Thrombocytopenia develops early in malaria, but the underlying mechanisms remain incompletely understood. We studied the aetiology of malaria-associated thrombocytopenia in volunteers experimentally infected with Plasmodium falciparum malaria, in Indonesian malaria patients and in ex vivo studies. In experimental human malaria, the decrease in platelet counts was associated with a concurrent rise in young platelets (immature platelet fraction) and thrombopoietin. D-dimer concentrations were moderately elevated without a prolongation in the activated partial thromboplastin time or decrease in fibrinogen. There was no increase in expression of the platelet surface markers CD62P, PAC-1 and CD63 and in plasma concentrations of the platelet factors P-selectin, CXCR4, CXCL7, RANTES and CD40L. In contrast, concentrations of soluble glycoprotein-1b (sGP1b), the external domain of the platelet receptor for von Willebrand factor (VWF), increased early. Indonesian malaria patients also had elevated concentrations of sGP1b, which correlated with VWF concentrations. Finally, incubation of platelets with parasitized erythrocytes in vitro failed to induce platelet aggregation or activation. We concluded that neither compromised platelet production nor platelet activation or consumptive coagulopathy were responsible for the early thrombocytopenia in malaria. We hypothesize that the increase in sGP1b concentrations results from VWF-mediated GP1b shedding; a process that may prevent excessive adhesion of platelets and parasitized erythrocytes.

OBJECTIVE

Platelets and neutrophils co-localize at sites of vessel injury, hemorrhage and thrombosis. Moreover, circulating platelets and leukocytes interact productively, and the formation of heterotypic aggregates is a feature of acute coronary syndromes, systemic inflammatory, neoplastic and autoimmune diseases. We have summarized the evidence suggesting a homeostatic function of the interaction, culminating in the removal of activated platelets from the bloodstream.

RESULTS

Anionic phospholipids, that is cell surface 'eat me' signals exposed both by activated platelets and dying cells, signals such as P-selectin (CD62P), specifically expressed by platelets, as well as of polarized clusters of neutrophils beta2 integrins play a role in the capture of platelets in vitro and in vivo.

CONCLUSIONS

A bona-fide synapse assembles as a consequence of the interaction between P-selectin and its counter-receptor on neutrophils, with clustering of activated beta2 integrins into membrane microdomains and reorganization of cytoskeleton components that control cell motility and phagocytosis. Actual engulfment of the tethered platelet depends on the recognition of phosphatidylserine and/or of phosphatidylserine-associated molecules. This event may have a physiologic role in the regulation of the hemostatic potential of circulating blood; conversely, a failure may contribute to persistent vascular inflammation and thrombosis.

Quantitation of immunofluorescence intensity serves to estimate the number of defined molecules expressed on or in cells. Clinical applications of this diagnostic tool are increasing, e.g., aberrant expression of various antigens (Ag) by leukemic blasts or lymphoma cells, intensity of CD38 expression by CD8+ T-lymphocytes to monitor activation status, and intensity of CD62P to detect platelet activation. In this report we discuss the quality-control measures required for quantitation of fluorescence intensity, and we review seven concepts that have been developed to quantify fluorescence intensity during the past 15 years. Initial work addressed the conversion of logarithmic channel numbers into units of relative fluorescence. The design and use of calibration beads labeled with predefined amounts of dye allowed instrument-independent expression of fluorescence intensity in units of molecules of equivalent soluble fluorochrome (MESF). This method was refined by the combined use of such standards with monoclonal antibodies (mAb) conjugated 1:1 with phycoerythrin (PE), allowing translation of fluorescence intensity into numbers of antibodies bound per cell. Alternatively, the use of 1:1 PE-conjugated mAb under the assumption that CD4+ lymphocytes reproducibly bind 50,000 CD4 mAb molecules was proposed to convert units of relative fluorescence intensity into units of antibodies bound per cell. The use of antibody-binding capacity as a surrogate marker for quantification of Ag expression was addressed more directly by the development of antibody-binding standards. The quantitative indirect immunofluorescence assay is based on beads labeled with various amounts of CD5 mAb that calibrate the binding of the secondary antibody in units of antibody-binding capacity. Alternatively, goat anti-mouse-labeled calibration beads have been developed. Published results obtained with the latter calibrators showed an unexpected inaccuracy. The different ways in which calibrators and cells under study bind mAb (i.e., Fab mediated versus Fc mediated) may have contributed to this variation. Recently, the use of stabilized cell populations expressing Ag in a specified range of concentrations has been proposed as an Ag-specific calibration system of mAb binding. We identify several issues on the level of instrumentation, reagents, and cells under study that should be solved to allow standardization of quantitative assessments of immunofluorescence intensity.

BACKGROUND

The commonest cause of mortality in patients with Type 2 diabetes is atherothrombosis, which can be related to abnormalities in the coagulation and fibrinolytic pathways, as well as in platelet function. Platelet microparticles (PMPs) may contribute to the prothrombotic state and may promote the progression of atherosclerosis. We hypothesized that PMPs are elevated in Type 2 diabetes and that patients with Type 2 diabetes and clinically apparent atherosclerosis would have the highest levels. Similarly, we hypothesized that soluble plasma P-selectin (sPsel) and CD40L (both molecules which are released by activated platelets), as well as %CD62P (P-selectin) and %CD63 positivity on platelets quantified by flow cytometry, would be highest in patients with Type 2 diabetes and clinically apparent atherosclerotic disease, and might be correlated to PMP levels.

METHODS

Venous blood was obtained from 21 Type 2 diabetic patients without atherosclerotic complications, 18 diabetic patients with clinically apparent atherosclerotic disease and 21 non-diabetic control subjects. PMPs, as well as %CD62P and %CD63 positivity on platelets, were quantified by flow cytometry. sPsel and CD40L were measured using ELISA.

RESULTS

Patients with Type 2 diabetes and clinically apparent atherosclerotic disease had the highest PMP (P=0.045) and sPsel (P=0.046) levels, compared with patients without complications (who had intermediate PMP levels) and control subjects. Control subjects had the lowest CD40L levels (P<0.001) when compared with patients with Type 2 diabetes, with no difference in sCD40L levels between the two diabetic subgroups. %CD62P and %CD63 positivity did not differ between the groups. PMP levels correlated with %CD62P positivity (P=0.026) but not to %CD63 positivity (P=0.089), sCD40L (P=0.407) or sP-sel (P=0.163); sCD40L levels did not correlate with any other marker of platelet activation.

CONCLUSIONS

PMPs are elevated in Type 2 diabetes. In addition, patients with clinically apparent atherosclerosis had the highest levels of PMPs and sPsel. Thus, PMPs may be a marker of symptomatic atherosclerotic vascular disease in Type 2 diabetes, and may both represent a useful risk stratification tool as well as a novel therapeutic target for anti-thrombotic drugs.

Podocalyxin is a membrane protein of rat podocytes and endothelial cells. It has not been described in other cell types, and no amino acid or DNA sequence data are available about it. Here we show that podocalyxin antigens are present in rat platelets and megakaryocytes. In resting platelets, the antigens are mainly intracellular but become surface exposed after thrombin stimulation, as shown by immunofluorescence and flow cytometry. By Western blotting, platelet podocalyxin has an apparent Mr of 140,000. Cytocentrifuge slides of rat bone marrow show that anti-podocalyxin antibodies recognize large polyploid cells also expressing CD62P, indicating that the cells are megakaryocytes. From a rat glomerular cDNA library we isolated a clone covering the carboxyl-terminal nucleotides of rat podocalyxin. Its putative transmembrane or intracellular domains are 100% or >93% identical, respectively, with the human and rabbit podocalyxin-like proteins. The truncated extracellular domain extends to include two of the four conserved cysteines shared by podocalyxin-like proteins. By Northern blotting, a 5.5-kb renal cortical transcript is seen. By in situ hybridization, cRNA probes recognize podocytes, endothelial cells, and megakaryocytes, and by reverse transcription polymerase chain reaction, platelets are shown to contain podocalyxin mRNA. Our results show that rat podocalyxin is a homologue of the previously cloned podocalyxin-like proteins and suggest that also in mammals podocalyxin has a role in hematopoiesis, as previously shown in the chicken.

We compared the levels of microparticles, platelet activation markers, soluble cell adhesion molecules, and soluble selectins between hypertensive patients with and without type 2 diabetes and control subjects. Binding of anti-glycoprotein IIb/IIIa and anti-glycoprotein Ib monoclonal antibodies to platelets did not differ significantly between the hypertensive patients and controls, but platelet expression of activation markers (CD62P, CD63, PAC-1, and annexin V) was higher in the hypertensive patients. Platelet-derived microparticle (PDMP) and monocyte-derived microparticle (MDMP) levels were significantly higher in the hypertensive patients than in the controls. Soluble ICAM-1, VCAM-1, P-selectin, and E-selectin levels were also higher in the hypertensive patients, and they were significantly higher in the hypertensive patients with diabetes. After treatment with efonidipine, the levels of PDMPs, CD62P-, CD63-, PAC-1-, and annexin V-positive platelets, sICAM-1, sVCAM-1, sP-selectin, and sE-selectin all decreased significantly. The MDMP levels decreased, and the decrease was significant in the hypertensive patients with diabetes. These findings suggest that administration of efonidipine to hypertension patients with diabetes may prevent the development of cardiovascular complications caused by cell adhesion molecules or activated platelets and monocytes.

BACKGROUND

Flow cytometry allows the analysis of multiple antigens in a single tube at a single cell level. We present a rapid and sensitive two tube flow cytometric protocol for the detection of multiple platelet antigens and activation markers gated on a pure platelet population.

METHODS

The presence of platelet specific antigens was analyzed in citrated whole blood of normal platelets and from patients diagnosed with platelet abnormalities. Quiescent platelets as well as stimulated platelets were analyzed using a gating strategy based on ubiquitously expressed platelet membrane markers. A ubiquitously expressed platelet marker was combined with antibodies against the activated alpha2b-beta3 (PAC-1), Lysosomal Activated Membrane Protein (CD63) and P-selectin (CD62P).

RESULTS

We were able to detect the platelet antigens CD36, CD41, CD42a, CD42b and CD61 in one single tube. Our approach allowed the single tube determination of PAC-1, CD63 and CD62P after activation of platelets by thrombin, collagen, ADP and PAR-1, and determination of platelet abnormalities.

CONCLUSIONS

Our two tube multi-parameter screening protocol is suited for the analysis of platelet antigens expressed on quiescent and activated platelets and allows the detection of aberrancies as found in blood of patients with thrombocytopathy such as Glanzmann Thrombasthenia, storage pool disease with diminished granule content and patients treated with clopidogrel and acetylsalicylic acid.

Evidence that platelets play a role in tumor metastasis includes the observation of circulating tumor cell-platelet aggregates and the anti-metastatic effect of thrombocytopenia and anti-platelet drugs. Platelets have recently been shown to contain vascular endothelial growth factor (VEGF) which is released during clotting. We therefore studied the effects of (1) tumor cell-platelet adherence and tumor cell TF activity on platelet VEGF release; and (2) the effects of GpIIb/IIIa blockade on tumor cell-induced platelet VEGF release, tumor cell-induced thrombocytopenia and experimental metastasis. Adherent A375 human melanoma cells (TF+) and KG1 myeloid leukemia (TF-) cells were cultured in RPMI containing 10% fetal bovine serum. Platelet-rich plasma was obtained from normal citrated whole blood and the presence of VEGF (34 and 44 kDa isoforms) confirmed by immunoblotting. Platelet-rich plasma with or without anti-GpIIb/IIIa (Abciximab) was added to A375 monolayers and supernatant VEGF measured by ELISA. Tumor cell-induced platelet activation and release were determined by CD62P expression and serotonin release respectively. In vitro, tumor cell-platelet adherence was evaluated by flow cytometry. In vivo, thrombocytopenia and lung seeding were assessed 30 min and 18 days, respectively, after i.v. injection of Lewis Lung carcinoma (LL2) cells into control or murine 7E3 F(ab')(2) (6 mg/ kg) athymic rats. Maximal in vitro platelet activation (72% serotonin release) occurred 30 min after adding platelets to tumor cells. At this time, 87% of the A375 cells had adhered to platelets. Abciximab significantly (P<0.05) reduced platelet adherence to tumor cells as evidenced by flow cytometry. Incubation of A375 cells with platelets induced VEGF release in a time-dependent manner. This release was significantly inhibited by Abciximab (81% at 30 min; P<0.05). In the presence of fibrinogen and FII, VEGF release induced by A375 (TF+) cells was significantly higher than that induced by KG1 (TF-) cells (105.5+/-24 vs. 42+/-7 pg/ml; P<0.001). Omitting fibrinogen or FII from the reaction mixture markedly decreased VEGF release. In vivo, GpIIb/IIIa blockade with murine 7E3 F(ab')(2) reduced LL2 tumor cell-induced thrombocytopenia by 90% (P<0.001) and lung seeding by 82% (P<0.05). We conclude that TF-bearing tumor cells can activate platelets largely via thrombin generation, and that such activation is associated with release of VEGF. This may enhance metastasis, possibly by increasing extravasation at points of adhesion to vascular endothelium.

We measured levels of platelet-derived microparticles (PMP), which have coagulative activity and are produced by platelet activation or physical stimulation, and CD62P/CD63-positive platelets in patients with diabetes mellitus to determine their clinical significance and effects on complications of diabetes including diabetic nephropathy. We also compared these levels before and after administration of the antiplatelet drug cilostazol. Plasma PMP and CD62P/CD63-positive platelet levels were significantly higher in patients with diabetes mellitus than normal controls. CD62P-positive platelet levels were significantly higher in patients with nephropathy than in patients without complications. After administration of cilostazol, PMP and CD62P/CD63-positive platelet levels were significantly decreased. The increases in platelet activity and its related procoagulant activity appear to account in part for the hypercoagulability observed in diabetes mellitus. Our findings suggest that activated platelets might play a role in the development of diabetic nephropathy. Furthermore, antiplatelet therapy with cilostazol for diabetic patients may be useful as antithrombin therapy including antiplatelet therapy, since it suppresses the production of intrinsic coagulants produced by platelet activation.

BACKGROUND

Monitoring of platelet activation by the ADVIA 120 Hematology System requires an anticoagulant and protocol that ensures that platelets are sphered and their activation status is not altered artifactually in vitro.

METHODS

Blood from healthy controls was collected into tripotassium EDTA; citrate, theophylline, adenosine, and dipyridamole (CTAD); or a combination of both (E/C) and stored at ambient temperature or at 4 degrees C (E/C only) and then analyzed between 0 and 180 min later on the ADVIA 120. In addition, immunofluorescent flow cytometry was used to identify activated platelets and platelet-leukocyte aggregates.

RESULTS

In blood stored with all three anticoagulants, the platelet count changed little, but the mean platelet volume (MPV) at first decreased and then increased, whereas the mean platelet component (MPC; an indicator of activation) changed in a reciprocal manner. The changes in MPV and MPC, which reflect platelet sphering and swelling, were greatest between 30 and 60 min in blood stored at ambient temperature, irrespective of which anticoagulant was used, and between 60 and 180 min when blood anticoagulated with E/C was stored at 4 degrees C. In all anticoagulants, the percentages of platelets expressing CD62P and of leukocytes in platelet-leukocyte aggregates increased significantly (P <0.01) over 180 min at ambient temperature. Only minimal (<2%) increases occurred when blood with E/C was stored at 4 degrees C.

CONCLUSIONS

When determining platelet activation ex vivo on the ADVIA 120, blood should be collected into E/C, stored at 4 degrees C, and analyzed between 60 and 180 min later; these conditions ensure maximum platelet sphering without concurrent artifactual platelet activation.

OBJECTIVE

The role of plasma monocyte-derived microparticles (MDMPs) and platelet-activation markers (platelet-derived microparticle [PDMP], platelet-bound CD62P [plt-CD62P], and platelet-bound CD63 [plt-CD63]) in diabetic vascular complications is not clear. We measured and compared plasma concentrations of MDMPs and the platelet-activation markers to investigate their possible contribution to diabetic vascular complications.

METHODS

Activated platelets and microparticles (PDMP and MDMP) were analysed by flow cytometry. Concentrations of serum sE-selectin were measured with enzyme-linked immunosorbent assay.

RESULTS

The concentration of MDMPs in diabetic patients was higher than in normal subjects. We found no differences in the binding of anti-GPIIb/IIIa and anti-GPIb monoclonal antibodies between groups. There were differences, however, in the concentrations of PDMPs, plt-CD62P, and plt-CD63 between Type II (non-insulin-dependent) diabetes mellitus patients and control subjects (PDMPs: 585 +/- 25 vs 263 +/- 9, p < 0.01; plt-CD62P: 28.1 % +/- 1.4 % vs 9.4 % +/- 0.6 %, p < 0.001; plt-CD63: 28.1 % +/- 1.4 % vs 8.6 % +/- 0.5 %, p < 0.001). Amounts of MDMPs correlated positively with these platelet activation markers, and the relation between PDMP and MDMP was the most significant. The concentration of MDMP in patients who had diabetes complicated with nephropathy, retinopathy, or neuropathy was higher than in those without diabetes-related complications. The increase in MDMP was particularly significant in patients with nephropathy. Concentrations of sE-selectin were higher in Type II diabetes patients than in control subjects, and correlated with MDMP, PDMP, plt-CD62P, and plt-CD63 levels in nephropathy patients.

CONCLUSIONS

In Type II diabetes patients, we detected increased activation of monocytes, which could have been stimulated by activated platelets and PDMPs. Because the activation of monocytes is associated with vascular endothelial damage, high concentrations of MDMPs could indicate vascular complications in diabetes patients, especially those who have diabetes-related nephropathy.

BACKGROUND

Interactions of endothelial progenitor cells (EPCs) with vascular and blood cells contribute to vascular homeostasis. Although platelets promote the homing of EPCs to sites of vascular injury and their differentiation into endothelial cells, the functional consequences of such interactions on platelets remain unknown. Herein, we addressed the interactions between EPCs and platelets and their impact on platelet function and thrombus formation.

RESULTS

Cultured on fibronectin in conditioned media, human peripheral blood mononuclear cells differentiated, within 10 days of culture, into EPCs, which uptake acetylated low-density lipoprotein, bind ulex-lectin, lack monocyte/leukocyte markers (CD14, P-selectin glycoprotein ligand-1, L-selectin), express progenitor/endothelial markers (CD34, vascular endothelial growth factor receptor-2, von Willebrand factor, and vascular endothelial cadherin), and proliferate in culture. These EPCs bound activated platelets via CD62P and inhibited its translocation, glycoprotein IIb/IIIa activation, aggregation, and adhesion to collagen, mainly via prostacyclin secretion. Indeed, this was associated with upregulation of cyclooxygenase-2 and inducible nitric oxide synthase. However, the effects on platelets in vitro were reversed by cyclooxygenase and cyclooxygenase-2 inhibition but not by nitric oxide or inducible nitric oxide synthase inhibition. Moreover, in a ferric chloride-induced murine arterial thrombosis model, injection of EPCs led to their incorporation into sites of injury and impaired thrombus formation, leading to an incomplete occlusion with 50% residual flow.

CONCLUSIONS

Peripheral blood mononuclear cell-derived EPCs bind platelets via CD62P and inhibit platelet activation, aggregation, adhesion to collagen, and thrombus formation, predominantly via upregulation of cyclooxygenase-2 and secretion of prostacyclin. These findings add new insights into the biology of EPCs and define their potential roles in regulating platelet function and thrombosis.

P-selectin was shown to bind committed human hematopoietic progenitors (colony-forming unit-granulocyte-macrophage [CFU-GM] and burst-forming unit-erythroid [BFU-E]) as identified by their expression of the CD34 antigen and by in vitro clonogenic assays. In addition, P-selectin bound all precursors (pre-CFU) of committed myeloid progenitors assayed by their ability to sustain hematopoiesis in both conventional stroma-containing and stroma-free, cytokine-dependent systems. Binding of CD34+ cells to P-selectin was temperature-independent and shear-resistant, occurred only in the presence of divalent cations, was protease sensitive, and was completely blocked by anti-P-selectin antibody. Neuraminidase treatment of CD34+ cells completely abrogated their binding to P-selectin, implying a prominent role for sialic acid in the structure and function of the P-selectin ligand on hematopoietic progenitors. Monoclonal antibodies (MoAbs) CSLEX-1 and HECA-452, which identify carbohydrate epitopes involving sialic acid, bound to 33% and 35% of CD34+ cells, respectively, and included the majority of CFU-GM and pre-CFU. Three-color flow cytometric analysis showed a precise codistribution of CSLEX-1 and HECA-452 antigens on CD34+ cells, implying recognition of the same glycoprotein antigen by the two MoAbs. Treatment of CD34+ cells with neuraminidase completely abolished binding of both MoAbs. In addition, HECA-452 partially blocked the adhesion of CD34+ cells to P-selectin. P-selectin glycoprotein ligand (PSGL-1), recently molecularly cloned from the promyelocytic leukemia cell line HL60, was expressed by CD34+ cells as determined by reverse transcription polymerase chain reaction. Combined with the functional and biochemical characteristics, these data suggest that PSGL-1 may comprise an important P-selectin ligand expressed by primitive hematopoietic cells, but do not preclude the existence of additional P-selectin ligands on these cells.

OBJECTIVE

Cardiac surgery in patients undergoing cardiopulmonary bypass (CPB) provokes a vigorous inflammatory response with substantial clinical implications. Once the inflammatory response is triggered by CPB, leukocytes and platelets are activated by multiple stimuli. The administration of a urinary trypsin inhibitor (ulinastatin) during CPB is hypothesized to reduce cytokine release and platelet activation and to decrease pulmonary injury. We performed a prospective randomized study to investigate the influence of high-dose ulinastatin on cytokines and platelet activation and on respiratory function during and after CPB.

METHODS

In this pilot, prospective, randomized and double-blinded study, 30 first-time three-vessel coronary artery disease (CAD) patients undergoing coronary artery bypass graft (CABG) were randomly divided into 2 groups: U group (n=15) received a total dose of 1000000 U ulinastatin and C group (n=15) received placebo. Blood samples were withdrawn from the central vein to measure polymorphonuclear neutrophil elastase (PMNE), tumour necrosis factor-alpha (TNF-a), interleukin-6 (IL-6) and interleukin-8 (IL-8), before induction, 30 min following clamping (T2), reperfusion 3 h (T3), reperfusion 6 h (T4) and reperfusion 12 h (T5). Whole blood samples were taken for CD62P immediately before induction (as baseline), at the end of CPB (before protamine administration), 1 h after heparin neutralization by protamine and 24 h after the operation. In addition, alveolo-arterial oxygen difference (A-aDO(2)) in pulmonary gas exchange function was calculated by obtaining arterial blood gas samples before and after CPB.

RESULTS

There were no differences in preoperative parameters between the groups. After CPB, the levels of PMNE, TNF-alfa, IL-6 and IL-8 increased in both groups over baseline values (P<0.01). The levels of PMNE, TNF-alfa, IL-6 and IL-8 in U group were significantly lower than those in C group (P<0.05). No significant differences in CD62p expression between the 2 groups during CPB were found. A-aDO(2) in U group significantly decreased compared with C group (P<0.05) and the duration of mechanical ventilation was shorter than C group (P<0.05).

CONCLUSIONS

Results suggest that ulinastatin may inhibit proinflammatory cytokine (PMNE, TNF-alfa, IL-6 and IL-8) release, reduce reperfusion lung injury and preserve pulmonary function but it fails to inhibit platelet activation and to prevent blood loss during CPB.

The isolation process exposes human pancreatic islets to exogenous isolation enzymes. Exposure to these enzymes, as a result of intraductal injection in the pancreas or simple contact of islets with enzyme components, causes internalization into the islet cells of enzymes and their by-products. Human islets exposed to Liberase-HI exhibit a decreased insulin secretory ability that correlates with the time of exposure. This phenomenon is paralleled by increased expression of adhesion molecules (CD106 and CD62p) and activation of apoptotic pathways (Bax and Bcl-2) in islet cells. Increased functional impairment is also observed after islet transplantation in diabetic immunodeficient mice. Experimental exposure of islet grafts to exogenous isolation enzymes causes intense inflammation (CD11b positive cells) at the transplant site and it was associated with sickness behavior and eventually death of mouse recipients. The extent of these adverse effects likely deceives the standard qualitative protocols currently in use to assess islet quality in vitro. Reducing the secondary effects of exogenous isolation enzymes on isolated human islets may be crucial to enhance the quality of islets as tissue grafts.

Neutrophil (polymorphonuclear neutrophil) migration across epithelial barriers is a common morphologic feature of many diseases. Previous studies show that PMN-epithelial interactions are dependent on the PMN beta 2-integrin CD11b/18; however, nothing is known about surface carbohydrates and PMN-epithelial interactions. Here we investigate the role of carbohydrates on PMN-epithelial interactions using PMN and cultured monolayers of the intestinal epithelial cell line T84. Addition of the carbohydrates mannose 6-phosphate (Man-6-P) and glucose 6-phosphate (Glu-6-P), but not fructose 1-phosphate (Fru-1-P) inhibited transmigration by>> or = 70%. Likewise, more complex carbohydrates, such as fucoidin and the Man-6-P-rich polysaccharide PPME selectively inhibited PMN transepithelial migration. These carbohydrates were found to be inhibitory in the apical-to-basolateral direction as well as the basolateral-to-apical direction, indicating a lack of polarity. This panel of related carbohydrates, however, was not effective in modulating short-term adhesion of PMN to epithelial monolayers, indicating that carbohydrate ligands may modulate different steps in the transmigration cascade. Finally, addition of functionally inhibitory monoclonal antibodies specific for the selectins (CD62E, CD62L, and CD62P) revealed no observable effect on PMN transmigration. These studies suggest that cell surface carbohydrates may play a role in inflammatory processes of the intestine.

OBJECTIVE

To investigate the variability in immunostaining for cytokines and cell adhesion molecules using multiple arthroscopically directed synovial biopsies from within a rheumatoid knee joint, quantitated by color video image analysis.

METHODS

Needle arthroscopic biopsies were taken from multiple sites (4-7 sites) around a knee joint in 8 patients with rheumatoid arthritis (RA). In 5 patients, immunoperoxidase staining for the cytokines tumor necrosis factor alpha (TNF-alpha), interleukin 8 (IL-8), and IL-1beta as well as the IL-1 receptor antagonist protein (IL-1ra) was performed. In 3 patients, immunoperoxidase staining for the cell adhesion molecules E-selectin (CD62E), P-selectin (CD62P), intercellular adhesion molecule 1 (ICAM-1, CD54), and platelet endothelial cell adhesion molecule (PECAM, CD31) was performed. Immunostaining was quantified using color video image analysis.

RESULTS

The overall probability of paired biopsies from the same RA knee joint being significantly different from each other due to sampling variation was at most 22% for cytokine staining (usually less than 10%). There were no significant differences between intrabiopsy and interbiopsy variability for cell adhesion molecule staining of the sublining and vessels.

CONCLUSIONS

The variability in cytokine and cell adhesion molecule staining within any single biopsy usually reflects the variability between biopsies taken from different sites in the same rheumatoid joint when the immunostaining is quantified using color video image analysis. Therefore, only a small number of synovial biopsies are required to accurately determine the cytokine and cell adhesion molecule expression in a single joint.

A reconstituted high density lipoprotein (rHDL) prepared for clinical use was tested for its influence on platelet activity modulated by various stimuli. In a first series of in vitro experiments, rHDL was added to blood in a concentration series, and platelet rich plasma (PRP) was isolated. Platelets were stimulated with arachidonic acid, collagen, epinephrine or ADP, and platelet aggregation was assessed. rHDL mediated a dose dependent inhibition of the platelet activity. With purified platelets rHDL inhibited the release reaction induced by collagen, but not by thrombin, as measured by CD62P (P-Selectin) expression on the plasma membrane. Ex vivo experiments were performed with PRP from volunteers, previously infused with 25 mg rHDL/kg body weight and 40 mg rHDL/kg body weight, respectively. Platelet activity in PRP was assessed before, and up to 30 h after the end of the rHDL infusion. A transient inhibition of the platelet aggregation induced by arachidonic acid and collagen was observed which was more pronounced in the group receiving 40 mg rHDL/kg body weight. In both groups of experiments, in vitro and ex vivo, the inhibition of the platelet activity was also dependent on the stimulus used.

Hemolytic uremic syndrome (HUS) is associated with acute renal failure in children and can be caused by Shiga toxin (Stx)-producing Escherichia coli. Thrombocytopenia and formation of renal thrombi are characteristic of HUS, suggesting that platelet activation is involved in its pathogenesis. However, whether Shiga toxin directly activates platelets is controversial. The present study evaluates if potential platelet sensitization during isolation by different procedures influences platelet interaction with Shiga toxin. Platelets isolated from sodium citrate anticoagulated blood were exposed during washing to EDTA and higher g forces than platelets prepared from acid-citrate-dextrose (ACD) plasma. Platelet binding of Stx was significantly higher in EDTA-washed preparations relative to ACD-derived platelets. Binding of Stx was also increased with ACD-derived platelets when activated with thrombin (1 U mL-1) and exposure of the Gb3 Stx receptor was detected only on platelets subjected to EDTA, higher g forces or thrombin. EDTA-exposed platelets lost their normal discoid shape and were larger. P-selectin (CD62P) exposure was significantly increased in EDTA-washed preparations relative to ACD-derived platelets, suggesting platelet activation. Taken together, these results suggest that direct binding of Stx occurs only on 'activated' platelets rather than on resting platelets. The ability of Stx to interact with previously activated platelets may be an important element in understanding the pathogenesis of HUS.

BACKGROUND

Changes in platelet count and function contribute to thrombo-hemorrhagic episodes in chronic myeloproliferative syndromes (MPS). We used flow cytometry to study platelet-leukocyte conjugates and markers of platelet activation in patients with MPS.

METHODS

Whole blood from patients with chronic myelogenous leukemia (CML), polycythemia vera (PV), chronic myelofibrosis (MF), and essential thrombocythemia (ET) and from healthy volunteers was prepared for flow cytometry. Platelet microparticles and platelet microaggregates were identified with anti-CD42b and forward scatter, activated platelets with anti-CD62p. Anti-CD42b, anti-CD14, and anti-CD45 were used to study platelet-leukocyte conjugates.

RESULTS

The percentage of CD62p-positive platelets was elevated in all myeloproliferate syndrome subtypes. The median percentage of platelet microparticles was 5.2% in controls and significantly higher in PV (12.0%), MF (11.0%), and ET (11.0%, all p<0.05). There was an increased percentage of platelet-neutrophil conjugates in patients with PV (8.3%) and ET (10.4%) compared to normal controls (6.8%, all p<0.05). Platelet-monocyte conjugates were 8.0% in controls and elevated in PV (15.4%) and ET (15.0%, all p<0.05). Patients with a history of venous or arterial thrombotic events had slightly less platelet-leukocyte conjugates and slightly more microparticles than patients without thrombosis; however, this difference was not statistically significant.

CONCLUSIONS

These findings suggest that platelet-leukocyte conjugate formation occurs in myeloproliferative syndromes and indicates platelet activation. Also, platelet microparticles are elevated and might provide a catalytic surface for thrombin generation. This could explain the clinical observation that patients with myeloproliferative syndromes have an increased risk to experience arterial or venous thrombotic events.

Severe dengue is characterised by thrombocytopenia, plasma leakage and bleeding. Platelets are important for preservation of endothelial integrity. We hypothesised that platelet activation with secondary platelet dysfunction contribute to plasma leakage. In adult Indonesian patients with acute dengue, we measured platelet activation status and the response to the platelet agonist TRAP using flow cytometer-based assays. Patients were monitored daily for plasma leakage by ultrasonography. Acute dengue was associated with platelet activation with an increased expression of the activated fibrinogen receptor (αIIbβ3), the lysosomal marker CD63 and the alpha-granule marker CD62P (P-selectin). Upon maximal platelet activation by TRAP, platelet function defects were observed with a significantly reduced maximal activated αIIbβ3 and CD63 expression and reduced platelet-monocyte and platelet-neutrophil complexes. Patients in the lowest tertile of activated αIIbβ3 and CD63 expression had an odds ratio for plasma leakage of 5.2 (95% confidence interval [CI] 1.3-22.7) and 3.9 (95% CI 1.1-13.7), respectively, compared to the highest tertile. Platelet-derived serotonin has previously been related to plasma leakage and we found increased intra-platelet serotonin concentrations in our patients. In conclusion, platelet activation with platelet function alterations can be found in patients with acute dengue and this may contribute to dengue-associated plasma leakage.

OBJECTIVE

Somatic stem cells can be obtained from a variety of adult human tissues. However, it was not clear whether human parathyroid glands, which secrete parathyroid hormones and are essential in maintaining homeostasis levels of calcium ions in the circulation, contained stem cells. We aimed to investigate the possibility of isolating such parathyroid-derived stem cells (PDSC).

METHODS

Surgically removed parathyroid glands were obtained with informed consent. Cell cytogenetics was used to observe chromosomal abnormalities. Surface phenotypes were characterized by flow cytometry. Telomerase repeat amplification protocol (TRAP) assay was performed to observe the telomerase activity. RT-PCR and real-time PCR was was used to detect gene expressions. Real-time calcium uptake imaging was performed for extent of calcium uptake and transmission electron microscopy and immunofluorecent staining for smooth muscle actin.

RESULTS

After enzymatic digestion and primary culture, plastic-adherent, fibroblast-like cells appeared in culture and a morphologically homogeneous population was derived from subsequent limiting dilution and clonal expansion. Karyotyping was normal and doubling time of clonal cell growth was estimated to be 70.7 +/- 14.5 h (mean +/- standard deviation). The surface phenotype of the cells was positive for CD73, CD166, CD29, CD49a, CD49b, CD49d, CD44, CD105, and MHC class I, and negative for CD34, CD133, CD117, CD114, CD31, CD62P, EGF-R, ICAM-3, CD26, CXCR4, CD106, CD90 and MHC class II, similar to mesenchymal stem cells (MSC). Detectable levels of telomerase activity along with pluripotency Sall4 gene expression were observed from the isolated PDSCs. Expression of calcium-sensing receptor gene along with alpha-smooth muscle actin was induced and cellular uptake of extracellular calcium ions was observed. Furthermore, PDSCs possessed osteogenic, chondrogenic and adipogenic differentiation potentials.

CONCLUSIONS

Our results reveal that PDSCs were similar phenotypically to MSCs and further studies are needed to formulate induction conditions to differentiate PDSCs into parathyroid hormone-secreting chief cells.

OBJECTIVE

Patients undergoing cardiopulmonary bypass frequently manifest generalized systemic inflammation and occasionally manifest serious multiorgan failure. Inflammatory responses of bypass are triggered by contact of blood with artificial surfaces of the bypass circuits, surgical trauma, and ischemia-reperfusion injury. We studied the effects of specific inhibition of the alternative complement cascade by using an anti-factor D monoclonal antibody (166-32) in extracorporeal circulation of human whole blood used as a simulated model of cardiopulmonary bypass.

METHODS

Five healthy blood donors were used in the study. Monoclonal antibody 166-32 was added to freshly collected, heparinized human blood recirculated in a pediatric cardiopulmonary bypass circuit at a final concentration of 18 microg/mL. An irrelevant monoclonal antibody was used as a negative control with the same donor blood in a parallel bypass circuit on the same day. Blood samples were collected at different time points during recirculation for measurement of activation of complement, neutrophils, and platelets by immunofluorocytometric methods and enzyme-linked immunosorbent assays.

RESULTS

Monoclonal antibody 166-32 inhibited the alternative complement activation and the production of Bb, C3a, sC5b-9, and C5a. Upregulation of CD11b on neutrophils and CD62P on platelets was also significantly inhibited by monoclonal antibody 166-32. This is consistent with the inhibition of the release of neutrophil-specific myeloperoxidase and elastase and platelet thrombospondin. The production of proinflammatory cytokine interleukin 8 was also suppressed by the antibody.

CONCLUSIONS

The alternative complement cascade is predominantly activated during extracorporeal circulation. Anti-factor D monoclonal antibody 166-32 is effective in inhibiting the activation of complement, neutrophils, and platelets. Inhibition of the alternative complement pathway by targeting factor D could be useful in reducing systemic inflammation in patients undergoing cardiopulmonary bypass.

OBJECTIVE

The aim of this work was to comprehensively study the role of platelets in atrial fibrillation (AF), in relation to the underlying cardiovascular diseases and type of AF, and to analyze the effect of antithrombotic treatment on different aspects of platelet activation.

BACKGROUND

Platelet activation is present in nonvalvular AF, but there is debate whether this is due to AF itself and/or to underlying cardiovascular diseases.

METHODS

A total of 121 AF patients were compared with 65 "healthy control subjects" and 78 "disease control subjects" in sinus rhythm. Platelet activation was assessed using 4 different aspects of platelet pathophysiology: 1) platelet surface expression of CD62P (P-selectin) and CD63 (a lysosomal glycoprotein) (by flow cytometry); 2) mean platelet volume (MPV) (by flow cytometry); 3) plasma levels of soluble P-selectin (sP-selectin, enzyme-linked immunoadsorbent assay); and 4) total amount of P-selectin per platelet (pP-selectin) ("platelet lysis" assay).

RESULTS

Both AF patients and "disease control subjects" had higher levels of CD62P (p < 0.001), CD63 (p < 0.001), and sP-selectin (p < 0.001) compared with "healthy control subjects," with no difference between AF patients and "disease control subjects." Patients with permanent AF had higher levels of sP-selectin (p = 0.014) and MPV (p = 0.025) compared with those with paroxysmal AF. The presence of AF independently affected the levels of CD62P expression, while "high-risk" AF patients (CHADS score>> or =2) had higher levels of CD62P compared with those with "low risk." Introducing warfarin resulted in a reduction of pP-selectin (p = 0.013).

CONCLUSIONS

There is a degree of excess of platelet activation in AF compared with "healthy control subjects," but no significant difference between AF patients and "disease control subjects" in sinus rhythm. Platelet activation may differ according to the subtype of AF, but this is not in excess of the underlying comorbidities that lead to AF. Platelet activation in AF may be due to underlying cardiovascular diseases, rather than due to AF per se.