scholarly journals Defective Ca(2+)-induced microvesiculation and deficient expression of procoagulant activity in erythrocytes from a patient with a bleeding disorder: a study of the red blood cells of Scott syndrome

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 380-388 ◽  
Author(s):  
EM Bevers ◽  
T Wiedmer ◽  
P Comfurius ◽  
SJ Shattil ◽  
HJ Weiss ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Calcium Ionophore ◽  
Coagulation Factors ◽  
Calcium Ionophore A23187 ◽  
Bleeding Disorder ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Factor Va ◽  
Resealed Ghosts

Abstract The erythrocytes from a patient with Scott syndrome, a bleeding disorder characterized by an isolated defect in expression of platelet procoagulant activity, have been studied. When incubated with the calcium ionophore A23187, Scott syndrome red blood cells (RBCs) expressed less than 10% of the prothrombinase (enzyme complex of coagulation factors Va and Xa) activity of A23187-treated RBCs obtained from normal controls. Consistent with the results from enzyme assay, the ionophore-treated Scott syndrome erythrocytes exhibited diminished membrane vesiculation and decreased exposure of membrane binding sites for factor Va compared with identically treated controls. When examined by scanning electron microscopy, untreated Scott syndrome RBCs were indistinguishable from normal cells. After incubation with A23187, however, the morphology of Scott syndrome RBCs contrasted markedly from normal erythrocytes. Whereas the Ca2+ ionophore induced marked echinocytosis and spiculation of normal RBCs, Scott syndrome RBCs remained mostly discoid under these conditions, with only an occasional echinocyte-like cell observed. These aberrant responses to intracellular Ca2+ were also observed for resealed ghosts prepared from Scott syndrome erythrocytes, indicating that they are related to a defect in the membrane or membrane-associated cytoskeleton. The finding that the erythrocytes of this patient share many of the membrane abnormalities reported previously for Scott syndrome platelets suggests that this defect is common to both cell lines and involves a membrane component required for vesicle formation and for expression of prothrombinase sites.

scholarly journals Defective Ca(2+)-induced microvesiculation and deficient expression of procoagulant activity in erythrocytes from a patient with a bleeding disorder: a study of the red blood cells of Scott syndrome

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 380-388 ◽  
Author(s):  
EM Bevers ◽  
T Wiedmer ◽  
P Comfurius ◽  
SJ Shattil ◽  
HJ Weiss ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Calcium Ionophore ◽  
Coagulation Factors ◽  
Calcium Ionophore A23187 ◽  
Bleeding Disorder ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Factor Va ◽  
Resealed Ghosts

The erythrocytes from a patient with Scott syndrome, a bleeding disorder characterized by an isolated defect in expression of platelet procoagulant activity, have been studied. When incubated with the calcium ionophore A23187, Scott syndrome red blood cells (RBCs) expressed less than 10% of the prothrombinase (enzyme complex of coagulation factors Va and Xa) activity of A23187-treated RBCs obtained from normal controls. Consistent with the results from enzyme assay, the ionophore-treated Scott syndrome erythrocytes exhibited diminished membrane vesiculation and decreased exposure of membrane binding sites for factor Va compared with identically treated controls. When examined by scanning electron microscopy, untreated Scott syndrome RBCs were indistinguishable from normal cells. After incubation with A23187, however, the morphology of Scott syndrome RBCs contrasted markedly from normal erythrocytes. Whereas the Ca2+ ionophore induced marked echinocytosis and spiculation of normal RBCs, Scott syndrome RBCs remained mostly discoid under these conditions, with only an occasional echinocyte-like cell observed. These aberrant responses to intracellular Ca2+ were also observed for resealed ghosts prepared from Scott syndrome erythrocytes, indicating that they are related to a defect in the membrane or membrane-associated cytoskeleton. The finding that the erythrocytes of this patient share many of the membrane abnormalities reported previously for Scott syndrome platelets suggests that this defect is common to both cell lines and involves a membrane component required for vesicle formation and for expression of prothrombinase sites.

Platelet microparticle membranes have 50- to 100-fold higher specific procoagulant activity than activated platelets

2007 ◽  
Vol 97 (03) ◽  
pp. 425-434 ◽  
Author(s):  
Dmitry Kireev ◽  
Nadezhda Popenko ◽  
Aleksei Pichugin ◽  
Mikhail Panteleev ◽  
Olga Krymskaya ◽  
...  
Keyword(s):  
Blood Platelets ◽  
Calcium Ionophore ◽  
Coagulation Factors ◽  
In Vitro Models ◽  
Calcium Ionophore A23187 ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Factor X ◽  
Activated Platelets

SummaryPlatelet microparticles (PMPs) are small vesicles released from blood platelets upon activation. The procoagulant activity of PMPs has been previously mainly characterized by theirability to bind coagulation factors VIII and Va in reconstructed systems. It can be supposed that PMPs can contribute to the development of thrombotic complications in the pathologic states associated with the increase of their blood concentration. In this study we compared procoagulant properties of calcium ionophore A23187-activated platelets and PMPs using several in-vitro models of hemostasis. Surface densities of phosphatidylserine, CD61, CD62P and factor X bound per surface area unit were determined by flow cytometry. They were 2.7-, 8.4-, 4.3-, and 13-fold higher for PMPs than for activated platelets, respectively. Spatial clot growth rate (Vclot) in the reaction-diffus ion experimental model and endogenous thrombin potential (ETP) were determined in plasma, which was depleted of phospholipid cell surfaces by ultra-centrifugation and supplemented with activated platelets or PMPs at different concentrations. Both Vcllot and ETP rapidly increased with the increase of PMP or platelet concentration until saturation was reached. The plateau values of Vclot and ETP for activated platelets and PMPs were similar. In both assays, the procoagulant activity of one PMP was almost equal to that of one activated platelet despite at least two-orders-of-magnitude difference in their surface areas. This suggests that the PMP surface is approximately 50- to 100-fold more procoagulant than the surface of activated platelets.

scholarly journals Complement proteins C5b-9 stimulate procoagulant activity through platelet prothrombinase

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 875-880 ◽  
Author(s):  
T Wiedmer ◽  
CT Esmon ◽  
PJ Sims
Keyword(s):  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Factor V ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Exogenous Factors

Abstract The capacity of platelets treated with nonlytic concentrations of the C5b-9 proteins to catalyze prothrombin activation and thereby trigger clot formation has been investigated. When suspended in the presence of exogenous factors Xa and Va, gel-filtered platelets treated with purified C5b-9 proteins catalyzed prothrombin to thrombin conversion at rates up to tenfold above controls, and exceeded by up to fourfold the prothrombinase activity observed for thrombin-stimulated platelets. In the absence of added factor Va, C5b-9 assembly on the platelet surface significantly shortened the lag period before prothrombinase expression that was observed for untreated platelets and increased the maximum catalytic rate of thrombin formation. A comparison with other platelet stimuli revealed that the C5b-9-induced activation of platelet prothrombinase closely paralleled the effects mediated by calcium ionophore A23187. Our data suggest that the C5b-9 proteins promote the release of platelet factor V and the assembly of the prothrombinase complex, thereby potentiating the effects of thrombin on the activation of prothrombinase. Membrane assembly of the C5b-9 proteins was also observed to markedly accelerate the rate of platelet-catalyzed plasma clotting, suggesting a direct link between C5b-9-mediated prothrombinase activation and procoagulant activity accompanying immunologic damage to the platelet.

scholarly journals Complement proteins C5b-9 stimulate procoagulant activity through platelet prothrombinase

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 875-880 ◽  
Author(s):  
T Wiedmer ◽  
CT Esmon ◽  
PJ Sims
Keyword(s):  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Factor V ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Exogenous Factors

The capacity of platelets treated with nonlytic concentrations of the C5b-9 proteins to catalyze prothrombin activation and thereby trigger clot formation has been investigated. When suspended in the presence of exogenous factors Xa and Va, gel-filtered platelets treated with purified C5b-9 proteins catalyzed prothrombin to thrombin conversion at rates up to tenfold above controls, and exceeded by up to fourfold the prothrombinase activity observed for thrombin-stimulated platelets. In the absence of added factor Va, C5b-9 assembly on the platelet surface significantly shortened the lag period before prothrombinase expression that was observed for untreated platelets and increased the maximum catalytic rate of thrombin formation. A comparison with other platelet stimuli revealed that the C5b-9-induced activation of platelet prothrombinase closely paralleled the effects mediated by calcium ionophore A23187. Our data suggest that the C5b-9 proteins promote the release of platelet factor V and the assembly of the prothrombinase complex, thereby potentiating the effects of thrombin on the activation of prothrombinase. Membrane assembly of the C5b-9 proteins was also observed to markedly accelerate the rate of platelet-catalyzed plasma clotting, suggesting a direct link between C5b-9-mediated prothrombinase activation and procoagulant activity accompanying immunologic damage to the platelet.

scholarly journals Platelet phosphatidylserine exposure and procoagulant activity in clotting whole blood – different effects of collagen, TRAP and calcium ionophore A23187

2003 ◽  
Vol 89 (01) ◽  
pp. 132-141 ◽  
Author(s):  
Mats Rånby ◽  
Tomas Lindahl ◽  
Sofia Ramström
Keyword(s):  
Whole Blood ◽  
Calcium Ionophore ◽  
Annexin V ◽  
Calcium Ionophore A23187 ◽  
Procoagulant Activity ◽  
Phospholipid Vesicles ◽  
Ionophore A23187 ◽  
Clotting Time ◽  
Phosphatidylserine Exposure ◽  
Free Plasma

SummaryWe have studied the effects of different platelet agonists on phosphatidylserine (PS) exposure and clotting times in blood without anticoagulants. Similar reductions in clotting time were obtained for collagen, TRAP-6 or calcium ionophore A23187 (50 μmol/L), in spite of huge differences in PS expression [6.7 ± 2.4%, 2.3 ± 0.5% and 99.9 ± 0.1%, respectively (mean ± SD, n = 5)]. Furthermore, the clotting times were much longer for samples with A23187 exposing the same amounts of PS as samples with collagen or TRAP-6. Annexin V reversed the clotting time reduction, but could not prevent coagulation. Addition of phospholipid vesicles containing 20% PS neither affected the clotting times nor induced clotting in recalcified, platelet-free plasma.We conclude that platelet PS exposure is necessary, but not sufficient, for the coagulation amplification observed when platelets are stimulated via physiological receptors in a whole blood environment.

Evidence of Different Subspecies of Microparticles (MP) Derived from Red Blood Cells (RMP) and Comparison of Their Phenotypes and Procoagulant Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1763-1763
Author(s):  
Wenche Jy ◽  
Jaehoon Bang ◽  
Loreta Bidot ◽  
Andrew Lin ◽  
Joaquin J. Jimenez ◽  
...  
Keyword(s):  
High Speed ◽  
Thrombin Generation ◽  
Calcium Ionophore ◽  
Lag Time ◽  
Peak Height ◽  
Calcium Ionophore A23187 ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Differential Centrifugation

Abstract BACKGROUND: The potential roles of cell derived microparticles (MP) such as those derived from platelets (PMP), endothelium (EMP), leukocytes (LMP), and red cells (RMP) have been receiving increasing attention in disorders of hemostasis/thrombosis and inflammation and they are emerging as valuable biomarkers. However among these MP, little is known about RMP. Our recent clinical studies indicate that RMP play a role in hemostasis and thrombosis in patients with thrombocytopenia and in thrombocytosis. However, the phenotypes and procoagulant activity of their subspecies remain unknown. We report evidence for heterogeneity of RMP following differential centrifugation. METHODS: RMP were prepared by exposure of washed RBC to the calcium ionophore, A23187, and the RBC were removed by low-speed centrifugation. The RMP were washed twice at 20,000xg for 15 min. Procoagulant activity of RMP was measured by the calibrated automated thrombogram (CAT) system (Hemker et al Pathophysiol Haemost Thromb.2002;32:249) using thrombin substrate Z-Gly-Gly-Arg-AMC on a fluorescence plate reader. The lag time and peak height (nM) of thrombin generation were recorded. Markers used for labeling RMP were PE-labeled anti-glycophorin (GlyP), FITC-anti-tissue factor (TF), FITC-annexin V (AnV), and/or FITC-lectin Ulex europeaus I (Ulex). RESULTS: In thrombin generation assay, RMP induced a long lag time (24±3 min) but high thrombin peak (330±37 nM). These data were consistent with the flow cytometric finding that RMP carried very little TF (<0.1%) but very high AnV binding (88±6%). By high speed centrifugation (15,000xg for 10 min), two populations of RMP were studied: the larger RMP in the pellet expressed GlyP, AnV and Ulex while the smaller or lighter RMP remaining in the supernatant, did not express GlyP and AnV but do express Ulex. The smaller RMP accounted for 30–40% of total Ulex+ RMP. These two subspecies (large and small) of RMP showed distinct thrombin generation profiles. The lag time and peak height of thrombin generation for large RMP (GlyP+/AnV+/Ulex+) was 23–28 min and 300–335 nM, respectively, which is close to values of whole RMP. On the other hand, the smaller RMP (Ulex+/GlyP−/AnV−) produced much longer lag time (31–38 min) and lower peak (60–75 nM), indicating that the majority of the procoagulant activity of RMP is associated with larger RMP. SUMMARY: The present study demonstrates that RMP are rich in anionic phospholipids and effective in generating thrombin in vitro. We have identified 2 distinct subpopulations of RMP by differential centrifugation: One larger RMP express binding of anti-GlyP, AnV and Ulex, and carry the majority of procoagulant activity. The smaller RMP expressing only Ulex binding exhibit much weaker procoagulant activity. The roles of these two species of RMP remain to be elucidated. We speculate that smaller RMP may represent the nanovesicles described by Allen et al [Biochem J 188:881, 1980] and that Ulex may be a novel and convenient means for the study of these small vesicles.

scholarly journals Increased expression of procoagulant activity on the surface of human platelets exposed to heavy-metal compounds

1995 ◽  
Vol 308 (1) ◽  
pp. 15-21 ◽  
Author(s):  
C A Goodwin ◽  
C P D Wheeler-Jones ◽  
S Namiranian ◽  
S Bokkala ◽  
V V Kakkar ◽  
...  
Keyword(s):  
Heavy Metal ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Anionic Phospholipid ◽  
Metal Compounds ◽  
Platelet Surface ◽  
Thrombin Formation

One of the essential roles for platelets in haemostasis is in the potentiation of blood clotting due to the contribution of anionic phospholipid from the surface of the cells, as an essential cofactor to the proteolytic reactions of coagulation (platelet procoagulant activity). Only a limited number of agonists are known to initiate platelet procoagulant activity. In this study the rate of thrombin formation on the platelet surface was observed to increase in a dose-dependent manner upon treatment of washed platelets with heavy-metal compounds. Unlike the immediate increase observed upon treatment of platelets with calcium ionophore, A23187, the change due to these agents was progressive, approaching a maximum after 10 min. The maximum-fold acceleration of the rate of thrombin formation compared with control platelets was calculated for HgCl2 (56-fold), AgNO3 (42-fold) phenylmercuriacetate (24-fold) and thimerosal (14-fold), compared with 70-fold observed for calcium ionophore. The increase in procoagulant activity due to HgCl2 coincided with a large increase in intracellular calcium and phosphorylation of 22 and 45 kDa proteins. It is considered that the mechanism responsible for the increase in procoagulant activity is exposure of anionic phospholipids. This was detected by a 2-fold increase in the binding of 125I-annexin V upon addition of HgCl2, compared with resting platelets (3-fold on treatment of platelets with calcium ionophore). In contrast to the generation of activity by A23187 and other known agonists of this reaction, heavy-metal compounds appeared to cause little or no release of microparticles from the platelet surface. Since HgCl2 did not cause aggregation of platelets or significant release of serotinin, these findings may give further support to the need for exposure and ligation of glycoprotein IIb:IIIa for vesiculization to occur. Treatment of platelets with heavy metals may constitute a new approach to investigating the early changes in the cell membrane which lead to increased expression of anionic phospholipid.

scholarly journals Formation of Leukotrienes From Calcium Ionophore-A23187 Stimulated Rabbit, Rat and Mice White Blood Cells

2002 ◽  
Vol 70 (3) ◽  
pp. 233-243
Author(s):  
Mansour Mahrnoud A. ◽  
AI-Jenoobi Fahad I. ◽  
Al-Suwayeh Saleh A. ◽  
Al-Majed Abdulhakeem A. ◽  
Al-Shabanah Othman
Keyword(s):  
Blood Cells ◽  
High Performance ◽  
Uv Spectroscopy ◽  
White Blood Cells ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Endogenous Substrate ◽  
Peripheral White Blood Cells ◽  
Human White Blood Cells

Leukotrienes (LTs) producing capacity was investigated in calcium ionophore A23 187- stimulated rabbit. rat and mice peripheral white blood cells suspension. A reverse phase high performance liquid chromatography technique and computerized UV spectroscopy were employed to isolate and quantitate the released LTs namely. LTC4 and LTB4. Preincubation of rabbit white blood cells at 37°C for 5 min followed by calcium ionophore-A23 187 (1 pM) stimulation for another 5 min produced an equal amounts of LTC4 as compared to LTC4 produced by human white blood cells (105±11 versus 95±9.5 pmol/107 cells respectively; mean ±SEM). In contrast rabbit white blood cells synthesized significantly lower LTB4 in comparison with LTB4 produced by peripheral white blood cells from healthy control (168±18 versus 228±19 pmol/107 cells respectively: mean ±SEM). When rat and mice white blood cells suspension were stimulated with calcium ionophore A23187 (1 µM) after preincubation at 37°C for 5 min, equivalent amounts of LTC4 and LTB4 were observed. However, LTB4 and LTC4 produced by rat and mice white blood cells were significantly lower in comparison with LTB4 and LTCj produced by human white blood cells stimulated with calcium ionophore-A23 187. These results demonstrate that rabbit. rat and mice white blood cells suspension possess the capacity to produce LTC4 and LTB4 from endogenous substrate after calcium ionophore-A23 187 stimulation.

Growth factors and the primary cilium in BALB/c 3T3 cells

1987 ◽  
Vol 45 ◽  
pp. 830-831
Author(s):  
R. W. Tucker ◽  
N. S. More ◽  
S. Jayaraman
Keyword(s):  
Growth Factors ◽  
Primary Cilium ◽  
Cultured Cells ◽  
Morphological Changes ◽  
Calcium Ionophore ◽  
Growth Stimulation ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
3T3 Cells ◽  
Microtubule Depolymerization

The mechanisms by which polypeptide growth factors Induce DNA synthesis in cultured cells is not understood, but morphological changes Induced by growth factors have been used as clues to Intracellular messengers responsible for growth stimulation. One such morphological change has been the transient disappearance of the primary cilium, a “9 + 0” cilium formed by the perinuclear centriole in interphase cells. Since calcium ionophore A23187 also produced both mitogenesis and ciliary changes, microtubule depolymerization might explain ciliary disappearance monitored by indirect immunofluorescence with anti-tubulin antibody. However, complete resorption and subsequent reformation of the primary cilium occurs at mitosis, and might also account for ciliary disappearance induced by growth factors. To settle this issue, we investigated the ultrastructure of the primary cilium using serial thin-section electron microscopy of quiescent BALB/c 3T3 cells before and after stimulation with serum.

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