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• W2019957066 abstract "SummaryBackground: Acute promyelocytic leukemia (APL) frequently causes disseminated intravascular coagulation that can worsen with cytotoxic chemotherapy but improve with the therapeutic differentiating agents, all trans retinoic acid (ATRA) and arsenic trioxide (As2O3). APL cells display tissue factor but the relationship of tissue factor and other procoagulant activity to phosphatidylserine (PS) exposure is largely unknown. Methods: Lactadherin, a milk protein with stereospecific binding to phosphatidyl‐L‐serine, was used as a probe for PS exposure on an immortalized APL cell line (NB4) and on the cells of eight patients with APL. PS exposure was evaluated with flow cytometry, confocal microscopy, coagulation assays, and purified prothrombinase and factor (F) Xase assays. Results: Plasma procoagulant activity of NB4 and APL cells increased approximately 15‐fold after exposure to etoposide or daunorubicin and decreased 80% after treatment with ATRA or As2O3. Procoagulant activity corresponded to exposed PS on viable APL cells. PS exposure decreased after treatment with ATRA or As2O3 and increased after treatment with daunorubicin or etoposide. Excess lactadherin inhibited 80–85% of intrinsic FXase, FVIIa‐tissue factor and prothrombinase activities on both NB4 cells and APL cells. Confocal microscopy identified membrane patches that stained with lactadherin, but not annexin V, demonstrating focal, low‐level PS exposure. Conclusions: PS is exposed on viable APL cells and is necessary for approximately 80% of procoagulant activity. Background: Acute promyelocytic leukemia (APL) frequently causes disseminated intravascular coagulation that can worsen with cytotoxic chemotherapy but improve with the therapeutic differentiating agents, all trans retinoic acid (ATRA) and arsenic trioxide (As2O3). APL cells display tissue factor but the relationship of tissue factor and other procoagulant activity to phosphatidylserine (PS) exposure is largely unknown. Methods: Lactadherin, a milk protein with stereospecific binding to phosphatidyl‐L‐serine, was used as a probe for PS exposure on an immortalized APL cell line (NB4) and on the cells of eight patients with APL. PS exposure was evaluated with flow cytometry, confocal microscopy, coagulation assays, and purified prothrombinase and factor (F) Xase assays. Results: Plasma procoagulant activity of NB4 and APL cells increased approximately 15‐fold after exposure to etoposide or daunorubicin and decreased 80% after treatment with ATRA or As2O3. Procoagulant activity corresponded to exposed PS on viable APL cells. PS exposure decreased after treatment with ATRA or As2O3 and increased after treatment with daunorubicin or etoposide. Excess lactadherin inhibited 80–85% of intrinsic FXase, FVIIa‐tissue factor and prothrombinase activities on both NB4 cells and APL cells. Confocal microscopy identified membrane patches that stained with lactadherin, but not annexin V, demonstrating focal, low‐level PS exposure. Conclusions: PS is exposed on viable APL cells and is necessary for approximately 80% of procoagulant activity. Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia caused by chromosome translocation t(15;17) [1Rowley J.D. Golomb H.M. Dougherty C. 15/17 translocation, a consistent chromosomal change in acute promyelocytic leukaemia.Lancet. 1977; 1: 549-50Abstract PubMed Scopus (548) Google Scholar] affecting the promyelocytic leukemia gene and the retinoic acid receptor gene [2De Thé H. Chomienne C. Lanotte M. Degos L. Dejean A. The t(15;17) translocation of acute promyelocytic leukaemia fuses the retinoic acid receptor alpha gene to a novel transcribed locus.Nature. 1990; 347: 558-61Crossref PubMed Scopus (1190) Google Scholar]. The resulting fusion protein arrests maturation of cells, causing accumulation of promyelocytes in the blood and bone marrow. A striking feature of APL is a life‐threatening coagulopathy (reviewed in [3Kwaan H.C. Wang J. Boggio L.N. Abnormalities in hemostasis in acute promyelocytic leukemia.Hematol Oncol. 2002; 20: 33-41Crossref PubMed Scopus (0) Google Scholar]). Patients may have petechiae, ecchymoses, epistaxis, hematuria and menometrorrhagia as well as major hemorrhage. As many as 20–30% of early deaths in APL are due to hemorrhagic complications. The coagulopathy of APL is characterized by continuous activation of the blood coagulation and fibrinolytic [4Menell J.S. Cesarman G.M. Jacovina A.T. McLaughlin M.A. Lev E.A. Hajjar K.A. Annexin II and bleeding in acute promyelocytic leukemia.N Engl J Med. 1999; 340: 994-1004Crossref PubMed Scopus (318) Google Scholar] mechanisms, with resulting consumption of clotting proteins. The coagulopathy of patients with APL has been a noteworthy challenge of clinical hematology, particularly as it can worsen with cytoxic chemotherapy [5Fenaux P. Le Deley M.C. Castaigne S. Archimbaud E. Chomienne C. Link H. Guerci A. Duarte M. Daniel M.T. Bowen D. Huebner G. Bauters F. Fegueux N. Fey M. Sanz M. Lowenberg B. Maloisel F. Auzanneau G. Sadoun A. Gardin C. et al.Effect of all transretinoic acid in newly diagnosed acute promyelocytic leukemia. Results of a multicenter randomized trial. European APL 91 Group.Blood. 1993; 82: 3241-9Crossref PubMed Google Scholar]. In recent years, it has become evident that APL is uniquely responsive to differentiating agents that work through stimulating the retinoic acid receptor rather than inducing apoptosis [6Wang Z.Y. Chen Z. Acute promyelocytic leukemia: from highly fatal to highly curable.Blood. 2008; 111: 2505-15Crossref PubMed Scopus (987) Google Scholar]. The differentiating agents that have entered clinical practise are all trans retinoic acid (ATRA) and arsenic trioxide (As2O3) [7Zhu J. Chen Z. Lallemand‐Breitenbach V. De The H. How acute promyelocytic leukaemia revived arsenic.Nat Rev Cancer. 2002; 2: 705-13Crossref PubMed Scopus (0) Google Scholar]. Treatment with either of these agents usually results in rapid correction of the coagulopathy preceding disappearance of the circulating leukemic promyelocytes [8Zhu J. Guo W.M. Yao Y.Y. Zhao W.L. Pan L. Cai X. Ju B. Sun G.L. Wang H.L. Chen S.J. Chen G.Q. Caen J. Chen Z. Wang Z.Y. Tissue factors on acute promyelocytic leukemia and endothelial cells are differently regulated by retinoic acid, arsenic trioxide and chemotherapeutic agents.Leukemia. 1999; 13: 1062-70Crossref PubMed Google Scholar]. Prior reports show that excessive procoagulant activity of APL is related to generation of factor (F) Xa via exposed tissue factor [9Gralnick H.R. Abrell E. Studies of the procoagulant and fibrinolytic activity of promyelocytes in acute promyelocytic leukaemia.Br J Haematol. 1973; 24: 89-99Crossref PubMed Google Scholar, 10Gouault Heilmann M. Chardon E. Sultan C. Josso F. The procoagulant factor of leukaemic promyelocytes: demonstration of immunologic cross reactivity with human brain tissue factor.Br J Haematol. 1975; 30: 151-8Crossref PubMed Google Scholar] as well as by a cysteine protease, named cancer procoagulant [11Falanga A. Consonni R. Marchetti M. Mielicki W.P. Rambaldi A. Lanotte M. Gordon S.G. Barbui T. Cancer procoagulant in the human promyelocytic cell line NB4 and its modulation by all‐trans‐retinoic acid.Leukemia. 1994; 8: 156-9PubMed Google Scholar]. However, exposed tissue factor is generally quiescent unless it resides in a membrane containing phosphatidylserine (PS) [12Bach R.R. Tissue factor encryption.Arterioscler Thromb Vasc Biol. 2006; 26: 456-61Crossref PubMed Scopus (220) Google Scholar]. In the context of membrane PS, tissue factor binds FVII and/or FVIIa and forms an active enzyme complex that cleaves the zymogen, FX to FXa. Factor Xa then combines with FVa, again on a PS‐containing membrane, to form the prothrombinase complex [13Mann K.G. Nesheim M.E. Church W.R. Haley P. Krishnaswamy S. Surface‐dependent reactions of the vitamin K‐dependent enzyme complexes.Blood. 1990; 76: 1-16Crossref PubMed Google Scholar]. The prothrombinase complex converts prothrombin to thrombin, the agent that converts fibrinogen to fibrin and activates platelets and nucleated cells. Prior studies have demonstrated that APL cells, like other cells, expose PS in the course of apoptosis [14Wang J. Weiss I. Svoboda K. Kwaan H.C. Thrombogenic role of cells undergoing apoptosis.Br J Haematol. 2001; 115: 382-91Crossref PubMed Scopus (85) Google Scholar]. However, they leave unanswered questions about PS exposure on the membranes of viable cells and the role of PS in the procoagulant activity. This study was focused on determining the extent of PS exposure by APL cells and the relationship to procoagulant activity. Lactadherin (also referred to as mfg‐e8) is a PS‐binding protein that exhibits stereoselective interaction with phospho‐L‐serine of PS [15Andersen M.H. Graversen H. Fedosov S.N. Petersen T.E. Rasmussen J.T. Functional analyses of two cellular binding domains of bovine lactadherin.Biochemistry. 2000; 39: 6200-6Crossref PubMed Scopus (168) Google Scholar, 16Shi J. Heegaard C.W. Rasmussen J.T. Gilbert G.E. Lactadherin binds selectively to membranes containing phosphatidyl‐L‐serine and increased curvature.Biochim Biophys Acta. 2004; 1667: 82-90Crossref PubMed Scopus (0) Google Scholar]. In contrast to annexin V (see Text S1 supporting information), membrane binding of lactadherin appears proportional to PS content, is independent of Ca++ concentration, and independent of membrane PE content [17Shi J. Gilbert G.E. Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid binding sites.Blood. 2003; 101: 2628-36Crossref PubMed Scopus (148) Google Scholar]. We have recently reported that fluorescence‐labeled lactadherin can be utilized, together with annexin V, to provide sensitive information about PS exposure on cells early in apoptosis [18Shi J. Shi Y. Waehrens L.N. Rasmussen J.T. Heegaard C.W. Gilbert G.E. Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death.Cytometry A. 2006; 69: 1193-201Crossref PubMed Scopus (94) Google Scholar] as well as in vivo on platelets participating in thrombosis [19Shi J. Pipe S.W. Rasmussen J.T. Heegaard C.W. Gilbert G.E. Lactadherin blocks thrombosis and hemostasis in vivo: correlation with platelet phosphatidylserine exposure.J Thromb Haemost. 2008; 6: 1167-74Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. In addition, we have found that the C2 domain of lactadherin is effective as an intracellular probe to localize PS [20Yeung T. Gilbert G.E. Shi J. Silvius J. Kapus A. Grinstein S. Membrane phosphatidylserine regulates surface charge and protein localization.Science. 2008; 319: 210-3Crossref PubMed Scopus (756) Google Scholar]. In the present study we have explored PS exposure on APL cells and the relationship to procoagulant activity. For materials and the following methods protein purification and labeling, cell culture, procoagulant activity, blood cell separation, leukemia cell separation, FXase assay, prothrombinase assay and confocal microscopy see Text S1, supporting information. Eight consecutive patients with newly diagnosed APL admitted to the First Affiliated Hospital of Harbin Medical University between March 2006 and July 2007 were included. Seven patients presented with hemorrhagic manifestations (Table 1). According to the FAB classification, six patients had the classical M3 APL, and two had the M3v. The diagnosis was established by morphologic characteristics, cytochemistry, cytogenetic analysis and immunophenotyping. Cytogenetic analysis indicated the t(15; 17) chromosomal marker in all cases and PML‐RARα gene rearrangement (Table 1). Eight sex‐ and age‐matched healthy donors acted as a control group for normal cells. All patients and healthy donors were advised of procedures and attendant risks, in accordance with institutional guidelines, and all patients gave informed consent consistent with the Helsinki Declaration.Table 1Patient characteristicsPatient (No.)Sex/age (years)DiagnosisWBC (× 109 L−1)Hgb (g L−1)Plts (× 109 L−1)Blasts (BM%)PT (s)aPTT (s)Fibrinogen (g L−1)D‐dimerHemorrhage (μg mL−1)1F/21M3/bcr136.469458817.528.31.32.1+2F/17M3/bcr14.066308218.431.51.50.88+3M/37M3/bcr15.858128319.237.81.13.19+4F/36M3/bcr38.967277516.735.12.50.92+5M/28M3v/bcr232.271258722.330.41.53.28+6M/32M3/bcr313.2100188019.240.11.40.72−7M/40M3v/bcr229.282229025.933.83.02.72+8F/24M3/bcr118.775339224.127.41.31.98+Ref. range4–10110–170100–3000–0.410.4–14.524–361.8–3.5≤ 0.3WBC, white blood cells; Hgb, hemoglobin; Plts, platelets; Blasts, promyelocytes + blasts; BM, bone marrow; bcr, breakpoint cluster region (bcr1 = intron 6, bcr2 = exon 6, bcr3 = intron3). Hemorrhage was manifested as mucosal oozing, spontaneous ecchymoses, petechiae, hematuria, or menorrhagia. Open table in a new tab WBC, white blood cells; Hgb, hemoglobin; Plts, platelets; Blasts, promyelocytes + blasts; BM, bone marrow; bcr, breakpoint cluster region (bcr1 = intron 6, bcr2 = exon 6, bcr3 = intron3). Hemorrhage was manifested as mucosal oozing, spontaneous ecchymoses, petechiae, hematuria, or menorrhagia. We first tested the procoagulant activity of immortalized APL cells (NB4 cell line [21Lanotte M. Martin‐Thouvenin V. Najman S. Balerini P. Valensi F. Berger R. NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3).Blood. 1991; 77: 1080-6Crossref PubMed Google Scholar]) in the absence and presence of differentiating agents and cytotoxic chemotherapy (Fig. 1A). A modified prothrombin time, in which NB4 cells provided the thromboplastin, was utilized. The procoagulant activity of NB4 cells was increased approximately 18‐fold by treatment with etoposide. In contrast, the cells treated with the differentiating agents, As2O3 and ATRA, had approximately 70% reduction of procoagulant activity. Thus, NB4 cells support procoagulant activity and the impact of chemotherapy parallels the overall pattern of procoagulant activity observed in patients with APL. We next evaluated the capacity of NB4 cells to support the individual procoagulant enzyme complexes that contribute to procoagulant activity (Fig. 1B–D). These assays, performed with NB4 cells in place of phospholipid vesicles, showed an overall pattern of support for the enzyme complexes that was similar to support for procoagulant activity. Support for the FVIIa‐TF complex increased 6‐fold following treatment with etoposide and decreased by half following treatment with As2O3 or ATRA (Fig. 1B). Support for the intrinsic FXase complex increased 3‐fold following treatment with etoposide and decreased approximately 80% following treatment by As2O3 or ATRA (Fig. 1C). Prothrombinase activity was increased 14‐fold by treatment with etoposide and reduced by approximately 20% and 60% by As2O3 and ATRA, respectively. Thus, the increase in procoagulant activity caused by etoposide and the decrease caused by differentiating agents relate to parallel changes in cellular support for all three procoagulant enzyme complexes. To evaluate the correlation between PS exposure and procoagulant activity, we utilized fluorescein‐labeled lactadherin and alexa 647‐labeled annexin V (Fig. 2). These PS‐binding reagents do not significantly compete with each other for membrane binding sites at the concentrations used [18Shi J. Shi Y. Waehrens L.N. Rasmussen J.T. Heegaard C.W. Gilbert G.E. Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death.Cytometry A. 2006; 69: 1193-201Crossref PubMed Scopus (94) Google Scholar]. Flow cytometry indicated that etoposide‐treated cells have a marked increase in PS exposure, with a wide range of values (Fig. 2A). Control experiments indicated that a polyclonal antibody directed against the peptide containing the RGD motif did not inhibit binding of lactadherin (not shown). In contrast, a polyclonal antibody against the C2 domain inhibited > 80% of lactadherin binding (not shown), indicating that most or all lactadherin binding was mediated by the PS‐binding C2 domain [15Andersen M.H. Graversen H. Fedosov S.N. Petersen T.E. Rasmussen J.T. Functional analyses of two cellular binding domains of bovine lactadherin.Biochemistry. 2000; 39: 6200-6Crossref PubMed Scopus (168) Google Scholar, 22Shao C. Novakovic V.A. Head J.F. Seaton B.A. Gilbert G.E. Crystal structure of lactadherin C2 domain at 1.7A resolution with mutational and computational analyses of its membrane‐binding motif.J Biol Chem. 2008; 283: 7230-41Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. Treatment with As2O3 and ATRA led to decreased average cellular PS exposure, as detected by lactadherin staining. Staining with annexin V detected the increase in PS exposure caused by etoposide treatment but not the decrease caused by differentiating agents (Fig. 2B). Confocal microscopy of untreated cells, co‐stained with lactadherin and annexin V, indicated the presence of scattered cells with PS exposed in patches and on cell protuberances (arrowheads) (Fig. 2C). Treatment with ATRA or As2O3 (Fig. 2D–E) led to a decrease in the number of cells with detectable PS exposure. Rare apoptotic cells (arrow) were seen in treated as well as untreated preparations. Annexin V was primarily identified in internal granules for non‐apoptotic cells, as previously reported [18Shi J. Shi Y. Waehrens L.N. Rasmussen J.T. Heegaard C.W. Gilbert G.E. Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death.Cytometry A. 2006; 69: 1193-201Crossref PubMed Scopus (94) Google Scholar, 23Kenis H. Van Genderen H. Bennaghmouch A. Rinia H.A. Frederik P. Narula J. Hofstra L. Reutelingsperger C.P. Cell surface‐expressed phosphatidylserine and annexin A5 open a novel portal of cell entry.J Biol Chem. 2004; 279: 52623-9Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar], rather than staining membrane structures. Following treatment with etoposide, nearly all cells stained with lactadherin and annexin V (Fig. 2F). Most cells exhibited a rim of lactadherin staining with a smooth, round contour. Annexin V was found both on the surface and on the interior. A smaller fraction of cells exhibited irregular morphology with condensed, fragmented nuclei, consistent with apoptosis. To evaluate the relationship between PS exposure and apoptosis, lactadherin and propidium iodide were incubated with cells. Rare cells stained with lactadherin and were permeable to propidium iodide in the absence of etoposide. Etoposide induced permeability to propidium iodide in the minority of cells that stained brightest with lactadherin (not shown). Thus, most cells exposed PS prior to loss of membrane integrity, consistent with apoptosis [24Koopman G. Reutelingsperger C.P. Kuijten G.A. Keehnen R.M. Pals S.T. Van Oers M.H. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.Blood. 1994; 84: 1415-20Crossref PubMed Google Scholar]. To investigate the relationship of PS exposure to procoagulant activity of NB4 cells we evaluated the inhibitory capacity of lactadherin (Fig. 3). Lactadherin inhibited overall procoagulant activity to the extent that plasma samples with added lactadherin took longer to clot than controls with no added NB4 cells (not shown). In defined assays, lactadherin inhibited 80–90% of the intrinsic FXase activity in a dose‐response manner (Fig. 3A). The procoagulant activity for cells treated by etoposide, As2O3 and ATRA differed (Fig. 1B–D) but are normalized here to allow comparison of the extent of inhibition. Lactadherin inhibited prothrombinase activity to a similar extent to FXase activity (Fig. 3B). Lactadherin also inhibited tissue factor dependent activity (Fig. 3C). The concentration of lactadherin required for half‐maximal inhibition was lower for etoposide‐treated cells, possibly indicating inhibition of the extrinsic FXase complex was more effective on membranes with higher PS content. By comparison with lactadherin, anti‐TF Ab also inhibited approximately 90% of FVIIa‐TF activity (Fig. 3D). Thus, PS exposure is required for at least 80% of procoagulant activity of NB4 cells for all three enzyme complexes that contribute to procoagulant activity. We evaluated the topographical relationship between exposed PS and exposed TF, utilizing confocal microscopy (Fig. 3E–F). The fluorescence signals from exposed TF were low on untreated cells and cells exposed to As2O3 or to ATRA (not shown) so that the image quality was not sufficient for conclusive interpretation. In contrast, cells treated with etoposide had strong fluorescence signals (Fig. 3E–F). Cells that had focal PS exposure had limited overlap between patches of TF and exposed PS. Overlap areas appeared at the borders of the two regions as well as in discrete areas that seemed to largely overlap. These results confirm that focal PS exposure overlaps with tissue factor exposure, consistent with a functional relationship. We next investigated the procoagulant activity of leukemic cells from eight patients with a recent diagnosis of APL (Table 1). Blood samples were collected after informed consent was obtained and prior to initiation of chemotherapy treatment. APL cells were partially purified and procoagulant activity was evaluated in a modified prothrombin time (Fig. 4A). Procoagulant activity was evaluated before and after the cells were supported with tissue culture conditions, with or without chemotherapy agents. As controls, the procoagulant activity of whole mononuclear cells and neutrophils from normal donors were compared. The results confirm that APL cells have procoagulant activity that exceeds control mononuclear cells or neutrophils. In vivo, the number of leukemic cells usually greatly exceeds the number of neutrophils and the number of monocytes. Thus, the net procoagulant activity is likely to be correspondingly greater than in our assay. Procoagulant activities were increased by exposure to daunorubicin or etoposide to a similar degree (not shown). Because the current treatment protocol calls for daunorubicin, results with this chemotherapy agent are displayed. Procoagulant activity decreased following exposure to ATRA or As2O3 to a level below mononuclear cells or neutrophils. Thus, procoagulant activity of authentic APL cells parallels evaluations with NB4 cells. The capacity of APL cells to support the individual enzyme complexes that contribute procoagulant activity was evaluated (Fig. 4B–D). The intrinsic FXase complex activity (Fig. 4B) was 5‐fold higher than mononuclear cells on a per cell basis. Activity increased nearly 5‐fold following treatment with daunorubicin and decreased approximately 65% following exposure to As2O3 or ATRA. Activity of the prothrombinase complex (Fig. 4C) was supported approximately 3‐fold above mononuclear cells and increased more than 8‐fold following exposure to daunorubicin. Support for prothrombinase activity decreased to the level of control cells following exposure to As2O3 or ATRA. Tissue factor activity in the FVIIa‐TF complex (Fig. 4D) was approximately 3‐fold above control mononuclear cells on a per cell basis. Activity increased 6‐fold following exposure to daunorubicin and decreased approximately 55% following exposure to As2O3 or ATRA. These results confirm that APL cells have increased support for all three of the procoagulant enzyme complexes. Support for all three complexes is increased to a similar degree by exposure to daunorubicin and diminished to a level comparable to control cells following exposure to ATRA or As2O3. Flow cytometry evaluation indicated that most fresh APL cells exposed only a low level of PS (Fig. 5A). Tissue factor exposure was below the detection threshold for this population. However, 1–2% of cells had much higher levels of PS exposure as well as clearly increased tissue factor antigen. After 24 h exposure to daunorubicin (Fig. 5B) the majority of cells had increased exposure of PS, with three different intensities of lactadherin staining (arrows). These intensities parallel the stages of PS exposure previously observed with immortalized leukemia cells [18Shi J. Shi Y. Waehrens L.N. Rasmussen J.T. Heegaard C.W. Gilbert G.E. Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death.Cytometry A. 2006; 69: 1193-201Crossref PubMed Scopus (94) Google Scholar]. Tissue factor antigen was increased on the majority of cells, with the highest intensity staining on cells with an intermediate level of PS exposure. Thus, both tissue factor and PS exposure increased on patient cells as observed on immortalized NB4 cells (Fig. 3). Confocal microscopy indicated that fresh and cultured APL cells had patches and small protuberances with exposed PS (arrowheads, Fig. 5C–D). Occasional cells had diffuse staining of the plasma membrane (arrows) giving a rim pattern. Most APL cells treated with daunorubicin stained diffusely with lactadherin but not with propidium iodide (Fig. 5E), indicating that increased PS exposure preceded loss of membrane integrity. Co‐staining the cells for PS and for tissue factor (Fig. 5F) indicated that patches of exposed PS overlap with patches staining for TF (yellow). These results are consistent with a supportive role for PS in the expression of tissue factor activity. To determine the necessity of exposed PS to support procoagulant reactions, inhibition studies were performed with lactadherin (Fig. 6). The inhibitory capacity of 200 nmol L−1 lactadherin, correlating to near‐maximal inhibition of procoagulant activity on NB4 cells (Fig. 3) was evaluated for cells treated with daunorubicin, ATRA, As2O3 and control cells. The displays are normalized to facilitate comparison of the extent of inhibition by lactadherin. Lactadherin inhibited 75–85% of prothrombinase activity. Lactadherin also inhibited 75–80% of tissue factor activity in the FVIIa‐TF complex. By comparison, an inhibitory antibody to tissue factor inhibited 85–90% of FVIIa‐TF activity. Likewise, lactadherin inhibited 75–80% of intrinsic FXase activity. The extent of inhibition was similar whether the cells had maximum activity induced by daunorubicin or minimal activity following treatment with ATRA or As2O3. Thus, these data confirm that exposed PS is required for at least 80% of procoagulant activity of human APL cells. We have found that PS exposure is a major mechanism through which APL cells support procoagulant activity. Exposed PS supports the prothrombinase and intrinsic FXase complexes, in addition to the FVIIa‐tissue factor complex. Differentiating agents, As2O3 and ATRA, which lead to termination of clinical coagulation abnormalities, cause diminished PS exposure on APL cells. Chemotherapy agents that cause worsening disseminated intravascular coagulation lead to increased PS exposure on APL cells. Thus, changes in PS exposure correlate to established clinical patterns when disseminated intravascular coagulation worsens or improves with therapy. This study clarifies the relationship between TF, PS exposure and procoagulant activity in APL. TF has been recognized as a major source of APL procoagulant activity though little was known about PS exposure on these cells. Plasma‐exposed TF is frequently encrypted, with little or no detectable procoagulant activity [12Bach R.R. Tissue factor encryption.Arterioscler Thromb Vasc Biol. 2006; 26: 456-61Crossref PubMed Scopus (220) Google Scholar, 25Bach R. Gentry R. Nemerson Y. Factor VII binding to tissue factor in reconstituted phospholipid vesicles: induction of cooperativity by phosphatidylserine.Biochemistry. 1986; 25: 4007-20Crossref PubMed Google Scholar]. TF gains activity upon association with PS and most activity is inhibited if PS is blocked [17Shi J. Gilbert G.E. Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid binding sites.Blood. 2003; 101: 2628-36Crossref PubMed Scopus (148) Google Scholar, 26Del Conde I. Nabi F. Tonda R. Thiagarajan P. Lopez J.A. Kleiman N.S. Effect of P‐selectin on phosphatidylserine exposure and surface‐dependent thrombin generation on monocytes.Arterioscler Thromb Vasc Biol. 2005; 25: 1065-70Crossref PubMed Scopus (0) Google Scholar, 27Wolberg A.S. Monroe D.M. Roberts H.R. Hoffman M.R. Tissue factor de‐encryption: ionophore treatment induces changes in tissue factor activity by phosphatidylserine‐dependent and ‐independent mechanisms.Blood Coagul Fibrinolysis. 1999; 10: 201-10Crossref PubMed Scopus (0) Google Scholar]. Thus, some degree of PS exposure was predicted to support previously reported TF activity. These results are consistent with several prior reports. Prior to treatment, the patients had characteristic bleeding, accompanied by depletion of fibrinogen, modest prolongation of the PT and variable prolongation of the aPTT. This pattern is consistent with the hypothesis that pretreatment bleeding is more closely linked to excessive fibrinolysis than depletion of coagulation factors through ongoing coagulation [4Menell J.S. Cesarman G.M. Jacovina A.T. McLaughlin M.A. Lev E.A. Hajjar K.A. Annexin II and bleeding in acute promyelocytic leukemia.N Engl J Med. 1999; 340: 994-1004Crossref PubMed Scopus (318) Google Scholar]. TF exposure on APL cells was previously reported together with up‐regulation by chemotherapy and down‐regulation following exposure to ATRA and As2O3 [8Zhu J. Guo W.M. Yao Y.Y. Zhao W.L. Pan L. Cai X. Ju B. Sun G.L. Wang H.L. Chen S.J. Chen G.Q. Caen J. Chen Z. Wang Z.Y. Tissue factors on acute promyelocytic leukemia and endothelial cells are differently regulated by retinoic acid, arsenic trioxide and chemotherapeutic agents.Leukemia. 1999; 13: 1062-70Crossref PubMed Google Scholar]. The exposure of PS on APL cells has previously been reported and correlated with TF activity, though only in the context of immortalized cells undergoing frank apoptosis [14Wang J. Weiss I. Svoboda K. Kwaan H.C. Thrombogenic role of cells undergoing apoptosis.Br J Haematol. 2001; 115: 382-91Crossref PubMed Scopus (85) Google Scholar, 28Langer F. Amirkhosravi A. Loges S. Meyer T. Eifrig B. Hossfeld D.K. Fiedler W. Francis J.L. An in vitro study on the mechanisms of coagulation activation in acute myelogenous leukemia (AML): role of tissue factor regulation by cytotoxic drugs and GM‐CSF.Thromb Haemost. 2004; 92: 1136-46Crossref PubMed Scopus (18) Google Scholar]. We have previously reported that immortalized leukemia cells expose PS in distinct stages early in apoptosis [18Shi J. Shi Y. Waehrens L.N. Rasmussen J.T. Heegaard C.W. Gilbert G.E. Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death.Cytometry A. 2006; 69: 1193-201Crossref PubMed Scopus (94) Google Scholar]. Early PS exposure was localized prior to the diffuse PS exposure that develops later in apoptosis (Fig. 5). This study goes beyond the prior reports, correlating the extent of PS exposure with support for the intrinsic FXase complex and prothrombinase complex as well as for TF. Limited PS exposure was demonstrated for viable cells as well as those entering apoptosis. Further, the pattern observed on immortalized cells was confirmed with fresh cells from patients with APL. Our photomicrographs indicate that there is limited overlap between TF‐rich regions of the plasma membrane and PS‐rich regions on etoposide‐treated NB4 cells. These data are consistent with reports that place TF primarily in sphingomyelin‐rich membrane ‘rafts’ [29Sevinsky J.R. Rao L.V. Ruf W. Ligand‐induced protease receptor translocation into caveolae: a mechanism for regulating cell surface proteolysis of the tissue factor‐dependent coagulation pathway.J Cell Biol. 1996; 133: 293-304Crossref PubMed Scopus (0) Google Scholar, 30Dietzen D.J. Page K.L. Tetzloff T.A. Lipid rafts are necessary for tonic inhibition of cellular tissue factor procoagulant activity.Blood. 2004; 103: 3038-44Crossref PubMed Scopus (0) Google Scholar]. PS content of the rafts is thought to be low based on both experimental and theoretical studies [31Hinderliter A. Almeida P.F. Creutz C.E. Biltonen R.L. Domain formation in a fluid mixed lipid bilayer modulated through binding of the C2 protein motif.Biochemistry. 2001; 40: 4181-91Crossref PubMed Scopus (84) Google Scholar, 32Samsonov A.V. Mihalyov I. Cohen F.S. Characterization of cholesterol‐sphingomyelin domains and their dynamics in bilayer membranes.Biophys J. 2001; 81: 1486-500Abstract Full Text Full Text PDF PubMed Google Scholar]. While PS is believed to reside primarily in the liquid crystalline portion of the membrane, the explanation for the observed PS‐rich patches of membrane is not known. Patches of PS exposure were also evident on NB4 cells in the absence of etoposide (Fig. 2C) but staining of TF was not sufficiently bright to obtain satisfactory images. Thus, it remains possible that the topographical distribution differs under those conditions. The current data suggest that active TF is most likely localized in the membrane regions where TF and PS overlap. Lactadherin inhibited approximately 80% of the FXase activity and prothrombinase activity of the NB4 cells and fresh patient APL cells, demonstrating that the major procoagulant activity was PS dependent [17Shi J. Gilbert G.E. Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid binding sites.Blood. 2003; 101: 2628-36Crossref PubMed Scopus (148) Google Scholar]. The degree of residual activity supported by APL cells contrasts with FVIIa‐TF and prothrombinase activity supported by platelets, where more than 98% of activity is inhibited by lactadherin [19Shi J. Pipe S.W. Rasmussen J.T. Heegaard C.W. Gilbert G.E. Lactadherin blocks thrombosis and hemostasis in vivo: correlation with platelet phosphatidylserine exposure.J Thromb Haemost. 2008; 6: 1167-74Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. This implies that on APL cells approximately 20% of activity is independent of PS. The most likely source of this residual activity is a cysteine protease of malignant cells called cancer procoagulant [33Falanga A. Gordon S.G. Isolation and characterization of cancer procoagulant: a cysteine proteinase from malignant tissue.Biochemistry. 1985; 24: 5558-67Crossref PubMed Google Scholar]. This enzyme is active on NB4 cells and is down‐regulated upon differentiation by ATRA [11Falanga A. Consonni R. Marchetti M. Mielicki W.P. Rambaldi A. Lanotte M. Gordon S.G. Barbui T. Cancer procoagulant in the human promyelocytic cell line NB4 and its modulation by all‐trans‐retinoic acid.Leukemia. 1994; 8: 156-9PubMed Google Scholar]. This underscores the fact that APL cells are procoagulant by several mechanisms, with approximately 80% of activity dependent on PS exposure. J. Shi, C. W. Heegaard, J. T. Rasmussen and G. E. Gilbert have applied for a patent on the use of lactadherin to detect phosphatidylserine. The other authors state that they have no conflict of interest. This research was supported by grants from the National Natural Science Foundation of China (30871227), Scientific Foundation of Heilongjiang Province (GB08C401–02), Scientific Research Fund of Heilongjiang Provincial Education Department (11531207), and a Department of Veterans Affairs Merit Grant to G.E. Gilbert. We thank J. O’Kelly (Los Angeles, CA) for providing NB4 cells, V.A. Novakovic, Y. Fu, W. Li, H. Li and X. Zhang for technical assistance, and H. Thatte for sharing expertise with confocal microscopy. Text S1. Phosphatidylserine exposure and procoagulant activity in acute promyelocytic leukemia. Please note: Wiley‐Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Download .doc (.08 MB) Help with doc files Supporting info item" @default.
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