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Background Phosphatidylserine (PS) is essential for inflammation-associated thrombogenesis, but the exact effect of PS on the prothrombotic state in periodontitis is uncertain. This study aimed to determine the PS-related procoagulant state in patients with periodontitis. Methods A total of 138 patients with periodontitis were examined compared with 42 healthy controls. PS-exposing cells and microvesicles in blood samples were detected by confocal microscopy and flow cytometry. The clotting time assay and prothrombinase complex formation assay were used to measure the procoagulant activity of microvesicles, blood cells and endothelial cells. Periodontal clinical parameters and laboratory characteristics of patients with severe periodontitis were recorded and analyzed at baseline and 6 months after non-surgical periodontal therapy. Results Total PS-positive (PS+ ) microvesicles and the percentage of PS+ blood cells increased in patients with severe periodontitis compared with patients with moderate/mild periodontitis or healthy controls. Endothelial cells cultured in serum from patients with severe periodontitis expressed more PS compared with those cultured in serum from healthy controls. Specifically, PS exposure on blood cells and endothelial cells significantly decreased after inhibiting the effect of inflammatory cytokines. The elevated levels of PS+ cells and microvesicles in severe periodontitis shortened clotting time and led to increased prothrombinase complex formation. Non-surgical periodontal therapy significantly attenuated the release of microvesicles and the PS exposure of blood cells in severe periodontitis. Conclusions The prothrombotic state of patients with periodontitis is mediated by PS+ cells and microvesicles stimulated by elevated levels of inflammatory cytokines.

Patients with COVID-19 often have hypoxemia, impaired lung function, and abnormal imaging manifestations in acute and convalescent stages. Alveolar inflammation, pulmonary vasculitis, and thromboembolism synergistically damage the blood-air barrier, resulting in increased pulmonary permeability and gas exchange disorders. The incidence of low platelet counts correlates with disease severity. Platelets are also involved in the impairment of pulmonary microcirculation leading to abnormal lung function at different phases of COVID-19. Activated platelets lose the ability to protect the integrity of blood vessel walls, increasing the permeability of pulmonary microvasculature. High levels of platelet activation markers are observed in both mild and severe cases, short and long term. Therefore, the risk of thrombotic events may always be present. Vascular endothelial injury, immune cells, inflammatory mediators, and hypoxia participate in the high reactivity and aggregation of platelets in various ways. Microvesicles, phosphatidylserine (PS), platelets, and coagulation factors are closely related. The release of various cell-derived microvesicles can be detected in COVID-19 patients. In addition to providing a phospholipid surface for the synthesis of intrinsic factor Xase complex and prothrombinase complex, exposed PS also promotes the decryption of tissue factor (TF) which then promotes coagulant activity by complexing with factor VIIa to activate factor X. The treatment of COVID-19 hypercoagulability and thrombosis still focuses on early intervention. Antiplatelet therapy plays a role in relieving the disease, inhibiting the formation of the hypercoagulable state, reducing thrombotic events and mortality, and improving sequelae. PS can be another potential target for the inhibition of hypercoagulable states.

Background: In normal cells, homologous recombination (HR) is tightly regulated and plays an important role in the maintenance of genomic integrity and stability through precise repair of DNA damage. RAD51 is a recombinase that mediates homologous base pairing and strand exchange during DNA repair by HR. Our previous data in multiple myeloma and esophageal adenocarcinoma (EAC) show that dysregulated HR mediates genomic instability. Purpose of this study was to investigate role of HR in genomic instability, chemoresistance and immune dysregulation in solid tumors including colon and breast cancers. Methods: The GEO dataset were used to investigate correlation of RAD51 expression with patient survival and expression of various immune markers in EAC, breast and colorectal cancers. RAD51 was inhibited in cancer cell lines using shRNAs and a small molecule inhibitor. HR activity was evaluated using a plasmid-based assay, DNA breaks assessed by evaluating expression of γ-H2AX (a marker of DNA breaks) and p-RPA32 (a marker of DNA end resection) using Western blotting. Genomic instability was monitored by investigating micronuclei (a marker of genomic instability). Impact of RAD51 inhibitor and/or a DNA-damaging agent was assessed on viability and apoptosis in EAC, breast and colon cancer cell lines in vitro and in a subcutaneous tumor model of EAC. Impact of RAD51 inhibitor on expression profile was monitored by RNA sequencing. Results: Elevated RAD51 expression correlated with poor survival of EAC, breast and colon cancer patients. RAD51 knockdown in cancer cell lines inhibited DNA end resection and strand exchange activity (key steps in the initiation of HR) as well as spontaneous DNA breaks, whereas its overexpression increased DNA breaks and genomic instability. Treatment of EAC, colon and breast cancer cell lines with a small molecule inhibitor of RAD51 inhibited DNA breaking agent-induced DNA breaks and genomic instability. RAD51 inhibitor potentiated cytotoxicity of DNA breaking agent in all cancer cell types tested in vitro as well as in a subcutaneous model of EAC. Evaluation by RNA sequencing demonstrated that DNA repair and cell cycle related pathways were induced by DNA breaking agent whereas their induction either prevented or reversed by RAD51 inhibitor. In addition, immune-related pathways such as PD-1 and Interferon Signaling were also induced by DNA breaking agent whereas their induction prevented by RAD51 inhibitor. Consistent with these observations, elevated RAD51 expression also correlated with that of genes involved in inflammation and other immune surveillance. Conclusions: Elevated expression of RAD51 and associated HR activity is involved in spontaneous and DNA damaging agent-induced DNA breaks and genomic instability thus contributing to chemoresistance, immune dysregulation and poor prognosis in cancer. Therefore, inhibitors of RAD51 have great potential as therapeutic agents for EAC, colon, breast and probably other solid tumors.

COVID-19 patients have a high incidence of thrombosis, and thromboembolic complications are associated with severe COVID-19 and high mortality. COVID-19 disease is associated with a hyper-inflammatory response (cytokine storm) mediated by the immune system. However, the role of the inflammatory response in thrombosis remains incompletely understood. In this review, we investigate the crosstalk between inflammation and thrombosis in the context of COVID-19, focusing on the contributions of inflammation to the pathogenesis of thrombosis, and propose combined use of anti-inflammatory and anticoagulant therapeutics. Under inflammatory conditions, the interactions between neutrophils and platelets, platelet activation, monocyte tissue factor expression, microparticle release, and phosphatidylserine (PS) externalization as well as complement activation are collectively involved in immune-thrombosis. Inflammation results in the activation and apoptosis of blood cells, leading to microparticle release and PS externalization on blood cells and microparticles, which significantly enhances the catalytic efficiency of the tenase and prothrombinase complexes, and promotes thrombin-mediated fibrin generation and local blood clot formation. Given the risk of thrombosis in the COVID-19, the importance of antithrombotic therapies has been generally recognized, but certain deficiencies and treatment gaps in remain. Antiplatelet drugs are not in combination with anticoagulant treatments, thus fail to dampen platelet procoagulant activity. Current treatments also do not propose an optimal time for anticoagulation. The efficacy of anticoagulant treatments depends on the time of therapy initiation. The best time for antithrombotic therapy is as early as possible after diagnosis, ideally in the early stage of the disease. We also elaborate on the possible mechanisms of long COVID thromboembolic complications, including persistent inflammation, endothelial injury and dysfunction, and coagulation abnormalities. The above-mentioned contents provide therapeutic strategies for COVID-19 patients and further improve patient outcomes.

Patients with pancreatic cancer (PC) are at increased risk of venous thrombosis, but the precise mechanisms of hypercoagulable state in PC remain unclear. We aimed to identify how phosphatidylserine positive (PS+) blood cells (BCs), PS+ microparticles (MPs) and neutrophil extracellular traps (NETs) regulate procoagulant activity (PCA) in PC, and to assess the relationship between PCA and PC staging. A total of 83 PC patients with different stages of disease were compared to 30 healthy controls, with confocal microscopy and flow cytometry used to assess MP and cellular PS exposure. MP and cell PCA was determined using both fibrin production assays and procoagulant enzyme complex analyses, and coagulation time was further measured. Patients with stage I PC and healthy controls exhibited significantly lower frequencies of PS+ MPs and BCs relative to those with more advanced disease, which may partly due to the increased levels of inflammation cytokines in advanced disease. Functional coagulation assays indicated that PS+ MPs and BCs derived from patients with stage II/III/IV PC directly contribute to elevated FXa, thrombin, and fibrin formation, and to more rapid coagulation relative to healthy control samples. In inhibition assays, lactadherin, which antagonizes PS, led to a roughly 80% inhibition of PCA. We further used isolated NETs to stimulate endothelial cells, revealing that this led to morphological changes including retraction from cell-cell junctions and a more pro-coagulative phenotype, with DNase I and activated protein C treatment reversing these changes. In patients with stage III PC, curative resection surgery significantly reduced PCA, whereas non-curative surgery did not have a marked impact based on studies of pre- and post-operative samples. These results highlight the pathogenic activity of PS+ cells, MPs, and NETs in promoting a prothrombotic environment within individuals suffering from advanced PC. Targeting PS and NETs in these patients may thus be a viable means of preventing pathological thrombosis.

Prior reports indicate that the convex membrane curvature of phosphatidylserine (PS)-containing vesicles enhances formation of binding sites for factor Va and lactadherin. Yet, the relationship of convex curvature to localization of these proteins on cells remains unknown. We developed a membrane topology model, using phospholipid bilayers supported by nano-etched silica substrates, to further explore the relationship between curvature and localization of coagulation proteins. Ridge convexity corresponded to maximal curvature of physiologic membranes (radii of 10 or 30 nm) and the troughs had a variable concave curvature. The benchmark PS probe lactadherin exhibited strong differential binding to the ridges, on membranes with 4% to 15% PS. Factor Va, with a PS-binding motif homologous to lactadherin, also bound selectively to the ridges. Bound factor Va supported coincident binding of factor Xa, localizing prothrombinase complexes to the ridges. Endothelial cells responded to prothrombotic stressors and stimuli (staurosporine, tumor necrosis factor-α [TNF- α]) by retracting cell margins and forming filaments and filopodia. These had a high positive curvature similar to supported membrane ridges and selectively bound lactadherin. Likewise, the retraction filaments and filopodia bound factor Va and supported assembly of prothrombinase, whereas the cell body did not. The perfusion of plasma over TNF-α–stimulated endothelia in culture dishes and engineered 3-dimensional microvessels led to fibrin deposition at cell margins, inhibited by lactadherin, without clotting of bulk plasma. Our results indicate that stressed or stimulated endothelial cells support prothrombinase activity localized to convex topological features at cell margins. These findings may relate to perivascular fibrin deposition in sepsis and inflammation.

The pandemic respiratory illness SARS-CoV-2 has increasingly been shown to be a systemic disease that can also have profound impacts on the cardiovascular system. Although associated cardiopulmonary sequelae can persist after infection, the link between viral infection and these complications remains unclear. There is now a recognized link between endothelial cell dysfunction and thrombosis. Its role in stimulating platelet activation and thrombotic inflammation has been widely reported. However, the procoagulant role of microparticles (MPs) in COVID-19 seems to have been neglected. As membrane vesicles released after cell injury or apoptosis, MPs exert procoagulant activity mainly by exposing phosphatidylserine (PS) on their lipid membranes. It can provide a catalytic surface for the assembly of the prothrombinase complex. Therefore, inhibiting PS externalization is a potential therapeutic strategy. In this paper, we describe the pathophysiological mechanism by which SARS-CoV-2 induces lung and heart complications through injury of endothelial cells, emphasizing the procoagulant effect of MPs and PS, and demonstrate the importance of early antithrombotic therapy. In addition, we will detail the mechanisms underlying hypoxia, another serious pulmonary complication related to SARS-CoV-2-induced endothelial cells injury and discuss the use of oxygen therapy. In the case of SARS-CoV-2 infection, virus invades endothelial cells through direct infection, hypoxia, imbalance of the RAAS, and cytokine storm. These factors cause endothelial cells to release MPs, form MPs storm, and eventually lead to thrombosis. This, in turn, accelerates hypoxia and cytokine storms, forming a positive feedback loop. Given the important role of thrombosis in the disease, early antithrombotic therapy is an important tool for COVID-19. It may maintain normal blood circulation, accelerating the clearance of viruses, waning the formation of MPs storm, and avoiding disease progression.

Genomic instability contributes to cancer progression and is at least partly due to dysregulated homologous recombination. Here, we show that an elevated level of ABL1 kinase overactivates the HR pathway and causes genomic instability in multiple myeloma (MM) cells. Inhibiting ABL1 with either shRNA or a pharmacological inhibitor (nilotinib) inhibits HR activity, reduces genomic instability, and slows MM cell growth. Moreover, inhibiting ABL1 rescues the HR dysregulation and genomic instability caused by melphalan, a chemotherapeutic agent used in MM treatment, and increases melphalan’s efficacy and cytotoxicity in vivo in a subcutaneous tumor model. In these tumors, nilotinib inhibits endogenous as well as melphalan-induced HR activity. These data demonstrate that inhibiting ABL1 using the clinically approved drug nilotinib reduces MM cell growth, promotes genome stability, increases the cytotoxicity of melphalan (and similar chemotherapeutic agents), and can potentially prevent or delay progression in MM patients.

Genomic instability fuels genomic alterations that befit cancer cells with necessary adaptations to keep proliferating and overcome the impact of host anti-tumor immunity and cytotoxic therapy. Since DNA breaks are required for genomic rearrangements to take place, we hypothesized that dysregulated nuclease activity mediates genomic instability in cancer. Using an integrated genomics protocol, we identified a four gene deoxyribonuclease signature correlating with genomic instability in six human cancers which included adenocarcinomas of esophagus (EAC), lung, prostate, stomach, pancreas and triple negative breast cancer. Functional screens confirmed the role of these nucleases in genomic instability and growth of cancer cells. Apurinic/apyrimidinic nuclease 1 (APE1), identified as top nuclease in functional screen, was further investigated in five cell lines representing four solid tumors (EAC, lung, prostate and breast cancer). We demonstrate that chemical as well as transgenic suppression of APE1 impaired growth/colony formation and increased cytotoxicity of chemotherapeutic agent, whereas inhibited spontaneous as well as chemotherapy-induced DNA breaks, homologous recombination (HR) activity and genomic instability in all cancer cell types tested. Treatment with APE1 inhibitor also impaired tumor growth and significantly increased efficacy of a chemotherapeutic agent in a subcutaneous mouse model of EAC. Overexpression of APE1 in normal esophageal epithelial cells increased DNA breaks and HR activity, leading to massive mutational, copy number as well as karyotypic instability. Evaluation of by whole genome sequencing identified HR as the top mutational process activated by APE1. Normal cells overexpressing APE1 grew as tumors in mice and tumors removed from mice displayed additional karyotypic changes, providing evidence of genomic instability in vivo. Overall, our data demonstrate that elevated APE1 dysregulates HR activity, G2/M checkpoint and genome stability thus contributing to tumorigenesis and chemoresistance in cancer. Therefore, inhibitors of APE1 have potential to inhibit growth and increase cytotoxicity of chemotherapeutic agents while minimizing spontaneous as well as chemotherapy-induced genomic damage and instability in EAC and other solid tumors.

The rate of complete remission of acute promyelocytic leukemia (APL) is currently over 90% because of the use of all-trans retinoic acid (ATRA) with arsenic trioxide (ATO). However, hemorrhagic mortality has emerged as the most significant barrier to APL-induced remission. Neutrophils extracellular traps (NETs/ETs) cause vascular leakage by damaging the integrity of endothelial cells. We have previously demonstrated that APL cells treated with ATRA/ATO undergo a cell death process, releasing extracellular chromatin, termed ETosis/NETosis. However, the mechanism underlying the involvement of ETs in endothelial injury in APL remain largely unknown. Here, we analysed the ability of mature and immature neutrophils to release ETs, and their interaction with platelets (PLTs) in APL. Importantly, the effect of ETs on vascular endothelium in APL was discussed. Our results showed that the ability of immature neutrophils to release ETs was impaired in APL, whereas mature neutrophils produced ETs, which were associated with activated PLTs. Moreover, ATRA+ATO induced immature neutrophil differentiation, as well as increased the release of ETs from mature neutrophils. The excessive ETs damaged endothelial cells, causing blood cell leakage. Removing ETs using DNase 1 alleviated endothelial damage and improved blood cells leakage. Our results indicate that vascular endothelial injury is at least partially associated with ETs in APL, and that targeting ETs production may be an effective approach for relieving vascular leakage and reducing the burden of bleeding in APL.

The intestinal tract, with high expression of angiotensin-converting enzyme 2 (ACE2), is a major site of extrapulmonary infection in COVID-19. During pulmonary infection, the virus enters the bloodstream forming viremia, which infects and damages extrapulmonary organs. Uncontrolled viral infection induces cytokine storm and promotes a hypercoagulable state, leading to systemic microthrombi. Both viral infection and microthrombi can damage the gut–blood barrier, resulting in malabsorption, malnutrition, and intestinal flora entering the blood, ultimately increasing disease severity and mortality. Early prophylactic antithrombotic therapy can prevent these damages, thereby reducing mortality. In this review, we discuss the effects of SARS-CoV-2 infection and intestinal thrombosis on intestinal injury and disease severity, as well as corresponding treatment strategies.

The link between hyperuricemia (HUA) and the risk of venous thromboembolism (VTE) has been well established. However, the mechanisms of thrombus generation and the effect of HUA on procoagulant activity (PCA) of erythrocytes remain unclear no matter in uremia or hyperuricemia. Here, phosphatidylserine (PS) exposure, microparticles (MPs) release, cytosolic Ca

Lung injury may persist during the recovery period of COVID-19 as shown through imaging, six-minute walk, and lung function tests. The pathophysiological mechanisms leading to long COVID have not been adequately explained. Our aim is to investigate the basis of pulmonary susceptibility during sequelae and the possibility that prothrombotic states may influence long-term pulmonary symptoms of COVID-19. The patient’s lungs remain vulnerable during the recovery stage due to persistent shedding of the virus, the inflammatory environment, the prothrombotic state, and injury and subsequent repair of the blood-air barrier. The transformation of inflammation to proliferation and fibrosis, hypoxia-involved vascular remodeling, vascular endothelial cell damage, phosphatidylserine-involved hypercoagulability, and continuous changes in serological markers all contribute to post-discharge lung injury. Considering the important role of microthrombus and arteriovenous thrombus in the process of pulmonary functional lesions to organic lesions, we further study the possibility that prothrombotic states, including pulmonary vascular endothelial cell activation and hypercoagulability, may affect long-term pulmonary symptoms in long COVID. Early use of combined anticoagulant and antiplatelet therapy is a promising approach to reduce the incidence of pulmonary sequelae. Essentially, early treatment can block the occurrence of thrombotic events. Because impeded pulmonary circulation causes large pressure imbalances over the alveolar membrane leading to the infiltration of plasma into the alveolar cavity, inhibition of thrombotic events can prevent pulmonary hypertension, formation of lung hyaline membranes, and lung consolidation.

Many discharged COVID-19 patients affected by sequelae experience reduced quality of life leading to an increased burden on the healthcare system, their families and society at large. Possible pathophysiological mechanisms of long COVID include: persistent viral replication, chronic hypoxia and inflammation. Ongoing vascular endothelial damage promotes platelet adhesion and coagulation, resulting in the impairment of various organ functions. Meanwhile, thrombosis will further aggravate vasculitis contributing to further deterioration. Thus, long COVID is essentially a thrombotic sequela. Unfortunately, there is currently no effective treatment for long COVID. This article summarizes the evidence for coagulation abnormalities in long COVID, with a focus on the pathophysiological mechanisms of thrombosis. Extracellular vesicles (EVs) released by various types of cells can carry SARS-CoV-2 through the circulation and attack distant tissues and organs. Furthermore, EVs express tissue factor and phosphatidylserine (PS) which aggravate thrombosis. Given the persistence of the virus, chronic inflammation and endothelial damage are inevitable. Pulmonary structural changes such as hypertension, embolism and fibrosis are common in long COVID. The resulting impaired lung function and chronic hypoxia again aggravates vascular inflammation and coagulation abnormalities. In this article, we also summarize recent research on antithrombotic therapy in COVID-19. There is increasing evidence that early anticoagulation can be effective in improving outcomes. In fact, persistent systemic vascular inflammation and dysfunction caused by thrombosis are key factors driving various complications of long COVID. Early prophylactic anticoagulation can prevent the release of or remove procoagulant substances, thereby protecting the vascular endothelium from damage, reducing thrombotic sequelae, and improving quality of life for long-COVID patients.

Early hemorrhagic death is still the main obstacle for the successful treatment of acute promyelocytic leukemia (APL). However, the mechanisms underlying hemostatic perturbations in APL have not been fully elucidated. Here, we report that CD44 on the membrane of APL blasts and NB4 cells ligated bound fibrinogen, resulting in in situ deposition of fibrin and abnormal fibrin distribution. Clots formed by leukemic cells in response to CD44 and fibrinogen interaction exhibited low permeability and resistance to fibrinolysis. Using flow cytometry and confocal microscopy, we found that CD44 was also involved in platelet and leukemic cell adhesion. CD44 bound activated platelets but not resting platelets through interaction with P-selectin. APL cell-coated fibrinogen-activated platelets directly induce enhanced procoagulant activity of platelets. In vivo studies revealed that CD44 knockdown shortened bleeding time, increased the level of fibrinogen, and elevated the number of platelets by approximately twofold in an APL mouse model. Moreover, CD44 expression on leukemic cells in an APL mouse model was not only associated with bleeding complications but was also related to the wound-healing process and the survival time of APL mice. Collectively, our results suggest that CD44 may be a potential intervention target for preventing bleeding complications in APL.

Tumor progression and cancer metastasis has been linked to the release of microparticles (MPs), which are shed upon cell activation or apoptosis and display parental cell antigens, phospholipids such as phosphatidylserine (PS), and nucleic acids on their external surfaces. In this review, we highlight the biogenesis of MPs as well as the pathophysiological processes of PS externalization and its involvement in coagulation activation. We review the available evidence, suggesting that coagulation factors (mainly tissue factor, thrombin, and fibrin) assist in multiple steps of tumor dissemination, including epithelial–mesenchymal transition, extracellular matrix remodeling, immune escape, and tumor angiogenesis to support the formation of the pre-metastatic niche. Platelets are not just bystander cells in circulation but are functional players in primary tumor growth and metastasis. Tumor-induced platelet aggregation protects circulating tumor cells (CTCs) from the blood flow shear forces and immune cell attack while also promoting the binding of CTCs to endothelial cells and extravasation, which activates tumor invasion and sustains metastasis. Finally, in terms of therapy, lactadherin can inhibit coagulation by competing effectively with coagulation factors for PS binding sites and may similarly delay tumor progression. Furthermore, we also investigate the therapeutic potential of coagulation factor inhibitors within the context of cancer treatment. The development of multiple therapies targeting platelet activation and platelet–tumor cell interactions may not only reduce the lethal consequences of thrombosis but also impede tumor growth and spread.

Multiple myeloma (MM) is a heterogeneous disease characterized by significant genomic instability. Recently, a causal role for the AID/APOBEC deaminases in inducing somatic mutations in myeloma has been reported. We have identified APOBEC/AID as a prominent mutational signature at diagnosis with further increase at relapse in MM. In this study, we identified upregulation of several members of APOBEC3 family (A3A, A3B, A3C, and A3G) with A3G, as one of the most expressed APOBECs. We investigated the role of APOBEC3G in MM and observed that A3G expression and APOBEC deaminase activity is elevated in myeloma cell lines and patient samples. Loss-of and gain-of function studies demonstrated that APOBEC3G significantly contributes to increase in DNA damage (abasic sites and DNA breaks) in MM cells. Evaluation of the impact on genome stability, using SNP arrays and whole genome sequencing, indicated that elevated APOBEC3G contributes to ongoing acquisition of both the copy number and mutational changes in MM cells over time. Elevated APOBEC3G also contributed to increased homologous recombination activity, a mechanism that can utilize increased DNA breaks to mediate genomic rearrangements in cancer cells. These data identify APOBEC3G as a novel gene impacting genomic evolution and underlying mechanisms in MM.

Background It is important to explore effective treatment for liver cancer. Photodynamic therapy (PDT) is a novel technique to treat liver cancer, but its clinical application is obstructed by limited depth of visible light penetration into tissue. The near-infrared (NIR) photosensitizer is a potential solution to the limitations of PDT for deep tumor tissue treatment. Purpose We aimed to investigate 808 nm NIR light-excited UCNPs@mSiO2-Ce6-GPC3 nanocomposites for PDT in liver cancer. Methods In our study, 808 nm NIR light-excited upconversion nanoparticles (UCNPs) were simultaneously loaded with the photosensitizer chlorin e6 (Ce6) and the antibody glypican-3 (GPC3), which is overexpressed in hepatocellular carcinoma cells. The multitasking UCNPs@mSiO2-Ce6-GPC3 nanoparticles under 808 nm laser irradiation with enhanced depth of penetration would enable the effective targeting of PDT. Results We found that the UCNPs@mSiO2-Ce6-GPC3 nanoparticles had good biocompatibility, low toxicity, excellent cell imaging in HepG2 cancer cells and high anti-tumor effect in vitro and in vivo. Conclusion We believe that the utilization of 808 nm NIR excited UCNPs@mSiO2-Ce6-GPC3 nanoparticles for PDT is a safe and potential therapeutic option for liver cancer.

Patients with colorectal cancer (CRC) are at increased risk of venous thrombosis, but the precise mechanisms of thrombogenesis in CRC remain largely unknown. We aimed to identify the novel role of neutrophil extracellular traps (NETs) in the induction of procoagulant activity (PCA) in CRC, and to evaluate its interactions with platelets and endothelial cells (ECs). In this study, we first showed that the levels of NETs in the peripheral blood of CRC patients were increased in parallel with cancer progression and reached significance in stage II patients compared to healthy subjects. In addition, neutrophils from CRC patients were more prone to produce NETs, resulting in shortened coagulation time, significantly increased thrombin-antithrombin (TAT) complexes and fibrin fibrils compared to healthy controls. Furthermore, platelets from CRC patients stimulated healthy neutrophils to extrude NETs, which could be inhibited by the depletion of HMGB1. Conversely, NETs from CRC patients could also induce the exposure of PS on platelets, leading to markedly enhanced PCA. Importantly, ECs were also converted to a procoagulant phenotype when exposed to NETs from CRC patients. The PCA of NETs-activated platelets or ECs could be inhibited either by the cleavage of NETs with DNase1 or the blockage of histone with activated protein C (APC). Our results reveal the complex interactions between neutrophils, platelets and ECs and their potential role in the hypercoagulable state in CRC. We propose that NETs may provide new therapeutic targets to combat the thrombotic consequences of CRC.

Background and Aims Despite the presence of neutrophil extracellular traps [NETs] in inflamed colon having been confirmed, the role of NETs, especially the circulating NETs, in the progression and thrombotic tendency of inflammatory bowel disease [IBD] remains elusive. We extended our previous study to prove that NETs constitute a central component in the progression and prothrombotic state of IBD. Methods In all 48 consecutive patients with IBD were studied. Acute colitis was induced by the treatment of C57BL/6 mice with 3.5% dextran sulphate sodium [DSS] in drinking water for 6 days. Peripheral blood neutrophils and sera were collected from IBD patients and murine colitis models. Exposed phosphatidylserine [PS] was analysed with flow cytometry and confocal microscopy. Procoagulant activity was evaluated using clotting time, purified coagulation complex, and fibrin formation assays. Results We observed higher plasma NET levels and presence of NETs in colon tissue in patients with active IBD. More importantly, NETs were induced in mice with DSS colitis, and inhibition of NET release attenuated colitis as well as colitis-associated tumorigenesis. NET degradation through DNase administration decreased cytokine levels during DSS-induced colitis. In addition, DNase treatment also significantly attenuated the accelerated thrombus formation and platelet activation observed in DSS-induced colitis. NETs triggered PS-positive microparticle release and PS exposure on platelets and endothelial cells partially through TLR2 and TLR4, converting them to a procoagulant phenotype. Conclusions NETs exacerbate colon tissue damage and drive thrombotic tendency during active IBD. Strategies directed against NET formation may offer a potential therapeutic approach for the treatment of IBD.

Relatively little information is known about the definitive role of phosphatidylserine (PS) in the hypercoagulability of heart failure (HF). Our objectives were to assess the levels of PS exposure on microparticles (MPs) and blood cells (BCs) in each group of HF patients and to evaluate their procoagulant activity (PCA). HF patients in each NYHA functional class II–IV (II n = 30, III n = 30, IV n = 30) and healthy controls (n = 25) were enrolled in the present study. PS exposure on MPs, BCs was analyzed with flow cytometry. MPs were classified based on their cellular origin: platelets (CD41a+), neutrophils (CD66b+), endothelial cells (CD31+CD41a−), erythrocytes (CD235a+), monocytes (CD14+), T lymphocytes (CD3+), and B lymphocytes (CD19+). PCA was evaluated by clotting time, extrinsic/intrinsic FXa and prothrombinase production assays, as well as fibrin formation assays. Inhibition assays of PCA of PS+ BCs and MPs were performed by lactadherin. There was no significant difference in MP cellular origin between healthy and HF subjects. However, the total number of PS+ MPs was significantly increased in HF patients compared with healthy controls. In addition, circulating PS+ BCs cooperated with PS+ MPs to markedly shorten coagulation time and dramatically increase FXa/thrombin generation and fibrin formation in each HF group. Moreover, blockade of exposed PS on BCs and MPs with lactadherin inhibited PCA by approximately 80%. Our results lead us to believe that exposing PS on the injured BCs and MPs played a pivotal role in the hypercoagulability state in HF patients.

Circulating neutrophil extracellular traps (NETs) resistant to t-PA have not been studied completely although NETs in thrombi may contribute to tissue plasminogen activator (t-PA) resistance. This research intended to elucidate whether circulating NETs are associated with t-PA resistance and the underlying mechanism. The levels of NETs were detected in the circulating neutrophils, ischemic brain tissue of acute ischemic stroke (AIS) patients, and transient middle cerebral artery occlusion (tMCAO) models. NET formation in blood, thrombi, and ischemic brain tissue of mice were analyzed by immunofluorescence. Exposed phosphatidylserine (PS) was assessed using flow cytometry and confocal microscopy. Procoagulant activity (PCA) was evaluated using fibrin formation assays, thrombin, and purified coagulation complex. The plasma levels of NETs in AIS patients were significantly higher than those in healthy individuals. After thrombolysis, a significant increase was noted in NET markers in no-improvement patients, while the changes in improvement patients were not significant. Importantly, NETs were decorated with von Willebrand factor (vWF) and plasminogen activator inhibitor-1 (PAI-1) in the blood and thrombi, which could reverse the fibrinolytic effects. In addition, NETs activated platelets (PLTs) and endothelial cells (ECs), stimulating a procoagulant phenotype and facilitating vWF and PAI-1 release. DNase I, activated protein C (APC), and sivelestat markedly inhibited these effects. Furthermore, targeting NETs protected mice from tMCAO-induced cerebral ischemia, possibly by regulating vWF and PAI-1. In summary, NETs may contribute to t-PA resistance in AIS through activation of PLTs and ECs. Strategies against NETs may present a promising therapeutic approach to improve the thrombolysis efficiency of t-PA in AIS patients.

The link between serum uric acid (SUA) and the risk of venous thromboembolism (VTE) is well established. Recent data suggested a causative role of UA in endothelial cells (ECs) dysfunction. However, the molecular mechanism of high UA on thrombogenesis is unknown. We investigate whether high UA induce phosphatidylserine (PS) externalization and microparticle (MP) shedding in cultured EC, and contribute to UA-induced hypercoagulable state. In the present study, we demonstrate that UA induces PS exposure and EMP release of EC in a concentration- and time-dependent manner, which enhances the procoagulant activity (PCA) of EC and inhibited over 90% by lactadherin in vitro. Furthermore, hyperuricemic rat model was used to evaluate the development of thrombi following by flow stasis in the inferior vena cava (IVC). Hyperuricemia group is more likely to form large and hard thrombi compared with control. Importantly, we found that TMEM16F expression is significantly upregulated in UA-treated EC, which is crucial for UA-induced PS exposure and MP formation. Additionally, UA increases the generation of reactive oxygen species (ROS), lipid peroxidation, and cytosolic Ca2+ concentration in EC, which might contribute to increased TMEM16F expression. Using confocal microscopy, we also observed disruption of the actin cytoskeleton, suggesting that depolymerization of actin filaments might be required for TMEM16F activation and followed by PS exposure and membrane blebbing in UA-treated EC. Our results demonstrate a thrombotic role of EC in hyperuricemia through TMEM16F-mediated PS exposure and MPs release.

COVID-19 has swept quickly across the world with a worrisome death toll. SARS-CoV-2 infection induces cytokine storm, acute respiratory distress syndrome with progressive lung damage, multiple organ failure, and even death. In this review, we summarize the pathophysiologic mechanism of neutrophil extracellular traps (NETs) and hypoxia in three main phases, focused on lung inflammation and thrombosis. Furthermore, microparticle storm resulted from apoptotic blood cells are central contributors to the generation and propagation of thrombosis. We focus on microthrombi in the early stage and describe in detail combined antithrombotic with fibrinolytic therapies to suppress microthrombi evolving into clinical events of thrombosis. We further discuss pulmonary hypertension causing plasmin, fibrinogen and albumin, globulin extruding into alveolar lumens, which impedes gas exchange and induces severe hypoxia. Hypoxia in turn induces pulmonary hypertension, and amplifies ECs damage in this pathophysiologic process, which forms a positive feedback loop, aggravating disease progression. Understanding the mechanisms paves the way for current treatment of COVID-19 patients.

COVID-19 is widely epidemic in the world and poses a great threat to our life. Coagulopathy is one of the major characteristics in the COVID-19 patients. A growing number of studies have found that the severe COVID-19 patients have thrombotic microangiopathy and thromboembolism. Coagulopathy associated with increased risk of death in the patients. Unfortunately, the mechanism of coagulopathy is not clearly addressed. Understanding the pathophysiological mechanism of COVID-19 thrombosis and improving the coagulopathy through efficient treatment may help to stop disease progression, reduce mortality and sequelae. In severe COVID-19 patients, inflammation, cytokine storm, and coagulation are closely related, which together cause blood congestion and thrombosis. Many cytokines activate blood cells, expressing activating factors or releasing activated microparticles, and then accelerating thrombosis. However, the role of blood cells is not well understood in COVID-19 patients. In addition, cytokines stimulate endothelial cells, transforming them into a procoagulant phenotype. Therefore, determine their role and propose new strategies for the prevention and treatment of thrombosis in severe COVID-19 patients. We outline the major events of coagulopathies, discuss the role of blood and endothelial cells in thrombosis, to formulate a new anticoagulation protocol.

Background: Neoadjuvant chemotherapy is relevant to the formation of thromboembolism and secondary neoplasms in triple-negative breast cancer (TNBC). Chemotherapy-induced breast cancer cell-derived microparticles (BCMPs) may have important thrombogenic and pro-metastatic effects on platelets and endothelium, which may be related to the expression and distribution of phosphatidylserine (PS). However, investigating these interactions is challenging due to technical limitations. Methods: A study was conducted in 20 healthy individuals and 18 patients who had been recently diagnosed with TNBC and were undergoing neoadjuvant chemotherapy with doxorubicin and cyclophosphamide. BCMPs were isolated from patient blood samples and doxorubicin-treated breast cancer cell lines. Their structure and morphology were studied by electron microscopy and antigen levels were measured by fluorescence-activated cell sorting. In an inhibition assay, isolated BCMPs were pretreated with lactadherin or tissue factor antibodies. Platelets isolated from healthy subjects were treated with BCMPs and coagulation time, fibrin formation, and expression of intrinsic/extrinsic factor Xase (FXa) and thrombin were evaluated. The effects of BCMPs on endothelial thrombogenicity and integrity were assessed by confocal microscopy, electron microscopy, measurement of intrinsic/extrinsic FXa, prothrombinase assay, and transwell permeability assay. Results: Neoadjuvant chemotherapy significantly increased the expression of PS+ BCMPs in patient plasma. Its expression was associated with a rapid increase in procoagulant activity. Treatment with lactadherin, a PS-binding scavenging molecule, markedly reduced the adhesion of BCMPs and abolished their procoagulant activity, but this was not observed with tissue factor antibody treatment. Intravenous injection of BCMPs in mice induced a significant hypercoagulable state, reducing the extent of plasma fibrinogen and promoting the appearance of new thrombus. Cancer cells incubated with doxorubicin released large numbers of PS+ BCMPs, which stimulated and transformed endothelial cells into a procoagulant phenotype and increased the aggregation and activation of platelets. Moreover, cancer cells exploited this BCMP-induced endothelial leakiness and showed promoted metastasis. Pretreatment with lactadherin increased uptake of both PS+ BCMPs and cancer cells by endothelial cells and limited the transendothelial migration of cancer cells. Conclusion: Lactadherin, a biosensor that we developed, was used to study the extracellular vesicle distribution of PS, which revealed a novel PS+ BCMPs administrative axis that initiated a local coagulation cascade and facilitated metastatic colonization of circulating cancer cells.

During the coronavirus disease 2019 (COVID‑19) pandemic, some patients with severe COVID‑19 exhibited complications such as acute ischemic stroke (AIS), which was closely associated with a poor prognosis. These patients often had an abnormal coagulation, namely, elevated levels of D‑dimer and fibrinogen, and a low platelet count. Certain studies have suggested that COVID‑19 induces AIS by promoting hypercoagulability. Nevertheless, the exact mechanisms through which COVID‑19 leads to a hypercoagulable state in infected patients remain unclear. Understanding the underlying mechanisms of hypercoagulability is of utmost importance for the effective treatment of these patients. The present review aims to summarize the current status of research on COVID‑19, hypercoagulability and ischemic stroke. The present review also aimed to shed light into the underlying mechanisms through which COVID‑19 induces hypercoagulability, and to provide therapies for different mechanisms for the more effective treatment of patients with COVID‑19 with ischemic stroke and prevent AIS during the COVID‑19 pandemic.

Background & aims Patients with obstructive jaundice (OJ) are considered to be prothrombotic with increased risk of thromboembolism complications. The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) and thrombosis risk in patients with OJ is unclear. In this study, we investigated NETs formation in OJ patients and the role of elevated unconjugated bilirubin (UCB) in inducing NETs, resulting in enhanced PCA and endothelial injury. Methods NETs of OJ patients and healthy controls were measured. NETs PCA was assessed via coagulation time (CT), fibrin formation and purified coagulation complex production assays. Visualization of NETs and mitochondrial reactive oxygen species (MitoROS) were performed with a fluorescence microscope. We further used confocal microscopy to quantify the exposure of phosphatidylserine (PS), fibrin strands and FVa/Xa on Human umbilical vein endothelial cells (HUVECs). Results Assessment of NETs components levels revealed greater NETs production in OJ patients than in healthy controls. Importantly, OJ-NETs were responsible for enhanced PCA. UCB induced NETs formation via MitoROS accumulation and mitochondrial mobilization. HUVECs cocultured with OJ NETs lost their cell-cell junctions and consequently converted to a procoagulant phenotype. The PCA was attenuated by using DNase I alone or in combination with lactadherin. Conclusions Our results suggest that UCB-induced NETs play a prominent role in promoting the hypercoagulable and prothrombotic state in OJ patients. The increased MitoROS accumulation in neutrophils initiated NETosis. NETs are promising targets for indicating or improving coagulation disorders in OJ patients.

Identification of genes driving genomic evolution can provide novel targets for cancer treatment and prevention. Here we show identification of a genomic instability gene signature, using an integrated genomics approach. Elevated expression of this signature correlated with poor survival in esophageal adenocarcinoma (EAC) as well as three other human cancers. Knockout and overexpression screens confirmed the relevance of this signature to genomic instability. Indepth evaluation of TTK (a kinase), TPX2 (spindle assembly factor) and RAD54B (recombination protein) further confirmed their role in genomic instability and tumor growth. Mutational signatures identified by whole genome sequencing and functional studies demonstrated that DNA damage and homologous recombination were common mechanisms of genomic instability induced by these genes. Consistently, a TTK inhibitor impaired EAC cell growth in vivo, and increased chemotherapy-induced cytotoxicity while inhibiting genomic instability in surviving cells. Thus inhibitors of TTK and other genes identified in this study have potential to inhibit/delay genomic evolution and tumor growth. Such inhibitors also have potential to increase chemotherapy-induced cytotoxicity while reducing its harmful genomic impact in EAC and possibly other cancers.

Background The definitive role of phosphatidylserine (PS) in the prothrombotic state of non-valvular atrial fibrillation (NVAF) remains unclear. Our objectives were to study the PS exposure on blood cells and microparticles (MPs) in NVAF, and evaluate their procoagulant activity (PCA). Methods NVAF patients without ( n = 60) and with left atrial thrombi ( n = 18) and controls ( n = 36) were included in our study. Exposed PS was analyzed with flow cytometry and confocal microscopy. PCA was evaluated using clotting time, factor Xa (FXa), thrombin and fibrin formation. Results PS + blood cells and MPs were significantly higher in NVAF patients without and with left atrial thrombi (both P + platelets, neutrophils, erythrocytes and MPs compared with patients without thrombi (all P + blood cells and MPs contributed to markedly shortened coagulation time and dramatically increased FXa/thrombin/fibrin (all P + platelets and MPs was positively correlated with thrombus diameter (all p Conclusions Our results suggest that exposed PS on blood cells and MPs play a procoagulant role in NVAF patients. Blockade of PS prior to thrombus formation might be a novel therapeutic approach in these patients.

BACKGROUND Nonalcoholic steatohepatitis (NASH) patients are at a high risk of developing venous thromboembolism, with a high rate of morbidity and mortality. The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) in patients with NASH remains unclear. Our study aimed to investigate the formation of NETs in NASH patients stimulated by specific pro-inflammatory factors. Moreover, we evaluated the pivotal role of NETs in the induction of hypercoagulability in NASH and the interaction between NETs and endothelial injury. METHOD The levels of the NETs biomarkers were evaluated in the plasma samples of 27 NASH patients and 18 healthy subjects. The formation of NETs was visualized using immunofluorescence microscopy. The PCA of the NETs was assessed using coagulation time, purified coagulation complex, and fibrin formation assays. Confocal microscopy was further used to evaluate the interactions between the NETs and HUVECs. RESULTS The levels of NETs markers in the plasma of NASH patients were significantly higher than healthy controls. NETs derived from NASH enhanced thrombin and fibrin formation and significantly reduced CT (p

Background/Aims: The mechanisms for thrombosis in diabetic retinopathy (DR) are complex and need to be further elucidated. The purpose of this study was to test phosphatidylserine (PS) exposure on microparticles (MPs) and MP-origin cells from the circulation and to analyze cell-/MP-associated procoagulant activity (PCA) in DR patients. Methods: PS-positive MPs and cells from healthy controls (n = 20) and diabetic patients (n = 60) were analyzed by flow cytometry and confocal microscopy. Clotting time and purified coagulation complex assays were used to measure PCA. Results: PS exposure on platelets and monocytes was higher in proliferative DR (PDR) patients than in non-PDR patients or controls. The highest levels of MPs (derived from platelets [30%], erythrocytes [13%], leukocytes [28%], and endothelial cells [10%]) were found in patients with PDR. In addition, PS exposure on blood cells and shed MPs in DR patients led to significantly increased FXa and FIIa generation, fibrin formation, and markedly shortened coagulation time. Moreover, lactadherin reduced 70% of PCA by blocking PS, while an anti-tissue factor antibody had a smaller effect. Conclusion: Our results confirmed that PCA in DR patients may be partly ascribed to PS exposure and MP release from blood and endothelial cells. Lactadherin may act as an efficient anticoagulant factor in this process.

Multiple myeloma (MM) is molecularly heterogenous disease with significant genomic instability. It carries number of mutations at diagnosis (median > 7000) and acquires additional changes overtime. With this background, we have evaluated the molecular intermediates of genomic instability in MM. Based on our large transcriptomic data we have identified apurinic/apyrimidinic deoxyribonuclease (APEX) as an important target whose elevated activity contributes to dysregulation of homologous recombination (HR) and genome stability in MM. Our investigation also demonstrates that transgenic overexpression of APEX nucleases induces genomic instability, leading to oncogenic transformation and tumorigenesis in a murine and Zebrafish models. Importantly, we have now observed that both transgenic as well as chemical inhibition of APEX1, reduces DNA breaks, HR activity and genomic instability as measured by micronucleus assay, and induces G2/M arrest in myeloma as well as esophageal cancer cells. To identify novel and effective inhibitors of APEX1, we optimized a high throughput APEX1 activity assay and screened a custom library of 100,000 small molecules. We identified API-93 as an effective APEX inhibitor in both the primary and secondary screens, and have now investigated it, alone as well as in combination, with existing myeloma drugs, for impact on different parameters of growth and genome maintenance. Although API-93 had minimal single agent cytotoxic effect on MM cells, it synergistically increased cytotoxicity of chemotherapeutic agent cyclophosphamide in both MM cell lines (MM1S and RPMI) tested, and also increased the efficacy of melphalan in several MM cell lines (RPMI, MM1R, KK1 and LR5) tested. A strong synergistic effect of API-93 was also observed in combination with lenalidomide in 5 MM cell lines tested (RPMI, MM1S, MM1R, KK1, H929; combination indexes < 1) as well as velcade in MM cells. For evaluation of impact on genomic changes, myeloma cells were treated with API-93, live cells purified and evaluated for impact on DNA breaks (by measuring levels of γ-H2AX), DNA end resection (a decisive step in the initiation of HR, by monitoring levels of p-RPA32), and genome stability by investigating micronuclei (the marker of genomic instability). Treatment with API-93 reduced spontaneous as well as melphalan-induced DNA breaks, DNA end resection as well as genomic instability (as assessed by significant reduction in micronuclei) in MM cells, in a dose-dependent manner. To further confirm the impact on genome stability, the MM cells were cultured in the presence or absence of API-93, and the acquisition of new copy number changes over 3 weeks were measured using SNP arrays, in the cultured relative to Day 0 cells (representing baseline genome). Relative to control cells, the cells treated with API-93 resulted in dose-dependent decrease in the acquisition of new copy number events, from 50% to > 90%. In conclusion, these data demonstrate APEX gene as being associated with MM cell survival and with genome instability. The novel APEX1 inhibitor, identified in a functional screen, provides an important tool to augment cytotoxicity of the current therapeutics while significantly decreasing genomic evolution in MM. Disclosures Munshi: Adaptive: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Legend: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy.

Background It is important to explore effective treatment for liver cancer. Photodynamic therapy (PDT) is a novel technique to treat liver cancer, but its clinical application is obstructed by limited depth of visible light penetration into tissue. The near-infrared (NIR) photosensitizer is a potential solution to the limitations of PDT for deep tumor tissue treatment. Purpose We aimed to investigate 808 nm NIR light-excited UCNPs@mSiO2-Ce6-GPC3 nanocomposites for PDT in liver cancer. Methods In our study, 808 nm NIR light-excited upconversion nanoparticles (UCNPs) were simultaneously loaded with the photosensitizer chlorin e6 (Ce6) and the antibody glypican-3 (GPC3), which is overexpressed in hepatocellular carcinoma cells. The multitasking UCNPs@mSiO2-Ce6-GPC3 nanoparticles under 808 nm laser irradiation with enhanced depth of penetration would enable the effective targeting of PDT. Results We found that the UCNPs@mSiO2-Ce6-GPC3 nanoparticles had good biocompatibility, low toxicity, excellent cell imaging in HepG2 cancer cells and high anti-tumor effect in vitro and in vivo. Conclusion We believe that the utilization of 808 nm NIR excited UCNPs@mSiO2-Ce6-GPC3 nanoparticles for PDT is a safe and potential therapeutic option for liver cancer.

Despite routine treatment of unselected acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA), early death because of hemorrhage remains unacceptably common, and the mechanism underlying this complication remains elusive. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, which involves release of extracellular chromatin. However, the role of promyelocytic extracellular chromatin in APL-associated coagulation remains unclear. Our objectives were to identify the novel role of ATRA-promoted extracellular chromatin in inducing a hypercoagulable and hyperfibrinolytic state in APL and to evaluate its interaction with fibrin and endothelial cells (ECs). Results from a series of coagulation assays have shown that promyelocytic extracellular chromatin increases thrombin and plasmin generation, causes a shortening of plasma clotting time of APL cells, and increases fibrin formation. DNase I but not anti-tissue factor antibody could inhibit these effects. Immunofluorescence staining showed that promyelocytic extracellular chromatin and phosphatidylserine on APL cells provide platforms for fibrin deposition and render clots more resistant to fibrinolysis. Additionally, coincubation assays revealed that promyelocytic extracellular chromatin is cytotoxic to ECs, converting them to a procoagulant phenotype. This cytotoxity was blocked by DNase I by 20% or activated protein C by 31%. Our current results thus delineate the pathogenic role of promyelocytic extracellular chromatin in APL coagulopathy. Furthermore, the remaining coagulation disturbance in high-risk APL patients after ATRA administration may be treatable by intrinsic pathway inhibition via accelerating extracellular chromatin degradation.

Background: In normal cells, RAD51-mediated homologous recombination (HR) is a precise DNA repair mechanism which plays a key role in the maintenance of genomic integrity and stability. However, elevated (dysregulated) RAD51 is implicated in genomic instability and is a potential target for treatment of certain cancers, including Barrett’s adenocarcinoma (BAC). In this study, we investigated genomic impact and translational significance of moderate vs. strong suppression of RAD51 in BAC cells. Methods: BAC cells (FLO-1 and OE33) were transduced with non-targeting control (CS) or RAD51-specific shRNAs, mediating a moderate (40-50%) suppression or strong (80-near 100%) suppression of the gene. DNA breaks, spontaneous or following exposure to DNA damaging agent, were examined by comet assay and 53BP1 staining. Genomewide expression was monitored by microarrays (Affymetrix). Genomic instability was evaluated by Alu-PCR and Homologous recombination (HR) and single strand annealing (SSA) activities measured using plasmid based assays. Results: We previously showed that suppression of RAD51 significantly reduces the acquisition of genomic changes over time, as assessed by SNP arrays (Affymetrix). Here, we show that although moderate suppression consistenly inhibits/reduces HR activity, the strong suppression is associated with increase in HR activity (by ~15 - ³ 50% in various experiments), suggesting activation of RAD51-independent pathway. Contrary to moderate suppression, a strong suppression of RAD51 is associated with a significant increase in spontaneous as well as UV-induced DNA breaks as well as altered expression of genes involved in detection/processing of DNA breaks and apoptosis. Stronger RAD51 suppression was also associated with mutagenic single strand annealing mediated HR with evidence of increased genomic instability. Suppression of RAD51C inhibited RAD51-independent (SSA-mediated) HR, indicating its role in this process. Conclusion: Elevated (dysregulated) RAD51 in BAC is implicated in both the repair of DNA breaks as well as ongoing genomic rearrangements. Moderate suppression of this gene reduces HR activity, whereas strong or near complete suppression of this gene activates RAD51C-dependent HR involving a mechanism known as single strand annealing (SSA). SSA-mediated HR, which is a mutagenic HR pathway, further disrupts genomic integrity by increasing DNA breaks and acquisition of new genomic changes in BAC cells. We, therefore, conclude that inhibition of RAD51C, alone or in combination with RAD51 suppression, has potential to make BAC cells static.

Background Relatively little is known about the role of phosphatidylserine (PS) in procoagulant activity (PCA) in patients with non-ST-elevated myocardial infarction (NSTEMI) after stent implantation. This study was designed to evaluate whether exposed PS on microparticles (MPs) and blood cells were involved in the hypercoagulable state in NSTEMI patients with stent implantation. Methods NSTEMI patients (n=90) and healthy controls (n=20) were included in our study. PS exposure on MPs and blood cells was analyzed with flow cytometer and confocal microscope. PCA was evaluated by clotting time, purified coagulation complex assays and fibrin production assays. Results Baseline levels of MPs and PS + blood cells were significantly higher (P + blood cells. Specifically, PS + MPs, PS + platelets and erythrocytes peaked at 18h following stent implantation, while PS + leukocytes peaked on day 2. In addition, circulating MPs (mostly derived from platelets, leukocytes, erythrocytes and endothelial cells) cooperating with PS + blood cells, contributed to markedly shortened coagulation time and markedly increased FXa/thrombin/fibrin (all P Conclusions Our results suggest that PS + MPs and blood cells play a procoagulant role in NSTEMI patients following stent implantation. Blockade of PS could become a novel therapeutic modality for the prevention of thrombosis in these patients.

The role of phosphatidylserine (PS)-mediated procoagulant activity (PCA) in stroke remains unclear. To ascertain this role, early dynamic evolution of PS exposure on blood cells and released microparticles (MPs) and the corresponding PCA were evaluated in patients with acute ischemic stroke (AIS). Flow cytometry analyses revealed that initial levels of PS exposure on erythrocyte, platelet, and leukocyte were 2.40-, 1.36-, and 1.38-fold higher, respectively, in AIS than the risk factor-matched (RF) controls. Concomitantly, total PS+ MPs were increased in AIS (1949 ± 483/μl) compared with the RF group (1674 ± 387/μl; P = 0.019) and healthy controls (1052 ± 179/μl; P

We evaluated cellular origin, numbers, and procoagulant activity of phosphatidylserine-positive microparticles (MPs) among subgroups in acute coronary syndromes (ACS).Parameters were measured on admission, days 1 (within 24 h of admission), 2, 3, and 7. All ST-elevated myocardial infarction (STEMI) patients presented more than 3 h from symptom onset and received fibrinolysis treatment; controls included unstable angina and non-STEMI patients as well as healthy controls. Phosphatidylserine-positive MPs were detected by flow cytometry, whereas procoagulant activity was assessed by coagulation time, purified coagulation complex assays, and fibrin formation. MP-induced fibrins were visualized by confocal microscopy.On admission, the total MP count was ∼2.5-fold higher in the ACS groups compared with the healthy controls (P0.05), primarily originating from platelets and endothelial cells, and there were no significant differences among ACS subgroups. Specifically, leukocyte-derived and erythrocyte-derived MPs were higher in the STEMI group compared with unstable angina and non-STEMI groups (both P0.05). Further, MPs from the ACS groups reduced coagulation time by 27.5% and induced intrinsic and extrinsic FXase, prothrombinase, and fibrin formation by 2.8-, 2.3-, 2.5-, and 1.7-fold, respectively (P0.05 for all), whereas blocking phosphatidylserine with lactadherin inhibited ∼70% of procoagulant activity. MP number and concomitant coagulation decreased significantly by day 2 and continued to decrease gradually during the recovery period.This study shows that MP characteristics from circulating blood may be used as prognostic indicators to reflect the origin cell of activation and thrombophilic states found in ACS subgroups.

Dysregulated homologous recombination (HR) contributes to the acquisition of genomic changes and development of drug resistance over time in multiple myeloma (MM). We have now further investigated the molecular intermediates that may play an important role in HR process. We observe that ABL kinase, which regulates RAD51 through its phosphorylation, contributes to increased HR activity and genomic instability in myeloma. Moreover, Cellular response after DSBs involves nuclear re-localization of ABL1 upon DNA damage. A cellular localization of ABL1 has been confirmed in MM. Consistently, we observe that ABL kinase inhibitor reduces HR activity and genomic instability (as assessed by micronucleus assay) in MM cells. Based on our observation that melphalan increases genomic instability (as assessed by micronuclei assay and by whole genome sequencing), we sought to investigate impact of melpahalan on HR and role of ABL1 kinase in this process. We show that treatment with melphalan leads to increase in RAD51 expression and HR activity in MM cells in a dose-dependent manner. Evaluation by RNA sequencing showed that treatment of MM1S cells with melphalan is associated with upregulation of p53 signaling (containing multiple genes involved in DNA damage, detection of damaged sites, recombination/repair and genomic instability) as the topmost pathway. These findings suggest that melphalan-induced DNA damage leads to a concerted overexpression of genes involved in DNA damage response, recombination, genomic instability and chemoresistance. With the role of ABL1-kinase following DSB, we investigated and report that treatment with melphalan induces micronuclei formation, whereas nilotinib significantly reduces both the basal and melphlalan-induced micronuclei in all MM cell lines tested. We have now confirmed these observations by evaluating copy number alterations using single nucleotide polymorphism (SNP) arrays. To evaluate the impact of nilotinib, melphalan and their combination on genomic instability, we cultured MM cells in the presence of nilotinib (2.5 µM), melphalan (1 µM) and combination of both drugs for three weeks and investigated the acquisition of new copy number events, relative to "day 0" cells (serving as baseline genome), using SNP arrays. Treatment with melphalan led to massive increase in the acquisition of amplification and deletion events, whereas nilotinib not only reduced the acquisition of copy number events under spontaneous condition but also almost completely reversed/prevented those induced by melphlalan. Importantly, the treatment with nilotinib could also significantly sensitized MM cell lines and bone marrow plasma cells from relapsed MM patients to melphalan treatment. These data confirm that ABL1 inhibition reduces spontaneous and melphalan-induced genomic instability in MM cells. Since components of cell cycle play critical role in the maintenance of genome stability and growth, we investigated the impact of ABL1-inhibitor nilotinib, alone and in combination with melphalan, on cell cycle. Treatment of MM1S cells with melphalan for 48hrs led to 2.5-fold increase in the accumulation of cells in S-phase, suggesting an increase in replication stress by melphalan. Nilotinib reduced both the spontaneous and melphalan-induced fraction of S-phase cells by 27% (±2%) and 30% (±1%), respectively. Combined treatment with nilotinib and melphalan also increased sub G1 fraction of cells by 2.3-fold compared to those treated with melphalan alone, suggesting increased apoptosis in these cells. These data are consistent with our observation that treatment of MM cells with melphalan increase the phosphorylation of RPA32, a marker of replication stress while nilotinib reduces both the endogenous and melphalan-induced phosphorylated RPA32 level, suggesting that nilotinib might also be helpful in combating replication stress-mediated genomic instability. Taken together, these data demonstrate the critical role of ABL1-kinase in both spontaneous and drug (melphalan)-induced genomic instability and its inhibition could reduce/delay genomic evolution while enhancing cytotoxicity in multiple myeloma. Disclosures Fulciniti: NIH: Research Funding. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy.

Multiple myeloma (MM) is a genomically heterogenous malignancy characterized by a number of copy number alterations (CNA). Moreover, there is a clear evolution of genomic changes that may affect prognosis. To identify the drivers of this inherent genomic instability, we applied an integrated genomics approach utilizing genomic data for copy number changes and transcriptomic profile. We first identified genes whose expression correlated with total copy number events in a patient dataset (gse26863, n=246). We then applied those genes to identify the genes whose elevated expression correlated with both overall and event free survival in two different datasets (IFM70, n=170; gse2408, n=559). Elevated expression of this 30 gene signature also correlated with poor overall as well as event free survival in a third myeloma dataset (MMRF; P In summary, we demonstrate that elevated ATAD2 contributes to dysregulation of DNA repair and genome stability and is required for MM cell survival, indicating that it is a promising target to inhibit growth and reduce genomic evolution in myeloma. Disclosures Munshi: BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company; Janssen: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy.

Background Essential thrombocythemia (ET) is characterized by thrombocytosis with increased platelet number and persistent activation. The mechanisms of thrombosis and the fate of these platelets are not clear. The aim of the present study is to explore the phagocytosis of platelets of ET patients by endothelial cells (ECs) in vitro and its relevance to the procoagulant activity (PCA). Methods Phosphatidylserine (PS) exposure on platelets was detected by flow cytometry. Phagocytosis of the platelets by ECs was performed using flow cytometry, confocal microscopy, and electron microscopy. The PCA of platelets was evaluated by coagulation time and purified coagulation complex assays. Results The PS exposure on platelets in ET patients is higher than that in healthy controls. The PS‐exposed platelets are highly procoagulant and lactadherin reduced 80% of the PCA by blockade of PS. When cocultured, the platelets of ET patients were sequestered by ECs in a time‐dependent fashion. Lactadherin enhanced phagocytosis by bridging the PS on activated platelets and the integrin αvβ3 on ECs, and P‐selectin played at least a partial role in this process. Furthermore, factor Xa and prothrombinase activity of PS‐exposed platelets were decreased after incubation with ECs. Conclusion Our results suggest that phagocytic clearance of platelets by ECs occurs in ET patients, thus representing a novel mechanism to remove activated platelets from the circulation; lactadherin and phagocytosis could cooperatively limit the thrombophilia in ET patients.

Background: The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) in stroke patients caused by thromboembolic occlusion of the internal carotid artery (ICA) remains unclear. Our objectives were to evaluate the critical role of NETs in the induction of hypercoagulability in stroke and to identify the functional significance of NETs during atherothrombosis. Methods: The levels of NETs, activated platelets (PLTs), and PLT-derived microparticles (PMPs) were detected in the plasma of 55 stroke patients and 35 healthy controls. NET formation and thrombi were analysed using immunofluorescence. Exposed phosphatidylserine (PS) was evaluated with flow cytometry and confocal microscopy. PCA was analysed using purified coagulation complex, thrombin, and fibrin formation assays. Findings: The plasma levels of NETs, activated PLTs, and PMP markers in the carotid lesion site (CLS) were significantly higher than those in the aortic blood. NETs were decorated with PS in thrombi and the CLS plasma of ICA occlusion patients. Notably, the complementary roles of CLS plasma and thrombin-activated PLTs were required for NET formation and subsequent PS exposure. PS-bearing NETs provided functional platforms for PMPs and coagulation factor deposition and thus increased thrombin and fibrin formation. DNase I and lactadherin markedly inhibited these effects. In addition, NETs were cytotoxic to endothelial cells, converting these cells to a procoagulant phenotype. Sivelestat, anti-MMP9 antibody, and activated protein C (APC) blocked this cytotoxicity by 25%, 39%, or 52%, respectively. Interpretation: NETs played a pivotal role in the hypercoagulability of stroke patients. Strategies that prevent NET formation may offer a potential therapeutic strategy for thromboembolism interventions. Funding: This study was supported by grants from the National Natural Science Foundation of China (61575058, 81873433 and 81670128) and Graduate Innovation Fund of Harbin Medical University (YJSKYCX2018-58HYD). Keywords: Neutrophil extracellular traps, Phosphotidylserine, Stroke, Hypercoagulable state, Thrombosis, Endothelial cells

Hypercoagulability is a major cancer-associated complication linked to poor patient prognosis. The production of neutrophil extracellular traps (NETs) is increasingly found to be linked with the development and metastasis of cancer, as well as with thrombi formation in cancer patients. We hypothesized that the neutrophil NET release may be triggered by specific cytokines in oral squamous cell carcinoma (OSCC) patients, thereby predisposing them to a hypercoagulable state. Moreover, we have evaluated the interaction between NETs and endothelial cells (ECs). NET procoagulant activity was assessed based on fibrin and purified coagulation complex production assays, as well as by measuring coagulation time (CT). We further used confocal microscopy to quantify the exposure of phosphatidylserine (PS), fibrin strands, and cell FVa/Xa binding. OSCC patients with stage III/IV exhibited elevated plasma NET levels compared to stage I/II or CTR (all P