Induction Of Thrombosis Research Articles

Compare efficacy and safety of autologous blood clot injection with C-clamp vascular closure device in treatment of iatrogenic pseudoaneurysm after femoral artery puncture.

Iatrogenic femoral artery pseudoaneurysm (FAP) is a complication following femoral artery puncture, with an incidence rate of 0.2%-5.5% post-cardiac catheterization procedures. Management typically involves observation or interventional treatments such as ultrasound-guided compression (UGC) or ultrasound-guided thrombin injections. This study compares the efficacy and safety of ultrasound-guided autologous blood clot injection (UGCI) with C-clamp vascular closure device-assisted UGC in treating FAP. Conducted at a high-volume training and research center, this prospective study enrolled patients with iatrogenic FAP post-femoral artery puncture. Patients were randomized into two treatments: UGC with a C-clamp device or UGCI. Primary endpoints were thrombosis induction within 24 h, average procedure time, and length of hospital stay (LOS). The study included 105 patients with 51 undergoing UGCI and 54 undergoing UGC. UGCI achieved a primary success rate of 96% versus 63% for UGC. The mean procedure duration for UGCI was significantly shorter (22 min) compared with UGC (49 min, p ⩽ 0.0001). LOS was also reduced in the UGCI group (1.5 days) compared to the UGC group (4.5 days). Complication rates were low for both procedures, with one deep vein thrombosis and one infection observed in the UGC group. UGCI demonstrated to be a safe, efficient, and faster alternative for treating iatrogenic FAPs with a higher success rate, reduced LOS, and similar low complication rates compared to UGC. This study suggests that UGCI could be considered a preferred method for the management of FAP post-femoral artery catheterization.

Extracellular RNA Induces Neutrophil Recruitment Via Toll-Like Receptor 3 During Venous Thrombosis After Vascular Injury.

Venous thromboembolism is associated with endothelial cell activation that contributes to the inflammation-dependent activation of the coagulation system. Cellular damage is associated with the release of different species of extracellular RNA (eRNA) involved in inflammation and coagulation. TLR3 (toll-like receptor 3), which recognizes (viral) single-stranded or double-stranded RNAs and self-RNA fragments, might be the receptor of these species of eRNA during venous thromboembolism. Here, we investigate how the TLR3/eRNA axis contributes to venous thromboembolism. Thrombus formation and size in wild-type and TLR3 deficient (-/-) mice were monitored by ultrasonography after venous thrombosis induction using the ferric chloride and stasis models. Mice were treated with RNase I, with polyinosinic-polycytidylic acid, a TLR3 agonist, or with RNA extracted from murine endothelial cells. Gene expression and signaling pathway activation were analyzed in HEK293T cells overexpressing TLR3 in response to eRNA or in human umbilical vein endothelial cells transfected with a small interference RNA against TLR3. Plasma clot formation on treated human umbilical vein endothelial cells was analyzed. Thrombosis exacerbated eRNA release invivo and increased eRNA content within the thrombus. RNase I treatment reduced thrombus size compared with vehicle-treated mice (P<0.05). Polyinosinic-polycytidylic acid and eRNA treatments increased thrombus size in wild-type mice (P<0.01 and P<0.05), but not in TLR3-/- mice, by reinforcing neutrophil recruitment (P<0.05). Mechanistically, TLR3 activation in endothelial cells promotes CXCL5 (C-X-C motif chemokine 5) secretion (P<0.001) and NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation (P<0.05). Finally, eRNA triggered plasma clot formation invitro (P<0.01). We show that eRNA and TLR3 activation enhance venous thromboembolism through neutrophil recruitment possibly through secretion of CXCL5, a potent neutrophil chemoattractant.

Abstract 2094: Sex Differences In Venous Thrombus Resolution In Mice With Metabolic Syndrome

Introduction: Metabolic syndrome (MS) is highly prevalent and have been shown to be associated with mitochondrial impairment and increased cardiovascular and thrombotic risk. It is less understood how MS impacts venous thrombosis and thrombus resolution and the effect of sex as a biological variable on these processes. We hypothesize that mice with MS will have impaired thrombus resolution, particularly males compared to females. Methods: We used a model of diet-independent MS where wildtype FVB mice that have been born to high fat diet fed mothers exhibit multiple organ manifestations of MS. 29 adult mice underwent surgical induction of deep vein thrombosis (DVT) via inferior vena cava ligation (stasis model) resulting in thrombi formation until day 3 and thrombus resolution until day 7. Immunohistochemistry was used to assess vascular tissue remodeling. Results: Comparing control mice to mice born to obese mothers after DVT, there were no significant differences in thrombus weights on day 3 (1.83±.62 vs 1.48±.53 g/kg, p =.186), suggesting a similar initial thrombus burden. However, mice born to obese mothers had significantly impaired thrombus resolution at day 7 (1.85±.53 vs 1.14±.42 g/kg, Student’s t-test p =.007) as compared to control mice born from mothers fed a regular diet. Importantly, thrombus weights were significantly larger in male offspring of obese mothers as compared to females (2.17±.67 vs 1.43±.26 g/kg, Student’s t test p =.013). Additionally, histology analyses showed associations with increased vein wall collagen deposition (Masson’s trichrome) (25±3 vs 13±4% area stained, Student’s t test, p =.0018) and neovascularization (smooth muscle cell actin) within the thrombi of mice with signs of MS as compared to control mice (70+/-10 vs 40+/-8% area stained, Student’s t test, p =.009). Conclusions: Wildtype mice with diet-independent MS demonstrate diminished thrombus resolution, with males having significantly larger thrombi than females. Impaired DVT resolution was associated with decreased tissue remodeling within the thrombi. These data suggest that MS may induce negative tissue remodeling decreasing thrombus involution and increasing vein wall fibrosis, factors that may increase the likelihood of post thrombotic syndrome.

Histological Features of In-Stent Restenosis after Iliac Vein Thrombus Removal and Stent Placement in a Goat Model

PurposeTo establish an animal model for in-stent restenosis (ISR) after postthrombotic iliac vein stent placement and characterize histopathological changes in tissue within the stented vein. Materials and MethodsIliac vein thrombosis was induced using balloon occlusion and thrombin injection in 8 male Boer goats. Mechanical thrombectomy and iliac vein stent placement were performed 3 days after thrombosis induction. Restenosis was evaluated by venography and optical coherence tomography (OCT) at 1 and 8 weeks after stent placement, and stent specimens were taken for pathological examination after the animals were euthanized. ResultsThrombosis induction was successful in all 8 goats, with >80% iliac vein occlusion. After thrombus removal, OCT revealed considerable venous intimal thickening and a small number of mural thrombi. Neointimal hyperplasia with thrombus formation was observed in all goats 1 week after stent implantation; the degree of ISR was 15%–33%. At 8 weeks, the degree of ISR was 21%–32% in 3 goats, and stent occlusion was observed in 1 goat. At 1 week, the neointima predominantly consisted of fresh thrombi. At 8 weeks, proliferplastic fibrotic tissue and smooth muscle cells (SMCs) were predominant, and the stent surfaces were endothelialized in 2 of 3 goats and partially endothelialized in 1 goat. ConclusionsIn the goat model, postthrombotic neointimal hyperplasia in the venous stent may result from time-dependent thrombus formation and organization, accompanied by migration and proliferation of SMCs, causing ISR. These results provide a basis to further explore the mechanism of venous ISR and promote the development of venous stents that reduce neointimal hyperplasia.

Sequential transcriptomic alterations in the cerebral cortex of mice after cerebral venous sinus thrombosis

To investigate the expression alterations of specific genes that occur after venous stroke, we identified differentially expressed genes (DEGs) between sham and damaged cortical tissues at 2 and 7 days after induction of cerebral venous sinus thrombosis (CVST) model. The profiles of DEGs were analyzed using GO, KEGG, GSEA, and PPI, and the crucial gene was further verified by western blot and immunofluorescence. We found 969 and 883 DEGs at 2 and 7 days after CVST, respectively. A marked increase in biological-process categories, such as immune system process and inflammatory response, and a decrease in neuropeptide signaling pathway were observed both at 2 and 7 days post-CVST. The KEGG pathway was enriched to varying degrees on complement and coagulation cascades, cytokine-cytokine receptor interaction, and multiple immune-inflammatory signaling pathways at 2 and 7 days post-CVST, separately. Furthermore, GSEA highlights the potential roles of the NOD-like receptor signaling pathway and cytokine-cytokine receptor interaction in CVST. Importantly, numerous genes related to KEGG pathways above featured prominently in the PPI network analysis, with IL1b being one of the most conspicuous. These time-dependent alterations in gene profiles and enrichment pathways reveal the unique pathophysiological characteristics of CVST and indicate novel therapeutic targets for venous stroke. SignificanceCerebral venous sinus thrombosis (CVST) is an underrated and potentially fatal cause of stroke with a reported mortality of 5–10% worldwide. Currently, in addition to anticoagulant and thrombolytic therapy, effective treatments targeting the injured brain parenchyma after CVST remain limited. Besides, accurate diagnostic markers are still sorely lacking. In the present study, we will detect the transcriptomic alterations of the cerebral cortex of mice post-CVST by RNA-sequencing, screen differentially expressed genes and abnormal pathways through bioinformatics methods, analyze the correlation of these signals and CVST pathology, and finally validate the key molecules through western blot and immunofluorescence assays. Collectively, the study aimed to offer a reference for the discovery of specific genes/pathway alterations in the damaged cortical tissues of CVST mice and further reveal the underlying pathogenesis, thereby providing evidence for the diagnosis and treatment of CVST.

Activation of Platelet mTORC2/Akt Pathway by Anti-β2GP1 Antibody Promotes Thrombosis in Antiphospholipid Syndrome.

Anti-β2GP1 (β2-glycoprotein 1) antibodies are the primary pathogenic antibody to promote thrombosis in antiphospholipid syndrome (APS), yet the underlying mechanism remains obscure. We aimed to explore the intracellular pathway that mediated platelet activation. Platelets were isolated from patients with APS and subjected to RNA sequencing. Platelet aggregation, the release of platelet granules, platelet spreading, and clot retraction were detected to evaluate platelet activation. We purified anti-β2GP1 antibodies from patients with APS and the total IgG from healthy donors to stimulate platelets with/without FcγRIIA (Fcγ receptor IIA) blocking antibody or Akt (protein kinase B) inhibitor. Platelet-specific Sin1 (stress-activated protein kinase-interacting protein) deficiency mice were established. The thrombus model of inferior vena cava flow restriction, ferric chloride-induced carotid injury model, and laser-induced vessel wall injury in cremaster arterioles model were constructed after administration of anti-β2GP1 antibodies. Combined RNA sequencing and bioinformatics analysis suggested that APS platelets exhibited increased levels of mRNA associated with platelet activation, which was in line with the hyperactivation of APS platelets in response to stimuli. Platelet activation in APS platelets was accompanied by upregulation of the mTORC2 (mammalian target of the rapamycin complex 2)/Akt pathway and increased levels of SIN1 phosphorylation at threonine 86. Anti-β2GP1 antibody derived from patients with APS enhanced platelet activation and upregulated the mTORC2/Akt pathway. Moreover, the Akt inhibitor weakened the potentiating effect of the anti-β2GP1 antibody on platelet activation. Notably, Sin1 deficiency suppresses anti-β2GP1 antibody-enhanced platelet activation in vitro and thrombosis in all 3 models. This study elucidated the novel mechanism involving the mTORC2/Akt pathway, which mediates the promotion of platelet activation and induction of thrombosis by the anti-β2GP1 antibody. The findings suggest that SIN1 may be a promising therapeutic target for the treatment of APS.

Endothelial P2X7 promotes venous thromboembolism

Venous thromboembolism (VTE) affects 117 people per 100,000 each year and is an important cause of morbidity and mortality. VTE can lead to 1) death through pulmonary embolism, 2) the post-thrombotic syndrome, or 3) pulmonary sequelae that dramatically impair quality of life. Thrombosis has long been characterized by the Virchow's triad encompassing hypercoagulability, venous stasis and vascular wall damage. However, inflammatory pathways are now well recognized mechanisms involved in the physiopathology of venous thromboembolism. Endothelial cells play critical roles in regulating immune functions in response to PAMPs and DAMPs. During inflammation, adenosine triphosphate (ATP) is released in the extracellular compartment and is recognized as a danger signal by endothelial cells. ATP can interact with the CD39/CD73 system involved in the metabolism of ATP into AMP. Recent data indicated that the CD39/CD73 system might protect against venous thrombosis by downregulating the NLRP3 inflammasome. ATP can also interact with the P2X7 receptor involved in a wide range of responses including NLRP3 inflammasome activation leading to IL1β production. However, the role of P2X7 receptor in VTE is unknown. To determine how the endothelial P2X7 receptor contributes to venous thromboembolism. After pretreatment with TNFα, HUVECs were incubated with BzATP alone or with thrombin. Immunofluorescence, western blot and real-time quantitative PCR analyses were used to study P2X7 expression in endothelial cells, activation of p38 and NFκB signaling pathways and gene expression, respectively. We confirmed that HUVECs expressed P2X7 receptor in vitro and in vivo after induction of venous thrombosis in an experimental model. BzATP and thrombin induced the activation of p38 and NFκB signaling pathways. Interestingly, this was associated with inflammasome assembly and increased IL1β expression. In addition, ICAM-1 and VCAM-1 expression was increased, while thrombomodulin expression was decreased. Our data suggest that ATP released in the extracellular space during venous thrombosis induced inflammasome activation in endothelial cell through P2X7 activation. P2X7 might have a pro-thrombotic role exacerbating venous thromboembolism.

New perspectives on the induction and acceleration of immune-associated thrombosis by PF4 and VWF.

Platelet factor 4 (PF4), also known as chemokine (C-X-C motif) ligand 4 (CXCL4), is a specific protein synthesized from platelet α particles. The combination of PF4 and heparin to form antigenic complexes is an important mechanism in the pathogenesis of heparin-induced thrombocytopenia (HIT), but vaccine-induced immune thrombotic thrombocytopenia (VITT) related to the COVID-19 vaccine makes PF4 a research hotspot again. Similar to HIT, vaccines, bacteria, and other non-heparin exposure, PF4 can interact with negatively charged polyanions to form immune complexes and participate in thrombosis. These anions include cell surface mucopolysaccharides, platelet polyphosphates, DNA from endothelial cells, or von Willebrand factor (VWF). Among them, PF4-VWF, as a new immune complex, may induce and promote the formation of immune-associated thrombosis and is expected to become a new target and therapeutic direction. For both HIT and VITT, there is no effective and targeted treatment except discontinuation of suspected drugs. The research and development of targeted drugs based on the mechanism of action have become an unmet clinical need. Here, this study systematically reviewed the characteristics and pathophysiological mechanisms of PF4 and VWF, elaborated the potential mechanism of action of PF4-VWF complex in immune-associated thrombosis, summarized the current status of new drug research and development for PF4 and VWF, and discussed the possibility of this complex as a potential biomarker for early immune-associated thrombosis events. Moreover, the key points of basic research and clinical evaluation are put forward in the study.

In vivo antithrombotic potential of protease from Bacillus thuringiensis HSFI-12

Background/Aim: Cardiovascular diseases (CVDs) are the primary noncommunicable disease at the global level due to abnormal platelet aggregation by fibrin forming clots in blood vessels called thrombus. The search for thrombolytic drugs is largely carried out to treat thrombosis. Crude extract and dialysate protease of Bacillus thuringiensis HSFI-12 is known to have thrombolytic activity in vitro. The in vivo thrombolytic activity evaluation of concentrated protease of the bacterium is yet to be done. This study aimed to evaluate in vivo thrombolytic activity of concentrated protease produced by ultrafiltration of crude B thuringiensis HSFI-12 protease using Rattus norvegicus as animal model. Methods: Carrageenan was used as thrombosis induction agent in rats. Intravenous injection of B thuringiensis HSFI-12 concentrated protease doses of 75, 150, 300, 600 µg/kg body weight (BW) was administered to rats, then induction of carrageenan was given intravenously to the rats' tails 30 min after injection of B thuringiensis HSFI-12 protease concentrate. The average length of the infarct area in the tail of the rat was shorter in the rats that were given various doses of B thuringiensis HSFI-12 protease concentrate compared to the negative control (rats induced by carrageenan 20 mg/kg BW). Results: The PT examination results showed a prolonged PT time at 300 µg/kg BW dose, while there was at risk of bleeding at 600 µg/kg BW dose. The activated partial thromboplastin time (aPTT) examination results showed that time elongation beyond the normal range did not occur in rats after treatment. The amount leukocytes (WBC) and erythrocytes (RBC) after treatment were within the normal range indicating that they did not affect the haemostasis mechanism, while the platelet count (PLT) assay showed decrease in the number of platelets (thrombocytopenia). However, after treatment the number of platelets (PLT) showed a positive response as seen from an increase in values close to normal range. As conclusion, induction of carrageenan conducted had successfully caused thrombosis in R norvegicus' tail used as the thrombosis model. Conclusion: Concentrated protease of B thuringiensis HSFI-12 showed in vivo antithrombotic potential with an effective dose of based on PT, aPTT and blood count evaluation at 150 µg/kg BW.

The arch remodelling stent for DeBakey I acute aortic dissection: experience with 100 implantations.

A novel hybrid non-covered stent was developed to treat malperfusion and prevent aneurysm formation following hemiarch procedure for DeBakey I acute aortic dissection (AAD). The present analysis investigates the performance of the device in 100 consecutive implantations. Between 2018 and 2021, 100 patients underwent surgical repair of DeBakey I AAD with implantation of a non-covered stent in the arch and descending aorta. The primary entry tear was located in the root or the ascending aorta. Clinical and imaging data were collected and analysed retrospectively. The endpoints of the study were 30-day mortality, neurological outcome and need of additional procedures due to postoperative malperfusion. Technical success was assessed in the first postoperative computed tomography in regard to the induction of false lumen thrombosis in the descending aorta. The median age was 61 (54-73) years. Preoperative malperfusion was present in 46 (46%) patients. The primary arterial cannulation strategy was the right axillary artery and an open distal anastomosis was performed in a median caudal circulatory arrest of 40 (34-52) min. In 48% of cases, a 55-40 tapered stent was implanted. The 30-day mortality was 18%, and the operation-related new postoperative neurological deficit was present in 8%. Technical success was achieved in 76% of patients. The novel non-covered stent can be safely applied to complement aortic repair with the hemiarch procedure for DeBakey I AAD. The expansion of the true lumen through the device may prevent postoperative malperfusion and induces positive vascular remodelling with the thrombosis of the false lumen.

Endothelial P2X7 promotes venous thromboembolism

Venous thromboembolism (VTE) affects 117 people per 100,000 each year and is an important cause of morbidity and mortality. VTE can lead to 1) death through pulmonary embolism, 2) the post-thrombotic syndrome, or 3) chronic pulmonary hypertension resulting in significant chronic respiratory compromise. Inflammatory pathways are intricately involved in thrombus formation and therefore in VTE mechanisms. In pathological conditions, adenosine triphosphate (ATP) is released in the extracellular compartment and is recognized as a danger signal by several cell types including endothelial cells. Recent data indicated that the CD39/CD73 system involved in the metabolism of ATP into AMP might protects against thrombosis by downregulating the pro-inflammatory pathway of the inflammasome. ATP can also interact with the P2X7 receptor involved in inflammation causing a wide range of responses. To determine how the endothelial P2X7 receptor contributes to venous thromboembolism. HUVECs were incubated with BzATP, thrombin or both. HUVECs were primed with TNF-alpha prior to stimulation. We used immunofluorescence, western blot and real-time quantitative PCR analyses to assess P2X7 expression by endothelial cells, activation of p38 and NFκB activation and expression of pro-inflammatory and pro-coagulant genes. We confirmed that endothelial cells expressed P2X7 in vitro in HUVECs and in vivo after induction of venous thrombosis by ligation of the inferior vena cava. Treatment of endothelial cells with BzATP and thrombin induced the activation of p38 and NFκB signaling pathways. This appears to be associated with an increased expression of IL-1β and downregulation of thrombomodulin expression. The expression of adhesion molecules, ICAM-1 and VCAM-1, tends to increase. Our data suggest that ATP released in the extracellular space following cell damage or activation induced a pro-inflammatory response in endothelial cells through P2X7 activation. P2X7 might have a pro-thrombotic role exacerbating venous thromboembolism.

Lipoprotein(a) and its Significance in Cardiovascular Disease

Lipoprotein(a) (Lp[a]) is a low-density lipoprotein (LDL) cholesterol-like particle bound to apolipoprotein(a). This novel marker of cardiovascular disease acts through induction of vascular inflammation, atherogenesis, calcification, and thrombosis. While an absolute risk threshold remains to be universally accepted, an estimated 20% to 25% of the global population have Lp(a) levels of 50 mg/dL or higher, a level noted by the European Atherosclerosis Society to confer increased cardiovascular risk. Compelling evidence from pathophysiological, observational, and genetic studies suggest a potentially causal association between high Lp(a) levels, atherosclerotic cardiovascular disease, and calcific aortic valve stenosis. Additional evidence has demonstrated that elevated Lp(a) levels are associated with a residual cardiovascular risk despite traditional risk factor optimization, including LDL cholesterol reduction. These findings have led to the formulation of the Lp(a) hypothesis, namely that Lp(a) lowering leads to cardiovascular risk reduction, intensifying the search for Lp(a)-reducing therapies. The ineffectiveness of lifestyle modification, statins, and ezetimibe to lower Lp(a); the modest Lp(a) reduction with proprotein convertase subtilisin/kexin type 9 inhibitors; the adverse effect profile and unclear cardiovascular benefit of pharmacotherapies such as niacin and mipomersen; and the impracticality of regular lipoprotein apheresis represent major challenges to currently available therapies. Nevertheless, emerging nucleic acid-based therapies, such as the antisense oligonucleotide pelacarsen and the small interfering RNA olpasiran, are generating interest because of their potent Lp(a)-lowering effects. Assessment of new-onset diabetes in patients achieving very low Lp(a) levels will be important in future trials. Epidemiologic and genetic studies suggest a potentially causal association between elevated Lp(a) levels, atherosclerotic cardiovascular disease, and aortic valve stenosis. Emerging nucleic acid-based therapies have potent Lp(a)-lowering effects and appear safe; phase 3 trials will establish whether they improve cardiovascular outcomes.

Endothelial P2X7 promotes venous thromboembolism

Venous thromboembolism (VTE) afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. VTE can lead to 1) death through pulmonary embolism, 2) the post-thrombotic syndrome, or 3) chronic pulmonary hypertension resulting in significant chronic respiratory compromise. Inflammatory pathways are intricately involved in venous thromboembolism mechanisms. In pathological conditions, adenosine triphosphate (ATP) is released in the extracellular compartment and is recognized as a danger signal. Recent data indicated that the CD39/CD73 system involved in the metabolism of ATP into AMP might protect against thrombosis by downregulating the pro-inflammatory pathway of the inflammasome. ATP can also interact with the P2X7 receptor involved in inflammation causing a wide range of responses. Determining how the endothelial P2X7 receptor contribute to venous thromboembolism. HUVEC were incubated with BzATP, thrombin or both. In some experiment, HUVEC were primed with TNFaprior to stimulation. We used immunofluorescence, western blot and real-time quantitative PCR analyses to assess P2X7 expression by endothelial cells, activation of p38 and NFkB activation and expression of pro-inflammatory and pro- coagulant genes. We confirmed that endothelial cells expressed P2X7 in vitro in HUVECs and in vivo after induction of venous thrombosis by ligation of the inferior vena cava. Treatment of endothelial cells with BzATP and thrombin induced the activation of p38 and NFkB signaling pathways. This was associated with an increased expression of IL-1b and tissue factor and downregulation of thrombomodulin expression. Our data suggest that ATP released in the extracellular space following cell damage or activation induced the inflammasome in endothelial through P2X7 activation. P2X7 might have a pro-thrombotic role exacerbating venous thromboembolism.

Modulation of interleukin-6 and its effect on late vein wall injury in a stasis mouse model of deep vein thrombosis

ObjectiveDeep vein thrombosis (DVT) and its sequela, post-thrombotic syndrome (PTS), remain a clinically significant problem. Interleukin-6 (IL-6) is a proinflammatory cytokine that is elevated in patients who develop PTS. We hypothesized that genetic deletion of IL-6 and the use of anti–IL-6 pharmacologic agents would be associated with decreased late vein wall injury. MethodsWild-type C57BL/6J (WT) and IL-6−/− mice underwent induction of stasis venous thrombosis by ligation of the infrarenal IVC. Vein wall inferior vena cava and thrombus were harvested at 21 days after ligation and analyzed by Western blot and immunohistochemistry of the vein wall using monocyte markers CCR2 and arginase 1, the endothelial marker CD31, and fibroblast markers DDR2 and FSP-1. Two anti–IL-6 pharmacologic agents (gp130 [glycoprotein 130] and tocilizumab) were tested and compared with low-molecular-weight heparin (LMWH) as the reference standard in WT mice. Plasma was collected at 4 and 48 hours to confirm the pharmacologic agents’ effects. ResultsLess fibrosis but no increase in luminal endothelialization was found in IL-6−/− mice compared with WT mice at 21 days. The IL-6−/− mice had fewer DDR2- and arginase 1-positive cells in the vein wall compared with the WT mice. However, no difference was found in the CCR2+ cells. Despite documented in vivo activity, exogenous gp130 and tocilizumab were not associated with decreased vein wall fibrosis or increased endothelial luminal coverage at 21 days. LMWH therapy, both before and after treatment, was not associated with decreased vein wall fibrosis at 21 days. ConclusionsIL-6 genetic deletion was associated with less fibrotic vein wall injury at a late time point, consistent with the PTS timeframe. However, neither the standard of care LMWH nor two available anti–IL-6 agents showed antifibrotic biologic effects in this model.

Investigating the role of receptor interacting protein kinase 3 in venous thrombosis

ObjectiveVenous thromboembolism is a disease that encompasses both deep vein thrombosis and pulmonary embolism. Recent investigations have shown that receptor interacting protein kinase 3 (RIPK3), a protein known for its role in the programmed form of cell death necroptosis, may play a role in thrombosis. Specifically, RIPK3 has been shown to promote platelet activation in arterial thrombosis and mixed lineage kinase domain-like pseudokinase (MLKL), a protein downstream of RIPK3 in the necroptosis pathway, has been shown to promote neutrophil extracellular trap formation in deep vein thrombosis. This investigation sought to comprehensively investigate the role of RIPK3 in deep vein thrombogenesis. MethodsThe inferior vena cava ligation and stenosis models of deep vein thrombosis were used in C57BL/6J, RIPK3 wild-type (Ripk3+/+) and RIPK3-deficient (Ripk3–/–) mice. Downstream tissue analyses included measurement of thrombus weight and histological and Western blot analysis of tissues for markers of necroptosis and cell death. A subset of C57BL/6J mice were treated with a RIPK3 inhibitor to determine the effect on venous thrombosis. ResultsC57BL/6J mice showed significant increases in thrombus weight from 6 to 48 hours. During the same time frame, RIPK3 progressively accumulated in the vein wall (a 35-fold increase from 0 to 48 hours). RIPK3 was present in the thrombus; however, it decreased with time. Although present in the thrombus, MLKL was nearly undetectable in the vein wall by Western blot at any timepoint. Immunostaining confirmed the high accumulation of RIPK3 in the vein wall, primarily colocalized to endothelial and smooth muscle cells. Phosphorylated MLKL, the active form of MLKL and executioner of necroptotic cell death, was detectable by immunostaining in the thrombus, but was present at low to undetectable levels in the vein wall. Propidium iodide and terminal deoxynucleotidyl transferase dUTP nick end labeling staining revealed a high burden of necrotic and apoptotic cells within the thrombus at 48 hours, but a relatively lower burden within the vein wall. Despite robust accumulation of RIPK3 within the vessel wall and the thrombus, knockout and inhibition of RIPK3 failed to impact thrombus incident or weight at 48 hours after inferior vena cava ligation. Neutrophil extracellular trap burden did not differ between Ripk3+/+ and Ripk3–/– mice. ConclusionsIn mice, the vein wall responded to deep vein thrombosis induction with elevation of RIPK3 without showing markers of necroptosis and apoptosis. Studies using genetic or pharmacological inhibition of RIPK3 suggest that this cell death mediator may not have a major role in the acute phase of venous thrombogenesis. Further investigation is needed to determine if RIPK3 plays a potentially non-necroptotic role within the vein wall during later stages of thrombus resolution and vein wall remodeling.

Abstract 9196: Innocuous Levels of Pulmonary Thrombosis Protect Against Inflammatory Lung Injury via Endothelial Arachidonate 15-Lipoxygenase and Hypoxia-Inducible Factor 1α

Introduction: Sepsis patients commonly suffer from lung thrombosis and sepsis-induced acute lung injury (ALI), but clinical trials of anti-coagulants in sepsis and ALI have failed, and the impact of varying severities of lung thrombosis on ALI is unknown. Hypothesis: Pulmonary thrombosis regulates the severity of sepsis-induced ALI through modulation of pulmonary endothelial cell (EC) viability. In this study, we aimed to determine the impact of varying levels of lung thrombosis on ALI and to delineate the underlying mechanisms. Methods: We assessed inflammatory lung injury in mouse models of pulmonary thrombosis combined with sepsis-induced lung injury and thrombocytopenia. We also used RNA sequencing and nanoparticle-mediated EC-targeted gene editing to delineate and target thrombosis-induced EC signaling pathways. Results: In wild type mice, excessive increases in lung thrombosis worsened ALI, but the induction of a mild and innocuous level of lung thrombosis instead reduced ALI and mortality. We then showed that this mild level of lung thrombosis increases the expression of multiple factors that regulate cell survival, including arachidonate 15-lipoxygenase (Alox15) and hypoxia-inducible factor (HIF) 1α in ECs. Next, we used nanoparticle-mediated EC gene editing to show that the protective impact of mild lung thrombosis on ALI is dependent upon EC-specific Alox15 and HIF1α. From a therapeutic perspective, the mild level of lung thrombosis could be induced up to 8 hours after sepsis and still maintain its protective impact on ALI. Furthermore, the induction of mild lung thrombosis completely prevented thrombocytopenia-induced increases and ALI and mortality. Conclusions: Mild levels of lung thrombosis protect against inflammatory lung injury via EC Alox15 and HIF1α. Promoting lung EC survival through Alox15 or HIF1α signaling represents a promising therapeutic strategy against ALI.

Inflammasome activation promotes venous thrombosis through pyroptosis

Crosstalk between coagulation and innate immunity contributes to the progression of many diseases, including infection and cardiovascular disease. Venous thromboembolism (VTE), including pulmonary embolism and deep vein thrombosis (DVT), is among the most common causes of cardiovascular death. Here, we show that inflammasome activation and subsequent pyroptosis play an important role in the development of venous thrombosis. Using a flow restriction-induced mouse venous thrombosis model in the inferior vena cava (IVC), we show that deficiency of caspase-1, but not caspase-11, protected against flow restriction-induced thrombosis. Interleukin-1β expression increased in the IVC following ligation, indicating that inflammasome is activated during injury. Deficiency of gasdermin D (GSDMD), an essential mediator of pyroptosis, protected against restriction-induced venous thrombosis. After induction of venous thrombosis, fibrin was deposited in the veins of wild-type mice, as detected using immunoblotting with a monoclonal antibody that specifically recognizes mouse fibrin, but not in the caspase-1-deficient or GSDMD-deficient mice. Depletion of macrophages by gadolinium chloride or deficiency of tissue factor also protected against venous thrombosis. Our data reveal that tissue factor released from pyroptotic monocytes and macrophages following inflammasome activation triggers thrombosis.

Endothelial P2X7 promotes venous thromboembolism

Venous thromboembolism (VTE) afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. VTE can lead to: – death through pulmonary embolism; – the post-thrombotic syndrome, or; – chronic pulmonary hypertension resulting in significant chronic respiratory compromise. Inflammatory pathways are intricately involved in venous thromboembolism mechanisms. In pathological conditions, adenosine triphosphate (ATP) is released in the extracellular compartment and is recognized as a danger signal. Recent data indicated that the CD39/CD73 system involved in the metabolism of ATP into AMP might protect against thrombosis by downregulating the pro-inflammatory pathway of the inflammasome. ATP can also interact with the P2X7 receptor involved in inflammation causing a wide range of responses. Determine how the endothelial P2X7 receptor contribute to venous thromboembolism. HUVEC were incubated with BzATP, thrombin or both. In some experiment, HUVEC were primed with TNFa prior to stimulation. We used immunofluorescence, western blot and real-time quantitative PCR analyses to assess P2X7 expression by endothelial cells, activation of p38 and NFkB activation and expression of pro-inflammatory and procoagulant genes. We confirmed that endothelial cells expressed P2X7 in vitro in HUVECs and in vivo after induction of venous thrombosis by ligation of the inferior vena cava. Treatment of endothelial cells with BzATP and thrombin induced the activation of p38 and NFkB signaling pathways. This was associated with an increased expression of IL-1b and tissue factor and downregulation of thrombomodulin expression. Our data suggest that ATP released in the extracellular space following cell damage or activation induced the inflammasome in endothelial through P2X7 activation. P2X7 might have a pro-thrombotic role exacerbating venous thromboembolism.

Development of a photochemical thrombosis investigation system to obtain a rabbit ischemic stroke model

Photochemical thrombosis is a method for the induction of ischemic stroke in the cerebral cortex. It can generate localized ischemic infarcts in the desired region; therefore, it has been actively employed in establishing an ischemic stroke animal model and in vivo assays of diagnostic and therapeutic techniques for stroke. To establish a rabbit ischemic stroke model and overcome the shortcoming of previous studies that were difficult to build a standardized photothrombotic rabbit model, we developed a photochemical thrombosis induction system that can produce consistent brain damage on a specific area. To verify the generation of photothrombotic brain damage using the system, longitudinal magnetic resonance imaging, 2,3,5-triphenyltetrazolium chloride staining, and histological staining were applied. These analytical methods have a high correlation for ischemic infarction and are appropriate for analyzing photothrombotic brain damage in the rabbit brain. The results indicated that the photothrombosis induction system has a main advantage of being accurately controlled a targeted region of photothrombosis and can produce cerebral hemisphere lesions on the target region of the rabbit brain. In conjugation with brain atlas, it can induce photochemical ischemic stroke locally in the part of the brain that is responsible for a particular brain function and the system can be used to develop animal models with degraded specific functions. Also, the photochemical thrombosis induction system and a standardized rabbit ischemic stroke model that uses this system have the potential to be used for verifications of biomedical techniques for ischemic stroke at a preclinical stage in parallel with further performance improvements.

Platelet α-granules are required for occlusive high-shear-rate thrombosis

Abstractvon Willebrand factor (VWF) is essential for the induction of arterial thrombosis. In this study, we investigated the critical role of platelet VWF in occlusive thrombosis formation at high shear in mice that do not express platelet VWF (Nbeal2−/−). Using in silico modeling, in vitro high-shear microfluidics, and an in vivo Folts model of arterial thrombosis we reproduced the platelet dynamics that occur under pathological flow in a stenosed vessel. Computational fluid dynamics (CFDs) simulated local hemodynamics in a stenosis based on arterial geometries. The model predicted shear rates, time course of platelet adhesion, and time to occlusion. These predictions were validated in vitro and in vivo. Occlusive thrombosis developed in wild-type control mice that had normal levels of plasma VWF and platelet VWF in vitro and in vivo. Occlusive thrombosis did not form in the Nbeal2−/− mice that had normal plasma VWF and an absence of platelet VWF. Occlusive thrombosis was corrected in Nbeal2−/− microfluidic assays by the addition of exogenous normal platelets with VWF. Combining model and experimental data, we demonstrated the necessary requirement of platelet VWF in α-granules in forming an occlusive thrombus under high shear. These results could inspire new pharmacological targets specific to pathological conditions and prevent arterial thrombosis.