2.1

Study design

The methods of ex vivo platelet activation applied in this study have been explained before. .

40 patients with peripheral artery disease (PAD) were enrolled in the study and divided into two groups. In the first group A the patients were treated with 100 mg ASA and an additional placebo for 4 weeks, and the second group B took 75 mg/d clopidogrel additionally to ASA 100 mg for 4 weeks. The placebo pills are composed of cellulose and lactose and have the same color and appearance as the clopidogrel pills. After obtaining blood at days 0, 7, and 28, the platelet activation was determined by measuring the protein concentration of CD63, CD62p and TSP after stimulation by TRAP, ADP, and collagen. The inflammation pathway of platelets was characterized by analyzing the release of the inflammatory chemokines RANTES and sCD40L from stimulated platelets by using ELISA. CRP was measured in the plasma of patients with ELISA. Inclusion criteria for patients were peripheral artery disease with an ankle arm index of < 0.9, ASA medication treatment more than 4 weeks duration and a minimum age of 18 years. Exclusion criteria were clopidogrel medication within 4 weeks before the study, oral anticoagulation, cancer disease, peptic ulcers, cerebral surgery or hematoma, cirrhosis of the liver with elevation of transaminase, thrombocytopenia < 100,000/μl, renal insufficiency with dialysis, surgery (including dental) in the previous 4 weeks and the following 6 weeks, or an influenza infection. At days 0, 7, and 28 of the study we took 10 ml venous blood from the patients of both groups for assessment of platelet reactivity.

The institutional review board of the hospital approved the study and written informed consent was obtained from each subject. All procedures were performed in accordance with the Declaration of Helsinki.

2.2

Ex vivo platelet stimulation

Blood was collected in acid citrate dextrose (ACD) and centrifuged at 2600 rpm for 80 s. Platelet rich plasma (PRP) was reacidified in ACD and again centrifugated at 3300 rpm for 2 min. The pellet was solved in HEPES buffered tyrode solution (NaCl 134 mM, NaHCO ₃ 12 mM, KCl 2.9 mM, NaH ₂ PO ₄ 0.36 mM, HEPES 5 mM, glucose 5 mM, bovine serum albumin 0.5 mg/ml). After adaption of the thrombocytes to 150,000/μl in HEPES buffered tyrode solution with calcium (final concentration 750 nM) and magnesium (200 mM) the thrombocytes were stimulated for 15 min with a calcium/magnesium buffer containing 20 μmol ADP, 20 μg/ml collagen and 10 μmol TRAP, or left unstimulated. After fixation with paraformaldehyde platelets were washed and then incubated with mouse IgG and monoclonal antibodies against CD62p, CD63, and thrombospondin, followed by an incubation with a fluorescein isothiocanate-labeled antibody (FITC). Finally a flow cytometry was performed (FACScan) .

2.3

sCD40L, RANTES and CRP expression after stimulating with ADP and TRAP

In a second experimental run the sCD40L and RANTES secretion of platelets was tested by ELISA. Therefore, the sCD40L and RANTES release from platelets was measured before the study, and 7 days after clopidogrel ( n = 20) or placebo ( n = 20) administration. Blood was collected and acidified with ACD, centrifuged at 2600 rpm for 75 s. Reacidified ACD platelets were centrifuged again at 3300 rpm. Platelets were solved in HEPES and diluted to 170,000/μl. They were stimulated with calcium (final concentration 500 nM) for 20 min in 20 μmol ADP, 20 μg/ml collagen or 10 μmol TRAP, or left unstimulated. After stopping the stimulation with heparin, a centrifugation at 4000 rpm followed. The supernatants were frozen at minus 80 °C. The measurement followed using a human sCD40L and RANTES ELISA Kit according to the manufacturers’ instructions. The sCD40 L Kit (BenderMedSystems BMS239MST) contains a coating antibody, a standard protein, HRP conjugate, and a sample diluent. For sample preparation frozen plasma was defrosted slowly and diluted 1:2 with sample diluent. The assay procedure starts with the coating. For this, 100 μl of coating antibody was pipetted in maxi sorb plates and was incubated at 4° followed by washing with 200 μl of buffer. For blockade, 250 μl of assay buffer was pipetted into the wells. After incubating for 2 h they were washed again. Samples were diluted with 100 μl of sample diluents, 100 μl probes and 200 μl HRP conjugate. 150 μl was pipetted on to the coated plates and was incubated for 2 h at room temperature or overnight at 4 °C. This was followed by washing three times, an addition of 100 μl TMB substrate, and an incubation for 30 min in darkness. After adding a stop solution (sulfate acid) the samples were measured at 620 nm with a spectrophotometer. For the RANTES data we took the R&D Systems # DRN00B Kit. First, frozen plasma was defrosted slowly, followed by a 1:4 diluent with calibrator diluent RD6-11. 100 μl of assay diluent was presented in all wells, 100 μl of diluent, standards and blanks were added and were incubated for 2 h at room temperature. After washing three times with buffer, 200 μl of conjugate solution was added to the wells and incubated again for 1 h at room temperature, followed by washing with buffer and incubation with 200 μl of substrate solution for 20 min. After stopping the procedure with stopping solution (H ₂ SO ₄ 2 N), measurement with a spectrophotometer at 450 nm followed.

2.4

Statistical analysis

Data were expressed as means ± SEM unless otherwise specified. Nominal data were primary tested by SSPS 11.0.1 and in a second row by GraphPad Prism 5.00. Differences within groups of placebo and treatment were compared using the Wilcoxon test. Comparison between the groups was done using the Mann–Whitney test. A probability value p < 0.05 was considered statistically significant.

2.1

Study design

The methods of ex vivo platelet activation applied in this study have been explained before. .

40 patients with peripheral artery disease (PAD) were enrolled in the study and divided into two groups. In the first group A the patients were treated with 100 mg ASA and an additional placebo for 4 weeks, and the second group B took 75 mg/d clopidogrel additionally to ASA 100 mg for 4 weeks. The placebo pills are composed of cellulose and lactose and have the same color and appearance as the clopidogrel pills. After obtaining blood at days 0, 7, and 28, the platelet activation was determined by measuring the protein concentration of CD63, CD62p and TSP after stimulation by TRAP, ADP, and collagen. The inflammation pathway of platelets was characterized by analyzing the release of the inflammatory chemokines RANTES and sCD40L from stimulated platelets by using ELISA. CRP was measured in the plasma of patients with ELISA. Inclusion criteria for patients were peripheral artery disease with an ankle arm index of < 0.9, ASA medication treatment more than 4 weeks duration and a minimum age of 18 years. Exclusion criteria were clopidogrel medication within 4 weeks before the study, oral anticoagulation, cancer disease, peptic ulcers, cerebral surgery or hematoma, cirrhosis of the liver with elevation of transaminase, thrombocytopenia < 100,000/μl, renal insufficiency with dialysis, surgery (including dental) in the previous 4 weeks and the following 6 weeks, or an influenza infection. At days 0, 7, and 28 of the study we took 10 ml venous blood from the patients of both groups for assessment of platelet reactivity.

The institutional review board of the hospital approved the study and written informed consent was obtained from each subject. All procedures were performed in accordance with the Declaration of Helsinki.

2.2

Ex vivo platelet stimulation

Blood was collected in acid citrate dextrose (ACD) and centrifuged at 2600 rpm for 80 s. Platelet rich plasma (PRP) was reacidified in ACD and again centrifugated at 3300 rpm for 2 min. The pellet was solved in HEPES buffered tyrode solution (NaCl 134 mM, NaHCO ₃ 12 mM, KCl 2.9 mM, NaH ₂ PO ₄ 0.36 mM, HEPES 5 mM, glucose 5 mM, bovine serum albumin 0.5 mg/ml). After adaption of the thrombocytes to 150,000/μl in HEPES buffered tyrode solution with calcium (final concentration 750 nM) and magnesium (200 mM) the thrombocytes were stimulated for 15 min with a calcium/magnesium buffer containing 20 μmol ADP, 20 μg/ml collagen and 10 μmol TRAP, or left unstimulated. After fixation with paraformaldehyde platelets were washed and then incubated with mouse IgG and monoclonal antibodies against CD62p, CD63, and thrombospondin, followed by an incubation with a fluorescein isothiocanate-labeled antibody (FITC). Finally a flow cytometry was performed (FACScan) .

2.3

sCD40L, RANTES and CRP expression after stimulating with ADP and TRAP

In a second experimental run the sCD40L and RANTES secretion of platelets was tested by ELISA. Therefore, the sCD40L and RANTES release from platelets was measured before the study, and 7 days after clopidogrel ( n = 20) or placebo ( n = 20) administration. Blood was collected and acidified with ACD, centrifuged at 2600 rpm for 75 s. Reacidified ACD platelets were centrifuged again at 3300 rpm. Platelets were solved in HEPES and diluted to 170,000/μl. They were stimulated with calcium (final concentration 500 nM) for 20 min in 20 μmol ADP, 20 μg/ml collagen or 10 μmol TRAP, or left unstimulated. After stopping the stimulation with heparin, a centrifugation at 4000 rpm followed. The supernatants were frozen at minus 80 °C. The measurement followed using a human sCD40L and RANTES ELISA Kit according to the manufacturers’ instructions. The sCD40 L Kit (BenderMedSystems BMS239MST) contains a coating antibody, a standard protein, HRP conjugate, and a sample diluent. For sample preparation frozen plasma was defrosted slowly and diluted 1:2 with sample diluent. The assay procedure starts with the coating. For this, 100 μl of coating antibody was pipetted in maxi sorb plates and was incubated at 4° followed by washing with 200 μl of buffer. For blockade, 250 μl of assay buffer was pipetted into the wells. After incubating for 2 h they were washed again. Samples were diluted with 100 μl of sample diluents, 100 μl probes and 200 μl HRP conjugate. 150 μl was pipetted on to the coated plates and was incubated for 2 h at room temperature or overnight at 4 °C. This was followed by washing three times, an addition of 100 μl TMB substrate, and an incubation for 30 min in darkness. After adding a stop solution (sulfate acid) the samples were measured at 620 nm with a spectrophotometer. For the RANTES data we took the R&D Systems # DRN00B Kit. First, frozen plasma was defrosted slowly, followed by a 1:4 diluent with calibrator diluent RD6-11. 100 μl of assay diluent was presented in all wells, 100 μl of diluent, standards and blanks were added and were incubated for 2 h at room temperature. After washing three times with buffer, 200 μl of conjugate solution was added to the wells and incubated again for 1 h at room temperature, followed by washing with buffer and incubation with 200 μl of substrate solution for 20 min. After stopping the procedure with stopping solution (H ₂ SO ₄ 2 N), measurement with a spectrophotometer at 450 nm followed.

2.4

Statistical analysis

Data were expressed as means ± SEM unless otherwise specified. Nominal data were primary tested by SSPS 11.0.1 and in a second row by GraphPad Prism 5.00. Differences within groups of placebo and treatment were compared using the Wilcoxon test. Comparison between the groups was done using the Mann–Whitney test. A probability value p < 0.05 was considered statistically significant.