BACKGROUND

Patients with bone marrow failure and severe thrombocytopenia are frequently given prophylactic platelet transfusion before interventions. The clinical effects of such transfusions, however, are poorly defined. We performed a prospective observational study on patients with bone marrow failure scheduled for prophylactic platelet transfusion before the insertion of a central venous catheter. The objectives were to evaluate the effect and duration of prophylactic platelet transfusions on central venous catheter insertion in thrombocytopenic patients with bone marrow failure.

METHODS

Thirty-nine adult patients with bone marrow failure and platelet counts below 50 × 10/L were consecutively enrolled before prophylactic platelet transfusion for subclavian central venous catheter insertion. Blood samples were drawn from the patients before platelet transfusion, 1 hour, and 4 hours after completion of the transfusion. The coagulation profile was assessed by conventional hematological tests, thromboelastometry (ROTEM) assays (EXTEM and FIBTEM), multiple electrode aggregometry (Multiplate) assays including adenosine diphosphate, collagen, and thrombin receptor agonist peptide, and by flow cytometry for the platelet expression of P-selectin (CD62P) and activated glycoprotein IIb-IIIa (PAC-1). Bleeding complications were classified with a 5-grade scale, according to the Common Terminology Criteria for Adverse Events.

RESULTS

Seventeen women and 22 men were included in the study. Platelet count was increased from 24 × 10/L (18-32) before to 42 × 10/L (31-50) 1 hour after transfusion (P < 0.0001) and was not significantly different 4 hours after transfusion (40 × 10/L (29-50), P = 0.047). Maximal clot firmness EXTEM was increased from 38 mm (32-45) before to 46 mm (41-52) 1 hour after transfusion (P < 0.0001) and did not change 4 hours after transfusion. Clotting time EXTEM was decreased from 58.5 seconds (50-78) beforehand to 53 seconds (45-61) 1 hour after transfusion (P = 0.0006) and was not significantly different 4 hours after transfusion (57 seconds (52-70, P = 0.025). FIBTEM results were all unchanged after transfusion. All Multiplate analyses were significantly increased after 1 hour and were not diminished 4 hours after transfusion. Four grade 1 bleeding episodes occurred, but no grade 2 to 5 bleeding could be detected. Flow cytometry analyses showed mixed results with no overall trend.

CONCLUSIONS

Prophylactic platelet transfusions in thrombocytopenic patients with bone marrow failure improve hemostatic parameters on ROTEM and Multiplate by increasing the number of platelets, and not through enhancement of platelet function. Improved clotting parameters on ROTEM and platelet aggregation on Multiplate appear to persist between 1 and 4 hours after transfusion.

OBJECTIVE

Platelet integrin alpha(IIb)beta3 plays a crucial role in platelet aggregation, and the affinity of alpha(IIb)beta3 for fibrinogen is dynamically regulated. Employing modified ligand-binding assays, we analyzed the mechanism by which alpha(IIb)beta3 maintains its high-affinity state.

RESULTS

Washed platelets adjusted to 50 x 10(3) microL(-1) were stimulated with 0.2 U mL(-1) thrombin or 5 microm U46619 under static conditions. After the completion of alpha(IIb)beta3 activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated alpha(IIb)beta3 was then detected by fluorescein isothiocyanate (FITC)-labeled PAC1. The addition of 1 mum AR-C69931MX (a P2Y12 antagonist) or 1 mm A3P5P (a P2Y1 antagonist) disrupted the sustained alpha(IIb)beta3 activation by approximately 92% and approximately 38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 microL(-1) also disrupted the sustained alpha(IIb)beta3 activation, and the disruption by such dilution was abrogated by the addition of exogenous adenosine 5'-diphosphate (ADP) in a dose-dependent fashion. The amounts of ADP released from activated platelets determined by high-performance liquid chromatography were compatible with the amounts of exogenous ADP required for the restoration. We next examined the effects of antagonists on protein kinase C (PKC) and Rap1B activation induced by 0.2 U mL(-1) thrombin. Thrombin induced long-lasting PKC and Rap1B activation. AR-C69931MX markedly inhibited Rap1B activation without inhibiting PKC activation.

CONCLUSIONS

Our data indicate that the continuous interaction between released ADP and P2Y12 is critical for the maintenance of alpha(IIb)beta3 activation.

The high incidence of cardiovascular disease in patients with moderate renal impairment is not fully explained by traditional atherothrombotic risk factors. Independently from these factors, blood platelet activation may increase the cardiovascular disease risk of patients with mild-to-moderate renal impairment. Blood platelet activation has not been studied in nondiabetic patients with mild-to-moderate renal impairment. Therefore, we measured the extent of platelet activation by means of fluorescence cytometry in 93 nondiabetic patients with MDRD-estimated creatinine clearance ranging from 13 - 63 ml/min/1.73 m2. As platelet activation parameters we used the expression of CD62P (P-selectin), CD 63 (glycoprotein 53), PAC-1 (activated fibrinogen receptor), CD42b (von Willebrand factor receptor) and CD41 (fibrinogen receptor) on the platelet surface membrane. The expression of CD62p, CD63 and PAC-1 was statistically significantly inversely related to the estimated glomerular filtration rate in these patients (standardized b -0.28, -0.32 and -0.39, respectively). We conclude that nondiabetic mild-to-moderate renal impairment is associated with blood platelet activation. Whether this contributes to the increased cardiovascular risk in these patients needs further study.

BACKGROUND

Platelet count is essential for the diagnosis and management of hemostasis abnormalities. Although existing platelet count methods installed in common hematology analyzers can correctly count platelets in normal blood samples, they tend to miscount platelets in some abnormal samples. The newly developed PLT-F channel in the XN-Series hematology analyzer (Sysmex) has been reported to be a reliable platelet count system, even in abnormal samples. However, how the PLT-F platelet counting system achieves such accuracy has not been described in scientific articles.

METHODS

Isolated platelets, erythrocytes, and fragmented erythrocytes were examined using an automated hematology analyzer. The samples were labeled by combining PLT-F reagents and anti-CD62p, CD63, Grp75, Calreticulin, CD41, or CD61 antibody, and analyzed using confocal laser scanning microscopy or flow cytometry.

RESULTS

The PLT-F system correctly discriminated platelets in erythrocytes. Its reagents strongly stained some intraplatelet organelles labeled with anti-Grp75, but only faintly stained the plasma membrane of both platelets and erythrocytes. Microscopic observation and flow cytometric examination revealed that all of these strongly stained cells were also labeled with platelet-specific anti-CD41 and anti-CD61 antibodies.

CONCLUSIONS

This study revealed that the staining property of the PLT-F reagents, by which platelets and fragmented erythrocytes are clearly distinguished, contributes to the platelet-counting accuracy of the PLT-F system.

This study aimed to examine the mechanisms of cellular activation by small-size platelet microparticles (sPMP) and to present the performance of high-resolution flow cytometry for the analysis of subcellular entities from different origins. Plasma counts of sPMP were analysed in coronary artery disease patients (n = 40) and healthy controls (n = 40). The effect of sPMP and platelet debris (PD) in pathophysiologically relevant doses on platelet and monocyte activation parameters and thrombogenesis was investigated via flow cytometry and thromboelastometry. New generation flow cytometry identifies differences in size, levels and surface molecules of sPMP derived in the absence of stimulus, thrombin activation and platelet disruption. Addition of sPMP resulted in platelet degranulation and P-selectin redistribution to the membrane (P = 0·019) in a dose and time-dependent manner. Blood clotting time decreased after addition of sPMP (P = 0·005), but was not affected by PD. Blocking P-selectin (CD62P) in sPMP markedly reverted the effect on thrombus kinetics (P = 0·035). Exposure to sPMP stimulated monocyte expression of intercellular adhesion molecule-1 (P < 0·03) and decreased monocyte interleukin-6 receptor density (P < 0·01). These results implicate sPMP as a direct source of downstream platelet and monocyte activation. In pathological coronary artery disease conditions, higher levels of sPMP favour a prothrombotic state, partly through P-selectin expression.

OBJECTIVE

To study the different therapy for different types of ulcerative colitis (UC) in China.

METHODS

Among 102 UC patients, 42 chronic relapse type UC patients were randomly divided into olsalazine sodium treatment group (n=21) and SASP group (n=21). Clinical effects and safety were observed in the 2 groups. Forty-two first episode type UC patients were randomly divided into Heartleaf houttuynia herb treatment group (n=21) and SASP group (n=21). Clinical effects were observed in the 2 groups while ultrastructure of colonic mucosa, ICAM-1 and the pressure of distant colon were studied in Heartleaf houttuynia herb group. Eighteen patients (8 males, 10 females) with refractory UC and unresponsive to high-dose prednisolone and sulfasalazine therapy more than one month were treated with Kangshuanling (7200 U/d). Prednisolone was gradually stopped and sulfasalazine was maintained. Stool frequency, rectal bleeding, colonoscopy, general well-being, histology were observed and CD62p, CD63, CD54, Pgp-170 (flow cytometry), TXA2 (RIA), blood platelet aggregation rate and thrombosis length in vitro were assessed.

RESULTS

In the 42 chronic relapse type UC patients, the overall clinical effects of olsalazine sodium group (complete remission in 16, improvement in 4, inefficiency in 1) were better than those of SASP group (complete remission in 10, improvement in 4, inefficiency in 7, P<0.05). Symptomatic remission of olsalazine sodium group (complete remission in 15, partial remission in 5, inefficiency in 1) was better than that of SASP group (complete remission in 10, partial remission in 5, inefficiency in 6, P<0.05). The colonoscopic remission of olsalazine sodium group(complete remission in 11, partial remission in 9, inefficiency in 1) was better than that of SASP group (complete remission in 7, partial remission in 8, inefficiency in 6, P<0.05). The histologic remission of olsalazine sodium group (complete remission in 13, partial remission in 7, inefficiency in in 1) was better than that of SASP group (complete remission in 6, partial remission in 10, inefficiency in 5, P<0.05). The side effects of gastrointestinal tract in olsalazine sodium group were less than those of SASP group except for frequency of watery diarrhea. No other side effects were observed in olsalazine sodium group while ALT increase, WBC decrease and skin eruption were observed in SASP group. Two patients relapsed in olsalazine sodium group while 8 cases relapsed in SASP group during the flow-up period (from six months to one year). In the 42 first episode type UC patients, the clinical effect of Heartleaf houttuynia herb group (complete remission in 20, 95.2%; improvement in 1, 4.8%) was better than that of SASP group (complete remission in 15, 72.4%, improvement in 5, 23.8%; inefficiency in 1, 3.8%, P<0.01). The time of stool frequency recovering to normal (5.6+/-3.3 d), and blood stool disappearance (6.7+/-3.8 d) and abdominal pain disappearance (6.1+/-3.5 d) in Heartleaf houttuynia herb group was all shorter than that in SASP group (9.5+/-4.9 d, 11.7+/-6.1 d, 10.6+/-5.3 d, P<0.01). Heartleaf houttuynia herb could inhibit the epithelial cell apoptosis of colonic mucous membrane and the expression of ICAM-1 (45.8+/-5.7% vs 30.7+/-4.1%, P<0.05). Compared with normal persons, the mean promotive speed of contraction wave stepped up (4.6+/-1.6 cm/min vs 3.2+/-1.8 cm/min, P<0.05) and the mean amplitude of the wave decreased (14.2+/-9.3 kPa vs 18.4+/-8.0 kPa, P<0.05) in active UC patients. After treatment with Heartleaf houttuynia herb, these 2 indexes improved significantly (17.3+/-8.3 kPa, 3.7+/-1.7 cm/min, P<0.05). In normal persons, the postprandial pressure of sigmoid (2.9 +/-0.9 kPa) was higher than that of descending colon (2.0+/-0.7 kPa) and splenic flexure (1.7+/-0.6 kPa), while the colonic pressure (1.5+/-0.5 kPa, 1.4+/-0.6 kPa, 1.3+/-0.6 kPa) decreased significantly (P<0.05) in active UC patients. After treatment with Heartleaf houttuynia herb, the colonic pressure (2.6+/-0.8 kPa, 1.8+/-0.6 kPa, 1.6+/-0.5 kPa) recovered to normal. The pain threshold Heartleaf houttuynia herb, the colonic pressure (2.6+/-0.8 kPa, 1.8+/-0.6 kPa, 1.6+/-0.5 kPa) recovered to normal. The pain threshold of distant colon (67.3+/-18.9 mL) in active UC patients decreased significantly compared with that of normal persons (216.2+/-40.8 mL, P<0.05) and recovered to normal after treatment with Heartleaf houttuynia herb(187.4+/-27.2 mL, P<0.05). In the 18 refractory UC patients with platelet activation, after more than 4 wk of combined Kangshuanling and sulfasalazine therapy, 16 patients achieved clinical remission, with a highly significant statistical difference (P<0.01) between pre-and post-treatment mean scores for all disease parameters: stool frequency (8.2/d vs 1.6/d), rectal bleeding (score 2.7 vs 0.3), colonoscopy (score 2.6 vs 1.1), histology (score 12.0 vs 5.0), general well being (score 4.0 vs 0.6) and CD62p (8.0+/-3.1% vs 4.1+/-1.8%), CD63 (6.3+/-2.1% vs 3.2+/-1.6%), TXA2 (548+/-85 ng/L vs 390+/-67 ng/L), platelet aggregation rate (43.2+/-10.7% vs 34.8+/-8.1%), thrombosis length in vitro (2.3+/-0.6 cm vs 1.8+/-0.3 cm), CD54 in blood (26.9+/-6.9% vs 14.4+/-5.1%), CD54 in tissues (51.1+/-6.2% vs 23.1+/-4.1%), Pgp-170 in blood (18.9+/-3.9% vs 10.4+/-2.7%), Pgp-170 in tissues (16.5+/-3.2% vs 10.2+/-2.3%, P<0.01 or 0.05).

CONCLUSIONS

Based on the characteristics of UC cases in China, different therapy should be given to different types of UC with expected satisfactory results.

BACKGROUND

The sum of undesirable side effects, occurring during haemodialysis (HD), is called bio-incompatibility. Concerning platelets, both an increase in the expression of the cell surface marker P-selectin (CD62p) and release of the intracellular granule product platelet factor 4 (PF4) have been described. However, as PF4 is also abundantly present on endothelium-bound proteoglycans, it is questionable whether the HD-induced increase is exclusively attributable to release from platelets. With respect to the cause of HD-induced bio-incompatibility, interest has been focused mainly on the extracorporeal circuit (ECC), especially the dialyser, whereas only little attention has been paid to other parts of the ECC and the mode of anticoagulation applied. To address the cause and origin of platelet activation and PF4 release during clinical HD, two complementary clinical studies were performed.

METHODS

In study I, the relative influence of the various parts of the ECC was evaluated by measuring the expression of CD62p, platelet aggregation and levels of PF4 and serotonin at various sampling points. In study II, low-molecular-weight heparin (LMWH) was administered 10 min before the actual start of HD, in order to separate the effects from LMWH and the ECC on platelet activation.

RESULTS

In study I, CD62p expression increased across the entire length of the ECC, including the roller pump and dialyser (median at t(5) from 26% to 43%, P = 0.008; median at t(30) from 28% to 48%, P = 0.007). Increments in PF4 and aggregation of platelets were relatively modest. Platelet serotonin content, which was below reference values in healthy controls, and plasma serotonin concentration, which was above reference values, did not change. In study II, PF4 levels increased markedly after the injection of LMWH (from 12 IU/ml at t(-10) to 75 IU/ml at t(0), P = 0.018), whereas CD62p expression remained stable until the start of HD.

CONCLUSIONS

Platelet activation, as measured by the up-regulation of CD62p, is an early process, occurring not only within the dialyser, but across the entire length of the ECC. As CD62p remained unaltered after the administration of LMWH 10 min before the actual start of HD, this kind of activation is independent of LMWH. Considering PF4 however, a sharp increment was observed after the administration of LMWH and before the start of HD. This finding suggests that the PF4 release observed early in clinical HD is largely independent from the ECC, and is probably the result of LMWH-induced detachment from the endothelium. As the platelet serotonin content was relatively reduced and the plasma serotonin levels were elevated, platelets from chronic HD patients might be depleted due to chronic repetitive activation. Based on these data, it appears first, that PF4 is an inferior marker of platelet activation in clinical HD and second, that LMWH is a major contributor to HD-induced bio-incompatibility.

OBJECTIVE

Leukocyte recruitment to the site of inflammation is a key event in a variety of cardiovascular pathologies. Infiltrating neutrophils constitute the first line of defense that precedes a second wave of emigrating monocytes reinforcing the inflammatory reaction. The mechanisms initiating this sequential process remained largely obscure.

RESULTS

Using advanced in vivo microscopy and in vitro/ex vivo techniques, we identified individual spatiotemporal expression patterns of selectins and their principal interaction partners on neutrophils, resident/inflammatory monocytes, and endothelial cells. Coordinating the intraluminal trafficking of neutrophils and inflammatory monocytes to common sites of extravasation, selectins assign different sites to these immune cells for their initial interactions with the microvascular endothelium. Whereas constitutively expressed leukocyte L-selectin/CD62L and endothelial P-selectin/CD62P together with CD44 and P-selectin glycoprotein ligand-1/CD162 initiate the emigration of neutrophils, de novo synthesis of endothelial E-selectin/CD62E launches the delayed secondary recruitment of inflammatory monocytes. In this context, P-selectin/CD62P and L-selectin/CD62L together with P-selectin glycoprotein ligand-1/CD162 and CD44 were found to regulate the flux of rolling neutrophils and inflammatory monocytes, whereas E-selectin/CD62E selectively adjusts the rolling velocity of inflammatory monocytes. Moreover, selectins and their interaction partners P-selectin glycoprotein ligand-1/CD162 and CD44 differentially control the intraluminal crawling behavior of neutrophils and inflammatory monocytes collectively enabling the sequential extravasation of these immune cells to inflamed tissue.

CONCLUSIONS

Our findings provide novel insights into the mechanisms initiating the sequential infiltration of the perivascular tissue by neutrophils and monocytes in the acute inflammatory response and might thereby contribute to the development of targeted therapeutic strategies for prevention and treatment of cardiovascular diseases.

The plasma concentration of soluble P-selectin (GMP-140/CD62P/PADGEM), a selectin produced by activated platelets and endothelial cells, was quantitated in a group of adults and East African negro children presenting with either non-severe or severe (cerebral) malaria caused by Plasmodium falciparum. Sixty percent of adults with non-severe malaria had immunoreactive levels of P-selectin above 200 ng/ml (the maximum recorded for any normal healthy adult in the assay) and 86% of all African children with malaria had concentrations above normal irrespective of their clinical categorization, and most exceeded the maximum limits of the assay >> 640 ng/ml). There was no correlation between P-selectin levels and parasitemia. These results raise the possibility that elevated soluble P-selectin in malaria may have an important beneficial anti-inflammatory function.

BACKGROUND

Current studies on CD62P have focused mainly on cardiovascular diseases, while only few studies have evaluated the effects of CD62P on the development of sepsis and the association between endothelial cell injury with inflammation and coagulation. This study attended to explore the association between endothelial cell injury with inflammation and coagulation by evaluating the expression of soluble CD62P (s-CD62P) in plasma and its mechanism in patients with sepsis, thus to provide the evidence of effective treatment of sepsis with anti-adhesion therapy targeted CD62P.

METHODS

A total of 70 critically ill patients with systemic inflammatory response syndrome (SIRS) admitted to intensive care unit (ICU) between September 2009 and February 2010 were enrolled for a prospective and control study. According to the diagnostic criteria of sepsis/SIRS, the patients were divided into two groups: a sepsis group (n=38) and a SIRS group (n=32). Another 20 healthy volunteers served as a control group. Patients in the sepsis group and SIRS group were matched by clinical signs of high blood pressure, diabetes and its complications. The demographics of the patients including age, sex, body mass index (BMI), smoking and alcohol addict were compared among the groups. Six mL peripheral blood samples were collected within 24-hour admission in ICU for enzymelinked immunosorbent assay (ELISA) to detect the plasma levels of s-CD62P, TNF-α, and hs-CRP. And variables of coagulation function such as platelet (PLT), prothrombin (PT), activated partial thromboplastin time (APTT), D-dimer and antithrombin-III (AT-III) were analyzed during 24 hours after admission to ICU. Meanwhile sequential organ failure assessment (SOFA) score of critically ill patients was evaluated. Data were expressed as mean±standard deviation and were statistically analyzed by using SPSS 17.0 statistical software. The differences in plasma levels of s-CD62P of patients in each group were analyzed by ANOVA and the Kruskal-Wallis test. The relations between s-CD62P and inflammatory cytokines as well as with coagulation were determined by Pearson's product moment correlation coefficient analysis. Changes were considered as statistically significant if P value was less than 0.05.

RESULTS

Compared with the control group and SIRS group, the sepsis group demonstrated significantly higher levels of s-CD62P, TNF-α and highly sensitive C-reactive protein (hs-CRP) (P<0.05). The plasma levels of D-dimer, PT, and APTT in the sepsis and SIRS groups were significantly higher than those in the control group, while the platelet count and the activity of AT-III were obviously lower (P<0.05). In the sepsis group, the plasma levels of hs-CRP and TNF-α were positively correlated with PT, APTT, and D-dimer, and negatively correlated with AT-III and PLT (P<0.05). The plasma levels of s-CD62P were significantly correlated with the plasma levels of TNF-α, hs-CRP, D-dimer, PT, and APTT, whereas they were correlated negatively well with PLT and AT-III (P<0.05).

CONCLUSIONS

The concentration of plasma s-CD62P is elevated as a early biomarker in patients with sepsis, and it serves as one of the pathogenic factors responsible for endothelial cell damage. Coagulation and mediators of inflammation promote each other, aggravating the severity of sepsis. Plasma s-CD62P may be an important factor for the development of coagulation and inflammatory reaction.

Protease-mediated degradation of proteins is critical in a plethora of physiological processes. Neutrophils secrete serine proteases including cathepsin G (CatG), neutrophile elastase (NE), and proteinase 3 (PR3) together with lactoferrin (LF) as a first cellular immune response against pathogens. Here, we demonstrate that LF increases the catalytic activity of CatG at physiological concentration, with its highest enhancing capacity under acidic (pH 5.0) conditions, and broadens the substrate selectivity of CatG. On a functional level, the enzymatic activity of CatG was increased in the presence of LF in granulocyte-derived supernatant. Furthermore, LF enhanced CatG-induced activation of platelets as determined by cell surface expression of CD62P. Consequently, LF-mediated enhancement of CatG activity might promote innate immunity during acute inflammation.

OBJECTIVE

This study was designed to investigate the effect of Panax notoginseng saponin (PNS) on platelet adhesion to injured endothelial cells (ECs) and platelet activation induced by injured ECs, and to explore its underlying mechanisms.

METHODS

Human umbilical vein endothelial cells (HUVECs) pretreated with aspirin (ASA,15μg/mL) or PNS (160μg/mL), or neither, were exposed to oxidized low-density lipoprotein (ox-LDL,80mg/L) for 16h. Platelets were then added and co-cultured with HUVECs for 5min. Platelet adhesion to ECs, platelet CD62p expression, and HUVEC apoptosis were assessed by fluorescence activated cell sorting (FACS)·Supernatant concentration of 6-keto-PGF1α and thromboxane 2 (TXB2) were measured by radioimmunoassay. Cyclooxygenase-1 (COX-1) and COX-2 protein expression were measured by western blotting.

RESULTS

The inhibitory effect of PNS on platelet activation was similar to ASA, but the inhibitory effect of PNS on platelet adhesion to ECs was superior to ASA. PNS modulated COX-2 expression, and increased 6-keto-PGF1α concentration in HUVECs, while down-regulated COX-1 expression and decreased supernatant TXB2 concentration in platelets. Co-culturing of injured HUVECs with platelets increased HUVEC apoptosis induced by ox-LDL compared with HUVECs cultured without platelets; ASA increased HUVEC apoptosis induced by ox-LDL when cultured without platelets, while decreased the apoptosis when co-cultured with platelets.

CONCLUSIONS

EC protection by ASA is closely associated with its inhibitory effect on platelet activation. PNS is superior to ASA in protecting ECs and in inhibiting platelet adhesion to injured ECs, and the regulation of COX pathway in both ECs and platelets might be the underlying mechanisms of PNS.

BACKGROUND

Platelet activation plays a key role in the pathogenesis of ischemic cerebrovascular diseases. Thus, it is very important to identify novel pharmacological targets for platelet inhibition to improve ischemic stroke treatment. The aim of the study was to assess the relationship between metabolic disorders and platelet activity markers in patients with acute ischemic stroke.

METHODS

Ninety-four patients with acute ischemic stroke were divided into four groups with: normolipidemia and normoglycemia (NL/NG), n = 25; normolipidemia and hyperglycemia (NL/HG), n = 21; hyperlipidemia and normoglycemia (HL/NG), n = 27; hyperlipidemia and hyperglycemia (NL/NG), n = 21. Twenty-one healthy subjects served as controls. We assessed the CD62P expression on resting and thrombin-activated blood platelets using the flow cytometer and anti-CD61 and anti-CD62P monoclonal antibodies. CD61-positive microparticles were defined as platelet-derived microparticles. The level of sP-selectin in serum was measured by the ELISA method.

RESULTS

We observed a significant influence of hyperlipidemia and hyperglycemia on sP-selectin concentration. A strong correlation between higher sP-selectin concentration and enhanced LDL (p = 0.001), total cholesterol (p = 0.02), HbA1c level (p < 0.001) was noticed. The level of sP-selectin and PDMPs (p < 0.001) were significantly higher in all groups of stroke patients compared with the controls. CD62P expression on resting and thrombin activated platelets were significantly lower in groups of patients with stroke.

CONCLUSIONS

Hyperlipidemia and hyperglycemia exert an equal stimulatory effect on tested platelet markers but with no synergistic action in stroke patients with both of the metabolic comorbidities. sP-selectin concentration in stroke patients best reflects the impact of hyperglycemia and hyperlipidemia on vascular lesions and platelet activation.

OBJECTIVE

Microvesicles (MVs) have been implicated in the pathomechanism of obstructive sleep apnoea (OSA); however, the results are inconsistent, possibly due to an unrevealed temporal variation in circulating MV levels. We aimed to investigate the diurnal changes of MV fractions in OSA.

METHODS

Peripheral blood was taken from 18 patients with OSA and 9 healthy subjects at different time points (11:00, 17:00, 21:00, 01:30 and 06:00). Samplings were repeated in nine OSA patients after 2 months of continuous positive airway pressure (CPAP) therapy. CD41+, CD62P+, glycophorin A+ and Annexin V+ MVs were determined with flow cytometry. Areas under the MV concentrations-time curves (AUC) were calculated and correlated with the severity of OSA.

RESULTS

A significant diurnal variability of plasma CD41+ and Annexin V+ MVs was observed only in OSA with a marked peak at 17:00. There was a direct correlation between CD41+ MV AUCs and the severity of OSA. CPAP treatment reduced diurnal variability in both CD41+ and Annexin V+ MV levels.

CONCLUSIONS

The relationship between the diurnal variability of CD41+ MVs and disease severity as well as the effect of CPAP treatment on MV levels support the role of MVs in the pathophysiology of OSA. More importantly, considering the significant diurnal variation in circulating MV levels, introduction of strict protocols for blood sampling is required for MV measurements.

Microparticles (MPs) are formed from platelets (PMPs), endothelial cells (EMPs) and monocytes (MMPs), and in acute myocardial infarction (MI), there is an increase of MPs in the culprit artery. In this study MPs were evaluated in whole blood in 105 patients with MI at five time-points during a two-year follow-up (FU). Patients with non-ST-elevated MI had higher concentrations of CD41+MPs compared to ST-elevated MI patients (p=0.024). The concentrations of PMPs in whole blood increased during the time period (p<0.001), but no significant change over time was found for EMPs and MMPs. CD62P+MP counts were higher in MI patients with diabetes (p=0.020), and patients with hypertension had increased levels of CD14+MPs (p=0.004). The amount of CD62P+TF+MPs increased significantly during FU (p<0.001). Patients with atherosclerosis in three arterial beds, i. e. coronary, carotid and peripheral arteries, had lower concentrations of CD62P+TF+MPs (p=0.035) and CD144+TF+MPs (p=0.004) compared to patients with atherosclerosis in one or two arterial beds. Higher concentrations of CD62P+MPs early after MI were associated with an increased risk of cardiovascular events during FU, hazard ratio 3.32 (95 %CI1.20-9.31). Only small variations in PMP, EMP and MMP concentrations were found during long-term FU after MI and their levels seem to reflect the underlying cardiovascular disease rather than the acute MI. PMPs expressing P-selectin might be a promising biomarker for predicting future cardiovascular events, but further studies are needed to confirm these results.

Thrombosis and thromboembolism are the life-threatening clinical complications for patients supported or treated with prosthetic cardiovascular devices. The high mechanical shear stress within these devices is believed to be the major contributing factor to cause platelet activation (PA) and function alteration, leading to thrombotic events. There have been limited quantitative data on how the high mechanical shear stress causes platelet activation. In this study, shear-induced PA in the ranges of well-defined shear stress and exposure time relevant to cardiovascular devices was quantitatively characterized for human blood using two novel flow-through Couette-type blood shearing devices. Four markers of platelet activation-surface P-selectin (CD62p), platelet-derived microparticles (PMPs), platelet-monocyte aggregation (PMA), and soluble P-selectin-were measured by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. The results indicated that PA induced by high shear stresses with short exposure time could be reliably detected with surface P-selectin, and, to a lesser extent, PMPs rather than soluble P-selectin. It was also verified that PMA can be a highly sensitive indirect marker of platelet activation. The quantitative relationship between percentage of activated platelets indicated by surface P-selectin expression and shear stress/exposure time follows well the power law functional form. The coefficients of the power law models of PA based on surface P-selectin expression were derived.

Heparanase is an endo-glucuronidase that specifically cleaves heparan sulfate (HS) and heparin polysaccharides. The enzyme is expressed at low levels in normal tissues, but is often upregulated under pathological conditions such as cancer and inflammation. Normal human platelets express exceptionally high levels of heparanase, but the functional consequences of this feature remain unknown. We investigated functional roles of heparanase by comparing the properties of platelets expressing high (Hpa-tg) or low (Ctr) levels of heparanase. Upon activation, Hpa-tg platelets exhibited a much stronger adhesion activity as compared to Ctr platelets, likely contributing to a higher thrombotic activity in a carotid thrombosis model. Furthermore, we found concomitant upregulated expression of both heparanase and CD62P (P-selectin) upon activation of mouse and human platelets. As platelets play important roles in tumor metastasis, these findings indicate contribution of the platelet heparanase to hyper-thrombotic conditions often seen in patients with metastatic cancer.

The platelet collagen receptor glycoprotein (GP) VI is critical for the formation of arterial thrombosis. GPVI platelet surface expression was examined in patients with stable angina and in patients with acute coronary syndrome (ACS). Surface expression of platelet activation markers such as P-selectin, GPIbalpha, and platelet GPVI was determined by flow cytometry. Patients with ACS showed a significantly enhanced GPVI expression compared with patients with stable angina and healthy controls. The expression of GPVI correlated well with CD62P. Elevated platelet GPVI expression was associated with ACS independent of markers of myocardial necrosis such as troponin and creatine kinase. In ACS, platelet surface GPVI expression was already elevated several hours before troponin and creatine kinase indicated myocardial injury. We conclude that the determination of the platelet-specific thrombotic marker GPVI may help to identify patients at risk before myocardial ischemia is evident.

BACKGROUND

Acute cerebral ischemia is caused by different pathophysiological mechanisms. The role of platelets and other blood cells can be different among the stroke subtypes.

METHODS

Seventy-two patients with acute ischemic cerebrovascular disease, including 31 patients with large vessel disease, 21 patients with cardioembolic disease, and 20 patients with small vessel disease, were evaluated. P-selectin (CD62P) expression and platelet leukocyte aggregates were measured with flow cytometry at the acute phase after the ischemic event. Markers were also measured in 37 control subjects. In all subjects, the serum high-sensitivity C-reactive protein (CRP) was also measured.

RESULTS

The platelet-monocyte aggregates (PMA) and platelet-granulocyte aggregates (PGA) in the large vessel disease group were higher than in control group (P=0.002, and P<0.0001, respectively). The PMA and PGA in the small vessel disease group were also higher than in the control group (P=0.004 and P<0.0001, respectively). In contrast, in the cardioembolic disease group, the PMA and PGA were not significantly different from the control group. CD62P expression was higher in all of the patient groups relative to the control group (P<0.05 for all comparisons). Serum CRP levels were also higher in all of the patient groups than in the control group (P<0.0001 for all comparisons).

CONCLUSIONS

In contrast to large vessel and small vessel disease, it seems that platelet-leukocyte association does not play a crucial role in the pathogenesis of cardioembolic stroke.

BACKGROUND

Platelets (PLTs) are currently stored at room temperature (22°C), which limits their shelf life, primarily due to the risk of bacterial growth. Alternatives to room temperature storage include PLT refrigeration (2-6°C), which inhibits bacterial growth, thus potentially allowing an extension of shelf life. Additionally, refrigerated PLTs appear more hemostatically active than conventional PLTs, which may be beneficial in certain clinical situations. However, the mechanisms responsible for this hemostatic function are not well characterized. The aim of this study was to assess the protein profile of refrigerated PLTs in an effort to understand these functional consequences.

METHODS

Buffy coat PLTs were pooled, split, and stored either at room temperature (20-24°C) or under refrigerated (2-6°C) conditions (n = 8 in each group). PLTs were assessed for changes in external receptor expression and actin filamentation using flow cytometry. Intracellular proteomic changes were assessed using two-dimensional gel electrophoresis and Western blotting.

RESULTS

PLT refrigeration significantly reduced the abundance of glycoproteins (GPIb, GPIX, GPIIb, and GPIV) on the external membrane. However, refrigeration resulted in the increased expression of high-affinity integrins (αIIbβ3 and β1) and activation and apoptosis markers (CD62P, CD63, and phosphatidylserine). PLT refrigeration substantially altered the abundance and localization of several cytoskeletal proteins and resulted in an increase in actin filamentation, as measured by phalloidin staining.

CONCLUSIONS

Refrigerated storage of PLTs induces significant changes in the expression and localization of both surface-expressed and intracellular proteins. Understanding these proteomic changes may help to identify the mechanisms resulting in the refrigeration-associated alterations in PLT function and clearance.

OBJECTIVE

To examine the effect of high doses of vitamin C (VitC) on ex vivo human platelets (PLTs).

METHODS

Platelet concentrates collected for therapeutic or prophylactic transfusions were exposed to: (1) normal saline (control); (2) 0.3 mmol/L VitC (Lo VitC); or (3) 3 mmol/L VitC (Hi VitC, final concentrations) and stored appropriately. The VitC additive was preservative-free buffered ascorbic acid in water, pH 5.5 to 7.0, adjusted with sodium bicarbonate and sodium hydroxide. The doses of VitC used here correspond to plasma VitC levels reported in recently completed clinical trials. Prior to supplementation, a baseline sample was collected for analysis. PLTs were sampled again on days 2, 5 and 8 and assayed for changes in PLT function by: Thromboelastography (TEG), for changes in viscoelastic properties; aggregometry, for PLT aggregation and adenosine triphosphate (ATP) secretion in response to collagen or adenosine diphosphate (ADP); and flow cytometry, for changes in expression of CD-31, CD41a, CD62p and CD63. In addition, PLT intracellular VitC content was measured using a fluorimetric assay for ascorbic acid and PLT poor plasma was used for plasma coagulation tests [prothrombin time (PT), partial thrombplastin time (PTT), functional fibrinogen] and Lipidomics analysis (UPLC ESI-MS/MS).

RESULTS

VitC supplementation significantly increased PLTs intracellular ascorbic acid levels from 1.2 mmol/L at baseline to 3.2 mmol/L (Lo VitC) and 15.7 mmol/L (Hi VitC, P < 0.05). VitC supplementation did not significantly change PT and PTT values, or functional fibrinogen levels over the 8 d exposure period (P>> 0.05). PLT function assayed by TEG, aggregometry and flow cytometry was not significantly altered by Lo or Hi VitC for up to 5 d. However, PLTs exposed to 3 mmol/L VitC for 8 d demonstrated significantly increased R and K times by TEG and a decrease in the α-angle (P < 0.05). There was also a fall of 20 mm in maximum amplitude associated with the Hi VitC compared to both baseline and day 8 saline controls. Platelet aggregation studies, showed uniform declines in collagen and ADP-induced platelet aggregations over the 8-d study period in all three groups (P>> 0.05). Collagen and ADP-induced ATP secretion was also not different between the three groups (P>> 0.05). Finally, VitC at the higher dose (3 mmol/L) also induced the release of several eicosanoids including thromboxane B2 and prostaglandin E2, as well as products of arachidonic acid metabolism via the lipoxygenases pathway such as 11-/12-/15-hydroxyicosatetraenoic acid (P < 0.05).

CONCLUSIONS

Alterations in PLT function by exposure to 3 mmol/L VitC for 8 d suggest that caution should be exerted with prolonged use of intravenous high dose VitC.

BACKGROUND

Microvesicles are small vesicles expressing specific antigens from their cells of origin. Elevated levels of microvesicles have been shown to be associated with coagulation disorders as well as with different types of malignancies. This study aims to evaluate a possible correlation of different microvesicle subpopulations with a positive history of venous thromboembolism (VTE) in patients with soft tissue sarcoma.

METHODS

Annexin V - positive microvesicles, leukocyte (CD45-positive), platelet (CD61-positive), activated platelet (CD62P-, CD63-positive), endothelium-derived (CD62E-positive) and tissue-factor (CD142-positive) microvesicles were identified in the peripheral blood of patients with soft tissue sarcoma (n = 39) and healthy controls (n = 17) using fluorescence-activated cell sorting (FACS).

RESULTS

Both the total amount of Annexin V-positive microvesicles and levels of endothelium-derived (CD62E-positive) microvesicles were shown to decrease significantly after tumor resection (n = 18, p = 0.0395 and p = 0.0109, respectively). Furthermore, the total amount of Annexin V - positive microvesicles as well as leukocyte (CD45-positive) and endothelium-derived (CD62E-positive) microvesicles were significantly higher in patients with grade 3 (G3) soft tissue sarcoma (n = 9) compared to healthy controls (n = 17) (p = 0.0304, p = 0.0254 and p = 0.0357, respectively). Moreover, patients with G3 soft tissue sarcoma (n = 9) presented higher levels of Annexin V-positive and endothelium-derived (CD62E-positive) microvesicles compared to patients with grade 2 (G2) soft tissue sarcoma (n = 8) (p = 0.0483 and p = 0.0045). Patients with grade 1 (G1) soft tissue sarcoma (n = 3) presented with significantly lower levels of platelet (CD61-positive) microvesicles than patients with G3 soft tissue sarcoma (n = 9) (p = 0.0150). In patients with a positive history of VTE (n = 11), significantly higher levels of activated platelet (CD62P- and CD63-positive) microvesicles (p = 0.0078 and p = 0.0450, respectively) were found compared to patients without a history of VTE (n = 28).

CONCLUSIONS

We found significantly higher levels of Annexin V-positive and endothelium-derived (CD62E-positive) microvesicles to be circulating in the peripheral blood of patients with G3 soft tissue sarcoma compared to patients with G2 soft tissue sarcoma. Furthermore, we showed that high counts of activated platelet-derived microvesicles correlate with the occurrence of VTE. Thus, the detection of these microvesicles might be an interesting new tool for early diagnosis of soft tissue sarcoma patients with increased risk for VTE, possibly facilitating VTE prevention by earlier use of thromboprophylaxis.

An upregulation of platelet CD40 ligand (CD40L) and CD62P has been described in atherosclerotic cardiovascular diseases and among patients with acute cerebral ischemia. Correlation between platelet and monocyte activation and the etiology of ischemic stroke were examined in 41 patients with acute ischemic stroke. Compared to 10 controls, all patients with stroke showed a significantly elevated platelet expression of CD40L (P < .001) and had significantly higher amounts of platelet-monocyte aggregates (P = .002). Plasma levels of interleukin 7 were significantly lower in patients with stroke compared to controls (P = .006). Patients with small artery disease had a significantly higher platelet CD40L expression than patients with cardioembolic stroke (P = .029). Plasma levels of soluble CD40L were significantly higher in patients with large artery disease compared to patients with cardioembolic stroke (P = .047). In conclusion, patients with acute ischemic stroke show an upregulation of platelet CD40L and an activation of cellular coagulation with highest activation in the large artery disease subgroup.