Evaluation of Whole Blood Flow Cytometric Detection of Platelet Bound Fibrinogen on Normal Subjects and Patients with Activated Platelets

Janes, Sarah L.; Wilson, Darren; Chronos, Nicolas; Goodall, Alison H.

doi:10.1055/s-0038-1649645

Cited by 103 publications

(85 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…When evaluated by flow cytometry in whole blood, platelets are sensitive to stimulation by ADP, especially with regard to fibrinogen binding. The threshold for ADP-induced enhancement of fibrinogen binding has been found to be 0.1 /imol/L, which agrees with previous data 24 - 25 and with proaggregatory effects of similar or even lower concentrations of ADP with filtragometry with whole blood in vitro. 27 The threshold for ADP-induced P-selectin expression is somewhat higher (=0.3 mol/L), but the maximum expression of this variable is also lower.…”

Section: Discussionsupporting

confidence: 91%

“…32 However, platelet contact and aggregation are avoided in the flow-cytometric assay. 25 It is possible that the thromboxane generation that occurs in citrated blood could enhance the effects of ADP, but we have shown that ADP-induced fibrinogen binding is unaffected by aspirin in citrated samples. 33 Alternatively, hirudin may inhibit low-level thrombin generation that augments the effects of ADP.…”

Section: Discussionmentioning

confidence: 69%

“…The flow-cytometric analysis of platelets from whole blood has been described 25 and is based on the methods of Shattil et al 26 and Warkentin et al 24 Briefly, within 10 minutes of collection 5 ^L of whole blood was added to 50 jiL of The fixed samples were analyzed in a Coulter EPICS Profile II flow cytometer (Coulter Electronics Ltd, Luton, UK) within 2 hours of collection. The flow cytometer, which produces a laser beam at 488 nm, was aligned daily with 10-^m Immuno Check and Standard Brite beads (Coulter Immunology) to calibrate the light-scatter and fluorescence parameters, respectively.…”

Section: Flow-cytometric Analysis Proceduresmentioning

confidence: 99%

See 2 more Smart Citations

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

Hjemdahl

Chronos

Wilson

et al. 1994

Arterioscler Thromb

View full text Add to dashboard Cite

Epinephrine (Epi) infusion influences platelet activation markers in vivo, but in vitro studies have mainly examined supraphysiologica] Epi concentrations and have yielded conflicting results. In this study whole-blood flowcytometric measurements of platelet fibrinogen binding and P-selectin expression were used to compare enhancement of ADP (0.1 to 10 /irnol/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 »»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 gfycoprotein Ilb/IIIa was unaffected by Epi infusion, but glycoprotein Ib expression per platelet was reduced (/*<.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.

show abstract

Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 69%

Section: Flow-cytometric Analysis Proceduresmentioning

confidence: 99%

See 1 more Smart Citation

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

Hjemdahl

Chronos

Wilson

et al. 1994

Arterioscler Thromb

View full text Add to dashboard Cite

show abstract

“…Platelets were isolated from EDTA-anticoagulated whole blood, resuspended in phosphate-buffered saline (PBS) containing 10 mM EDTA and 0.25% bovine serum albumin (BSA), and incubated with saturating concentration of mAb; the excess antibody was removed by 3 washes, followed by the addition of FITCconjugated goat anti-mouse Ig reagent (Dako). Platelet activation markers were analyzed in a whole-blood flow cytometric assay as previously described 18 using FITC-antifibrinogen (Dako), FITC-PAC-1 (BD Biosciences), or PE-anti-CD62P (Sanquin). Data were collected and analyzed using a Beckman Coulter XL-MCL flow cytometer (Beckman Coulter, High Wycombe, United Kingdom).…”

Section: Flow Cytometrymentioning

confidence: 99%

A nonsynonymous SNP in the ITGB3 gene disrupts the conserved membrane-proximal cytoplasmic salt bridge in the αIIbβ3 integrin and cosegregates dominantly with abnormal proplatelet formation and macrothrombocytopenia

Ghevaert

Salsmann

Watkins

et al. 2008

Blood

108

View full text Add to dashboard Cite

We report a 3-generation pedigree with 5 individuals affected with a dominantly inherited macrothrombocytopenia. All 5 carry 2 nonsynonymous mutations resulting in a D723H mutation in the ␤ 3 integrin and a P53L mutation in glycoprotein (GP) Ib␣. We show that GPIb␣-L53 is phenotypically silent, being also present in 3 unaffected pedigree members and in 7 of 1639 healthy controls. The ␤ 3 -H723 causes constitutive, albeit partial, activation of the ␣ IIb ␤ 3 complex by disruption of the highly conserved cytoplasmic salt bridge with arginine 995 in the ␣ IIb integrin as evidenced by increased PAC-1 but not fibrinogen binding to the patients' resting platelets. This was confirmed in CHO ␣ IIb ␤ 3 -H723 transfectants, which also exhibited increased PAC-1 binding, increased adhesion to von Willebrand factor (VWF) in static conditions and to fibrinogen under shear stress. Crucially, we show that in the presence of fibrinogen, ␣ IIb ␤ 3 -H723, but not wild-type ␣ IIb ␤ 3 , generates a signal that leads to the formation of proplatelet-like protrusions in transfected CHO cells. Abnormal proplatelet formation was confirmed in the propositus's CD34 ؉ stem cell-derived megakaryocytes. We conclude that the constitutive activation of the ␣ IIb ␤ 3 -H723 receptor causes abnormal proplatelet formation, leading to incorrect sizing of platelets and the thrombocytopenia observed in the pedigree. IntroductionInherited thrombocytopenias are a rare group of diseases with a wide spectrum of clinical phenotypes. Because of their rare occurrence, patients with an inherited low platelet count may be misdiagnosed with autoimmune thrombocytopenia (ITP) and receive inappropriate therapy. 1,2 Among the inherited thrombocytopenias, macrothrombocytopenia constitutes a subgroup in which Bernard-Soulier Syndrome (BSS), caused by mutations in the GP1BA, GP1BB, and GP9 genes, is the most common one, with large platelets and severe bleeding. 3 The molecular mechanisms of some of the rarer syndromes which are accompanied by macrothrombocytopenia have also been elucidated. Mutations in the myosin heavy-chain protein (MYH9) were identified in groups of patients with a spectrum of platelet disorders, such as the MayHegglin anomaly and the Epstein, Fechtner, and Sebastian syndromes. 4,5 The mode of inheritance of these disorders is generally autosomal recessive, but autosomal-dominant forms of a BSS-like disorder caused by nonsynonymous single-nucleotide polymorphisms (nsSNPs) in the GP1BA gene have also been reported. 6 More recently, mutations in the transcription factor GATA1 were defined as the cause of X-linked macrothrombocytopenia. 7 The most frequent autosomal-recessive platelet bleeding disorder is Glanzmann thrombasthenia (GT), which is caused by mutations in the ITGA2B or ITGB3 genes that encode for the integrin ␣ IIb ␤ 3 . This integrin, also named platelet glycoprotein (GP) IIbIIIa, is the the most abundantly expressed platelet membrane glycoprotein, 8 and its role is pivotal for platelet function. 9,10 Qualitative and quantitative defects...

show abstract

“…14,15 Briefly, fibrinogen binding to platelets was measured in unstimulated blood samples and in samples stimulated with ADP (1ϫ10 Ϫ6 mol/L; Sigma) by the binding of an FITCconjugated rabbit antibody to fibrinogen (Dako Ltd) to the platelets.…”

Section: Whole-blood Flow Cytometrymentioning

confidence: 99%

Beneficial Effects of Clopidogrel Combined With Aspirin in Reducing Cerebral Emboli in Patients Undergoing Carotid Endarterectomy

et al. 2004

View full text Add to dashboard Cite

Background-Postoperative thromboembolic stroke affects 2% to 3% of patients undergoing carotid endarterectomy (CEA) and is preceded by 1 to 2 hours of increasing cerebral embolization. Previous work has demonstrated that high rates of postoperative embolization are associated with increased platelet reactivity to adenosine 5Ј-diphosphate (ADP). Our hypothesis was that preoperative administration of the platelet ADP antagonist clopidogrel could reduce postoperative embolization. Methods and Results-One hundred CEA patients on routine aspirin therapy (150 mg) were randomized to 75 mg clopidogrel (nϭ46) or placebo (nϭ54) the night before surgery. Platelet response to ADP was assessed by whole-blood flow cytometry. The number of emboli detected by transcranial Doppler within 3 hours of CEA was independently quantified. Time taken from flow restoration to skin closure was used as an indirect measure of the time to secure hemostasis. In comparison with placebo, clopidogrel produced a small (8.8%) but significant reduction in the platelet response to ADP (PϽ0.05) while conferring a 10-fold reduction in the relative risk of those patients having Ͼ20 emboli in the postoperative period (odds ratio, 10.23; 95% CI, 1.3 to 83.3; Pϭ0.01, Fisher's exact test). However, in the clopidogrel-treated patients, the time from flow restoration to skin closure (an indirect marker of hemostasis) was significantly increased (Pϭ0.04, Fisher's exact test), although there was no increase in bleeding complications or blood transfusions. Conclusions-This is the first study to show that a CEA patient's postoperative thromboembolic potential can be significantly reduced by targeted preoperative antiplatelet therapy without increasing the risk of bleeding complications.

show abstract

Evaluation of Whole Blood Flow Cytometric Detection of Platelet Bound Fibrinogen on Normal Subjects and Patients with Activated Platelets

Cited by 103 publications

References 11 publications

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

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

A nonsynonymous SNP in the ITGB3 gene disrupts the conserved membrane-proximal cytoplasmic salt bridge in the αIIbβ3 integrin and cosegregates dominantly with abnormal proplatelet formation and macrothrombocytopenia

Beneficial Effects of Clopidogrel Combined With Aspirin in Reducing Cerebral Emboli in Patients Undergoing Carotid Endarterectomy

Contact Info

Product

Resources

About