Phosphatidylserine exposing-platelets and microparticles promote procoagulant activity in colon cancer patients

Background: Colon cancer is invariably accompanied by altered coagulation activity; however, the precise role of phosphatidylserine (PS) in the hypercoagulable state of colon cancer patients remains unclear. We explored the exposure of PS on platelets and microparticles (MPs), and evaluate its role in procoagulant activity in colon cancer patients. Methods: PS-positive platelets and MPs, mainly from platelets and endothelial cells, were detected by flow cytometry and confocal microscopy, and their procoagulant activity was assessed with purified coagulation complex assays, clotting time, and fibrin turbidity. Results: Plasma levels of PS-positive platelets increased gradually from stage I to IV and were higher in all stages of the patients than in the healthy control, while PS-positive platelet-derived MPs only increased significantly in stage III/IV patients. Meanwhile, PS-positive MPs and endothelial-derived MPs in stage II/III/IV patients were markedly higher than ones in controls but no difference with stage I. Tissue factor positive MPs were higher in all 4 stages of colon cancer patients than in the healthy control. Platelets and MPs from the patients demonstrated significantly enhanced intrinsic/extrinsic FXa and thrombin generation, greatly shortened coagulation time, and increased fibrin formation. Combined treatment with PS antagonist lactadherin, strongly prolonged the coagulation time and reduced fibrin formation by inhibiting factor tenase and prothrombinase complex activity. In contrast, pretreatment with anti tissue factor antibody played a lesser role in suppression of procoagulant activity. Conclusion: Our results suggest that PS-positive platelets and MPs contribute to hypercoagulability and represent a potential therapeutic target to prevent coagulation in patients with colon cancer

Prognostic implications and procoagulant activity of phosphatidylserine exposure of blood cells and microparticles in patients with atrial fibrillation treated with pulmonary vein isolation

The present study aimed to evaluate the procoagulant effects of phosphatidylserine (PS) exposure on blood cells and microparticles (MPs), and examine its role in predicting early recurrence atrial fibrillation (ERAF) in patients with atrial fibrillation (AF) treated with pulmonary vein isolation (PVI). Blood samples were obtained from 40 healthy controls and 56 patients with AF at baseline (prior to PVI), and 0, 1 h, 1 day, 3 days and 7 days following PVI. The exposure of PS (PS+) to blood cells (platelets, erythrocytes and leukocytes) and MPs was detected using flow cytometry. The procoagulant activity was evaluated by coagulation time, and the formation of factor Xa (FXa) and thrombin. In addition, independent factors associated with PS+ blood cells and MPs, and significant predictors of ERAF following PVI were investigated by statistical analyses. The numbers of PS+ blood cells and MPs were significantly increased by PVI (P355/µl were identified as independent predictors of ERAF (P<0.05). The increased numbers of PS+ platelets, erythrocytes, leukocytes and MPs contributed to the procoagulant state of AF, and hs-CRP and EMPs were able to predict ERAF following PVI

Arsenic trioxide promoting ETosis in acute promyelocytic leukemia through mTOR-regulated autophagy

Despite the high efficacy and safety of arsenic trioxide (ATO) in treating acute promyelocytic leukemia (APL) and eradicating APL leukemia-initiating cells (LICs), the mechanism underlying its selective cytotoxicity remains elusive. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, through autophagy. However, the role of ETosis in ATO-induced APL LIC eradication remains unclear. For this study, we evaluated the effects of ATO on ETosis and the contributions of drug-induced ETosis to APL LIC eradication. In NB4 cells, ATO primarily increased ETosis at moderate concentrations (0.5–0.75 μM) and stimulated apoptosis at higher doses (1.0–2.0 μM). Furthermore, ATO induced ETosis through mammalian target of rapamycin (mTOR)-dependent autophagy, which was partially regulated by reactive oxygen species. Additionally, rapamycin-enhanced ATO-induced ETosis in NB4 cells and APL cells from newly diagnosed and relapsed patients. In contrast, rapamycin had no effect on apoptosis in these cells. We also noted that PML/RARA oncoprotein was effectively cleared with this combination. Intriguingly, activation of autophagy with rapamycin-enhanced APL LIC eradication clearance by ATO in vitro and in a xenograft APL model, while inhibition of autophagy spared clonogenic cells. Our current results show that ATO exerts antileukemic effects at least partially through ETosis and targets LICs primarily through ETosis. Addition of drugs that target the ETotic pathway could be a promising therapeutic strategy to further eradicate LICs and reduce relapse

Publisher Correction: Phosphatidylserine-mediated platelet clearance by endothelium decreases platelet aggregates and procoagulant activity in sepsis

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper

Thrombotic Role of Blood and Endothelial Cells in Uremia through Phosphatidylserine Exposure and Microparticle Release.

The mechanisms contributing to an increased risk of thrombosis in uremia are complex and require clarification. There is scant morphological evidence of membrane-dependent binding of factor Xa (FXa) and factor Va (FVa) on endothelial cells (EC) in vitro. Our objectives were to confirm that exposed phosphatidylserine (PS) on microparticle (MP), EC, and peripheral blood cell (PBC) has a prothrombotic role in uremic patients and to provide visible and morphological evidence of PS-dependent prothrombinase assembly in vitro. We found that uremic patients had more circulating MP (derived from PBC and EC) than controls. Additionally, patients had more exposed PS on their MPs and PBCs, especially in the hemodialysis group. In vitro, EC exposed more PS in uremic toxins or serum. Moreover, reconstitution experiments showed that at the early stages, PS exposure was partially reversible. Using confocal microscopy, we observed that PS-rich membranes of EC and MP provided binding sites for FVa and FXa. Further, exposure of PS in uremia resulted in increased generation of FXa, thrombin, and fibrin and significantly shortened coagulation time. Lactadherin, a protein that blocks PS, reduced 80% of procoagulant activity on PBC, EC, and MP. Our results suggest that PBC and EC in uremic milieu are easily injured or activated, which exposes PS and causes a release of MP, providing abundant procoagulant membrane surfaces and thus facilitating thrombus formation. Blocking PS binding sites could become a new therapeutic target for preventing thrombosis

PS exposure on the plasma membrane of blood cells.

<p>Comparison of PS exposure on blood cells among Non-D patients, CAPD patients, HD patients and healthy subjects. Cells were incubated with Alexa Fluro 488 -lactadherin separately in the dark for 10 min at room temperature before evaluation by flow cytometry. (<b>A</b>) We measured the percent of RBCs, platelets, PMNs, MNCs that bound lactadherin from healthy subjects (n = 20), Non-D (n = 25), CAPD (n = 18) and HD patients (n = 23). (<b>B</b>) Lactadherin -binding RBC number per liter of plasma according their count in each person. Data are expressed as mean ± SD (***P < 0.001, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001). PS exposure on the plasma membrane of blood cells was observed by confocal microscopy with LSM 510 3.2 SP2 software. Platelets, RBCs and WBCs of healthy subjects and uremic patients were incubated with Alexa Fluro 488 -lactadherin and PI in the dark 10 min at room temperature. Cells were then washed very gently to remove unbound dye. Cell membrane displayed green fluorescence when labeled with lactadherin and nucleus displayed red fluorescence. Lactadherin staining (green) is observed on platelets membrane and MPs <b>(D)</b> and RBC <b>(F)</b>/WBC <b>(G)</b> in uremic patient but no staining in healthy subjects <b>(C, E)</b>. The inset bar equals 5 μm. PS: phosphatidylserine; PMN: polymorphomuclear cell; MNC: mononuclear cell; Non-D: Non-dialysis; CAPD: continuous ambulatory peritoneal dialysis; HD: haemodialysis.</p

Circulating MPs from uremic patients and ECs treated with uremic serum support FVa/FXa binding and fibrin deposition.

<p>PS on circulating MPs, isolated from uremic patients, were co-stained (yellow) with lactadherin (green) and annexin V (red) <b>(A)</b> and co-bound with (yellow) factor Va-fluorescein-maleimide (green) and factor Xa-EGRck-biotin (red) <b>(B)</b> and visualized using confocal microscopy. <b>(C)</b> MPs were incubated with MDP in the presence of calcium for 30 min, stained with lactadherin (green), fixed, and stained with Alexa Fluro 647-anti-fibrin for 30 minutes. Converted fibrin networks were detected around uremic MPs. <b>(D)</b> Fibrin production, supported by normal-subject derived MPs (ΔMPs) and uremic patient derived MPs (UMPs), was measured by turbidity at 405 nm in the presence of recalcified MDP with or without 128 nM lactadherin or 25.6 μg/ml anti-TF using a SpectraMax 340PC plate reader. <b>(E)</b> ECs and nuclei were visualized by actin (green) and DAPI (blue). <b>(F)</b> FVa and FXa simultaneous staining (yellow) is observed on filopods near the retracted edges of uremic ECs similar to the binding sites for lactadherin. <b>(G)</b> Considerable fibrin was deposited radially along the filopodia between uremic ECs that were incubated with recalcified MDP for 30 min. <b>(H)</b> After adding recalcified MDP, dynamic fibrin formation on normal (ΔECs) and uremic serum cultured- ECs (UECs) was measured in the absence or presence of 128 nM lactadherin. Images were obtained by LSM 510 3.2 SP2 software. The inset bar equals 2 μm in A and B, 5 μm in C; 5 μm in E-G. PS: phosphatidylserine; MDP: MP-depleted plasma; UMPs: microparticles from uremic patient; ECs: Endothelial Cells; UECs: uremic serum cultured-EC.</p

Microparticles per microliter of plasma in Non-D, CAPD, HD and controls.

<p>MP, microparticle; Non-D, non-dialyzed; CAPD: continuous ambulatory peritoneal dialysis; HD, hemodialysis. Corrected for the number of events with isotype controls,</p><p>***P < 0.001 versus controls.</p><p>^#P < 0.05,</p><p>^##P < 0.01,</p><p>^###P < 0.001 versus Non-D and CAPD.</p><p>Microparticles per microliter of plasma in Non-D, CAPD, HD and controls.</p

PS exposure and reconstitution experiments of cultured HUVECs.

<p>ECs were cultured in medium with normal serum (●, ○), uremic serum (●, ○), and normal serum with mixed uremic toxins (●, ○) for 24h, respectively (filled circles). Then the cells were washed and incubated with normal serum for another 24h (open circles). At indicated time points (0, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48h), ECs were collected and incubated with lactadherin -Alexa Fluor 488 for 10 min in the dark before evaluation by flow cytometry. ECs cultured in normal serum for 48h were used as control. Each point represents mean ± SD for triplicate samples of independent experiments. <b>(A)</b> Kinetic mode of PS reversal on EC with different treatments. PS exposure on the outer membrane surface of EC occurred mainly from 12 to 24h when cultured with uremic serum or mixed toxins. After washing ECs, about 80% of PS reverted to the inner leaflet during the first 8h of culture with normal serum. ECs cultured for 24 h with normal or uremic serum were stained with CD31-Alexa Fluor 647 and PS exposure was detected using lactadherin-Alexa Fluor 488 and visualized using confocal microscopy. <b>(B)</b> Almost no lactadherin staining was observed on ECs cultured in normal serum. <b>(C)</b> Treatment of ECs with uremic serum led to retraction of cell margins, extension of filopods, and lactadherin (green) binding on filopods. The inset bar equals 10 μm.</p

Flow cytometry analyses of MPs in a sample from a HD patient.

<p>A representative set of scatter grams in a sample from a HD patient is shown to illustrate MPs and subpopulation definition. Results were acquired and analyzed with BD FACS Diva Software. (A) Forward and side scatter were used to define the events with a size of smaller than 1 μm which were gated in the P1 window. P2 and P3 are drawn around 1.0-μm and Trucount beads, respectively. (B) Events were then selected for their lactadherin binding, determined by positivity for lactadherin-Alexa Fluor 488 (on the x-axis). (C) Lactadherin-positive MPs were further examined for expression of other antigens by co-labeling with Alexa Fluor 488- and Alexa Fluor 647-labeled antibodies as is shown here for PMPs (Alexa Fluro 647-CD41a⁺) and EMPs (Alexa Fluro 488-CD31⁺/ Alexa Fluor 647-CD41a^-). (D) LMPs (pan-leukocyte, Alexa Fluor 647-CD45⁺) and monocyte origin -MPs (Alexa Fluor-488 CD14⁺). (E) MPs derived from lymphocyte (Alexa Fluro 647-CD3⁺) and neutrophil (Alexa Fluor 488-CD66b⁺). (F) RMPs (Alexa Fluor 488-CD235a⁺) and the TF expressing MPs (Alexa Fluor 647-CD142⁺). HD, haemodialysis; MP, microparticle; PMPs, platelet MPs; EMPs, endothelial cell MPs; LMPs, leukocyte MPs; RMPs, RBC MPs; TF, tissue factor; neg, negative; pos, positive.</p