Whole blood aggregometry and lumiaggregometry were performed using two 2-channel Chrono-Log 700 aggregometers (Havertown, PA). Citrated venous blood (800 μL) was introduced in plastic cuvettes and pre-incubated for 5 min at 37°C with 130 nM enalaprilat, then stimulated in stirring conditions (1,200 rev/min) with 60 μM ADP or 4 μg/mL collagen (Chrono-Log) for 5, 15 or 30 min. Luciferase was added to each sample to monitor ATP release (final total volume: 1 ml) and platelet aggregation was recorded by impedance measurement. The reaction was stopped by adding 4 mL of cold ethanol. Samples were then centrifuged for 15 min at 1,000 g and supernatants were stored at −80°C until analysis. Alternatively, citrated whole blood was preincubated with higher concentrations of enalaprilat (1 or 10 μM) and stimulated with 20% v/v of Kaolin (CK-Prest reagent, Stago, Asnières sur Seine, France), 4 μg/ml collagen, 10 μM of thrombin receptor PAR-1 activating peptide (TRAP-6, Bachem AG, Bubendorf, Germany) or 4 μg/mL collagen + 10 μM TRAP-6.
Trap 6
Manufactured by Bachem
Sourced in United States, Switzerland, Germany, United Kingdom
TRAP-6 is a synthetic peptide that activates platelets by targeting the protease-activated receptor 4 (PAR4). It is a common tool used in platelet research and ex vivo experiments.
Automatically generated - may contain errors
Lab products found in correlation
Adenosine diphosphate (adp), by Merck Group (4 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Pac 1 fitc, by BD (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Alexa fluor 488 labeled human fibrinogen, by Thermo Fisher Scientific (2 mentions)
Anti human cd62p phycoerythrin labeled murine antibodies, by BD (2 mentions)
Facscan flow cytometer, by BD (2 mentions)
Fitc anti p selectin mab, by BD (2 mentions)
Adenosine diphosphate (adp), by Chrono-Log (2 mentions)
Fluostar optima, by BMG Labtech (2 mentions)
Cellfix, by BD (1 mentions)
Vacutainer citrate 4.5 ml tubes, by BD (1 mentions)
Flow check, by Beckman Coulter (1 mentions)
Flow set fluorospheres, by Beckman Coulter (1 mentions)
Cd42b apc, by Beckman Coulter (1 mentions)
Cd62p pe, by Beckman Coulter (1 mentions)
Epinephrine, by Chrono-Log (1 mentions)
Cellquest pro software, by BD (1 mentions)
Blebbistatin, by Merck Group (1 mentions)
Reopro, by Eli Lilly (1 mentions)
29 protocols using trap 6
1
Platelet Aggregation Assay by Whole Blood Impedance
2
Platelet Activation Assay Protocol
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Vacutainer Citrate 4.5 mL tubes, Cellfix and antibodies PAC1-FITC, MsIgM-FITC and CD63-V450 BD were from BD Biosciences (Franklin Lakes, NJ, USA). Flow-Check, Flow-Set Fluorospheres and antibodies CD62P-PE, CD42b-APC and MsIgG-PE were from Beckman Coulter (Brea, CA, USA). Microtiter plates were from Bio-RAD (cat number CON960; CA, USA). Bovine serum albumin (BSA) was from Sigma-Aldrich (St Louis, MO, USA). ADP was from Sigma-Aldrich (St Louis, MO, USA), CRP-XL was purchased from Prof. Farndale (University of Cambridge, UK) and TRAP-6 was from Bachem (Bubendorf, Switzerland). Collagen and Epinephrine were from Chrono-Log Corp. (Haverton, PA, USA) and Ristocetin was from (American Biochemical & Pharmaceutical Corporation, Middlesex, U.K.). HEPES-Bovine serum albumin (BSA) buffer (20 mM Hepes, 137 mM NaCl, 2.7 mM KCL, 1 mM MgCl and 1% BSA).
3
Platelet Functionality Assay in Rheumatoid Arthritis
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Platelet functionality was assessed in 27 RA patients and 15 healthy individuals in parallel experiments (one control for one or more RA samples analyzed at a time) by expression of P-selectin (CD62p) and active integrin αIIbβ3 (determined by its fibrinogen-binding capacity) before and after activation with the PAR1-specific thrombin receptor-activating peptide 6 (TRAP-6)(Bachem Americas Inc., Torrance, CA, USA). TRAP-6 was added to isolated platelets at 50 μM and incubated for 3 min at room temperature. Then platelets (200,000 in 50 μL in Tyrode’s buffer) were mixed with 0.045 μg/mL anti-human-CD62p phycoerythrin-labeled murine antibodies (BD Biosciences, San Jose, CA, USA) or 5 μg/mL Alexa Fluor 488-labeled human fibrinogen (ThermoFisher Scientific, Waltham, MA, USA) and incubated for 10 min. After incubation with the labeled ligands, the platelets were analyzed using a FacsCalibur flow cytometer equipped with BD Cell-Quest software (Table 1 and Figure S1 ). Platelets were gated based on their size and granularity and 5000 platelets were counted in each sample. FlowJo X software was used for data analysis.
4
Platelet Activation Assay with Anti-PF4 Sera
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The ability of anti-PF4–positive sera to stimulate platelets was tested with platelet-rich plasma (PRP) prepared by centrifugation (150 g for 20 minutes at 23 °C) of blood from a group O donor, selected on the basis of not having prothrombotic diseases and not receiving antiplatelet or anticoagulation medication. PRP was diluted 1:5 with HEPES buffer (10 mmol/L HEPES, 145 mmol/L NaCl, 5 mmol/L KCl, and 1 mmol/L MgSO4, pH 7.4). Heat-inactivated serum (56ºC, 30 minutes) from the participants of control and intervention groups whose anti-PF4 Abs levels were above the cutoff value in any of the visits was added in a ratio 1:5 to diluted PRP and incubated with FITC-anti P-selectin mAb (BD Pharmingen). Platelet activation using the participants' serum was also tested on PRP stimulated with a suboptimal concentration of thrombin receptor-activating peptide (TRAP)-6 (Bachem AG) (10 μmol/L, Supplementary Figure 2 ) at room temperature (RT) before the addition of FITC-anti P-selectin mAb.
After incubation, platelets were diluted in the HEPES buffer for flow cytometry analysis with a FACScan flow cytometer (BD Biosciences) and 10,000 events in the platelet region were acquired and analyzed using BD CellQuest Pro software (BD Biosciences).
After incubation, platelets were diluted in the HEPES buffer for flow cytometry analysis with a FACScan flow cytometer (BD Biosciences) and 10,000 events in the platelet region were acquired and analyzed using BD CellQuest Pro software (BD Biosciences).
5
Platelet Function Assessment by LTA
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Platelet function and activation by LTA was performed using PAP 8E (MoeLab, Langenfeld, Germany) as previously described [23 (link),24 (link)]. In short, the PCs were diluted to a final concentration of 250 × 109/L with solvent-detergent plasma (Octaplas; blood group AB; Octapharma, Vienna, Austria). The aggregation response to collagen (final concentration, 190 µg/mL; BioData Corporation, Horsham, PA, USA) or TRAP-6 (final concentration of 25 µmol; Bachem, Bubendorf, Switzerland) was continuously recorded for 5 min. The maximal aggregation (MA) was used for further statistics.
6
Platelet Aggregation Assay Protocol
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Platelet aggregation was monitored by recording changes in light transmission with the use of an aggregometer APACT 4S Plus (Dyasys Greiner Gmb, Flacht, Germany) as previously described57 . Either platelet-rich plasma prepared as described58 (link) or washed platelets (3 × 105 platelets/μl) were used. TRAP 6 (20 μg/ml) (Bachem, Bubendorf, Switzerland) as a control, or Horm collagen (5 μg/ml), or collagen G (5-, and 100 μg/ml), or PLL (100 μg/ml) was added to platelet suspension at 37 oC and platelet aggregation trace (%) was continuously recorded. For comparison, the same blood donors were used for every set of measurement.
7
Platelet Agonists and Antagonists Effects
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Many assays in the current study were performed using PRP containing various platelet agonists/antagonists. These treatments include 10 μg mL−1 Abciximab (anti-GpIIb/IIIa, Reopro, Eli Lilly), 10 μM blebbistatin (Sigma), 20 μg mL−1 anti-CD42b antibody (#14-0429-80, HIP1 clone, ThermoFisher), and 15 μM acetylsalicylic acid (ASA, TCI America). Here, mAbs and ASA were incubated with citrated PRP for 30 min before flow and blebbistatin was incubated for 5 min before flow. In some cases, 15 µM TRAP-6 (#4017752, Bachem, Torrance, CA) was added to PRP prepared using PPACK just before flow. ADP (10 μM) and thrombin (5 U mL−1) were diluted in Dulbecco’s phosphate-buffered saline and infused at 500 s−1 shear rate for 10 min over microclots previously formed under citrated PRP flow (Fig. 4b−e ). In some cases, ADP (5 μM) and thrombin (2 U mL−1) were directly added to citrate PRP before flow to study their effects on clot formation (Supplementary Fig. 21 ). For all the agonist and antagonist treatments, at least three donors with at least seven samples per donor were measured.
8
Quantifying Platelet Activation in PRP
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PRP was isolated from fresh human blood using either 70 µM PPACK or 3.8% sodium citrate (1:9) as anticoagulant. In some cases, PRP was recalcified by addition of 10 mM CaCl2. 50 µL PRP was incubated with a mixture of anti-human CD41/CD61 activation-specific mAb PAC-1 (Alexa647 labeled, #362805, Biolegend, San Diego, CA) and PE-labeled anti-CD62P mAb AC1.2 (#550561, BD, San Jose, CA) for 5 min at room temperature. This mixture was then stimulated with either 15 µM TRAP-6 (#4017752, Bachem, Torrance, CA), 5 U mL−1 Thrombin (#T7009, Sigma, Burlington, MA), 20 µM ADP (Sigma, Burlington, MA) or 5 µgmL−1 collagen (Chrono-Log, Haverton, PA). A portion of the sample was diluted 200-fold at various times and analyzed using a BD Fortessa X-20 flow cytometer (Supplementary Fig. 16 ).
9
Platelet Function in Venous Thromboembolism
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Platelet functionality was analyzed in 11 VTE patients and 11 healthy individuals in parallel by expression of P-selectin (CD62p) and active integrin αIIbβ3 (determined by its fibrinogen-binding capacity) before and after activation with a thrombin-receptor activation peptide (TRAP-6, the PAR-1-specific hexapeptide Ser-Phe-Leu-Leu-Arg-Asn; Bachem Americas Inc., Torrance, California, United States). TRAP-6 was added to isolated platelets at 50 µM for 3 minutes at room temperature. Then platelets (200,000 in 50 µL) were incubated for 10 minutes with anti-human-CD62p phycoerythrin-labeled murine antibodies (BD Biosciences, San Jose, California, United States) (0.045 µg/mL) or Alexa fluor 488-labeled human fibrinogen (ThermoFisher Scientific, Waltham, Massachusetts, United States) (5 µg/mL). After incubation with the labeled ligands, the platelets were analyzed using a FacsCalibur flow cytometer equipped with BD CellQuest software. Platelets were gated based on their size and granularity and 5,000 platelets were counted in each sample. FlowJo X software was used for data analysis.
10
ATP Measurement in Activated Platelets
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Freshly isolated platelets (0.1mLat 107 cells/mL) in Tyrode’s buffer were incubated for 30, 45, and 60 min without or with 50 μM thrombin receptor activated peptide (TRAP-6, Bachem Americas). Following incubation, platelets were spun down for 5minat 2,000 g and lysed with a lysis RIPA buffer (pH 7.4). Debris was removed from the lysates by centrifugation at 8,000 g for 10 min. ATP concentration in the platelet lysates was measured with a plate reader, Infinite 200 PRO (Tecan, Switzerland) using an ATP Bioluminescent Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions.
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