Tirofiban-associated thrombocytopenia is due to drug-dependent antibodies (DDAbs) directed against the GPIIb/IIIa complex which can bind after drug-induced conformational changes to the receptor complex. In such cases a higher incidence of myocardial infarction and mortality has been reported raising the possibility of platelet activation. We followed consecutive cases treated with tirofiban to determine the incidence of thrombocytopenia and confirmed that this was due to tirofiban-dependent antibodies. We then tested if these antibodies could cause platelet activation in vitro and correlated this with clinical outcome. In 871 treated patients, severe thrombocytopenia was observed in 11 cases, an incidence of 1.26%. Tirofiban dependent antibodies were confirmed in all cases using a flow cytometric assay. There were two distinct presentations of thrombocytopenia, one occurring acutely, and the second a delayed thrombocytopenia occurring after several days of tirofiban exposure and in keeping with a primary immune response. The effects of DDAbs on platelet activation was analysed by measuring P-selectin (CD62p) and annexin V, in the presence or absence of tirofiban, by flow cytometry. In addition, platelet activation was sought using the serotonin release assay. In six cases there was evidence of platelet activation and this was significantly associated with further coronary ischaemic events experienced at the time of acute thrombocytopenia. Tirofiban-induced thrombocytopenia due to DDAbs is a common occurrence and can lead to platelet activation and increased thrombotic events.

In platelets, thrombin receptor signaling depends upon the release of adenosine diphosphate and subsequent activation at purinergic subtype Y (P2Y) receptors. The purpose of this study is to evaluate the influence of specific P2Y12 polymorphisms on platelet reactivity in healthy subjects mediated by thrombin receptor activating peptide (TRAP). We recruited a total of 29 healthy volunteers who had been previously genotyped for two polymorphisms of the P2Y12 receptor: the H2 haplotype (rs2046934) and 34C>T (rs6785930). Flow cytometry and the VerifyNow assay were used to assess platelet activation and aggregation stimulated by TRAP in the presence and absence of specific receptor antagonists for the P2Y1, P2Y12, and thromboxane A2 receptors. We identified a significant recessive effect of the P2Y12-receptor H2 haplotype on TRAP-induced flow cytometry. Specifically, H2/H2 carriers (n = 5) demonstrated a significant reduction in both glycoprotein IIb/IIIa receptor activation (p < 0.001) and CD62P expression (p = 0.035). While the VerifyNow assay did not reveal any effect of haplotype on TRAP-mediated platelet aggregation (p = 0.72), the H2/H2 subjects demonstrated greater platelet inhibition in the presence of cangrelor, a specific receptor antagonist for the P2Y12 receptor (p = 0.023). No consistent effects of the separate 34C>T genotype (rs6785930) were demonstrated under the conditions evaluated. The findings of this study suggest a potential association between P2Y12-receptor H2/H2 carriers and reduced platelet function mediated by TRAP in healthy volunteers.

OBJECTIVE

In multicomponent collection, various blood components are prepared during one apheresis process. The aim of this prospective crossover study was to compare the function, metabolic parameters and activation state of fresh and stored platelets (PLTs) collected by two different cell separators.

METHODS

Twenty-four donors underwent apheresis on each of two cell separators (Fenwal Amicus(®) and CaridianBCT Trima Accel(®)) with an interval of at least 2 months between donations. Per donation, one double dose of PLT concentrate (PC) and one unit of packed red-blood-cells were collected. In total, 48 single unit PCs were tested for pH, glucose, bicarbonate, lactate, potassium and LDH concentration during 7 days of storage. PLT function was analysed by aggregometry, rotation thrombelastometry and hypotonic shock response. The PLT surface expression of P-selectin (CD62P) and LAMP-3 (CD63) was estimated by flow cytometry.

RESULTS

During storage, metabolic parameters were well maintained in both groups, but levels of glucose and pH were significantly lower, while lactate and LDH were significantly higher in Amicus(®)-PCs. Amicus(®)-derived PLTs were significantly more activated as evidenced by higher CD62P and CD63 expression. In parallel, the in vitro function of Amicus(®)-PLTs was significantly reduced compared to Trima(®)-PLTs.

CONCLUSIONS

In multicomponent apheresis, standardized PLT collection is effective and well tolerated. The higher activation of Amicus(®)-derived PLTs may be because of the divergent centrifugation modalities during collection. Possible consequences for the clinical outcome of thrombocytopenic patients will be evaluated in further trials.

Sulfonated polyethersulfone (SPES) and poly (acrylonitrile-co-acrylic acid-co-vinyl pyrrolidone) (P(AN-AA-VP)), which provided sulfonic acid (-SO(3)H) and carboxylic acid groups (-COOH), respectively, were used to modify polyethersulfone (PES) membrane with a heparin-like surface by blending method. The SPES was prepared by sulfonation of PES using chlorosulfonic acid as the sulfonating agent, while the P(AA-AN-VP) was prepared through a free radical polymerization. The PES and modified PES membranes were prepared by a phase-inversion technique; the modified membranes showed lowered protein (bovine serum albumin, BSA; bovine serum fibrinogen, FBG) adsorption and suppressed platelet adhesion. For the modified membranes, significant decreases in thrombin-antithrombin (TAT) generation, percentage platelets positive for CD62p expression, and the complement activation on C3a and C5a levels were observed compared with those for the pure PES membrane. Due to the similar negatively charged groups as heparin, the modified membranes effectively prolonged the activated partial thromboplastin time (APTT). Furthermore, the modified membranes showed good cytocompatibility. Hepatocytes cultured on the modified materials exhibited improved functional profiles in terms of scanning electron microscope (SEM) observation and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay compared with those on the pure PES membrane. It could be concluded that the modified membranes with sulfonic acid and carboxylic acid groups were endowed with excellent biocompatibility, and the heparin-like surface modification seemed to be a promising approach to improve the biocompatibility of materials.

BACKGROUND

Nitrogen (N2) microbubbles activate the blood platelets and coagulaltion system.

OBJECTIVE

Breathing nitrox rather than air may reduce the level of platelet activation associated with decompression.

METHODS

We tested platelet counts and the expression of functional membrane molecules on platelets in 10 divers subjected to saturated compression in nitrox at 4 ATA and in 9 divers subjected to compression in air at 2.8 ATA. Blood samples were taken before and immediately after the test. We measured the percentages of microplatelets, platelet aggregates and platelets bearing the activation marker C-D62P, and bearing molecules forming receptors for fibrinogen (CD61) and for von Willebrand factor (CD42b) using flow cytometry and specific monoclonal antibodies. Symptoms for DCS were also evaluated.

RESULTS

DCS symptoms were not noted in either the nitrox or air group. In both groups we observed a marked increase in the percentage of activated platelets bearing CD62P molecules and an enhanced number of microplatelets and a marked drop in the platelets count in the blood of (divers in the air group.

CONCLUSIONS

In all divers we observed certain changes in the platelet system, nevertheless decompression in nitrox resulted in a lesser degree of platelet activation. Though this study cannot exclude platelet activation as an etiological factor in DCS, the findings suggest platelet activation can occur in the absence of observable sign of DCS. Thus, platelet activation may be too sensitive a marker to serve as a predictor of DCS.

The mechanism of the transient beneficial effect of 1-deamino(8-D-arginine) vasopressin (dDAVP) infusion in the hemostasis of some BSS patients is not fully understood. We have studied the effect of dDAVP infusion in a BSS patient using an ex vivo perfusion system. Additional coagulation and flow cytometry studies were also performed. Prolonged bleeding time >> 30 min) was not affected by dDAVP infusion. However, perfusion experiments performed with low molecular weight heparin anticoagulated blood (which permits the study of fibrin deposition on perfused subendothelium) showed a significant increase in platelet deposition (6.2% before dDAVP infusion; 20.3% after) and fibrin formation. dDAVP infusion also caused an increase in prothrombin consumption compared with base line values (33 vs 46%). Flow cytometry studies of the patients platelets showed no changes in binding of monoclonal antibodies against CD41, CD36, CD62P or CD63. The increase in thrombus formation observed in perfusions may be dependent on FVIII since it could be reproduced by adding purified free or von Willebrand factor (vWf)-associated FVIII to the patient's blood in vitro. The shortening effect of dDAVP on bleeding time observed in some Bernard-Soulier syndrome patients might be related to an increase in factor FVIII levels induced by dDAVP infusion.

OBJECTIVE

Partially replacing plasma with additive solutions in platelet (PLT) concentrates (PCs) may help to reduce transfusion reactions. Constituents of PLT additive solutions (PASs) have been revealed to affect the quality of PCs. Previous studies involved pairwise comparison of identical PLTs with two different PASs or multicomparison using random PLTs with three or more PASs. In this study, we performed parallel comparison using PCs from identical donors with four PASs. In addition to traditional parameters, the release of bioactive substances and plasma proteins was assessed.

METHODS

Platelets collected four times by apheresis from three donors were suspended in Intersol, SSP+, Composol or M-sol with 35% autologous plasma. The PC parameters, including PLT activation markers, glucose consumption, chemokines and plasma proteins, were assessed during 5-day storage.

RESULTS

Mean PLT volumes were decreased in SSP+, Composol and M-sol after 5-day storage, with significant differences, whereas the hypertonic shock response (HSR) was decreased only in Intersol. Glucose consumption was faster in Intersol and M-sol than in SSP+ or Composol. PLT activation, determined as CD62P, sCD62P, sCD40L and RANTES, was significantly higher in Intersol than the other three PASs. No marked change was observed in fibrinopeptide A and C3a in any PASs.

CONCLUSIONS

M-sol, SSP+ and Composol effectively preserved the quality of PCs. PLT activation was significantly enhanced in Intersol compared with the other three PASs. These effects seem to depend on magnesium and potassium as a constituent. Parallel comparison further verified that the PC quality largely depended on PASs but not donors.

BACKGROUND

This study aimed to compare platelet (PLT) quality during storage of buffy coat (BC) PLT concentrates (PCs), prepared either manually or by the automated OrbiSac system (Gambro BCT).

METHODS

Following overnight storage at 20 to 22 degrees C, five BCs were pooled with 300 mL of PLT additive solution. Twenty-one PCs were produced manually (M-PCs) and 21 by the automated OrbiSac system (A-PCs). Swirling, PLT count, mean PLT volume, blood gas analyses, potassium, glucose, and lactate were assessed. Expression of the activation markers CD42a, CD62P, and PAC-1 was analyzed by flow cytometry on resting PLTs and PLTs stimulated with thrombin receptor agonist peptide (TRAP). Levels of CCL5 and transforming growth factor-beta1 (TGF-beta1) were measured by enzyme-linked immunosorbent assay.

RESULTS

The A-PCs had significantly larger volume and higher PLT yield, PLT recovery, and white blood cell concentration than the M-PCs, whereas the red blood cell content was significantly highest in the M-PCs. pH levels were between 6.9 and 7.2 in all PCs. Neither glucose consumption nor lactate production differed significantly over time. A-PCs had, compared to M-PCs, significantly higher expression of CD62P on resting PLTs, lower capacity for up regulating CD62P on TRAP-stimulated PLTs, and higher levels of CCL5 during storage. TRAP-stimulated A-PCs had a significantly higher potential for down regulation of CD42a than M-PCs. No difference was found in TGF-beta1 levels or TRAP-induced up regulation of PAC-1.

CONCLUSIONS

The levels of CCL5 and the expression of CD62P in resting and stimulated PLTs indicate that PLTs in A-PCs are slightly more activated than in M-PCs, but the clinical importance of this finding is yet unknown.

BACKGROUND

Previously, we evaluated the Mirasol pathogen reduction technology (PRT) system on platelet (PLT) function before resuspension. We now evaluated this system in the presence of PLT additive solution (PAS).

METHODS

Double-dose PLTs (n = 15) were generated using a commercially available apheresis system (Trima, Version 5.2, CaridianBCT) allowing for the resuspension in SSP+ (MacoPharma) immediately after collection. Paired units (n = 30) were PRT treated (M) or remained untreated (C) and analyzed for metabolism (pH, pO(2) , glucose, lactate, adenosine triphosphate [ATP]), swirl, hypotonic shock response (HSR), turbidometric aggregation, CD62P expression, annexin A5 and lactate dehydrogenase (LDH) release, mitochondrial enzymatic reduction activity (MTS), transmembrane mitochondrial potential (Δψ), and surface coverage (SC) during shear-induced adhesion throughout 8 days of storage.

RESULTS

As seen previously, PRT treatment of PLT units, containing a mean of 3.9 × 10(11) ± 0.3 × 10(11) PLTs in 397 ± 10 mL with a 32% to 34% plasma carryover, was associated with significantly (p < 0.001) increased cell activation, acidity, and glycolytic flux. PRT treatment appeared to up regulate both oxidative pathway and adhesional properties as evidenced by significantly higher MTS reduction, oxygen consumption, and shear-induced SC on Day 1 (p ≤ 0.016). While no significant differences were found for LDH release and ATP content (except for Day 8), M units were significantly inferior (p ≤ 0.021) for aggregation (TRAP-6); for Δψ and annexin A5 release (by Day 5); and for swirl, HSR, and MTS reduction (by Day 7).

CONCLUSIONS

PRT treatment in the presence of PAS was comparable to PRT treatment before resuspension preserving ATP content and mitochondrial function.

Interaction between polymorphonuclear leukocyte (PMN) and platelets is important in the pathogenesis of thrombosis and inflammation. This study investigates how strenuous, acute exercise affects PMN oxidative burst activity under adherence to surface-adherent platelets. Thirty sedentary healthy men exercised strenuously (up to maximal oxygen consumption) on a bicycle ergometer. Before and immediately after exercise, the kinetics of oxidant production, phosphorylation of various protein kinase C (PKC) isoforms, and translocation of p47(phox) in PMNs under adherence to surface-adherent platelets were measured using fluorescence microscopy combined with computerized image analysis. Analytical results can be summarized as follows: (i) either treating the platelet with P-selectin (CD62P) and glycoprotein IIb/IIIa (CD41) antibodies or treating the PMN with beta 2-integrin (CD18) and Mac-1 (CD11b) anti-bodies and PKC zeta pseudosubstrate effectively inhibits platelet-promoted oxidant production of PMN; (ii) PMNs adhesion to surface-adherent platelets is associated with a higher amount of phospho-PKC zeta and a larger ratio of membrane to cytosolic p47(phox) than suspended PMNs; (iii) strenuous, acute exercise decreases platelet-promoted oxidant production of PMN and is accompanied by suppressed phosphorylation of PKC zeta, translocation of p47(phox), and inhibition of PKC zeta pseudosubstrate to oxidant production; (iv) no significant changes occur in PKC alpha/beta II and delta phosphorylation of adherent PMNs following this exercise. Therefore, we conclude that strenuous, acute exercise suppresses platelet-promoted oxidative burst of PMN, possibly by reducing phosphorylation of PKC zeta and translocation of the cytosolic p47(phox) to the plasma membrane, thus inhibiting the assembly and activation of NADPH oxidase in PMN.

BACKGROUND

To improve platelet (PLT) quality, hyperconcentrated PLT concentrates (hcPCs) were compared to standard PLT concentrates (stdPCs) in two different PLT additive solutions, T-Sol and PAS-27a. PAS-27a differs from T-Sol by containing glucose, phosphate, potassium, magnesium, and bicarbonate.

METHODS

PLTs were harvested by apheresis twice from 14 donors; each unit was divided into two. Four units from each donor were produced: hcPCs, 2000 x 10(9) per L in T-Sol or PAS-27a; and stdPCs, 1400 x 10(9) per L in 65 percent T-Sol or PAS-27a and 35 percent acid citrate dextrose-plasma. On Days 1 through 4, swirling was scored and PLT count, mean PLT volume, pH, blood gas, glucose, and lactate were measured. Expression of CD42a, CD62P, CD63, and PAC-1 was analyzed by flow cytometry on resting PLTs and PLTs stimulated with thrombin receptor agonist peptide (TRAP).

RESULTS

Glucose consumption and lactate production were significantly higher in hcPCs stored in PAS-27a than in T-Sol. Both stdPC and hcPC PLTs in T-Sol expressed CD62P and PAC-1 significantly higher than in PAS-27a. Over time the T-Sol hcPCs revealed highest expression of CD62P and CD63. A significantly higher capacity for up regulation of CD62P, CD63, and PAC-1 upon TRAP stimulation was found for stdPCs and hcPCs in PAS-27a compared to PLTs in T-Sol. TRAP-stimulated PLTs in stdPCs and hcPCs suspended in PAS-27a showed significantly higher potential for down regulation of CD42a than the T-Sol concentrates.

CONCLUSIONS

PLTs appear better preserved in vitro in PAS-27a than in T-Sol, and this suggests that storage of hcPCs in PAS-27a could be extended beyond 24 hours.

Rabbit is one of several species that depend on development of B lymphocytes in gut-associated lymphoid tissues for primary immunoglobulin-repertoire diversification. The rabbit appendix is an important site of early B-lymphocyte development. We previously reported that peripheral lymph node addressin detected by monoclonal antibody (mAb) MECA-79 played a role in recruitment of immature blood-borne B cells into neonatal rabbit appendix. Here, we report expression of an approximately 127 000 MW O-linked sulphated proteoglycan on developing B cells in appendix and Peyer's patches recognized by the mAb MECA-79. Binding of the mAb to B lymphocytes was sensitive to enzyme treatment with O-sialoglycoprotease and expression was partially inhibited by sodium chlorate, a metabolic inhibitor of sulphation. The proportions of MECA-79(+) B lymphocytes gradually increased from < 0.5% at 3 days to>> 70% at 6 weeks in appendix and Peyer's patches. The proportions of MECA-79(+) B lymphocytes in spleen and peripheral blood were very low (0.5-2%). However, the MECA-79 determinant was detected on B cells in splenic germinal centres after immunization. In situ labelling of appendix cells showed that the MECA-79 determinant was expressed on fluorescein-labelled B lymphocytes that migrated from appendix into mesenteric lymph nodes. B-cell MECA-79 may be involved in interactions with T cells and/or dendritic cells. Alternatively, because we found that lymphatic endothelium in the thymus-dependent area of appendix, a site for lymphocyte exit, expressed P-selectin (CD62P), interaction of the MECA-79 determinant on B cells with CD62P may have a role in the exit of B lymphocytes from rabbit appendix.

Leprosy is an infectious-contagious disease whose clinical evolution depends on the immune response pattern of the host. Adhesion molecules and leukocyte migration from blood to tissue are of the utmost importance for the recognition and elimination of infectious pathogens. Selectins are transmembrane glycoproteins that share a similar structural organization and can be divided into three types according to their site of expression. The biopsies were cut into 5μm thick sections and submitted to immunohistochemistry using antibodies against E-selectin and P-selectin. The number of E-selectin-positive cells was significantly higher in the tuberculoid form than in the lepromatous form. The immunostaining pattern of P-selectin differed from that of E-selectin. Analysis showed a larger number of endothelial cells expressing CD62P in the lepromatous form compared to the tuberculoid form. The presence of these adhesins in the endothelium contributing to or impairing the recruitment of immune cells to inflamed tissue and consequently influences the pattern of immune response and the clinical presentation of the disease.

P-selectin (CD62P) is expressed on activated platelets and on stimulated endothelial cells. It interacts with P-selectin glycoprotein ligand-1 (PSGL-1; CD162) for adhesion of activated platelets on leukocytes and for rolling of leukocytes on stimulated endothelial cells. Recently, resting and activated platelets have been shown to roll on endothelial P-selectin, indicating that platelets express (a) ligand(s) for P-selectin. Here we show that P-selectin specifically precipitated one 28-kDa glycoprotein from the whole cell lysates and the membrane lysates of human platelets in a Ca2+-dependent manner. Further, the purified 28-kDa molecule could inhibit the binding of P-selectin to human resting and activated platelets. In contrast, KPLI (a leukocyte adhesion blocking MoAb to PSGL-1) did not neutralize the binding of P-selectin to human platelets, even though it abolished the binding of P-selectin to human promyeloid HL-60 cells. Our results thus indicate that the 28-kDa glycoprotein may function as an important platelet ligand for P-selectin.

BACKGROUND

Interactions between platelets and endothelial cells under inflammatory conditions lead to an increased expression of various activity markers of atherosclerosis in the vessel wall. The purpose of this study was to investigate possible protective effects of nicotinic acid in an in vitro endothelial cell model.

METHODS

After a 24-hour incubation period with nicotinic acid (1 mmol/l), human umbilical vein endothelial cells were stimulated for 1 h with lipopolysaccharide and were then incubated in direct contact with activated platelets. Following this incubation, the expression of CD40L and CD62P on platelets and the expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, uPAR, and MT1-MMP on endothelial cells were measured by flow cytometry. Supernatants were analyzed by ELISA for soluble MCP-1 and MMP-1.

RESULTS

The increased expression of VCAM-1 on endothelial cells by proinflammatory stimulation with activated platelets was significantly reduced through preincubation with nicotinic acid (P<.05). Furthermore, platelets in direct contact with preincubated endothelial cells showed a significant reduction in their CD62P and CD40L expression when compared to platelets incubated with untreated endothelial cells (P<.05). Treatment with nicotinic acid did not have a significant effect on ICAM-1, uPAR, and MT1-MMP expression on endothelial cells. Levels of soluble MCP-1 and MMP-1 in supernatants were lower after preincubation with nicotinic acid.

CONCLUSIONS

Nicotinic acid inhibits platelet activation after platelets contacted nicotinic acid treated endothelial cells and inhibits VCAM-1 expression on human endothelial cells under inflammatory conditions. These findings suggest a possible pleiotropic therapeutic relevance of nicotinic acid in atherosclerosis.

Our aim was to investigate the CD40-CD40 ligand system in preeclamptic women. We also studied CD62P and platelet-monocyte aggregates, which have been closely linked to the CD40-CD40L system. Platelet expression of CD40L and CD62P and expression of CD40 on monocytes and platelet-monocyte aggregates were determined by flow cytometry in whole blood from 23 preeclamptic women, 23 normotensive pregnant women, and 23 nonpregnant women. The preeclamptic women showed a significant increase in CD40L and CD62P on platelets and in CD40 on monocytes when compared with normotensive pregnant women and nonpregnant women (all P < .001). There was a significant increase in platelet-monocyte aggregates in preeclamptic women (P < .001) and normotensive pregnant women (P = .003) compared with nonpregnant women. Preeclampsia is associated with activation of the CD40-CD40L system. The activation of this system may contribute to the development or maintenance of the proinflammatory and prothrombotic milieu found in preeclampsia.

BACKGROUND

With the implementation of universal WBC reduction in the United Kingdom, in-process WBC-reduction filters for pooled buffy coat (BC)-derived platelet concentrates (PCs) are used in routine production. The effects of three filter/storage bag combinations on platelet activation and microvesiculation and on the activation of coagulation were investigated.

METHODS

Using pooled BCs from the same donors, three filter/storage bag combinations (Autostop BC/CLX, Pall Biomedical; Sepacell PLX5/PL2410, Asahi Medical; and Imugard III-PL 4P/Teruflex, Terumo) were compared with unfiltered controls for their effects on microvesiculation and other storage-induced changes in platelets. Process efficiency was measured by platelet yield and residual WBC count. The storage changes were assessed: pH, activation of platelets measured by CD62P on the platelet surface and in supernatant plasma, quantitation of platelet-derived and RBC-derived microvesicles, cellular injury measured by annexin V in the supernatant plasma, and activation of the coagulation system measured by kallikrein-like and thrombin-like activities, prothrombin fragment 1+2, and thrombin-antithrombin complex.

RESULTS

All three filters were comparable in terms of platelet recovery and WBC removal, and none induced immediate platelet activation or microvesiculation. With storage, platelet activation or microvesiculation increased in platelets prepared by all three filters and in unfiltered controls, but these effects were significantly less in the Imugard PCs than in controls. These findings were consistent with those for annexin V in the supernatant plasma, which were lower in Imugard PCs than in other products. Sepacell and Imugard filters reduced RBC-derived microvesicles to 50 percent of control levels, but the Autostop filter had no effect. On storage, levels of RBC-derived microvesicles in filtered products remained static, but levels in the unfiltered control doubled. Kallikrein- and thrombin-like activities were generated only by the Autostop filter without any further increment on storage.

CONCLUSIONS

WBC-reduced pooled BC-PCs prepared by various filter/bag combinations were equivalent on Day 1 but differed during storage in terms of platelet activation or microvesiculation.

OBJECTIVE

An increased platelet activation status is present in patients with preeclampsia. Our purpose was (1) to establish by means of flow cytometry whether platelets circulate in an activated state during the first and second trimesters of pregnancy and (2) to establish whether early platelet activation predicts the onset of preeclampsia.

METHODS

Consecutively, 244 pregnant women were included in a prospective study design. Platelets in whole blood samples from the pregnant women in the first trimester, the second trimester, and after delivery were labeled with the following antibodies associated with platelet activation: anti-CD62P (P-selectin, alpha-granule secretion), anti-CD63 (GP53, lysosomal secretion), anti-CD31 (GPIIa', platelet endothelial cell adhesion molecule-1). The surface antigen exposure was determined by double-label flow cytometry with anti-CD42b (GPIb, a platelet-specific monoclonal glycoprotein) to select platelets and platelet-derived materials. Preeclampsia was defined as a diastolic blood pressure>> or = 90 mm Hg and proteinuria>> or = 0.3 gm in a 24-hour urine sample (International Society for Study of Hypertension in Pregnancy criteria).

RESULTS

Seventeen of 244 patients had preeclampsia (6.9%). Only first-trimester CD63 expression had an area under the curve>> 0.5 by receiver-operator characteristic curve analysis and was selected as a possible predictor of preeclampsia. We found a sensitivity of 47% and a specificity of 76% with use of a percentage of activated platelets above 2% as a positive test. Likelihood ratios were 1.94 for positive likelihood and 0.69 for negative likelihood. Univariate logistic regression analysis results were odds ratio 2.8 (95% confidence interval 1.0 to 7.6). Multivariate logistic regression analysis results were odds ratio 2.9 (95% confidence interval 0.92 to 8.9). However, the odds ratio of first antenatal diastolic blood pressure was two to four times higher than the odds ratio of first-trimester CD63 expression. The combination of first-trimester CD63 and first antenatal diastolic blood pressure increases the positive likelihood ratio from 1.94 to 9.4, with a sensitivity of 41%, a specificity of 96%, and a negative likelihood ratio of 0.62.

CONCLUSIONS

Increased first-trimester CD63 expression is an independent risk factor for development of preeclampsia. CD63 expression might be useful to identify a subgroup of patients with a high risk for development of preeclampsia, especially in combination with first-trimester antenatal diastolic blood pressure. This method of patient selection may enable more efficient intervention studies in patients at risk than do the selection methods used so far.

Preeclampsia, a common complication of pregnancy, contributes significantly to maternal and fetal morbidity and mortality. It may lead to both quantitative and qualitative defects of maternal and neonatal platelets. In this prospective study, flow cytometry has been used to study expression of platelet-surface glycoproteins (GPs) on maternal and neonatal platelets of both healthy and preeclamptic subjects. We studied 15 preeclamptic women, 20-44 y of age, and their newborns (median gestational age, 32 wk; range, 26-38) and seven healthy women (aged 26-41 y) and their healthy newborns (median gestational age, 38 wk; range, 38-42). Compared with their healthy and preeclamptic mothers, resting platelets from neonates expressed significantly less CD41 and CD9. Thrombin activation resulted in significant increases in platelet-surface expression of CD62P, CD63, CD41, CD9, and CD36 in neonates and their healthy mothers. Compared with neonates of healthy mothers, platelets from neonates of preeclamptic mothers expressed lower levels of CD62P, CD63, CD9, and CD36 on activated platelets. These findings suggest that preeclampsia influences the expression of platelet-surface GPs on neonatal and maternal platelets, which may affect platelet function, leading to an additional risk for bleeding in thrombocytopenic neonates of mothers with preeclampsia.

BACKGROUND

The air bubbles and foam that develop during the preparation of platelet units have traditionally been considered to interact with the platelets, causing activation and release reactions. However, there actually seems to be no data available concerning the platelet damage that may occur as a result of air bubbles and foam present in the final unit. In this in vitro study we, therefore, investigated the effects of not removing air bubbles/foam from final platelet units, by measuring in vitro parameters during a 7-day storage period.

METHODS

Platelet samples (n=8) from eight pools of 12 buffy-coats were aliquoted and prepared with the OrbiSac system for storage with (test) or without (reference) air bubbles/foam included in the final units. The metabolic, cellular and activation parameters of all units, comprising approximately 30% plasma and 70% SSP+ platelet additive solution, were analysed during the 7-day storage period.

RESULTS

Differences in platelet counts and contents between the test and reference units were detected throughout storage (p<0.05 at day 5 and p<0.01 at day 7). Lactate dehydrogenase increased during storage in the test units and was significantly higher than in the reference units (p<0.01 from day 5). The hypotonic shock response was greater in the reference units (p<0.05 on day 2 and p<0.01 from day 5). The extent of shape changes was less in the test units (p<0.05 until day 5 and p<0.01 on day 7). CD62P was higher in the test units (p<0.05 on day 7). CD42b decreased in all units but was lower in the test units (p<0.01 on day 5). CD41, CD61 and PAC-1 showed no difference throughout storage between the units (p=NS). Aggregates were visible (day 7) and occurred in three of the test units. pH was maintained at >6.8 (day 7) and swirling remained at the highest level (score =2) for all units throughout storage.

CONCLUSIONS

This study shows that storage with air bubbles/foam causes considerable enhancement of disintegration of platelets. In addition, various in vitro parameters of the platelets remaining seem to be negatively affected. The results of this study suggest that platelets should be stored without air bubbles/foam, given that these cause increased disintegration of platelets.

Platelet aggregation contributes to the pathogenesis of infective endocarditis, and aggregation of platelets induced by lactobacilli is thought to be an important contributory factor in the development and progression of Lactobacillus endocarditis. The main purpose of this study was to examine the effect of immunity-enhancing probiotic strains Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019 on the activation and aggregation of human blood platelets. Whole blood samples from healthy individuals were incubated in vitro with HN001 or HN019 and subsequently labeled with platelet-specific monoclonal antibodies, fluorescein isothiocyanate-conjugated anti-CD41a (expressed on normal platelets), and phycoerythrin-streptavidin-conjugated anti-CD62p (expressed on activated platelets) before analysis by flow cytometry. Platelet-rich plasma was used to assist the gating of the platelet cluster. ADP and epinephrine were used as the physiological platelet activation agonists. Platelet aggregation-inducing strain Streptococcus sanguis 133-79 was used as a positive control strain. The mean fluorescence intensity of phycoerythrin and the percentage of platelets expressing the CD62p marker were used to assess the degree of platelet activation. The percentage of CD62p-positive platelets and the light scatter profiles of the agonist-activated platelets were used to identify the occurrence and degree of platelet aggregation. HN001 and HN019 had no effect on spontaneous platelet activation and aggregation; they also failed to exacerbate the platelet aggregation activity induced by ADP and epinephrine. Therefore, these test probiotic strains HN001 and HN019 are less likely to participate in the pathogenesis of infective endocarditis or other thrombotic disorders with regard to platelet aggregation factors.

Thrombocytopenia is a common problem in critically ill patients, which is associated with increased mortality. Recently, Drotrecogin alfa (activated) (recombinant human activated protein C (APC)) was shown to reduce mortality in patients with severe sepsis. Only minimal effect of APC on coagulation markers was demonstrated. Nevertheless, low platelet count was identified as a risk factor for bleeding with use of this drug. We conducted this study to evaluate possible influence of APC on in vitro expression of platelet receptors at therapeutic and supra-therapeutic concentrations. Blood samples of volunteers and patients with severe sepsis were adjusted with APC to final concentrations of 0.045 microg mL(-1) APC (APC-45, therapeutic dose) and 0.225 microg mL(-1) APC (APC-225, five-fold therapeutic dose), respectively. The activation of platelets was mediated by two different agonists. APC had no significant influence on platelet activation, with or without stimulation at both concentrations. In group APC-225, CD62P showed a non-significant decrease. This in vitro study demonstrates that therapeutic plasma concentrations of Drotrecogin alfa (activated) have neither influence on expression of platelet activation markers nor on platelet-granulocyte complexes in blood of volunteers and patients with severe sepsis. Thus, a direct drug-platelet interaction seems unlikely.

Cardiopulmonary bypass (CPB) is associated with impaired platelet function and a systemic inflammatory response. The present study was designed to evaluate whether any correlation between platelet activation and inflammatory response during CPB exists. The results obtained from 8 patients undergoing hypothermic CPB for cardiac surgery showed the occurrence of a moderate degree of platelet activation during CPB, demonstrated by an increase of platelet CD62P expression in correlation with an increase of beta-thromboglobulin levels, with a concomitant decrease of in vitro platelet response. Plasma IL-1beta levels significantly increased during CPB, with a peak between 1 and 4 h after CPB. Similarly, IL-6 levels were elevated 30 min from CPB starting, peaked at 4 h, and remained elevated after 24 h. A direct correlation was found between plasma IL-1beta and IL-6 levels. A significant correlation between plasma IL-1beta and beta-thromboglobulin levels was also found. In turn, plasma beta-thromboglobulin levels correlated with CD62P expression on activated platelets. An inverse correlation was found between in vitro platelet aggregation and plasma IL-1beta or IL-6 levels. From the present results it may be speculated that platelet activation during CPB may contribute, through the release of IL-1beta, to activation of endothelial cells and subsequent release of other cytokines with chemotactic and pro-inflammatory properties, thus playing an important role in the inflammatory response associated with CPB.