Mouse Model Of Thrombosis Research Articles

The gut microbiota in thrombosis.

The gut microbiota has emerged as an environmental risk factor that affects thrombotic phenotypes in several cardiovascular diseases. Evidence includes the identification of marker species by sequencing studies of the gut microbiomes of patients with thrombotic disease, the influence of antithrombotic therapies on gut microbial diversity, and preclinical studies in mouse models of thrombosis that have demonstrated the functional effects of the gut microbiota on vascular inflammatory phenotypes and thrombus formation. In addition to impaired gut barrier function promoting low-grade inflammation, gut microbiota-derived metabolites have been shown to act on vascular cell types and promote thrombus formation. Therefore, these meta-organismal pathways that link the metabolic capacities of gut microorganisms with host immune functions have emerged as potential diagnostic markers and novel drug targets. In this Review, we discuss the link between the gut microbiota, its metabolites and thromboembolic diseases.

Parvovirus B19 Infection Is Associated with the Formation of Neutrophil Extracellular Traps and Thrombosis: A Possible Linkage of the VP1 Unique Region.

Neutrophil extracellular traps (NETs) formation, namely NETosis, is implicated in antiphospholipid syndrome (APS)-related thrombosis in various autoimmune disorders such as systemic lupus erythematosus (SLE) and APS. Human parvovirus B19 (B19V) infection is closely associated with SLE and APS and causes various clinical manifestations such as blood disorders, joint pain, fever, pregnancy complications, and thrombosis. Additionally, B19V may trigger the production of autoantibodies, including those against nuclear and phospholipid components. Thus, exploring the connection between B19V, NETosis, and thrombosis is highly relevant. An in vitro NETosis model using differentiated HL-60 neutrophil-like cells (dHL-60) was employed to investigate the effect of B19V-VP1u IgG on NETs formation. A venous stenosis mouse model was used to test how B19V-VP1u IgG-mediated NETs affect thrombosis in vivo. The NETosis was observed in the dHL-60 cells treated with rabbit anti-B19V-VP1u IgG and was inhibited in the presence of either 8-Br-cAMP or CGS216800 but not GSK484. Significantly elevated reactive oxygen species (ROS), myeloperoxidase (MPO), and citrullinated histone (Cit-H3) levels were detected in the dHL60 treated with phorbol myristate acetate (PMA), human aPLs IgG and rabbit anti-B19V-VP1u IgG, respectively. Accordingly, a significantly larger thrombus was observed in a venous stenosis-induced thrombosis mouse model treated with PMA, human aPLs IgG, rabbit anti-B19V-VP1u IgG, and human anti-B19V-VP1u IgG, respectively, along with significantly increased amounts of Cit-H3-, MPO- and CRAMP-positive infiltrated neutrophils in the thrombin sections. This research highlights that anti-B19V-VP1u antibodies may enhance the formation of NETosis and thrombosis and implies that managing and treating B19V infection could lower the risk of thrombosis.

Antipolyphosphate monoclonal antibodies derived from autoimmune mice

BackgroundInorganic polyphosphates (polyPs) are linear chains of phosphates that accelerate blood clotting. Targeting polyP in vivo has been shown to reduce thrombosis. ObjectivesTo identify and characterize anti-polyP monoclonal antibodies that could be used as analytical tools and as antithrombotic agents. MethodsHybridomas were prepared from spleen cells from autoimmune NZBWF1/J female mice and screened for anti-polyP antibodies. Antibodies that bound polyP using enzyme-linked immunosorbent assay and pull-down assays were further characterized with plate binding, surface plasmon resonance, and plasma-based clotting assays. Antithrombotic potential was evaluated in a murine ferric chloride–induced carotid artery thrombosis model. ResultsOf 4 antibodies that bound polyP in our pull-down assay, 2 (PP2069 and PP2099) were available for further characterization. While analyzing these anti-polyP antibodies, we found secretory leukocyte peptidase inhibitor (SLPI) to be a common contaminant of these antibodies and that SLPI binds polyP. We removed SLPI quantitatively from our purified immunoglobulin G. Both PP2069 and PP2099 immunoglobulin G displayed high affinity for polyP but also bound to other polyanions such as DNA, heparin, and certain other glycosaminoglycans, indicating limited specificity. Both antibodies inhibited polyP-initiated plasma clotting in vitro. When tested in vivo in a mouse thrombosis model, however, neither PP2069 nor PP2099 exhibited a significant antithrombotic effect. ConclusionAutoimmune mice spontaneously produce antibodies against polyP. The 2 examples of anti-polyP monoclonal antibodies studied here not only bound to polyP with high affinity but also cross-reacted with DNA and heparin. Neither antibody protected against thrombosis in a mouse model, but they might have some utility for in vitro studies of polyP.

Inhibition of neutrophil rolling and migration by caADAMTS13 in vitro and in mouse models of thrombosis and inflammation

Recent investigation of a constitutively active ADAMTS13 variant (caADAMTS13) in murine models of acute ischaemic stroke (AIS) have revealed a potential anti-inflammatory mechanism of action contributing to its protective effect. However, it remains unclear whether these observations are a direct result of VWF proteolysis by caADAMTS13. We have implemented state of the art in vitro assays of neutrophil rolling and transmigration to quantify the impact of caADAMTS13 on these processes. Moreover, we have tested caADAMTS13 in two in vivo assays of neutrophil migration to confirm the impact of the treatment on the neutrophil response to sterile inflammation. Neutrophil rolling, over an interleukin-1β stimulated hCMEC/D3 monolayer, is directly inhibited by caADAMTS13, reducing the proportion of neutrophils rolling to 9.5 ± 3.8 % compared to 18.0 ± 4.5 % in untreated controls. Similarly, neutrophil transmigration recorded in real-time, was significantly suppressed in the presence of caADAMTS13 which reduced the number of migration events to a level like that in unstimulated controls (18.0 ± 4.5 and 15.8 ± 7.5 cells/mm2/h, respectively). Brain tissue from mice undergoing experimental focal cerebral ischaemia has indicated the inhibition of this process by caADAMTS13. This is supported by caADAMTS13’s ability to reduce neutrophil migration into the peritoneal cavity in an ischaemia-independent model of sterile inflammation, with the VWF-dependent mechanism by which this occurs being confirmed using a second experimental stroke model. These findings will be an important consideration in the further development of caADAMTS13 as a potential therapy for AIS and other thromboinflammatory pathologies, including cardiovascular disease.

Neuroprotective and vasoprotective effects of herb pair of Zhiqiao-Danggui in ischemic stroke uncovered by LC-MS/MS-based metabolomics approach.

Ischemic stroke is the most important cause of disability and death worldwide, but current treatments remain limited. Traditional Chinese medicine (TCM) including the herb pair of Zhiqiao-Danggui (ZD) offers a multifaceted treatment approach through promoting blood circulation, yet its specific anti-ischemic mechanism remains unclear. This study used the photochemically induced thrombosis (PIT) mouse model and the oxygen glucose deprivation/reoxygenation (OGD/R) cell model to explore the therapeutic effect of ZD on ischemic stroke. Mice were treated with high and low doses of ZD extract or positive control. Behavior was assessed using the grid test. The brain tissue was then subjected to infarct volume assessment, histopathology, oxidative stress marker detection, LC/MS metabolomic analysis and qRT-PCR validation. The therapeutic effect of ZD-medicated serum on OGD/R model was tested on cells. Experimental results show that ZD can improve motor function, reduce infarct size, neuronal damage and apoptosis as well as alleviate oxidative stress in mice. ZD-medicated serum promotes endothelial cell proliferation, improves cell survival against OGD/R-induced injury, reduces oxidative damage and protects mitochondrial function. Metabolomics reveals ZD regulation of metabolites in energy metabolism, amino acid metabolism, TCA cycle, and angiogenesis signaling pathways. qRT-PCR results also showed that ZD could attenuate abnormal conduction of angiogenic signals and enhance vessel stability. This study confirmed the neuroprotective and vasoprotective effects of ZD, highlighted its potential in treating ischemic stroke, and provided a scientific basis for the traditional use of ZD.

Ferumoxytol-enhanced MRI assessment of venous Thrombus resolution and macrophage content in a murine deep vein thrombosis model

BackgroundImaging evaluation of acute deep vein thrombosis (DVT) or post-thrombotic syndrome (PTS) in animal or clinical models is limited to anatomical assessment of the location and extent of thrombi. We hypothesize that Fe-MRI, used to evaluate macrophage content in other inflammatory diseases, can be useful to evaluate the thromboinflammatory features after DVT over time. MethodsNineteen wild-type CD-1 mice underwent surgical IVC ligation to induce DVT. Mice received either saline or 5 mg/kg of 14E11, a Factor XI inhibitor, before the procedure. Fe-MRI was performed on days 6–7 after ligation to evaluate thrombus volume, perfusion, and macrophage content via T2-weighted images. Mice were euthanized at days 3–15 after surgery. The thrombi and adjacent vein walls were excised, weighed, formalin-fixed, and paraffin-embedded for immunohistological analysis. Specimens were stained with specific antibodies to evaluate macrophage content, collagen deposition, neovascularization, and recanalization. Significance was determined using the Mann-Whitney U or Student's t-test. ResultsAfter IVC-ligation in control mice, thrombus weights decreased by 59 % from day 3 to 15. Thrombus volumes peaked on day 5 before decreasing by 85 % by day 13. FXI inhibition led to reduced macrophage content in both thrombi (p = .008) and vein walls (p = .01), decreased thrombus volume (p = .03), and decreased thrombus mass (p = .01) compared to control mice. CCR2+ staining corroborated these findings, showing significantly reduced macrophage presence in the thrombi (p = .002) and vein wall (p = .002). ConclusionsFe-MRI T2 relaxation times can be used to characterize and quantify post-thrombotic changes of perfusion, macrophage content, and thrombus volume over time in a surgical mouse model of venous thrombosis. This approach could lead to better quantification of in vivo inflammation correlating monocyte and macrophage content within resolving thrombi and veins and may serve as a useful tool for research and clinically in the evaluation of the post-thrombotic environment.

Abstract 2093: Quantitative Assessment Of Venous Thrombus Resolution And Macrophage Content In Mice Using Ferumoxytol-enhanced Magnetic Resonance Imaging

Objective: The objective of this study is to explore the feasibility of ferumoxytol-enhanced magnetic resonance imaging (Fe-MRI) for noninvasive assessment of deep venous thrombus (DVT) resolution and macrophage content in resolving thrombi in a surgical murine model of inferior vena cava (IVC) ligation. Background: Imaging evaluation of acute DVT or post-thrombotic syndrome (PTS) in animal or clinical models is limited to assessment of anatomy. We hypothesize that Fe-MRI, used to evaluate macrophage content in other inflammatory diseases, can be useful to evaluate thromboinflammatory features after DVT. Methods: Nineteen wild-type CD-1 mice underwent surgical IVC ligation to induce DVT. Mice received either saline or administered 5 mg/kg of 14E11, a Factor XI inhibitor, before the procedure. Fe-MRI was performed from days 6-7 after ligation to evaluate thrombus volume, perfusion and macrophage content via T2, and T2 *-weighted images. Mice were euthanized at days 3-15 after surgery. The thrombi and adjacent vein walls were excised, weighed, formalin fixed, and paraffin embedded for histological analysis. Specimens were stained with specific antibodies to evaluate macrophage activity, collagen deposition, neovascularization, and recanalization. Significance was determined using the Mann-Whitney U or Student’s t-test. Results: After IVC-ligation in control mice, thrombus weights decreased by 59% from day 3 to day 15. FXI inhibition led to reduced macrophage content in both thrombus (p = .008) and vein wall (p = .01), decreased thrombus volume (p = .03), decreased thrombus mass (p = .01) and impaired thrombus perfusion (p = .01) compared to control mice. Iba1 staining supported these findings, showing significantly reduced macrophage presence in thrombi (p = .02). Conclusions: Fe-MRI T2 and T2 * relaxation times can be used to characterize and quantify post-thrombotic changes of perfusion, macrophage activity, and volume over time in a surgical mouse model of venous thrombosis. This approach could lead to better quantification of in vivo inflammation correlating monocyte and macrophage activity within resolving thrombi and veins and may serve as a useful tool in research and clinically in the evaluation of DVT or PTS.

Dual Inhibition of Factor XIIa and Factor XIa Produces a Synergistic Anticoagulant Effect.

Clinical practice shows that a critical unmet need in the field of thrombosis prevention is the availability of anticoagulant therapy without bleeding risk. Inhibitors against FXIa or FXIIa have been extensively studied because of their low bleeding risk. However, whether these compounds produce synergistic effects has not yet been explored. In this study, analyses of activated partial thromboplastin time in combination with the FXIa inhibitor PN2KPI and the FXIIa inhibitor Infestin4 at different proportions were performed using the SynergyFinder tool identifying synergistic anticoagulation effects. Both an FeCl 3 -induced carotid artery thrombosis mouse model and a transient occlusion of the middle cerebral artery mouse model showed that the combination of PN2KPI and Infestin4, which are 28.57% and 6.25% of the effective dose, respectively, significantly prevents coagulation, and furthermore, dual inhibition does not cause bleeding risk.

Antithrombotic pharmacodynamics and metabolomics study in raw and processed products of Whitmania pigra Whitman

ObjectiveAs a traditional Chinese medicine, leech has obvious pharmacological activities in anticoagulantion and antithrombosis. Whitmania pigra Whitman (WP) is the most commonly used leech in the Chinese market. It is often used in clinical applications after high-temperature processing by talcum powder to remove the fishy taste and facilitate crushing. The anticoagulant and thrombolytic active ingredients are protein and polypeptide, which may denaturate and lose activity after high-temperature processing. The rationality of its processing has been questioned in recent years. This study aims to investigate the effect of talcum powder scalding on the antithrombotic activity of WP in vivo and to discuss its pharmacodynamic mechanism in vivo. MethodsRaw and talcum-powdered processed WP were administered intragastrically for 14 days, and carrageenan was injected intraperitoneally to prepare a mouse model of tail vein thrombosis. The incidence rate of tail vein thrombosis and the thrombus area under pathological tissue sections were calculated to evaluate the antithrombotic effect between raw and processed WP. Non-targeted metabolomics was conducted using UPLC-Q-TOF/MS technology to analyze the changes of small molecule metabolites in the body after administration of WP. ResultsAfter intragastric administration, both the raw product and the processed product of WP could inhibit the thrombosis induced by carrageenan, and the processed product had a more apparent antithrombotic effect than the raw product. The administration of WP could regulate the changes of some small molecular metabolites, such as amino acids, lipids, and steroids, in Sphingolipid metabolism and Glycerophospholipid metabolism. ConclusionsBased on the results of pharmacodynamics and metabolomics, processed WP will not reduce the antithrombotic activity of WP. This study provided a scientific basis for the rational use of leeches.

Involvement of peptidylarginine deiminase 4 in eosinophil extracellular trap formation and contribution to citrullinated histone signal in thrombi

BackgroundExtracellular traps formed by neutrophils (NETs) and eosinophils (EETs) have been described in coronary thrombi, contributing to thrombus stability. A key mechanism during NET formation is histone modification by the enzyme PAD4. Citrullinated histones, the product of PAD4 activity, are often attributed to neutrophils. Eosinophils also express high levels of PAD4. ObjectivesWe aimed to explore the contribution of PAD4 to EET formation. MethodsWe performed immunohistological analyses on thrombi, including a large, intact, and eosinophil-containing thrombus retrieved from the right coronary artery using an aspiration catheter and stroke thrombi from thrombectomy retrieval. We studied eosinophils for their capability to form PAD4-dependent EETs in response to strong ET-inducing agonists as well as activated platelets and bacteria. ResultsHistopathology and immunofluorescence microscopy identified a coronary thrombus rich in platelets and neutrophils, with distinct areas containing von Willebrand factor and citrullinated histone H3 (H3Cit). Eosinophils were also identified in leukocyte-rich areas. The majority of the H3Cit+ signal colocalized with myeloperoxidase, but some colocalized with eosinophil peroxidase, indicating EETs. Eosinophils isolated from healthy volunteers produced H3Cit+ EETs, indicating an involvement of PAD4 activity. The selective PAD4 inhibitor GSK484 blocked this process, supporting PAD4 dependence of H3Cit+ EET release. Citrullinated histones were also present in EETs produced in response to live Staphylococci. However, limited evidence for EETs was found in mouse models of venous thrombosis or infective endocarditis. ConclusionAs in NETosis, PAD4 can catalyze the formation of EETs. Inhibition of PAD4 decreases EET formation, supporting the future utility of PAD4 inhibitors as possible antithrombotic agents.

Characterization of active peptides derived from three leeches and comparison of their anti-thrombotic mechanisms using the tail vein thrombosis model in mice and metabonomics

Background and aims: The increasing incidence of cardiovascular diseases has created an urgent need for safe and effective anti-thrombotic agents. Leech, as a traditional Chinese medicine, has the effect of promoting blood circulation and removing blood stasis, but its real material basis and mechanism of action for the treatment of diseases such as blood stasis and thrombosis have not been reported.Methods: In this study, Whitmania Pigra Whitman (WPW), Hirudo nipponica Whitman (HNW) and Whitmania acranutata Whitman (WAW) were hydrolyzed by biomimetic enzymatic hydrolysis to obtain the active peptides of WPW (APP), the active peptides of HNW (APH) and the active peptides of WAW (APA), respectively. Then their structures were characterized by sykam amino acid analyzer, fourier transform infrared spectrometer (FT-IR), circular dichroism (CD) spectrometer and LC-MS. Next, the anti-thrombotic activities of APP, APH and APA were determined by carrageenan-induced tail vein thrombosis model in mice, and the anti-thrombotic mechanisms of high-dose APP group (HAPP), high-dose APH group (HAPH) and high-dose APA group (HAPA) were explored based on UHPLC-Q-Exactive Orbitrap mass spectrometry.Results: The results showed that the amino acid composition of APP, APH and APA was consistent, and the proportion of each amino acid was few different. The results of FT-IR and CD showed that there were no significant differences in the proportion of secondary structures (such as β-sheet and random coil) and infrared absorption peaks between APP, APH and APA. Mass spectrometry data showed that there were 43 common peptides in APP, APH and APA, indicating that the three have common material basis. APP, APH and APA could significantly inhibit platelet aggregation, reduce black-tail length, whole blood viscosity (WBV), plasma viscosity (PV), and Fibrinogen (FIB), and prolong coagulation time, including activated partial thrombin time (APTT), prothrombin time (PT) and thrombin time (TT). In addition, 24 metabolites were identified as potential biomarkers associated with thrombosis development. Among these, 19, 23, and 20 metabolites were significantly normalized after administration of HAPP, HAPH, and HAPA in the mice, respectively. Furthermore, the intervention mechanism of HAPP, HAPH and HAPA on tail vein thrombosis mainly involved in linoleic acid metabolism, primary bile acid biosynthesis and ether lipid metabolism.Conclusion: Our findings suggest that APP, APH and APA can exert their anti-blood stasis and anti-thrombotic activities by interfering with disordered metabolic pathways in vivo, and there is no significant difference in their efficacies.

Lycopene inhibits carrageenan-induced thrombi by regulating AKT/FoxO3a and TLR4/NF-κB pathways

Lycopene (LYC) is a natural phytonutrient with various bioactivities, such as anti-oxidative stress and anti-inflammation. In this study, human endothelial cells (HUVECs), macrophages and human platelets, and carrageenan-induced thrombosis mouse model were used to evaluate the antithrombosis effects of LYC. In vitro, LYC reduced reactive oxygen species (ROS) production by activating antioxidant enzymes and inhibiting cellular inflammatory factors. LYC inhibited platelets activation by enhancing antioxidant enzymes expression through reducing AKT/FoxO3a signaling pathway. In vivo, LYC effectively improved carrageenan-induced thrombosis in mouse tail, liver and lung tissues. Additionally, in mouse liver and lung tissues, LYC reduced inflammation by inhibiting toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) pathway, and inhibited oxidative stress by increasing antioxidant enzymes expression. In conclusion, LYC can inhibit thrombosis in mouse model, which suggesting that LYC could act as a potential functional food for prevention or treatment of thrombus.

Venous Thrombosis Assay in a Mouse Model of Cancer.

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis (CAT). Deep venous thrombosis is a common complication in cancer survivors and can be potentially fatal. The current murine venous thrombosis models typically involve a complete or partial mechanical occlusion of the inferior vena cava (IVC) using a suture. This procedure induces a total or partial stasis of blood and endothelial damage, triggering thrombogenesis. The current models have limitations such as higher variability in clot weights, significant mortality rate, and prolonged learning curve. This report introduces surgical refinements using vascular clips to address some of these limitations. Using a syngeneic colon cancer xenograft mouse model, we employed customized vascular clips to ligate the infrarenal vena cava. These clips allow residual lip space similar to a 5-0 polypropylene suture after IVC ligations. Mice with the suture method served as controls. The vascular clip method resulted in a consistent reproducible partial vascular occlusion and greater clot weights with less variability than the suture method. The larger clot weights, greater clot mass, and clot to the IVC luminal surface area were expected due to the higher pressure profile of the vascular clips compared to a 6-0 polypropylene suture. The approach was validated by gray scale ultrasonography, which revealed consistently greater clot mass in the infrarenal vena cava with vascular clips compared to the suture method. These observations were further substantiated with the immunofluorescence staining. This study offers an improved method to generate a venous thrombosis model in mice, which can be employed to deepen the mechanistic understanding of CAT and in translational research such as drug discovery.

Endogenous SIRT6 in platelets negatively regulates platelet activation and thrombosis.

Thromboembolism resulting from platelet dysfunction constitutes a significant contributor to the development of cardiovascular disease. Sirtuin 6 (SIRT6), an essential NAD+-dependent enzyme, has been linked to arterial thrombosis when absent in endothelial cells. In the present study, we have confirmed the presence of SIRT6 protein in anucleated platelets. However, the precise regulatory role of platelet endogenous SIRT6 in platelet activation and thrombotic processes has remained uncertain. Herein, we present compelling evidence demonstrating that platelets isolated from SIRT6-knockout mice (SIRT6-/-) exhibit a notable augmentation in thrombin-induced platelet activation, aggregation, and clot retraction. In contrast, activation of SIRT6 through specific agonist treatment (UBCS039) confers a pronounced protective effect on platelet activation and arterial thrombosis. Moreover, in platelet adoptive transfer experiments between wild-type (WT) and SIRT6-/- mice, the loss of SIRT6 in platelets significantly prolongs the mean thrombus occlusion time in a FeCl3-induced arterial thrombosis mouse model. Mechanistically, we have identified that SIRT6 deficiency in platelets leads to the enhanced expression and release of proprotein convertase subtilisin/kexin type 9 (PCSK9), subsequently activating the platelet activation-associated mitogen-activated protein kinase (MAPK) signaling pathway. These findings collectively unveil a novel protective role of platelet endogenous SIRT6 in platelet activation and thrombosis. This protective effect is, at least in part, attributed to the inhibition of platelet PCSK9 secretion and mitogen-activated protein kinase signaling transduction. Our study provides valuable insights into the intricate interplay between SIRT6 and platelet function, shedding light on potential therapeutic avenues for managing thrombotic disorders.

Identification and characterization of a versatile keratinase, KerZJ, from Stenotrophomonas sp. LMY.

Keratinases have drawn increasing attention in recent decades owing to their catalytic versatility and broad applications from agriculture to medicine. In the present study, we isolated a highly keratinolytic and fibrinolytic bacterium from the campus soil and named it Stenotrophomonas sp. LMY based on genetic information. To identify the potential keratinase genes, the genome sequence of the strain was obtained and analyzed. Sequence alignment and comparison revealed that the protein 1_737 (KerZJ) had the highest sequence homology to a reported keratinase KerBL. We recombinantly expressed KerZJ in Escherichia coli Origami™ (DE) pLysS and purified it to homogeneity. KerZJ showed the highest activity at 40°C and pH 9.0, and metal ions exhibited no significant effects on its activity. Although reducing agents would break the disulfide bonds in KerZJ and reduce its activity, KerZJ still exhibited the ability to hydrolyze feather keratin in the presence of β-ME. KerZJ could efficiently digest human prion proteins. In addition, KerZJ showed fibrinolytic activity on fibrin plates and effectively eliminated blood clots in a thrombosis mouse model without side effects. Our results suggest that KerZJ is a versatile keratinase with significant potential for keratin treatment, decontamination of prions, and fibrinolytic therapy.

The Mechanism Underlying Lenalidomide-Induced Thrombosis Susceptibility

Introduction: Lenalidomide, a well-established drug for the treatment of multiple myeloma, significantly enhances patients' survival. Previous clinical studies have demonstrated that its main side effect is an increased risk of thrombotic events. However, the underlying mechanism remains unexplored. Therefore, this study aims to elucidate the mechanism and offer insights into the selection of clinical thrombotic prophylaxis drugs. Methods: Firstly, we conducted a retrospective analysis of clinical data from 169 newly diagnosed multiple myeloma patients who received lenalidomide. To confirm the impact of lenalidomide on thrombosis formation, FeCl3-induced thrombosis and deep venous thrombosis models in mice were established. To investigate the effects of lenalidomide on platelet function, both in vivo and in vitro experiments were designed. Results From January 2013 to June 2021, 169 NDMM patients with VRD as the first-line treatment were included in this study. During our follow-up, 8 patients had TEs . TEs included 8 VTE and 1 ATE. There were 7 DVT and 1 PE including in 8 VTE. Among the 8 patients suffering from thrombus, 2 patients did not receive thromboprophylaxis because of low platelet count, and the other 6 patients took aspirin, warfarin and rivaroxaban. Lenalidomide (25 mg/kg, 50 mg/kg, per day) or vehicle (DMSO) for 14 days were administrated for 2 weeks to healthy male mice. Compared with DMSO group, lenalidomide 25 mg/kg group and lenalidomide 50 mg/kg group significantly affected the formation of venous thrombosis , and there were significant statistical differences in both the length ( p=0.002; p=0.0005) and weight ( p<0.0001; p<0.0001) of thrombosis. In contrast, the formation of arterial thrombosis was not affected by lenalidomide administration in vivo, occlusion time of experiment group had control group did not have difference, which is consistent with the low incidence of ATE in clinical practice. To further explore the mechanism, we firstly focused on whether lenalidomide affects platelet function. Compared to baseline data, after taking one cycle of lenalidomide, the mean platelet volume (MPV, p<0.0001), platelet distribution width (PDW, p<0.0001), large platelet ratio ( p<0.0001), platelet hematocrit ( p=0.0005), and fibrinogen ( p=0.0008) of patients increased significantly. To evaluate the effect of lenalidomide on NDMM patient's platelet activation in vitro, we pretreated the washed platelets from NDMM with lenalidomide (5 or 10 um) or DMSO for platelet function assays . There was no difference between the three groups. To clarify the effect of lenalidomide on platelet function and parameters in vivo, lenalidomide (25 mg/kg, once per day), (50 mg/kg, once per day) or DMSO were administrated for 2 weeks to mice . The last dose was taken the night before narcotism, and platelet function was measured the next morning. We found that different from the changes in platelet related parameters in patients, in vivo data of mice showed no significant changes in platelet count , MPV , PDW , large platelet ratio , and platelet hematocrit after taking lenalidomide.Then we prepared washed platelets from mice for functional experiments. There was no difference between the three groups. In order to further explore the impact of lenalidomide on coagulation function in NDMM patients, we compared the coagulation parameters of NDMM patients who were enrolled at the time of diagnosis and after one cycle of taking lenalidomide. We found significant differences in prothrombin time, thrombin time, and prothrombin time ratio ( PTR, p<0.0001), while there was no statistical difference in some changes in activated partial thrombin time ( APTT, p=0.0881). These results may suggest that lenalidomide promotes the formation of venous thrombosis in vivo by influencing the coagulation pathway. Conclusions: The administration of lenalidomide had no significant impact on platelet function, which may affect venous thrombus formation by affecting coagulation. Therefore, anticoagulant drugs may be superior to antiplatelet drugs in the selection of clinical thrombus prophylaxis.

CS585 Demonstrates Favorable Selectivity and Sustained In Vivo Action in Preventing Platelet Activation and Thrombosis Compared to Existing IP Receptor Agonists

Anti-platelet therapeutics are common tools in the treatment of thrombotic diseases, decreasing morbidity and mortality resulting from cardiovascular complications. However, many patients remain at risk for a cardiovascular event while others experience bleeding due to hemostatic dysregulation. The need for target specificity and lasting stability presents a major challenge in the development of novel antithrombotic therapeutics. While increasing platelet cAMP levels through activation of the prostacyclin (IP) receptor is a viable therapeutic approach, the IP receptor is currently considered a challenging target due to the non-selectivity and instability of existing IP agonists. Our lab has developed a novel IV and orally available IP receptor agonist, CS585, with selectivity towards the IP receptor and sustained in vivo protection from injury-induced thrombosis without observed bleeding. CS585 demonstrates IP receptor-dependent activity, leading to activation of protein kinase A (PKA), a key player in inhibitory signaling. While indications of current IP receptor agonists are limited to pulmonary arterial hypertension and temporary relief of peripheral artery disease, CS585 presents a new approach to selectively regulate platelet activation and thrombosis. CS585 and other IP receptor agonists, iloprost and selexipag, were assessed by direct comparison to elucidate potential differences in selectivity and sustained action. Selectivity was determined using aggregation and VASP phosphorylation. The effects of CS585 are inhibited by pharmacological inhibition of the IP receptor, but not by inhibition of the DP1, EP2, or EP4 prostaglandin receptors. Meanwhile, the activities of iloprost and selexipag were affected by other prostaglandin inhibitors, supporting the improved selectivity toward activation of the IP receptor with CS585. Arterial thrombosis was measured and quantified in a laser-induced cremaster thrombosis assay in IP agonist-treated WT mice to assess platelet activation and thrombus formation in vivo in mouse models of thrombosis. In WT mice treated with CS585, we observed sustained effects of CS585 following both IV and oral administration. Platelets and fibrin were labeled and accumulation at the site of injury was measured using intravital microscopy. In contrast to the FDA-approved IP receptor agonists (iloprost and selexipag) which showed short-term protection from thrombosis, treatment with CS585 resulted in sustained inhibition of clot and fibrin formation at the site of injury in the same model between 24 and 48 hours post-administration. To determine if the anti-thrombotic effects of CS585 impact coagulation, coagulation parameters were assessed in whole blood using thromboelastography (TEG). Treatment with CS585 did not impact coagulation parameters in whole blood, while a significant increase in lysis was observed with iloprost. Here, we show for the first time a head-to-head comparison of CS585, a novel IP receptor agonist, with selexipag and iloprost. Our preclinical results with CS585 indicate a favorable profile for inhibiting platelet activation and clot formation and demonstrate a sustained duration of action in mice in the ability of CS585 to inhibit platelet activation through multiple routes of administration. Importantly, CS585 does not affect coagulation parameters and previous studies demonstrated no potential for increased bleeding in mouse models. Overall, CS585 provides a new option of activating the IP receptor to decrease platelet reactivity and could represent the first viable option for targeting the IP receptor on platelets for primary inhibition of thrombosis with a reduced risk of bleeding.

Immune Checkpoint Blockade Promotes Thrombosis Via T-Cell and Neutrophil Activation, and Tumor-Cell Associated Tissue Factor (TF) in a Murine Model of Colorectal Cancer

Background: Immune checkpoint blockade (ICB) is a paradigm shift in cancer therapy. Despite clinical benefit, various adverse events are associated with ICB using immune checkpoint inhibitors (ICI). Venous thrombosis is an emerging clinical problem in cancer patients receiving ICI. Little is known about how ICI promotes thrombosis in cancer. To define the underlying mechanisms of ICI-associated thrombosis (IAT), we assessed the ICI impact on blood hypercoagulability and tumor TF expression using a mouse thrombosis model of colorectal cancer. Methods: We developed a CT26 mouse thrombosis model with ICI (aPD-1+aCTLA4) administration. Systemic hypercoagulability was assessed by measurement of circulating nucleosomes (Nu.Q H3.1 Immunoassay), neutrophil extracellular traps (NETs) (CitH3 immunofluorescence), circulating neutrophil-platelet aggregates (immunophenotyping), and levels of thrombin-antithrombin (TAT) complexes in ICI-treated mice. Tumor-associated TF and TF activity in circulating extracellular vesicles (EV) were assessed by western blotting and Factor Xa generation assays. To gain mechanistic insights into TF induction by ICI, tumor cytokines were also profiled (proteome profiler array). The role of cancer cell-derived TF in IAT was determined using mice implanted with CRISPR/Cas9-mediated TF knockout (TFKO) CT26 tumor cells. Results: ICI treated tumor-bearing mice developed larger thrombi than control mice and demonstrated elevated levels of circulating nucleosomes (114 vs 82 ng/ml), NET (15.8 vs 6.8%), platelet-neutrophil aggregates (44.8 vs 22.3%), and TAT complexes (12.6 vs 7.9 ng/ml) compared to IgG-treated mice. Tumor TF expression was increased in tumor extracts. Cytokine profiling suggested enhanced production of IFNɣ (2-fold) and TNFα (5-fold), as well as CXCL11 (6.8-fold), an effector T cell-recruiting chemokine. IFNɣ-increased TF expression in cultured CT26 cells, suggesting that T-cell derived IFNɣ may play a role in enhancing tumor TF expression in vivo. Circulating EV TF antigen and procoagulant activity were increased by ICI. CRISPR/Cas9-mediated TF KO in CT26 cells resulted in complete loss of TF expression, and mice bearing these cells showed reduced tumor and EV-associated TF (Fig. A), associated with a significant decrease in thrombus formation after ICI treatment (24.2 vs 20.5 mg, Fig. B). There was no significant difference in growth of wild-type and TF KO CT26 tumors. Conclusions: Our findings provide insights into cellular and molecular mechanisms associated with ICI-induced thrombosis, and suggest a role for neutrophil and granulocyte activation, and tumor-associated TF. Additional studies are ongoing to further define these pathways in the murine model and in patients.

C-type lectin-like receptor-2 (CLEC-2) is a key regulator of kappa-carrageenan-induced tail thrombosis model in mice

Kappa-carrageenan (KCG), which is used to induce thrombosis in laboratory animals for antithrombotic drug screening, can trigger platelet aggregation. However, the cell-surface receptor and related signaling pathways remain unclear. In this study, we investigated the molecular basis of KCG-induced platelet activation using light-transmittance aggregometry, flow cytometry, western blotting, and surface plasmon resonance assays using platelets from platelet receptor-deficient mice and recombinant proteins. KCG-induced tail thrombosis was also evaluated in mice lacking the platelet receptor. We found that KCG induces platelet aggregation with α-granule secretion, activated integrin αIIbβ3, and phosphatidylserine exposure. As this aggregation was significantly inhibited by the Src family kinase inhibitor and spleen tyrosine kinase (Syk) inhibitor, a tyrosine kinase-dependent pathway is required. Platelets exposed to KCG exhibited intracellular tyrosine phosphorylation of Syk, linker activated T cells, and phospholipase C gamma 2. KCG-induced platelet aggregation was abolished in platelets from C-type lectin-like receptor-2 (CLEC-2)-deficient mice, but not in platelets pre-treated with glycoprotein VI-blocking antibody, JAQ1. Surface plasmon resonance assays showed a direct association between murine/human recombinant CLEC-2 and KCG. KCG-induced thrombosis and thrombocytopenia were significantly inhibited in CLEC-2-deficient mice. Our findings show that KCG induces platelet activation via CLEC-2.

Superiority of CS585 as a selective prostacyclin receptor agonist in prevention of thrombosis

Abstract Background A major challenge with development of new targets for antithrombotic treatment is the sustained effect on the platelet and the target selectivity. While increasing cAMP in the platelet through activation of the prostacyclin (IP) receptor represents a viable antiplatelet target for prevention of thrombosis, current IP receptor agonists lack stability and selectivity. Development of a selective IP receptor agonist that is stable in the blood and selective for the IP receptor would represent a new approach for prevention of platelet activation and thrombosis in the blood and expand the utility of IP receptor agonists which are currently used for the treatment of pulmonary arterial hypertension (PAH). Purpose Develop an IV and oral IP receptor agonist, CS585, with sustained activity in vivo in the blood and eliminating the off-target effects observed with other IP agonists not having selective activation of the IP receptor. These characteristics would make CS585 an attractive new therapeutic approach for limiting platelet activation and thrombosis. Methods We assessed platelet activation and thrombosis in human blood ex vivo and mouse models in vivo. For mouse models, platelets and fibrin were labelled and accumulation at the site of injury was measured using intravital microscopy. Thrombosis in the vessel was assessed in the laser-induced cremaster thrombosis assay. Selectivity was assessed in human blood pharmacologically using prostaglandin receptor inhibitors and in mouse with an IP receptor knockout mouse. Flow cytometry, aggregometry, and western blot techniques were used to assess selectivity. Results IV and oral administration of CS585 resulted in sustained inhibited clot formation and fibrin formation at the site of injury in the laser-induced cremaster arteriole thrombosis assay in wild-type mice but was unable to alter platelet function in IP knockout mice. CS585 inhibition of platelet activation was fully prevented in platelets from IP knockout mice as assessed by flow cytometry (integrin activation and granule secretion) and by VASP phosphorylation. Inhibition of human platelet activation by CS585 was inhibited by pharmacological inhibition of the IP receptor, but not by inhibition of the DP1, EP2, or EP4 receptors as assessed by aggregation, flow cytometry, and VASP phosphorylation. Conclusions We have shown for the first time that CS585, a novel IP receptor agonist, not only inhibits platelet activation and clot formation, but gives sustained inhibition through multiple routes of administration in an in vivo mouse model of thrombosis. Importantly, the challenge in selectivity observed with other IP receptor agonists is not observed with CS585 supporting its development for a number of diseases in the cardiovascular system including thrombosis, VTE, secondary prevention after MI and stroke, and PAH.CS585 inhibition of thrombosis in vivoCS585 selectively signals through IP