Scholar]
16.Bagoly Z, Koncz Z, Harsfalvi J, Muszbek L. Factor XIII, clot structure, thrombosis. Thrombosis research. 2012;129(3):382–7. Epub 2011/12/27. doi: 10.1016/j.thromres.2011.11.040 . [DOI] [PubMed] [Google Scholar]
17.Kattula S, Bagoly Z, Toth NK, Muszbek L, Wolberg AS. The factor XIII-A Val34Leu polymorphism decreases whole blood clot mass at high fibrinogen concentrations. J Thromb Haemost. 2020;18(4):885–94. doi: 10.1111/jth.14744 . [DOI] [PMC free article] [PubMed] [Google Scholar]
18.European Stroke Organisation Executive C, Committee ESOW. Guidelines for management of ischaemic stroke and transient ischaemic attack 2008. Cerebrovascular diseases. 2008;25(5):457–507. doi: 10.1159/000131083 . [DOI] [PubMed] [Google Scholar]
19.Aviv RI, Mandelcorn J, Chakraborty S, Gladstone D, Malham S, Tomlinson G, et al. Alberta Stroke Program Early CT Scoring of CT perfusion in early stroke visualization and assessment. AJNR American journal of neuroradiology. 2007;28(10):1975–80. doi: 10.3174/ajnr.A0689 . [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Brott T, Adams HP Jr., Olinger CP, Marler JR, Barsan WG, Biller J, et al. Measurements of acute cerebral infarction: a clinical examination scale. Stroke. 1989;20(7):864–70. doi: 10.1161/01.str.20.7.864 . [DOI] [PubMed] [Google Scholar]
21.Adams HP Jr., Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, et al. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke. 1993;24(1):35–41. doi: 10.1161/01.str.24.1.35 . [DOI] [PubMed] [Google Scholar]
22.Hacke W, Kaste M, Fieschi C, Toni D, Lesaffre E, von Kummer R, et al. Intravenous thrombolysis with recombinant tissue plasminogen activator for acute hemispheric stroke. The European Cooperative Acute Stroke Study (ECASS). Jama. 1995;274(13):1017–25. . [PubMed] [Google Scholar]
23.National Institute of Neurological D, Stroke rt PASSG. Tissue plasminogen activator for acute ischemic stroke. N Engl J Med. 1995;333(24):1581–7. doi: 10.1056/NEJM199512143332401 . [DOI] [PubMed] [Google Scholar]
24.Simonsen CZ, Schmitz ML, Madsen MH, Mikkelsen IK, Chandra RV, Leslie-Mazwi T, et al. Early neurological deterioration after thrombolysis: Clinical and imaging predictors. International journal of stroke: official journal of the International Stroke Society. 2016;11(7):776–82. doi: 10.1177/1747493016650454 . [DOI] [PubMed] [Google Scholar]
25.van Swieten JC, Koudstaal PJ, Visser MC, Schouten HJ, van Gijn J. Interobserver agreement for the assessment of handicap in stroke patients. Stroke. 1988;19(5):604–7. doi: 10.1161/01.str.19.5.604 . [DOI] [PubMed] [Google Scholar]
26.Shemirani AH, Muszbek L. Rapid detection of the factor XIII Val34Leu (163 G—>T) polymorphism by real-time PCR using fluorescence resonance energy transfer detection and melting curve analysis. Clin Chem Lab Med. 2004;42(8):877–9. doi: 10.1515/CCLM.2004.142 . [DOI] [PubMed] [Google Scholar]
27.Bereczky Z, Balogh E, Katona E, Pocsai Z, Czuriga I, Szeles G, et al. Modulation of the risk of coronary sclerosis/myocardial infarction by the interaction between factor XIII subunit A Val34Leu polymorphism and fibrinogen concentration in the high risk Hungarian population. Thrombosis research. 2007;120(4):567–73. doi: 10.1016/j.thromres.2006.12.013 . [DOI] [PubMed] [Google Scholar]
28.Treurniet KM, Yoo AJ, Berkhemer OA, Lingsma HF, Boers AM, Fransen PS, et al. Clot Burden Score on Baseline Computerized Tomographic Angiography and Intra-Arterial Treatment Effect in Acute Ischemic Stroke. Stroke. 2016;47(12):2972–8. doi: 10.1161/STROKEAHA.116.014565 . [DOI] [PubMed] [Google Scholar]
29.Szegedi I, Nagy A, Szekely EG, Czuriga-Kovacs KR, Sarkady F, Lanczi LI, et al. PAI-1 5G/5G genotype is an independent risk of intracranial hemorrhage in post-lysis stroke patients. Ann Clin Transl Neurol. 2019;6(11):2240–50. Epub 2019/10/23. doi: 10.1002/acn3.50923 ; PubMed Central PMCID: PMC6856768. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Pikija S, Trkulja V, Mutzenbach JS, McCoy MR, Ganger P, Sellner J. Fibrinogen consumption is related to intracranial clot burden in acute ischemic stroke: a retrospective hyperdense artery study. J Transl Med. 2016;14(1):250. doi: 10.1186/s12967-016-1006-6 ; PubMed Central PMCID: PMC5006507. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Lim BC, Ariens RA, Carter AM, Weisel JW, Grant PJ. Genetic regulation of fibrin structure and function: complex gene-environment interactions may modulate vascular risk. Lancet. 2003;361(9367):1424–31. Epub 2003/05/03. doi: 10.1016/S0140-6736(03)13135-2 . [DOI] [PubMed] [Google Scholar]
32.Boekholdt SM, Sandhu MS, Wareham NJ, Luben R, Reitsma PH, Khaw KT. Fibrinogen plasma levels modify the association between the factor XIII Val34Leu variant and risk of coronary artery disease: the EPIC-Norfolk prospective population study. J Thromb Haemost. 2006;4(10):2204–9. doi: 10.1111/j.1538-7836.2006.02154.x . [DOI] [PubMed] [Google Scholar]
33.Szekely EG, Czuriga-Kovacs KR, Bereczky Z, Katona E, Mezei ZA, Nagy A, et al. Low factor XIII levels after intravenous thrombolysis predict short-term mortality in ischemic stroke patients. Sci Rep. 2018;8(1):7662. doi: 10.1038/s41598-018-26025-z ; PubMed Central PMCID: PMC5955963. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Coutinho JM, Liebeskind DS, Slater LA, Nogueira RG, Clark W, Davalos A, et al. Combined Intravenous Thrombolysis and Thrombectomy vs Thrombectomy Alone for Acute Ischemic Stroke: A Pooled Analysis of the SWIFT and STAR Studies. JAMA Neurol. 2017;74(3):268–74. doi: 10.1001/jamaneurol.2016.5374 . [DOI] [PubMed] [Google Scholar]
35.Choi JH, Im SH, Lee KJ, Koo JS, Kim BS, Shin YS. Comparison of Outcomes After Mechanical Thrombectomy Alone or Combined with Intravenous Thrombolysis and Mechanical Thrombectomy for Patients with Acute Ischemic Stroke due to Large Vessel Occlusion. World Neurosurg. 2018;114:e165–e72. doi: 10.1016/j.wneu.2018.02.126 . [DOI] [PubMed] [Google Scholar]
36.Bembenek JP, Niewada M, Siudut J, Plens K, Czlonkowska A, Undas A. Fibrin clot characteristics in acute ischaemic stroke patients treated with thrombolysis: the impact on clinical outcome. Thromb Haemost. 2017;117(7):1440–7. doi: 10.1160/TH16-12-0954 . [DOI] [PubMed] [Google Scholar]
37.Staessens S, Denorme F, Francois O, Desender L, Dewaele T, Vanacker P, et al. Structural analysis of ischemic stroke thrombi: histological indications for therapy resistance. Haematologica. 2020;105(2):498–507. doi: 10.3324/haematol.2019.219881 ; PubMed Central PMCID: PMC7012484. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Donkel SJ, Benaddi B, Dippel DWJ, Ten Cate H, de Maat MPM. Prognostic Hemostasis Biomarkers in Acute Ischemic Stroke. Arterioscler Thromb Vasc Biol. 2019;39(3):360–72. Epub 2019/02/01. doi: 10.1161/ATVBAHA.118.312102 ; PubMed Central PMCID: PMC6392207. [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0254253.r001

Decision Letter 0

Arijit Biswas

6 May 2021

PONE-D-21-10757

Decreased clot burden is associated with factor XIII Val34Leu polymorphism and better functional outcomes in acute ischemic stroke patients treated with intravenous thrombolysis

PLOS ONE

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Additional Editor Comments:

The manuscript by Szegedi et al. aims at assessing the relevance of clot burden score and other parameters on the outcome of thrombolysis in acute ischemic stroke patient. Two reviewers have critically analyzed the manuscript and proposed some changes. Reviewer 1 has suggested minor revision while Reviewer 2 suggests major revisions. I agree with the suggestions made by both reviewers. However, none of the reviewers have suggested any major new experimentation to be performed and the changes suggested are more in the form of formatting or extending the discussion in certain context (like the context of Val34Leu polymorphism). Similarly certain questions raised by the reviewers can also be answered by the author as a point-wise reply or with specific edits in the manuscript and does not require wholesome changes to the manuscript itself. In my view therefore, the proposed changes fall in the purview of minor revisions only, although ofcourse the authors need to make these changes and answer the questions absolutely. From my personal perspective I find the article of novel relevance and quite well written and well reported. Apart from the questions raised by the two reviewers, I have a small query (more like a comment) which is that the authors have focussed significantly on the val34leu polymorphism and ofcourse for good reason. Could the authors briefly mention the relevance (or the lack of it) of other background genetic polymorphisms of FXIII (if any) in the context of thrombolytic outcomes in acute ischemic stroke? If there are no studies in this regard, there may be a suggestion for investigating them in this context unless they are simply in strong linkage disequilibrium with this (or any other functional polymorphism).

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Reviewer #1: This manuscript by Szegedi et al. aims at assessing the relevance of clot burden score and other parameters on the outcome of thrombolysis in acute ischemic stroke patient.

This manuscript is very nicely written, with well-presented and analysed data.

The reviewers only has one main comment: Would the author be able to speculate on which clot burden score would be a reasonable threshold in determining the appropriate treatment (thrombolysis or thrombectomy) in AIS patients.

Minor comment: in your results section, you only have one subtitle “Baseline characteristics of patients according to CBS”. You should either take this out, or add other subsections.

Reviewer #2: The present study demonstrates for the first time that in acute ischemic stroke patients with a common genetic polymorphism, FXIII-A Leu34, smaller thrombus burden is observed. The study has novel aspects with regard to search for factors affecting thrombus size and thrombolysis outcomes in acute stroke. The laboratory methods used were standard. Clinical evaluation of the patients enrolled was sufficient.

My concerns are as follows:

1. The frequency of the 3 allelic variants should be presented. Were they in Hardy Weinberg equilibirium?

2. The Lancet study of Ariens' group in 2003 demonstrated a strong impact of plasma fibrinogen concentration on the FXIII-A Leu34 allele on fibrin properties. Did the authors assess their fidings with respect to fibrinogen?

3. The authors even in the conclusions of the abstract addressed "the in vitro described whole blood clot mass reducing effects" of the Leu34 allele. To the knowledge of this reviewer, the vitro data mentioned have not been performed in acute thromboembolism where activation of blood coagulation and enhanced inflammatory state along with oxidative stress occur. Since in the current study no investigations to support the above statement in acute stroke were presented, that conclusion is overly speculative. This is a major limitation which should be ackowledged; in fact the section of Study limiatations is missing in the masnuscript and should be added including comment on the risk of underpowered analysis in allele-associated analysis).

4. The study shows that the FXIII-A Leu34 polymorphism has no impact on thrombolysis outcomes in acute stroke despite association with thrombus burden. This aspect should be discussed in more detail.

5.Table 2 did not show relevant intergroup differences and a brief comment would be sufficient. The same holds true for Table 4. Tables 1 and 3 could be combined since most variables presented were identical in both.

6. Did the authors follow the patients for a longer period of time? Were any data on recurrent stroke available?

Minor comments

In table 1 BMI should be replaced by obesity; BMI is not a risk factor for stroke, only its increased values.

In the first paragraph of the discussion, the authors stated that "thrombus size directly

relates to major coagulation or fibrinolysis proteins regulating clot structure and lysis". Obviously, apart from fibrin network, red blood cells and platelets are important components of each thrombus. Hemoglobin and erythrocytes should be added to tables.

The authors claimed that they showed "major fibrinolysis parameters". PAI_1 is of key importance, but it has not been determined, therefore more precision is suggested while discussing fibrinolysis.

Reference style requires correction.

**********

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PLoS One. 2021 Jul 7;16(7):e0254253. doi: 10.1371/journal.pone.0254253.r002

Author response to Decision Letter 0

18 Jun 2021

Response to Reviewers

We are grateful to the Reviewers for the critical reading and overall positive assessment of our manuscript and the valuable remarks.

We addressed all comments raised and we believe that the changes introduced in the manuscript resulted in an improved version of the paper.

Response to the Editor

„Apart from the questions raised by the two reviewers, I have a small query (more like a comment) which is that the authors have focussed significantly on the val34leu polymorphism and ofcourse for good reason. Could the authors briefly mention the relevance (or the lack of it) of other background genetic polymorphisms of FXIII (if any) in the context of thrombolytic outcomes in acute ischemic stroke? If there are no studies in this regard, there may be a suggestion for investigating them in this context unless they are simply in strong linkage disequilibrium with this (or any other functional polymorphism).”

We are thankful to the Editor for the critical reading of our manuscript and for the valuable remark. The relevance of other major FXIII polymorphisms was investigated in detail in another paper by our group (Szekely et al, Sci Rep 2018; 8:7662). In that report including 132 consecutive AIS patients receiving i.v thrombolysis, other major FXIII polymorphisms, including FXIII-A p.Tyr204Phe (c.614 A > T; rs3024477), FXIII-B p.His95Arg (c.344 G > A; rs6003) and FXIII-B Intron K (IVS11 c.1952 + 144 C > G; rs12134960) were also investigated and they showed no association with stroke severity, unfavorable outcomes of therapy, therapy-associated symptomatic intracranial hemorrhage and mortality in that cohort.

This is now stated in the Discussion section of the manuscript, page 18, lines 22-28.

Szekely EG, Czuriga-Kovacs KR, Bereczky Z, Katona E, Mezei ZA, Nagy A, et al. Low factor XIII levels after intravenous thrombolysis predict short-term mortality in ischemic stroke patients. Sci Rep. 2018;8(1):7662. doi: 10.1038/s41598-018-26025-z. PubMed PMID: 29769590; PubMed Central PMCID: PMCPMC5955963.

Response to Reviewer 1

We would like to thank the Reviewer the overall positive comments of our manuscript.

Response to the main comment:

„Would the author be able to speculate on which clot burden score would be a reasonable threshold in determining the appropriate treatment (thrombolysis or thrombectomy) in AIS patients.”

This is a very interesting question indeed. As the study was not planned to answer this specific question, the wording „speculate” is very appropriate here and unfortunately we cannot provide a valid statistical analysis to answer the question. We performed an analysis where the studied population was layered based on their clot burden score (e.g. CBS 10 vs. 9-0; 10-9 vs. 8-0; 10-8 vs. 7-0; etc.) and the score where the benefit of thrombolysis was not statistically significant anymore was at CBS 2. This would mean that the only cases where thrombolysis would not have a benefit is CBS ≤2, although it must be emphasized that bleeding complications were not incorporated in this analysis for the lack of statistical power. Based on the 2018 Guideline of AHA/ASA guideline for the early management of patients with acute ischemic stroke, it is an IA level of evidence and recommendation that patients eligible for IV alteplase should receive IV alteplase even if EVTs are being considered [1]. Therefore, at least based on current evidence, CBS itself does not alter this decision. On the other hand, a low CBS could indicate the high probability of unsuccessful thrombolysis to the clinicians and suggests the need to organize and perform mechanical thrombectomy after thrombolysis.

1. Powers WJ, Rabinstein AA, Ackerson T, Adeoye OM, Bambakidis NC, Becker K, et al. 2018 Guidelines for the Early Management of Patients With Acute Ischemic Stroke: A Guideline for Healthcare Professionals From the American Heart Association/American Stroke Association. Stroke. 2018;49(3):e46-e110. Epub 2018/01/26. doi: 10.1161/STR.0000000000000158. PubMed PMID: 29367334.

Minor comment:

„ in your results section, you only have one subtitle “Baseline characteristics of patients according to CBS”. You should either take this out, or add other subsections. „

Thank you for noticing this, we deleted the subtitle of the subsection.

Response to Reviewer 2

We would like to thank the Reviewer the overall positive comments of our manuscript.

Major comments:

The frequency of the 3 allelic variants should be presented. Were they in Hardy Weinberg equilibirium?

Genotype frequencies of FXIII-A Val34Leu polymorphism were consistent with Hardy-Weinberg equilibrium in the total cohort (FXIII-A Val34Val: n=112 (56%), FXIII-A Val34Leu: n=78 (39%) and FXIII-A Leu34Leu: n= 10 (5%). This information is now included in the Results section of the manuscript, page 6, ultimate sentence.

In order to better reply to this comment, we performed a statistical analysis in which the specific relationships between admission fibrinogen concentration (below or above 3.5 g/L) and the CBS for each FXIII-A genotype was studied, but no significant difference was found between groups. Results are described on page 7, lines 6-10 and Table S1 is added to the manuscript.

Given the modifying effects of fibrinogen concentration on FXIII-A Val34Leu genotype dependent clot structure and thrombus burden, we further looked at the specific relationships between admission fibrinogen concentration (below or above 3.5 g/L) and the CBS for each FXIII-A genotype, but no significant difference was found between groups (Table S2).

Also, the Discussion section is now supplemented with the following reasoning (page 18, lines 10-17):

In the current cohort of AIS patients, median fibrinogen level on admission was above 3.5 g/L in both groups of patients (3.88 g/L vs. 3.91 g/L in CBS 0-9 vs. CBS 10), therefore the effect of Leu34 allele could potentially prevail. Further statistical analysis of the FXIII-A Leu34 allele’s effect on clot burden with respect to fibrinogen levels did not reveal statistically significant differences among groups, which might be explained by the influence of the acute event on fibrinogen levels.

We also included the mentioned reference as a citation.

Lim BC, Ariens RA, Carter AM, Weisel JW, Grant PJ. Genetic regulation of fibrin structure and function: complex gene-environment interactions may modulate vascular risk. Lancet. 2003;361(9367):1424-31. Epub 2003/05/03. doi: 10.1016/S0140-6736(03)13135-2. PubMed PMID: 12727396.

This is very important point, indeed. A Limitation section is now included in the manuscript, acknowledging the above mentioned limitations of the study (page 19):

Limitations of the study

Results of the present study should be interpreted in the context of its limitations and strengths. Due to the single-centered study design, the sample size is limited, however, as compared to other published studies measuring hemostasis or fibrinolysis biomarkers in acute ischemic stroke patients from pre-thrombolysis and post-thrombolysis samples, it is among the largest studies as yet. [1] Being single-centered, our study had the advantages of uniform sample handling and uniform patient care, moreover, the proportion of patients that were lost to follow-up was remarkably low (2% and 3.5% for short-term and long-term follow up, respectively) as compared to that observed in other studies involving post-stroke patients [1]. It must be noted, however, that the relatively low sample size together with this drop-out rate at follow-up might have influenced the results to a certain extent and thus larger clinical studies are needed to confirm and to validate our data.

1. Donkel SJ, Benaddi B, Dippel DWJ, Ten Cate H, de Maat MPM. Prognostic Hemostasis Biomarkers in Acute Ischemic Stroke. Arterioscler Thromb Vasc Biol. 2019;39(3):360-72. Epub 2019/02/01. doi: 10.1161/ATVBAHA.118.312102. PubMed PMID: 30700129; PubMed Central PMCID: PMCPMC6392207.

4. The study shows that the FXIII-A Leu34 polymorphism has no impact on thrombolysis outcomes in acute stroke despite association with thrombus burden. This aspect should be discussed in more detail.

We would like to thank the Reviewer for this useful comment. This aspects is now discussed in more detail in the Discussion section: page 18, lines 18-23):

As thrombolysis outcomes are influenced by several factors, most importantly by the severity and localization of stroke, it is plausible that the FXIII-A Val34Leu polymorphism is not a significant contributor of the overall functional outcome of patients. This is in line with our previous report where FXIII-A Val34Leu had no effect on outcomes in a smaller AIS patient cohort undergoing thrombolysis treatment [3].

5. Table 2 did not show relevant intergroup differences and a brief comment would be sufficient. The same holds true for Table 4. Tables 1 and 3 could be combined since most variables presented were identical in both.

Table 2 was deleted from the manuscript as requested and added as a Supplemental Table. We kept Table 4 in the manuscript as we believe that it indeed has relevant, significant intergroup differences.

Unfortunately, Tables 1 and 3 cannot be combined as the colums of these Tables represent completely different groups (i.e. Table 1: CBS 0-9 vs.CBS 10 while Table 3 (Table 2 in the revised version of the manuscipt): favorable vs. unfavorable short term outcomes).

6. Did the authors follow the patients for a longer period of time? Were any data on recurrent stroke available?

Unfortunately, we did not have the opportunity to follow the patients for a longer period of time. However, in most clinical studies on acute ischemic stroke, it is a standard practice to follow the patients for 90 days and to determine the long-term functional outcome of the acute event by the mRS at this point. Beyond this time, outcomes might be determined by several factors unrelated to the acute event itself (including co-morbidities, treatment of risk factors, socio-economic factors, etc), which also potentially influence recurrent stroke, therefore this aspect was not investigated and was out of the scope of this study.

Minor comments:

„In table 1 BMI should be replaced by obesity; BMI is not a risk factor for stroke, only its increased values.”

Thank you for noticing this error, BMI was removed in every Table from the section „Cerebrovascular risk factors” and was placed below as an individual parameter.

„In the first paragraph of the discussion, the authors stated that "thrombus size directly

Hemoglobin and red blood cell count were added to the Tables 1,2, and 4. Slightly, but significantly decreased red blood cell count and hemoglobin concentration was found in patients with higher thrombus burden. Results were discussed at the end of the first paragraph of the Discussion (page 17, lines 20-27):

Apart from the fibrin and fibrinolytic network, thrombus size might be influenced by red blood cells and platelets that are important components of thrombi. Here we show that slightly, but significantly decreased red blood cell count and hemoglobin concentration was found in patients with higher thrombus burden (CBS<10). Without further experiments, it is difficult to interpret whether this difference might be the result of consumption, but overall, red blood cell count was not associated with outcomes in this cohort. Platelet count did not show an association with thrombus burden or outcomes.

„The authors claimed that they showed "major fibrinolysis parameters". PAI_1 is of key importance, but it has not been determined, therefore more precision is suggested while discussing fibrinolysis.

Reference style requires correction.”

We would like to thank the reviewer for noticing the fact that we did not include or mention PAI-1 in this paper. In a previous study by our group involving 131 AIS patients, we found that PAI-activity and antigen levels before thrombolysis, 1 hour and 24 hours after thrombolysis showed no association with short‐term or long‐term functional outcomes (Szegedi et al, Ann Clin Transl Neurol, 2019;6(11):2240-50), thus levels of this marker were not measured in this cohort. We are thankful to the reviewer for noticing that we haven’t explained this circumstance in the manuscript and now this exact reasoning is mentioned on page 17, lines 6-9.

„Reference style requires correction.”

In case of all references, EndNote Style for Plos was used when citing relevant papers.

Once again, we are grateful to the Reviewer for the overall positive evaluation of our manuscript and the helpful criticism.

Attachment

Submitted filename: Response to reviewers_Plos one_rev.docx

Click here for additional data file.^{(31.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0254253.r003

Decision Letter 1

Arijit Biswas

24 Jun 2021

Decreased clot burden is associated with factor XIII Val34Leu polymorphism and better functional outcomes in acute ischemic stroke patients treated with intravenous thrombolysis

PONE-D-21-10757R1

Dear Dr. Bagoly,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Arijit Biswas

Academic Editor

PLOS ONE

PLoS One. doi: 10.1371/journal.pone.0254253.r004

Acceptance letter

Arijit Biswas

28 Jun 2021

PONE-D-21-10757R1

Decreased clot burden is associated with factor XIII Val34Leu polymorphism and better functional outcomes in acute ischemic stroke patients treated with intravenous thrombolysis

Dear Dr. Bagoly:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Effects of fibrinogen and FXIII-A Val34 or Leu34 alleles on clot burden score.

(DOCX)

Click here for additional data file.^{(14.9KB, docx)}

S2 Table. Levels of different hemostasis markers according to CBS, median (IQR).

(DOCX)

Click here for additional data file.^{(16.1KB, docx)}

Attachment

Submitted filename: Response to reviewers_Plos one_rev.docx

Click here for additional data file.^{(31.7KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

[pone.0254253.ref001] 1.Donkor ES. Stroke in the 21(st) Century: A Snapshot of the Burden, Epidemiology, and Quality of Life. Stroke research and treatment. 2018;2018:3238165. doi: 10.1155/2018/3238165 ; PubMed Central PMCID: PMC6288566. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0254253.ref002] 2.Powers WJ, Rabinstein AA, Ackerson T, Adeoye OM, Bambakidis NC, Becker K, et al. 2018 Guidelines for the Early Management of Patients With Acute Ischemic Stroke: A Guideline for Healthcare Professionals From the American Heart Association/American Stroke Association. Stroke. 2018;49(3):e46–e110. doi: 10.1161/STR.0000000000000158 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref003] 3.Lees KR, Emberson J, Blackwell L, Bluhmki E, Davis SM, Donnan GA, et al. Effects of Alteplase for Acute Stroke on the Distribution of Functional Outcomes: A Pooled Analysis of 9 Trials. Stroke. 2016;47(9):2373–9. doi: 10.1161/STROKEAHA.116.013644 ; PubMed Central PMCID: PMC5024752. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0254253.ref004] 4.Mehrpour M, Afrakhte M, Shojaei SF, Sohrabi A, Ashayeri R, Esmaeili S, et al. Factors predicting the outcome of intravenous thrombolysis in stroke patients before rt-PA administration. Caspian journal of internal medicine. 2019;10(4):424–30. doi: 10.22088/cjim.10.4.424 ; PubMed Central PMCID: PMC6856912. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0254253.ref005] 5.Smith WS, Lev MH, English JD, Camargo EC, Chou M, Johnston SC, et al. Significance of large vessel intracranial occlusion causing acute ischemic stroke and TIA. Stroke. 2009;40(12):3834–40. doi: 10.1161/STROKEAHA.109.561787 ; PubMed Central PMCID: PMC2796543. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0254253.ref006] 6.Rennert RC, Wali AR, Steinberg JA, Santiago-Dieppa DR, Olson SE, Pannell JS, et al. Epidemiology, Natural History, and Clinical Presentation of Large Vessel Ischemic Stroke. Neurosurgery. 2019;85(suppl_1):S4–S8. doi: 10.1093/neuros/nyz042 ; PubMed Central PMCID: PMC6584910. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0254253.ref007] 7.Puetz V, Dzialowski I, Hill MD, Subramaniam S, Sylaja PN, Krol A, et al. Intracranial thrombus extent predicts clinical outcome, final infarct size and hemorrhagic transformation in ischemic stroke: the clot burden score. International journal of stroke: official journal of the International Stroke Society. 2008;3(4):230–6. doi: 10.1111/j.1747-4949.2008.00221.x . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref008] 8.Bagoly Z, Szegedi I, Kalmandi R, Toth NK, Csiba L. Markers of Coagulation and Fibrinolysis Predicting the Outcome of Acute Ischemic Stroke Thrombolysis Treatment: A Review of the Literature. Front Neurol. 2019;10:513. Epub 2019/07/19. doi: 10.3389/fneur.2019.00513 ; PubMed Central PMCID: PMC6611415. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0254253.ref009] 9.Hudak R, Szekely EG, Kovacs KR, Nagy A, Hofgart G, Berenyi E, et al. Low thrombin generation predicts poor prognosis in ischemic stroke patients after thrombolysis. PLoS One. 2017;12(7):e0180477. Epub 2017/07/12. doi: 10.1371/journal.pone.0180477 ; PubMed Central PMCID: PMC5503253. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0254253.ref010] 10.Toth NK, Szekely EG, Czuriga-Kovacs KR, Sarkady F, Nagy O, Lanczi LI, et al. Elevated Factor VIII and von Willebrand Factor Levels Predict Unfavorable Outcome in Stroke Patients Treated with Intravenous Thrombolysis. Front Neurol. 2017;8:721. Epub 2018/02/08. doi: 10.3389/fneur.2017.00721 ; PubMed Central PMCID: PMC5787073. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0254253.ref011] 11.Muszbek L, Yee VC, Hevessy Z. Blood coagulation factor XIII: structure and function. Thrombosis research. 1999;94(5):271–305. doi: 10.1016/s0049-3848(99)00023-7 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref012] 12.Mikkola H, Syrjala M, Rasi V, Vahtera E, Hamalainen E, Peltonen L, et al. Deficiency in the A-subunit of coagulation factor XIII: two novel point mutations demonstrate different effects on transcript levels. Blood. 1994;84(2):517–25. . [PubMed] [Google Scholar]

[pone.0254253.ref013] 13.Wartiovaara U, Mikkola H, Szoke G, Haramura G, Karpati L, Balogh I, et al. Effect of Val34Leu polymorphism on the activation of the coagulation factor XIII-A. Thrombosis and haemostasis. 2000;84(4):595–600. . [PubMed] [Google Scholar]

[pone.0254253.ref014] 14.Ariens RA, Lai TS, Weisel JW, Greenberg CS, Grant PJ. Role of factor XIII in fibrin clot formation and effects of genetic polymorphisms. Blood. 2002;100(3):743–54. doi: 10.1182/blood.v100.3.743 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref015] 15.Voko Z, Bereczky Z, Katona E, Adany R, Muszbek L. Factor XIII Val34Leu variant protects against coronary artery disease. A meta-analysis. Thrombosis and haemostasis. 2007;97(3):458–63. . [PubMed] [Google Scholar]

[pone.0254253.ref016] 16.Bagoly Z, Koncz Z, Harsfalvi J, Muszbek L. Factor XIII, clot structure, thrombosis. Thrombosis research. 2012;129(3):382–7. Epub 2011/12/27. doi: 10.1016/j.thromres.2011.11.040 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref017] 17.Kattula S, Bagoly Z, Toth NK, Muszbek L, Wolberg AS. The factor XIII-A Val34Leu polymorphism decreases whole blood clot mass at high fibrinogen concentrations. J Thromb Haemost. 2020;18(4):885–94. doi: 10.1111/jth.14744 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0254253.ref018] 18.European Stroke Organisation Executive C, Committee ESOW. Guidelines for management of ischaemic stroke and transient ischaemic attack 2008. Cerebrovascular diseases. 2008;25(5):457–507. doi: 10.1159/000131083 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref019] 19.Aviv RI, Mandelcorn J, Chakraborty S, Gladstone D, Malham S, Tomlinson G, et al. Alberta Stroke Program Early CT Scoring of CT perfusion in early stroke visualization and assessment. AJNR American journal of neuroradiology. 2007;28(10):1975–80. doi: 10.3174/ajnr.A0689 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0254253.ref020] 20.Brott T, Adams HP Jr., Olinger CP, Marler JR, Barsan WG, Biller J, et al. Measurements of acute cerebral infarction: a clinical examination scale. Stroke. 1989;20(7):864–70. doi: 10.1161/01.str.20.7.864 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref021] 21.Adams HP Jr., Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, et al. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke. 1993;24(1):35–41. doi: 10.1161/01.str.24.1.35 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref022] 22.Hacke W, Kaste M, Fieschi C, Toni D, Lesaffre E, von Kummer R, et al. Intravenous thrombolysis with recombinant tissue plasminogen activator for acute hemispheric stroke. The European Cooperative Acute Stroke Study (ECASS). Jama. 1995;274(13):1017–25. . [PubMed] [Google Scholar]

[pone.0254253.ref023] 23.National Institute of Neurological D, Stroke rt PASSG. Tissue plasminogen activator for acute ischemic stroke. N Engl J Med. 1995;333(24):1581–7. doi: 10.1056/NEJM199512143332401 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref024] 24.Simonsen CZ, Schmitz ML, Madsen MH, Mikkelsen IK, Chandra RV, Leslie-Mazwi T, et al. Early neurological deterioration after thrombolysis: Clinical and imaging predictors. International journal of stroke: official journal of the International Stroke Society. 2016;11(7):776–82. doi: 10.1177/1747493016650454 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref025] 25.van Swieten JC, Koudstaal PJ, Visser MC, Schouten HJ, van Gijn J. Interobserver agreement for the assessment of handicap in stroke patients. Stroke. 1988;19(5):604–7. doi: 10.1161/01.str.19.5.604 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref026] 26.Shemirani AH, Muszbek L. Rapid detection of the factor XIII Val34Leu (163 G—>T) polymorphism by real-time PCR using fluorescence resonance energy transfer detection and melting curve analysis. Clin Chem Lab Med. 2004;42(8):877–9. doi: 10.1515/CCLM.2004.142 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref027] 27.Bereczky Z, Balogh E, Katona E, Pocsai Z, Czuriga I, Szeles G, et al. Modulation of the risk of coronary sclerosis/myocardial infarction by the interaction between factor XIII subunit A Val34Leu polymorphism and fibrinogen concentration in the high risk Hungarian population. Thrombosis research. 2007;120(4):567–73. doi: 10.1016/j.thromres.2006.12.013 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref028] 28.Treurniet KM, Yoo AJ, Berkhemer OA, Lingsma HF, Boers AM, Fransen PS, et al. Clot Burden Score on Baseline Computerized Tomographic Angiography and Intra-Arterial Treatment Effect in Acute Ischemic Stroke. Stroke. 2016;47(12):2972–8. doi: 10.1161/STROKEAHA.116.014565 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref029] 29.Szegedi I, Nagy A, Szekely EG, Czuriga-Kovacs KR, Sarkady F, Lanczi LI, et al. PAI-1 5G/5G genotype is an independent risk of intracranial hemorrhage in post-lysis stroke patients. Ann Clin Transl Neurol. 2019;6(11):2240–50. Epub 2019/10/23. doi: 10.1002/acn3.50923 ; PubMed Central PMCID: PMC6856768. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0254253.ref030] 30.Pikija S, Trkulja V, Mutzenbach JS, McCoy MR, Ganger P, Sellner J. Fibrinogen consumption is related to intracranial clot burden in acute ischemic stroke: a retrospective hyperdense artery study. J Transl Med. 2016;14(1):250. doi: 10.1186/s12967-016-1006-6 ; PubMed Central PMCID: PMC5006507. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0254253.ref031] 31.Lim BC, Ariens RA, Carter AM, Weisel JW, Grant PJ. Genetic regulation of fibrin structure and function: complex gene-environment interactions may modulate vascular risk. Lancet. 2003;361(9367):1424–31. Epub 2003/05/03. doi: 10.1016/S0140-6736(03)13135-2 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref032] 32.Boekholdt SM, Sandhu MS, Wareham NJ, Luben R, Reitsma PH, Khaw KT. Fibrinogen plasma levels modify the association between the factor XIII Val34Leu variant and risk of coronary artery disease: the EPIC-Norfolk prospective population study. J Thromb Haemost. 2006;4(10):2204–9. doi: 10.1111/j.1538-7836.2006.02154.x . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref033] 33.Szekely EG, Czuriga-Kovacs KR, Bereczky Z, Katona E, Mezei ZA, Nagy A, et al. Low factor XIII levels after intravenous thrombolysis predict short-term mortality in ischemic stroke patients. Sci Rep. 2018;8(1):7662. doi: 10.1038/s41598-018-26025-z ; PubMed Central PMCID: PMC5955963. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0254253.ref034] 34.Coutinho JM, Liebeskind DS, Slater LA, Nogueira RG, Clark W, Davalos A, et al. Combined Intravenous Thrombolysis and Thrombectomy vs Thrombectomy Alone for Acute Ischemic Stroke: A Pooled Analysis of the SWIFT and STAR Studies. JAMA Neurol. 2017;74(3):268–74. doi: 10.1001/jamaneurol.2016.5374 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref035] 35.Choi JH, Im SH, Lee KJ, Koo JS, Kim BS, Shin YS. Comparison of Outcomes After Mechanical Thrombectomy Alone or Combined with Intravenous Thrombolysis and Mechanical Thrombectomy for Patients with Acute Ischemic Stroke due to Large Vessel Occlusion. World Neurosurg. 2018;114:e165–e72. doi: 10.1016/j.wneu.2018.02.126 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref036] 36.Bembenek JP, Niewada M, Siudut J, Plens K, Czlonkowska A, Undas A. Fibrin clot characteristics in acute ischaemic stroke patients treated with thrombolysis: the impact on clinical outcome. Thromb Haemost. 2017;117(7):1440–7. doi: 10.1160/TH16-12-0954 . [DOI] [PubMed] [Google Scholar]

[pone.0254253.ref037] 37.Staessens S, Denorme F, Francois O, Desender L, Dewaele T, Vanacker P, et al. Structural analysis of ischemic stroke thrombi: histological indications for therapy resistance. Haematologica. 2020;105(2):498–507. doi: 10.3324/haematol.2019.219881 ; PubMed Central PMCID: PMC7012484. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0254253.ref038] 38.Donkel SJ, Benaddi B, Dippel DWJ, Ten Cate H, de Maat MPM. Prognostic Hemostasis Biomarkers in Acute Ischemic Stroke. Arterioscler Thromb Vasc Biol. 2019;39(3):360–72. Epub 2019/02/01. doi: 10.1161/ATVBAHA.118.312102 ; PubMed Central PMCID: PMC6392207. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Decreased clot burden is associated with factor XIII Val34Leu polymorphism and better functional outcomes in acute ischemic stroke patients treated with intravenous thrombolysis

István Szegedi

Rita Orbán-Kálmándi

Attila Nagy

Ferenc Sarkady

Nikolett Vasas

Máté Sik

Levente István Lánczi

Ervin Berényi

László Oláh

Alexandra Crișan

László Csiba

Zsuzsa Bagoly

Roles

Abstract

Background

Methods

Results

Conclusions

Introduction

Materials and methods

Patients

Blood sampling and laboratory measurements

Statistical analysis

Informed consent

Results

Table 1. Baseline characteristics of patients according to their CBS.

Table 2. Baseline parameters and specific hemostasis markers according to short-term functional outcomes in the studied cohort.

Table 3. Independent predictors of poor short-term outcomea in the studied cohort.

Table 4. Baseline parameters and specific hemostasis markers according to long-term functional outcomes in the studied cohort.

Table 5. Independent predictors of unfavorable long-term outcomea (mRS) in the total cohort.

Discussion

Limitations of the study

Conclusions

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Arijit Biswas

Roles

Author response to Decision Letter 0

Decision Letter 1

Arijit Biswas

Roles

Acceptance letter

Arijit Biswas

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Table 3. Independent predictors of poor short-term outcome^a in the studied cohort.

Table 5. Independent predictors of unfavorable long-term outcome^a (mRS) in the total cohort.