Article ID: CJ-17-0375
Background: The composition of intra-arterial clots might influence the efficacy of mechanical thrombectomy (MT) in ischemic stroke (IS) due to the acute occlusions within large cerebral arteries. The aims were to assess the factors associated with blood clot structure and the impact of thromboembolus structure on MT using stent-retrievers in patients with acute large artery IS in the anterior circulation.
Methods and Results: In an observational cohort study, we studied the components of intra-arterial clots retrieved from large cerebral arteries in 80 patients with acute IS treated with MT with or without i.v. thrombolysis (IVT). Histology of the clots was carried out without knowledge of the clinical findings, including the treatment methods. The components of the clots, their age, origin and semi-quantitative graded changes in the architecture of the fibrin components (e.g., “thinning”) were compared via neuro-interventional, clinical and laboratory data. The most prominent changes in the architecture of the fibrin components in the thromboemboli were associated with IVT (applied in 44 patients; OR, 3.50; 95% CI: 1.21–10.10, P=0.02) and platelet count (OR, 2.94; 95% CI: 1.06–8.12, P=0.04).
Conclusions: In patients with large artery IS treated with the MT using stent-retrievers, bridging therapy with IVT preceding MT and higher platelet count were associated with significant changes of the histological structure of blood clots.
Five randomized controlled trials have recently reported the superiority of thrombectomy using stent-retrievers compared with i.v. thrombolysis (IVT) for the treatment of large artery stroke in the anterior circulation.1–5 Despite recent, significant, technical advances in radiological methods that have better identified suitable patients and enhanced diagnostic and treatment processes, recanalization remains difficult in certain patients. Apart from technical grounds, the characteristics of a clot could constitute one of the reasons for failure. Endovascular methods were first used to analyze clot characteristics by making it possible to obtain clot material for examination. Unlike acute myocardial ischemia,6–13 the knowledge obtained via endovascular treatment regarding the components of clots from cerebral arteries has been limited. Moreover, the nature of the occlusions is different. While in situ thromboses typically result in occlusions of coronary arteries, cerebral arteries are most commonly occluded by emboli originating in the heart or proximal parts of cerebral arteries. Previously published studies have presented histological analyses of cerebral emboli for small groups of patients (between 10 and 50) with controversial results.14–18 For example, Boeckh-Behrens et al found that a higher percentage of white blood cells (WBC) in the thrombus was associated with cardioembolic etiology, extended mechanical recanalization time, and less favorable recanalization and clinical outcome.18 In contrast, Marder et al concluded that thromboemboli had similar histological components, whether derived from cardiac or arterial sources,14 and, in the Liebeskind et al study, no correlation was found between thrombus histopathology and stroke etiology, success of thrombus extraction and stroke outcome.15 In contrast, in the Hashimoto et al study involving 83 patients, thrombus composition differed in patients who achieved reperfusion (with less frequently present atheromatous gruel and a higher proportion of red blood cell [RBC] components) vs. those without reperfusion.19 Boeckh-Behrens et al recently carried out the largest study so far, involving 145 consecutive stroke patients, in whom histopathology of thrombi retrieved during mechanical thrombectomy (MT) showed that cardioembolic thrombi had higher proportions of fibrin/platelets (fibrin/PLT), fewer RBC and more WBC (confirming their previous finding) than non-cardioembolic ones, and that cryptogenic stroke showed strong overlap with cardioembolic stroke but not with non-cardioembolic stroke, in terms of both thrombus histology and interventional and clinical outcome parameters.20
The aims of this prospective study were therefore to assess (1) factors associated with blood clot structure (evaluated on histology); and (2) the impact of thromboembolus structure on MT using stent-retrievers in patients with acute ischemic large artery stroke.
In an observational cohort study, 92 consecutive acute ischemic stroke patients (male, n=35; mean age, 77.0±12.5 years) from the Department of Neurology, Comprehensive Stroke Centre, University Hospital, Hradec Králové, Czech Republic underwent MT with or without IVT between May 2014 and October 2015 for acute occlusion of the large artery in the anterior or posterior circulation. Basic routine investigations included neurological, physical and laboratory examinations (including fibrinogen, RBC count, hematocrit [HCT], PLT count and mean platelet volume [MPV]), National Institutes of Health Stroke Scale (NIHSS)21 assessments (performed by a certified neurologist), brain imaging with native computed tomography (CT), and CT angiography (CTA). Collateral circulation on CTA was assessed using a 3-grade scale: presence of >70% arteries was assessed as very good; 40–70% as good; and <40% as poor.22 CT-perfusion (CTP) was performed in select patients (with unknown time of onset or at the end of the therapeutic time window). MT was indicated in patients fulfilling criteria according to the guidelines of the American Heart Association/American Stroke Association (AHA/ASA).23 Patients with unknown time of onset or at the end of the therapeutic time window were indicated for MT if the Alberta Stroke Program Early Computed Tomography Score (ASPECTS)24 was ≥7 or if CTP indicated a small ischemic core and the presence of ischemic penumbra. We obtained materials via endovascular procedures for histology. We evaluated the clinical results using the modified Rankin Scale (mRS)25 after 3 months, with good clinical outcome (self-sufficiency) defined as mRS score 0–2. Evaluation of mRS was performed during follow-up clinical visits by a certified neurologist blinded to the histology and entered into the patient medical record. The study was conducted in accordance with the ethics standards laid down in the 1964 Declaration of Helsinki and its later amendments and approved by the Ethics Committee of the University Hospital in Hradec Králové. All procedures were performed in accordance with institutional guidelines. All conscious patients signed informed consent forms. Independent witnesses verified the signatures in the case of technical problems.
I.v. ThrombolysisI.v. thrombolysis with a standard dose of 0.9 mg/kg (maximum dose, 90 mg) of recombinant tissue plasminogen activator (rtPA; Actilyse®, Boehringer Ingelheim, Ingelheim am Rhein, Germany) was used in all patients with known stroke onset time fulfilling the inclusion (i.e., treatment ≤4.5 h from stroke onset) and exclusion criteria according to the AHA/ASA and Czech guidelines. Ten percent of the rtPA dose was given as i.v. bolus, followed by a 60-min infusion of the remaining 90% of the dose.23,26 According to the local protocol, IVT was performed also in patients with unknown time of onset with ASPECTS ≥7 or with a small ischemic core and ischemic penumbra on CTP.
Endovascular ProcedureWe did not wait for the effects of the IVT treatment, and endovascular intervention was initiated as soon as possible. We used a stent-retriever for clot extraction. The choice of the particular stent-retriever(s) was at the discretion of the treating physician. Recanalization in post-treatment angiograms was assessed by a certified radiologist blinded to the histology, using the Thrombolysis in Cerebral Infarction (TICI) grading scale.27 TICI scores of 2b and 3 indicated successful recanalization.
HistologyClots retrieved from the cerebral arteries were immediately fixed in neutral formalin. After 1–2 days, the clots were processed, cut, and stained with hematoxylin and eosin (Merck, Darmstadt, Germany) and examined on immunohistochemistry using primary monoclonal antibodies anti-CD68 against histiocytes and macrophages and anti-CD34 against endothelia (both DAKO, Glostrup, Denmark).
Histology was carried out by a blinded experienced investigator (I.Š.) with no knowledge of the clinical procedure. The histological examination had 4 aims. The first aim was to differentiate between a locally formed thrombus and a lodged thromboembolus. The only indication for the former would be an admixture of components of underlying atheromatous plaque (e.g., cholesterol crystals, foamy lipophages or calcific debris). The second aim was to determine the composition of the blood clot, that is, the ratios of erythrocytes, fibrin and thrombocytes (WBC were omitted). The third aim was to assess the age of the blood clot (i.e., fresh or older). As a criterion for classifying an older clot (more than approximately 2 days old), the presence of histiocytes and macrophages (Figure 1A) and/or endothelia (Figure 1B) indicated early organization.14,18,28,29 Finally, the fourth aim was to note any histological changes to the fibrin component of the clots as a presumed effect of IVT. Here, we observed a transformation of normally solid fibrin into a loose web-like state; we described this phenomenon as “thinning” and regarded it as a stage of fibrinolysis. The thinning was graded semiquantitatively (Figure 2): 0, not present; 1, thinning present in <1/3 of the total clot volume; 2, thinning present in 1/3–2/3 of the total clot volume; 3, thinning present in >2/3 of the total clot volume.
(A) Older thromboembolus: multiple macrophages/histiocytes on the surface of the thromboembolus (anti-CD68); (B) older thromboembolus: several endothelia (arrows), mostly on the surface of the thromboembolus (anti-CD34).
Clot thinning. (A) Grade 1, most of the clot fibrin has a solid or coarsely trabecular structure; there is an area of thinning in the center; (B) grade 2, the majority of the clot shows thinning; there are remnants of solid fibrin in the center; (C) grade 3, total thinning of fibrin. (A–C, hematoxylin-eosin stain.)
We analyzed the associations between thinning and the various predictors in the overall patient group. TICI scores were dichotomized and defined as successful recanalization (TICI 2b–3) vs. no–poor recanalization (TICI 0–2a). We used an ordinal logistic regression model wherein the thinning was the dependent variable and fibrinogen, RBC, HCT, PLT, MPV, history of ischemic heart disease and diabetes mellitus, pretreatment with acetylsalicylic acid, anticoagulants and statins, and IVT were the predictors. Thinning was dichotomized into grades 0–2 and grade 3. The model was adjusted for age and baseline NIHSS. Quantitative variables did not follow a standard normal distribution (as indicated by the Kolmogorov-Smirnov test). Mon-parametric Mann-Whitney U-test was used to test the null hypothesis, while for statistical analysis of categorical variables, exact Fisher test (with its Freeman-Halton extension for 4×2 and 3×2 tables) was used. The MATLAB Statistical Toolbox (MathWorks, Natick, MA, USA) was used for statistical analysis.
Of the 92 consecutive acute ischemic stroke patients, 12 patients were excluded from the study: 10 without the achievement of recanalization (TICI 0 in 8 and TICI 1 in 2 patients) and 2 due to incomplete histological data on the thromboembolus (thromboembolus disintegration in 1 and insufficient amount of material for analysis in 1 patient). Thus, the set consisted of 80 patients, including 30 men (37.5%), enrolled between May 2014 and October 2015. Forty-four patients (55%) were treated with a combination of both IVT and MT, and 36 patients (45%) were treated with MT alone (MT-A; 10 patients with unknown stroke onset time and 26 patients with contraindications of IVT represented by anticoagulation therapy, recent bleeding or higher bleeding risk). Patient age ranged from 19 to 94 years; 27 patients (33.8%) were older than 80 years. Mean age in the 2 treatment groups was similar. The NIHSS median of the whole group was 14.9 points and did not significantly differ between the treatment groups. Observed cerebrovascular risk factors also did not significantly differ between the 2 treatment groups (Table 1).
| Factor | All (n=80) |
IVT+MT (n=44) |
MT-A (n=36) |
P-value |
|---|---|---|---|---|
| Age (years) | 74.5±12.3 (19–94) | 74.8±9.7 (45–88) | 74.2±15.0 (19–94) | 0.83 |
| >80 years | 27 (33.8) | 13 (29.5) | 14 (38.9) | 0.66 |
| Male | 30 (37.5) | 19 (43.2) | 11 (30.6) | 0.52 |
| Cerebrovascular risk factor | ||||
| Atrial fibrillation | 47 (58.8) | 23 (52.3) | 24 (66.7) | 0.58 |
| Ischemic heart disease | 26 (32.5) | 9 (20.5) | 17 (47.2) | 0.11 |
| Arterial hypertension | 61 (76.3) | 34 (77.3) | 27 (75.0) | 1.00 |
| Diabetes mellitus | 18 (22.5) | 9 (20.5) | 9 (25.0) | 0.80 |
| Hyperlipidemia | 30 (37.5) | 18 (40.9) | 12 (33.3) | 0.67 |
| Unknown stroke onset time | 18 (22.5) | 6 (13.6) | 12 (33.3) | 0.12 |
| NIHSS at time of treatment | 14.9±5.6 | 14.9±5.1 | 15.0±6.3 | 0.94 |
| mRS 0–2 on day 90 | 46 (57.5) | 26 (59.1) | 20 (55.6) | 0.32 |
Data given as mean±SD (range) or n (%). IVT, i.v. thrombolysis; mRS, modified Rankin Scale; MT, mechanical thrombectomy; MT-A, MT alone; NIHSS, National Institutes of Health Stroke Scale.
Using digital subtraction angiography, occlusions in the anterior circulation were identified in 70 patients (87.5%). An occlusion of the M1/2 segment of the middle cerebral artery (MCA) was most often detected. Ten patients (12.5%) had posterior circulation occlusions, including 9 patients (11.3%) with a basilar artery (BA) occlusion with or without occlusion to the P1 segment of the posterior cerebral artery (PCA; Table 2). In 58 patients (72.5%), MT was initiated ≤6 h from the onset of the first symptoms. Based on CTP and ASPECTS, we treated 4 patients (5%) after 6 h and 18 patients (22.5%) with an unknown stroke onset time. We used a stent-retriever in all patients (both Solitaire and Trevo stents were used in one of the patients; Table 2). Despite the obvious tendency for greater number of necessary passages in the IVT+MT group (mean, 1.75±1.04 vs. 1.42±0.77 in the MT-A group), the difference was not statistically significant (P=0.12). The maximum number of passages was 5 and was required in 2 cases. For 60% of the patients, a single passage was sufficient. Eight patients (10%) with tandem pathology were concurrently treated with intracranial thrombectomy and carotid artery stenting. IVT did not delay the initiation of endovascular treatment (onset-to-groin puncture time, 208.0 min in IVT+MT group and 205.9 min in MT-A group). When compared with patients treated with IVT+MT, the endovascular intervention (i.e., from groin puncture to recanalization) took less time in patients treated with MT alone (38.1 min vs. 45.8 min), and the percentage of successful recanalizations was higher (TICI 2b/3, 91.7% vs. 81.8%), but these differences were not statistically significant (P>0.05 in both cases). When evaluated after 3 months, self-sufficiency (mRS 0–2) was achieved by 57.5% of the patients (i.e., 59.1% in the IVT+MT group and 55.6% in the MT-A group; Tables 1,2).
| Observed parameter | All (n=80) |
IVT+MT (n=44) |
MT-A (n=36) |
P-value |
|---|---|---|---|---|
| Localization of arterial occlusion | ||||
| MCA: M1/2 segment | 51 (63.8) | 30 (68.2) | 21 (58.3) | 0.72 |
| ICA: T occlusion | 11 (13.8) | 7 (15.9) | 4 (11.1) | 0.75 |
| ICA: tandem occlusion | 8 (10.0) | 5 (11.4) | 3 (8.3) | 1.00 |
| BA | 9 (11.3) | 2 (4.5) | 7 (19.4) | 0.08 |
| PCA: isolated P1 segment | 1 (1.3) | 0 | 1 (2.8) | 0.46 |
| Grade of collateral circulation | 1.88±0.40 | 2.00±0.47 | 0.22 | |
| Onset-to-IVT start time (min) | 127.8 | NA | ||
| IVT-to-groin puncture time (min) | 71.4 | NA | ||
| Primary transport to CSC (n=30) | 38.7 | NA | ||
| Onset-to-groin puncture time (min) | 207.2 | 208.0 | 205.9 | 0.91 |
| Groin puncture-to-recanalization time (min) | 42.3 | 45.8 | 38.1 | 0.22 |
| Device type | 0.56 | |||
| Solitaire | 45 (56.3) | 25 (56.8) | 20 (55.6) | |
| Trevo | 33 (41.3) | 18 (40.9) | 15 (41.7) | |
| Eric 4 | 2 (2.5) | 2 (4.5) | 0 | |
| Preset | 1 (1.3) | 0 | 1 (2.8) | |
| No. passages | 1.75±1.04 | 1.42±0.77 | 0.12 | |
| TICI 2b/3 | 69 (86.3) | 36 (81.8) | 33 (91.7) | 0.74 |
Data given as n (%), median, or mean±SD. BA, basilar artery; CSC, Comprehensive Stroke Center; Eric 4, ERIC® 4 Retrieval Device, MicroVention Terumo, Saint-Germain-en-Laye, France; ICA, internal carotid artery; MCA, middle cerebral artery; NA, not applicable; PCA, posterior cerebral artery; Preset, pREset®, Phenox, Bochum, Germany; Solitaire, SolitaireTM, Covidien, Dublin, Ireland; TICI, Thrombolysis in Cerebral Infarction; Trevo, Trevo® ProVueTM, Concentric Medical, Mountain View, CA, USA. Other abbreviations as in Table 1.
The histological findings are listed in Tables 3,4. Seventy-eight cases were classified as thromboemboli. A single case of cerebral artery thrombosis (i.e., a mixture of amorphous atheromatous material) was detected in a woman treated with MT alone. Another case of atheromatous mass emboli was detected in a male patient who had undergone previous IVT. There were occlusions in the M2 segment of the MCA in both cases. In 58 patients (72.5%), typical proportions of blood constituents in the clots were observed. In 22 patients (27.5%), there was a predominance of thrombocytes. Among the 78 thromboemboli, 60 were classified as fresh clots (76.9%), 7 were classified as older clots (9.0%), and 11 clots (14.1%) were a mixture of fresh and older particles. Twenty-two clots predominantly had fresh thrombocytes. Regarding the older emboli, the organization process typically started at the surfaces of the clots, that is, near the histiocytes, macrophages and (less frequently) endothelia (Figure 1). Based on the histology alone, however, it was difficult to determine whether the organization process took place at the site of the formation of thrombus, that is, in the left heart, or in the cerebral artery. Unlike mostly a solid structure of fibrin, present in typical thrombi or thromboemboli, the thinning phenomenon in the described cases typically started in the superficial layers of fibrin masses. In 32 cases (41.0%), there was a total (grade 3) thinning transformation of fibrin clots (Figure 2).
| Parameter | All (n=78) |
IVT+MT (n=43) |
MT-A (n=35) |
P-value |
|---|---|---|---|---|
| Thinning grade | 1.78±1.13 | 2.16±1.02 | 1.31±1.11 | 0.001 |
| Clot freshness | 0.016 | |||
| Fresh | 60 (76.9) | 38 (88.4) | 22 (62.9) | |
| Older | 7 (9.0) | 1 (2.3) | 6 (17.1) | |
| Mixed | 11 (14.1) | 4 (9.3) | 7 (20.0) |
Data given as mean±SD or n (%). Abbreviations as in Table 1.
| Parameter | Thinning grade 0–2 (n=46) |
Thinning grade 3 (n=32) |
P-value |
|---|---|---|---|
| Clot freshness | 0.041 | ||
| Fresh | 30 (65.2) | 29 (90.6) | |
| Older | 7 (15.2) | 1 (3.1) | |
| Mixed | 9 (19.6) | 2 (6.3) | |
| Sex | 0.476 | ||
| Male | 19 (41.3) | 10 (31.3) | |
| Female | 27 (58.7) | 22 (68.8) |
Data given as n (%).
Thinning of grade 3 was observed in 55.8% of clots (24 patients) in the IVT+MT group and in 22.9% of clots (8 patients) in the MT-A group, respectively (P=0.06). The results of logistic regression analysis are presented in Table 5. Thinning of grade 3 was associated with IVT (OR, 3.50; 95% CI: 1.21–10.10, P=0.02) and PLT (OR, 2.94; 95% CI: 1.06–8.12, P=0.04) in the whole group. No correlation was found between the grades of thinning and the time between IVT and clot removal (R=−0.0019; 95% CI: −0.30 to 0.30, P=0.99). In spite of the fact that fibrinogen level was significantly different between the low and high thinning grades (Table 6), its predictive value was not significant (OR, 0.54; 95% CI: 0.25–1.16, P=0.11). Pronounced thinning in the IVT+MT group was associated with a longer lasting endovascular intervention compared with the MT-A group (45.8 min vs. 38.1 min) and worse recanalization (TICI 2b/3: IVT+MT, 81.8% vs. MT-A, 91.7%). As for the ratio of patients with TICI 2b/3, however, the time difference from groin puncture to recanalization between the two treatment groups was not statistically significant. No statistically significant correlation was observed between the grades of thinning and of collateral circulation (R=0.12; 95% CI: −0.12 to 0.35, P=0.33).
| Outcome: Thinning grade 3 |
Regression coefficient (95% CI) |
t statistics | P-value | OR (95% CI) |
|---|---|---|---|---|
| Intercept | 0.72 (−2.40 to 3.83) | 0.45 | 0.65 | 2.05 (0.09–45.99) |
| Fibrinogen | −0.62 (−1.39 to 0.14) | −1.59 | 0.11 | 0.54 (0.25–1.16) |
| PLT | 1.08 (0.06–2.09) | 2.08 | 0.04 | 2.94 (1.06–8.12) |
| IVT | 1.25 (0.20–2.31) | 2.32 | 0.02 | 3.50 (1.21–10.10) |
IVT, i.v. thrombolysis; PLT, platelets.
| Parameter | All (n=78) |
Thinning grades 0–2 (n=46) |
Thinning grade 3 (n=32) |
P-value |
|---|---|---|---|---|
| Fibrinogen (g/L) | 3.97±0.90 | 3.52±0.67 | 0.02 | |
| RBC (×1012/L) | 4.23±0.61 | 4.42±0.42 | 0.13 | |
| Hematocrit | 0.38±0.14 | 0.40±0.03 | 0.43 | |
| PLT (×109/L) | 221.60±80.38 | 247.44±79.22 | 0.16 | |
| MPV (fL) | 10.75±0.90 | 10.79±1.05 | 0.86 | |
| History of | ||||
| Ischemic heart disease | 24 (30.8) | 15 (32.6) | 9 (28.1) | 0.82 |
| Diabetes mellitus | 20 (25.6) | 13 (28.3) | 7 (21.9) | 0.80 |
| Pretreatment with | ||||
| ASA | 29 (37.2) | 18 (39.1) | 11 (34.4) | 0.93 |
| Warfarin | 7 (9.0) | 7 (15.2) | 0 (0) | 0.04 |
| INR | 1.77±0.42 (1.40–2.64) | |||
| Statins | 28 (35.9) | 18 (39.1) | 10 (31.3) | 0.66 |
| IVT | 44 (56.4) | 20 (43.5) | 24 (75.0) | 0.19 |
Data given as mean±SD (range) or n (%). ASA, acetylsalicylic acid; INR, international normalized ratio; IVT, i.v. thrombolysis; MPV, mean platelet volume; PLT, platelets; RBC, red blood cells.
Fresh thromboemboli were significantly more frequent among those with thinning of grade 3 vs. grade 0–2 (90.6% vs. 65.2%, P=0.041; Table 4).
To our knowledge this is the first study to describe the novel finding of dissociation of fibrin fibers in the intra-arterial clots on MT with stent-retrievers from large cerebral arteries in acute ischemic stroke patients and its association with IVT. Patients who underwent MT at Comprehensive Stroke Centre had an 89.1% success rate in terms of recanalization (TICI 2b/3) and achieved a 54.3% rate of self-sufficiency (mRS 0-2). These results agree with other recent studies in clinical practice.1–5,30,31
The changes in fibrin fiber compositions in some of the observed clots are of special note. As a thrombus develops, all thrombus component compositions (including fibrin) are altered. During initial phases, fibrin binds to platelets and constitutes a high proportion of the forming thrombus. Furthermore, fibrin fibers form a porous network. In subsequent phases, the fibrin fibers and adjacent fibrin monomers are cross-linked by factor XIIIa (thrombin-activated FXIII) via amide or isopeptide bonds.32 At this stage, the thrombus is compact and has smaller pores, and the clot is retracted and stabilized. The fact that older thrombi contain greater amounts of fibrin has been demonstrated by experimental studies and by analyses of clots obtained from coronary and cerebral arteries.9,17,33,34 Clots collected from thrombectomy patients contain various levels of von Willebrand factor (vWF),35 a large multimeric plasma glycoprotein that links platelets together and with fibrin (thereby, organizing the clots). The main function of vWF is to mediate initial platelet adhesion and activation at sites of vascular injury. High plasma vWF has been considered as a strong predictor for ischemic stroke.36 Along with faulty patient selection and prolonged onset-recanalization time, these factors could constitute additional reasons for so-called futile recanalization, that is, successful recanalization followed by poor clinical outcome, which occurs in approximately 30% of patients. In the present group, this situation was observed in 34.8% of the patients (TICI 2b/3, 89.1%; mRS 0–2, 54.3%). Reperfusion of the ischemic area can activate vWF and retroactively induce thrombus formation in small arteries downstream. In mice, the inhibition of vWF significantly reduces stroke reperfusion injury.35,37 rtPA exerts fibrinolytic actions on the surface of the thrombus and within the thrombus (via diffusion), but the tightly packed fibrin fibers in older clots prohibits rtPA diffusion, thereby limiting the fibrinolytic action. Thus, aged thrombi may be more resistant to rtPA than newer thrombi.7,16,38–40 Changes to the fibrin fiber architecture may also result in limited rtPA efficiency. Varjú et al showed in 2011 that mechanical stresses (i.e., axial shear forces) in stenotic arteries may longitudinally straighten fibrin fibers on the surface of the thrombus and result in tighter packing in the transverse direction.16 Mechanical stress confers proteolytic resistance to fibrin fibers as a result of impaired plasminogen activation and a lower plasmin sensitivity of the dense fibrin network. Stretched fibrin fibers are not as readily activated by rtPA. Similarly, light fibrin fibers that formed under elevated thrombin concentration are resistant to fibrinolysis.41
In a previous study, we examined the histology of thrombi collected from coronary arteries. Using similar methods, in the present study we determined thromboemboli age using immunohistochemistry.42 As a criterion for classifying clot age, we considered the presence of histiocytes, macrophages and/or endothelia to indicate an older clot. Most thromboemboli (76.9%) were fresh. This indicates that the highest risk of embolization appears shortly after the formation of the thrombus. In 2013, Singh et al used similar immunohistochemical methods to detect endothelial cells in 48 cerebral thromboemboli. The presence of endothelial cells suggests intimal damage during thrombectomy. On histology, however, MT with stent-retrievers did not cause the corresponding intimal damage observed during acute ischemic stroke treatment.43
On histology in the present study, we observed the novel finding of the dissociation of fibrin fibers, which we termed “thinning”. This phenomenon was significantly pronounced in patients who had undergone IVT (P=0.02). In contrast, no correlation was found between the grades of thinning and the time between IVT and clot removal, although one may assume that more thinning might be observed in those clots retrieved from patients with extended time between IVT and clot removal (i.e., with a longer effective time during which rtPA could exert its activity on the clot). Less frequent occurrence of thinning in patients who had not undergone IVT may be explained by spontaneous fibrinolysis. Theoretically, there might be a possibility of a reverse change (from grade 3 to grade 1), that is, fibrin aggregation, but the histological finding of a network of very fine fibrin threads observed sometimes in the early stages of thrombogenesis in general, differs significantly from the thinning phenomenon. Dissociation of fibrin fibers was also associated with PLT, for which we have no explanation. In contrast, fibrin clot architecture was not influenced by MPV in the present study, although in previous studies elevated MPV was associated with worse outcome in patients with acute myocardial infarction,44 increased mortality and restenosis following coronary angioplasty.45 Although the fibrinogen level was significantly different between the low and high thinning grades, its predictive value was not significant, and differences in the thromboembolus structure were not associated with the presence of comorbidities such as diabetes or ischemic heart disease in the present study. Even though one may assume that patients with better collaterals might have more distal blood flow (including rtPA) that reaches the distal part of the thrombus and that better accessibility for rtPA could promote thinning, we observed no statistically significant correlation between the grades of thinning and of collateral circulation. One may also speculate that the presence of thinning might be influenced by other factors, such as time interval. For example, patients with unknown stroke onset time would have a longer time before intervention and might be more likely to have spontaneous fibrinolysis. Nevertheless, in the present study, these patients were treated (although statistically non-significantly) more frequently with MT-A than with IVT+MT. Also onset-to-groin puncture and groin puncture-to-recanalization times did not differ between the 2 treatment groups. Fresh thromboemboli were significantly more frequent in those with thinning of grade 3. This phenomenon could possibly be explained by a higher susceptibility of fresh thromboemboli to the treatment, while older, more compact blood clots may be more resistant to thinning.
When comparing the present 2 treatment groups, efficacy was similar with only a statistically non-significant trend towards better procedural success in the patients who did not receive rtPA (e.g., lower number of passages, shorter duration of endovascular intervention and higher proportion of successful recanalization). Crushed thrombi may be harder to extract. This would account for the more pronounced thinning observed in the present patients after IVT and the higher number of necessary passages, longer duration of endovascular intervention and poorer recanalization outcome. Thus, one may speculate that the clot thinning might be viewed as a result of multiple stent retriever passes disrupting the clot rather than IVT. From a purely histological standpoint, however, the clot changes do not feature a mechanical origin. The thinning is a fine process affecting usually a major part of the clot, proceeding from its surface inwards, thus featuring a stage of fibrinolysis.
The aforementioned poorer recanalization outcomes observed in the IVT+MT group were not influenced by the localization of arterial occlusions, because the IVT+MT group had a non-significantly smaller proportion of worse recanalizable occlusions in the BA and also a non-significantly greater proportion of recanalizable proximal occlusions in the MCA (M1/2). This indicates the possible influence of thinning on intervention with stent-retrievers. Clinical outcome differed non-significantly between the 2 groups (mRS 0–2, 59.1% in the IVT+MT group vs. 55.6% in the MT-A group).
The main limitation of this study was that we were not able to perform histological analysis in patients without achieved recanalization (TICI 0), in whom no clot was retrieved. Second, patients were not randomized into IVT+MT and MT-A groups, which could cause a bias. Third, some other factors, previously reported as biomarkers or predictors of stroke or thromboembolism, such as fibrin degradation products, D-dimers (fibrinolysis markers), thrombin-antithrombin complex (thrombogenesis marker) or brain natriuretic peptide (cardiac overload marker), were not assessed in the present study.
In conclusion, in patients with ischemic large artery stroke treated with MT using stent-retrievers, bridging therapy with IVT preceding MT and higher PLT were associated with significant changes of the histological structure of blood clots. Nevertheless, these clot changes did not significantly influence the procedural success or clinical outcome.
This research was supported in part by grants from the Ministry of Health (RVO – FNHK, 00179906) and the Ministry of Education, Youth and Sports of the Czech Republic (PRVOUK: P37/08).
The authors declare no conflict of interest.
