LETTERS TO THE EDITORS
levels have been associated with an increased risk of thrombosis, which increases during surgery [1,2,10]. Subjects with VWD are usually dosed on the basis of VWF:RCo. Therefore, maintaining lower postinfusion FVIII levels could possibly lower the risk of thrombosis. To date, there have been no adverse reactions with a thrombotic aetiology from either formal clinical trials or reporting of spontaneous adverse drug reactions with Optivate [2,6–9]. In addition, thrombogenicity data gathered from a separate Optivate study in 11 VWD patients (unpublished data) concluded there was no clinical evidence of thrombosis. Therefore, the data presented suggests that Optivate would be as safe and effective as other VWF/FVIII products in treating patients with VWD.
References 1 European Medicines Agency (EMA). Committee for Medicinal Products for Human Use (CHMP) guidelines on using human plasma-derived von Willebrand Factor (VWF) in clinical trials (CPMP/BPWG/220/ 02) Draft Jul 03. 2 Coppola A, Franchini M, Makris M, Santagostino E, Di Minno G, Mannucci PM. Thrombotic adverse events to coagulation factor concentrates for treatment of patients with haemophilia and von Willebrand disease: a systematic article of prospective studies. Haemophilia 2012; 18: e173–87. 3 Marshall PJ, Winkelman L, Lloyd J, Hardway C, Worthington N. Characterisation of Optivate, a high purity factor VIII concentrate with von Willebrand factor. Haemophilia 2004; 10: 136.
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Acknowledgements We are grateful to investigator and staff at Haematology Dept., Institute of Thrombosis and Hemostasis, Sheba Medical Centre, Israel for enrolling and managing the patients into the cross-over clinical PK study. In addition, BPL thanks all the staff at this centre for processing the study coagulation samples. CD and RS-Z were involved the management and oversight of the clinical trial. All the authors were involved in the preparation of this article.
Disclosures This article presents data from trials funded by Bio Products Laboratory Ltd (BPL). AI (Principal Investigator) and AL (Co-Investigator) participated in the clinical study funded by BPL. CD has worked as an independent Medical Director for BPL. RS-Z has been contracted by BPL to work as a Clinical Research Consultant. All the authors have no other potential conflicts of interest and have been involved in the preparation of this article.
4 Bolsa H, Kingsland S, Feldman P. Analysis of the composition of a factor VIII concentrate, Optivateâ. Haemophilia 2012; 18: 33 PO-TU-030. 5 Riddell A, McKinnon T, Vinayagam S, Gomez K Evaluation of von Willebrand factor (VWF) concentrates by platelet adhesion to type III collagen using a high shear in vitro flow assay. J Thromb Hemost 2011; 9: PTH-453, 910–1. 6 Dmoszynska A, Hellmann A, Baglin T et al. Pharmacokinetics of Optivateâ, a high-purity concentrate of factor VIII with von Willebrand factor, in patients with severe haemophilia A. Haemophilia 2011; 17: 185–90. 7 Dmoszynska A, Kuliczkowski K, Hellmann A et al. Clinical assessment of Optivateâ, a high-purity concentrate of factor VIII with von Willebrand factor, in the management
of patients with haemophilia A. Haemophilia 2011; 17: 456–62. 8 Hay C, Hellmann A, Dmoszynska A et al. Experience with Optivateâ, a new high purity concentrate of factor VIII with von Willebrand factor, in patients undergoing surgery. Haemophilia 2011; 17: 428–32. 9 Matysiak M, Bobrowska H, Balwierz W et al. Clinical experience with Optivateâ, high-purity factor VIII (FVIII) product with von Willebrand factor (VWF) in young children with Haemophilia A. Haemophilia 2011; 17: 737–42. 10 Kessler C, Friedman K, Schwartz B, Gill J, Powell J. The pharmacokinetic diversity of two con Willebrand factor (VWF)/factor VIII (FVIII) concentrates in subjects with congenital von Willebrand disease. J Thromb Hemost 2011; 106: 279–88.
Coated platelet assay: a feasible approach to a complicated science B. M. MOHAMMED,*† D. CONTAIFER JR.,* K. K. LASTRAPES,*‡ E. J. MARTIN,* M. A. MAZEPA,§ M. HOFFMAN,¶** D. M. MONROE** and D. F. BROPHY* *Coagulation Advancement Laboratory, Department of Pharmacotherapy & Outcomes Science, Virginia Commonwealth University, Richmond, VA, USA; †Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Giza, Egypt; ‡Department of Pediatric Hematology/Oncology, Children’s Hospital of Richmond and Virginia Commonwealth University Health System, Richmond, VA; §Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC; ¶Department of Pathology, Duke University and Durham Veterans Affairs Medical Centers, Durham, NC; and **Department of Medicine, University of North Carolina, Chapel Hill, NC, USA
Correspondence: Bassem M. Mohammed, PhD, Virginia Commonwealth University, PO Box 980533, Richmond, VA, USA. Tel.: 804 827 1455; fax: 804 828 0343; e-mail: [email protected] Accepted after revision 23 September 2015 DOI: 10.1111/hae.12845 © 2015 John Wiley & Sons Ltd
Coated platelets (CP) are a subpopulation of platelets with enhanced prothrombinase complex activity that develop following simultaneous dual activation with agonists (collagen and thrombin) [1,2]. Previous methods used to quantify CP have been described in the literature [3,4], however, they involved non-clinical standard blood collection procedures, multistep plateHaemophilia (2016), 22, e36--e79
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LETTERS TO THE EDITORS
let preparation techniques including several labelling and washing steps, and/or the use of agonists of animal origin. In addition, these reports often lack completeness and/or clarity thus limiting reproducibility of the assay. Herein, we report a simple, reproducible assay that can bring CP screening closer to clinical laboratory practice and help standardize the methodology. We used clinic-standard blood collection procedures, minimum platelet manipulation techniques including a single-step platelet labelling procedure. Moreover, unlike previous methods that used bovine thrombin to boost CP signal detection, we used human thrombin, which is more physiologically relevant, to obtain a detectable CP signal. There has been interest in monitoring CP levels clinically as various reports link them to procoagulant disorders such as stroke and perhaps a mild bleeding phenotype in haemophilia [5,6]. Anatomically CPs localize preferentially at sites of collagen exposure and initiate thrombin generation. Hence, the in vitro use of the dual agonists (thrombin and collagen) confers physiological relevance [7]. Nevertheless, other collagen and thrombin receptor agonists, as well as single agonist/agent have also been reported to successfully produce similar CP population in vitro [1,2]. Unlike traditionally activated platelets, CP display a strong affinity in retaining factor V (FV), FVIIa, FVIII, FIX, FX [8] and other procoagulant components of platelets a-granules (e.g. fibrinogen, von-Willebrand factor and fibronectin) onto their surfaces. CP have a AV
characteristic prominent phosphatidylserine exposure on their surfaces and inactivation of integrin GP IIb/ IIIa [1,2]. These two characteristics formulated the basis for our CP flow-cytometry assay. By virtue of its surface features, CPs interactions are deemed critical for thrombus formation and structure [2,7]. The average percentage of CPs in healthy volunteers is reported to be 30% (range 15–55%) of the total platelet population [1]. In addition, the percentage of CP formed by a given individual tends to be stable over time as long as their baseline characteristics prevail. Some medications have been found to modulate CP levels. For instance, desmopressin was found to correct platelet dysfunction in part through increasing CP formation, whereas clopidogrel partially attenuates CP formation as a part of its antithrombotic properties [9,10]. Because CP may present a unique pathophysiology of thrombosis as well as a novel target for pharmacotherapy, we developed the following simple flow cytometric screening assay method.
Reagents and materials Human thrombin (T7009), Gly-Pro-Arg-Pro (GPRP) tetra-peptide inhibitor of fibrin polymerization, serum bovine albumin, magnesium chloride and calcium chloride dehydrate were all obtained from Sigma Aldrich (St. Louis, MO, USA). Convulxin was obtained from Enzo Life Sciences (Farmingdale, NY,
PAC-1
PERCENTAGE COATED PLATELETS (%)
(a) 70.00% 60.00%
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40.00%
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Percentage COATed plarelets(%)
(b)
Donor 1
DONOR 10
DONOR 11 DONOR 12
Donor 2
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10.00%
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Haemophilia (2016), 22, e36--e79
6
7 1 Timepoint
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Fig. 1. (a) Percentage coated platelets for the twelve donors as determined using the single marker approach. Positive Annexin-V binding (blue) and negative PAC-1 binding (red). (b) Percentage coated platelets obtained by averaging Annexin-V and PAC-1 CP findings over seven time points for donor 1 (brown) and donor 2 (green).
© 2015 John Wiley & Sons Ltd
LETTERS TO THE EDITORS
USA); sodium chloride from Fisher Scientific (Pittsburg, PA, USA); HEPES-buffered solution (pH 7.2–7.4) from Boston BioProducts (Ashland, MO, USA); phosphate-buffered saline and formalin from Beckman Coulter (Fullerton, CA, USA). Fluorescent conjugated Annexin V-FITC (marker for highly exposed phosphatidylserine) and PAC1-FITC (marker for GPIIb/IIIa activation) were obtained from BD Biosciences (San Jose, CA, USA). Coated platelet buffer (250 mM NaCl, 16.67 mM HEPES, 3.33 mM CaCl2 and 1.67 mM MgCl2 in deionized water) buffer, was prepared fresh on the week of participants’ enrolment. Convulxin was reconstituted in 1 mL 10 mM HEPES (pH 7.3) containing 1% bovine serum albumin (BSA) to a final concentration 50 lg mL 1. GPRP tetra-peptide was reconstituted in deionized water to give a 10 mM stock solution and was then aliquoted and stored at 20°C. Human thrombin was reconstituted to a final concentration of 50 U mL 1 using deionized water and 0.1 M NaCl (ratio 1:4). Both thrombin and convulxin aliquoted stocks were further diluted (1:10), before use, using deionized water containing 1% BSA. We found it essential to verify convulxin potency with each new lot and to avoid freeze and thaw cycles. Comparing stored convulxin and freshly prepared convulxin, showed that stored aliquots at 80°C are stable for ~1 month. Fluorescent reagents, salt solutions and buffers were stored at 4°C, except for GPRP which was stored at 20°C, all other aliquots were stored at 80°C.
10 10 10 10 10
(a)
10 10 10 10
6
PLT 97.2%
5
10
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4
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Annexin-V (+ve) 60.4%
200
3
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4
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300
6
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Blood was obtained from 12 healthy human volunteers (>18 years, with no bleeding diathesis) into standard sodium citrate (3.2%) BD vacutainersâ (Franklin Lakes, NJ, USA) using a 21-gauge butterfly blood collection set. Two donors of the 12 were sampled seven times, each at least 1 month apart from the respective donor previous sampling time point. Blood was centrifuged (100 g, 10 min) to obtain platelet-rich plasma (PRP). The platelet count was adjusted to 100 000 lL 1 using platelet-poor plasma (PPP) from the respective subject. PPP was obtained by further spinning the blood tubes at higher speed (2000 g for 10 min). Adjusted PRP was diluted (1:10) using sterile normal saline. Two tubes were prepared per participant; one for Annexin V-FITC and the other for PAC-1-FITC. 60 lL of coated platelet buffer was added to each tube followed by 5 lL of the GPRP and 10 lL of each of the diluted agonists, namely thrombin and convulxin, followed by 5 lL of the respective fluorescent reagent. 10 lL of diluted adjusted PRP was then added last to assure simultaneous activation with the agonists. The 100 lL assay had the following final concentrations 150 mM NaCL, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 0.5 mM GPRP, 0.5 U mL 1 human thrombin, 500 ng mL 1 convulxin and 5 lL Annexin
7
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Coated platelet assay
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Fig. 2. Representative flow Cytometric analysis of coated platelets using Annexin-V-FITC and PAC-1-FITC staining for donor 10. (a) shows the pre-set gate used to identify platelets by their forward and side scatter characteristics. Events falling within the platelet gate were then identified as coated platelets by staining positive for Annexin V-FITC binding (b and c) or negative for PAC-1-FITC binding (d and e). (PLT, Platelet).
© 2015 John Wiley & Sons Ltd
Haemophilia (2016), 22, e36--e79
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V-FITC or PAC-1-FITC. Tubes were incubated in the dark at 37°C for 5 min for Annexin V-FITC and for 20 min for PAC-1-FITC. 100 lL of fixative (2% formalin) was then added and the tubes were processed immediately on the flow cytometer (BD AccuriTM C6, San Jose, CA, USA). The assay was performed under the following conditions: Fluidics: Fast; Forward scatter threshold: 30 000; and 10 000 events were collected in a preset platelet gate using standard methods including CD41a conjugated with PE-Cy5 (mouse anti-human: BD Pharmingen, Franklin Lakes, NJ, USA) as a platelet marker. The collected data were analysed using FlowJo version 7.6.5. Using Annexin-V-FITC binding alone, donors had an average (SD) of 43.3% (13) CP as detected by positive Annexin-V-FITC binding. Using PAC-1-FITC binding alone, donors had an average (SD) of 44.2% (11) as detected by negative PAC-1-FITC binding. Figure 1a shows the %CP for each of the 12 donors using a single marker approach. For the two donors that were sampled over time, using Annexin-V-FITC binding alone, donors 1 and 2 had an average (SD) of 43.4% (2.9) and 22.4% (3.3) CP as detected by positive Annexin-V-FITC binding, respectively, when sampled overtime. Using PAC-1-FITC binding alone, donors 1 and 2 had an average (SD) of 45.3% (3.8) and 26.9% (2.6) of CP as detected by negative PAC1-FITC binding, respectively, when sampled overtime. A more conservative approach was to average the percentage CP detected using both markers. Using the later approach donor 1 was found to have 44.3%
References 1 Dale G. Coated-platelets: an emerging component of the procoagulant response. J Thromb Haemost 2005; 3: 2185–92. 2 Mazepa M, Hoffman M, Monroe D. Superactivated platelets: thrombus regulators, thrombin generators, and potential clinical targets. Arterioscler Thromb Vasc Biol 2013; 33: 1747–52. 3 Prodan C, Joseph P, Vincent A, Dale G. Coated-platelet levels are influenced by smoking, aspirin, and selective serotonin reuptake inhibitors. J Thromb Haemost 2007; 5: 2149–51. 4 Knudsen T, Kjalke M, Tranholm M, Nichols TC, Jensen AL, Kristensen AT. Development of a flow cytometric assay for detection of coated platelets in dogs
Haemophilia (2016), 22, e36--e79
(2.7), whereas donor 2 had 24.3% (2.7) CP over an 8 to 10 month period. We found that averaging % CP using the two markers approach to be more stable and resistant to non-interventional and/or nonpathological variability over time (Fig. 1b). The coefficient of variation for the single marker assays overtime averaged (range) 10% (6.6–14.6); while it was 8.5% (6–11) when using the percentage CP calculated as average of the two markers. Figure 2 is a representative flow cytometric analysis of CP population from donor 10. In conclusion, we developed a rigorous yet simple assay that involved minimal platelet manipulation, thus minimizing assay-induced artefacts. The average CP observed in this report is in line with the documented average in the literature. The added feasibility may take this assay a step closer from the bench to the clinical laboratory, providing a fast and accurate screening tool that meets the growing need to rapidly quantify CP.
Author contributions BM, DC, KL, and MM performed the research; BM and DB wrote the manuscript; BM performed the data analysis; EM and KL coordinated the study; DB supervised the study and MH and DM assessed the assay and critically revised the important content of this article.
Disclosures Drs Brophy, Hoffman and Monroe have received research funding from Novo Nordisk A/S. The authors stated that they had no interests which might be perceived as posing a conflict or bias.
and evaluation of binding of coated platelets to recombinant human coagulation factor VIIa. Am J Vet Res 2011; 72: 1007–14. 5 Kirkpatrick AC, Tafur AJ, Vincent AS, Dale GL, Prodan CI. Coated-platelets improve prediction of stroke and transient ischemic attack in asymptomatic internal carotid artery stenosis. Stroke 2014; 45: 2995–3001. 6 Saxena K, Pethe K, Dale G. Coated-platelet levels may explain some variability in clinical phenotypes observed with severe hemophilia. J Thromb Haemost 2010; 8: 1140–2. 7 Shi J, Pipe SW, Rasmussen JT, Heegaard CW, Gilbert GE. Lactadherin blocks thrombosis and hemostasis in vivo: correlation with platelet phosphatidylserine
exposure. J Thromb Haemost 2008; 6: 1167–74. 8 Kempton CL, Hoffman M, Roberts HR, Monroe DM. Platelet heterogeneity: variation in coagulation complexes on platelet subpopulations. Arterioscler Thromb Vasc Biol 2005; 25: 861–6. 9 Colucci G, Stutz M, Rochat S, et al. The effect of desmopressin on platelet function: a selective enhancement of procoagulant COAT platelets in patients with primary platelet function defects. Blood 2014; 123: 1905–16. 10 Norgard NB, Saya S, Hann CL, Hennebry TA, Schechter E, Dale GL. Clopidogrel attenuates coated-platelet production in patients undergoing elective coronary catheterization. J Cardiovasc Pharmacol 2008; 52: 536–9.
© 2015 John Wiley & Sons Ltd
levels have been associated with an increased risk of thrombosis, which increases during surgery [1,2,10]. Subjects with VWD are usually dosed on the basis of VWF:RCo. Therefore, maintaining lower postinfusion FVIII levels could possibly lower the risk of thrombosis. To date, there have been no adverse reactions with a thrombotic aetiology from either formal clinical trials or reporting of spontaneous adverse drug reactions with Optivate [2,6–9]. In addition, thrombogenicity data gathered from a separate Optivate study in 11 VWD patients (unpublished data) concluded there was no clinical evidence of thrombosis. Therefore, the data presented suggests that Optivate would be as safe and effective as other VWF/FVIII products in treating patients with VWD.
References 1 European Medicines Agency (EMA). Committee for Medicinal Products for Human Use (CHMP) guidelines on using human plasma-derived von Willebrand Factor (VWF) in clinical trials (CPMP/BPWG/220/ 02) Draft Jul 03. 2 Coppola A, Franchini M, Makris M, Santagostino E, Di Minno G, Mannucci PM. Thrombotic adverse events to coagulation factor concentrates for treatment of patients with haemophilia and von Willebrand disease: a systematic article of prospective studies. Haemophilia 2012; 18: e173–87. 3 Marshall PJ, Winkelman L, Lloyd J, Hardway C, Worthington N. Characterisation of Optivate, a high purity factor VIII concentrate with von Willebrand factor. Haemophilia 2004; 10: 136.
e67
Acknowledgements We are grateful to investigator and staff at Haematology Dept., Institute of Thrombosis and Hemostasis, Sheba Medical Centre, Israel for enrolling and managing the patients into the cross-over clinical PK study. In addition, BPL thanks all the staff at this centre for processing the study coagulation samples. CD and RS-Z were involved the management and oversight of the clinical trial. All the authors were involved in the preparation of this article.
Disclosures This article presents data from trials funded by Bio Products Laboratory Ltd (BPL). AI (Principal Investigator) and AL (Co-Investigator) participated in the clinical study funded by BPL. CD has worked as an independent Medical Director for BPL. RS-Z has been contracted by BPL to work as a Clinical Research Consultant. All the authors have no other potential conflicts of interest and have been involved in the preparation of this article.
4 Bolsa H, Kingsland S, Feldman P. Analysis of the composition of a factor VIII concentrate, Optivateâ. Haemophilia 2012; 18: 33 PO-TU-030. 5 Riddell A, McKinnon T, Vinayagam S, Gomez K Evaluation of von Willebrand factor (VWF) concentrates by platelet adhesion to type III collagen using a high shear in vitro flow assay. J Thromb Hemost 2011; 9: PTH-453, 910–1. 6 Dmoszynska A, Hellmann A, Baglin T et al. Pharmacokinetics of Optivateâ, a high-purity concentrate of factor VIII with von Willebrand factor, in patients with severe haemophilia A. Haemophilia 2011; 17: 185–90. 7 Dmoszynska A, Kuliczkowski K, Hellmann A et al. Clinical assessment of Optivateâ, a high-purity concentrate of factor VIII with von Willebrand factor, in the management
of patients with haemophilia A. Haemophilia 2011; 17: 456–62. 8 Hay C, Hellmann A, Dmoszynska A et al. Experience with Optivateâ, a new high purity concentrate of factor VIII with von Willebrand factor, in patients undergoing surgery. Haemophilia 2011; 17: 428–32. 9 Matysiak M, Bobrowska H, Balwierz W et al. Clinical experience with Optivateâ, high-purity factor VIII (FVIII) product with von Willebrand factor (VWF) in young children with Haemophilia A. Haemophilia 2011; 17: 737–42. 10 Kessler C, Friedman K, Schwartz B, Gill J, Powell J. The pharmacokinetic diversity of two con Willebrand factor (VWF)/factor VIII (FVIII) concentrates in subjects with congenital von Willebrand disease. J Thromb Hemost 2011; 106: 279–88.
Coated platelet assay: a feasible approach to a complicated science B. M. MOHAMMED,*† D. CONTAIFER JR.,* K. K. LASTRAPES,*‡ E. J. MARTIN,* M. A. MAZEPA,§ M. HOFFMAN,¶** D. M. MONROE** and D. F. BROPHY* *Coagulation Advancement Laboratory, Department of Pharmacotherapy & Outcomes Science, Virginia Commonwealth University, Richmond, VA, USA; †Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Giza, Egypt; ‡Department of Pediatric Hematology/Oncology, Children’s Hospital of Richmond and Virginia Commonwealth University Health System, Richmond, VA; §Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC; ¶Department of Pathology, Duke University and Durham Veterans Affairs Medical Centers, Durham, NC; and **Department of Medicine, University of North Carolina, Chapel Hill, NC, USA
Correspondence: Bassem M. Mohammed, PhD, Virginia Commonwealth University, PO Box 980533, Richmond, VA, USA. Tel.: 804 827 1455; fax: 804 828 0343; e-mail: [email protected] Accepted after revision 23 September 2015 DOI: 10.1111/hae.12845 © 2015 John Wiley & Sons Ltd
Coated platelets (CP) are a subpopulation of platelets with enhanced prothrombinase complex activity that develop following simultaneous dual activation with agonists (collagen and thrombin) [1,2]. Previous methods used to quantify CP have been described in the literature [3,4], however, they involved non-clinical standard blood collection procedures, multistep plateHaemophilia (2016), 22, e36--e79
e68
LETTERS TO THE EDITORS
let preparation techniques including several labelling and washing steps, and/or the use of agonists of animal origin. In addition, these reports often lack completeness and/or clarity thus limiting reproducibility of the assay. Herein, we report a simple, reproducible assay that can bring CP screening closer to clinical laboratory practice and help standardize the methodology. We used clinic-standard blood collection procedures, minimum platelet manipulation techniques including a single-step platelet labelling procedure. Moreover, unlike previous methods that used bovine thrombin to boost CP signal detection, we used human thrombin, which is more physiologically relevant, to obtain a detectable CP signal. There has been interest in monitoring CP levels clinically as various reports link them to procoagulant disorders such as stroke and perhaps a mild bleeding phenotype in haemophilia [5,6]. Anatomically CPs localize preferentially at sites of collagen exposure and initiate thrombin generation. Hence, the in vitro use of the dual agonists (thrombin and collagen) confers physiological relevance [7]. Nevertheless, other collagen and thrombin receptor agonists, as well as single agonist/agent have also been reported to successfully produce similar CP population in vitro [1,2]. Unlike traditionally activated platelets, CP display a strong affinity in retaining factor V (FV), FVIIa, FVIII, FIX, FX [8] and other procoagulant components of platelets a-granules (e.g. fibrinogen, von-Willebrand factor and fibronectin) onto their surfaces. CP have a AV
characteristic prominent phosphatidylserine exposure on their surfaces and inactivation of integrin GP IIb/ IIIa [1,2]. These two characteristics formulated the basis for our CP flow-cytometry assay. By virtue of its surface features, CPs interactions are deemed critical for thrombus formation and structure [2,7]. The average percentage of CPs in healthy volunteers is reported to be 30% (range 15–55%) of the total platelet population [1]. In addition, the percentage of CP formed by a given individual tends to be stable over time as long as their baseline characteristics prevail. Some medications have been found to modulate CP levels. For instance, desmopressin was found to correct platelet dysfunction in part through increasing CP formation, whereas clopidogrel partially attenuates CP formation as a part of its antithrombotic properties [9,10]. Because CP may present a unique pathophysiology of thrombosis as well as a novel target for pharmacotherapy, we developed the following simple flow cytometric screening assay method.
Reagents and materials Human thrombin (T7009), Gly-Pro-Arg-Pro (GPRP) tetra-peptide inhibitor of fibrin polymerization, serum bovine albumin, magnesium chloride and calcium chloride dehydrate were all obtained from Sigma Aldrich (St. Louis, MO, USA). Convulxin was obtained from Enzo Life Sciences (Farmingdale, NY,
PAC-1
PERCENTAGE COATED PLATELETS (%)
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Haemophilia (2016), 22, e36--e79
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Fig. 1. (a) Percentage coated platelets for the twelve donors as determined using the single marker approach. Positive Annexin-V binding (blue) and negative PAC-1 binding (red). (b) Percentage coated platelets obtained by averaging Annexin-V and PAC-1 CP findings over seven time points for donor 1 (brown) and donor 2 (green).
© 2015 John Wiley & Sons Ltd
LETTERS TO THE EDITORS
USA); sodium chloride from Fisher Scientific (Pittsburg, PA, USA); HEPES-buffered solution (pH 7.2–7.4) from Boston BioProducts (Ashland, MO, USA); phosphate-buffered saline and formalin from Beckman Coulter (Fullerton, CA, USA). Fluorescent conjugated Annexin V-FITC (marker for highly exposed phosphatidylserine) and PAC1-FITC (marker for GPIIb/IIIa activation) were obtained from BD Biosciences (San Jose, CA, USA). Coated platelet buffer (250 mM NaCl, 16.67 mM HEPES, 3.33 mM CaCl2 and 1.67 mM MgCl2 in deionized water) buffer, was prepared fresh on the week of participants’ enrolment. Convulxin was reconstituted in 1 mL 10 mM HEPES (pH 7.3) containing 1% bovine serum albumin (BSA) to a final concentration 50 lg mL 1. GPRP tetra-peptide was reconstituted in deionized water to give a 10 mM stock solution and was then aliquoted and stored at 20°C. Human thrombin was reconstituted to a final concentration of 50 U mL 1 using deionized water and 0.1 M NaCl (ratio 1:4). Both thrombin and convulxin aliquoted stocks were further diluted (1:10), before use, using deionized water containing 1% BSA. We found it essential to verify convulxin potency with each new lot and to avoid freeze and thaw cycles. Comparing stored convulxin and freshly prepared convulxin, showed that stored aliquots at 80°C are stable for ~1 month. Fluorescent reagents, salt solutions and buffers were stored at 4°C, except for GPRP which was stored at 20°C, all other aliquots were stored at 80°C.
10 10 10 10 10
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Blood was obtained from 12 healthy human volunteers (>18 years, with no bleeding diathesis) into standard sodium citrate (3.2%) BD vacutainersâ (Franklin Lakes, NJ, USA) using a 21-gauge butterfly blood collection set. Two donors of the 12 were sampled seven times, each at least 1 month apart from the respective donor previous sampling time point. Blood was centrifuged (100 g, 10 min) to obtain platelet-rich plasma (PRP). The platelet count was adjusted to 100 000 lL 1 using platelet-poor plasma (PPP) from the respective subject. PPP was obtained by further spinning the blood tubes at higher speed (2000 g for 10 min). Adjusted PRP was diluted (1:10) using sterile normal saline. Two tubes were prepared per participant; one for Annexin V-FITC and the other for PAC-1-FITC. 60 lL of coated platelet buffer was added to each tube followed by 5 lL of the GPRP and 10 lL of each of the diluted agonists, namely thrombin and convulxin, followed by 5 lL of the respective fluorescent reagent. 10 lL of diluted adjusted PRP was then added last to assure simultaneous activation with the agonists. The 100 lL assay had the following final concentrations 150 mM NaCL, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 0.5 mM GPRP, 0.5 U mL 1 human thrombin, 500 ng mL 1 convulxin and 5 lL Annexin
7
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(e)
10
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PAC-1 (–ve) 56.0%
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FL1-A::FL1-A
Fig. 2. Representative flow Cytometric analysis of coated platelets using Annexin-V-FITC and PAC-1-FITC staining for donor 10. (a) shows the pre-set gate used to identify platelets by their forward and side scatter characteristics. Events falling within the platelet gate were then identified as coated platelets by staining positive for Annexin V-FITC binding (b and c) or negative for PAC-1-FITC binding (d and e). (PLT, Platelet).
© 2015 John Wiley & Sons Ltd
Haemophilia (2016), 22, e36--e79
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LETTERS TO THE EDITORS
V-FITC or PAC-1-FITC. Tubes were incubated in the dark at 37°C for 5 min for Annexin V-FITC and for 20 min for PAC-1-FITC. 100 lL of fixative (2% formalin) was then added and the tubes were processed immediately on the flow cytometer (BD AccuriTM C6, San Jose, CA, USA). The assay was performed under the following conditions: Fluidics: Fast; Forward scatter threshold: 30 000; and 10 000 events were collected in a preset platelet gate using standard methods including CD41a conjugated with PE-Cy5 (mouse anti-human: BD Pharmingen, Franklin Lakes, NJ, USA) as a platelet marker. The collected data were analysed using FlowJo version 7.6.5. Using Annexin-V-FITC binding alone, donors had an average (SD) of 43.3% (13) CP as detected by positive Annexin-V-FITC binding. Using PAC-1-FITC binding alone, donors had an average (SD) of 44.2% (11) as detected by negative PAC-1-FITC binding. Figure 1a shows the %CP for each of the 12 donors using a single marker approach. For the two donors that were sampled over time, using Annexin-V-FITC binding alone, donors 1 and 2 had an average (SD) of 43.4% (2.9) and 22.4% (3.3) CP as detected by positive Annexin-V-FITC binding, respectively, when sampled overtime. Using PAC-1-FITC binding alone, donors 1 and 2 had an average (SD) of 45.3% (3.8) and 26.9% (2.6) of CP as detected by negative PAC1-FITC binding, respectively, when sampled overtime. A more conservative approach was to average the percentage CP detected using both markers. Using the later approach donor 1 was found to have 44.3%
References 1 Dale G. Coated-platelets: an emerging component of the procoagulant response. J Thromb Haemost 2005; 3: 2185–92. 2 Mazepa M, Hoffman M, Monroe D. Superactivated platelets: thrombus regulators, thrombin generators, and potential clinical targets. Arterioscler Thromb Vasc Biol 2013; 33: 1747–52. 3 Prodan C, Joseph P, Vincent A, Dale G. Coated-platelet levels are influenced by smoking, aspirin, and selective serotonin reuptake inhibitors. J Thromb Haemost 2007; 5: 2149–51. 4 Knudsen T, Kjalke M, Tranholm M, Nichols TC, Jensen AL, Kristensen AT. Development of a flow cytometric assay for detection of coated platelets in dogs
Haemophilia (2016), 22, e36--e79
(2.7), whereas donor 2 had 24.3% (2.7) CP over an 8 to 10 month period. We found that averaging % CP using the two markers approach to be more stable and resistant to non-interventional and/or nonpathological variability over time (Fig. 1b). The coefficient of variation for the single marker assays overtime averaged (range) 10% (6.6–14.6); while it was 8.5% (6–11) when using the percentage CP calculated as average of the two markers. Figure 2 is a representative flow cytometric analysis of CP population from donor 10. In conclusion, we developed a rigorous yet simple assay that involved minimal platelet manipulation, thus minimizing assay-induced artefacts. The average CP observed in this report is in line with the documented average in the literature. The added feasibility may take this assay a step closer from the bench to the clinical laboratory, providing a fast and accurate screening tool that meets the growing need to rapidly quantify CP.
Author contributions BM, DC, KL, and MM performed the research; BM and DB wrote the manuscript; BM performed the data analysis; EM and KL coordinated the study; DB supervised the study and MH and DM assessed the assay and critically revised the important content of this article.
Disclosures Drs Brophy, Hoffman and Monroe have received research funding from Novo Nordisk A/S. The authors stated that they had no interests which might be perceived as posing a conflict or bias.
and evaluation of binding of coated platelets to recombinant human coagulation factor VIIa. Am J Vet Res 2011; 72: 1007–14. 5 Kirkpatrick AC, Tafur AJ, Vincent AS, Dale GL, Prodan CI. Coated-platelets improve prediction of stroke and transient ischemic attack in asymptomatic internal carotid artery stenosis. Stroke 2014; 45: 2995–3001. 6 Saxena K, Pethe K, Dale G. Coated-platelet levels may explain some variability in clinical phenotypes observed with severe hemophilia. J Thromb Haemost 2010; 8: 1140–2. 7 Shi J, Pipe SW, Rasmussen JT, Heegaard CW, Gilbert GE. Lactadherin blocks thrombosis and hemostasis in vivo: correlation with platelet phosphatidylserine
exposure. J Thromb Haemost 2008; 6: 1167–74. 8 Kempton CL, Hoffman M, Roberts HR, Monroe DM. Platelet heterogeneity: variation in coagulation complexes on platelet subpopulations. Arterioscler Thromb Vasc Biol 2005; 25: 861–6. 9 Colucci G, Stutz M, Rochat S, et al. The effect of desmopressin on platelet function: a selective enhancement of procoagulant COAT platelets in patients with primary platelet function defects. Blood 2014; 123: 1905–16. 10 Norgard NB, Saya S, Hann CL, Hennebry TA, Schechter E, Dale GL. Clopidogrel attenuates coated-platelet production in patients undergoing elective coronary catheterization. J Cardiovasc Pharmacol 2008; 52: 536–9.
© 2015 John Wiley & Sons Ltd
