Transcriptomic Analysis of Extracellular Vesicles in the Search for Novel Plasma and Thrombus Biomarkers of Ischemic Stroke Etiologies.

Accurate etiologic diagnosis provides an appropriate secondary prevention and better prognosis in ischemic stroke (IS) patients; still, 45% of IS are cryptogenic, urging us to enhance diagnostic precision. We have studied the transcriptomic content of plasma extracellular vesicles (EVs) (n = 21) to identify potential biomarkers of IS etiologies. The proteins encoded by the selected genes were measured in the sera of IS patients (n = 114) and in hypertensive patients with (n = 78) and without atrial fibrillation (AF) (n = 20). IGFBP-2, the most promising candidate, was studied using immunohistochemistry in the IS thrombi (n = 23) and atrium of AF patients (n = 13). In vitro, the IGFBP-2 blockade was analyzed using thromboelastometry and endothelial cell cultures. We identified 745 differentially expressed genes among EVs of cardioembolic, atherothrombotic, and ESUS groups. From these, IGFBP-2 (cutoff > 247.6 ng/mL) emerged as a potential circulating biomarker of embolic IS [OR = 8.70 (1.84-41.13) p = 0.003], which was increased in patients with AF vs. controls (p < 0.001) and was augmented in cardioembolic vs. atherothrombotic thrombi (p < 0.01). Ex vivo, the blockage of IGFBP-2 reduced clot firmness (p < 0.01) and lysis time (p < 0.001) and in vitro, diminished endothelial permeability (p < 0.05) and transmigration (p = 0.06). IGFBP-2 could be a biomarker of embolic IS and a new therapeutic target involved in clot formation and endothelial dysfunction.

Role of factor XIII in ischemic stroke: a key molecule promoting thrombus stabilization and resistance to lysis.

BACKGROUND: Active coagulation factor XIII (FXIII) catalyzing crosslinking of fibrin and other hemostatic factors plays a key role in clot stability and lysis. OBJECTIVES: To evaluate the effect of FXIII inhibition in a mouse model of ischemic stroke (IS) and the role of activated FXIII (FXIIIa) in clot formation and lysis in patients with IS. METHODS: A ferric chloride IS murine model was performed before and after administration of a FXIIIa inhibitor (FXIIIinh). Thromboelastometry in human and mice blood was used to evaluate thrombus stiffness and lysis with FXIIIinh. FXIIIa-dependent fibrin crosslinking and lysis with fibrinolytic drugs (tissue plasminogen activator and tenecteplase) were studied on fibrin plates and on thrombi and clotted plasma of patients with IS. Finally, circulating and thrombus FXIIIa were measured in 85 patients with IS. RESULTS: FXIIIinh administration before stroke induction reduced infarct size, α2-antiplasmin (α2AP) crosslinking, and local microthrombosis, improving motor coordination and fibrinolysis without intracranial bleeds (24 hours). Interestingly, FXIII blockade after stroke also reduced brain damage and neurologic deficit. Thromboelastometry in human/mice blood with FXIIIinh showed delayed clot formation, reduced clot firmness, and shortened tissue plasminogen activator lysis time. FXIIIa fibrin crosslinking increased fibrin density and lysis resistance, which increased further after α2AP addition. FXIIIinh enhanced ex vivo lysis in stroke thrombi and fibrin plates. In patients with IS, thrombus FXIII and α2AP were associated with inflammatory and hemostatic components, and plasma FXIIIa correlated with thrombus α2AP and fibrin. CONCLUSION: Our results suggest a key role of FXIIIa in thrombus stabilization, α2AP crosslinking, and lysis resistance, with a protective effect of FXIIIinh in an IS experimental model.

The thrombus proteome in stroke reveals a key role of the innate immune system and new insights associated with its etiology, severity, and prognosis.

BACKGROUND: Nowadays little is known about the molecular profile of the occluding thrombus of patients with ischemic stroke. OBJECTIVES: To analyze the proteomic profile of thrombi in patients who experienced an ischemic stroke in order to gain insights into disease pathogenesis. METHODS: Thrombi from an exploratory cohort of patients who experienced a stroke were obtained by thrombectomy and analyzed by sequential window acquisition of all theoretical spectra-mass spectrometry. Unsupervised k-means clustering analysis was performed to stratify patients who experienced a stroke. The proteomic profile was associated with both the neurological function (National Institute of Health Stroke Scale [NIHSS]) and the cerebral involvement (Alberta Stroke Program Early CT Score [ASPECTS]) prior to thrombectomy and the clinical status of patients at 3 months using the modified Rankin Scale. In an independent cohort of 210 patients who experienced a stroke, the potential role of neutrophils in stroke severity was interrogated. RESULTS: Proteomic analysis identified 580 proteins in thrombi, which were stratified into 4 groups: hemostasis, proteasome and neurological diseases, structural proteins, and innate immune system and neutrophils. The thrombus proteome identified 3 clusters of patients with distinctive severity, prognosis, and etiology of the stroke. A protein signature clearly distinguished atherothrombotic and cardioembolic strokes. Several proteins were significantly correlated with the severity of the stroke (NIHSS and ASPECTS). Functional proteomic analysis highlighted the prominent role of neutrophils in stroke severity. This was in line with the association of neutrophil activation markers and count with NIHSS, ASPECTS, and the modified Rankin Scale score 90 days after the event. CONCLUSION: The use of sequential window acquisition of all theoretical spectra-mass spectrometry in thrombi from patients who experienced an ischemic stroke has provided new insights into pathways and players involved in its etiology, severity, and prognosis. The prominent role of the innate immune system identified might pave the way for the development of new biomarkers and therapeutic approaches in this disease.

Impact on functional outcome of an adaptive Stroke Unit based system of care for patients undergoing endovascular treatment during pandemic times.

Introduction: The COVID19 pandemic collapsed intensive care units (ICUs) all around the world, conditioning systems of care (SOC) for other critical conditions such as severe ischemic stroke requiring endovascular treatment (EVT). Our aim was to evaluate the impact of an adaptive Stroke Unit (SU) based SOC on functional outcomes, with the goal of avoiding both general anesthesia (GA) and ICU admission in stroke patients treated with EVT. Material and methods: We performed an observational study comparing data from our traditional ICU-GA based SOC and the adaptive SU-Conscious Sedation (CS) based SOC (consecutive patients undergoing EVT 1 year prior and after onset of the pandemic). Primary outcome was 90-days modified Rankin Scale (mRS), and secondary outcomes included, among others, in-hospital complications, and hospital length of stay (LOS). Results: A total of 210 EVT were performed during the study period (107 under the traditional-SOC and 103 under the adaptive-SOC). A significantly greater proportion of patient was treated under CS (15.9% vs 57.3%; p < 0.001) and admitted for post-procedural care at SU (15% vs 66%; p < 0.001) in the adaptive SOC. Rates of in-hospital complications were similar in both periods, with reduced hospital LOS in the adaptive SOC (10 (7-15) vs 8 (6-12); p = 0.005). The adaptive SOC was associated with higher odds for 90 days favorable outcome (mRS 0-2) (aOR 3.15 (1.34-7.39); p = 0.008). Conclusion: In our case, an adaptive SOC that combined both preference for CS and postprocedural care in SU was associated with better functional outcomes and reduced healthcare resource use for patients undergoing EVT.

CM-352 Efficacy in a Mouse Model of Anticoagulant-Associated Intracranial Hemorrhage.

BACKGROUND: Intracranial hemorrhage (ICH) is one of the major devastating complications of anticoagulation. Matrix metalloproteinase (MMP) inhibition has been proposed as a novel pharmacological approach for ICH treatment. OBJECTIVES: We evaluated the effects of CM-352 (MMP-fibrinolysis inhibitor) in an experimental ICH model associated with oral anticoagulants as compared with clinically used prothrombin complex concentrate (PCC). METHODS: ICH was induced by collagenase injection into the striatum of wild type (C57BL/6J) anticoagulated mice (warfarin or rivaroxaban) and Mmp10 -/- mice. Hematoma volume and neurological deficits were measured 24 hours later by diaminobenzidine staining and different behavioral tests. Circulating plasminogen activator inhibitor-1 (PAI-1) activity and interleukin-6 (IL-6) were measured in plasma samples and local inflammation was assessed by neutrophil infiltration. Finally, fibrinolytic effects of MMP-10 and rivaroxaban were evaluated by thromboelastometry and thrombin-activatable fibrinolysis inhibitor (TAFI) activation assays. RESULTS: Only PCC reduced hemorrhage volume and improved functional outcome in warfarin-ICH, but both PCC and CM-352 treatments diminished hemorrhage volume (46%, p < 0.01 and 64%, p < 0.001, respectively) and ameliorated functional outcome in rivaroxaban-ICH. We further demonstrated that CM-352, but not PCC, decreased neutrophil infiltration in the hemorrhage area at 24 hours. The effect of CM-352 could be related to MMP-10 inhibition since Mmp10 -/- mice showed lower hemorrhage volume, better neurological score, reduced IL-6 levels and neutrophil infiltration, and increased PAI-1 after experimental ICH. Finally, we found that CM-352 reduced MMP-10 and rivaroxaban-related fibrinolytic effects in thromboelastometry and TAFI activation. CONCLUSION: CM-352 treatment, by diminishing MMPs and rivaroxaban-associated fibrinolytic effects, might be a novel antihemorrhagic strategy for rivaroxaban-associated ICH.

Inside the Thrombus: Association of Hemostatic Parameters With Outcomes in Large Vessel Stroke Patients.

Background: Actual clinical management of ischemic stroke (IS) is based on restoring cerebral blood flow using tissue plasminogen activator (tPA) and/or endovascular treatment (EVT). Mechanical thrombectomy has permitted the analysis of thrombus structural and cellular classic components. Nevertheless, histological assessment of hemostatic parameters such as thrombin-activatable fibrinolysis inhibitor (TAFI) and matrix metalloproteinase 10 (MMP-10) remains unknown, although their presence could determine thrombus stability and its response to thrombolytic treatment, improving patient's outcome. Methods: We collected thrombi (n = 45) from large vessel occlusion (LVO) stroke patients (n = 53) and performed a histological analysis of different hemostatic parameters [TAFI, MMP-10, von Willebrand factor (VWF), and fibrin] and cellular components (erythrocytes, leukocytes, macrophages, lymphocytes, and platelets). Additionally, we evaluated the association of these parameters with plasma levels of MMP-10, TAFI and VWF activity and recorded clinical variables. Results: In this study, we report for the first time the presence of MMP-10 and TAFI in all thrombi collected from LVO patients. Both proteins were localized in regions of inflammatory cells, surrounded by erythrocyte and platelet-rich areas, and their content was significantly associated (r = 0.41, p < 0.01). Thrombus TAFI was lower in patients who died during the first 3 months after stroke onset [odds ratio (OR) (95%CI); 0.59 (0.36-0.98), p = 0.043]. Likewise, we observed that thrombus MMP-10 was inversely correlated with the amount of VWF (r = -0.30, p < 0.05). Besides, VWF was associated with the presence of leukocytes (r = 0.37, p < 0.05), platelets (r = 0.32, p < 0.05), and 3 months mortality [OR (95%CI); 4.5 (1.2-17.1), p = 0.029]. Finally, plasma levels of TAFI correlated with circulating and thrombus platelets, while plasma MMP-10 was associated with cardiovascular risk factors and functional dependence at 3 months. Conclusions: The present study suggests that the composition and distribution of thrombus hemostatic components might have clinical impact by influencing the response to pharmacological and mechanical therapies as well as guiding the development of new therapeutic strategies.

Association of calprotectin with other inflammatory parameters in the prediction of mortality for ischemic stroke.

BACKGROUND: Inflammatory response plays an important role in many processes related to acute ischemic stroke (AIS). Calprotectin (S100A8/S100A9), released by monocytes and neutrophils, is a key protein in the regulation of inflammation and thrombosis. The purpose of this study is to evaluate the association of circulating calprotectin with other inflammatory biomarkers and AIS prognosis, as well as the calprotectin content in stroke thrombi. METHODS: Among the 748 patients treated at a comprehensive stroke center between 2015 and 2017, 413 patients with confirmed acute ischemic injury were prospectively evaluated. Patients with systemic inflammation or infection at onset were excluded. Plasma calprotectin was measured by ELISA in blood samples of AIS patients within the first 24 h. Univariate and multivariate logistic regression models were performed to evaluate its association with mortality and functional independence (FI) at 3 months (defined as modified Rankin Scale < 3) and hemorrhagic transformation (HT) after ischemic stroke. Further, S100A9 was localized by immunostaining in stroke thrombi (n = 44). RESULTS: Higher calprotectin levels were associated with 3-month mortality, HT, and lower 3-month FI. After adjusting for potential confounders, plasma calprotectin remained associated with 3-month mortality [OR (95% CI) 2.31 (1.13-4.73)]. Patients with calprotectin ≥ 2.26 µg/mL were 4 times more likely to die [OR 4.34 (1.95-9.67)]. Addition of calprotectin to clinical variables led to significant improvement in the discrimination capacity of the model [0.91 (0.87-0.95) vs 0.89 (0.85-0.93); p < 0.05]. A multimarker approach demonstrated that patients with increased calprotectin, CRP, and NLR had the poorest outcome with a mortality rate of 42.3% during follow-up. S100A9 protein, as part of the heterodimer calprotectin, was present in all thrombi retrieved from AIS patients. Mean S100A9 content was 3.5% and tended to be higher in patients who died (p = 0.09). Moreover, it positively correlated with platelets (Pearson r 0.46, p < 0.002), leukocytes (0.45, p < 0.01), and neutrophil elastase (0.70, p < 0.001) thrombus content. CONCLUSIONS: Plasma calprotectin is an independent predictor of 3-month mortality and provides complementary prognostic information to identify patients with poor outcome after AIS. The presence of S100A9 in stroke thrombi suggests a possible inflammatory mechanism in clot formation, and further studies are needed to determine its influence in resistance to reperfusion.

Circulating TIMP-1 is associated with hematoma volume in patients with spontaneous intracranial hemorrhage.

Matrix metalloproteinases (MMPs) are proteolytic zinc-endopeptidases regulated by tissue Inhibitors of matrix metalloproteinases (TIMPs). We evaluated the potential of MMPs and TIMPs as clinical tools for Intracranial Haemorrhage (ICH). Spontaneous non-traumatic ICH patients were recruited from two hospitals: Complejo Hospitalario de Navarra (CHN = 29) and Vall d´Hebron (VdH = 76). Plasmatic levels of MMP-1, -2, -7, -9, -10 and TIMP-1 and their relationship with clinical, radiological and functional variables were evaluated. We further studied the effect of TIMP-1 (0.05-0.2 mg/Kg) in an experimental tail-bleeding model. In CHN, TIMP-1 was associated with admission-hematoma volume and MMP-7 was elevated in patients with deep when compared to lobar hematoma. In VdH, admission-hematoma volume was associated with TIMP-1 and MMP-7. When data from both hospitals were combined, we observed that an increase in 1 ng/ml in TIMP-1 was associated with an increase of 0.14 ml in haemorrhage (combined ß = 0.14, 95% CI = 0.08-0.21). Likewise, mice receiving TIMP-1 (0.2 mg/Kg) showed a shorter bleeding time (p < 0.01). Therefore, the association of TIMP-1 with hematoma volume in two independent ICH cohorts suggests its potential as ICH biomarker. Moreover, increased TIMP-1 might not be sufficient to counterbalance MMPs upregulation indicating that TIMP-1 administration might be a beneficial strategy for ICH.