Current strategies in tissue engineering seek to obtain a functional tissue analogue by either seeding acellular scaffolds with cells ex vivo or repopulating them with cells in vivo, after implantation in patients. To function properly, the scaffold should be non-thrombogenic and biocompatible. Especially for the case of in vivo cell repopulation, the scaffold should be prepared in a manner that protects the tissue against platelet activation and adhesion. Anti-thrombogenicity can be achieved by chemical or physical surface modification. The aim of our study was to evaluate the platelet activation and thrombogenic properties of an acellular tissue scaffold that was surface modified with reduced graphene oxide (rGO). Graphene oxide was prepared by a modified Hummers method. For the study, an acellular pulmonary valve conduit modified with rGO was used. The rGO modified tissue samples were subjected to in vitro testing through interaction with whole blood under simulated laminar flow conditions. The following cellular receptors were then analysed: CD42a, CD42b, CD41a, CD40, CD65P and PAC-1. In parallel, the adhesion of platelets (CD62P positive), leukocytes (CD45 positive) and platelet-leukocyte aggregates (CD62P/CD45 positive) on the modified surface was evaluated. As a reference, non-coated acellular tissue, Poly-l lysine and fibronectin coated tissue were also tested. The rGO surface was also analysed for biocompatibility by performing a cytotoxicity test, TUNEL assay and Cell Cycle analysis. There was no significant difference in platelet activation and adhesion between the study groups. The only significant difference was observed for the PAC-1 receptor between Poly-l lysine group and rGO and the percentage of PAC-1 positive cells was 6% and 18% respectively. The average number of activated platelets (CD62P) in the field of view was 1, while the average number of leukocytes in the field of view was 3. No adherent platelet-leukocyte aggregates were observed. There were no significant differences in the DNA fragmentation. No significant effect of rGO on the amount of cells in different phases of the cell cycle was observed. Cytotoxicity indicates that the rGO can damage cells in direct contact but have no effect on the viability of fibroblasts in indirect contact.

OBJECTIVE

To evaluate platelet activation markers in psoriasis patients, compared to controls, and investigate their association with the inflammatory burden of psoriasis.

METHODS

Forty psoriatic patients without cardiovascular disease, and 12 healthy controls were subjected to measurement of baseline platelet CD62P, CD63 and CD42b expression, platelet-leukocyte complexes, i.e., platelet-monocyte complexes (PMC), platelet-neutrophil complexes (PNC) and platelet-lymphocyte complexes, and concentrations of platelet-derived microparticles (PMPs) using flow cytometry. Both larger-size (0.5-0.9 μm) and smaller-size (< 0.5 μm) PMPs were determined. Serum interleukin (IL)-12 and IL-17 levels were also measured by enzyme-linked immunosorbent assay. The severity of psoriasis was evaluated by the Psoriasis Area Severity Index (PASI).

RESULTS

PMP concentrations were significantly higher in psoriasis patients than controls [mean ± standard error of mean (SEM): 22 ± 5/μL vs 11 ± 6/μL; P = 0.018), for both smaller-size (10 ± 2/μL vs 4 ± 2/μL; P = 0.033) and larger-size (12 ± 3/μL vs 6 ± 4/μL; P = 0.014) PMPs. Platelet CD62P, CD63 and CD42b expression and circulating PMC and PNC were similar between the two groups. Lower circulating PLC were observed in psoriasis patients compared to controls (mean ± SEM: 16% ± 3% vs 23% ± 6%; P = 0.047). Larger-size PMPs were related with IL-12 levels (P < 0.001) and smaller-size PMPs with both IL-12 and IL-17 levels (P < 0.001). Total PMPs also correlated with IL-12 (P < 0.001). CD63 expression was positively correlated with both IL-12 and IL-17 (P < 0.05). Increased PASI score was associated with increased levels of larger-size PMPs (r = 0.45; P = 0.011) and increased CD63 expression (r = 0.47; P < 0.01).

CONCLUSIONS

PMPs, known to be predictive of cardiovascular outcomes, are increased in psoriasis patients, and associated with high inflammatory disease burden. Enhanced platelet activation may be the missing link leading to cardiovascular events in psoriatic patients.

Inflammatory cytokines have been shown to mediate organ damage by their action on vascular endothelia and leukocytes, in part by upregulating the expression of adhesion molecules, which in turn convey transmigration of leukocytes into tissue. The upregulation and activation of vascular cell adhesion molecules on the endothelial cells avail firm leukocyte adhesion to the vascular endothelium and enhance their transmigration and consecutive tissue injury. The aim of this study was to evaluate the expression of vascular adhesion molecules CD 31 (PECAM-1), CD 106 (VCAM-1), CD 62E (E-Selectin) and CD 62P (P-Selectin) in the pancreas and distant organs of pigs suffering from acute necrotizing pancreatitis (AP). AP was induced in 13 pigs by a combination of intravenous cerulein and intraductal glycodeoxycholic acid. For immunostaining of vascular adhesion molecules slides of porcine pancreas, lung, kidney and liver tissue were stained with monoclonal antibodies (Ab) against PECAM-1-1, VCAM-1 E- and P- SELECTIN. The endothelial cell expression of CD 31 (PECAM-1), CD 106 (VCAM), CD 62E (E-Selectin) and CD 62P (P-SELECTIN) in severe porcine pancreatitis is detectable and upregulation is partly significantly.

OBJECTIVE

Changes in hemostasis after off-pump coronary artery bypass grafting are still being discussed. There is a lack of information about platelet activity and aspirin efficacy after coronary artery bypass grafting. The aim of this study was to assess and compare platelet activity and aspirin efficacy early and late after off-pump and on-pump coronary artery bypass grafting.

METHODS

Eighty patients were enrolled in a prospective randomized study. Platelet activity was determined based on membrane expression of antigen CD62P (P-selectin) by means of flow cytometric analysis. Aspirin efficacy was assessed by using arachidonic acid-induced platelet aggregation. Blood samples were collected before the operation, immediately postoperatively, and on days 1, 2, 5, and 30.

RESULTS

In the off-pump group expression of P-selectin was markedly increased in comparison with preoperative values, with a maximum difference observed on day 2 (+53%, P = .02), and it was significantly higher compared with that seen in the on-pump group on days 2 and 5 (+53% vs +4%, P = .004, and +20% vs -16%, P = .005). On day 30, P-selectin expression was similar both between the groups and in comparison with the preoperative values. Arachidonic acid-induced platelet aggregation was gradually decreasing until day 30, but on day 2, there was an unexpected increase in aggregation that was more expressed in the off-pump group.

CONCLUSIONS

The platelet activity is higher in the early postoperative period in off-pump compared with on-pump coronary artery bypass grafting. The present aspirin strategy seems to be insufficient in the early postoperative period, irrespective of the surgical technique used.

Xueshuantong capsule (XST) is a patented traditional Chinese medicine used for the prevention and treatment of thrombosis. The molecular mechanism of anti-thrombotic effect of XST was investigated through the cross-talk among the platelets/leukocytes, endothelial cells (ECs), and flow shear stress. The Bioflux 1000 system was used to generate two levels of shear stress conditions: 0.1 and 0.9 Pa. Bioflux Metamorph microscopic imaging system was used to analyze the adhesion cell numbers. Protein expressions were detected by western blotting and flow cytometry. The flow-cytometry results showed that under 0.1 Pa flow, XST decreased ADP induced platelets CD62p surface expression in a concentration-dependent manner. Under 0.9 Pa flow, XST at a concentration of 0.15 g⋅L^-1 reduced the platelets activation by 29.5%, and aspirin (ASA) showed no inhibitory effects. XST showed similar efficiency on monocytes adhesion both under 0.1 and 0.9 Pa flow conditions, and the inhibition rate was 30.2 and 28.3%, respectively. Under 0.9 Pa flow, the anti-adhesive effects of XST might be associated with the suppression of VE-cadherin and Cx43 in HUVECs. Blood flow not only acts as a drug transporter, but also exerts its effects to influence the pharmacodynamics of XST. Effects of XST on inhibiting platelets activation and suppressing platelets/leukocytes adhesion to injured ECs are not only concentration-dependent, but also shear stress-dependent. The mechanic forces combined with traditional Chinese medicine may be used as a precise treatment for cardiovascular diseases.

OBJECTIVE

We investigated whether an ultrasonic echo contrast agent containing microbubbles (Levovist [SH U 508A]; Schering AG, Berlin, Germany) could in routine use activate platelets.

METHODS

Levovist and its main component, galactose, were mixed with separate samples of whole blood (1.5-75 mg/mL) from 5 healthy volunteers to form a 1-mL suspension sample. After in vitro exposure to ultrasound emitted from a commercial ultrasonic scanner at a pulse frequency of 3.5 MHz with a mechanical index of 1.9 and an exposure duration of 5 minutes, 5 microL of the sample was incubated for 20 minutes with the fluorescein isothiocyanate-labeled CD61 antibody, which is a platelet-specific antigen, and the phycoerythrin-labeled CD62P (P-selectin) antibody, an activation-specific antigen, both on the platelet surface. After more than 30 minutes of fixing in 1% paraformaldehyde, flow cytometric analysis was performed.

RESULTS

The percentage of CD62P-expressing platelets increased according to the concentrations of Levovist and galactose, which showed almost equal effects. Ultrasound exposure did not enhance the effect except at the highest concentration of Levovist (75 mg/mL).

CONCLUSIONS

In vitro, a galactose-based echo contrast agent could not activate the platelets at its routine concentration.

BACKGROUND

The novel TACSI system is designed for automated preparation of platelets (PLTs) from pooled buffy coats (BCs). One TACSI device will handle 6 units at the same time. The aim of our in vitro study is to investigate the effects of using this automated equipment with subsequent storage in two different plastic containers and to compare these results with PLTs prepared by the OrbiSac system.

METHODS

Buffy-coat-derived PLTs (n=8) were prepared by using the TACSI system, including storage in polyvinyl chloride (PVC)-based plastic containers with di, n-decyl phthalate (DnDP) (TACSI R) and BTHC (TACSI T)-based plasticizers. As a reference, the OrbiSac System was used to prepare PLTs (n=8) with subsequent storage in a PVC plastic container with a citrate-based plasticizer (BTHC). In total, 16 TACSI and eight reference units, supplied by approximately 30% plasma and 70% SSP+, were analysed for various in vitro variables during the 7-day storage period.

RESULTS

No significant difference in PLT counts, LDH, mean platelet volume (MPV) and adenosine triphosphate between the groups was detected. Glucose was lower (P<0·05) and lactate was higher (P<0·05) in TACSI R vs. OrbiSac. With exception of day 7 (P<0·05 TACSI R vs. OrbiSac), HSR reactivity were not different between groups. Extent of shape change was lower and CD62P higher in TACSI T when compared with TACSI R and OrbiSac units (P<0·05). pH was maintained at >6·8 (day 7) and swirling remained at the highest level (score=2) for all units throughout storage.

CONCLUSIONS

Platelets prepared by the TACSI system with subsequent storage in two different PVC-based plastic containers were equivalent to reference PLTs with regard to in vitro characteristics during 7 days of storage.

Perfluorinated chemicals (PFCs) have been widely used in a variety of products worldwide. Our previous study has documented a close association of higher serum level of perfluorooctane sulfonate (PFOS) with an increased carotid intima-media thickness (CIMT) in a cohort of adolescents and young adults. Herein, we further investigated the association of oxidative stress, circulating endothelial microparticles (EMPs) and platelet microparticles (PMPs) with PFCs and CIMT in humans. We recruited 848 subjects (12-30years old) from a population-based sample to determine the relationship between serum levels of PFCs, EMPs (CD62E and CD31+/CD42a-), PMPs (CD62P and CD31+/CD42a+), and the urine levels of 8-hydroxydeoxyguanosine (8-OHdG) and CIMT. The results showed that CD31+/CD42a- (endothelial apoptosis marker) and CD31+/CD42a+ (platelet apoptosis marker) increased significantly across quartiles of PFOS in multiple linear regression analysis. Furthermore, the elevation of CD31+/CD42a- and CD31+/CD42a+ corresponded to the increase of the odds ratios of thicker CIMT (greater than 50th percentile) with higher serum PFOS concentration (greater than 50%) (OR=2.86, 95% C.I.=1.69-4.84, P<0.001) in logistic regression models. There was no association between PFC concentration and 8-OHdG. In conclusion, we found the positive association between PFOS and CIMT that was more evident when serum levels of EMPs (CD31+/CD42a-) and PMPs (CD31+/CD42a+) were elevated. Further studies are warranted to investigate the causal inference of PFOS exposure on endothelial cell damage and atherosclerosis.

It has been suggested that platelet function in chronic immune thrombocytopenic purpura (ITP) may be abnormal. Thrombopoietin mimetics used for treatment can affect it, but the data remain limited. We investigated platelet function of 20 children diagnosed with severe ITP (aged 1-16 years, 12 females and eight males). Platelet functional activity in whole blood was characterized by flow cytometry before and after stimulation with SFLLRN plus collagen-related peptide. Levels of CD42b, PAC1, and CD62P, but not CD61 or annexin V, were significantly increased (P < 0.05) in resting platelets of patients before treatment compared with healthy donors. On average, PAC1 and CD62P in patients after activation were also significantly elevated, although some patients failed to activate integrins. Romiplostim (1-15 μg/kg/week s.c.) was prescribed to seven patients, with clinical improvement in six. Interestingly, one patient had clinical improvement without platelet count increase. Eltrombopag (25-75 mg/day p.o.) was given to four patients, with positive response in one. Others switched to romiplostim, with one stable positive response, one unstable positive response, and one non-responding. Platelet quality improved with romiplostim treatment, and their parameters approached the normal values. Our results suggest that platelets in children with severe ITP are pre-activated and abnormal, but improve with treatment.

OBJECTIVE

The etiology of biliary atresia (BA) is still elusive. Inflammation plays a key role in bile duct and liver injury. The recruitment and accumulation of inflammatory cells is largely dependent on adhesion molecules. We aimed to investigate P-selectin (CD62P) expression in liver tissue in patients with BA compared with other neonatal cholestatic disorders.

METHODS

The study included 63 infants with neonatal cholestasis in 2 groups: BA group (n = 32) and non-BA group (n = 31) with non-BA cholestatic disorders as controls. Demographic, clinical, laboratory, ultrasonographic, and histopathological parameters were collected. P-selectin immunostaining was performed. Immunostaining in bile duct epithelium, cellular infiltrate, and vascular endothelial cells were scored as positive or negative.

RESULTS

The frequency of P-selectin-positive endothelium, platelets, and bile duct epithelium was significantly higher in the BA group (72%, 72%, and 63%, respectively) than in the non-BA group (32%, 16%, and 13%, respectively) with P of 0.002, <0.0001, and <0.0001, respectively. Few mononuclear cellular infiltrates in portal tract expressed P-selectin and were comparable in both groups (P = 0.932). Of interest, the platelet count was significantly higher in the BA group (532 ± 172) than in the non-BA group (406 ± 158), and 68.8% of the BA group had thrombocytosis versus 25% in the controls (P = 0.001 for both).

CONCLUSIONS

The significant expression of P-selectin in endothelium, platelets, and bile duct epithelium in patients with BA suggests a potential role for this adhesion molecule in the pathogenesis of this devastating neonatal hepatic disorder. It further suggests that platelets in BA are activated and may have a role in the inflammatory process in BA.

BACKGROUND

There are various hydroxyethyl starch (HES) solutions with different degrees of hydroxylation and different molecular weights. HES200/0.5 solution is most commonly used. HES130/0.4 is a new HES solution and is the 'state-of-the-art' in volume substitution. However, the mechanism of the observed anticoagulation action of HES has not been fully delineated. The objective of this study was to further investigate the effect of HES200/0.5 and HES130/0.4 on platelet coagulation.

METHODS

Sixty ASA I-II patients undergoing elective minor surgery were randomly allocated to receive an intravenous infusion (20 ml/kg) of lactated Ringer's solution (group L), HES200/0.5 (group H) or HES130/0.4 (group V) after the induction of anesthesia. The expression of CD42b, CD41/61 and CD62p in vivo was assessed on non-stimulated platelets and adenosine diphosphate (ADP) agonist-activated platelets using flow cytometry.

RESULTS

Resting glycoprotein expression of the non-stimulated platelets was observed. HES200/0.5 and HES130/0.4 reduced the CD42b, CD41/61 and CD62p expression of ADP-agonist-activated platelets at 15 min after intravenous infusion. At 6 h after intravenous infusion, the trend of decreasing expression of activated CD42b, CD41/61 and CD62p was maintained in group H. However, CD42b, CD41/61 and CD62p expression returned to the pre-operative level in group V.

CONCLUSIONS

This study showed that both HES200/0.5 and HES130/0.4 can inhibit platelet coagulation. Platelet dysfunction experienced a faster recovery after the infusion of HES130/0.4 than after HES200/0.5. Liquid resuscitation with HES130/0.4 may decrease the risk of hemorrhage in the operative period.

(RADA)4 peptides are promising biomaterials due to their high degree of hydration (<99.5% (w/v)), programmability at the molecular level, and their subsequent potential to respond to external stimuli. Interestingly, these peptides have also demonstrated the ability to cause rapid (∼15s) hemostasis when applied directly to wounds. General hemocompatibility of (RADA)4 nanofibers was investigated systematically using clot formation kinetics, C3a generation, and platelet activation (morphology and CD62P) studies. (RADA)4 nanofibers caused a rapid clot formation, but yielded a low platelet activation and low C3a activation. The study suggests that the rapid hemostasis observed when these materials are employed results principally from humoral coagulation, despite these materials having a net neutral charge and high hydration at physiological conditions. The observed rapid hemostasis may be induced due to the available nanofiber surface area within the hydrogel construct. In conclusion, our experiments strongly support further development of (RADA)4 peptide based biomaterials.

UNASSIGNED

Biomedicine based applications of (RADA)4 peptides are being extensively studied for the purpose of improving drug carriers, and 3D peptide nanofiber scaffolds. However, this peptide's biocompatibility has not been investigated till now. One particular study has reported a revolutionary and very desirable ability of (RADA)4 peptide to achieve complete and rapid hemostasis, nevertheless, the literature remains inconclusive on the underlying molecular mechanism. In this manuscript we bridge these two main knowledge gaps by providing the much needed systematic biocompatibility analysis (morphology analysis, platelet and C3a activation) of the (RADA)4 based hydrogels, and also investigate the underlying hemostatic mechanism of this peptide-induced hemostasis. Our work not only provides the much-needed biocompatibility of the peptide for applicative research, but also explores the molecular mechanism of hemostasis, which will help us design novel biomaterials to achieve hemostasis.

The aim of the present study was to examine and understand changes in platelet functions prior to and after the treatment of primary immune thrombocytopenia (ITP) in children. An automatic hematology analyzer and whole blood flow cytometry were used to detect immature platelet fraction (IPF), IPC and membrane glycoproteins (CD62p, PAC-1 and CD42b) in ITP children (ITP group), children with complete response after ITP treatment (ITP-CR group) and children with elective surgery (normal control group). The results showed that, levels of platelet count (PLT) and plateletcrit in the ITP group were lower alhtough the levels of mean platelet volume, platelet distribution width and platelet-large cell ratio (P-LCR) were higher than those in the normal control and ITP-CR groups. PLT in the ITP-CR group was lower than that in the normal controls. Additionally, IPF% was higher in the normal control and ITP-CR groups, IPC was lower in the ITP group compared to the normal control and ITP-CR groups. Furthermore, prior to ADP activation, the expression levels of CD62p, PAC-1 and CD42b in the ITP group were lower in ITP group than those in the normal control and ITP-CR groups. The expression level of PAC-1 was lower in the ITP-CR and normal control groups. No differences were identified in CD62p and CD42b expression levels. Following ATP activation, CD62p, PAC-1 and CD42b expression in the ITP group was lower than that in the normal control and ITP-CR groups. PAC-1 expression was lower while CD62p expression was higher in the ITP-CR group compared to the normal control group. In conclusion, the activation of platelets in ITP children was low. Decreased platelet function, platelet parameters and platelet glycoproteins may be used as markers for monitoring the treatment efficacy in ITP children.

Essentials Successful outcome of platelet transfusion depends on specific antiplatelet therapy in use. We assessed if ticagrelor, clopidogrel or prasugrel impacts on donor platelet activity ex vivo. Ticagrelor and/or its active metabolite in plasma or bound to platelets can inhibit donor platelets. This might compromise the effectiveness of platelet transfusion therapy.

CONCLUSIONS

Background Platelet transfusion is the conventional approach to restore platelet function during acute bleeds or surgery, but successful outcome depends on the specific antiplatelet therapy. Notably ticagrelor is associated with inadequate recovery of platelet function after platelet transfusion. We examined whether plasma and/or platelets from ticagrelor-treated patients influence donor platelet function, in comparison with clopidogrel and prasugrel. Methods Platelet transfusion was mimicked ex vivo by mixing naïve donor platelet-rich plasma (PRP) or gel-filtered platelets (GFP) in defined proportions with PRP, plasma or GFP from cardiovascular patients receiving standard care including medication with prasugrel, clopidogrel or ticagrelor (n = 20 each). Blood was taken 4 h after the previous dose. HLA2/HLA28 haplotyping let us distinguish net (all platelet) and individual patient/donor platelet reactivity in mixtures of patient/donor platelets, measured by flow cytometry analysis of ADP-induced fibrinogen binding and CD62P expression. Results ADP responsiveness of donor platelets was dramatically reduced by even low (10%) concentrations of PRP or plasma from ticagrelor-treated patients. Clopidogrel and prasugrel were associated with more modest donor platelet inhibition. GFP from ticagrelor-treated patients but not patients receiving clopidogrel or prasugrel also suppressed donor GFP function upon mixing, suggesting the transfer of ticagrelor from patient platelets to donor platelets. This transfer did not lead to recovery of ADP responsiveness of patient's platelets. Conclusion Collectively, these observations support the concept that ticagrelor and/or its active metabolite in plasma or bound to platelets can inhibit donor platelets, which might compromise the effectiveness of platelet transfusion therapy.

There is convincing evidence that cell adhesion plays an important role in cardiovascular pathology and is frequently associated to "in vivo" cellular activation. This study involves patients with mechanical heart valve replacement (MHVR patients) who have increased platelet polymorphonuclear leukocyte (PMN) reactivity. Dual-color cytometry was used to determine the expression of adhesive molecules on cellular surfaces, platelet, and PMN-bound fibrinogen as well as the presence of circulating platelet/PMN mixed-cell aggregates (MCA) in 55 MHVR patients, 49 control patients under oral anticoagulant therapy, and 22 healthy volunteers. The results demonstrated that (a) PMN from MHVR patients showed an increased PMN-bound fibrinogen (mean +/- SEM: 1,420 +/- 169 anti-fibrinogen fluorescence intensity, P= 0.0012), when compared to controls (mean +/- SEM: 747 +/- 32 anti-fibrinogen fluorescence intensity) and healthy volunteers (mean +/- SEM: 692 +/- 25 anti-fibrinogen fluorescence intensity; (b) platelet activation in MHVR patients was evidenced by the higher expression of CD62P (mean +/- SEM: 128 +/- 19 anti-CD62P fluorescence intensity, P = 0.003) compared to controls (mean +/- SEM: 65 +/- 15 and 50 +/- 10 anti CD62P fluorescence intensity) and by increased levels of platelet-bound fibrinogen (mean +/- SEM: 625 +/- 20 anti-fibrinogen fluorescence intensity, P = 0.0043 versus 496 +/- 45 and 480 +/- 30 for control patients and for healthy volunteers, respectively); and (c) the proportion of MCA in MHVR patients (15 +/- 2%) was significantly higher (P = 0.009) compared to controls (7 + 1%) and healthy volunteers (6 +/- 2%). The results indicate that the presence of stable circulating MCA represents another marker of "in vivo" PMN activation in MHVR patients.

OBJECTIVE

Oxidative stress with free radicals plays a crucial role in acute pancreatitis (AP). Pantoprazole (PPZ), widely used as a proton pump inhibitor, possesses reactivity towards hydroxyl radicals. The aim of the study was to examine the effect of PPZ on the course of experimental AP.

METHODS

Mild AP was induced in rats by caerulein (n=12). Severe AP was induced by infusion of glycodeoxycholic acid (10mM) into the pancreatic duct combined with caerulein (n=12). Both AP models were randomized to PPZ treatment (20mg/kg at baseline and after 12h) or placebo. Control animals received Ringer solution (n=6) without AP induction. After 24h severity of AP was examined by histology, enzyme levels, edema and inflammatory markers (myeloperoxidase, protein profiling). Furthermore, CD62P and CD31 for leukocyte and platelet activation were investigated.

RESULTS

Histology showed that PPZ treatment reduced tissue infiltration of inflammatory cells and acinar cell necrosis in severe AP. After PPZ treatment CD62P expression in mild AP and CD31 expression in severe pancreatitis decreased, indicating an inhibition of platelet activation. In mild and severe AP, PPZ significantly decreased amylase, LDH, edema and myeloperoxidase activity. Protein profile of pancreatic juice and serum revealed different spectra and less pancreatic juice proteins in PPZ treated groups indicating less acinar cell leakage.

CONCLUSIONS

PPZ possesses anti-inflammatory in vivo properties and attenuates the course of AP. This is mediated via a reduced expression of inflammatory and adhesive proteins with a consecutive decrease in platelet and leukocyte activation as key steps in the pathogenesis of AP.

We investigated expression of several antigens on neutrophils and monocytes, involved in cell adhesion, from patients hemodialyzed with cellulosic and polyacrylonitrile membranes. Among the antigens tested only the expression of CD15s and CD11b was significantly increased on neutrophils and monocytes in patients dialyzed with cellulosic membranes. No changes occurred with polyacrylonitrile membranes. Leukocyte counts from patients dialyzed with cuprophane membranes decreased at the same time as expression of cellular CD15s increased, resulting in a significant negative correlation at all time points tested. No correlation was found between the drop of monocytes and their expression of CD11b. When CD15s expression increased on neutrophils and monocytes, we observed a concomitant increase of CD62P, a specific selectin of activated platelets. When whole blood cells were incubated with complement activated serum both antigens increased but not when cells were incubated with hrC5a. We also observed that CD61, a platelet phenotypic antigen, was present on leukocytes incubated with complement activated serum. At the time when platelet-leukocyte coaggregates decreased, CD62P expression remained stable on leukocytes, suggesting that both neutrophils and monocytes are able to trap either CD62P shed by activated platelets or soluble CD62P present in normal human serum. The present study documents a major role of P-selectin (CD62P)/sialyl-Lewis x (CD15s) interaction in the transient leukocyte margination during hemodialysis.

Early detection of the platelet storage lesion is still a challenge in transfusion practice. Using flow cytometry, we evaluated the appearance of the storage lesion, based on the expression of platelet activation markers, in total platelets and platelet populations.

Buffy-coat-derived platelet concentrates were stored under standard conditions for 5 days. The expression of activation antigens CD42b, CD36, CD62p and phosphatidylserine on total platelets and populations of small, medium-sized and large platelets was analysed by flow cytometry on storage days 1, 3 and 5.

The activation/lesion on total platelets and each platelet population was detected on storage day 3, by the increased expression of CD36. On the same day, increased expression of CD42b and CD62p was detected, but only on large platelets. Small and medium-sized platelets had increased CD62p expression only on day 5. Externalisation of phosphatidylserine was not detected.

Evaluation of the level of expression of various activation markers on different platelet populations could be an additional valid analysis in cell quality control of platelet concentrates, and in the assessment of novel approaches to platelet concentrate manipulation.

BACKGROUND

Inconsistent findings are reported about the relation of platelet activity to disease severity in stable patients with chronic coronary artery disease (CAD). Nevertheless, most reports studied only very small groups of patients. The purpose aim of our study was to assess the relation of platelet activity to disease severity in sufficient number of patients with chronic CAD.

METHODS

One hundred and sixty stable patients with chronic CAD were studied (25 with single-, 63 with double- and 72 with triple-vessel disease). 91% of them were on aspirin, 1.6% on clopidogrel medication. Platelet activity was determined as membrane expression of antigens CD62P (P-selectin, as % of positive cells) and CD41 (part of GpIIb/IIIa integrin, as mean fluorescence intensity) by flow cytometry. Platelet aggregability was measured by ADP-optical aggregometry. Data sets were compared by Kruskal-Wallis test, correlation by Spearman test. Data are shown as median with 25-75 percentiles.

RESULTS

Membrane CD62P expression correlated with vessel severity (P < 0.001, Kruskal-Wallis test). Patients with triple-vessel disease had the highest CD62P expression (1.6; 1.1-2.0) followed by patients with double-vessel (1.2; 0.63-1.95) and single-vessel (0.7; 0.30-0.84) disease. Positive correlation was found between CD62P expression with triglycerides (r = 0.49, P < 0.05) and CD41 with fasting glucose (r = 0.48, P < 0.05). No differences in CD41 expression or ADP aggregability were found between groups.

CONCLUSIONS

Higher platelet activity is present in patients with more severe CAD. More aggressive anti-platelet treatment in these patients should be considered, especially when metabolic syndrome is simultaneously present.

The human corpus luteum is a unique endocrine organ that is periodically constructed from the ovulated follicle. During human corpus luteum formation, which is well known as a pathophysiological model for tissue remodeling, the precise mechanisms by which centripetal vascular development is regulated remain unknown. Recently platelets were reported to contain chemoattractive substances with the potential to induce endothelial migration. In this study, we examined the involvement of platelets in the early tissue remodeling process of the human corpus luteum. An immunohistochemical study demonstrated that considerable amounts of red blood cells and CD41-positive platelets were localized at extravascular sites among luteinizing granulosa cells after ovulation. Platelet deposition gradually decreased and became limited near the central cavity toward which microvessels were extending. Platelets were hardly observed in the midluteal phase when the vascular network had already been established. These platelets expressed CD62P/P-selectin and were colocalized with extracellular matrix, suggesting that platelets had been activated by the extracellular matrix. Progesterone production by luteinizing granulosa cells that were isolated from patients undergoing in vitro fertilization therapy was significantly promoted by direct contact with platelets during 4-d culture. Platelet-derived soluble factors induced spreading in granulosa cell morphology. These factors also increased the migration of human umbilical vein endothelial cells, whereas luteinizing granulosa cells attenuated platelet-induced endothelial cell migration. These findings lead us to propose the novel concept that platelets are regulators of endothelial cell migration and granulosa cell luteinization in the remodeling process of the human corpus luteum.

OBJECTIVE

NMSO3, a sulfated derivative of sialic acid, is a specific inhibitor for P-selectin (CD62P)-mediated cell adhesion. We attempted to apply liposomes modified with NMSO3 for selective targeting of activated platelets.

METHODS

The binding of fluorescently labeled NMSO3-containing liposomes (NMSO3-liposomes) to CHO cells expressing P-selectin (CHO-P cells) and activated platelets were examined. The distribution of NMSO3-liposomes incorporated into the cells was observed by fluorescence microscopy.

RESULTS

The binding assay revealed that NMSO3-liposomes specifically bound to immobilized P-selectin and CHO-P cells in a dose-dependent manner. The binding of NMSO3-liposomes to CHO-P cells was much stronger than that to the parental CHO-K1 cells. Fluorescence microscopic observation showed that NMSO3-liposomes were incorporated into CHO-P cells after the binding and distributed throughout the cytoplasm of the cell. NMSO3-liposomes bound more strongly to thrombin-activated platelets than to resting platelets, as assessed by flow cytometry.

CONCLUSIONS

These results suggest that NMSO3-liposomes can be applied for selective drug delivery to activated platelets.

Currently, there are several animal models of diabetes mellitus and hypertension, but relatively little is known about blood platelet function in these models. The aim of this work was to characterise and compare platelet reactivity and activation in db/db mice (mouse model of diabetes) and mice receiving L-NAME (model of chronic inhibition of NO synthesis), using various platelet function assays. We found higher platelet activation (circulating resting platelets) in db/db mice than in db/+heterozygotes, as evidenced by elevated expressions of CD62P and CD40L and a lower expression of CD42b. The expression of COX-1 was significantly increased, and the phosphorylation of vasodilator stimulated phosphoprotein (VASP) Ser(157) significantly reduced in platelets from db/db mice. Similarly, we observed platelet hyperreactivity in db/db mice following the in vitro responses to 20μg/ml collagen (reflected by increased expressions of CD62P and CD40L, and reduced CD42b), 20μM ADP (reduced CD42b) and lower concentrations of thrombin (0.025 U/ml) (increased CD62P, JON/A, bound vWF, and bound fibrinogen). Otherwise, platelet hyporeactivity was revealed for higher thrombin (0.25 U/ml) (reduced CD62P and bound vWF), while hyperreactivity occurred for CD40L and bound Fg in db/db mice compared to non-diabetic control, db/+. Plasma levels of sCD40L, but not of sCD62P, were increased in db/db mice; also plasma TXB2 concentrations were over 3.5-fold higher in this group than in the heterozygous db/+mice (P<0.01). In contrast, in the mice administered with L-NAME, no statistical differences in expressions of platelet activation markers were found between mice supplemented with L-NAME and controls. Likewise, the TXB2 level did not differ between L-NAME mice and controls, but L-NAME mice had significantly higher plasma levels of sCD62P and sCD40L than controls. In conclusion, these two studied models differ in the overall picture of blood platelet activation and reactivity, as they demonstrated opposite time sequence patterns of platelet activation in circulating blood. More generally, our study provides another argument for the opinion that multiparametric analysis of platelet function offers a much better tool for investigation and minimizes the likelihood of artefacts.

Essential thrombocythemia (ET) patients are at risk of developing thrombotic events. Qualitative platelet (PLT) abnormalities and activation of endothelial cells (ECs) and PLTs are thought to be involved. Microparticles (MPs) can originate from PLTs (PMPs), ECs (EMPs), or red cells (RMPs). Previous studies have indicated that MPs contribute to ET pathophysiology. Endothelial modulators (eg, nitric oxide [NO], adrenomedullin [ADM], and endothelin-1 [ET-1]) are also involved in the pathophysiology of this condition. We hypothesized that treatments for reducing PLT count might also indirectly affect MP generation and endothelial activity by altering endothelial modulator production. The rationale of this study was that hydroxyurea (HU), a cytostatic drug largely used in ET, induces the production of a potent vasoactive agent NO in ECs. An observational retrospective study was designed to investigate the relationship between MPs, NO, ADM, and ET-1 in ET patients on treatment with HU, anagrelide (ANA), aspirin (ASA), and a group of patients before treatment. A total of 63 patients with ET diagnosis: 18 on HU + ASA, 15 on ANA + ASA, 19 on ASA only, and 11 untreated patients, and 18 healthy controls were included in this study. Blood samples were analyzed for MP (absolute total values) and functional markers (percentage values) by flow cytometry. PLT-derived MPs were studied using CD61, CD62P, CD36, and CD63, whereas endothelial-derived MPs were studied using CD105, CD62E, and CD144. Endothelial modulator markers (NO, ADM, and ET-1) were measured by ELISA. Total MP count was higher in the group treated with ANA + ASA (P < 0.01). MP markers modified in ET patients returned to levels of healthy controls following treatment, in particular, in patients on ANA treatment. NO and ADM values were higher in the HU group (P < 0.001). HU and ANA treatment also affected MP production in a cell origin-specific manner. HU and ANA, although acting via different pathways, have similar final effects. For instance, HU causes vasodilatation by increasing NO and ADM levels, whereas ANA impairs vasoconstriction by reducing ET-1. In conclusion, therapy with HU cytostatic drugs and ANA can reduce PLT count in ET, and also affect endothelial modulatory agents, with HU sustaining vasodilation and prothrombotic MP concentration, whereas ANA decreases vasoconstriction.

The opening of mitochondrial permeability transition pore (MPTP) plays a critical role in platelet activation. However, the potential trigger of the MPTP opening in platelet activation remains unknown. Inflammation is the crucial trigger of platelet activation. In this study, we aimed to explore whether and how the important inflammatory cytokine IL-17 is associated with MPTP opening in platelets activation by using MPTP inhibitor cyclosporine-A (CsA). The mitochondrial membrane potential (ΔΨm) was detected to reflect MPTP opening levels. And the platelet aggregation, activation, and the primary signaling pathway were also tested. The results showed that the MPTP opening levels were increased and Δψm reduced in platelets administrated with IL-17. Moreover, the levels of aggregation, CD62P, PAC-1, P53 and the phosphorylation of ERK2 were enhanced along with the MPTP opening in platelets pre-stimulated with IL-17. However, CsA attenuated these effects triggered by IL-17. It was suggested that IL-17 could induce MPTP opening through ERK2 and P53 signaling pathway in platelet activation and aggregation.