Randomized trial of red cell washing for the prevention of transfusion-associated organ injury in cardiac surgery.

BACKGROUND: Experimental studies suggest that mechanical cell washing to remove pro-inflammatory components that accumulate in the supernatant of stored donor red blood cells (RBCs) might reduce inflammation and organ injury in transfused patients. METHODS: Cardiac surgery patients at increased risk of large-volume RBC transfusion were eligible. Participants were randomized to receive either mechanically washed allogenic RBCs or standard care RBCs. The primary outcome was serum interleukin-8 measured at baseline and at four postsurgery time points. A mechanism substudy evaluated the effects of washing on stored RBCs in vitro and on markers of platelet, leucocyte, and endothelial activation in trial subjects. RESULTS: Sixty adult cardiac surgery patients at three UK cardiac centres were enrolled between September 2013 and March 2015. Subjects received a median of 3.5 (interquartile range 2-5.5) RBC units, stored for a mean of 21 ( sd 5.2) days, within 48 h of surgery. Mechanical washing reduced concentrations of RBC-derived microvesicles but increased cell-free haemoglobin concentrations in RBC supernatant relative to standard care RBC supernatant. There was no difference between groups with respect to perioperative serum interleukin-8 values [adjusted mean difference 0.239 (95% confidence intervals -0.231, 0.709), P =0.318] or concentrations of plasma RBC microvesicles, platelet and leucocyte activation, plasma cell-free haemoglobin, endothelial activation, or biomarkers of heart, lung, or kidney injury. CONCLUSIONS: These results do not support a hypothesis that allogenic red blood cell washing has clinical benefits in cardiac surgery. CLINICAL TRIAL REGISTRATION: ISRCTN 27076315.

Platelet responses to agonists in a cohort of highly characterised platelet donors are consistent over time.

BACKGROUND AND OBJECTIVES: Platelet function shows significant inheritance that is at least partially genetically controlled. There is also evidence that the platelet response is stable over time, but there are few studies that have assessed consistency of platelet function over months and years. We aimed to measure platelet function in platelet donors over time in individuals selected from a cohort of 956 donors whose platelet function had been previously characterised. MATERIALS AND METHODS: Platelet function was assessed by flow cytometry, measuring fibrinogen binding and P-selectin expression after stimulation with either cross-linked collagen-related peptide or adenosine 5'-diphosphate. Eighty-nine donors from the Cambridge Platelet Function Cohort whose platelet responses were initially within the lower or upper decile of reactivity were retested between 4 months and five and a half years later. RESULTS: There was moderate-to-high correlation between the initial and repeat platelet function results for all assays (P ≤ 0·007, r2 0·2961-0·7625); furthermore, the range of results observed in the initial low and high responder groups remained significantly different at the time of the second test (P ≤ 0·0005). CONCLUSION: Platelet function remains consistent over time. This implies that this potential influence on quality of donated platelet concentrates will remain essentially constant for a given donor.

Low molecular weight heparin significantly reduces embolisation after carotid endarterectomy--a randomised controlled trial.

OBJECTIVES: The administration of unfractionated heparin (UFH) prior to carotid clamping during carotid endarterectomy (CEA) transiently increases the platelet aggregation response to arachidonic acid (AA) despite the use of aspirin. We hypothesized that this phenomenon might be reduced by using low molecular weight heparin (LMWH) resulting in fewer emboli in the early post-operative period. METHODS: 183 aspirinated patients undergoing CEA were randomised to 5000 IU UFH (n=91) or 2500 IU LMWH (dalteparin, n=92) prior to carotid clamping. End-points were: transcranial Doppler (TCD) measurement of embolisation, effect on bleeding and platelet aggregation to AA and adenosine 5'-diphosphate (ADP). RESULTS: Patients randomised to UFH had twice the odds of experiencing a higher number of emboli in the first 3h after CEA, than those randomised to LMWH (p=0.04). This was not associated with increased bleeding (mean time from flow restoration to operation end: 23 min (UFH) vs. 24 min (LMWH), p=0.18). Platelet aggregation to AA increased significantly following heparinisation, but was unaffected by heparin type (p=0.90). The platelets of patients randomised to LMWH exhibited significantly lower aggregation to ADP compared to UFH (p<0.0001). CONCLUSIONS: Intravenous LMWH is associated with a significant reduction in post-operative embolisation without increased bleeding. The higher rate of embolisation seen with UFH may be mediated by increased platelet aggregation to ADP, rather than to AA.

Effects of apoptosis and lipid peroxidation on T-lymphoblastoid phospholipid-dependent procoagulant activity.

BACKGROUND: Coagulation has an absolute requirement for macromolecular complexes to be assembled on a negatively charged phospholipid (PL) surface. Previously, we reported that malignant T-lymphoblastoid cells have the ability to support procoagulant activity (PCA) independently of tissue factor by providing such a surface. OBJECTIVE: To explore the effect of two pathophysiologic processes, apoptosis and lipid peroxidation (LP), on this PL-dependent PCA. METHODS: Three different assays for PL-dependent PCA (factor IXa-initiated FXa and thrombin generation and prothrombinase activity) were used to investigate this PCA after exposing three T-lymphoblastoid cell lines to either apoptotic stimuli (1 microM staurosporine) or oxidative stress (4 mm H(2)O(2) and 40 microM CuSO(4)). Surface exposure of anionic PL was measured by flow cytometry using annexin A5(FITC) and an antibody (3G4) specific for native, but not oxidized, phosphatidylserine (PS). RESULTS AND CONCLUSIONS: Both apoptosis and LP significantly enhanced the PCA of cells, to a level that was greater than that observed following calcium ionophore treatment, suggesting that the increased activity was not solely due to anionic PL exposure. Whereas cells undergoing apoptosis bound both annexin A5(FITC) and 3G4, only annexin A5(FITC) bound to cells undergoing LP. This implies that apoptosis increases PCA by causing the translocation of oxidized/native PS to the outer membrane, whereas LP appears to increase the PCA, possibly due to malondialdehyde adducts altering the net charge on the cell surface, which allows PLs other than PS to participate in thrombin generation.

Mapping the platelet profile for functional genomic studies and demonstration of the effect size of the GP6 locus.

BACKGROUND: Evidence suggests the wide variation in platelet response within the population is genetically controlled. Unraveling the complex relationship between sequence variation and platelet phenotype requires accurate and reproducible measurement of platelet response. OBJECTIVE: To develop a methodology suitable for measuring signaling pathway-specific platelet phenotype, to use this to measure platelet response in a large cohort, and to demonstrate the effect size of sequence variation in a relevant model gene. METHODS: Three established platelet assays were evaluated: mobilization of [Ca(2+)](i), aggregometry and flow cytometry, each in response to adenosine 5'-diphosphate (ADP) or the glycoprotein (GP) VI-specific crosslinked collagen-related peptide (CRP). Flow cytometric measurement of fibrinogen binding and P-selectin expression in response to a single, intermediate dose of each agonist gave the best combination of reproducibility and inter-individual variability and was used to measure the platelet response in 506 healthy volunteers. Pathway specificity was ensured by blocking the main subsidiary signaling pathways. RESULTS: Individuals were identified who were hypo- or hyper-responders for both pathways, or who had differential responses to the two agonists, or between outcomes. 89 individuals, retested three months later using the same methodology, showed high concordance between the two visits in all four assays (r(2) = 0.872, 0.868, 0.766 and 0.549); all subjects retaining their phenotype at recall. The effect of sequence variation at the GP6 locus accounted for approximately 35% of the variation in the CRP-XL response. CONCLUSION: Genotyping-phenotype association studies in a well-characterized, large cohort provides a powerful strategy to measure the effect of sequence variation in genes regulating the platelet response.

Characterization of the cell-surface procoagulant activity of T-lymphoblastoid cell lines.

The procoagulant activity (PCA) of four T-lymphoblastoid cell lines (CEM-CCRF, Jurkat, Molt-4 and A3.01) at different stages of differentiation has been characterized and compared with that of a monocytoid cell line (THP-1). Four assay systems were employed; the activated partial thromboplastin time (APTT); prothrombin time/tissue factor (TF) activity; a purified factor (F)Xa generation system and cancer procoagulant. High levels of TF activity were seen only with the monocytic cells. However the more differentiated of the T-lymphoblastoid cells (Molt-4 and A3.01) were more active than monocytic cells in supporting FXa generation. This pattern was not repeated for the APTT assay, which was related to cell-surface TF activity, since it was partially inhibited by antiTF antibody. Annexin V totally inhibited the activity observed in all three assay systems, indicating that the PCA of T-lymphoblastoid cells is primarily due to expression of negatively charged phospholipids. However, antiphosphatidylserine antibody even at a high concentration gave only partial inhibition of the activity observed in the APTT and FXa generation systems for the cells compared with almost total inhibition for the phospholipid standard, suggesting either that cellular phosphatidylserine (PS) is less accessible to the antibody, or that PS is not the sole negatively charged phospholipid responsible for this activity. Flow cytometry studies using propidium iodide and annexin V showed that the PCA, although linked to PS exposure, was not the result of apoptosis.

Combined therapy with clopidogrel and aspirin significantly increases the bleeding time through a synergistic antiplatelet action.

OBJECTIVE: Many thromboembolic events occur in patients taking aspirin. Dual therapy with aspirin and clopidogrel may prove effective at reduction of thromboembolic complications. However, the extent to which these two drugs interact may significantly increase the risk of bleeding in open surgery. Because of the increased use of combination antiplatelet therapy in populations with significant atherosclerotic disease, this risk needs to be evaluated by the assessment of the combined effect in vivo of clopidogrel and aspirin on bleeding time and platelet function. OUTCOMES: In seven healthy subjects, addition of low dose clopidogrel (2 x 75 mg) to aspirin (150 mg) therapy significantly increased bleeding time (from 7.6 +/- 3.4 minutes to 17.5 +/- 8.6 minutes; P <.05), with concomitant falls in adenosine diphosphate (ADP)-induced platelet fibrinogen binding and aggregation (P <.05). Increasing the dose of clopidogrel to 300 mg increased bleeding time (to 24.9 +/- 8.5 minutes; P <.05) without significant additional platelet inhibition. There was considerable variability in the individual subject platelet response to the lower dose of clopidogrel. Those patients with the highest ADP response at baseline had the least response, and subjects with a weak response to ADP at baseline achieved maximal platelet inhibition with the low dose of clopidogrel. CONCLUSION: The increases in bleeding times should be considered in combination antiplatelet therapy in patients who undergo open vascular surgery.

Characterization and regulation of the receptor tyrosine kinase Tie-1 in platelets.

The receptor tyrosine kinase Tie-1 is expressed predominantly on endothelial cells where it has an essential role in blood vessel formation. Targeted disruption of the Tie-1 gene results in a lethal phenotype with severe disruption to the normal integrity of the vasculature. In an examination of Tie-1 in vivo, we observed a significant pool of the receptor present in the circulation associated with the platelet fraction. Western blotting reveals the platelet form of Tie-1 to be a protein of approximately 110 kDa, this contrasts with the 135/125-kDa doublet found in endothelial cells. Platelet activation results in increased surface expression of Tie-1. The closely related receptor tyrosine kinase Tie-2/Tek is not present in platelets. Endothelial Tie-1 undergoes metalloprotease-mediated ectodomain cleavage in response to phorbol ester and other agonists. Tie-1 cleavage leads to release of the extracellular domain and generation of a cell-associated intracellular domain with signalling capacity. The potential for cleavage was investigated in platelets. In contrast to endothelial Tie-1, phorbol ester does not stimulate truncation of the platelet receptor, suggesting these cells lack one or more components of the regulated metalloprotease system controlling Tie-1. These data demonstrate the Tie-1 receptor tyrosine kinase is present on platelets and its surface expression is regulated. Furthermore, platelet Tie-1 differs significantly from the endothelial receptor. Platelet Tie-1 has the potential to modulate endothelial function by competing for any Tie ligands and may have signalling roles important in controlling aspects of platelet behaviour.

Stent implantation reduces restenosis in patients with suboptimal results following coronary angioplasty.

BACKGROUND: Primary intracoronary stenting reduces the rate of restenosis when compared with balloon angioplasty (PTCA) in selected patients. The strategy of PTCA followed by provisional stent placement for suboptimal PTCA results may be preferable to universal stenting but has not yet been tested in a randomized trial. METHODS: An attempt was made to obtain an optimal result with PTCA alone in 143 patients. Stenting was required in 50 patients (35%) for significant coronary dissection or PTCA failure. In the remaining 93 patients, the angiographic result was assessed immediately using on-line quantitative coronary angiography and classified as either optimal (<15% residual stenosis) or suboptimal (>/=15% residual stenosis). Sixteen patients (11%) had an optimal result from PTCA. The remaining 77 (54%) patients had a suboptimal result and were immediately randomized either to no further treatment or to the placement of a stent. The primary end-point was the rate of restenosis (>50% stenosis), assessed by quantitative coronary angiography, at 6 months. RESULTS: Angiographic follow-up was completed in 132 patients. Restenosis occurred in 53 (36,69)% of patients with a suboptimal result randomized to PTCA alone compared with 24 (12,41)% of patients randomized to stent (P=0.023). There was no significant difference in minimal luminal diameter at follow-up between the randomized groups. The rate of restenosis was 14 (2,43)% in patients with an optimal PTCA result and 14 (5,28)% in those that required stenting. CONCLUSIONS: Optimal angiographic results following conventional PTCA are rare and the restenosis rate following suboptimal results is high. The strategy of stenting suboptimal results is associated with a significant reduction in the rate of stenosis.

Increased platelet responsiveness following coronary stenting. Heparin as a possible aetiological factor in stent thrombosis.

AIMS: Platelet activation may be a determinant of thrombotic and restenotic complications following intracoronary stenting. In order to measure the effect of stenting on platelet activation antigen expression we used whole blood flow cytometry in 18 patients undergoing Palmaz-Schatz stenting (treated with full anticoagulation) and compared these with a group of 18 patients undergoing elective angioplasty. The effects of low molecular weight heparin and unfractionated heparin on platelet behaviour were also studied, both in vitro and in vivo to determine the contribution of prolonged heparin therapy to platelet activation following stenting. METHODS AND RESULTS: Fibrinogen binding to activated GPIIb-IIIa, and surface expression of P-selectin, GPIb and GPIIb-IIIa antigens were measured in unstimulated peripheral blood samples (rest) and on stimulation with adenosine diphosphate (0.1-10 micromol x 1(-1)) and thrombin (0.02-0.16 U x ml(-1)). No changes were seen in resting samples following angioplasty or stenting. Agonist responsiveness was unaltered after angioplasty, but in stented patients antigen expression in response to thrombin was significantly reduced (P< or =0.04), whilst the adenosine diphosphate response was significantly increased (P=0.01). Similar effects were observed in patients with unstable angina treated with either low molecular weight heparin or unfractionated heparin in vivo. In vitro, both unfractionated and low molecular weight heparin inhibited thrombin-induced platelet activation, but stimulation of adenosine diphosphate responses was more marked with unfractionated than low molecular weight heparin. CONCLUSIONS: There was a significant increase in platelet responsiveness to adenosine diphosphate following intracoronary stenting in patients treated with conventional anticoagulants. This was probably a consequence of treatment with heparin. Activation of platelets by heparin may explain the increased rate of stent thrombosis in patients treated with anticoagulant therapy. Low molecular weight heparins stimulate platelets less than unfractionated heparin.

Altered platelet function detected by flow cytometry. Effects of coronary artery disease and age.

Platelet activation state and responsiveness to physiological agonists were measured in 65 patients with documented coronary artery disease (54 male and 11 female; mean age, 58 years). Twelve patients (mean age, 52 years), selected at random from the male cohort, were compared with 12 age-matched male control subjects (mean age, 52 years) and with 10 normal, young male subjects (mean age, 25 years). Whole-blood flow cytometry was used to measure platelet activation status ex vivo and platelet responsiveness to physiological agonists in vitro. Peripheral blood samples were analyzed for bound fibrinogen and expression of P-selectin, GPIb, and GPIIb-IIIa at rest and in response to ADP (0.1 to 10 mumol/L) and thrombin (0.02 to 0.32 mu/mL). No significant differences were seen in the basal levels of fibrinogen binding between any of the groups, but P-selectin expression was significantly lower in patients compared with age-matched control subjects (P = .0005). When stimulated with agonists, patients' platelets had significantly decreased fibrinogen binding (P < .03) but no difference in P-selectin expression compared with the age-matched group. Both agonist-induced fibrinogen binding and P-selectin expression were, however, higher in the young subjects compared with either the older control group or the patients (P < .05). GPIb and GPIIb-IIIa expression were lowest in the patients with angina and highest in the young control subjects, with levels in the age-matched control subjects falling between these values. Data from the total patient cohort (n = 65) were identical to those in the smaller cohort (n = 12). In conclusion, atherosclerosis impairs platelet aggregatory responses (fibrinogen binding) over and above the decreased response seen with age. Platelet degranulation (P-selectin expression) is also impaired in patients with coronary artery disease, but only in comparison with younger subjects, not age-matched controls.

A sensitive flow cytometric assay for circulating platelet-leucocyte aggregates.

A whole blood flow cytometric method for studies of platelet-leucocyte aggregates (PLAs) in vivo, involving neither fixation nor centrifugation, is described. With this method, PLAs in the total leucocyte population (PLA/L) were 15.3 +/- 8.5% in 36 healthy volunteers. Blocking antibodies had little effect on PLAs in the absence of in vitro stimulation, suggesting that the aggregates were preformed in vivo. Fixation with formaldehyde or paraformaldehyde increased PLA/L significantly. Similarly, prefixation of the blood or red blood cell lysis, with repeated washing and centrifugation, caused artefactual 3-5-fold increases in PLAs (P<0.05). ADP, thrombin, platelet activating factor (PAF) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) each increased PLA formation in unfixed whole blood dose-dependently: additive effects were found when they were combined. Experiments with blocking MAbs suggested that different ligand-receptor systems mediate PLA formation by different agonists. PLA formation by the platelet agonist ADP was inhibited by P-selectin blockade, but enhanced by GPIIb/IIIa blockade (which inhibits platelet-platelet interactions). PLA formation by the leucocyte agonist fMLP was inhibited by GPIIb/IIIa blockade, suggesting linking via fibrinogen. Platelet-leucocyte aggregate analysis by this whole blood method appears to reflect in vivo conditions, and enables investigations of the mechanisms involved in their formation.

Flow cytometric detection of activated platelets in pregnant women prior to the development of pre-eclampsia.

Pre-eclampsia is a common complication of pregnancy, in which platelets may have an early pathogenetic role. In this prospective study a whole blood flow cytometric method has been used to detect circulating activated platelets in pregnant women prior to the development of pre-eclampsia. Activated platelets were identified by bound fibrinogen or by CD63 antigen expression. Of 121 healthy primiparous women studied at 28 weeks of pregnancy, 18 (15%) developed clinical pre-eclampsia six to thirteen weeks later. The platelets of these women showed increased fibrinogen binding ex vivo (5.1% platelets positive, compared with 3.4% in those who completed a normal pregnancy, p < 0.02), and increased CD63 antigen expression (0.73% positive compared to 0.45%, p = 0.01). In contrast, no differences between the women with different outcomes were detected at 28 weeks in platelet counts, or plasma beta-thromboglobulin levels. These findings confirm that whole blood flow cytometry is a sensitive technique for investigating platelet activation in a clinical setting and support the hypothesis that platelets have a critical role in the pathogenesis of pre-eclampsia.

Aspirin does not affect the flow cytometric detection of fibrinogen binding to, or release of alpha-granules or lysosomes from, human platelets.

1. Aspirin inhibits the conversion of arachidonic acid to thromboxane A2 which reinforces the effects of weak agonists such as ADP in platelets. 2. In this study the effect of aspirin (300 mg/day) on platelet agonist response was measured by whole blood flow cytometry of unfixed blood samples from normal subjects (n = 10), an assay that investigates aggregation-independent changes in the platelet. 3. Fibrinogen binding to unstimulated platelets or to platelets stimulated with ADP or thrombin was unaffected by aspirin. 4. Under the conditions of this assay, platelets undergo a partial degranulation of alpha-granules and lysosomes (evidenced by expression of P-selectin and CD63, respectively) in response to ADP, and full degranulation in response to thrombin. P-selectin expression was paralleled by release of beta-thromboglobulin. None of these events was affected by aspirin. 5. Thromboxane formation was totally prevented by the aspirin treatment, as shown by Born aggregometry in which the platelet aggregatory response to arachidonic acid was abolished and secondary aggregation by ADP was inhibited. 6. The flow cytometric assay can therefore be used to investigate platelets in patients, regardless of aspirin therapy. 7. These findings suggest that platelet fibrinogen binding and the release of platelet alpha-granule and lysosomal contents, in response to stimulation with physiological agonists, can continue in patients despite aspirin therapy. This may help to explain why aspirin is only partially effective in preventing thrombotic events.

Bone marrow transplantation for Glanzmann's thrombasthenia.

Allogeneic matched bone marrow transplantation (BMT) was performed in a patient with type I Glanzmann's thrombasthenia, a rare, inherited bleeding disorder caused by a deficiency in the platelet membrane glycoprotein IIb-IIIa complex. The patient was a 2-year-old girl with a history of frequent hospitalisation. She was successfully transplanted with BM from her HLA-identical sibling. Engraftment was monitored by analysis of the platelet GPIIb-IIIa complex and by RFLP analysis using a minisatellite probe. Complete engraftment was seen at day +25. The patient has been clinically stable for 19 months. It is proposed that BMT is a suitable treatment for this condition where a matched, related donor is available and at an early stage, before the development of anti-platelet antibodies as a result of repeated transfusions.

Flow cytometric detection of circulating activated platelets and platelet hyper-responsiveness in pre-eclampsia and pregnancy.

1. Platelet activation status and response to stimulation with agonists ex vivo were studied by whole blood flow cytometry in 15 women with pre-eclampsia, 20 age- and gestational-age-matched women who completed a normal pregnancy, and 20 age-matched non-pregnant women. 2. Women with proteinuric pre-eclampsia showed evidence of activated, degranulated platelets in the circulation, with increased platelet-bound fibrinogen, increased expression of the lysosomal granule membrane antigen, CD63, and raised plasma levels of beta-thromboglobulin. 3. CD63 expression and beta-thromboglobulin per platelet were also significantly higher in normal pregnant women than in non-pregnant women, but in these subjects fibrinogen binding was normal. 4. There was good correlation for all subjects in degranulation, measured by CD63 antigen expression, and by plasma beta-thromboglobulin levels corrected for platelet count (r = 0.65; P < 0.01). 5. Platelet responsiveness to ADP in vitro showed a heightened degranulation response (CD63 expression) in normal pregnancy compared with the non-pregnant control group, which was increased further in women with non-proteinuric and proteinuric pre-eclampsia. 6. However, this response was not accompanied by an increased binding of fibrinogen to GPIIb-IIIa. Fibrinogen binding in response to 'weak' agonist stimulation, by low concentrations of ADP or, in a subgroup by adrenaline, was in fact lower in the normal pregnant women than in the non-pregnant women. 7. It is postulated that women at risk of developing pre-eclampsia may have hyper-reactive platelets, primed to undergo release by passage through the abnormal placenta.

ADP causes partial degranulation of platelets in the absence of aggregation.

Whole blood flow cytometry has revealed that platelets undergo partial degranulation in response to ADP, in the absence of aggregation, as evidenced by the expression of the P-selectin and CD63 antigens of the alpha-granule and lysosomal membranes respectively. With maximum ADP (10(-5) M) fibrinogen bound to 76.1 +/- 7.2% of platelets but P-selectin and CD63 antigen were expressed on 26.9 +/- 9.8% and 8.6 +/- 3.5% of platelets respectively. Maximum fibrinogen binding, P-selectin and CD63 expression induced by alpha-thrombin were 96.1 +/- 1.4%, 92.8 +/- 2.3% and 77.6 +/- 9.7% respectively. beta-thromboglobulin release from the ADP-stimulated platelets correlated closely with the expression of P-selectin and CD63 (r = 0.98 +/- 0.02 for both antigens). No platelet aggregates were seen by flow cytometry and the absence of aggregation was confirmed by single cell counting. Addition of the GPIIb-IIIa antagonist echistatin, at concentrations that totally blocked fibrinogen binding to ADP-stimulated platelets, had no effect on the expression of the granule membrane antigens. The partial degranulation of normal platelets was independent of thrombin generation since it was not inhibited by hirudin (5 units/ml). In conclusion, ADP is capable of causing partial degranulation of platelets independently of aggregation, fibrinogen binding or thrombin generation. Thus release of potent procoagulant, vasoactive and mitogenic substances from the platelets could continue in the presence of thrombin inhibitors and GPIIb-IIIa antagonists.

Epinephrine sensitizes human platelets in vivo and in vitro as studied by fibrinogen binding and P-selectin expression.

Epinephrine (Epi) infusion influences platelet activation markers in vivo, but in vitro studies have mainly examined supraphysiological Epi concentrations and have yielded conflicting results. In this study whole-blood flow-cytometric measurements of platelet fibrinogen binding and P-selectin expression were used to compare enhancement of ADP (0.1 to 10 mumol/L)-induced platelet activation by Epi infusion in vivo (0.1 and 0.4 nmol.kg-1.min-1) and by Epi in vitro (10 and 50 nmol/L) in nine healthy volunteers. ADP caused concentration-dependent increases in the percentage of platelets that bound fibrinogen (from 4.4 +/- 0.9% to 69.9 +/- 4.2%) and that expressed P-selectin (from 4.5 +/- 0.5% to 44.2 +/- 3.8%). Fibrinogen and P-selectin binding indices (FgBI and PSBI; calculated from mean fluorescence intensity and percentage of positive cells) also increased from 0.18 +/- 0.03 to 11.70 +/- 1.99 for FgBI and from 0.22 +/- 0.03 to 2.34 +/- 0.29 for PSBI. Epi concentration-dependently enhanced fibrinogen binding and P-selectin expression in vitro (by approximately 30% at the midportion of the ADP curve at 10 nmol/L Epi; P < .001 for both by ANOVAs). High-dose Epi infusion enhanced FgBI similarly and increased maximal P-selectin expression by 38%. Epi (50 nmol/L in vitro) enhanced platelet activation further, whether samples were taken with or without prior Epi infusion. Total expression of glycoprotein IIb/IIIa was unaffected by Epi infusion, but glycoprotein Ib expression per platelet was reduced (P < .05). These in vivo and in vitro effects of Epi on platelet responses to agonist stimulation indicate a prothrombotic potential for sympathoadrenal activation in humans.

Evaluation of whole blood flow cytometric detection of platelet bound fibrinogen on normal subjects and patients with activated platelets.

Activated platelets can be detected by measuring platelet-bound fibrinogen in a whole blood, flow cytometric assay, using a fluorescently-conjugated polyclonal antibody. Fibrinogen binding to unstimulated platelets from normal subjects was low in this assay, as was expression of the CD63 antigen. Single cell counting of samples prepared for flow cytometric analysis showed platelet aggregates do not form during the assay procedure. Immune complexes were not seen, and fibrinogen binding to the platelets was unaffected by the CD32 MAb, IV.3. Artefactual activation of the unfixed samples could be minimised by control of phlebotomy, time and temperature of incubation. Variations in platelet count in the range 140-430 x 10(9) 1(-1) and in plasma fibrinogen in the range 2-6 g 1(-1) did not affect the assay results. Comparison of fibrinogen binding with expression of CD63 antigen on normal platelets, stimulated with agonists in vitro, demonstrated that fibrinogen binding detects an earlier stage of platelet activation. Platelet bound fibrinogen was shown to be sensitive in detecting small numbers of activated platelets in clinical samples in twelve patients on intensive care, four undergoing haemofiltration. The patients had a significantly higher median percentage of circulating platelets with bound fibrinogen (p < 0.005), but fibrinogen binding was significantly lower (p < 0.02) in response to 10(-5) M ADP, compared to twelve age-matched normal controls.

Platelet activation during preparation and storage of concentrates: detection by flow cytometry.

This paper describes a rapid, whole blood micro-method for assessing the activation status of platelets that can be used to monitor changes occurring during the preparation and storage of platelet concentrates. Platelets are examined for the expression of markers of early activation (fibrinogen binding) and of degranulation (GP53 and GMP-140 expression), using flow cytometry. Fibrinogen binding can be detected in a number of blood packs even before processing. Platelet concentrates prepared by the washed platelet method become further activated during centrifugation and separation. More than 90% of platelets in freshly prepared concentrates carried bound fibrinogen (90.9 +/- 7.7%) whilst the GP53 and GMP-140 antigens were expressed on 11.3 +/- 3.3% and 13.7 +/- 5.1 of these cells respectively (mean +/- SD). Fibrinogen binding fell on storage of the platelets for four days, attributed to shedding of the fibrinogen molecule.