Human Thrombi Research Articles

Cellular and enzymatic features of thrombi in humans are vascular bed dependent

AbstractMechanisms behind vascular remodeling following thrombosis are unclear. Although acute arterial thrombosis in the cerebrovascular circulation has devastating consequences and requires immediate attention, the management of venous thromboembolism (VTE) varies significantly. Our goal was to determine the molecular signatures and cellular content of thrombus extracted using a catheter to gain insight into vascular remodeling. Twenty-five patients underwent catheter-directed thrombectomy (CDT); 13 for cerebrovascular accident (CVA), 8 for pulmonary embolism, and 4 for deep vein thrombosis. Protein and RNA were extracted from thrombi to enable immunoblotting, RNA sequencing, and quantification of gene expression. The time from symptom onset to thrombus extraction was 7.7 ± 1.9 hours for CVA and 109 ± 55 hours for VTE. Protein concentration, white blood cell content (monocytes), and red blood cell content were greater in venous thrombus than in arterial thrombus, whereas the platelet content was similar. Both venous and arterial thrombi contained several zinc endopeptidases belonging to the matrix metalloproteinase (MMP) family. MMP9 activity in venous thrombus was greater than arterial thrombus (61 ± 9 ng/mL per μg protein vs 25 ± 6 ng/mL per μg protein; P = .005). Arterial and venous thrombi displayed surprisingly different phenotypes, with biologically active enzymes promoting blood vessel remodeling and enzymatic activity proportional to thrombus age extracted from the veins. These mechanistic data may support the role of early CDT in venous circulation to avoid irreversible vascular remodeling.

Identification of nitrated proteins in human thrombi from ischemic cerebrovascular events

Identification of nitrated proteins in human thrombi from ischemic cerebrovascular events

Abstract 1069: Novel Approaches Dissecting Human Thrombus Composition

Introduction: Thrombus formation is a key contributor to ischemic stroke and cardiovascular diseases. Variations in thrombus composition involving platelets, fibrin, and leukocytes play a pivotal role in the pathophysiology, impacting clinical outcomes. Understanding its character and the interplay with Neutrophil Extracellular Traps (NETs) is clinically vital due to treatment response and outcome. This study introduces light-sheet microscopy and holotomography to dissect clot composition, aiming to enhance clinical insights into thrombosis. Unraveling these complexities holds promise for targeted interventions and improved patient outcomes. Aims: This study aimed to enhance our understanding of human blood thrombus composition by employing a novel approach using light-sheet microscopy and holotomography. Methods: Human thrombus samples were obtained by mechanical thrombectomy from acute ischemic stroke patients. To access the thrombus structure, the samples were cleared by a tissue-clearing rapid system, and light-sheet microscope imaging was obtained with CitH3, CD45, P-selectin, and fibrin. Holotomography images were obtained from cryosectioned thrombus at 5μm thickness and stained with DAPI and CitH3. Holotomography provided quantitative information on the refractive index of thrombus components. Comparative analysis with traditional imaging methods was performed to assess the advantages of the novel approaches. Results: The results revealed a comprehensive visualization of the spatial arrangement of human thrombus composition through light-sheet microscopy imaging. Holotomography successfully acquired a three-dimensional image of a thrombus section sample. HT images were used to qualitatively distinguish differences in neutrophil RI distribution by NETosis and differences in RI values within the immune cell population. Conclusion: This study demonstrates the efficacy of combining light sheet microscopy and holotomography to comprehensively dissect human thrombus composition. The integration of these advanced imaging techniques provides an understanding of thrombosis, surpassing the limitations of traditional methods.

Identification of nitrated proteins in human thrombi from ischemic cerebrovascular events

Identification of nitrated proteins in human thrombi from ischemic cerebrovascular events

The inhibitory effects of glabridin on human platelet aggregation and thrombus formation

The inhibitory effects of glabridin on human platelet aggregation and thrombus formation

PB0594 Factor XI Localization in Human Deep Venous Thrombus and Function of Activated Factor XI on Mural Thrombus Formation Under Low-Shear Condition

PB0594 Factor XI Localization in Human Deep Venous Thrombus and Function of Activated Factor XI on Mural Thrombus Formation Under Low-Shear Condition

PB0875 A Parallel Kinetic Monte Carlo Simulation of Human Stenotic Thrombus Growth with Single Platelet Resolution from Plaque Rupture to Occlusion

PB0875 A Parallel Kinetic Monte Carlo Simulation of Human Stenotic Thrombus Growth with Single Platelet Resolution from Plaque Rupture to Occlusion

Correction to: "Spatial Proteomics Analysis of Soft and Stiff Regions in Human Acute Arterial Thrombus".

Correction to: "Spatial Proteomics Analysis of Soft and Stiff Regions in Human Acute Arterial Thrombus".

Platelet accumulation in an endothelium-coated elastic vein valve model of deep vein thrombosis is mediated by GPIbα-VWF interaction.

Deep vein thrombosis is a life-threatening disease that takes millions of people's lives worldwide. Given both technical and ethical issues of using animals in research, it is necessary to develop an appropriate in vitro model that would recapitulate the conditions of venous thrombus development. We present here a novel microfluidics vein-on-a-chip with moving valve leaflets to mimic the hydrodynamics in a vein, and Human Umbilical Vein Endothelial Cell (HUVEC) monolayer. A pulsatile flow pattern, typical for veins, was used in the experiments. Unstimulated human platelets, reconstituted with the whole blood, accumulated at the luminal side of the leaflet tips proportionally to the leaflet flexibility. Platelet activation by thrombin induced robust platelet accrual at the leaflet tips. Inhibition of glycoprotein (GP) IIb-IIIa did not decrease but, paradoxically, slightly increased platelet accumulation. In contrast, blockade of the interaction between platelet GPIbα and A1 domain of von Willebrand factor completely abolished platelet deposition. Stimulation of the endothelium with histamine, a known secretagogue of Weibel-Palade bodies, promoted platelet accrual at the basal side of the leaflets, where human thrombi are usually observed. Thus, platelet deposition depends on the leaflet flexibility, and accumulation of activated platelets at the valve leaflets is mediated by GPIbα-VWF interaction.

Spatial Proteomics Analysis of Soft and Stiff Regions in Human Acute Arterial Thrombus.

The soft regions of a thrombus tend to be more susceptible to r-tPA (recombinant tissue-type plasminogen activator)-mediated thrombolysis and are more easily removed by mechanical thrombectomy than the stiff counterpart. This study aimed to understand the molecular pathological differences between the soft and stiff regions of human arterial thrombus. We developed a spatial proteomic workflow combining proteomics with laser-captured microdissection to analyze human arterial thrombi with Masson trichrome staining to identify stiff and soft regions from 2 independent cohorts of patients with acute myocardial or cerebral infarction. Dysregulated proteins in a C57BL6/J male mouse model of arterial thrombosis were identified by pathway enrichment and pairwise analyses from the common gene ontology enrichment and dysregulated proteins between carotid and coronary arterial thrombi, and validated by immunohistochemistry. Spatial proteomics of the coronary arterial thrombi collected from 7 patients with myocardial infarct revealed 7 common dysregulated proteins in 2 cohorts of patients, and upregulation of TGF-β1 (transforming growth factor β1) was the most prominent fibrosis-related protein. Inhibition of TGF-β1 resulted in delayed arterial thrombosis and accelerated blood flow restoration in mouse model. We further expanded the spatial proteomic workflow to the carotid artery thrombi collected from 11 patients with cerebral infarction. Pairwise proteomic analysis of stiff and soft regions between carotid and coronary arterial thrombi further revealed 5 common gene ontology clusters including features of platelet activation, and a common dysregulated protein COL1A1 (collagen type 1 alpha 1) that was reported to be influenced by TGF-β1. We also verified the expression in human and mice carotid arterial thrombi. This study demonstrates the spatially distinct composition of proteins in the stiff and soft regions of human arterial thrombi, and suggests that TGF-β1 is a key therapeutic target for promoting arterial thrombolysis.

Soluble endoglin reduces thrombus formation and platelet aggregation via interaction with αIIbβ3 integrin

BackgroundThe circulating form of human endoglin (sEng) is a cleavage product of membrane-bound endoglin present on endothelial cells. Because sEng encompasses an RGD motif involved in integrin binding, we hypothesized that sEng would be able to bind integrin αIIbβ3, thereby compromising platelet binding to fibrinogen and thrombus stability. MethodsIn vitro human platelet aggregation, thrombus retraction, and secretion-competition assays were performed in the presence of sEng. Surface plasmon resonance (SPR) binding and computational (docking) analyses were carried out to evaluate protein-protein interactions. A transgenic mouse overexpressing human sEng (hsEng+) was used to measure bleeding/rebleeding, prothrombin time (PT), blood stream, and embolus formation after FeCl3-induced injury of the carotid artery. ResultsUnder flow conditions, supplementation of human whole blood with sEng led to a smaller thrombus size. sEng inhibited platelet aggregation and thrombus retraction, interfering with fibrinogen binding, but did not affect platelet activation. SPR binding studies demonstrated that the specific interaction between αIIbβ3 and sEng and molecular modeling showed a good fitting between αIIbβ3 and sEng structures involving the endoglin RGD motif, suggesting the possible formation of a highly stable αIIbβ3/sEng. hsEng+ mice showed increased bleeding time and number of rebleedings compared to wild-type mice. No differences in PT were denoted between genotypes. After FeCl3 injury, the number of released emboli in hsEng+ mice was higher and the occlusion was slower compared to controls. ConclusionsOur results demonstrate that sEng interferes with thrombus formation and stabilization, likely via its binding to platelet αIIbβ3, suggesting its involvement in primary hemostasis control.

Feasibility of Microbubble-Accelerated Low-Dose Thrombolysis of Peripheral Arterial Occlusions Using an Ultrasound Catheter.

Intra-arterial administration of microbubbles (MBs) through an ultrasound (US) catheter increases the local concentration of MBs into the thrombus and may further enhance outcomes of contrast-enhanced sonothrombolysis (CEST). The objective of this study was to evaluate the feasibility and lytic efficacy of intra-arterial infusion of MBs during US-enhanced thrombolysis in both in vitro and in vivo peripheral arterial occluded models. SonoVue and Luminity MBs were infused at a flow rate of 20 mL/h through either the drug delivery lumen of the US catheter (DDC, n=20) or through the tube lumen of the vascular phantom (systematic infusion, n=20) during thrombolysis with a low-dose urokinase (UK) protocol (50 000 IU/h) with(out) US application to assess MB survivability and size by pre-treatment and post-treatment measurements. A human thrombus was placed into a vascular phantom of the flow system to examine the lytic effects of CEST by post-treatment D-dimer concentrations measurements of 5 treatment conditions (saline, UK, UK+US, UK+US+SonoVue, and UK+US+Luminity). Thrombolytic efficacy of localized MBs and US delivery was then investigated in vivo in 5 porcine models by arterial blood flow, microcirculation, and postmortem determined thrombus weight and remaining length. US exposure significantly decreased SonoVue (p=0.000) and Luminity (p=0.000) survivability by 37% and 62%, respectively. In vitro CEST treatment resulted in higher median D-dimer concentrations for the SonoVue (0.94 [0.07-7.59] mg/mL, p=0.025) and Luminity (0.83 [0.09-2.53] mg/mL, p=0.048) subgroups when compared with thrombolysis alone (0.36 [0.02-1.00] mg/mL). The lytic efficacy of CEST examined in the porcine model showed an improved median arterial blood flow of 21% (7%-79%), and a median thrombus weight and length of 1.02 (0.96-1.43) g and 2.25 (1.5-4.0) cm, respectively. One allergic reaction and 2 arrhythmias were observed due to the known allergic reaction on lipids in the porcine model. SonoVue and Luminity can be combined with an US catheter and could potentially accelerate thrombolytic treatment of peripheral arterial occlusions. Catheter-directed thrombolysis showed to be an effective alternative to surgery for acute peripheral arterial occlusions, but this technique is still associated with several limb and life-threatening complications. The effects of thrombolysis on clot dissolution may be further enhanced by intra-arterial administration of microbubbles through an ultrasound catheter. This study demonstrates the feasibility and lytic efficacy of intra-arterial infusion of microbubbles during US-enhanced thrombolysis in both in vitro and in vivo peripheral arterial occluded models.

Amine-modified nanoplastics promote the procoagulant activation of isolated human red blood cells and thrombus formation in rats

BackgroundMicroplastics (MPs) and nanoplastics (NPs) formed from decomposed plastic are increasing environmental threats. Although MPs and NPs exposed through various routes enter the systemic circulation, the potential toxicity of those is largely unknown. We investigated whether polystyrene NPs (PS-NPs) promote the coagulation activity of red blood cells (RBCs).ResultsWe tested several types of PS-NPs using human RBCs and found that amine-modified 100 nm PS-NPs were the most potent. We measured the uptake of PS-NPs using flow cytometry and confocal microscopy. Electron microscopy revealed morphological changes of RBCs by PS-NPs. PS-NPs induced the externalization of phosphatidylserine, generation of microvesicles in RBCs, and perturbations in the intracellular microenvironment. PS-NPs increased the activity of scramblases responsible for phospholipid translocation in RBCs. PS-NPs modulated the functional interaction to adjacent tissues and coagulation cascade, enhancing RBC adhesion and thrombin generation. Our observations in human RBCs were consistent with those in isolated rat RBCs, showing no inter-species differences. In rat venous thrombosis models, the intravenous administration of PS-NPs enhanced thrombus formation.ConclusionAmine-modified PS-NPs induce the prothrombotic activation of RBCs causing thrombus formation. We believe that our study will contribute to understanding the potential toxicity of amine-modified polystyrene particles in blood cells and cardiovascular systems.

Heterocomplexes between the atypical chemokine MIF and the CXC-motif chemokine CXCL4L1 regulate inflammation and thrombus formation

To fulfil its orchestration of immune cell trafficking, a network of chemokines and receptors developed that capitalizes on specificity, redundancy, and functional selectivity. The discovery of heteromeric interactions in the chemokine interactome has expanded the complexity within this network. Moreover, some inflammatory mediators, not structurally linked to classical chemokines, bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified macrophage migration inhibitory factor (MIF) as an ACK that binds to chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine–chemokine interactions extend to ACKs and that MIF forms heterocomplexes with classical chemokines. We tested this hypothesis by using an unbiased chemokine protein array. Platelet chemokine CXCL4L1 (but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12) was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, also establishing high-affinity binding. We next determined whether heterocomplex formation modulates inflammatory/atherogenic activities of MIF. Complex formation was observed to inhibit MIF-elicited T-cell chemotaxis as assessed by transwell migration assay and in a 3D-matrix-based live cell-imaging set-up. Heterocomplexation also blocked MIF-triggered migration of microglia in cortical cultures in situ, as well as MIF-mediated monocyte adhesion on aortic endothelial cell monolayers under flow stress conditions. Of note, CXCL4L1 blocked binding of Alexa-MIF to a soluble surrogate of CXCR4 and co-incubation with CXCL4L1 attenuated MIF responses in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. Because MIF and CXCL4L1 are platelet-derived products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM-FRET, and proximity-ligation assay visualized heterocomplexes in platelet aggregates and in clinical human thrombus sections obtained from peripheral artery disease (PAD) in patients undergoing thrombectomy. Moreover, heterocomplexes inhibited MIF-stimulated thrombus formation under flow and skewed the lamellipodia phenotype of adhering platelets. Our study establishes a novel molecular interaction that adds to the complexity of the chemokine interactome and chemokine/receptor-network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be target structures that can be exploited to modulate inflammation and thrombosis.

An in vitro model for Extracellular DNA Traps (ETs)-rich Human Thrombus Analogs

BackgroundExtracellular DNA traps (ETs) have important implications in both thrombosis and thrombolysis. Thus, developing benchtop thrombus analogs that recapitulate clinical ETs is potentially of great value for preclinical development and...

Impedance-based sensors discriminate among different types of blood thrombi with very high specificity and sensitivity

BackgroundIntracranial occlusion recanalization fails in 20% of endovascular thrombectomy procedures, and thrombus composition is likely to be an important factor. In this study, we demonstrate that the combination of electrical...

Metagenomics Analysis of Thrombus Samples Retrieved from Mechanical Thrombectomy.

PurposeThe purpose of this study was to assess the microbiota in middle cerebral artery thrombi retrieved in mechanical thrombectomy arising out of symptomatic carotid plaque within 6 hours of acute ischemic stroke. Thrombi were subjected to next-generation sequencing for a bacterial signature to determine their role in atherosclerosis.Materials and MethodsWe included 4 human middle cerebral artery thrombus samples (all patients were male). The median age for the patients was 51±13.6 years. Patients enrolled in the study from Pacific Medical University and Hospital underwent mechanical thrombectomy in the stroke window period. All patients underwent brain magnetic resonance angiography (MRA) and circle of Willis and neck vessel MRA along with the standard stroke workup to establish stroke etiology. Only patients with symptomatic carotid stenosis and tandem lesions with ipsilateral middle cerebral artery occlusion were included in the study. Thrombus samples were collected, stored at –80 degrees, and subjected to metagenomics analysis.ResultsOf the 4 patients undergoing thrombectomy for diagnosis with ischemic stroke, all thrombi recovered for bacterial DNA in qPCR were positive. More than 27 bacteria were present in the 4 thrombus samples. The majority of bacteria were Lactobacillus, Stenotrophomonas, Pseudomonas, Staphylococcus, and Finegoldia.ConclusionGenesis of symptomatic atherosclerotic carotid plaque leading to thromboembolism could be either due to direct mechanisms like acidification and local inflammation of plaque milieu with lactobacillus, biofilm dispersion leading to inflammation like with pseudomonas fluorescence, or enterococci or indirect mechanisms like Toll 2 like signaling by gut microbiota.

Antiplatelet effect of cudraxanthone B is related to inhibition of calcium mobilization, \u03b1IIb\u03b23 activation, and clot retraction

Cudrania tricuspidata (C. tricuspidata) is widespread throughout Asia and has known to have various physiological activities such as, inflammation, diabetes, obesity and tumor. Cudrania tricuspidata, a rich source of xanthones and flavonoids, have been investigated phytochemically and biologically. However, research of these compounds on platelets is limited. Therefore, we searched for a new substance from various xanthones and flavonoids in C. tricuspidata. We confirmed the results that steppogenin and isoderrone suppress human platelets among the various components isolated from C. tricuspidata, and as a result of analyzing the antiplatelet effect using additional new samples, we found that cudraxanthone B (CXB) has the effect of suppressing human platelets. Therefore, we studied the potential efficacies of CXB on human platelet aggregation and its inhibitory mechanism. Inhibitory effects of CXB on platelet aggregation were assessed using washed platelets, followed by measurement of [Ca2+]i mobilization and dense granule release, fibrinogen binding, fibronectin adhesion assay, and clot retraction. Our data showed that CXB suppressed collagen-induced human platelet aggregation, [Ca2+]i mobilization, fibrinogen binding, fibronectin adhesion and clot retraction without cytotoxicity. Thus, our results show that inhibitory effects of CXB on human platelet activation and thrombus formation, suggesting its potential use as a natural substance for preventing platelet-induced thrombosis.

The resistance of swine blood clots to alteplase-induced thrombolysis in vitro is concentration-dependent

BackgroundSwine have been used as a large animal translational research model to investigate the effectiveness of fibrinolytic agents. However, swine thrombi have different characteristics than human thrombi, which may confer more resistance to fibrinolysis. MethodsIn this study, we performed an in-vitro clot lysis assay to compare both human and swine blood clots lysis induced by alteplase, a recombinant tissue plasminogen-activator agent. Human and swine whole blood were allowed to clot for 3 ​h at 37 ​°C. Increasing concentrations of alteplase (250–580,000 IU/mL) or phosphate-buffered saline (PBS, pH 7.4) were added into the clots. Clot lysis was determined by calculating the difference between the clot mass pre- and post-lysis. ResultsAt low alteplase concentrations (250–2000 IU/mL) we observed significantly less swine blood clot lysis (14 ​± ​1.7% - 35 ​± ​4.9%) compared to the lysis found to human blood clots (52 ​± ​4.8% - 68 ​± ​3.3%, ∗p ​< ​0.05). In contrast, we did not find lysis differences between human and swine clots at higher alteplase concentrations (5000–580,000 IU/mL). ConclusionsIn conclusion, our results suggest that the swine clot resistance to alteplase-induced thrombolysis is concentration-dependent. A high concentration of alteplase allows equivalent thrombolysis of swine and human blood clots in vitro.

Abstract 1: Optical Coherence Tomography Imaging in Acute Ischemic Stroke: Preliminary Animal and Human Results

Background: Studies evaluating endothelial injury after EVT have been done by means of retrieved human thrombus, MR vessel-wall imaging, and animal histopathologic studies. These techniques have limitations, as MR imaging has insufficient spatial resolution to directly visualize endothelium, and histopathologic examinations are ex-vivo and unable to provide real-time patterns of injury. Objective: Endovascular imaging after EVT using optical coherence tomography (OCT) to examine for vessel injury in real-time. Methodology: Three swine weighing 35-40kg were selected for the animal model. Autologous venous whole-blood was used to create thrombus. A second-generation stent retriever was used for EVT. Next, three consecutive patients with basilar artery occlusion underwent EVT and endovascular OCT imaging. Results: In the animal model, revascularization and OCT imaging was successful for all 9 vessels. Endothelial injury was observed in 4/9 (44%) of vessels, and residual thrombus was observed in 4/9 (44%) of vessels despite complete angiographic revascularization. All vessels undergoing EVT after 6 hours had evidence of endothelial injury, and 2/3 (66%) had residual thrombus. Two basilar stroke patients (2/3) 66% had significant residual thrombus despite complete angiographic revascularization. The residual thrombus was also not visible on CT angiography or MR imaging done within 24 hours of EVT. Conclusions: Endothelial injury and residual thrombus despite complete revascularization is present after EVT and can be observed in real-time using OCT. It is possible that the longer occlusive thrombus is present, the more endothelial injury will occur during EVT.