Clinical trial NCT03331666
Conditions: Familial Hypercholesterolemia
Keywords: Hyperlipidemia, Hypercholesterolemia, atherothrombosis
Sponsor: Columbia University
Start date: 2018-11-16; Completion date: 2020-02-04
Enrollment: 4 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
The primary goal is to assess the impact of Evolocumab therapy on platelet function of familial hypercholesterolemia (FH) patients in a randomized, double blind study. Evolocumab is a humanized monoclonal antibody that targets circulating PCSK9, increases hepatic LDL receptor, decreases plasma LDL cholesterol and reduces risk of cardiovascular events. Evolocumab (brand name Rapatha) has been approved by FDA along with diet and maximally tolerated statin therapy in adults with FH or atherosclerotic heart or blood vessel problems, who need additional lowering of LDL cholesterol.

The secondary goal is to determine if platelet activation or the response to Evolocumab therapy is modified by rs3184504 polymorphism. The investigators believe that these investigations will complement ongoing studies to demonstrate that Evolocumab reduces athero-thrombotic risk and aid the decision-making as to whether Evolocumab can reduce the atherothrombotic risk in acute coronary syndrome (ACS) patients.

Detailed description:
Hyperlipidemia as exemplified by familial hypercholesterolemia is associated with increased platelet activation and an underlying pro-coagulant state. Hyperlipidemia primes platelets and increases platelet activation in response to various agonists. Plasma cholesterol levels appear to have a critical role in modulating platelet activity as hypercholesterolemia increases platelet activation more potently than hypertriglyceridemia. Increased platelet reactivity may contribute to the increased risk of atherothrombosis associated with hypercholesterolemia. Plasma levels of platelet activation markers such as thrombin-antithrombin complex (TAT), soluble P-selectin (sP-selectin), soluble CD40L (sCD40L) or P-selectin exposure at surface of platelets are increased in hypercholesterolemic patients. Increased levels of the platelet activation markers are associated with increased platelet membrane cholesterol content in hypercholesterolemia.Statins may show antithrombotic properties.

Arms: arm 1: Subjects will start with placebo and will receive Evolocumab 140 mg every 14 days starting Day 14 until Day 196. arm 2: Subjects will start with placebo and will receive Evolocumab 140 mg every 14 days starting Day 28 until Day 196.
Interventions: intervention 1: Evolocumab
Intervention descriptions: intervention 1: 140 mg every 14 days A monoclonal antibody designed for the treatment of hyperlipidemia

Protocol documents:
Prot_SAP_004.pdf: 
Title: Impact of LDL-cholesterol lowering on platelet activation  
NCT number: NCT03331666 
Date: 05-29-2019 
Introduction 
Hyperlipidemia as exemplified by familial hypercholesterolemia is associated with increased 
platelet activation and an underlying pro-coagulant state1-3. Hyperlipidemia primes platelets and 
increases platelet activation in response to various agonists1, 4. Plasma cholesterol levels 
appear to have a critical role in modulating platelet activity as hypercholesterolemia increases 
platelet activation more potently than hypertriglyceridemia1, 4. Plasma levels of platelet activation 
markers such as thrombin-antithrombin complex (TAT), soluble P-selectin (sP-selectin), soluble 
CD40L (sCD40L) or P-selectin exposure at surface of platelets are increased in 
hypercholesterolemic patients5-7. Increased levels of the platelet activation markers are 
associated with increased platelet membrane cholesterol content in hypercholesterolemia8. 
Hyperlipidemia increases platelet activation likely via multiple mechanisms9, 10. Oxidized LDL or 
oxidized phospholipids, which are increased in hyperlipidemia9, 11, serve as ligands of platelet 
CD36 and activate platelets9, 12. In vitro cholesterol-loading increases 13 while cholesterol-
unloading decreases human platelet activation.14, 15  Recently, we showed that cholesterol 
enrichment of platelets increased platelet activation via a mechanism involving ↓LYN Kinase 
activation→↓SHIP-1 activity→↑PIP3→↑AKT activity15. 
Statins may show antithrombotic properties16. The PROVE-IT trial, a study to assess the impact 
of statins on acute coronary syndrome (ACS), demonstrated an early beneficial effect after 30 
days of follow-up that significantly reduced the risk of death, MI or rehospitalization17. A 
retrospective study of 1616 patients (PRISM) also showed significant reduction of the risk during 
30 day follow-up in ACS patients with statin therapy18. Additional human studies suggest that 
the benefits may come partly from the antithrombotic properties of statins16. Statins reduced the 
level of TAT, sP-selectin, sCD40L and P-selectin exposure at surface of platelets8, 19-21. 
Reduced levels of P-selectin at the platelet surface were associated with reduced platelet 
cholesterol content in subjects treated with statins8 . These studies suggest an early direct effect 
of statins on platelets and a late prolonged effect that is associated with progressive reduction of 
plasma LDL-cholesterol levels16, 22. Both the early and late effects are proposed to lower platelet 
activation and contribute to the reduced risk. Consistent with the idea that cholesterol 
enrichment increases platelet activation, infusions of a reconstituted HDL (rHDL) preparation 


reduced ex vivo platelet activation in diabetic subjects, likely by promoting cholesterol efflux 
from platelets14.  
Human genome wide association studies have revealed novel genetic loci associated with 
coronary heart disease. One such locus resides in LNK/SH2B3 which in mice is expressed in 
hematopoietic cells and suppresses thrombopoietin signaling via its receptor MPL.  Using 
human cord blood, we recently showed that the common TT risk genotype (R262W) of LNK is 
associated with expansion of hematopoietic stem cells and enhanced megakaryopoiesis, 
demonstrating reduced LNK function and increased MPL signaling.  To model the human 
reduced LNK function associated with the TT risk genotype, we used Lnk-/- mice.   In mice, 
hematopoietic Lnk deficiency led to accelerated arterial thrombosis, but only in the setting of 
hypercholesterolemia. In platelets LNK deficiency increased MPL signaling, while cholesterol 
loading decreased SHIP-1 activation, acting convergently to increase AKT and platelet 
activation. Increased platelet activation combines with hypercholesterolemia induced 
myelopoiesis to promote pro-thrombotic platelet/leukocyte aggregate formation and accelerated 
atherogenesis. Atherosclerotic lesions were characterized by larger size, increased necrotic 
cores and increased leukocyte-derived DNA nets.  
These findings suggest the possibility that in humans, the common LNK variant rs3184504 
impacts platelet activation in a fashion dependent on plasma cholesterol levels. In light of these 
findings, we propose the following studies as an investigator initiated study. The primary goal is 
to assess the impact of Evolocumab therapy on platelet function of FH patients in a randomized, 
double blind study. The secondary goal is to determine if platelet activation or the response to 
Evolocumab therapy is modified by rs3184504 polymorphism. We believe that these 
investigations will complement ongoing studies to demonstrate that Evolocumab reduces 
athero-thrombotic risk and aid the decision-making as to whether Evolocumab can reduce the 
atherothrombotic risk in ACS patients.  
Proposed studies. 
A. Sample collection: All adult male and female subjects who have a clinical diagnosis of 
familial hypercholesterolemia (FH) and who are referred to Dr. Ginsberg’s Lipid Practice 
for treatment with PCSK9 inhibitor, with LDL cholesterol levels ≥70 mg/dl on baseline 


treatment with statins and/or ezetimibe or intolerant to statin and/or ezetimibe. The platelet 
counts in peripheral blood should be ≥100,000 per microliter. The subjects that meet these 
criteria will be recruited 
for the study. 
Blood will be collected as 
shown in Fig. 1 in the 
morning from the resting 
and fasting subjects. An 
aliquot will be collected 
for genomic DNA and 
platelet-rich 
plasma 
(PRP) preparation.  
B. Power Calculation:  
The effect of PCSK9 
inhibitors, including evolocumab, on platelet activation measures has not been assessed 
previously. However, our hypothesis is based on the LDL-cholesterol lowering effect of 
evolocumab, and evolocumab has been demonstrated to have at least as potent an effect 
on LDL lowering as statins23, 24. Statin therapy in hypercholesterolemia patients results in 
50% reduction in LDL-cholesterol levels and add-on evolocumab therapy indicated an 
additional 50-60% reduction23-25. Therefore, we base our power estimates on platelet 
activation data available for statins.  
Primary measure/platelet aggregation - The primary measurement will be ADP-stimulated 
platelet aggregation as assessed by the commercially-available VerifyNow P2Y12 assay 
(Accriva Diagnostics). The assay read-outs are 1) PRU, platelet reactivity units (specific 
for P2Y12 receptors); 2) BASE, total platelet function despite P2Y12 receptor blockage; 
and 3) IPA, percent platelet P2Y12 inhibition calculated as [(BASE - PRU) / BASE] X 100. 
Two reports involving patients on antiplatelet therapy with high residual platelet reactivity 
demonstrated an effect of statins on lowering platelet aggregation via the VerifyNow assay. 
In CAD patients treated with clopidogrel, the addition of atorvastatin for 30 days resulted 
in an absolute mean difference of 102 ± 54 PRU compared to baseline, corresponding to 
a 37 ± 14% IPA25. In a separate study of CAD/diabetic patients treated with clopidogrel, 
the addition of atorvastatin for 30 days resulted in an absolute mean difference of 106 ± 
75 PRU compared to baseline, corresponding to a 33 ± 20% IPA8. Similar effect sizes and 
variabilities of statins on reductions in platelet aggregation were observed in type II 


hyperlipidemic patients using light transmittance aggregometry21. The relative reductions 
were reported as 30 ± 23% (pravastatin), 19 ± 16% (atorvastatin), and 22 ± 22% 
(simvastatin)7. 
The reference range for the VerifyNow assay in individuals not on antiplatelet therapy is 
180-376 PRU. These data are continuous and normally distributed5-6. Thus, means and 
standard deviations will be used to describe reductions in platelet aggregation. Based on 
reported literature indicating additional effects of statins on platelet function following 30-
day vs shorter periods of treatment, we have revised our experimental design to have a 
primary endpoint of 30-day treatment26,21. Within-group thirty day evolocumab treatment 
will be tested by one-group paired t-test (two-sided) using each subject as its own control 
(evolocumab treatment vs vehicle pre-treatment control). Between-group thirty day 
evolocumab treatment will be tested by standard two-group t-test (two-sided, evolocumab 
vs vehicle control group run in parallel). The sample size estimates have been run using 
one-group, paired t-test published data. Using PRU mean difference = 10226, SD = 5426, 
power = 0.8 and alpha = 0.05, we estimate 6 subjects. Since the effects of statins on 
platelet aggregation may be mediated by both LDL-lowering and non-LDL-lowering 
mechanisms, we can calculate the sample size based on 50% reduced mean difference 
(PRU = 51). Then, we estimate 11 subjects. An equal number of subjects will be required 
for the placebo group run in parallel. Target enrollment will include all patients screened; 
we estimate that 10% may not be eligible based on exclusion criteria and/or may not return 
for scheduled blood draws. Thus, for 80% power, the target enrollment is 24 subjects over 
two years. 
Secondary measures – Secondary measures will include various cellular and circulating 
markers of platelet activation measured by flow cytometry or commercially-available 
enzyme-linked immunoassays (ELISAs). The target enrollment of 24 subjects should 
provide at least 80% power to detect differences between treatment groups based on data 
reported in the literature. For example, platelet P-selectin level measured by flow 
cytometry and reported as % positive cells (out of 50,000 cells counted) showed a mean 
reduction ± SD of 10.9 ± 6.0 (simvastatin), 10.9 ± 5.6 (atorvastatin), 7.2 ± 6.8 (fluvastatin) 
and 9.0 ± 6.8 (pravastatin) following 30 days of statin treatment in hypercholesterolemic 
patients compared with untreated controls27. In a separate study of stroke patients with 
hypercholesterolemia, platelet P-selectin showed a mean reduction of 41 ± 27% positive 
cells in response to thrombin following 6 months of treatment with simvastatin compared 
to baseline measurements in the same subjects before statin treatment28. An additional 


effect of simvastatin treatment was observed for soluble P-selectin serum concentrations 
measured by ELISA. Soluble P-selectin showed a mean reduction of 46.4 ± 49 ng/ml 
compared to baseline measurements in the same subjects before statin treatment28. Other 
secondary measures include VerifyNow IIb/IIIa and VerifyNow Aspirin assays. 
C. Study Design: Amgen will provide Evolocumab and placebo as open label. An 
independent biostatistician will generate a randomization table and provide it to Research 
Pharmacy of CUMC and Research Pharmacy will randomize the subjects accordingly. All 
patients recruited will start with placebo and blood and urine samples will be collected at 
day 7 and day 14. Then, the subjects will be randomized so that half will continue to 
receive placebo and half will receive Evolocumab via subcutaneous injection of 140 mg 
every 14 days. Blood and urine samples will be collected at day 21 and day 28. Then, all 
patients will receive Evolocumab 140 mg every 14 days and the treatment will continue. 
Blood and urine samples 
will 
be 
collected 
following 
Evolocumab 
therapy at day 84 and 
196 (Fig. 2). While every 
effort will be made to 
collect the blood and 
urine samples on the 
indicated time points, 
variation for the sample 
collection 
within 
1-3 
days at the indicated 
time 
point 
will 
be 
allowed. The power of this design is that we can compare any changes due to Evolocumab 
therapy relative to a placebo control. 
Based on the power calculations as detailed above, recruitment of total 20 participants 
into this study will give ~80% power for statistical significance of 0.05. We target to recruit 
24 subjects over 2 year period. However, we anticipate that some participants will drop 
out from the study and incomplete data will be obtained. We will continue to recruit the 
subjects until the complete data sets for 22 subjects are obtained. Incomplete data from 
the dropout may not be used unless data up to 28 days at least are collected. 
D. Genotyping: rs3184504 genotyping will be performed using the genomic DNA samples 


and rs3184504 specific PCR genotyping primers from Invitrogen.  
E. Assays: We propose to use a clinically validated system, VerifyNow, as the assay for 
assessment of platelet activation state. Compared with other assays, this system requires 
~2 ml whole blood, with minimal ex vivo manipulations, minimal hands-on time and no 
pipetting or sample preparation. It is fast (10 min for result presentation) and approved as 
a clinical lab test of platelet reactivity. Thus, the results obtained this way should be more 
reliable when the data need to be compared with results from other studies using the same 
assay. In principle, this assay is designed to measure platelet function based upon the 
ability of activated platelets to bind fibrinogen.  Fibrinogen-coated microparticles 
aggregate in whole blood in proportion to the number of unblocked platelet GP IIb/IIIa 
receptors.  Light transmittance increases as activated platelets bind and aggregate 
fibrinogen-coated beads. The instrument measures this change in optical signal caused 
by aggregation. We propose to use VerifyNow IIb/IIIa, VerifyNow PRUTest, and VerifyNow 
Aspirin assays, testing platelet responses to thrombin-like peptide, ADP and arachidonic 
acid, respectively. In addition to the VerifyNow assay, basal level of P-selectin and active 
form of GPIIbIIIa at surface of platelets will be determined by flow cytometry. In order to 
reduce the variation generated by manipulation of blood samples in vitro, we will use 1% 
paraformaldehyde to fix an aliquot of the whole blood sample and basal levels of P-selectin 
and active GPIIbIIIa at surface of platelets will be determined by flow cytometry after 
staining with specific P-selectin or PAC-1 antibody (PAC-1 specific for active GPIIbIIIa)29, 
30. Plasma levels of TAT, sP-selectin and sCD40L will be determined by ELISA assays. 
Levels of plasma LDL cholesterol, HDL cholesterol, triglyceride and platelet total 
cholesterol content will be determined. All these assays are validated assays. 
F. Analysis: The primary hypothesis is that Evolocumab will reduce thrombin-peptide 
stimulated platelet activation as determine by VerifyNow. Secondary hypothesis is related 
to basal and ADP or arachidonic acid stimulated activation, direct measurements of 
platelet activation by flow cytometry or levels of the surrogate markers. This will help 
assess the validity of these markers for larger ongoing studies that we are performing in 
collaboration with Amgen using EDTA plasma samples obtained in phase 2 development 
studies of Evolocumab. Even though the statistical power is limited by small sample size, 
the potential modification of these phenotypes by rs3184504 polymorphism will be 
examined.  
Time Frame: two years 


Reference 
 
1. 
Carvalho AC, Colman RW and Lees RS. Platelet function in hyperlipoproteinemia. The New 
England journal of medicine. 1974;290:434-8. 
2. 
Connor WE, Hoak JC and Warner ED. Massive thrombosis produced by fatty acid infusion. The 
Journal of clinical investigation. 1963;42:860-6. 
3. 
Owens AP, 3rd, Passam FH, Antoniak S, Marshall SM, McDaniel AL, Rudel L, Williams JC, Hubbard 
BK, Dutton JA, Wang J, Tobias PS, Curtiss LK, Daugherty A, Kirchhofer D, Luyendyk JP, Moriarty PM, 
Nagarajan S, Furie BC, Furie B, Johns DG, Temel RE and Mackman N. Monocyte tissue factor-dependent 
activation of coagulation in hypercholesterolemic mice and monkeys is inhibited by simvastatin. The 
Journal of clinical investigation. 2012;122:558-68. 
4. 
Harmon JT, Tandon NN, Hoeg JM and Jamieson GA. Thrombin binding and response in platelets 
from patients with dyslipoproteinemias: increased stimulus-response coupling in type II 
hyperlipoproteinemia. Blood. 1986;68:498-505. 
5. 
Wada H, Mori Y, Kaneko T, Wakita Y, Nakase T, Minamikawa K, Ohiwa M, Tamaki S, Tanigawa M, 
Kageyama S and et al. Elevated plasma levels of vascular endothelial cell markers in patients with 
hypercholesterolemia. American journal of hematology. 1993;44:112-6. 
6. 
Davi G, Romano M, Mezzetti A, Procopio A, Iacobelli S, Antidormi T, Bucciarelli T, Alessandrini P, 
Cuccurullo F and Bittolo Bon G. Increased levels of soluble P-selectin in hypercholesterolemic patients. 
Circulation. 1998;97:953-7. 
7. 
Garlichs CD, John S, Schmeisser A, Eskafi S, Stumpf C, Karl M, Goppelt-Struebe M, Schmieder R 
and Daniel WG. Upregulation of CD40 and CD40 ligand (CD154) in patients with moderate 
hypercholesterolemia. Circulation. 2001;104:2395-400. 
8. 
Puccetti L, Pasqui AL, Pastorelli M, Bova G, Cercignani M, Palazzuoli A, Angori P, Auteri A and 
Bruni F. Time-dependent effect of statins on platelet function in hypercholesterolaemia. Eur J Clin Invest. 
2002;32:901-8. 
9. 
Podrez EA, Byzova TV, Febbraio M, Salomon RG, Ma Y, Valiyaveettil M, Poliakov E, Sun M, Finton 
PJ, Curtis BR, Chen J, Zhang R, Silverstein RL and Hazen SL. Platelet CD36 links hyperlipidemia, oxidant 
stress and a prothrombotic phenotype. Nature medicine. 2007;13:1086-95. 
10. 
Korporaal SJ, Meurs I, Hauer AD, Hildebrand RB, Hoekstra M, Cate HT, Pratico D, Akkerman JW, 
Van Berkel TJ, Kuiper J and Van Eck M. Deletion of the high-density lipoprotein receptor scavenger 
receptor BI in mice modulates thrombosis susceptibility and indirectly affects platelet function by 
elevation of plasma free cholesterol. Arteriosclerosis, thrombosis, and vascular biology. 2011;31:34-42. 
11. 
Boullier A, Bird DA, Chang MK, Dennis EA, Friedman P, Gillotre-Taylor K, Horkko S, Palinski W, 
Quehenberger O, Shaw P, Steinberg D, Terpstra V and Witztum JL. Scavenger receptors, oxidized LDL, 
and atherosclerosis. Annals of the New York Academy of Sciences. 2001;947:214-22; discussion 222-3. 
12. 
Magwenzi S, Woodward C, Wraith KS, Aburima A, Raslan Z, Jones H, McNeil C, Wheatcroft S, 
Yuldasheva N, Febbriao M, Kearney M and Naseem KM. Oxidized LDL activates blood platelets through 
CD36/NOX2-mediated inhibition of the cGMP/protein kinase G signaling cascade. Blood. 2015;125:2693-
703. 
13. 
Shattil SJ, Anaya-Galindo R, Bennett J, Colman RW and Cooper RA. Platelet hypersensitivity 
induced by cholesterol incorporation. J Clin Invest. 1975;55:636-43. 
14. 
Calkin AC, Drew BG, Ono A, Duffy SJ, Gordon MV, Schoenwaelder SM, Sviridov D, Cooper ME, 
Kingwell BA and Jackson SP. Reconstituted high-density lipoprotein attenuates platelet function in 
individuals with type 2 diabetes mellitus by promoting cholesterol efflux. Circulation. 2009;120:2095-
104. 


15. 
Wang W, Tang Y, Wang Y, Tascau L, Balcerek J, Tong W, Levine RL, Welch C, Tall AR and Wang N. 
LNK/SH2B3 Loss of Function Promotes Atherosclerosis and Thrombosis. Circ Res. 2016;119:e91-e103. 
16. 
Violi F, Calvieri C, Ferro D and Pignatelli P. Statins as antithrombotic drugs. Circulation. 
2013;127:251-7. 
17. 
Ray KK, Cannon CP, McCabe CH, Cairns R, Tonkin AM, Sacks FM, Jackson G, Braunwald E and 
Investigators PI-T. Early and late benefits of high-dose atorvastatin in patients with acute coronary 
syndromes: results from the PROVE IT-TIMI 22 trial. J Am Coll Cardiol. 2005;46:1405-10. 
18. 
Heeschen C, Hamm CW, Laufs U, Snapinn S, Bohm M, White HD and Platelet Receptor Inhibition 
in Ischemic Syndrome Management I. Withdrawal of statins increases event rates in patients with acute 
coronary syndromes. Circulation. 2002;105:1446-52. 
19. 
Cipollone F, Mezzetti A, Porreca E, Di Febbo C, Nutini M, Fazia M, Falco A, Cuccurullo F and Davi 
G. Association between enhanced soluble CD40L and prothrombotic state in hypercholesterolemia: 
effects of statin therapy. Circulation. 2002;106:399-402. 
20. 
Undas A, Brummel-Ziedins KE, Potaczek DP, Stobierska-Dzierzek B, Bryniarski L, Szczeklik A and 
Mann KG. Atorvastatin and quinapril inhibit blood coagulation in patients with coronary artery disease 
following 28 days of therapy. Journal of thrombosis and haemostasis : JTH. 2006;4:2397-404. 
21. 
Pawelczyk M, Chmielewski H, Kaczorowska B, Przybyla M and Baj Z. The influence of statin 
therapy on platelet activity markers in hyperlipidemic patients after ischemic stroke. Arch Med Sci. 
2015;11:115-21. 
22. 
Pignatelli P, Carnevale R, Pastori D, Cangemi R, Napoleone L, Bartimoccia S, Nocella C, Basili S 
and Violi F. Immediate antioxidant and antiplatelet effect of atorvastatin via inhibition of Nox2. 
Circulation. 2012;126:92-103. 
23. 
Writing C, Lloyd-Jones DM, Morris PB, Ballantyne CM, Birtcher KK, Daly DD, Jr., DePalma SM, 
Minissian MB, Orringer CE and Smith SC, Jr. 2016 ACC Expert Consensus Decision Pathway on the Role of 
Non-Statin Therapies for LDL-Cholesterol Lowering in the Management of Atherosclerotic Cardiovascular 
Disease Risk: A Report of the American College of Cardiology Task Force on Clinical Expert Consensus 
Documents. J Am Coll Cardiol. 2016;68:92-125. 
24. 
Sabatine MS, Giugliano RP, Wiviott SD, Raal FJ, Blom DJ, Robinson J, Ballantyne CM, Somaratne 
R, Legg J, Wasserman SM, Scott R, Koren MJ, Stein EA and Open-Label Study of Long-Term Evaluation 
against LDLCI. Efficacy and safety of evolocumab in reducing lipids and cardiovascular events. N Engl J 
Med. 2015;372:1500-9. 
25. 
Sikora J, Kostka B, Marczyk I, Krajewska U, Chalubinski M and Broncel M. Effect of statins on 
platelet function in patients with hyperlipidemia. Arch Med Sci. 2013;9:622-8. 
26. 
Leoncini M, Toso A, Maioli M, Angiolillo DJ, Giusti B, Marcucci R, Abbate R and Bellandi F. High-
dose atorvastatin on the pharmacodynamic effects of double-dose clopidogrel in patients undergoing 
percutaneous coronary interventions: The ACHIDO (Atorvastatin and Clopidogrel HIgh DOse in stable 
patients with residual high platelet activity) study. JACC Cardiovasc Interv. 2013;6:169-79. 
27. 
Malinin A, Pokov A, Swaim L, Kotob M and Serebruany V. Validation of a VerifyNow-P2Y12 
cartridge for monitoring platelet inhibition with clopidogrel. Methods Find Exp Clin Pharmacol. 
2006;28:315-22. 
28. 
Corash L. Measurement of platelet activation by fluorescence-activated flow cytometry. Blood 
Cells. 1990;16:97-106; discussion 107-8. 
29. 
Michelson AD. Methods for the measurement of platelet function. Am J Cardiol. 2009;103:20A-
26A. 
30. 
Kuhne T, Hornstein A, Semple J, Chang W, Blanchette V and Freedman J. Flow cytometric 
evaluation of platelet activation in blood collected into EDTA vs. Diatube-H, a sodium citrate solution 
supplemented with theophylline, adenosine, and dipyridamole. Am J Hematol. 1995;50:40-5.