Puncture Wound Hemostasis and Preparation of Samples for Montaged Wide-Area Electron Microscopy Analysis.

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds from time to time. In hemostasis, self-limiting platelet aggregation leads to the formation of a structured thrombus in which bleeding cessation comes from capping the hole from the outside. Detailed characterization of this structure could lead to distinctions between hemostasis and thrombosis, a case of excessive platelet aggregation leading to occlusive clotting. An imaging-based approach to puncture wound thrombus structure is presented here that draws upon the ability of thin-section electron microscopy to visualize the interior of hemostatic thrombi. The most basic step in any imaging-based experimental protocol is good sample preparation. The protocol provides detailed procedures for preparing puncture wounds and platelet-rich thrombi in mice for subsequent electron microscopy. A detailed procedure is given for in situ fixation of the forming puncture wound thrombus and its subsequent processing for staining and embedding for electron microscopy. Electron microscopy is presented as the end imaging technique because of its ability, when combined with sequential sectioning, to visualize the details of the thrombus interior at high resolution. As an imaging method, electron microscopy gives unbiased sampling and an experimental output that scales from nanometer to millimeters in 2 or 3 dimensions. Appropriate freeware electron microscopy software is cited that will support wide-area electron microscopy in which hundreds of frames can be blended to give nanometer-scale imaging of entire puncture wound thrombi cross-sections. Hence, any subregion of the image file can be placed easily into the context of the full cross-section.

Analysis of fibrin networks using topological data analysis - a feasibility study.

Blood clot formation, a crucial process in hemostasis and thrombosis, has garnered substantial attention for its implications in various medical conditions. Microscopic examination of blood clots provides vital insights into their composition and structure, aiding in the understanding of clot pathophysiology and the development of targeted therapeutic strategies. This study explores the use of topological data analysis (TDA) to assess plasma clot characteristics microscopically, focusing on the identification of the elements components, holes and Wasserstein distances. This approach should enable researchers to objectively classify fibrin networks based on their topologic architecture. We tested this mathematical characterization approach on plasma clots formed in static conditions from porcine and human citrated plasma samples, where the effect of dilution and direct thrombin inhibition was explored. Confocal microscopy images showing fluorescence labeled fibrin networks were analyzed. Both treatments resulted in visual differences in plasma clot architecture, which could be quantified using TDA. Significant differences between baseline and diluted samples, as well as blood anticoagulated with argatroban, were detected mathematically. Therefore, TDA could be indicative of clots with compromised stability, providing a valuable tool for thrombosis risk assessment. In conclusion, microscopic examination of plasma clots, coupled with Topological Data Analysis, offers a promising avenue for comprehensive characterization of clot microstructure. This method could contribute to a deeper understanding of clot pathophysiology and thereby refine our ability to assess clot characteristics.

Aortic Valve Thrombus, Stroke, and Endovascular Thrombectomy in a Child With APML and Trisomy 21.

APML, a subtype of acute myeloid leukemia, is highly curable, with cure rates over 90%. Despite its therapeutic success, APML poses elevated bleeding risks due to frequent prior disseminated intravascular coagulation. Less commonly recognized but critical is the thrombotic risk. We document a unique pediatric case: a 13-year-old with trisomy 21 diagnosed with APML had an asymptomatic aortic valve thrombus leading to thromboembolic arterial ischemic stroke. Through endovascular thrombectomy, cerebral circulation was re-established, extracting a fibrin thrombus with APML cells. Neurological recovery was swift. This report underscores the importance of vigilance for thrombotic complications in APML, highlighting the potential severity of overlooked risks.

The role of asymmetry and volume of thrombotic masses in the formation of local deformation of the aneurysmal-altered vascular wall: An in vivo study and mathematical modeling.

Currently, the primary factor indicating the necessity of an operation for an abdominal aortic aneurysm (AAA) is the diameter at its widest part. However, in practice, a large number of aneurysm ruptures occur before reaching a critical size. This means that the mechanics of aneurysm growth and remodeling have not been fully elucidated. This study presents a novel method for assessing the elastic properties of an aneurysm using an ultrasound technique based on tracking the oscillations of the vascular wall as well as the inner border of the thrombus. Twenty nine patients with AAA and eighteen healthy volunteers were considered. The study presents the stratification of a group of patients according to the elastic properties of the aneurysm, depending on the relative volume of intraluminal thrombus masses. Additionally, the neural network analysis of CT angiography images of these patients shows direct (r = 0.664271) correlation with thrombus volume according to ultrasound data, the reliability of the Spearman correlation is p = 0.000215. The use of finite element numerical analysis made it possible to reveal the mechanism of the negative impact on the AAA integrity of an asymmetrically located intraluminal thrombus. The aneurysm itself is considered as a complex structure consisting of a wall, intraluminal thrombus masses, and areas of calcification. When the thrombus occupies > 70% of the lumen of the aneurysm, the deformations of the outer and inner surfaces of the thrombus have different rates, leading to tensile stresses in the thrombus. This poses a risk of its detachment and subsequent thromboembolism or the rupture of the aneurysm wall. This study is the first to provide a mechanistic explanation for the effects of an asymmetrical intraluminal thrombus in an abdominal aortic aneurysm. The obtained results will help develop more accurate risk criteria for AAA rupture using non-invasive conventional diagnostic methods.

Cathepsin K deficiency prevented stress-related thrombosis in a mouse FeCl3 model.

BACKGROUND: Exposure to chronic psychological stress (CPS) is a risk factor for thrombotic cardiocerebrovascular diseases (CCVDs). The expression and activity of the cysteine cathepsin K (CTSK) are upregulated in stressed cardiovascular tissues, and we investigated whether CTSK is involved in chronic stress-related thrombosis, focusing on stress serum-induced endothelial apoptosis. METHODS AND RESULTS: Eight-week-old wild-type male mice (CTSK+/+) randomly divided to non-stress and 3-week restraint stress groups received a left carotid artery iron chloride3 (FeCl3)-induced thrombosis injury for biological and morphological evaluations at specific timepoints. On day 21 post-stress/injury, the stress had enhanced the arterial thrombi weights and lengths, in addition to harmful alterations of plasma ADAMTS13, von Willebrand factor, and plasminogen activation inhibitor-1, plus injured-artery endothelial loss and CTSK protein/mRNA expression. The stressed CTSK+/+ mice had increased levels of injured arterial cleaved Notch1, Hes1, cleaved caspase8, matrix metalloproteinase-9/-2, angiotensin type 1 receptor, galactin3, p16IN4A, p22phox, gp91phox, intracellular adhesion molecule-1, TNF-α, MCP-1, and TLR-4 proteins and/or genes. Pharmacological and genetic inhibitions of CTSK ameliorated the stress-induced thrombus formation and the observed molecular and morphological changes. In cultured HUVECs, CTSK overexpression and silencing respectively increased and mitigated stressed-serum- and H2O2-induced apoptosis associated with apoptosis-related protein changes. Recombinant human CTSK degraded γ-secretase substrate in a dose-dependent manor and activated Notch1 and Hes1 expression upregulation. CONCLUSIONS: CTSK appeared to contribute to stress-related thrombosis in mice subjected to FeCl3 stress, possibly via the modulation of vascular inflammation, oxidative production and apoptosis, suggesting that CTSK could be an effective therapeutic target for CPS-related thrombotic events in patients with CCVDs.

Histidine-Rich Glycoprotein Modulates the Toxic Effects of High-Dose Polyphosphate in Mice.

BACKGROUND: Polyphosphate (polyP), a procoagulant released from platelets, activates coagulation via the contact system and modulates cardiomyocyte viability. High-dose intravenous polyP is lethal in mice, presumably because of thrombosis. Previously, we showed that HRG (histidine-rich glycoprotein) binds polyP and attenuates its procoagulant effects. In this study, we investigated the mechanisms responsible for the lethality of intravenous polyP in mice and the impact of HRG on this process. METHODS: The survival of wild-type or HRG-deficient mice given intravenous synthetic or platelet-derived polyP in doses up to 50 mg/kg or saline was compared. To determine the contribution of thrombosis, the effect of FXII (factor XII) knockdown or enoxaparin on polyP-induced fibrin deposition in the lungs was examined. To assess cardiotoxicity, the ECG was continuously monitored, the levels of troponin I and the myocardial band of creatine kinase were quantified, and the viability of a cultured murine cardiomyocyte cell line exposed to polyP in the absence or presence of HRG was determined. RESULTS: In HRG-deficient mice, polyP was lethal at 30 mg/kg, whereas it was lethal in wild-type mice at 50 mg/kg. Although FXII knockdown or enoxaparin administration attenuated polyP-induced fibrin deposition in the lungs, neither affected mortality. PolyP induced dose-dependent ECG abnormalities, including heart block and ST-segment changes, and increased the levels of troponin and myocardial band of creatine kinase, effects that were more pronounced in HRG-deficient mice than in wild-type mice and were attenuated when HRG-deficient mice were given supplemental HRG. Consistent with its cardiotoxicity, polyP reduced the viability of cultured cardiomyocytes in a dose-dependent manner, an effect attenuated with supplemental HRG. CONCLUSIONS: High-dose intravenous polyP is cardiotoxic in mice, and HRG modulates this effect.

An age-progressive platelet differentiation path from hematopoietic stem cells causes exacerbated thrombosis.

Platelet dysregulation is drastically increased with advanced age and contributes to making cardiovascular disorders the leading cause of death of elderly humans. Here, we reveal a direct differentiation pathway from hematopoietic stem cells into platelets that is progressively propagated upon aging. Remarkably, the aging-enriched platelet path is decoupled from all other hematopoietic lineages, including erythropoiesis, and operates as an additional layer in parallel with canonical platelet production. This results in two molecularly and functionally distinct populations of megakaryocyte progenitors. The age-induced megakaryocyte progenitors have a profoundly enhanced capacity to engraft, expand, restore, and reconstitute platelets in situ and upon transplantation and produce an additional platelet population in old mice. The two pools of co-existing platelets cause age-related thrombocytosis and dramatically increased thrombosis in vivo. Strikingly, aging-enriched platelets are functionally hyper-reactive compared with the canonical platelet populations. These findings reveal stem cell-based aging as a mechanism for platelet dysregulation and age-induced thrombosis.

Mechanisms of Pulmonary Vasculopathy in Acute and Long-Term COVID-19: A Review.

Despite the end of the pandemic, coronavirus disease 2019 (COVID-19) remains a major public health concern. The first waves of the virus led to a better understanding of its pathogenesis, highlighting the fact that there is a specific pulmonary vascular disorder. Indeed, COVID-19 may predispose patients to thrombotic disease in both venous and arterial circulation, and many cases of severe acute pulmonary embolism have been reported. The demonstrated presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within the endothelial cells suggests that direct viral effects, in addition to indirect effects of perivascular inflammation and coagulopathy, may contribute to pulmonary vasculopathy in COVID-19. In this review, we discuss the pathological mechanisms leading to pulmonary vascular damage during acute infection, which appear to be mainly related to thromboembolic events, an impaired coagulation cascade, micro- and macrovascular thrombosis, endotheliitis and hypoxic pulmonary vasoconstriction. As many patients develop post-COVID symptoms, including dyspnea, we also discuss the hypothesis of pulmonary vascular damage and pulmonary hypertension as a sequela of the infection, which may be involved in the pathophysiology of long COVID.

The Etiology of the Thrombotic Phenomena Involved in the Process of Coronary Artery Disease-What Is the Role of Thrombophilic Genes in the Development of This Pathology?

Cardiovascular diseases, among which includes coronary artery disease, represent one of the most important causes of mortality and morbidity worldwide. Research aimed at determining the risk factors involved recognizes a group of "traditional" risk factors, but also more recent studies identified over 100 "novel" ones which may have a role in the disease. Among the latter is the thrombophilia profile of a patient, a pathology well-established for its involvement in venous thromboembolism, but with less studied implications in arterial thrombosis. This paper reviews the literature, explaining the pathophysiology of the thrombophilia causes associated most with coronary thrombosis events. Results of several studies on the subject, including a meta-analysis with over 60,000 subjects, determined the significant involvement of factor V Leiden, prothrombin G20210A mutation, plasminogen activator inhibitor-1 and antiphospholipid syndrome in the development of coronary artery disease. The mechanisms involved are currently at different stages of research, with some already established and used as therapeutic targets.

The Role of TLR-2 in Lethal COVID-19 Disease Involving Medullary and Resident Lung Megakaryocyte Up-Regulation in the Microthrombosis Mechanism.

Patients with COVID-19 have coagulation and platelet disorders, with platelet alterations and thrombocytopenia representing negative prognostic parameters associated with severe forms of the disease and increased lethality. METHODS: The aim of this study was to study the expression of platelet glycoprotein IIIa (CD61), playing a critical role in platelet aggregation, together with TRL-2 as a marker of innate immune activation. RESULTS: A total of 25 patients were investigated, with the majority (24/25, 96%) having co-morbidities and dying from a fatal form of SARS-CoV-2(+) infection (COVID-19+), with 13 men and 12 females ranging in age from 45 to 80 years. When compared to a control group of SARS-CoV-2 (-) negative lungs (COVID-19-), TLR-2 expression was up-regulated in a subset of patients with deadly COVID-19 fatal lung illness. The proportion of Spike-1 (+) patients found by PCR and ISH correlates to the proportion of Spike-S1-positive cases as detected by digital pathology examination. Furthermore, CD61 expression was considerably higher in the lungs of deceased patients. In conclusion, we demonstrate that innate immune prolonged hyperactivation is related to platelet/megakaryocyte over-expression in the lung. CONCLUSIONS: Microthrombosis in deadly COVID-19+ lung disease is associated with an increase in the number of CD61+ platelets and megakaryocytes in the pulmonary interstitium, as well as their functional activation; this phenomenon is associated with increased expression of innate immunity TLR2+ cells, which binds the SARS-CoV-2 E protein, and significantly with the persistence of the Spike-S1 viral sequence.

Material strengths of shear-induced platelet aggregation clots and coagulation clots.

Arterial occlusion by thrombosis is the immediate cause of some strokes, heart attacks, and peripheral artery disease. Most prior studies assume that coagulation creates the thrombus. However, a contradiction arises as whole blood (WB) clots from coagulation are too weak to stop arterial blood pressures (> 150 mmHg). We measure the material mechanical properties of elasticity and ultimate strength for Shear-Induced Platelet Aggregation (SIPA) type clots, that form under stenotic arterial hemodynamics in comparison with coagulation clots. The ultimate strength of SIPA clots averaged 4.6 ± 1.3 kPa, while WB coagulation clots had a strength of 0.63 ± 0.3 kPa (p < 0.05). The elastic modulus of SIPA clots was 3.8 ± 1.5 kPa at 1 Hz and 0.5 mm displacement, or 2.8 times higher than WB coagulation clots (1.3 ± 1.2 kPa, p < 0.0001). This study shows that the SIPA thrombi, formed quickly under high shear hemodynamics, is seven-fold stronger and three-fold stiffer compared to WB coagulation clots. A force balance calculation shows a SIPA clot has the strength to resist arterial pressure with a short length of less than 2 mm, consistent with coronary pathology.

Myocardial ischemia-reperfusion injury released cellular fibronectin containing domain A (CFN-EDA): A destructive positive loop amplifying arterial thrombosis formation and exacerbating myocardial reperfusion injury.

Previous research has identified intravascular platelet thrombi in regions affected by myocardial ischemia-reperfusion (MI/R) injury and neighbouring areas. However, the occurrence of arterial thrombosis in the context of MI/R injury remains unexplored. This study utilizes intravital microscopy to investigate carotid artery thrombosis during MI/R injury in rats, establishing a connection with the presence of prothrombotic cellular fibronectin containing extra domain A (CFN-EDA) protein. Additionally, the study examines samples from patients with coronary artery disease (CAD) both before and after coronary artery bypass grafting (CABG). Levels of CFN-EDA significantly increase following MI with further elevation observed following reperfusion of the ischemic myocardium. Thrombotic events, such as thrombus formation and growth, show a significant increase, while the time to complete cessation of blood flow in the carotid artery significantly decreases following MI/R injury induced by ferric chloride. The acute infusion of purified CFN-EDA protein accelerates in-vivo thrombotic events in healthy rats and significantly enhances in-vitro adenosine diphosphate and collagen-induced platelet aggregation. Treatment with anti-CFN-EDA antibodies protected the rat against MI/R injury and significantly improved cardiac function as evidenced by increased end-systolic pressure-volume relationship slope and preload recruitable stroke work compared to control. Similarly, in a human study, plasma CFN-EDA levels were notably elevated in CAD patients undergoing CABG. Post-surgery, these levels continued to rise over time, alongside cardiac injury biomarkers such as cardiac troponin and B-type natriuretic peptide. The study highlights that increased CFN-EDA due to CAD or MI initiates a destructive positive feedback loop by amplifying arterial thrombus formation, potentially exacerbating MI/R injury.

Elevated plasma levels of factor VIII enhance arterial thrombus formation on erosive smooth muscle cell-rich neointima but not normal intima in rabbits.

BACKGROUND: Plaque erosion, a type of coronary atherothrombosis, involves superficial injury to smooth muscle cell (SMC)-rich plaques. Elevated levels of coagulation factor VIII (FVIII) correlate with an increased ischemic heart disease risk. FVIII may contribute to thrombus formation on eroded plaques. AIMS: We aimed to elucidate the role of elevated FVIII in arterial thrombus formation within SMC-rich neointima in rabbits. METHODS AND RESULTS: We assessed the effect of recombinant human FVIII (rFVIII) on blood coagulation in vitro and platelet aggregation ex vivo. An SMC-rich neointima was induced through balloon injury to the unilateral femoral artery. Three weeks after the first balloon injury, superficial erosive injury and thrombus formation were initiated with a second balloon injury of the bilateral femoral arteries 45 min after the administration of rFVIII (100 IU/kg) or saline. The thrombus area and contents were histologically measured 15 min after the second balloon injury. rFVIII administration reduced the activated partial thromboplastin time and augmented botrocetin-induced, but not collagen- or adenosine 5'-diphosphate-induced, platelet aggregation. While rFVIII did not influence platelet-thrombus formation in normal intima, it increased thrombus formation on SMC-rich neointima post-superficial erosive injury. Enhanced immunopositivity for glycoprotein IIb/IIIa and fibrin was observed in rFVIII-administered SMC-rich neointima. Neutrophil count in the arterial thrombus on the SMC-rich neointima correlated positively with thrombus size in the control group, unlike the rFVIII group. CONCLUSIONS: Increased FVIII contributes to thrombus propagation within erosive SMC-rich neointima, highlighting FVIII's potential role in plaque erosion-related atherothrombosis.

Multimaterial decomposition in dual-energy CT for characterization of clots from acute ischemic stroke patients.

BACKGROUND: Nowadays, there is no method to quantitatively characterize the material composition of acute ischemic stroke thrombi prior to intervention, but dual-energy CT (DE-CT) offers imaging-based multimaterial decomposition. We retrospectively investigated the material composition of thrombi ex vivo using DE-CT with histological analysis as a reference. METHODS: Clots of 70 patients with acute ischemic stroke were extracted by mechanical thrombectomy and scanned ex vivo in formalin-filled tubes with DE-CT. Multimaterial decomposition in the three components, i.e., red blood cells (RBC), white blood cells (WBC), and fibrin/platelets (F/P), was performed and compared to histology (hematoxylin/eosin staining) as reference. Attenuation and effective Z values were assessed, and histological composition was compared to stroke etiology according to the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) criteria. RESULTS: Histological and imaging analysis showed the following correlation coefficients for RBC (r = 0.527, p < 0.001), WBC (r = 0.305, p = 0.020), and F/P (r = 0.525, p < 0.001). RBC-rich thrombi presented higher clot attenuation in Hounsfield units than F/P-rich thrombi (51 HU versus 42 HU, p < 0.01). In histological analysis, cardioembolic clots showed less RBC (40% versus 56%, p = 0.053) and more F/P (53% versus 36%, p = 0.024), similar to cryptogenic clots containing less RBC (34% versus 56%, p = 0.006) and more F/P (58% versus 36%, p = 0.003) than non-cardioembolic strokes. No difference was assessed for the mean WBC portions in all TOAST groups. CONCLUSIONS: DE-CT has the potential to quantitatively characterize the material composition of ischemic stroke thrombi. RELEVANCE STATEMENT: Using DE-CT, the composition of ischemic stroke thrombi can be determined. Knowledge of histological composition prior to intervention offers the opportunity to define personalized treatment strategies for each patient to accomplish faster recanalization and better clinical outcomes. KEY POINTS: • Acute ischemic stroke clots present different recanalization success according to histological composition. • Currently, no method can determine clot composition prior to intervention. • DE-CT allows quantitative material decomposition of thrombi ex vivo in red blood cells, white blood cells, and fibrin/platelets. • Histological clot composition differs between stroke etiology. • Insights into the histological composition in situ offer personalized treatment strategies.

Fitusiran reduces bleeding in factor X-deficient mice.

ABSTRACT: Factor X (FX) deficiency is a rare bleeding disorder manifesting a bleeding tendency caused by low FX activity levels. We aim to explore the use of fitusiran (an investigational small interfering RNA that silences antithrombin expression) to increase thrombin generation and the in vivo hemostatic potential under conditions of FX deficiency. We therefore developed a novel model of inducible FX deficiency, generating mice expressing <1% FX activity and antigen (f10low mice). Compared with control f10WT mice, f10low mice had sixfold and fourfold prolonged clotting times in prothrombin time and activated partial prothrombin time assays, respectively (P < .001). Thrombin generation was severely reduced, irrespective of whether tissue factor or factor XIa was used as an initiator. In vivo analysis revealed near-absent thrombus formation in a laser-induced vessel injury model. Furthermore, in 2 distinct bleeding models, f10low mice displayed an increased bleeding tendency compared with f10WT mice. In the tail-clip assay, blood loss was increased from 12 ± 16 µL to 590 ± 335 µL (P < .0001). In the saphenous vein puncture (SVP) model, the number of clots generated was reduced from 19 ± 5 clots every 30 minutes for f10WT mice to 2 ± 2 clots every 30 minutes (P < .0001) for f10low mice. In both models, bleeding was corrected upon infusion of purified FX. Treatment of f10low mice with fitusiran (2 × 10 mg/kg at 1 week interval) resulted in 17 ± 6% residual antithrombin activity and increased thrombin generation (fourfold and twofold to threefold increase in endogenous thrombin potential and thrombin peak, respectively). In the SVP model, the number of clots was increased to 8 ± 6 clots every 30 minutes (P = .0029). Altogether, we demonstrate that reduction in antithrombin levels is associated with improved hemostatic activity under conditions of FX deficiency.

Predominance of M2 macrophages in organized thrombi in chronic thromboembolic pulmonary hypertension patients.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a debilitating disease characterized by thrombotic occlusion of pulmonary arteries and vasculopathy, leading to increased pulmonary vascular resistance and progressive right-sided heart failure. Thrombotic lesions in CTEPH contain CD68+ macrophages, and increasing evidence supports their role in disease pathogenesis. Macrophages are classically divided into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages, which are involved in wound healing and tissue repair. Currently, the phenotype of macrophages and their localization within thrombotic lesions of CTEPH are largely unknown. In our study, we subclassified thrombotic lesions of CTEPH patients into developing fresh thrombi (FT) and organized thrombi (OT), based on the degree of fibrosis and remodeling. We used multiplex immunofluorescence histology to identify immune cell infiltrates in thrombotic lesions of CPTEH patients. Utilizing software-assisted cell detection and quantification, increased proportions of macrophages were observed in immune cell infiltrates of OT lesions, compared with FT. Strikingly, the proportions with a CD206+INOS- M2 phenotype were significantly higher in OT than in FT, which mainly contained unpolarized macrophages. Taken together, we observed a shift from unpolarized macrophages in FT toward an expanded population of M2 macrophages in OT, indicating a dynamic role of macrophages during CTEPH pathogenesis.

Disturbed flow in the juxta-anastomotic area of an arteriovenous fistula correlates with endothelial loss, acute thrombus formation, and neointimal hyperplasia.

Clinical failure of arteriovenous neointimal hyperplasia (NIH) fistulae (AVF) is frequently due to juxta-anastomotic NIH (JANIH). Although the mouse AVF model recapitulates human AVF maturation, previous studies focused on the outflow vein distal to the anastomosis. We hypothesized that the juxta-anastomotic area (JAA) has increased NIH compared with the outflow vein. AVF was created in C57BL/6 mice without or with chronic kidney disease (CKD). Temporal and spatial changes of the JAA were examined using histology and immunofluorescence. Computational techniques were used to model the AVF. RNA-seq and bioinformatic analyses were performed to compare the JAA with the outflow vein. The jugular vein to carotid artery AVF model was created in Wistar rats. The neointima in the JAA shows increased volume compared with the outflow vein. Computational modeling shows an increased volume of disturbed flow at the JAA compared with the outflow vein. Endothelial cells are immediately lost from the wall contralateral to the fistula exit, followed by thrombus formation and JANIH. Gene Ontology (GO) enrichment analysis of the 1,862 differentially expressed genes (DEG) between the JANIH and the outflow vein identified 525 overexpressed genes. The rat jugular vein to carotid artery AVF showed changes similar to the mouse AVF. Disturbed flow through the JAA correlates with rapid endothelial cell loss, thrombus formation, and JANIH; late endothelialization of the JAA channel correlates with late AVF patency. Early thrombus formation in the JAA may influence the later development of JANIH.NEW & NOTEWORTHY Disturbed flow and focal endothelial cell loss in the juxta-anastomotic area of the mouse AVF colocalizes with acute thrombus formation followed by late neointimal hyperplasia. Differential flow patterns between the juxta-anastomotic area and the outflow vein correlate with differential expression of genes regulating coagulation, proliferation, collagen metabolism, and the immune response. The rat jugular vein to carotid artery AVF model shows changes similar to the mouse AVF model.