Thromboembolic events (TEE) are a serious clinical problem in multiple myeloma (MM) patients receiving thalidomide (T). Thirty-one MM patients were tested on diagnosis and after 2 and 4 weeks of therapy with T alone, or T in combination with dexamethasone (TD). Closure time (CT) in PFA-100 and P-selectin expression were assessed, as well as plasma levels of thrombin-antithrombin complexes (TAT), D-dimer (DD), soluble thrombomodulin (sTM) and von Willebrand factor antigen (vWF:Ag), along with the activity of coagulation factor VII and factor VIII. The concentration of vascular endothelial growth factor and its type 1 and 2 receptors were also assayed. On diagnosis, significantly prolonged median CT with both used cartridges, elevated P-selectin expression, DD concentration, TAT, vWF:Ag and factor VIII and factor VII activity were seen in the patient group as compared to controls. Therapy with these regimens caused marked shortening of CT with both cartridges. Treatment with TD leads to the significant increase in CD62P expression on platelets. Median TAT value increased significantly in relation to baseline after therapy with both regimens. Factor VIII activity exceeded 150 % in all patients after 2 weeks of TD therapy and was markedly elevated compared to baseline. One month of TD therapy significantly increased sTM concentration. These results demonstrate the enhanced platelet and coagulation system activation already present in MM patients on diagnosis, which is further increased by antimyeloma therapy. These changes are more pronounced after TD therapy and may promote TEE. Tested angiogenesis marker levels are elevated already on diagnosis, do not change after therapy and have no significant impact on the coagulation system in patients with MM.

In the present work, the time sequence of blood activation by alumina membranes with different porosities (20 and 200 nm in diameter) was studied. The membranes were incubated with whole blood from 2 min to 4 h. Platelet adhesion and activation in addition to complement activation was monitored at different time points. Evaluation of platelet adhesion and activation was done by determining the change in platelet number and the levels of thrombospondin-1 (TSP-1) in the fluid phase. Scanning electron microscopy studies were done to further evaluate platelet adhesion and morphology. Immunocytochemical staining was used to evaluate the presence of CD41 and CD62P antigens on the material surface. Complement activation was monitored by measuring C3a and sC5b-9 in plasma samples by means of enzyme immunoassays. Both alumina membranes displayed similar complement activation time profiles, with levels of C3a and sC5b-9 increasing with incubation time. A statistically significant difference between the membranes was found after 60 min of incubation. Platelet activation characteristics and time profile were different between the two membranes. Platelet adhesion increased over time for the 20 nm surface, while the clusters of microparticles on the 200 nm surface did not appreciably change during the course of the experiment. The release of TSP-1 increased with time for both membranes; however, much later for the 200 nm alumina (240 min) as compared to the 20 nm membrane (60 min). The surface topography of the alumina most probably influence protein transition rate, which in turn affects material platelet activation kinetics.

Medical-grade polytetrafluoroethylene (PTFE), polydimethylsiloxane (PDMS), polyetherurethane (PEU) and ultrahigh molecular weight polyethylene (UHMWPE) were plasma treated with O2, Ar, N2 and NH3. Their surface properties were characterised using X-ray photoelectron spectroscopy (XPS), static secondary ion mass spectroscopy (SSIMS), atomic force microscopy (AFM) and dynamic contact angle (DCA) analysis. Platelet adhesion, aggregation, activation and release of microparticles were determined after contact with whole blood in a cone and plate viscometer. Activation of the coagulation system was quantified in a static environment using a partial thromboplastin time (PTT) assay. The chemical compositions of the untreated surfaces were found to be very similar to those of the bulk material except for PEU, whose surface was comprised almost entirely of soft ether segments. For all materials, the different plasma treatments resulted in moderate etching with the incorporation of functional groups and removal of side groups: defluorination, dehydrogenation, cleavage of methyl side groups and soft segments for PTFE, UHMWPE, PDMS and PEU, respectively. Consequently, plasma treatment resulted in increased wettability in all cases. Blood contact with the virgin materials resulted in activation of platelets and the clotting cascade. Plasma treatment resulted in a significant reduction in platelet adhesion for all materials and all treatments. In the case of PTFE and PEU, the activation status of these cells was also reduced. Plasma treatment of all materials reduced fluid-phase CD62P expression. Platelet aggregate size correlated well with degree of aggregate formation, but many treatments increased the degree of aggregation, as was the case for microparticle shedding. There was no correlation between CD62P expression, aggregate formation and platelet microparticle (PMP) shedding. It is concluded that despite incorporation of the same chemical groups, the pattern of response to blood in vitro is not the same across different polymers.

OBJECTIVE

Glucose and acetate have been proposed to be required elements in platelet storage media. This study investigated the role of these compounds on the varied elements that comprise the platelet storage lesion.

METHODS

For each replicate, four pooled and split ABO group-specific buffy coat-derived platelet concentrates were suspended in an in-house additive solution with minimal plasma and varying final concentrations of acetate or glucose. Units were sampled on days 2, 3, 6, 8 and 10 and tested for markers of platelet morphology, activation, function, metabolism and indicators of cell death.

RESULTS

The absence of glucose was associated with a decrease in ATP, falling to a mean of 1·1 ± 0·1 μmol/10(11) plts in units with no added glucose compared with 4·2 ± 0·6 μmol/10(11) plts (P < 0·001) in units with 30 mm glucose. As glucose became depleted, the decrease in ATP to levels below 3 μmol/10(11) plts was associated with an increase in both annexin V binding and intracellular free calcium. In units lacking exogenous acetate, ATP levels on day 10 were 5·2 ± 1·5 μmol/10(11) plts compared with 2·7 ± 0·9 μmol/10(11) plts in units with 56 mm acetate (P = 0·006). Higher concentrations of exogenous acetate were associated with a lower hypotonic shock response and higher surface expression of CD62P suggestive of a dose dependency.

CONCLUSIONS

Under current physical storage conditions, glucose appears necessary for the maintenance of platelets stored as concentrates in minimal volumes of plasma. The addition of acetate was associated with increased platelet activation and reduced ATP levels.

BACKGROUND

Climacteric increases the risk of thrombotic events by alteration of plasmatic coagulation. Up to now, less is known about changes in platelet- (PMP) and endothelial cell-derived microparticles (EMP).

METHODS

In this prospective study, plasma levels of microparticles (MP) were compared in 21 premenopausal and 19 postmenopausal women.

RESULTS

No altered numbers of total MP or EMP were measured within the study groups. However, the plasma values of CD61-exposing MP from platelets/megakaryocytes were higher in premenopausal women (5,364 × 10(6)/l, range 4,384-17,167) as compared to postmenopausal women (3,808 × 10(6)/l, range 2,009-8,850; p = 0.020). This differentiation was also significant for the subgroup of premenopausal women without hormonal contraceptives (5,364 × 10(6)/l, range 4,223-15,916; p = 0.047; n = 15). Furthermore, in premenopausal women, higher plasma levels of PMP exposing CD62P were also present as compared to postmenopausal women (288 × 10(6)/l, range 139-462, vs. 121 × 10(6)/l, range 74-284; p = 0.024). This difference was also true for CD63+ PMP levels (281 × 10(6)/l, range 182-551, vs. 137 × 10(6)/l, range 64-432; p = 0.015).

CONCLUSIONS

Climacteric lowers the level of PMP but has no impact on the number of EMP in women. These data suggest that PMP and EMP do not play a significant role in enhancing the risk of thrombotic events in healthy, postmenopausal women.

OBJECTIVE

To investigate the effects of ulinastatin (U) on coagulation, platelet function, and postoperative bleeding in patients undergoing coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB).

METHODS

Thirty-six selective patients undergoing CABG with CPB were randomly assigned to two groups: Group U (n=18) in which ulinastatin 4 x 10(6) U in 100 ml normal saline (NS) was infused intravenously for 30 min since skin incision with 4 x 10(6) U added to the CPB pump prime, and then 2 x 10(6) U of ulinastatin was infused intravenously at a rate of 4-6 x 10(4) U x h(-1) until the chest was closed; and control group (Group C, n=18) in which 100 ml NS was infused without ulinastatin. Peripheral blood samples were collected 1 min before operation (T0), 1 min before heparinization (T1), at the end of operation (T2), 6 hour after operation (T3), and 24 hour after operation (T4). Platelet membrane glucoprotein IIb/IIIa (GP IIb/IIIa) and platelet alpha granule membrane protein-140 (CD62p) were measured by flow cytometry. Activated partial thromboplasin time (APTT), activated coagulation time of whole blood (ACT), prothrombin time (PT), fibrinogen, and platelet number were also measured. Postoperative blood loss and allogeneic transfusion were recorded.

RESULTS

There were no statistical differences in CD26p, GP IIb/IIIa, and PT between the 2 groups (all P>> 0.05). The APTT levels was significantly shortened in Group U at T1, T3 and T4 compared to T0. The APTT levels of Group U from T1 to T4 were all significantly lower than those of Group C (all P < 0.05). The ACT levels after heparinization and during CPB in Group U were significantly shorter than those of Group C (all P < 0.01), and the amount of added heparin during CPB of Group U was significantly higher than that of Group C (P < 0.01). There were not significant differences in platelet amount, fibrinogen, total amount of blood loss in 24 h after operation was 960 (420, 1500) ml in group C, and 850 (380, 1600) ml in group U (P>> 0.05). The platelet count, CD26p, GPIIb/IIIa, total amounts of blood loss and blood infusion 24 h after operation, and hemoglobin concentration between these 2 groups (all P>> 0.05).

CONCLUSIONS

Ulinastatin shortens the APTT and ACT after heparinization, increases the dose of heparin during CPB, has no effect on the expression of GP IIb-IIIa and CD62p, and does not reduce postoperative blood loss.

BACKGROUND

The aim of our study was to assess the influence of polyclonal antithymocyte globulins (ATGs) on the expression of adhesion molecules on lymphocytes, neutrophils, and thrombocytes by means of flow cytometry. ATGs are employed in various regimens for solid organ transplantation. Immunosuppression with ATGs may influence the expression of adhesion molecules on thrombocytes, lymphocytes, and neutrophils due to nonspecific antibodies directed against myeloid and nonmyeloid cells.

METHODS

Depletion, activation, and expression of adhesion molecules on thrombocytes (CD41, CD42, CD62p and CD107a), neutrophils, and lymphocytes (CD11, CD18, CD62L) were studied in vitro in whole blood of healthy volunteers by means of flow cytometry after incubation with different dosages of three ATGs.

RESULTS

Our data showed no ATG-mediated cytotoxic activity against platelets. ATGs were able, however, to induce activation of platelets through increased expression of P-selectin and hLAMP-1. ATGs also influenced the expression of adhesion molecules on lymphocytes and neutrophils by reducing the expression of CD62L. Furthermore, the effects of ATG on CD11/CD18 were dependent on the dosage and type of ATG.

CONCLUSIONS

Polyclonal ATGs induced expression of adhesion molecules and activation of unstimulated thrombocytes as well as reduced the expression of adhesion molecules on lymphocytes and neutrophils. Increased adhesion of thrombocytes may be responsible for the undesirable side effects observed in clinical practice such as thrombocytopenia. However, reduction in the expression of adhesion molecules on lymphocytes and neutrophils may decrease the effects of ischemia/reperfusion injury.

Induction therapy with polyclonal antithymocyte-globulin (ATG) is widely used in the prophylaxis and treatment of acute cardiac-allograft rejection. Thrombocytopenia, however, is a major side-effect of ATG therapy and its mechanisms are poorly understood. The influence of ATG on platelet aggregation was studied aggregometrically, expression of platelet surface activation antigens CD62P and CD63 was determined by flow cytometry analysis, and electron microscopy was utilized to determine thrombocyte morphology. Treatment of platelets with ATG markedly induced aggregation, whereas OKT3 or anti-IL-2R antibodies did not. Furthermore, platelets incubated with ATG featured an up-regulation of the surface activation markers CD62P and CD63, secretion of platelet-bound sCD40L (CD154) and increased signs of aggregation in electron microscopy analysis. The capacity of ATG to induce platelet aggregation was completely blocked by antibodies against the low-affinity Fc IgG receptor (CD32). Since blocking of CD32 abrogates platelet aggregation, we suggest that CD32 plays a crucial role in ATG-induced thrombocytopenia.

During storage of platelet concentrates (PCs), the quality of the platelets deteriorates gradually, partially dependent on gas exchange. UPX80 (JMS, Japan) 1-L platelet storage PVC containers with increased gas transport capacity were compared with 1- and 1. 5-L polyolefin (PO) containers (NPBI, the Netherlands) with filtered PCs stored either in GAC (gluconate-acetate-citrate, < 10% plasma) or in plasma, for 8 days. In total 32 PCs were made (260-330 x 109 platelets per concentrate), equally divided over different bags and storage media. During storage, gas exchange, metabolic, physical and activation parameters were measured. No consistent differences for all parameters were observed between UPX80 and PO containers (1-L or 1.5-L). Blood gas parameters indicated better gas exchange for UPX80 containers compared with PO containers. Good morphology was observed in UPX80 and metabolic functions were not significantly different compared with PO containers. During prolonged storage (after day 6), some significant differences in CD62P and CD63 expression were found, indicating a higher degree of platelet activation in UPX80 containers, especially in GAC. UPX80 PC containers are suitable for storage of PCs. Although in UPX80 better gas exchange is demonstrated, as compared with PO containers, this does not improve the platelet quality during storage for 6 days, indicating that gas exchange above the level of PO containers has no effect on the switch to aerobic metabolism in platelets.

OBJECTIVE

To investigate the effect of Helicobacter pylori (Hp) on platelet activation and coagulation function in patients with acute cerebral infarction.

METHODS

Sixty-six patients with acute cerebral infarction and 50 health individuals were enrolled in the study. Hp antibody,expression of CD62p on platelets and clotting indexes were measured and compared between two groups.

RESULTS

The positive rate of Hp-IgG and Hp-CagA in cerebral infarction patients were higher than that in controls (P<0.05). The positive rate of CD62p in patients with positive Hp-IgG and Hp-CagA was significantly higher than that in negative patients and also controls (P<0.05). The APTT and TT were lower and FIB was higher in patients with positive Hp antibody than those in patients with negative Hp antibody (P<0.05),but there was no difference in PT,PTR and INR (P>0.05).

CONCLUSIONS

Hp infection can activate platelets and affect coagulation function,which may be involved in the development of cerebral infarction.

OBJECTIVE

To explore the flow cytometric 3-color analysis of circulating activated platelets (CAP) and its clinical significance in ischemic cerebrovascular diseases.

METHODS

The fibrinogen receptor (FIB-R) and P-selectin (CD62P) were used for molecular marker of CAP, which were analyzed by flow cytometric 3-color immunofluorescence.

RESULTS

Expression of FIB-R and CD62P on the resting platelet membrane surface was very lower in normal individuals, the median of FIB-R and CD62P positive percent was 1.40% and 0.70% respectively. Platelet can be obviously activated by 0.1 micromol/L ADP, and 10.0 micromol/L ADP leaded to the peak expression of FIB-R and CD62P on the activated platelet membrane surface, FIB-R and CD62P positive percent of 87.8% and 81.34% respectively. That monoclonal antibody of the FIB-R combined with ADP activating platelets was inhibited by synthetic peptide (RGDS). The quantity of CAP with FIB-R expression was markedly higher (P < 0.01) and the amount of CAP with CD62P expression was not significantly higher (P>> 0.05) in blood of 112 patients with thrombotic tendency and 120 patients with ischemic cerebrovascular diseases than in healthy individuals. The expression of CAP with FIB-R of in front and behind treatment of acute cerebral infarction continuously decreased (P < 0.05), and the quantity of CAP with FIB-R expression was related negatively to Europe stroke score (ESS) of patients with acute cerebral infarction.

CONCLUSIONS

The FIB-R may be regarded as sensitive and specific molecular marker in detection of CAP. The level of platelet activation in vivo can be reflected accurately by flow cytometric 3-color analysis of CAP, which has an important clinical significance for the study of pathogenesis, estimation of prognosis and supervision of curative effect in ischemic cerebrovascular diseases.

BACKGROUND

The aim of the study was to compare histopathologic and immunohistochemical markers between survivors and nonsurvivors of surgical necrotizing enterocolitis (NEC).

METHODS

With appropriate ethical approval, archived resection specimens were identified for patients with NEC (Bell Stages II and III) for whom outcome data (survival yes/no) were available. For each specimen, a severely affected part of the bowel and the least affected area, usually the margin, were analyzed. Histologic findings were scored as no necrosis/mucosal necrosis/full-thickness necrosis and immunohistochemistry staining for inflammatory markers vascular cell adhesion protein (VCAM), CD68, CD20, intercellular adhesion molecule (ICAM), human leukocyte antigen (HLA-DR), CD3, Cleaved Caspase-3 (CC3), forkhead box P3 (FOXP3), CD62p, and C4d were performed and scored on a semiquantitative scale (0; no staining to 10, strong extensive staining). All samples were identified by only their study number throughout and the samples were analyzed completely blinded to all clinical information. Data were compared using chi-square test for trend (histologic data) or Mann-Whitney U test.

RESULTS

A total of 123 slides from 60 patients (birth weight 1.3 ± 0.1 kg, gestational age at birth 29.3 ± 0.6 weeks) were examined. Seventy-four specimens (60%) were from survivors and 49 specimens (40%) were from those who subsequently died. There was no relationship between histologic severity of necrosis (none/mucosal/full thickness) and mortality (p = 0.58). VCAM (adhesion molecule; p = 0.005) and CC3 (a marker of apoptosis, p = 0.008) expression was significantly elevated in nonsurvivors, whereas there were no differences in CD68, CD20, ICAM, HLA-DR, CD3, FOXP3, CD62p, or C4d expression.

CONCLUSIONS

There is a poor relationship between histologic severity of bowel necrosis and patient survival in infants undergoing bowel resection for NEC. There is statistically increased expression of VCAM reflecting severity of systemic inflammatory response and evidence of increased apoptosis in the form of CC3 expression in those who subsequently die, but no histologic features can predict outcome.

OBJECTIVE

Several investigators have reported decreased expression of glycoprotein Ib on the platelet surface during coronary artery bypass grafting, but others could not confirm this finding. Because platelet glycoprotein Ib functions as an adhesion receptor for von Willebrand factor and other adhesive proteins, this decreased expression may explain excessive postoperative blood loss. In this study the expressions of glycoprotein Ib and other platelet activation markers were studied in the systemic and pericardial blood of seven patients undergoing coronary artery bypass grafting. Pericardial blood was recently shown to have high activation levels of fibrinolytic and coagulation pathways; we hypothesized that this local blood activation might be paralleled by extensive platelet activation and associated disappearance of glycoprotein Ib.

METHODS

Expression of platelet surface antigens was determined by whole-blood double-label flow cytometry.

RESULTS

Glycoprotein Ib expression in systemic blood decreased 10% (p = 0.03) from preoperative levels at the start of cardiopulmonary bypass and 30% (p = 0.04) before release of the aortic crossclamp. Expression in pericardial blood at these times decreased by 50% and 51%, respectively (p = 0.003, p = 0.009). No changes were observed in the expression of the platelet activation antigens CD62P (P-selectin, indicating platelet alpha-granular release) and CD63 (indicating lysosomal release) or in binding of monoclonal antibody PAC-1 (detecting the fibrinogen-binding receptor conformation of the glycoprotein IIb-IIIa complex).

CONCLUSIONS

Glycoprotein Ib disappeared from the platelet surface during bypass grafting, most notably in pericardial blood. No increased expression of CD62P, CD63, or PAC-1 was found, indicating the absence of general platelet activation.

OBJECTIVE

In forensic autopsies it is important to determine the age of early vital skin wounds as accurate as possible. In addition to inflammation, coagulation is also induced in vital wounds. Analysis of blood coagulation markers in wound hemorrhage could therefore be an important additional discriminating factor in wound age determination. The aim of this study was to develop a wound age probability scoring system, based on the immunohistochemical expression levels of Fibronectin, CD62p and Factor VIII in wound hemorrhage.

METHODS

Tissue samples of (A) non injured control skin (n=383), and samples of mechanically induced skin injuries of known wound age, (B) injuries inflicted shortly before death (up to a few minutes old) (n=382), and (C) injuries inflicted 15-30 min before death (n=42) were obtained at autopsy in order to validate wound age estimation. Tissue slides were stained for Fibronectin, CD62p and Factor VIII and were subsequently scored for staining intensity (IHC score) in wound hemorrhage (1=minor, 2=moderate, 3=strong positive). Finally, probability scores of these markers were calculated.

RESULTS

In at most 14% of the non-injured control samples, hemorrhage was found, with mean±standard deviation IHC scores of 0.1±0.4, 0.2±0.4 and 0.2±0.5 for Fibronectin, CD62p, and Factor VIII, respectively. Expression of these markers significantly increased to mean IHC scores 1.4±0.8 (Fibronectin), 1.2±0.6 (CD62p), and 1.6±0.8 (Factor VIII) in wounds inflicted shortly before death (few minutes old) and to 2.6±0.5 (Fibronectin), 2.5±0.6 (CD62p), and 2.8±0.4 (Factor VIII) in 15-30 min old wounds. The probabilities that a wound was non vital in case of an IH score 0 were 87%, 88% and 90% for Fibronectin, CD62p, and Factor VIII, respectively. In case of an IHC score 1 or 2, the probabilities that a wound was a few minutes old were 82/90%, 82/83% and 72/93%. Finally, in case of an IHC score 3, the probabilities that a wound was 15-30 min old were 65%, 76% and 55%.

CONCLUSIONS

Based on the expression of Fibronectin, CD62p and Factor VIII in wound hemorrhage, we developed a probability scoring system that can be used in forensic autopsies to improve wound age estimation in early skin injuries.

The relationship between platelet aging and early markers of membrane activation have not been defined clearly. Activation markers expressed during prolonged storage are similar if not identical to those that appear after exposure to thrombin. Using flow cytometry, we investigated platelet membrane expression of CD62P, CD63, and annexin V binding (i.e., loss of membrane asymmetry) in platelets stored for up to 11 days under standard blood banking conditions. We compared five apheresis platelets to two random donor platelet concentrates, and to one pooled platelet preparation from six single platelet concentrates before and after exposure to thrombin. CD62P, CD63 expression, and annexin V binding increased during storage albeit with different kinetics. The differential increments observed between resting and thrombin (1 unit/ml) activated platelets showed an inverse correlation to storage time for CD62P, CD63, and annexin V binding, which was identical to published survival curves. A difference between apheresis platelets and platelet concentrates was observed only on day 1. Our data indicate that the in vitro platelet reserve activity to thrombin activation mirrors that of radiolabeled platelet survival in vivo and that platelet cross-sectional residual life span can explain their diminished capacity to respond to thrombin as a function of viability.

Thrombosis is a hallmark of the fatal fungal infection mucormycosis. Yet, the platelet activation pathway in response to mucormycetes is unknown. In this study we determined the platelet aggregation potential of Mucor circinelloides (M. circinelloides) NRRL3631, characterized the signaling pathway facilitating aggregation in response to fungal spores, and identified the influence of the spore developmental stage upon platelet aggregation potential. Using impedance and light-transmission aggregometry, we showed that M. circinelloides induced platelet aggregation in whole blood and in platelet-rich plasma, respectively. The formation of large spore-platelet aggregates was confirmed by light-sheet microscopy, which showed spores dispersed throughout the aggregate. Aggregation potential was dependent on the spore's developmental stage, with the strongest platelet aggregation by spores in mid-germination. Inhibitor studies revealed platelet aggregation was mediated by the low affinity IgG receptor FcγRIIA and integrin αIIbβ3; Src and Syk tyrosine kinase signaling; and the secondary mediators TxA2 and ADP. Flow cytometry of antibody stained platelets showed that interaction with spores increased expression of platelet surface integrin αIIbβ3 and the platelet activation marker CD62P. Together, this is the first elucidation of the signaling pathways underlying thrombosis formation during a fungal infection, highlighting targets for therapeutic intervention.

OBJECTIVE

To develop a sensitive and reproducible technique for measuring the adherence of blood lymphocytes to vessel walls exposed in sections of human retina and for examining the role of lymphocyte and vascular adhesion molecules in these events.

METHODS

Cryostat sections of human retina were overlaid with blood lymphocytes from healthy subjects, and experimental conditions were sought by which preferential attachment of the cells occurred to blood vessel walls in the retinal sections. Adherent lymphocytes were identified by staining with methyl green-thionine, and transected blood vessels were identified by their structure and by staining of basement membranes with periodic acid-Schiff. The adherence of enriched preparations of CD4+ (T-helper) and CD8+ (T-cytotoxic) lymphocytes, of interleukin-2 (IL-2)-activated cells, and of lymphocytes from patients with ocular Behçet's disease was examined. The distribution of adhesion molecules on retinal vessel walls was determined by immunohistochemistry, and the contribution of leukocyte integrins to lymphocyte binding was studied by blocking experiments with monoclonal antibodies.

RESULTS

The optimal selectivity of blood lymphocyte attachment to retinal vessel walls occurred when purified lymphocytes were suspended in culture medium with 10% fetal calf serum and overlaid onto retinal sections for 30 minutes at 23 degrees C with gentle agitation. Under these conditions, 92% of the lymphocytes that adhered to the section were confined to the retinal microvasculature, and CD4+ T cells were more adherent than CD8+ T cells (P < 0.01). Prior exposure of normal lymphocytes to IL-2 enhanced their binding to retinal blood vessels, and lymphocytes from patients with Behçet's disease showed supranormal vascular adherence (P < 0.005). Many transected vessels stained positively for CD31; PECAM (mean 62%), CD54; ICAM-1 (mean 73%), CD62E; E-selectin (mean 35%), CD62P; P-selectin (mean 61%), and CD106; VCAM-1 (mean 42%). However, these vascular adhesion molecules occupied < 20% of the area of the blood vessel walls. Lymphocyte adhesion to the retinal vessels was more dependent on CD29 (the common chain of the beta 1 integrins) expression than either CD11a/CD18 or CD49d.

CONCLUSIONS

This technique allows measurements to be made of lymphocyte adherence to vascular and nonvascular structures of retina ex vivo. Extension of this approach to the study of leukocyte adherence to sections of pathologic retina may be of clinical and experimental applicability in understanding mechanisms of retinal inflammation.

Platelet-leukocyte interactions represent an important determinant of the inflammatory response. Although mechanisms of platelet-neutrophil adhesion were studied extensively, little is known on the mechanisms of platelet-eosinophil interactions. The aim of the present study was to analyze the involvement of adhesion molecules and lipid mediators in platelet-eosinophil adhesion as compared to platelet-neutrophil adhesion. For that purpose human platelets, eosinophils and neutrophils were isolated and platelet-eosinophil and platelet-neutrophil adhesion induced by thrombin (30 mU/ml), LPS (0.01 microg/ml) and fMLP (1 microM) was quantified using the "rosettes" assay. The involvement of adhesion molecules such as selectin P, glycoprotein IIb/IIIa (GPIIb/IIIa) and lipid mediators such as of thromboxane A2 (TXA2), platelet activating factor (PAF) and cysteinyl leukotrienes (cysLTs) were studied using monoclonal antibodies and pharmacological inhibitors, respectively. Thrombin (30 mU/ml), LPS (0.01 microg/ml) and fMLP (1 microM) each of them induced platelet-eosinophil adhesion that was even more pronounced as compared with platelet-neutrophil adhesion induced by the same stimulus. Anti-CD62P antibody (1 microg/ml) and anti-GP IIb/IIIa antibody (abciximab-3 microg/ml) strongly inhibited platelet-eosinophil as well as platelet-neutrophil adhesion. Aspirin inhibited platelet-eosinophil adhesion, while MK 886-a FLAP inhibitor (10 microM), or WEB 2170-a PAF receptor antagonist (100 microM) were less active. On the other hand aspirin, MK 886 and WEB 2170 all three of them inhibited platelet-neutrophil adhesion. In summary, platelets adhered avidly to eosinophils both after activation of platelets by thrombin, eosinophils by fMLP or simultaneous activation of platelets and eosinophils by LPS. Similarly to platelet-neutrophil interaction adhesion of platelets to eosinophils involved not only adhesion molecules (selectin P, GPIIb/IIIa), but also lipid mediators such as TXA2. The involvement of PAF and cysteinyl leukotrienes in platelet-eosinophil adhesion was less pronounced as compared to platelet-neutrophil adhesion.

It has been hypothesized that the cytosolic esterase-induced fluorescence intensity (CEIFI) from carboxy dimethyl fluorescein diacetate (CMFDA) in platelets may related to platelet functions. In the present study, we measured the change of CEIFI in platelets during storage, and examined the correlations of CEIFI with the in vitro functionality of stored platelets, including the ADP-induced aggregation activity, hypotonic shock response, expression of CD62P as well as platelet apoptosis. The CEIFI of fresh platelets, when tested at 10 μM CMFDA, the mean fluorescence intensity index (MFI) was 305.9 ± 49.9 (N = 80). After 1-day storage, it was 203.8 ± 34.4, the CEIFI of the stored platelets started to decline significantly, and reduced to 112.7 ±27.7 after 7-day storage. The change in CEIFI is highly correlated to all four functional parameters measured, with the correlation coefficients being 0.9813, 0.9848, -0.9945 and -0.9847 for the ADP-induced aggregation activity, hypotonic shock response (HSR), expression of CD62P and platelet apoptosis respectively. The above results show that the CEIFI measurement of platelets represents well the viability and functional state of in vitro stored platelets. This may be used as a convenient new method for quality evaluation for stored platelets if this result can be further validated by the following clinical trials.

Hypercoagulability and excessive platelet activation account for a significant percentage of mortality and morbidity in cancer patients. In order to test the hypothesis that preloading infusion (PLI) with 6% hydroxyethyl starch 200/0.5 (HES 200), or 6% hydroxyethyl starch 130/0.4 (HES 130) solution can attenuate the hypercoagulable state and inhibit excessive platelet activation of patients with colon cancer, we selected 35 colon cancer patients undergoing laparoscopic-assisted radical colectomy. They were received randomly a test of 15 ml/kg of either HES 200 (n=17), or HES 130 (n=18) over a 30-min period preoperatively. In addition, fifteen healthy volunteers were selected as normal control group. Coagulation function was assessed by thrombelastography (TEG), platelet glycoprotein IIb/IIIa and CD62P was analyzed by flow cytometry before PLI, the end of PLI, 1 h after PLI, and 1 h after the end of surgery. Results demonstrated that hypercoagulable state indicated by TEG and excessive platelet activation was found in patients with colon cancer. We found that preloading infusion with HES 200/0.5 can inhibit platelet activation, and the two solutions, especially HES 200/0.5, compromised TEG parameters that indicated hypercoagulability of patients with colon cancer during perioperative period.

Platelets and monocytes play a pivotal role in the initiation and progression of large-vessel atherosclerosis. An up-regulation of various platelet and coagulation activation markers has been described in cardiovascular diseases and in patients with acute cerebral ischemia. In the present study the role of platelets and cellular coagulation activation in cerebral small-vessel disease (cSVD) was assessed. In 24 patients with cSVD but without established large-vessel disease, whole blood samples were obtained. Patients were divided into three subgroups (Fazekas 1, 2 and 3) according to extent of cSVD based on morphological magnetic resonance imaging criteria. Surface expression of CD40L and CD62P on platelets, tissue-factor exposition on monocytes and platelet-monocyte aggregates were measured with flow cytometry. Plasma levels of soluble CD40L, interleukin (IL)-6 and IL-7 were assessed by ELISA. Patients with cSVD show a significantly elevated expression of platelet CD40L (P < 0.001) and CD62P (P < 0.023), significantly elevated amounts of platelet-monocyte aggregates (P < 0.004), a significantly enhanced tissue-factor exposition on monocytes (P < 0.019) and significantly lower plasma levels of IL-7 compared to 10 healthy controls. However, this platelet and monocyte activation did not correlate with the severity of cSVD. Patients with cSVD show an up-regulation of the platelet CD40L and CD62P system and an activation of cellular coagulation which might contribute to the initiation and progression of cSVD.

To assess whether platelets are activated in transient global amnesia (TGA) and TIA, blood samples were analyzed by fluorescence-activated cytoscan using antibodies specific for platelet fibrinogen receptor (PAC1) and P-selectin (CD62P). Samples from TIA contained high levels of CD62P compared with age-matched control subjects, whereas those from TGA did not. The authors suggest that activated platelets are involved in brain ischemia, whereas ischemia appears not to be associated with most TGA.

Microsomal prostaglandin E2 synthase-1 (mPGES-1) constitutes an essential player of inflammation and is involved in the pathogenesis of rheumatoid arthritis (RA). Platelets participate in the regulation of inflammatory processes by the release of pro-inflammatory mediators and platelet-derived microparticles (PMPs). However, the role of the inducible mPGES-1/PGE2 pathway on platelet functions has not been investigated. Here we report a significant impact of mPGES-1 on platelet functions during inflammation. Wild-type (WT) and mPGES-1-/- (KO) mice were stimulated with lipopolysaccharide (LPS) for 24 hours. Platelet counts and activation were assessed by flow cytometry analyzing CD62P - CD154 expression, PMPs number, platelet-leukocyte aggregates and platelet aggregation. The accumulation of platelets and fibrinogen in the liver was analyzed by immunofluorescent staining. In native platelets from WT and mPGES-1 KO mice, there were no differences among the investigated functions. After LPS treatment, the number of platelets were significantly decreased in WT mice, but not in KO. Platelet activation, platelet-leukocyte aggregates, and PMP numbers were all significantly lower in KO mice compared to WT after LPS-treatment. In addition, KO mice displayed a significant reduction in platelet aggregation ex vivo In the liver of LPS-stimulated WT and KO mice, there were no differences in platelet accumulation, although, the percentage of total vessel area in the KO liver was significantly lower compared to WT. Our results demonstrate that systemic inhibition of mPGES-1 prevents platelet activation, which should have important implications regarding the cardiovascular safety of mPGES-1 inhibitors.