Tetsuya Miyake

Platelet-derived microparticles may influence the development of atherosclerosis in diabetes mellitus.

We investigated the association between low-density lipoprotein (LDL), triglycerides, and platelet activation in 18 patients with hypertension age 41-64 years and 18 with diabetes mellitus aged 43-70 years. Platelet P-selectin positivity and the microparticle level (indicators of activation) were both significantly higher in the diabetics than in healthy controls (P-selectin: 28.0% +/- 7.5% vs. 7.3% +/- 4.2%, P < 0.001; microparticles: 1900 +/- 966 vs. 526 +/- 158/10(4) platelets, P < 0.01). In contrast, there was no significant increase of either parameter in the patients with hypertension. Plasma microparticle levels were also significantly greater in the diabetics with high LDL levels than in those with low LDL levels (2375 +/- 949 vs. 1519 +/- 796/10(4) platelets, P < 0.05), and in those with high rather than low triglyceride levels (2188 +/- 845 vs. 1492 +/- 783/10(4) platelets, P < 0.05). However, platelet positivity for P-selectin was not significantly different between these two subgroups. Microparticle and P-selectin levels both showed no significant difference between the hypertensive patients with high and low LDL or triglyceride levels. These results suggest that platelet-derived microparticles may participate in the development or progression of atherosclerosis in patients with diabetes mellitus.

Expression of Functional Tissue Factor on Small Vesicles of Lipopolysaccharide- Stimulated Human Vascular Endothelial Cells

We examined tissue factor expression on lipopolysaccharide-stimulated endothelial cells and their small vesicles by using specific antibodies and flow cytometry. Tissue factor functional activity was also assessed by activation of factor X. Endothelial cells were stimulated with 10 microg/ml of lipopolysaccharide in M-199/bovine serum albumin. Flow cytometry showed that expression of tissue factor on endothelial cells reached a maximum at 6 hours after stimulation, whereas that on small vesicles reached a maximum after 12 hours. Factor X activation mediated by factor VIIa and tissue factor was observed over a similar time course and was inhibited by the addition of antitissue factor antibody. Immunoelectron microscopy suggested that small vesicles with expression of some tissue factor were produced from the surface of endothelial cells. Our findings thus showed that tissue factor on endothelial cells produced by lipopolysaccharide stimulation was partly released to small vesicles. This may cause disseminated intravascular coagulation and related coagulation disorders.

Relationship of microparticles withβ2-glycoprotein I and P-selectin positivity to anticardiolipin antibodies in immune thrombocytopenic purpura

We investigated the association ofβ2-glycoprotein I and P-selectin with platelet-derived microparticles in 48 patients with immune thrombocytopenic purpura and 20 normal controls using two-color flow cytometric analysis. In addition, anticardiolipin antibodies were detected by an enzyme-linked immunosorbent assay. Platelet microparticles from the patients showed a higher positivity forβ2-glycoprotein I than those from the normal controls (23.1±15.4% vs. 5.3±3.1%, p<0.01), but this positivity was not related to the presence of platelet-associated IgG or to the severity of thrombocytopenia. In the 18 patients with more than 20% P-selectin-positive microparticles,β2-glycoprotein I positivity was significantly higher than in the 30 patients with less than 20% P-selectin-positive microparticles (37.1±20.5% vs. 21.5±17.3%, p<0.01). In addition, anticardiolipin antibodies were detected in eight patients, and they had a significantly higher level ofβ2-glycoprotein I-positive microparticles than the patients without such antibodies (42.0±22.9% vs. 22.6±18.9%, p<0.05). Our results suggest that anticardiolipin antibodies activate platelets in immune throm bocytopenic purpura and cause the generation of microparticles rich inβ2-glycoprotein I and P-selectin. These microparticles may then act to regulate coagulation abnormalities in patients with anticardiolipin antibodies.

Participation of αIIbβ3 in platelet microparticle generation by collagen plus thrombin

We investigated the role of αIIbβ3 in microparticle generation by normal and thrombasthenic platelets stimulated with collagen plus thrombin. Microparticle generation by normal p

Significance of cytokines and CD68‐positive microparticles in immune thrombocytopenic purpura

Abstract: We investigated the significance of cytokines (soluble interleukin‐2 receptor, granulocyte‐macrophage colony‐stimulating factor, interleukin‐6, and interferon‐gamma) and CD68‐positive microparticles in immune thrombocytopenic purpura. Cytokines were measured by enzyme‐linked immunosorbent assay and microparticles were detected by flow cytometry. CD68 expression by histiocytic U937 cells incubated with lipopolysaccharide or cytokines was also assessed in a control study. The level of CD68‐positive microparticles was significantly higher in the patients with thrombocytopenia than in normal controls (p<0.01). The soluble interleukin‐2 receptor level was also significantly higher in patients than in controls (p<0.01), but the other cytokines did not show a significant difference. However, patients with severe thrombocytopenia (platelet count >20000/μl) had significantly higher levels of granulocyte‐macrophage colony‐stimulating factor and interleukin‐6 than the controls (p<0.05). When opsonized platelets were incubated with activated U937 cells, lipopolysaccharide and granulocyte‐macrophage colony‐stimulating factor caused an increase of CD68‐positive microparticles in the supernatant. These results suggest that granulocyte‐macrophage colony‐stimulating factor is released by activated T cells in immune thrombocytopenic purpura and activates monocyte/macrophage phagocytosis, resulting in an increase of circulating CD68‐positive microparticles and enhanced platelet destruction.

Platelet-derived microparticles in alloxan-induced diabetes in rabbits.

To study platelet-derived microparticle generation in diabetes mellitus, we injected alloxan into male Japanese white rabbits. Injection of alloxan induced diabetes, but did not cause any significant change in various biochemical and hematological parameters. However, diabetic rabbits showed a significant elevation of platelet-derived microparticles from 8 weeks after alloxan injection (week 0: 0.45 +/- 0.24%; week 8: 1.12 +/- 0.61%, p < 0.005). These microparticles are known to have prothrombinase activity, suggesting that they may promote vascular complications in diabetes and may be used as a marker of vascular disease.

EFFECT OF CEPHARANTHIN AND CYTOCHALASIN D ON PLATELET INTERNALIZATION OF ANTI-GLYCOPROTEIN IIb/IIIa ANTIBODIES

The effects of cepharanthin and cytochalasin D on the internalization of anti-glycoprotein IIb/IIIa antibodies by platelets were investigated in 13 patients with chronic immune thrombocytopenic purpura who had circulating anti-glycoprotein IIb/IIIa autoantibodies. Unfixed platelets were incubated with a monoclonal anti-glycoprotein IIb/IIIa antibody (NNKY1-32) or with platelet-binding IgG from the patients (which contained anti-glycoprotein IIb/IIIa antibodies). Flow cytometry showed that the binding of NNKY1-32 to platelets was markedly decreased after incubation for 120 min compared with incubation for 10 min. This decrease was inhibited by cepharanthin but not by cytochalasin D. Platelet-binding IgG also showed markedly reduced binding after incubation for 120 min compared with 10 min, and this decrease was inhibited by both cepharanthin and cytochalasin D. Cytochalasin D inhibits platelet cytoskeletal activity while cepharanthin does not. Therefore, our results suggest that the internalization of anti-glycoprotein IIb/IIIa antibodies from the plasma of patients with immune thrombocytopenic purpura is related to platelet cytoskeletal reorganization, while the cytoskeleton did not participate in internalization of the monoclonal anti-glycoprotein IIb/IIIa antibody (NNKY1-32). Cepharanthin may be useful for studying the internalization and cycling of glycoprotein IIb/IIIa in human platelets, and it may also be potentially useful for the treatment of immune thrombocytopenic purpura.

Expression of prothrombinase activity and CD9 antigen on the surface of small vesicles from stimulated human endothelial cells

We employed flow cytometry and monoclonal antibodies (MoAb) to study the surface membrane protein of shed particles (small vesicles, SV) that were released from vascular endothelial cells (EC) by agonists such as a Ca ionophore (A23187) and thrombin. After stimulation of EC by A23187, CD9 antigens disappeared entirely from the EC surface in a time- and concentration-dependent manner; they subsequently moved onto the SV surface. Von Willebrand factor (vWF) and P-selectin from Weibel-Palade (W-P) bodies were expressed rapidly on the EC surface after thrombin stimulation, but not on the SV surface. P-selectin may have some effect on maintenance of hemostasis on the EC surface. We demonstrated that the surfaces of SV and EC significantly supported prothrombinase activity and confirmed that A23187-induced SV from EC express binding sites for factors IXa and Xa. These results suggest that the SV are an important factor in a novel controlling mechanism of the coagulation system on the EC surface.

Microparticle release from platelets by leukemic cell lines

To the Editor: Microparticles are vesicles released from the plate!et plasma membrane during activation, which are rich in membrane receptors for coagulation factor Va and provide a catalytic surface for the prothrombinase reaction (1,2). Therefore, the detection of microparticles derived from activated platelets might help to identify thrombotic disorders. Various coagulation abnormalities occur in cancer patients, such as hypercoagulability and thrombosis ( 3 ) , and many authors have reported elevated clotting factor levels in association with malignancy (4). In addition, some leukemic cell lines have been reported to induce procoagulant activity ( 5 ) . We investigated microparticle generation from platelets by leukemic cell lines in order to determine whether microparticles could have a role in the coagulation abnormalities related to malignancy. The three leukemic cell lines (U937 (6), K562 (7), and HL60 (8)) used in this study were obtained from the American Type Culture Collection. Cells were grown in RPMI1640 medium (Gibco Laboratories, New York) supplemented with 10 7; heat-inactivated fetal calf serum at 37°C in a humidified 5 9 , CO, atmosphere. The cells were routinely passaged with medium changes once or twice a week. Nonleukemic leukocytes from healthy volunteers were used for the control study. Washed platelets from healthy volunteers were incubated in RPMI-1640 with or without one of the leukemic cell lines or non-leukemic leukocytes. After incubation, the platelet samples were centrifuged at 200 g for 10 min at room temperature and the supernatant was collected. The supernatant was fixed with 1 0; paraformaldehyde (final concentration) without centrifugation and a fluorescein isothiocyanate (F1TC)-conjugated monoclonal anti-glycoprotein I b antibody (NNKY5-5(9)) was added, after which the microparticles in the supernatant were detected by flow cytometry. Forward and side light scatter were measured as an index of particle size and FITC fluorescence using a Becton Dickinson FACScan. The size of all particles detected was estimated by comparing their forward light scatter with that of FITC-labeled reference particles ranging from 0.1 to 2.0 pm in diameter. The percentage of microparticles was calculated as follows: [number of microparticles/(number of platelets + microparticles)] x 100. To obtain the percentage of plateletspecific microparticles, the percentage of microparticles positive for monoclonal antibody NNKY 5-5 was multiplied by the percentage of all microparticles. Students t-test was used for statistical comparisons and p < 0.05 was taken to indicate significance. Microparticle release increased when washed platelets were incubated any of the leukemic cell lines, but not when platelets were incubated with nonleukemic leukocytes. The increase was timedependent, and the most prominent change was noted with U937 cells (Table 1). In contrast, when washed platelets were incubated in the absence of leukemic cells, little microparticle release occurred. Hemorrhage or thrombosis often occur for obscure reasons in patients with malignancy, although the mechanisms involved are increasingly being defined. The present study showed that three leukemic cell lines could stimulate microparticle release from platelets in v i m unlike nonleukemic leukocytes, suggesting that some of the coagulation abnormalities in leukemia patients may be related to microparticle generation. Leukemic cell blasts contain various procoagulant materials (lo), and the existence of both platelet microparticles and such procoagulant mate-

Uptake of fibrinogen by circulating platelets.

Whether platelets obtain fibrinogen by biosynthesis or endocytosis has been a controversial issue. Recently, Harrison et al. [I] reported that platelets and/or megakaryocytes from a patient with afibrinogenemia took up fibrinogen after replacement therapy with cryoprecipitate [l]. In addition, our studies have suggested that platelet glycoprotein Ilbi IIla mediates such fibrinogen uptake [2]. However, the evidence has been stronger for megakaryocytes, and whether or not platelets actually participate in fibrinogen uptake has remained unclear. We therefore investigated in vitro and in vivo fibrinogen uptake by platelets from a patient with symptomatic afibrinogenemia. Platelets from this patient were washed and resuspended in autologous plasma, normal plasma, or normal plasma containing a monoclonal anti-glycoprotein IIb/Illa antibody. After incubation for 48 h gt 37°C with agitation, platelet fibrinogen levels were determined by rocket immunoelectrophoresis as reported previously [2]. Then dried human fibrinogen (Fibrinogen HT-Green Cross@) Uptake of Fibrinogen by Circulating Platelets