Blockade Of Phosphatidylserine Research Articles

Phosphatidylserine-binding molecules induce the formation of tumor cell-derived microvesicles for enhanced cancer immunotherapy

Engineered microvesicles (MVs) derived from tumor cells, which preserve tumor-specific characteristics, have been extensively investigated as delivery systems or vaccines for cancer therapy. However, the negatively charged phosphatidylserine (PS) exposed on the surface of tumor-derived MVs can induce immune tolerance, evade immune responses and dampen immunotherapy effectiveness. Here, we report a functional molecule (FaZn) that not only actively targets PS exposed on tumor cell surfaces, inducing membrane blebbing and producing PS-blocked tumor cell-derived MVs (FaZn-MVs) but also directly generates stable PS-blocked MVs in situ within tumors, serving as an antigen repository. FaZn-MVs not only activate T-cell-mediated immune responses akin to a prophylactic vaccine, demonstrating robust protection against tumors but also suppress tumor growth in mice and offer long-term immune surveillance. Furthermore, when FaZn-MVs are combined with programmed death ligand 1 (PD-L1) inhibitors, they enhance potent antitumor immune responses, leading to significant improvements in survival and immune memory. This work establishes a promising method for the in vivo generation of personalized tumor microvesicle vaccines with PS blockade, revealing a promising strategy for activating antitumor immune responses.

Platelet phosphatidylserine is the critical mediator of thrombosis in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a severe immune-mediated prothrombotic disorder caused by antibodies (Ab) reactive to complexes of platelet factor 4 and heparin. Platelets (PLT) and their interaction with different immune cells contribute to prothrombotic conditions in HIT. However, the exact mechanisms and the role of different PLT subpopulations in this prothrombotic environment remain poorly understood. In this study, we observed that HIT patient Ab induce a new PLT population that is characterized by increased P-selectin expression and phosphatidylserine (PS) externalization. Formation of this procoagulant PLT subpopulation was dependent on engagement of PLT Fc-γ-RIIA by HIT Ab and resulted in a significant increase of thrombin generation on the PLT surface. Using an ex vivo thrombosis model and multi-parameter assessment of thrombus formation, we observed that HIT Ab-induced procoagulant PLT propagated formation of large PLT aggregates, leukocyte recruitment and most importantly, fibrin network generation. These prothrombotic conditions were prevented via the upregulation of PLT intracellular cAMP with Iloprost, a clinically approved prostacyclin analogue. Additionally, the functional relevance of P-selectin and PS was dissected. While inhibition of P-selectin did not affect thrombus formation, the specific blockade of PS prevented HIT Ab-mediated thrombin generation and most importantly procoagulant PLT-mediated thrombus formation ex vivo. Taken together, our findings indicate that procoagulant PLT are critical mediators of prothrombotic conditions in HIT. Specific PS targeting could be a promising therapeutic approach to prevent thromboembolic events in HIT patients.

Phase 2 trial of bavituximab with chemoradiation and adjuvant temozolomide in newly diagnosed glioblastoma.

2030 Background: Glioblastoma (GBM) and tumor endothelial cells express phosphatidylserine (PS), a highly immunosuppressive membrane phospholipid. PS receptors engage with immune cells, leading to expansion of myeloid-derived suppressor cells (MDSCs) which promote an immunosuppressive and pro-angiogenic tumor microenvironment. Bavituximab (BAV) – a chimeric monoclonal antibody – binds to β2-glycoprotein 1 (β2-GP1) to form a complex of β2-GP1 with PS, resulting in immune activation against tumor cells and anti-angiogenic effects. Pre-clinical data in GBM models suggest synergistic effects of PS blockade, radiation (RT), and temozolomide (TMZ). Here, we present results from a phase II trial (NCT03139916) of BAV, RT and TMZ in GBM patients. Methods: 33 adults with newly diagnosed IDH-wild-type GBM were enrolled and received 6 weeks of RT+TMZ, followed by 6 cycles of TMZ. BAV (3 mg/kg) was given weekly, starting at week 1 of RT+TMZ, for 18 weeks with the option to continue if tolerated. The primary endpoint was the proportion of patients alive at 12 months (OS-12). The null hypothesis would be rejected if OS-12 was ≥ 72%. As an exploratory endpoint, the immune profile in tumor tissue and peripheral blood mononuclear cells (PBMCs) was assessed using nanoString and multispectral immunofluorescence, with the goal to assess on-target effects of BAV in longer vs. shorter surviving patients (split based on median survival). Relative cerebral blood flow (rCBF) from dynamic susceptibility contrast MRI was also obtained. Results: 24 patients were alive at 12 months and OS-12 was 73% (95% CI 59-90%) so the study met its primary endpoint. Median OS was 15.4 months. As best response, 79% of patients had stable disease, 12% had a partial response and 9% had progressive disease. Eight grade 3 or 4 adverse events were seen (no grade 5 AEs). Ten pre-treatment and 7 post-treatment tissue samples were available. Analysis of RNA from pre-treatment tumor specimens showed a significantly positive shift in myeloid-related gene expression in patients with longer survival, with enrichment of 116 and 120 transcripts as well as downregulation of 2 and 1 gene for PFS and OS, respectively. There was no differential expression in PBMCs. Including all tissue samples, there was a marked reduction of MDSCs after BAV compared to time of diagnosis (p = 0.011). Decreased rCBF post-RT/pre-cycle 1 TMZ was associated with improved OS (HR 4.63, p = 0.029). Conclusions: OS-12 was 73%, meeting the primary endpoint and suggesting potential activity of BAV in newly diagnosed GBM. BAV leads to on-target depletion of intratumoral immunosuppressive MDSCs and anti-angiogenic effects. As expected, based on the mechanism of action of BAV, there was no difference in PBMC gene expression profile in patients with long and short survival. Combining BAV with immune checkpoint inhibitors in the future may augment tumor immune response. Clinical trial information: NCT03139916.

Inflammatory cytokines enhance procoagulant activity of platelets and endothelial cells through phosphatidylserine exposure in patients with essential hypertension.

The exact mechanism of the prothrombotic state of essential hypertension (EH) patients remains elusive. Our objective was to assess whether phosphatidylserine (PS) exposure on endothelial cells (ECs), platelets, and microparticles (MPs) can account for the hypercoagulability in EH patients. PS exposure on cells and MPs, mainly from platelets and ECs was analyzed with flow cytometry. Procoagulant activity (PCA) was evaluated by purified coagulation complex assays, clotting time, and fibrin turbidity. We found that EH patients exhibited elevated levels of PS+ platelets, serum-cultured ECs, MPs, endothelial-derived MPs and platelet-derived MPs compared to the controls (all P < 0.05). Moreover, platelets and MPs from the patients and their sera-cultured ECs showed markedly enhanced intrinsic/extrinsic FXa, thrombin, and fibrin generation, and greatly shortened coagulation time. This PCA could be blocked approximately 80%, by the addition of lactadherin. Furthermore, we detected elevated levels of IL-8, IL-6, and TNF-α in EH patients could activate platelets/ECs and induce elevated PS exposure on their membranes. Our results suggest that inflammatory cytokines could enhance procoagulant activity of platelets and endothelial cells via their PS exposure in EH patients. As such, a PS blockade may be a viable therapeutic strategy for treating such patients.

MRI changes in patients with newly diagnosed glioblastoma treated as part of a phase II trial with bavituximab, radiation, and temozolomide.

2546 Background: Glioblastoma and tumor endothelial cells express phosphatidylserine (PS), a highly immunosuppressive membrane phospholipid. Bavituximab – a chimeric monoclonal antibody – binds to β2-glycoprotein 1 (β2-GP1) to form a complex of β2-GP1 with PS, resulting in immune activation against tumor cells and anti-angiogenic effects. Phase I/II trials in other solid cancers demonstrated response rates up to 75% when bavituximab was given with cytotoxic chemotherapy. Pre-clinical data in glioblastoma models suggested synergistic effects of PS blockade, radiation, and temozolomide. Methods: 33 adult patients with IDH-wild-type, MGMT-methylated or -unmethylated newly diagnosed glioblastoma were enrolled in this phase II trial (NCT03139916) and received 6 weeks of chemoradiation, followed by 6 cycles of adjuvant temozolomide (C1-C6 aTMZ). Bavituximab (3 mg/kg) was given weekly, starting week 1 of chemoradiation, for 18 weeks with the option to continue if tolerated. Physiologic MRIs were performed pre-treatment, pre-C1, pre-C3, and pre-C5 aTMZ. Within the enhancing tumor region, median tumor Ktrans (reflecting vascular permeability) and relative cerebral blood flow (rCBF) were measured. Median percent changes during treatment were compared to pre-treatment values. Results: Median progression-free survival (mPFS) was 8 months. Based on a median overall survival (mOS) of 17.1 months, patients were categorized into above-median survivors (AMS) and below-median survivors (BMS). All patients had pre-treatment scans. 31 had evaluable pre-C1, 25 had pre-C3, and 7 had pre-C5 scans. Compared to BMS, AMS had a greater reuction in enhancing tumor volume and rCBF, and a greater increase in Ktrans during treatment (table). One patient remains on study; 23 patients have died. Bavituximab was well tolerated. Conclusions: mPFS and mOS in patients treated with bavituximab, radiation and temozolomide were comparable to standard chemoradiation and aTMZ. Lower rCBF in AMS may reflect decreased tumor perfusion while higher Ktrans could imply enhanced drug delivery to the tumor. Bavituximab induces changes in tumor vasculature that may improve survival in a subset of patients. Clinical trial information: NCT03139916 . [Table: see text]

Protective effect of phosphatidylserine blockade in sepsis induced organ dysfunction

BackgroundPhosphatidylserine is usually an intracellularly oriented cell membrane phospholipid. Externalized phosphatidylserine on activated cells is a signal for phagocytosis. In sepsis, persistent phosphatidylserine exposure is also a signal for activation of the coagulation and inflammatory cascades. As such, phosphatidylserine may be a key molecule in sepsis induced cellular and organ injury. We hypothesize that phosphatidylserine blockade provides a protective effect in sepsis induced organ dysfunction. MethodsSepsis was induced in adult female rats using an endotoxin model. Diannexin, a homodimer of annexin A5, was administered for phosphatidylserine blockade. Rats were allocated to control (n = 5), sepsis (n = 6), or sepsis and phosphatidylserine blockade (n = 9) groups. Gut, pulmonary, renal, and hematologic dysfunctions were evaluated by mesenteric microvascular fluid leak, partial pressure of oxygen, serum creatinine, activated clotting time, and glomerular fibrin deposition, respectively. ResultsRats in the sepsis group demonstrated gut, renal, and hematologic dysfunction. Phosphatidylserine blockade reversed signs of gut dysfunction and mesenteric microvascular leak (P < .01). In addition, phosphatidylserine blockade corrected systemic coagulopathy, as measured by activated clotting time (P = .03) and glomerular fibrin deposition (P = .008). There was no difference in renal dysfunction (P = .1) or pulmonary dysfunction in any of the groups (P = .6). ConclusionIn sepsis, phosphatidylserine blockade had a protective effect on gut dysfunction and coagulopathy. Increased phosphatidylserine exposure may be a key mediator of organ dysfunction and coagulopathy during sepsis. These data may provide insights into novel treatment options for septic patients.

Abstract 530: Phosphatidylserine-targeting antibody combined with chemoradiation enhances systemtic anti-tumor immunity

Abstract The discovery of immune checkpoint blockades have resulted in significant clinical benefits for patients with metastatic cancers including non-small cell lung cancer (NSCLC). Nevertheless, a large proportion of patients do not respond to such therapy. Strategies to enhance the therapeutic efficacy of cancer immunotherapy among a larger group of patients are urgently needed. Increasing the recognition of cancer cells by immune system may enhance response to immunotherapies. Tumor-associated macrophage (TAM) is the most abundant immune cell in most solid tumors, and is critical in bridging innate and adaptive immune system functions. Cancer cells overexpress multiple immune suppressive molecules including phosphatidylserine (PS) to inhibit immunological functions of TAMs. PS expression can be enhanced by cancer therapeutics such as radiation to induce immune suppression. Our results show that radiation increased expression of PS on lung cancer cells. The combination of PS blocking antibody with radiation decreased immune-suppressive phagocytosis and enhanced antigen presentation of macrophages. In murine lung cancer models, combination treatment of PS blockade and chemoradiation more effectively controlled tumor growth and prolonged survival than each treatment used alone. In a bilateral lung cancer tumor model, addition of PS blockade to chemoradiation promoted cytotoxic T cell infiltration and induced tumor growth inhibition of the non-irradiated tumors. Together, our results demonstrate that PS-targeting antibodies may be combined with chemoradiation to enhance intrinsic tumor immunogenicity and activate systemic anti-tumor immune responses. This approach may provide new routes for improving the efficacy of modern immunotherapeutics. Citation Format: Yifan Wang, Zhigang Liu, Weiye Deng, Betty Y. Kim, Steven H. Lin, Wen Jiang. Phosphatidylserine-targeting antibody combined with chemoradiation enhances systemtic anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 530.

Increased Phosphatidylserine on Blood Cells in Oral Squamous Cell Carcinoma

The specific function of phosphatidylserine (PS) in the context of the development of a hypercoagulable state among individuals with oral squamous cell carcinoma (OSCC) is uncertain. The goal of this study was therefore to assess the exposure of PS on microparticles (MPs) as well as on endothelial and blood cells and to assess procoagulant activity (PCA) as a function of the stage of OSCC progression. We recruited patients with OSCC (n = 63) as well as healthy controls (n = 26) to participate in this study. PS exposure was then assessed via confocal microscopy and flow cytometry, revealing that patients with stage III/IV OSCC exhibited higher frequencies of PS-exposing blood cells, MPs, and serum-cultured endothelial cells (ECs) than did patients with stage I/II OSCC or healthy controls. When we conducted functional coagulation assays, we discovered that PS+ blood cells, MPs, and serum-cultured ECs from patients with stage III/IV OSCC mediated more rapid coagulation and more substantial production of FXa, thrombin, and fibrin as compared with controls. When samples were treated with the PS antagonist lactadherin, this resulted in an 80% disruption of PCA. Strikingly, when pre- and postoperative samples were compared from patients with stage III/IV OSCC undergoing resective surgery, PCA was significantly reduced in the postoperative samples. After stimulating ECs with inflammatory cytokines, we found by confocal microscopy that they expose PS on their cell membranes, thus generating FVa and FXa binding sites and mediating the formation of fibrin. Together our findings provide evidence that PS+ blood cells and MPs are important mediators of the development of a hypercoagulable and prothrombotic state among individuals afflicted by advanced-stage OSCC. As such, a PS blockade may be a viable therapeutic strategy for treating such patients.

The Role of Exposed Phosphatidylserine on Microparticles and Blood Cells in Coagulant Activity in Acute Kidney Injury Patients on Continuous Renal Replacement Therapy

Introduction: Serving as the preferred treatment for patients with acute kidney injury (AKI), continuous renal replacement therapy (CRRT) is mostly interrupted by clotting caused by procoagulant state despite of persistent improvements in anticoagulant technology, reducing therapy time, enhancing cost and resulting in blood lost. Due to hemodynamics disorders and foreign material circuit, blood cells undergo definite activation. However, considered as markers of cell activation, the role of phosphatidylserine (PS) and microparticles (MPs) in the hypercoagulability remains largely unexplored. The aim of this study is to measure PS exposure on blood cells and MPs at baseline and after different therapy duration in AKI patients.Methods: Fifty AKI patients admitted to the First Affiliated Hospital of Harbin Medical University between March 2017 and May 2018 and forty healthy controls were enrolled in this study. Plasma samples were collected at baseline from patients were diagnosed with AKI and after receiving CRRT (Mode: CVVH; dose: 25mL/kg/h; Citrate which has relative little influence on PS exposure and MPs count was used as regional anticoagulant.) for 6hours, 12hours, 18hours, as well as from healthy controls. After being isolated, MPs were measured by flow cytometry (FCM) while procoagulant activity (PCA) of erythrocytes, platelets and MPs was assessed through coagulation time and purified coagulation complex. Lactadherin was applied as a probe and inhibitor of PS.Results: Between AKI baseline group and healthy controls, there was no obvious difference in lactadherin+ MPs, exposure on platelets and erythrocytes (Table 1,2), coagulation time and protein production (Fig. 1). While with therapy time prolonged to 6h, 12h and 18h, lactadherin+ MPs and blood cells showed a significant increase compared with baseline group (Table 1,2). Additionally, coagulation time shortened significantly (Fig. 1A) and purified coagulation complex increased visibly in parallel with total therapy time (Fig. 1B-D). Based on previous analysis, we used lactadherin to suppress PS exposure at time point 18h. Compared with the baseline, lactadherin+ MPs count declined by 75.5% (P < 0.001), coagulation time for MPs, platelets and erythrocytes was shortened by 74.6%, 77.2% and 71.9% (all p < 0.01) and procoagulant enzyme complexes were also reduced by 73.8, 78.7% and 72.9% (all p < 0.01). (Figure. 1)Conclusions: Our results first reveal that upon AKI patients on CRRT, a procoagulant role of PS-driven PCA and an immediate time-dependent elevation in PS+ MPs and blood cells is observed. What's more, suppression of PS obviously inhibits the PCA. Accordingly, PS shows potential to be a new predictor and new strategies focusing on blockade of PS may provide a novel way of anticoagulants in CRRT process. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Intravascular Cells and Circulating Microparticles Induce Procoagulant Activity Via Phosphatidylserine Exposure in Venous Thromboembolism and Contribute to Altered Plasma Fibrin Clot Properties

Introduction:Venous thromboembolism(VTE), encompassing pulmonary embolism (PE) and deep vein thrombosis (DVT), is a major contributor to global morbidity and mortality, especially among cancer patients. However, the factors that contribute to PE, a prevalent and fatal complication of DVT, remain poorly understood.Despite a common pathology relating PE to DVT, this leaves a grey area in the differences among DVT, confirmed PE, or DVT with PE, and the definitive relationship between VTE and this hypercoagulable state.However, relatively little is known about the definitive role of phosphatidylserine (PS), one of the blood constituents in the coagulant pathway, in the hypercoagulability of VTE and their altered plasma fibrin clot properties. Our objectives were to assess the levels of PS exposure on microparticles (MPs), blood cells, endothelial cells(ECs) and cell-derived MPs in each group of VTE and to evaluate their procoagulant activity (PCA), as well as the correlation between initial PS exposure and their altered plasma fibrin clot properties.Methods:DVT alone (n=27), definitely PE alone (n=22), DVT with PE (n=19) and healthy controls (n=30) were enrolled in the present study. PS exposure on MPs, blood cells and cultured ECs treated with VTE serum in vitro was analyzed with flow cytometry and confocal microscopy. MPs were classified based on their cellular origin: platelets (CD41a+, PMPs), neutrophils (CD66b+, NMPs), endothelial cells(CD31+CD41a-, EMPs), erythrocytes (CD235a+, RMPs), monocytes (CD14+, MMPs), T lymphocytes (CD3+, T LyMPs), and B lymphocytes (CD19+, B LyMPs). 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+MPs, blood cells and ECs were performed by lactadherin. Abnormal fibrin clot properties measured ex vivo in plasma was examined by fibrin clot analysis and scanning electron microscopy (SEM).Results: There was no significant difference in MP cellular origin between healthy and VTE subjects. However, the total number of PS+MPs was significantly increased (P < 0.001 for total MP and all MP subtypes) in VTE patients compared with healthy controls. In addition, circulating PS+ MPs cooperated with PS+blood cells and cultured ECs to markedly shorten coagulation time (Figure 1A) and dramatically increase FXa/thrombin generation and fibrin formation in each VTE group (all P < 0.05). Confocal microscopy images showed that the FVa/FXa complex and fibrin strands co-stained with PS on ECs. Moreover, blockade of exposed PS on MPs and intravascular cells with lactadherin inhibited PCA by approximately 80% (Figure 1B). Fibrin clot structure was evaluated in 15 VTE patients (DVT=5, PE=5 and DVT with PE=5) and 5 healthy subjects.Compared with those from DVT and PE, clots from plasma of DVT with PE subjects showed lower fiber density and faster clot lysis time, whereas there were no differences in lag time, rate of clot formation and maximum absorbance of turbidity.Conclusions: Our results suggest that compared with healthy subjects, each group of VTE patients had markedly higher levels of circulating PS+MPs, PS+blood cells. Additional, ECs treated with VTE patient serum had elevated percentages of PS+cells versus those treated with serum from healthy controls. Blockade of PS with lactadherin significantly inhibits PCA of blood cells, MPs and VTE serum-treated ECs. In addition, differences in fibrin clot properties and clot structure among DVT, PE and DVT with PE subjects may provide important insights into the pivotal mechanisms that regulate embolization. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Role of Prothrombinase Complex Assembly and in Situ Fibrin Deposition on the Surface of Acute Promyelocytic Leukemia Cells in Hemorrhage

Background: Despite dramatic improvement in treatment for acute promyelocytic leukemia (APL), early death due to hemorrhage remains a major obstacle to achieving a complete cure. In contrast to classical disseminated intravascular coagulation, APL-associated coagulopathy is characterized by rare microvascular fibrin thrombi. Thus, it is attractive to speculate whether other unknown mechanisms depleting coagulation factors and unrecognized fibrin-deposition location exist. Procoagulant activity associated with APL cells plays a direct role in bleeding complicationsin. We have shown that exposed phosphatidylserine (PS) on APL cells supports purified prothrombinase (Zhou J et al, JTH 2010) and fibrin preferentially deposits on promyelocytic chromatin from ETosis or apoptosis (Cao M et al, Blood 2017). However, relatively little is known about the PS-driven prothrombinase complex assembly and in situ fibrin deposition on APL cells.Aims: Our objectives were to determine how APL cells promote thrombin generation and modulate fibrin formation and distribution, as well as to explore the relationship between in situ fibrin deposition and consumptive hemorrhage in APL patients.Methods: Twenty-seven newly diagnosed APL patients were included. Fresh APL blasts were obtained from bone marrow specimens by centrifugation through Ficoll-Hypaque. Lactadherin was used as a probe for PS exposure on the fresh APL blasts and on an immortalized APL cell line (NB4). PS exposure and fluorescein-labeled FV/X binding were evaluated by flow cytometry. Thrombin generation was measured by modifed thrombin generation test. Fibrin production was quantified by turbidity. The distribution of PS, prothrombinase complex and in situ fibrin deposition were imaged by confocal microscopy. For the inhibition assay, APL cells were pre-treated with lactadherin, DNase I or anti-TF antibody for 10 min at 37 °C before incubation with plasma.Results: Thrombin generation and fibrin formation supported by NB4 and APL cells increased approximately 1.5-fold after exposure to daunorubicin and decreased 80% after treatment with all-trans retinoic acid (ATRA) or arsenic trioxide (ATO). Procoagulant activity corresponded to exposed PS on viable APL cells. PS exposure increased approximately 2.7-fold after treatment with daunorubicin, while ATRA and ATO initially led to a 70% reduction in PS exposure, which rose again on day 3 and 5 (P<0.001), respectively. Levels of externalized PS on APL cells paralleled levels of FV/FX binding, lag time, peak thrombin, endogenous thrombin potential and fibrin formation. Lactadherin inhibited the above parameters by approximately 80%, while anti-tissue factor antibody or DNase I produced no effect. Interestingly, confocal imaging showed that fibrin preferentially deposited on the surface of APL cells, which we defined as “in situ fibrin”. Untreated viable APL and NB4 cells displayed discrete or occasionally annular fibrin deposition on the membrane. Moreover, fibrin formation supported by apoptotic APL cells displayed the following progression: (i) patchy deposition, (ii) diffuse rim, (iii) “fibrin coat”, and (iv) network. The dynamic changes in fibrin formation paralleled the kinetics of PS exposure and prothrombinase assembly. Furthermore, initial percentage of PS-positive fresh APL cells was negatively correlated with plasma levels of fibrinogen and factor II, V, VIII, X in APL patients on admission (all P<0.01).Conclusion: PS-driven prothrombinase complex assembly and in situ fibrin deposition on the surface of APL cells consume massive coagulation factors, providing a novel explanation for consumptive hemorrhage in APL patients. Blockade of PS might be a novel therapeutic approach for preventing bleeding in APL via inhibiting invisible “in situ coagulation”, especially in high-risk APL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Phosphatidylserine on microparticles and associated cells contributes to the hypercoagulable state in diabetic kidney disease.

Relatively little is known about the role of phosphatidylserine (PS) in procoagulant activity (PCA) in patients with diabetic kidney disease (DKD). This study was designed to evaluate whether exposed PS on microparticles (MPs) and MP-origin cells were involved in the hypercoagulability in DKD patients. DKD patients (n = 90) were divided into three groups based on urinary albumin excretion rate, defined as normoalbuminuria (No-A) (<30 mg/24 h), microalbuminuria (Mi-A) (30-299 mg/24 h) or macroalbuminuria (Ma-A) (>300 mg/24 h), and compared with healthy controls (n = 30). Lactadherin was used to quantify PS exposure on MPs and their original cells. Healthy blood cells (BCs) and human umbilical vein endothelial cells (HUVECs) were treated with 25, 5 or 2.5 mmol/L glucose as well as 3-12 mg/dL uric acid and cells were evaluated by clotting time and purified coagulation complex assays. Fibrin production was determined by turbidity. PS exposure and fibrin strands were observed using confocal microscopy. Using flow cytometry, we found that PS+ MPs (derived from platelets, erythrocytes, HUVECs, neutrophils, monocytes and lymphocytes) and BCs were significantly higher in patients than in controls. Furthermore, the number of PS+ MPs and BCs in patients with Ma-A was significantly higher than in patients with No-A. Similarly, we observed markedly elevated PS exposure on HUVECs cultured with serum from patients with Ma-A versus serum from patients with Mi-A or normoalbuminuria. In addition, circulating PS+ MPs cooperated with PS+ cells, contributing to markedly shortened coagulation time and dramatically increased FXa/thrombin generation and fibrin formation in each DKD group. Confocal microscopy images demonstrated colocalization of fibrin with PS on HUVECs. Moreover, blockade of exposed PS on MPs and cells with lactadherin inhibited PCA by ∼80%. In vitro, BCs and endothelial cells exposed more PS in hypoglycemia or hyperglycemia. Interestingly, reconstitution experiments showed that hypoglycemia-treated cells could be further activated or injured when recovery is obtained reaching hyperglycemia. Moreover, uric acid induced PS exposure on cells (excluding platelets) at concentrations >6 mg/dL. Linear regression analysis showed that levels of PS+ BCs and microparticles were positively correlated with uric acid and proteinuria, but negatively correlated with glomerular filtration rate. Our results suggest that PS+ MPs and MP-origin cells play procoagulant roles in patients with DKD. Blockade of PS could become a novel therapeutic modality for the prevention of thrombosis in these patients.

Abstract 1651: Targeting phosphatidylserine in combination with adoptive T cell transfer eliminates advanced tumors without off-target toxicities in a melanoma preclinical model

Abstract A viable strategy to treat advanced cancers includes transferring of tumor-specific T cells. T cells that recognize tumor antigens can be expanded and reinvigorated ex-vivo. Furthermore, autologous T cells can be genetically modified to express anti-tumor T cell receptors or chimeric antigen receptors. Although the potency and specificity of tumor-specific T cells can be manipulated ex-vivo, once re-infused into patients, the T cells are subjected to immunosuppressive mechanisms established by the tumor. An important immune checkpoint regulator within tumors is phosphatidylserine (PS). Innate immune cells exposed to PS secrete suppressive cytokines and chemokines that can significantly impair the function and activation of anti-tumor T cells. Therefore, monoclonal antibodies that block PS activity can increase the anti-tumor potency of transferred T cells to treat aggressive cancers. Here we show that a PS targeting monoclonal antibody in combination with CD4+ T cells that recognize the melanoma antigen Trp1 can regress very advanced melanomas in all treated mice. Combination of anti-Trp1 CD4+ T cells with other immunomodulatory modalities such as anti-OX40 antibodies, can achieve equivalent treatment rates but these are typically accompanied by severe immune related adverse events. In contrast, in this setting, PS blockade did not show any off-target toxicities. Flow cytometry analysis revealed lower levels of CD206 expression concomitant with higher activation markers in macrophages and neutrophils in tumors from anti-PS treated mice. These results suggest that diminishing suppressive mechanisms locally in adoptive transfer protocols is a highly desirable strategy that can eliminate tumors while minimizing related adverse events. Citation Format: Daniel Hirschhorn-Cymerman, Sara Sara Schad, Sadna Budhu, Zhong Hong, Xia Yang, Hutchins T. Jeff, Bruce D. Freimark, Michael J. Gray, Jedd Wolchok, Taha Merghoub. Targeting phosphatidylserine in combination with adoptive T cell transfer eliminates advanced tumors without off-target toxicities in a melanoma preclinical model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1651. doi:10.1158/1538-7445.AM2017-1651

Stimulated Platelets but Not Endothelium Generate Thrombin Via a Factor XIIa-Dependent Mechanism Requiring Phosphatidylserine Exposure

To inhibit pathological thrombus formation without impairing hemostasis is the holy grail of anticoagulant therapy. Recent data from animal models have indicated that factor XII (FXII) may be a promising new antithrombotic target that is particularly intriguing due to the longstanding clinical observation that severe congenital FXII deficiency is not associated with a bleeding diathesis in humans. FXII is thought to participate in thrombus formation after being activated in high shear arterial environments. FXIIa then initiates downstream activation of the contact pathway, culminating in thrombin generation. However, the relevant cell surface for FXII activation remains unclear. Here we compare the role of platelets versus endothelial cells in FXII activation and study the function of surface phospholipids in this process.To explore the effect of FXII inhibition on thrombus formation in vivo, we used antibody X210-C01, a novel human IgG1 developed using phage-display technology that blocks both mouse and human FXIIa. Using a mouse laser injury model of arterial thrombosis, we showed that X210-C01 inhibited both fibrin formation and platelet accumulation at sites of vascular injury. Plasma removed from animals after completion of these experiments was used to quantify the concentrations of X210-C01 achieved in vivo at a given dose. FXII inhibition was somewhat more potent in preventing platelet accumulation (IC50 dose = 27 mg/kg, R2=0.93) than fibrin formation (IC50 dose = 43 mg/kg, R2=0.95). Importantly, treatment with X210-C01 at 100 mg/kg did not prolong bleeding times or increase total blood loss in a tail bleeding assay.To evaluate the mechanism underlying our in vivo observations, we studied the differential role of FXII in thrombin generation by stimulated platelets and endothelium. X210-C01 did not globally inhibit SFLLRN-induced platelet aggregation or granule release. We next performed a fluorogenic thrombin generation assay (TGA) using human platelets treated with the peptide agonist SFLLRN. X210-C01 inhibited platelet-based thrombin generation in a dose-dependent fashion, whereas anti-tissue factor (TF) and anti-factor VIIa (FVIIa) antibodies did not. By contrast, in a similar TGA using SFLLRN-stimulated endothelial cells, X210-C01 had no effect, while anti-TF antibodies abrogated thrombin generation. These results indicate that stimulated endothelium generates thrombin by a mechanism distinct from that of platelets.FXII is known to be activated in vitro by anionic surfaces. Because phosphatidylserine (PS) is a negatively-charged phospholipid expressed on the surface of stimulated platelets, we reasoned that PS may serve as the platelet-based activator of FXII. To test this hypothesis, we used lactadherin, a potent and specific inhibitor of PS, in the platelet-based TGA and showed that PS blockade inhibited platelet-based thrombin generation at concentrations as low as 10 nM. We then used a chromogenic FXIIa activity assay to test the ability of PS-containing liposomes to activate FXII. Liposomes containing 80% phosphatidylcholine (PC) and 20% PS (PC-PS 80/20) failed to activate FXII at concentrations as high as 100 µM. In this assay, SFLLRN-stimulated platelets led to significantly greater FXII activation than either resting platelets or the PC-PS 80/20 liposomes tested.In summary, we have made the unexpected finding that thrombin generation on the surface of stimulated platelets proceeds by a FXIIa-dependent pathway and does not require FVIIa or TF. By contrast, thrombin generation on endothelium requires TF but not FXIIa. Additionally, surface PS is necessary but not sufficient for platelet-based FXII activation and thrombin generation, pointing to the involvement of a second platelet component. Further studies will be directed towards investigating the in vivo role of platelet-based FXII activation in arterial thrombus formation. DisclosuresMason:Shire Pharmaceuticals: Employment. Kenniston:Shire Pharmaceuticals: Employment.

1074P - Antibody mediated blockade of phosphatidylserine improves immune checkpoint blockade by repolarizing immune suppressive mechanisms of the tumor microenvironment

1074P - Antibody mediated blockade of phosphatidylserine improves immune checkpoint blockade by repolarizing immune suppressive mechanisms of the tumor microenvironment

Microparticles and blood cells induce procoagulant activity via phosphatidylserine exposure in NSTEMI patients following stent implantation

BackgroundRelatively 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. MethodsNSTEMI 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. ResultsBaseline levels of MPs and PS+ blood cells were significantly higher (P<0.001) in the patients than in controls. After stent implantation, a remarkable increase was observed in both MPs and PS+ 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<0.01) generation in patient group. Moreover, blockade of exposed PS on MPs and cells with lactadherin inhibited PCA by approximately 70%. ConclusionsOur 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.

Abstract PR09: Antibody-mediated blockade of phosphatidylserine synergizes with immune checkpoint blockade by inhibiting multiple immune suppressive mechanisms

Abstract Background: The expression of phosphatidylserine (PS) on cell surfaces drives immunosuppressive mechanisms associated with tolerogenic cell death. In the tumor microenvironment, PS is exposed on tumor cells and tumor vascular endothelial cells treated with conventional therapies. PS signals through multiple immune cell signaling receptors where it drives the expansion of myeloid-derived suppressor cells (MDSCs), regulatory T cells, M2 macrophages, and stimulates their production of immunosuppressive cytokines (e.g., TGFβ and IL-10), rendering the tumor microenvironment non-responsive to immune activation. PS targeting antibodies have significant anti-tumor effects in multiple tumor xenografts, syngeneic tumors, and genetically engineered preclinical tumor models through mechanisms that re-program the tumor microenvironment from immunosuppressive to immune potentiating. Methods: The combination of PS-targeting and anti-PD1 antibodies was compared to single agent therapy in multiple pre-clinical mouse models, including syngeneic melanoma and breast tumors, and a genetically engineered mouse model (GEMM) of pancreatic cancer. Mice were treated weekly IP at 5 mg/kg with a PS targeting antibody, anti-PD1, or the combination for the duration of each experiment; tumor and spleen tissue microenvironments were immune profiled by FACS, ELISPOT, and immunohistochemistry. Results: In all tumor models examined, the anti-tumor effect of combination therapy was significantly superior to single agent therapy. In B16 and K1735 melanoma tumors, the combination more than doubled the anti-tumor effect of anti-PD-1 alone. Combination therapy significantly inhibited tumor growth by over 90% in E077.1 breast tumors and prolonged animal survival by 30% (n&amp;gt;25 each group) in the GEMM of pancreatic cancer despite the fact that anti-PD1 therapy alone was ineffective in each of these models. Analysis of immune cell subsets in the tumor microenvironment indicated that the combination of antibody-mediated blockade of PS and PD-1 significantly enhanced the effector function of tumor infiltrating CD8+ T cells and increased the ratio of T effector to T regulatory cells, as demonstrated by significant increases in TILs producing IFNγ, TNFα, IL-2, granzyme B, and Ki-67. Combination therapy re-conditioned the tumor microenvironment to favor immune potentiation, as demonstrated by significant decreases in the frequency of MDSC, the ratio of M2 to M1 macrophages, the expression of surface PD-L1, and reductions in TGFβ and IL-10. Furthermore, combination treatment induced systemic tumor specific CD8 T cell immunity, as mice demonstrating complete responses (in the K1735 and E077.1 models) rejected tumor cells upon re-challenge. Splenocytes from these mice had significantly higher numbers of tumor-specific IFNγ-producing cells in ELISPOT assay. Studies were completed without toxicity in any setting. Conclusions: Antibody-mediated PS blockade inhibits PS-mediated immune suppression and stimulates FcRγ activation. This results in immune activation that enables an otherwise non-responsive tumor microenvironment to respond to checkpoint inhibition. This abstract is also presented as Poster B08. Citation Format: Xianming Huang, Jian Gong, Michael Gray, Van Nguyen, Ryan Parks, Chris Hughes, Jeff Hutchins, Rolf Brekken, Bruce Freimark. Antibody-mediated blockade of phosphatidylserine synergizes with immune checkpoint blockade by inhibiting multiple immune suppressive mechanisms. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR09.

Antibody-Mediated Phosphatidylserine Blockade Enhances the Antitumor Responses to CTLA-4 and PD-1 Antibodies in Melanoma.

In tumor-bearing animals, the membrane phospholipid phosphatidylserine (PS) suppresses immune responses, suggesting that PS signaling could counteract the antitumor effect of antibody-driven immune checkpoint blockade. Here, we show that treating melanoma-bearing mice with a PS-targeting antibody enhances the antitumor activity of downstream checkpoint inhibition. Combining PS-targeting antibodies with CTLA-4 or PD-1 blockade resulted in significantly greater inhibition of tumor growth than did single-agent therapy. Moreover, combination therapy enhanced CD4(+) and CD8(+) tumor-infiltrating lymphocyte numbers; elevated the fraction of cells expressing the proinflammatory cytokines IL2, IFNγ, and TNFα; and increased the ratio of CD8 T cells to myeloid-derived suppressor cells and regulatory T cells in tumors. Similar changes in immune cell profiles were observed in splenocytes. Taken together, these data show that antibody-mediated PS blockade enhances the antitumor efficacy of immune checkpoint inhibition. Cancer Immunol Res; 4(6); 531-40. ©2016 AACR.

Antibody mediated blockade of phosphatidylserine to enhance immune checkpoint blockade by repolarizing immune suppressive mechanisms of the tumor microenvironment.

e23195Background: The expression of phosphatidylserine (PS) on cell surfaces drives immunosuppressive mechanisms associated with tolerogenic cell death. In the tumor microenvironment, PS is exposed...

Phosphotidylserine exposure and neutrophil extracellular traps enhance procoagulant activity in patients with inflammatory bowel disease.

Inflammatory bowel disease (IBD)-associated thromboembolic event often lacks precise aetiology. The aim of this study was to investigate the contribution of phosphatidylserine (PS) exposure and neutrophil extracellular traps (NETs) towards the hypercoagulable state in IBD. We demonstrated that the levels of PS exposed MPs and the sources of MP-origin, platelets, erythrocytes, leukocytes and cultured endothelial cells (ECs) were higher in IBD groups than in healthy controls using flow cytometry and confocal microscopy. Wright-Giemsa and immunofluorescence staining demonstrated that the elevated NETs were released by activated IBD neutrophils or by control neutrophils treated with IBD sera obtained from patients with the active disease. MPs and MP-origin cells in IBD groups, especially in active stage, markedly shortened coagulation time and had increased levels of fibrin, thrombin and FXa production as assessed by coagulation function assays. Importantly, we found that on stimulated ECs, PS rich membranes provided binding sites for FXa and FVa, promoting fibrin formation while TNF blockage or IgG depletion attenuated this effect. Treatment of control neutrophils with TNF and isolated IgG from PR3-ANCA-positive active IBD patients also resulted in the release of NETs. Blockade of PS with lactadherin prolonged coagulation time, decreased fibrin formation to control levels, and inhibited the procoagulant enzymes production in the MPs and MP-origin cells. NET cleavage by DNase I partly decreased PCA in IBD or stimulated neutrophils. Our study reveals a previously unrecognised link between hypercoagulable state and PS exposure or NETs, and may further explain the epidemiological association of thrombosis within IBD patients.