An 11-year-old spayed-female German Shepherd dog was presented to the Veterinary Medical Teaching Hospital at Kansas State University with a history of weight loss, anorexia, depression, and lethargy for 2-3 weeks. Radiographic examination revealed a mass in the spleen and several round radiodense foci in the liver. CBC results included normocytic normochromic anemia, marked thrombocytopenia, and low numbers of neoplastic cells that frequently had cytoplasmic projections or blebs. A bone marrow aspirate contained about 80% neoplastic megakaryoblasts with the same microscopic features as those observed in peripheral blood. Using flow cytometry, cells of large size were identified in peripheral blood that expressed CD41/61, CD45, CD61, and CD62P (P-selectin) and were negative for markers of T cells, B cells, monocyte/macrophages, and dendritic cells. Because of the poor prognosis, euthanasia and subsequently necropsy were performed. On histopathologic examination, neoplastic megakaryoblasts were identified in spleen, liver, mesenteric lymph node, and the pulmonary vasculature. Using immunohistochemistry, the neoplastic megakaryoblasts weakly expressed von Willebrand factor. Based on microscopic and immunophenotypic findings, a diagnosis of acute megakaryoblastic leukemia (AMegL) was made. To our knowledge, this is the first report of AMegL in a domestic animal in which immunophenotyping by flow cytometry and a panel of antibodies against CD41/61, CD61, and CD62P were used to support the diagnosis.

OBJECTIVE

Platelet function abnormalities have been reported in blood donors who have not consumed aspirin. Our objective was to identify factors other than aspirin that may contribute to impaired platelet function in qualified volunteer blood donors.

METHODS

Blood samples were obtained from 24 donors following routine blood donation. Donors completed a study questionnaire that included questions about recent food consumption, medication and medical history. Platelet activation was measured using monoclonal antibodies and flow cytometry. CD62P expression and PAC-1 binding on platelets were used as indicators of platelet activation. Platelet function was measured on a platelet function analyser (PFA-100) using both collagen/epinephrine (cEPI) and collagen/ADP (cADP) cartridges.

RESULTS

Fifty-four per cent of donors (13 of 24) had normal platelet function. Thirty-eight per cent (nine of 24) had prolonged cEPI closure times, of whom four (17%) had no cEPI closure >> 300 seconds). No closure was associated with aspirin use (two donors) or chocolate consumption (two donors) before donation. Two donors (8%) had either a shortened cEPI or cADP closure time.

CONCLUSIONS

Platelet dysfunction in qualified blood donors is underestimated. Platelet function screening can identify donors with diet-related platelet dysfunction or with poor recollection of aspirin use.

SUMMARY: BACKGROUND: The Mirasol® pathogen reduction technology (PRT) for platelet concentrates (PC) uses riboflavin and UV light (270-360 nm). We evaluated the impact of PRT on platelets in comparison to standard single-donor PC. MATERIAL AND METHODS: Platelets were resuspended in autologous plasma. After 2 h rest without agitation, PC were split into an untreated control unit (C-PC) and an immediately treated unit (T-PC) (series I). In series IV, split PC were stored under agitation over night before PRT was carried out. Platelet quality was assessed by pH, glucose consumption, lactate production rate, LDH, soluble sCD62p and CD62p expression with and without TRAP (thrombin receptor-activating peptide) over 7 days. RESULTS: SERIES I: On day 5, pH values were lower for T-PC (6.8 ± 0.2 vs. 7.4 ± 0.1, C-PC), accompanied by a higher glucose consumption rate of 0.069 ± 0.016 vs. 0.035 ± 0.006 mmol/10(12) platelets/h and lactate production rate of 0.126 ± 0.031 vs. 0.063 ± 0.011 mmol/10(12) platelets/h. CD62p using TRAP was lower for T-PC (50 ± 11 vs. 62 ± 14%). Baseline activation was higher in T-PC (35 ± 12 vs. 28 ± 15%). Longer initial rest time had no impact on these results (series II/III/IV). CONCLUSION: PRT leads to an increase of platelet metabolism and activation independent of the length of the initial rest times. PC resuspended in autologous plasma should be stored at maximum up to day 5.

BACKGROUND

In Japan, no platelet (PLT) additive solutions (PASs) are officially approved for clinical use although blood centers often receive requests for washed PLTs to reduce adverse reactions. Recently, we developed a novel PAS called BRS-A based on clinically available bicarbonated Ringer's solution (BRS), Bicanate and acid-citrate-dextrose formula A (ACD-A), which has been shown to maintain the in vitro properties of PLTs in the condition of <5% residual plasma during 7-day storage. The aim of this study was to evaluate whether another clinically available BRS, Bicarbon with different electrolyte concentrations can be used as a PAS.

METHODS

Two types of BRS-As were prepared by adding 25 mL of ACD-A to 500 mL of Bicanate or Bicarbon BRSs. Bicanate-based BRS-A and Bicarbon-based BRS-A contain 0.9 or 0.5 mmol/L of magnesium chloride, 95.2 or 100.1 mmol/L of sodium chloride, 4.2 or 5.1 mmol/L of trisodium citrate, and 26.6 or 23.8 mmol/L of sodium bicarbonate, respectively; the other components were identical. Apheresis PLTs stored in these solutions with less than 5% plasma for 7-day storage were compared with regard to their in vitro properties.

RESULTS

The pH levels of all units were above 7 throughout storage. The mean PLT volume, hypotonic shock response, glucose consumption, lactate production, swirling, and CD62P and CD42b expression were similar during 7-day storage. The bicarbonate levels in Bicarbon-based BRS-A were lower than those in Bicanate-based BRS-A.

CONCLUSIONS

Differences in concentrations of electrolytes such as magnesium, sodium, citrate, and bicarbonate salts in BRS-A do not affect the in vitro properties of PLTs during 7-day storage. These results indicate that the use of another type of BRS-A based on Bicarbon as a PAS is feasible. Thus, BRS-A can be used in hospitals that do not stock Bicanate but have Bicarbon.

Platelet recovery, and viability, and function is strongly dependent on the method of the preparation of platelet concentrate (PC). The glucose consumption, decrease of pH, release of alpha granules during storage in platelet concentrate impair their clinical effectiveness.

OBJECTIVE

To compare of the quality of buffy-coat-derieved platelet concentrates prepared using automatic system terumo automated centrifuge and separator integration (TACSI) and stored over 7 days.

METHODS

PCs were prepared from buffy coats using manual method (group I), or automatic system TACSI (group II). Fifteen PCs prepared from the 5 buffy coats each were stored over 7 days in 22-24 degrees C and tested. Samples were taken from the PCs container on days 1 and 7. The following laboratory tests were performed: number of platelets, platelets derived microparticles, CD62P expression, platelet adhesion, pH, glucose, lactate dehydrogenase activity.

RESULTS

We have observed higher expression of CD62P in PCs prepared using manual method compared to the PCs produced automatically Platelet recovery was significantly higher in PCs prepared using automatic systems compare to manual method.

CONCLUSIONS

Compared to manual methods, automatic system for preparation of buffy coats, is more efficient and enable production of platelets concentrates of higher quality.

The purpose of this investigation was to obtain information on platelet-leukocyte conjugate formation in whole blood and on factors that affect it. We also measured platelet and leukocyte activation by quantitating the expression of CD62P and CD11b. In both cases a flow cytometric approach was used. The results show that platelet-monocyte and platelet-polymorphonuclear leukocyte (PMNL) conjugate formation is enhanced by simply stirring blood, with optimum conjugate formation occurring after 10 min. In the case of monocytes,conjugate formation was enhanced by adenosine diphosphate (ADP). Both monocyte and PMNL conjugate formation was enhanced by phorbol myristate acetate (PMA), but L-formyl methionyl lysyl proline (FMLP) was either without effect (monocytes) or inhibitory (PMNL). EDTA also inhibited conjugate formation (implying involvement of divalent cations), as did dextran sulphate (implying involvement of P-selectin = CD62P). Interestingly GR144053F, which acts at GpIIb-IIIa on platelets to interfere with fibrinogen binding, and also glycyl prolyl arginyl proline (GPRP), a peptide that interferes with the interaction between CD11c on leukocytes and fibrinogen, did not inhibit platelet-monocyte conjugate formation, but did inhibit the platelet-PMNL interaction; this indicates that GpIIb-IIIa on platelets and CD11c on leukocytes and fibrinogen are involved in mediating the interaction between platelets and PMNL but not platelets and monocytes. Surprisingly arginyl-glycyl aspartyl serine (RGDS) inhibited the formation of both types of conjugate but this may be because it also inhibited both platelet and leukocyte activation as measured by CD62P and CD11b exposure and/or interferes with the binding of adhesion molecules other than fibrinogen. The results show that a flow cytometric procedure can be effective in obtaining rapid information on platelet-leukocyte conjugate formation in whole blood and on factors that are involved in its regulation. It is suggested that the technique may be applicable to the study of platelet-leukocyte conjugate formation in whole blood in disease, and also to study the effects of drugs interfering with conjugate formation.

BACKGROUND

To investigate the changes of platelet microparticle (PMPs), monocyte-platelet aggregation (MPAs), and the platelet membrane glycoprotein GPIIb/IIIa ligands (PAC-1) and P-hormone (CD62P) activation ratio changes in acute coronary syndrome (ACS) patients.

METHODS

92 patients were divided into ACS group (54 cases) and coronary angiography negative group (38 cases). 30 cases of age/gender matched healthy control group were recruited. The flow cytometry analysis in each group of PMPs, the MPAs expression of CD62P, GPIIb/IIIa activation ratio, and ROC curve were performed to evaluate the sensitivity and specificity of each parameter.

RESULTS

The healthy control group showed MPAs 5.94 +/- 1.93%, PMPs 1.89 +/- 0.53%, and PAC-1 2.86 +/- 0.93%, the coronary angiography-negative group showed MPAs 11.97 +/- 4.92%, PMPs 3.08 +/- 1.38%, and PAC-1 3.38 +/- 0.92%, and the ACS group showed MPAs 46.27 +/- 17.74%, PMPs 5.28 +/- 2.44%, and PAC-1 5.34 +/- 2.44%. In the ACS group, the area under the ROC curve of each indicator for identifying suspected ACS patients were MPAs (0.952), PMPs (0.807), PAC-1 (0.770), and CD62p (0.656). MPAs showed the highest sensitivity (94.4%) and specificity (84.2%) for the diagnosis of ACS.

CONCLUSIONS

acute coronary syndrome, platelet microparticle, monocyte-platelet aggregation, CD62P, GPIIb/IIIa.

Tethering of PMNL by platelets via CD62P has been shown to cause PMNL activation. Co-incubation of purified PMNL with platelets that were activated with thrombin and then fixed and washed, resulted in the formation of platelet-PMNL conjugates as well as in a generation of reactive oxygen species that were measured as luminol-enhanced chemiluminescence. When platelets were thrombin activated in the presence of RGDS to prevent binding of fibrinogen to membrane receptors, they had a reduced capacity to adhere to PMNL, but ROS generation was enhanced. In samples of citrated whole blood RGDS as well as the more specific platelet fibrinogen receptor antagonist GR144053F or a dissociation of the platelet glycoprotein IIb/IIIa complex markedly enhanced ROS generation that was induced by stirring the samples for 10 min at 1000 rpm, by 175%, 95% and 138%, respectively. Removal of platelets from the whole blood samples also resulted in an enhancement of stirring-induced ROS generation, which was inversely correlated to the platelet count. These data provide some evidence that platelets are capable of inhibiting ROS generation in PMNL by a mechanism that involves platelet-bound fibrinogen and probably depends on fibrinogen-mediated platelet-PMNL contact.

BACKGROUND

The influence, extent, and duration of changes in platelet antigen expression caused by blood-biomaterial interaction in plateletpheresis were assessed.

METHODS

Twenty-two apheresis donors were studied by using two automated continuous-flow apheresis devices. Blood samples were taken before, during, and for 4 days after extracorporeal circulation. The platelet surface expression of glycoproteins CD41a, CD42b, CD62p, and CD63 was analyzed by flow cytometry.

RESULTS

Over the course of plateletpheresis, there was a significant increase in mean channel fluorescence intensity (MCFI) of CD62p, from 25.1 +/- 7.9 (mean +/- SD) to 50.4 +/- 28.9, and of CD63, from 22.3 +/- 6.5 to 33.3 +/- 13.2. There was a significant decrease in CD41a expression as measured by the MCFI, from 1129.8 +/- 125.0 to 1066.6 +/- 102.2, and in CD42b MCFI, from 329.6 +/- 49.4 to 321.4 +/- 52.0. The two apheresis devices showed different platelet activation kinetics, but the overall MCFI of CD62p and CD63 did not significantly diverge after 60 minutes of apheresis. CD62p and CD63 expression as measured by the MCFI returned to preapheresis levels during the follow-up period in 25 and 25 of 44 procedures, respectively, within 24 hours; in 10 and 13 of 44 procedures after 48 hours; in 7 and 3 of 44 procedures after 72 hours; and in 2 and 3 of 44 procedures on Day 5.

CONCLUSIONS

The varying kinetics of expression, as measured by the MCFI, of platelet antigens CD62p, CD63, CD41a, and CD42b during extracorporeal circulation may be useful for biocompatibility testing. Activated platelets continue to circulate in donors for several days after cytapheresis, which suggests that a sufficient interval between apheresis procedures is necessary to avoid the collection of activated platelets.

Novel analytical measures are needed to accurately monitor the properties of platelet concentrates (PCs). Since activated platelets produce platelet-derived extracellular vesicles (EVs), analyzing EVs of PCs may provide additional information about the condition of platelets. The prospect of using EVs as an auxiliary measure of platelet activation state was investigated by examining the effect of platelet additive solutions (PASs) on EV formation and platelet activation during PC storage. The time-dependent activation of platelets in PCs with PAS-B or with the further developed PAS-E was compared by measuring the exposure of CD62P by flow cytometry and the content of soluble glycoprotein V (sGPV) of PCs by an immunoassay. Changes in the concentration and size distribution of EVs were determined using nanoparticle tracking analysis. A time-dependent increase in platelet activation in PCs was demonstrated by increased CD62P ex-posure, sGPV content, and EV concentration. Using these strongly correlating parameters, PAS-B platelets were shown to be more activated compared to PAS-E platelets. Since the EV concentration correlated well with the established platelet activation markers CD62P and sGPV, it could potentially be used as a complementary parameter for platelet activation for PCs. More detailed characterization of the resulting EVs could help to understand how the PC components contribute the functional effects of transfused PCs.

Using a diluted whole blood method of flow cytometric analysis, we have shown that platelets could be activated in vitro in the presence of high concentrations (100 nmol/l) of recombinant factor (F) VIIa (rFVIIa; NovoSeven(R)) and 2.5 mmol/l calcium chloride. This was demonstrated by a significant increase in the mean percentage of platelets expressing CD62P and their mean fluorescent intensity (MFI) after 30 min versus platelets incubated with calcium or rFVIIa alone or diluted blood alone. The presence of rFVIIa and calcium increased the exposure of the PAC-1 activation epitope of glycoprotein (Gp) IIb/IIIa. This effect was equally influenced by the presence of calcium alone but not by rFVIIa. The effect of rFVIIa was time and concentration dependent. Thrombin generation was also necessary, as the effect of rFVIIa was completely abrogated by the additional presence of hirudin. Furthermore, soy bean trypsin inhibitor (SBTI) but not corn trypsin inhibitor (CTI) abrogated CD62P exposure, suggesting that thrombin was derived via FX but not FXII activation. Exposure of CD62P demonstrated a significant lag phase, sometimes of the order of>> 30 min, as well as large intersubject variation. Significant platelet activation was observed at a concentration as low as 25 nmol/l rFVIIa. Platelet-leucocyte aggregation was also increased in the presence of 25 nmol/l rFVIIa and calcium. No significant difference was observed between levels of CD62P in diluted whole blood and platelet-rich plasma adjusted to an identical platelet count after their exposure to rFVIIa and calcium for 30 min.

Di-(2-ethylhexyl) phthalate (DEHP) has been used worldwide in various products for many years. In vitro studies have shown that exposure to DEHP and its metabolite mono(2-ethylhexyl) phthalate (MEHP) induces endothelial cell apoptosis. Moreover, exposure to DEHP had been linked to cardiovascular risk factors and cardiovascular diseases in epidemiological studies. Circulating microparticles have been known to be indicators of vascular injury. However, whether DEHP or its metabolites are independently associated with microparticles in humans remains unknown. From 2006 to 2008, we recruited 793 subjects (12-30years) from a population-based sample to participate in this cardiovascular disease prevention examination. Each participant was subjected to interviews and biological sample collection to determine the relationship between concentrations of DEHP metabolites MEHP, mono(ethyl-5-hydroxyhexyl) phthalate, and mono(2-ethly-5-oxoheyl) phthalate in urine and concentrations of endothelial microparticles (CD62E and CD31+/CD42a-), platelet microparticles (CD62P and CD31+/CD42a+), and CD14 in serum. Multiple linear regression analysis revealed that an ln-unit increase in MEHP concentration in urine was positively associated with an increase in serum microparticle counts/μL of 0.132 (±0.016) in CD31+/CD42a- (endothelial apoptosis marker), 0.117 (±0.023) in CD31+/CD42a+ (platelet apoptosis marker), and 0.026 (±0.007) in CD14 (monocyte, macrophage, and neutrophil activation marker). There was no association between DEHP metabolite concentration and CD62E or CD62P. In conclusion, a higher MEHP concentration in urine was associated with an increase in endothelial and platelet microparticles in this cohort of adolescents and young adults. Further studies are warranted to clarify the causal relationship between exposure to DEHP and atherosclerosis.

X-linked adrenoleukodystrophy (X-ALD) is an inherited disorder of peroxisomal beta-oxidation, which results in accumulation of very long-chain fatty acids, causing damage to the nervous system, adrenal cortex and testis. The two most frequent phenotypes are childhood cerebral adrenoleukodystrophy (CCALD) and adrenomyeloneuropathy (AMN). Some affected males demonstrate no clinical signs (asymptomatic ALD), whereas female carriers can also be affected. Patients with X-ALD have been treated with Lorenzo's oil, a 4:1 combination of oleic acid and erucic acid, with thrombocytopenia as the main side effect and sometimes leading to a hemorrhagic diathesis. We studied platelet count, size and membrane surface exposure of platelet activation antigens in 17 adult X-ALD patients. Eight patients used the prescribed amount of erucic acid (as glyceroltrierucate) or more (very compliant), five used less(compliant), and four did not use the diet. All eight very compliant patients had highly enlarged platelets and seven manifested thrombocytopenia. An enhanced in vivo platelet activation status was established by increased platelet surface expression of P-selectin (CD62P, PADGEM, GMP-140) in five of the seven thrombocytopenic patients, and of increased fibrinogen receptor exposure (measured with the antibody PAC-1) in three of these five patients. The other nine compliant or untreated patients had normal platelet counts and, generally, normal P-selection and fibrinogen receptor expression. A diet-induced 7- to 27-fold enrichment of erucic acid was observed in the platelets of the four patients studied. We conclude that the thrombocytopenia in AMN patients using Lorenzo'soil is associated with circulating platelets that have an increased erucic acid content, size and activation status. We hypothesize that the erucic acid in some way induces the increased size and thus, directly or indirectly, increased platelet activation or instability in vivo. This then causes the thrombocytopenia, with circulating platelets representing a population that has not yet been sufficiently changed to be removed, but has clear signs of activation.

BACKGROUND

Risk factors for thrombosis (TB) in thrombocythaemia (TC) associated with myeloproliferative disorder (MPD) are not well defined.

METHODS

We measured antiphospholipid antibodies (APLA) in 35 patients with TC associated with MPD. Fourteen had TB and 21 did not. We assayed IgG and IgM APLA by ELISA for 6 antigens: beta2GP1, cardiolipin (CL), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE) and FVII/VIIa, together with markers of activation of platelets (CD62P) and endothelium [endothelial microparticles (EMP)].

RESULTS

At least one positive APLA was detected in 66% of TC patients overall. The incidence was significantly higher in the TB subgroup (92.8%) than non-TB (47.6%, p < 0.05). Multiple APLA (positive for more than one antigen) were also more frequent in TB, for both IgG and IgM, for all 6 antigens tested (p < 0.05). However, IgM APLA predominated, being about 2-fold more frequently positive than IgG for all 6 antigens. Platelet CD62P was significantly higher in the TB group (p < 0.05). EMP did not differ between TB and non-TB. The most frequent thrombotic complication was recurring ischemic cerebral vascular accidents (ICVA), leading to progressive cognitive impairment. Venous TB often developed at unusual sites. Recurring and reversible TB were common features in TC.

CONCLUSIONS

This study suggests that APLA and platelet activation are risk factors for TB in TC. APLA are prevalent in TC, and IgM APLA predominated over IgG. Activation of platelets but not of endothelium may be consistent with the reversible and recurrent features of TB in TC.

While it has been proved that centrifugal conditions for pure platelet-rich plasma (P-PRP) preparation influence the cellular composition of P-PRP obtained, the optimal centrifugal conditions to prepare P-PRP have not yet been identified. In the present study, platelet-containing plasma (PCP) was prepared with the first-spin of different double-spin methods and P-PRP was prepared with different double-spin methods. Whole-blood analysis was performed to evaluate the cellular composition of PCP and P-PRP. The basal and ADP-induced CD62P expression rates of platelets were assessed by flow cytometry to evaluate the function of platelets in PCP and P-PRP. Enzyme-linked immune sorbent assay was performed to quantify interleukin-1β, tumor necrosis factor-α, platelet-derived growth factor AB and transforming growth factor β1 concentrations of PCP and P-PRP. Correlations between the cellular characteristics and cytokine concentrations of P-PRP were analyzed by Pearson correlation analysis. Effects of P-PRP on the proliferation, survival and migration of human bone marrow-derived mesenchymal stem cells and human articular chondrocytes were evaluated by a Cell Counting Kit-8 assay, live/dead staining and Transwell assay, respectively. The results showed that centrifugation at 160 × g for 10 min and 250 × g for 15 min successively captured and concentrated platelets and growth factors significantly more efficiently with preservation of platelet function compared with other conditions (P<0.05). The correlation analysis showed that the similar leukocyte concentrations and leukocyte-reducing efficiencies resulted in similar pro-inflammatory cytokine concentrations in P-PRP (P>0.05) and the maximization of platelet concentration, platelet enrichment factor, platelet capture efficiency and platelet function resulted in the maximization of growth factor concentrations in P-PRP obtained using the optimal conditions (P<0.05). Compared with P-PRP obtained under other conditions, P-PRP obtained under the optimal conditions significantly promoted the proliferation and migration of cells (P<0.05) and did not alter cell survival (P>0.05). Therefore, centrifugation at 160 × g for 10 min and 250 × g for 15 min successively with removal of the buffy coat as a crucial step may provide an optimal preparation system of P-PRP for clinical application.

Bone marrow transplantation (BMT) is the only curable option for thalassemia major, β-thalassemia/HbE. However, some patients still have the risk of hypercoagulable complications. We used a whole blood flow cytometric analysis to measure the circulating microparticle (MP) levels, activated platelets, and leukocyte-platelet aggregates in 59 young β-thalassemia/HbE patients compared with 20- and 28-matched healthy and patients receiving regular blood transfusion (RT), respectively. Results from the studies showed that blood samples from BMT group contained a significantly higher numbers of circulating MPs originated from platelets (ann-V+CD41a+), leukocyte (ann-V+CD45+) and endothelial cells (ann-V+CD146+) when compared to samples from healthy subjects and RT patients. In contrast, the percentages of activated/procoagulant platelets (CD62P and CD142 expressing platelets) were decreased in BMT group. In addition, monocytes forming microaggregates were the major population among other leukocyte-platelet complexes. Different patterns of CD11b, CD62P and CD142 expression on platelet-leukocyte microaggregate surface were also found. These data suggest that circulating MPs together with leukocyte-platelet aggregates may be responsible, in part, in pathogenesis of hypercoagulable state in β-thalassemia/HbE patients who undergone BMT.

Acute graft-versus-host disease (aGVHD) is a major complication associated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). Interleukin (IL)-35 is a novel anti-inflammatory cytokine that suppresses the immune response. This prospective study explored IL-35 plasma levels in 65 patients after HSCT. The results revealed that the peripheral blood of patients with grades III-IV aGVHD (23.46 ng/ml) had reduced IL-35 compared to transplanted patients with grades I-II aGVHD (40.26 ng/ml, p < 0.01) or patients without aGVHD (41.40 ng/ml, p < 0.05). Allografts, including granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cell (PBPC) and G-CSF-primed bone marrow (GBM), from 38 patients were analyzed for IL-35 levels with respect to aGVHD. The patients who received lower levels of IL-35 cells in the GBM (28.0 ng/ml, p = 0.551) or lower levels of IL-35 in PBPC (53.46 ng/ml, p = 0.03) exhibited a higher incidence of aGVHD. Patients with aGVHD have increased platelet aggregation. IL-35 was added to patient blood in vitro, and platelet aggregation was inhibited by IL-35 in a dose-dependent manner. The markers of platelet activation (CD62P/PAC-1) can also be inhibited by IL-35. The results indicate that IL-35 may affect the development of aGVHD by inhibiting platelet activation and aggregation. Our data suggests that IL-35 represents a potentially effective therapeutic agent against aGVHD after allo-HSCT.

BACKGROUND

Cell-surface adhesion molecules regulate multiple intercellular and intracellular processes and play important roles in inflammation by facilitating leukocyte endothelial transmigration. Whether cardiomyocytes express surface-adhesion molecules related to inflammation and the effect of pro-inflammatory mediators remain unknown.

METHODS

In the present study, the expression of different cell-adhesion molecules (CD11a, CD11b, CD31, CD62P, CD162, F11 receptor and mucosal vascular addressin cell adhesion molecule 1 (MADCAM1)) and the effect of pro-inflammatory mediators were investigated in an in vitro model of human cardiomyocytes. Cells were supplied as a primary culture of cardiac alpha actin-positive cells from human heart tissue. The cells were incubated for 24 h with 1 U/ml thrombin or 700 ng/ml lipopolysaccharide (LPS) or with a combination of both. The expression of the cell adhesion molecules was measured by flow cytometry.

RESULTS

In cultured human cardiomyocytes, 22.8% of cells expressed CD31, 7.1% MADCAM1 and 2.6% F11R. CD11a, CD11b, CD62P and CD162 were expressed by fewer than 2% of the cells at baseline. CD31 expression increased on incubation of cardiomyocytes with thrombin by 26% (p<0.05) and with LPS by 26% (p=0.06). The combination of thrombin and LPS did not result in increased levels of CD31 (p>0.10). The pro-inflammatory agents LPS and thrombin had no effect on the expression of MADCAM1 and F11R.

CONCLUSIONS

Inflammation-related cell-adhesion molecules CD31, MADCAM1 and F11R were shown to be expressed on the surface of human cardiomyocytes in an in vitro model. Incubation with LPS or thrombin resulted in increased expression of CD31, however, it did not modify the expression of the cell adhesion molecules MADCAM1 and F11R.

The development of deep venous thrombosis (DVT) is a sterile inflammatory process related to cytokines, such as interleukin (IL)-6 or IL-17. IL-9 is a cytokine involved in many inflammatory diseases, including cystic fibrosis, ulcerative colitis, psoriasis and psoriatic arthritis. However, it remains unknown whether IL-9 is related to DVT. In this study, we characterized the role and mechanism of IL-9 in DVT. Analysis of the data of patients with and without DVT revealed that stasis, venous surgery as well as elevated IL-9 and sP-selectin levels were related to the development of DVT. We also showed for the first time that IL-9 receptor was expressed in mouse platelets, and it dramatically promoted the aggregation rate and expression of P-selectin (CD62P) in the presence of adenosine diphosphate, but otherwise exhibited no effect on platelets. This study also revealed that Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signalling pathway, not phosphoinositide 3-kinase/AKT pathway, was involved in the process. We also showed in a mouse model of stasis that the thrombus size (weight and length) and CD62P expression in the thrombus were higher and lower in the IL-9 group and IL-9 antibody group, respectively, than in the control group. All these findings indicated that IL-9 facilitated platelet function through the JAK2/STAT3 pathway, thus promoting the development of DVT.

BACKGROUND

There is an increased risk of thromboembolic complications in inflammatory bowel disease. Activated platelets play a crucial role in the pathogenesis of this disease.

OBJECTIVE

To evaluate platelet activation in inflammatory bowel disease.

METHODS

This study comprised 20 healthy control subjects and a total of 20 patients. Out of them, 4 patients and 16 patients had suffered from Crohn's disease and ulcerative colitis, respectively. Nine patients were in active phase and 11 were in inactive phase of the disease. To evaluate platelet activation, we used the monoclonal antibodies of mouse anti-human CD42a-Fluorescein isothiocyanate (FITC), CD42b-FITC and mouse anti-human CD62P-phycoerythrin. We assessed the activation of platelets in peripheral blood using flow cytometric analysis.

RESULTS

The platelet activation was found to be statistically significantly higher in the active-phase patient group when compared with the control subjects group. On the other hand, it was insignificant in the inactive patient group.

CONCLUSIONS

The results of our study might suggest that the elevation of CD62P expression in patients with inflammatory bowel disease could be used as a criterion of disease activation. Furthermore, agents with properties to diminish the platelet activation could prevent the development of thromboembolic complications in a patient with inflammatory bowel disease (Fig. 1, Ref. 15).

OBJECTIVE

Initial elevated and prolonged high carbon dioxide levels related to mitochondrial dysfunction are recently being suggested as a contributing factor to rapid pH decline in platelet (PLT) units. The use of different storage environments may influence this phenomenon. This study has two objectives (i) to investigate the relationship of mitochondrial function and apoptotic events with different storage environments capability of pH control and (ii) to examine the cause and relationship between pH decline in PLT units, carbon dioxide levels and mitochondrial function.

METHODS

Platelet units were prepared for storage in (A) 70% SSP+, 300-400 × 10(9) /unit, (B) 70% SSP+, 550-600 × 10(9) /unit, (C) 100% plasma, 550-600 × 10(9) /unit, and (D) additional 100% plasma, >600 × 10(9) /unit. In vitro variables including mitochondrial function (JC-1), reactive oxygen species (ROS) and caspase 3 activity were analysed on days 2, 5 and 7.

RESULTS

Glucose/lactate was higher, pH, ATP, Hypotonic shock response (HSR) and extent of shape change (ESC) decreased (P < 0·001 on day 7), CD62P (P < 0·001 on day 7) increased, the JC-1-positive PLTs were lower (P < 0·001 on day 7), and ROS was higher (P < 0·001 days 2-7) in the plasma (C) units as compared with the SSP+ (A) and (B) units. All plasma (D) units showed rapid pH and pCO(2) decline from day 2 but by means of >80% maintenance of mitochondrial function until day 7.

CONCLUSIONS

The use of SSP+ instead of plasma may reduce the risk of triggering pro-apoptotic events in high-yield PLT units. A rapid decline in pH in PLT units cannot be explained with initial elevated and prolonged high carbon dioxide levels and mitochondrial dysfunction.

OBJECTIVE

Several in vitro laboratory tests to assess the quality control of platelet concentrates (PC) are available. Some of them have a good correlation with the platelet recovery index. To assess the quality control of standard PC prepared in our blood bank, we measured the blood gas and the degree of platelet activation.

METHODS

SPC were prepared by the PRP method. Fifty-five SPC (45 SPC at day one of storage and 20 SPC at day five of storage) were analysed. Blood gas (pH, PO(2), PCO(2) and bicarbonate concentration) in the SPC were measured by blood gas automate. Platelet activation profile were determined by measuring the percentage of platelet expressing the CD62p (% CD62) and the percentage of platelet-leukocyte aggregate (% PLA).

RESULTS

The pH values of all studied SPC were comprised between 7.0 and 7.6. SPC at day 1 of storage have a significantly higher pH than those at day 5 of storage (7.5+/-0.05 versus 7.3+/-0.14; p<0.001). The % CD62p were higher in SPC at day five compared to the SCP at day one without reaching a statistical significance (28.4+/-15% versus 24.3+/-9.7%, p=0.052). The percentage of PLA were higher in SPC at day one compared to SCP at day five although this difference is not statistically significant (22.2+/-7.5% versus 17.9+/-8.0%; p=0.23).

CONCLUSIONS

Preparation and storage procedure adopted in our centre did not significantly affect the quality SPC. Our study is the first to assess the PLA in PC. Studies assessing the PLA are warranted to appreciate the clinical impact of this parameter.

OBJECTIVE

To evaluate the influence of treatment with ultralow-dose aspirin (ULDAsp) on platelet aggregation, P-selectin (CD62P) expression, and formation of platelet-leukocyte aggregates in clinically normal dogs.

METHODS

18 clinically normal dogs.

METHODS

Studies were conducted before and 24 hours after ULDAsp administration (0.5 mg/kg, PO, q 24 h, for 2 days). Whole blood impedance aggregometry for the assessment of platelet function was performed with sodium citrate-anticoagulated blood and aggregation agonists (ADP at 20, 10, and 5 μmol/L; collagen at 10, 5, and 2 μg/mL). Onset, maximum response, and rate of platelet aggregation were recorded. Flow cytometric assays were configured to detect thrombin-induced CD62P expression and platelet-leukocyte aggregates in EDTA-anticoagulated whole blood. Externalized platelet CD62P and constitutive CD61 (GPIIIa) were labeled with antibodies conjugated to phycoerythrin (PE) and fluorescein isothiocyanate (FITC), respectively. Red blood cell-lysed paraformaldehyde-fixed EDTA-anticoagulated whole blood was dual labeled with CD61-FITC and a panleukocyte antibody (CD18-PE) to characterize platelet-leukocyte aggregates.

RESULTS

ULDAsp significantly delayed platelet aggregation onset with ADP at 20 μmol/L by 54% to 104%, attenuated maximum aggregation with various concentrations of ADP and collagen by ≥ 41%, and slowed aggregation rate with the highest ADP and collagen concentrations by ≥ 39%. Depending on the parameter tested, up to 30% of dogs failed to have an ULDAsp effect. Thrombin stimulation significantly increased CD62P expression in platelets and platelet-leukocyte aggregates, but ULDAsp did not alter basal or thrombin-stimulated CD62P expression.

CONCLUSIONS

ULDAsp treatment of clinically normal dogs impaired platelet aggregation in most dogs, but did not influence CD62P platelet membrane expression.