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ABSTRACT: Total elevated plasma homocysteine (Hcy) is a risk factor for thromboembolism. Vascular endothelium is important to regulate coagulation, but the impact of Hcy on the clot-promoting activity (CPA) of endothelial cells has not been fully understood. In our study, human umbilical vein endothelial cells (HUVECs) were treated with Hcy (8, 20, 80, 200, 800 μmol/L) for 24 h. Annexin V was utilized to detect phosphatidylserine (PS) externalization and endothelial microparticles (MPs) formation. CPA was assessed by recalcification time and purified clotting complex tests. We found that Hcy enhanced the externalized PS and consequent CPA of HUVECs in a dose-dependent fashion, effect of Hcy had statistical significance at 800 μmol/L. In addition, Hcy also increased the shedding of procoagulant endothelial MPs. Blocking of PS with 128 nmol/L annexin V reduced approximately 70% CPA of HUVECs and endothelial MPs, but human anti-tissue factor antibody had little inhibitive effect. Our results showed that Hcy increased CPA of HUVECs via PS externalization and MPs release. Our present study has implications for hyperhomocysteinemia-related hypercoagulability.
Amino Acids 12/2011; 43(3):1243-50. · 3.25 Impact Factor
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ABSTRACT: The coagulopathy of acute promyelocytic leukemia (APL) is mainly related to procoagulant substances and fibrinolytic activators of APL blasts, but the fate of these leukemic cells is unknown. The aim of this study was to investigate the removal of APL blasts by macrophages and endothelial cells in vitro and consequent procoagulant and fibrinolytic activity of APL cells. We found that human umbilical vein endothelial cells as well as THP-1 and monocyte-derived macrophages bound, engulfed, and subsequently degraded immortalized APL cell line NB4 and primary APL cells. Lactadherin promoted phagocytosis of APL cells in a time-dependent fashion. Furthermore, factor Xa and prothrombinase activity of phosphatidylserine-exposed target APL cells was time-dependently decreased after incubation with phagocytes (THP-1-derived macrophages or HUVECs). Thrombin production on target APL cells was reduced by 40%-45% after 2 hours of coincubation with phagocytes and 80% by a combination of lactadherin and phagocytes. Moreover, plasmin generation of target APL cells was inhibited 30% by 2 hours of phagocytosis and ∼ 50% by lactadherin-mediated engulfment. These results suggest that engulfment by macrophages and endothelial cells reduce procoagulant and fibrinolytic activity of APL blasts. Lactadherin and phagocytosis could cooperatively ameliorate the clotting disorders in APL.
Blood 12/2011; 119(10):2325-34. · 9.90 Impact Factor
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ABSTRACT: Coagulopathy is a major cause of early death when arsenic trioxide (As(2)O(3)) therapy fails. In addition to the procoagulant properties of blast cells, the cytotoxic therapy may contribute to the coagulation disorders. The aim of the present study was to evaluate the possible impact of As(2)O(3) on membrane alterations, including phosphatidylserine (PS) exposure and microparticle generation, and the consequent procoagulant properties of endothelial cells.
Procoagulant activity (PCA) of human umbilical vein endothelial cells (HUVECs) was assessed by measuring clotting time and through purified coagulation complex assays. PS exposure on HUVEC membrane was observed by confocal microscopy and quantified with flow cytometry. In addition, counts and PCA of endothelial microparticles were determined by flow cytometry and plasma coagulation assay.
As(2)O(3) increased the ability of HUVECs to accelerate coagulation process and promote formation of coagulation complexes. Procoagulant activity corresponded to PS exposed on HUVECs. In coincidence with the PS externalization, As(2)O(3) increased the production of PS-bearing microparticles, which then accelerated fibrin strand formation significantly. By blocking PS, lactadherin was able to inhibit over 90% of the intrinsic tenase/prothrombinase activity of As(2)O(3)-treated HUVECs, and restored coagulation times of As(2)O(3)-treated cells and microparticles to control levels.
As(2)O(3) increases PCA of HUVECs through PS exposure and PS-bearing microparticle generation, which might cause thrombosis and act as a contributing factor in As(2)O(3) therapy-related coagulopathy.
Thrombosis Research 01/2011; 127(5):466-72. · 2.44 Impact Factor
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ABSTRACT: Cytotoxic chemotherapy induces or worsens hemostatic disorders in patients with acute myeloid leukemia. Procoagulant release and tissue factor expression on tumor cells are considered to contribute to chemotherapeutic agents-associated coagulopathy. The role of red blood cells during this process has not been determined; although they lack tissue factor, they may contribute suitable membranes for the amplification phase of blood coagulation. The present study aims to evaluate the possible impact of daunorubicin on phosphatidylserine exposure and consequent procoagulant property of red blood cells.
Red blood cells from acute myeloid leukemia patients and healthy donors were treated with daunorubicin as well as all-trans-retinoic acid, arsenic trioxide or etoposide for 0-48 h. Procoagulant activity was assessed by measurement of a clotting time and by purified coagulation complex assays. Lactadherin was used as a probe for phosphatidylserine.
Daunorubicin treatment increased procoagulant activity of red blood cells regardless of the cell origin. Moreover, coagulation complexes assays supported daunorubicin-evoked procoagulant response. The procoagulant property of red blood cells was not affected by the other three agents. The modulating effect of procoagulant activity was concomitant with and dependent on level of phosphatidylserine on the outer surface. Blockade of phosphatidylserine with lactadherin inhibited over 90% of tenase generation and prothrombinase activity and prolonged the coagulation time.
We conclude that daunorubicin interacts with red blood cells in a manner that increases phosphatidylserine exposure and consequent procoagulant activity. Lactadherin is an efficient anticoagulant of this process.
Thrombosis Research 10/2009; 125(2):178-83. · 2.44 Impact Factor
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ABSTRACT: Amniotic fluid (AF) may induce disseminated intravascular coagulation (DIC) when it enters maternal circulation by breaching the placental-maternal circulation barrier. The precise mechanism of the procoagulant activity of AF is unclear, but tissue factor (TF) has been proposed to be the main cause. As one constituent of AF, AF cells accumulate and undergo apoptosis continuously. Therefore, we speculate that AF cells have procoagulant activity due to the externalisation of phosphatidylserine (PS). The present study aims to demonstrate that, in addition to TF, the PS that is externalised on AF cells is important for the procoagulant activity of AF. Ten AF samples from parturient women were analysed using lactadherin as the probe for PS. Anti-TF antibody also was used to identify TF and its associated coagulation functions in AF cells. Normal platelets, neutrophils, and lymphocytes were harvested as controls. Confocal microscopy and flow cytometry was used to assess PS expression on AF cells. The procoagulant activity of AF cells was demonstrated by a plasma coagulation assay and further confirmed by factor Xase/prothrombinase assays. PS and TF were present on most AF cells, providing substantial procoagulant activity. Furthermore, factor Xase and prothrombinase assays showed that AF cells substantially enforced the activation of factor X and prothrombin. PS on AF cells is an important procoagulant source for AF. Lactadherin is an ideal anticoagulant for inhibiting the procoagulant activity of AF cells.
Thrombosis and Haemostasis 06/2009; 101(5):845-51. · 5.04 Impact Factor
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ABSTRACT: Electrostatic interactions with negatively charged membranes contribute to the subcellular targeting of proteins with polybasic clusters or cationic domains. Although the anionic phospholipid phosphatidylserine is comparatively abundant, its contribution to the surface charge of individual cellular membranes is unknown, partly because of the lack of reagents to analyze its distribution in intact cells. We developed a biosensor to study the subcellular distribution of phosphatidylserine and found that it binds the cytosolic leaflets of the plasma membrane, as well as endosomes and lysosomes. The negative charge associated with the presence of phosphatidylserine directed proteins with moderately positive charge to the endocytic pathway. More strongly cationic proteins, normally associated with the plasma membrane, relocalized to endocytic compartments when the plasma membrane surface charge decreased on calcium influx.
Science 02/2008; 319(5860):210-3. · 31.20 Impact Factor
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ABSTRACT: Phosphatidylserine (PS) appears on the outer membrane leaflet of cells undergoing programmed cell death and marks those cells for clearance by macrophages. Macrophages secrete lactadherin, a PS-binding protein, which tethers apoptotic cells to macrophage integrins.
We utilized fluorescein-labeled lactadherin together with the benchmark PS Probe, annexin V, to detect PS exposure by flow cytometry and confocal microscopy. Immortalized leukemia cells were treated with etoposide, and the kinetics and topology of PS exposure were followed over the course of apoptosis.
Costaining etoposide-treated leukemoid cells with lactadherin and annexin V indicated progressive PS exposure with dim, intermediate, and bright staining. Confocal microscopy revealed localized plasma membrane staining, then diffuse dim staining by lactadherin prior to bright generalized staining with both proteins. Annexin V was primarily localized to internal cell bodies at early stages but stained the plasma membrane at the late stage. Calibration studies suggested a PS content less, less than or approximately equal to 2.5%-8% for the membrane domains that stained with lactadherin but not annexin V.
Macrophages may utilize lactadherin to detect PS exposure prior to exposure of sufficient PS to bind annexin V. The methodology enables detection of PS exposure at earlier stages than established methodology.
Cytometry Part A 01/2007; 69(12):1193-201. · 3.73 Impact Factor
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Pierfrancesco Tassone,
Victor S Goldmacher,
Paola Neri,
Antonella Gozzini,
Masood A Shammas,
Kathleen R Whiteman,
Linda L Hylander-Gans,
Daniel R Carrasco,
Teru Hideshima,
Reshma Shringarpure, Jialan Shi,
Charles K Allam,
John Wijdenes,
Salvatore Venuta,
Nikhil C Munshi,
Kenneth C Anderson
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ABSTRACT: We tested the in vitro and in vivo antitumor activity of the maytansinoid DM1 (N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine), a potent antimicrotubule agent, covalently linked to the murine monoclonal antibody (mAb) B-B4 targeting syndecan-1 (CD138). We evaluated the in vitro activity of B-B4-DM1 against a panel of CD138(+) and CD138(-) cell lines, as well as CD138(+) patient multiple myeloma (MM) cells. Treatment with B-B4-DM1 selectively decreased growth and survival of MM cell lines, patient MM cells, and MM cells adherent to bone marrow stromal cells. We further examined the activity of B-B4-DM1 in 3 human MM models in mice: (1) severe combined immunodeficient (SCID) mice bearing subcutaneous xenografts; (2) SCID mice bearing green fluorescent protein-positive (GFP(+)) xenografts; and (3) SCID mice implanted with human fetal bone (SCID-hu) and subsequently injected with patient MM cells. Tumor regression and inhibition of tumor growth, improvement in overall survival, and reduction in levels of circulating human paraprotein were observed in mice treated with B-B4-DM1. Although immunohistochemical analysis demonstrates restricted CD138 expression in human tissues, the lack of B-B4 reactivity with mouse tissues precludes evaluation of its toxicity in these models. In conclusion, B-B4-DM1 is a potent anti-MM agent that kills cells in an antigen-dependent manner in vitro and mediates in vivo antitumor activity at doses that are well tolerated, providing the rationale for clinical trials of this immunoconjugate in MM.
Blood 01/2005; 104(12):3688-96. · 9.90 Impact Factor
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ABSTRACT: Lactadherin, a milk protein, contains discoidin-type lectin domains with homology to the phosphatidylserine-binding domains of blood coagulation factor VIII and factor V. We have found that lactadherin functions, in vitro, as a potent anticoagulant by competing with blood coagulation proteins for phospholipid binding sites [J. Shi and G.E. Gilbert, Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid binding sites, Blood 101 (2003) 2628-2636]. We wished to characterize the membrane-binding properties that correlate to the anticoagulant capacity. We labeled bovine lactadherin with fluorescein and evaluated binding to membranes of composition phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine, 4:20:76 supported by 2 mum diameter glass microspheres. Lactadherin bound saturably with an apparent KD of 3.3+/-0.4 nM in a Ca++ -independent manner. The number of lactadherin binding sites increased proportionally to the phosphatidylserine content over a range 0-2% and less rapidly for higher phosphatidylserine content. Inclusion of phosphatidylethanolamine in phospholipid vesicles did not enhance the apparent affinity or number of lactadherin binding sites. The number of sites was at least 4-fold higher on small unilamellar vesicles than on large unilamellar vesicles, indicating that lactadherin binding is enhanced by membrane curvature. Lactadherin bound to membranes with synthetic dioleoyl phosphatidyl-L-serine but not dioleoyl phosphatidyl-D-serine indicating stereoselective recognition of phosphatidyl-L-serine. We conclude that lactadherin resembles factor VIII and V with stereoselective preference for phosphatidyl-L-serine and preference for highly curved membranes.
Biochimica et Biophysica Acta 12/2004; 1667(1):82-90. · 4.66 Impact Factor
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ABSTRACT: Lactadherin, a glycoprotein of the milk-fat globule membrane, contains tandem C domains with homology to discoidin-type lectins and to membrane-binding domains of blood-clotting factors V and VIII. We asked whether the structural homology confers the capacity to compete for the membrane-binding sites of factor VIII and factor V and to function as an anticoagulant. Our results indicate that lactadherin competes efficiently with factor VIII and factor V for binding sites on synthetic phosphatidylserine-containing membranes with half-maximal displacement at lactadherin concentrations of 1 to 4 nM. Binding competition correlated to functional inhibition of factor VIIIa-factor IXa (factor Xase) enzyme complex. In contrast to annexin V, lactadherin was an efficient inhibitor of the prothrombinase and the factor Xase complexes regardless of the degree of membrane curvature and the phosphatidylserine content. Lactadherin also inhibited the factor VIIa-tissue factor complex efficiently whereas annexin V was less effective. Because the inhibitory concentration of lactadherin was proportional to the phospholipid concentration, and because lactadherin was not an efficient inhibitor in the absence of phospholipid, the major inhibitory effect of lactadherin relates to blocking phospholipid sites rather than forming inhibitory protein-protein complexes. Lactadherin was also an effective inhibitor of a modified whole blood prothrombin time assay in which clotting was initiated by dilute tissue factor; 60 nM lactadherin prolonged the prothrombin time 150% versus 20% for 60 nM annexin V. These results indicate that lactadherin can function as a potent phospholipid-blocking anticoagulant.
Blood 05/2003; 101(7):2628-36. · 9.90 Impact Factor
