Alison H. Goodall

Activation during preparation of therapeutic platelets affects deterioration during storage : a comparative flow cytometric study of different production methods

Three different separation methods, all using centrifugation, are routinely used to prepare therapeutic platelet concentrates from human donor blood. Platelet concentrates derived from platelet‐rich plasma (PRP‐PC), buffy coat (BC‐PC) and apheresis (AP‐PC) were investigated at the end of production, and over an 8 d storage period. Change in platelet surface markers were measured by flow cytometry, using fluorescein‐conjugated antibodies to fibrinogen, P‐selectin (CD62P), GPIIb–IIIa (CD41), GPIbα (CD42b) and GPV (CD42d), and fluorescein‐conjugated Annexin V was used to measure expression of anionic phospholipid.

Profound platelet degranulation is an important side effect of some types of contrast media used in interventional cardiology.

BackgroundThrombotic complications occurring during coronary angiography and percutaneous transluminal coronary angioplasty (PTCA) are relatively frequent and can be influenced by the type of radiographic contrast media used. Low osmolar contrast media (LOCM), both ionic and nonionic, have been considered to be safer than the older high osmolar contrast media (HOCM), causing less haemodynamic and symptomatic side effects. Recently, however, nonionic LOCM have been associated with an increased incidence of thrombotic events, including coronary occlusion and stroke. Methods and ResultsThe effects of commonly used contrast media on platelets in native blood were investigated using immunolabeling and flow cytometry to detect platelet activation in vitro. A nonionic LOCM (Omnipaque) caused profound platelet degranulation in nearly 80%o of platelets compared with 2 to 3% of platelets in the control. Conversely, an ionic HOCM (Urografin) caused only 25% degranulation, whereas an ionic LOCM (Hexabrix) caused no platelet activation and, furthermore, it inhibited the effects of thrombin on platelets. Platelet degranulation, quantified by immunolabeling, was paralleled by release of fthromboglobulin and platelet factor 4 from platelet a-granules. Blood from patients anticoagulated with heparin and pretreated with standard-dose aspirin in preparation for PICA showed the same pattern of contrast media-induced platelet activation as normal subjects. ConclusionsThese results suggest that the type of contrast media used during invasive imaging of the vasculature could have a significant effect on platelets. Platelet degranulation within a PTCA-damaged vessel would be increased by a nonionic contrast medium, releasing procoagulant molecules and platelet-derived growth factors into the damaged vessel lumen, which might contribute to acute thrombosis and the initiation of the restenosis process.

Alimentary lipemia enhances the membrane expression of platelet P-selectin without affecting other markers of platelet activation

The present study was conducted to determine whether alimentary lipemia alters platelet activity in vivo. Normolipidemic volunteers were given a fatty meal and platelet function was assessed before, and 3 and 6 h after the meal. Platelet aggregability and secretion was determined using whole blood flow cytometry (expression of platelet P-selectin and fibrinogen binding), filtragometry ex vivo (reflecting platelet aggregability in vivo) and by measurements of platelet specific products in plasma (beta-thromboglobulin and platelet factor 4). Plasma triglycerides increased from 0.8 (0.6:1.1; median, 25th and 75th percentiles) to 1.7 (1.0:2.3) mmol/l at 3 h and returned to baseline after 6 h (P < 0.001, one-way ANOVA). Apo B-100 and apo B-48 were both markedly increased 3 h postprandially in the Sf 60-400 fraction (large VLDLs, P < 0.001 for both), whereas the Sf 20-60 (small VLDLs) and Sf 12-20 fractions (IDL) did not change. The platelet function assessments revealed that the percentage of platelets expressing P-selectin increased by 40% (5%; 64%) after 3 h and by 51% (- 7%; 85%) 6 h postprandially in unstimulated samples (P < 0.05 for both). In samples stimulated by ADP in vitro P-selectin expression increased by 45% (6%; 58%) after 3 h and by 30% (12%; 58%) (P<0.01 for both) after 6 h at 0.1 microM. Platelet P-selectin expression was less influenced at higher ADP concentrations. The plasma levels of beta-thromboglobulin (approximately 20 ng/ml) and platelet factor 4 (approximately 0.3 ng/ml) were not affected by the fat load. Flow cytometric analyses of fibrinogen binding and filtragometry measurements also failed to reveal any postprandial alterations. The present finding of enhanced platelet P-selectin expression suggests that platelets are mildly sensitized postprandially. Whether this is of importance for thrombus formation and atherosclerosis needs to be studied further.

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.

Different Effects of Calcium Antagonists, Nitrates, and β-Blockers on Platelet Function Possible Importance for the Treatment of Unstable Angina

BACKGROUND The three major classes of antianginal drug all inhibit platelet aggregation at high concentrations in vitro, but detecting clinically relevant effects has proved to be more difficult. We used whole-blood flow cytometry, a sensitive method that allows direct measurement of activation antigens on the surface of individual platelets in whole unfixed blood, to evaluate the effect of representative antianginal drugs on platelet function in vivo in healthy volunteers. METHODS AND RESULTS The effects of glyceryl trinitrate (GTN), amlodipine, and atenolol were studied in nine normal volunteers. Fibrinogen binding to activated GP IIb/IIIa and expression of P-selectin, GP Ib, and GP IIb/IIIa on the platelet surface were measured. In addition, fibrinogen binding and P-selectin expression were measured in response to ex vivo stimulation with the agonists ADP and thrombin. The three drugs had very different effects on platelets. GTN inhibited platelet fibrinogen binding and expression of P-selectin at rest and in response to agonist stimulation, whereas amlodipine enhanced P-selectin expression and atenolol increased fibrinogen binding in response to agonists. Atenolol did not block the stimulatory effects of epinephrine on ADP-induced platelet activation. GTN neutralized the proactivatory effects of amlodipine, whereas the effects of atenolol and amlodipine were not additive. CONCLUSIONS The three main classes of antianginal medication have different and possible clinically relevant effects on platelet behavior in vivo, nitrates causing inhibition of aggregation (fibrinogen binding) and degranulation (P-selectin expression), calcium antagonists enhancing degranulation, and beta-blockers enhancing aggregation.Background The three major classes of antianginal drug all inhibit platelet aggregation at high concentrations in vitro, but detecting clinically relevant effects has proved to be more difficult. We used whole-blood flow cytometry, a sensitive method that allows direct measurement of activation antigens on the surface of individual platelets in whole unfixed blood, to evaluate the effect of representative antianginal drugs on platelet function in vivo in healthy volunteers. Methods and Results The effects of glyceryl trinitrate (GTN), amlodipine, and atenolol were studied in nine normal volunteers. Fibrinogen binding to activated GP IIb/IIIa and expression of P-selectin, GP Ib, and GP IIb/IIIa on the platelet surface were measured. In addition, fibrinogen binding and P-selectin expression were measured in response to ex vivo stimulation with the agonists ADP and thrombin. The three drugs had very different effects on platelets. GTN inhibited platelet fibrinogen binding and expression of P-selectin at rest...

A rapid one-step radiometric assay for hepatitis b surface antigen utilising monoclonal antibodies.

A two-site antigen assay for HBsAg has been developed that employs 3 monoclonal antibodies. The antibodies were selected for their high affinity and their particular epitope specificity to establish an assay with a sensitivity for the antigen comparable with that of a conventional assay with heterologous antisera. In addition, by selecting a monoclonal antibody for use as a tracer which does not compete for antigenic binding sites with the solid-phase monoclonal antibodies, it has been possible to perform a two-site assay in a single 1 h incubation step, achieving the same degree of sensitivity. This principle of using monoclonal antibodies in a one-step assay therefore gives advantages of speed and simplicity over assays using heterologous antisera and would be applicable to a variety of antigen assays for which appropriate monoclonal antibodies are available.

An immunoradiometric assay for human factor VIII/von Willebrand factor (VIII:vWF) using a monoclonal antibody that defines a functional epitope

Summary A murine monoclonal antibody has been produced (RFF‐VIII:R/2) that binds specifically to human factor VIII‐related antigen (VIII:RAg) in plasma and in vascular endothelial cells but has no reactivity with factor VIII procoagulant antigen (VIII:cAg). This antibody is a potent inhibitor of von Willebrand factor activity (VIII:vWF) in that it can totally neutralize ristocetin‐induced aggregation of platelet rich plasma and inhibit platelet adhesion at high flow rates. RFF‐VIII:R/2 can be used in a one‐stage, fluid phase immunoradiometric assay that can detect VIII:RAg at concentrations of 0.001 u/ml. This method has been used to analyse plasma from patients with von Willebrands disease (vWD). Results obtained in these patients showed a high degree of correlation between the monoclonally‐defined epitope and VIII:vWF levels measured by ristocetin‐induced aggregation of washed platelets. This correlation was maintained in those patients with the ‘variant’ types of vWD who exhibit highly disparate VIII:vWF and VIII: RAg levels when the latter is determined using polyclonal antisera. It appears that this monoclonal antibody recognizes a site on the VIII:RAg molecule which is associated with its interaction with the platelet membrane. Immunoradiometric assays using RFF‐VIII:R/2 offer a simplified, reproducible means of detecting functionally‐active VIII:RAg as an alternative or supplement to techniques involving platelet interactions.

ADP causes partial degranulation of platelets in the absence of aggregation

Summary. 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 α‐granule and lysosomal membranes respectively. With maximum ADP (10‐5m) 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 α‐thrombin were 96·1±1·4%, 92·8±2·3% and 77·6±9·7% respectively. β‐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.

A flow cytometric analysis of fibronectin binding to platelets from patients with peripheral vascular disease

Flow cytometry using fluorescently-conjugated antibodies has been used as a sensitive technique to study the expression of glycoproteins on the surface of resting and activated platelets (8-10). This technique permits analysis of single cells and thus can be used to detect heterogeneous expression of cell surface antigens or ligands (11). This paper reports the results of a flow cytometric analysis of platelet bound fibronectin to resting and thrombin-stimulated platelets from a group of 13 patients with peripheral vascular disease.

Identification of six functional clotting factor VIII:C epitopes by analysis of cross-reactive public idiotypes in murine monoclonal VIII:C inhibitors

Six factor VIII:C epitopes which can elicit clotting factor inhibitory antibodies were demonstrated by analysis of public idiotypes of murine monoclonal anti-VIII:C inhibitors. Anti-idiotypic immunoradiometric assays were developed with rabbit antibodies to murine VIII:C inhibitors: Synbiotics, Hybritech, C7F7 and RFF-VIIIC/8. Crossreactions among 10 murine monoclonal antibodies (MoAbs) to VIII:C were tested, showing 6 functional and immunospecific VIII:C epitopes. One epitope on the C-terminal, 80,000 dalton fragment of VIII:C was identified with cross-reaction among 3 MoAbs (Synbiotics, Chemicon, and IB3). Another unique site on this same fragment was recognized with C7F7. Two MoAbs (RFF-VIIIC/6 and RFF-VIIIC/8) defined another site with cross-reactive idiotypes on the N-terminal, 90,000 dalton fragment. Hybritech MoAb identified a fourth functionally distinct site to which no other cross-reacting MoAbs bound. A fifth functional locus was defined with RFF-VIIIC/2 which reacted with an N-terminal site (distinct from the RFF-VIIIC/6 X RFF-VIIIC/8-defined site). A sixth functional locus was recognized with RFF-VIIIC/5 which reacted with a C-terminal site (distinct from the Synbiotics/Chemicon/IB3-defined site but possibly near the C7F7-defined site). RFF-VIIIC/10 identified a non-functional locus on the middle region of VIII:C. These MoAb-based assays resolve six sites to which high-titered inhibitors are directed and offer a path to further study the immunoregulation of human anti-VIII:C inhibitors.