Determining Correlations between Intraocular Pressure
and Platelet Aggregation in Patients Suffering from Type 2 Diabetes
Joshua Clineburg, Department of Biology, York College of Pennsylvania
Proposal Summary
Research has suggested that aspirin increases the level of platelet
aggregation, while aspirin combined with clopidogrel decreases aggregation
effectively in type 2 diabetics (Angiolillo et al. 2005).
Specific interest arises from possible correlations between the
application of aspirin and clopidogrel and their resulting effects upon
platelet aggregation, as well as platelet aggregation and its contribution to
intraocular pressure in type 2 diabetic patients.
Objective/Hypotheses
Introduction
Diabetes mellitus is known to effect platelet production and function in
humans. Many studies have been conducted with the intent of analyzing
the effects of specific aspects of platelet production in patients suffering
from diabetes.
It has been suggested that platelet production is increased in patients
suffering from diabetes. This increased platelet aggregation has been
shown to increase the occurrence of many cardiovascular ailments in both
healthy and diabetic individuals alike (Michno et al. 2007). Retinopathy and
vision loss is a major occurrence in type 2 diabetics. Retinopathy may be
attributed to the increased levels of platelet aggregation.
Aspirin, when combined with clopidogrel, has been shown to suppress
platelet aggregation in type 2 diabetics. The goal of this study is to compare
groups of individuals suffering from type 2 diabetes and increased
intraocular pressures with groups of type 2 diabetics introduced to aspirin
and clopidogrel as a means to combat excessive platelet aggregation.
By performing this study, both the effectiveness of aspirin and
clopidogrel as an anti platelet aggregate, as well as the role that this
aggregation plays in affecting intraocular pressure will be assessed.
The primary objective of this study is to determine whether any
correlation exists between the administration of aspirin and clopidogrel and
possible effects upon intraocular pressure. Additionally, this study will
reinforce, or contest, previous precedents in correlations between the
administration of aspirin and clopidogrel and platelet aggregation, as well as
platelet aggregation and intraocular pressure.
H1: There is a negative correlation between aspirin and clopidogrel use, platelet
aggregation, and intraocular pressures.
This result would suggest that platelet aggregation is effectively mediated through the
use of aspirin and clopidogrel, as well as reinforcing the idea that platelet aggregation is
a contributing factor to intraocular pressure
H0: There is no correlation between the two factors.
This result would indicate that: 1.) Platelet aggregation is not effectively mediated by
aspirin and clopidogrel, 2.) Platelet aggregation levels do not play a significant role in
intraocular pressures, or 3.) Both 1 and 2 are apparent
Research Design
Intraocular Pressure (mmHg)
Platelet Aggregation vs Intraocular Pressure
30
20
10
0
50
55
60
65
70
Platelet Aggregation (%)
20
10
0
50
55
60
65
70
Commence Pilot Study n=30
Review of Literature
Studies have generally shown that platelet aggregation levels in patients
suffering from diabetes are significantly greater than those in healthy
individuals. It has been suggested that one may measure the degree of
platelet hyperactivity by monitoring the body’s levels of ATP and ADP
(Michno et al. 2007).
Glucose initiates acetyl-CoA production and increases enzyme activity.
Excessive glucose levels, hyperglycemia, stimulates enzymes, which in turn
trigger excessive levels of acetyl-CoA synthesis. Increased acetyl-CoA levels
promote the initiation of ADP platelet aggregation, or collagen aggregation
(Michno et al. 2007). This collagen aggregation has been shown to involve
FcyRIIA (a platelet) interacting with 807TT to increase collagen-platelet
sensitivity, resulting in elevated platelet aggregation levels (Calverley et al.
2005).
Increased platelet aggregation stimulates Müller cells within the eye,
causing them to exert tractional force. This tractional force plays a great
role in retinopathy (Guidry et al. 2004). Additionally, the restriction of blood
flow to the retina, which may be ascribed to platelets, induces further stress
upon Müller cells. This in turn triggers the Müller cells to release angiogenic
growth factor, leading to increased intraocular pressure, and retinopathy.
Platelet aggregation has been correlated to the contraction of Müller cells,
and therefore possibly retinopathy (Guidry et al. 2004).
Figure 1a
30
Platelet Aggregation (%)
•
•
•
•
75
Platelet Aggregation vs Intraocular Pressure
when Mediated with Aspirin and Clopidogrel
Intraocular Pressure (mmHg)
Employment of this study will prove to be beneficial in reinforcing
previous studies pertaining to the nature of diabetes and vascular disease.
Very little research has been conducted which observes relationship of
vascular complications and retinopathy in diabetic subjects. The proposed
study will also offer possible direction in the conception of new treatments
in response to retinopathy, in both healthy and diabetic subjects.
Additionally, this project will warrant the examination of a new possible link
in causal origin to retinopathy in general, namely platelet aggregation.
Cumulatively, this project will further validate, or contest, previous
research/correlations in the fields of vascular medicine, and ophthalmology,
while possibly detecting a new correlation between the two in their
contribution to disease.
Expected Results
75
Figure 1b
Figures 1a and 1b. Expected results, comparing platelet aggregation and intraocular pressure in a single type 2 diabetic
subject prior to treatment with aspirin and clopidogrel (Figure 1a), and post treatment (Figure 1b). Treatment is expected
to correlate negatively with platelet aggregation levels; additionally, platelet aggregation should negatively correlate with
intraocular pressures. The primary objective of this study will be to assess the correlation between treatment and
intraocular pressures.
Collect initial blood samples from designated groups
Group A: 10 healthy individuals
Group B: 10 type 2 diabetics
Group C: 10 type 2 diabetics introduced to 100mg of aspirin and
75mg of clopidogrel per day 10 days prior to collection of blood
samples
Platelet Isolation
Measurement of
Platelet Aggregation
ADP/ATP Isolation
• Collect blood samples of 10
ml
• Introduce 10 mL of
tripotassium EDTA per
blood sample, the sample
centrifuged at 4o C at 150g
for 15 minutes
• Add 140/1 NaCl solution
• Transfer to plastic tube and
centrifuge at 500g for 15
minutes
• Wash with buffer solution
• Analyze samples with HMX
hematologic analyzer
• Suspend platelets in 0.3 ml
of suspension medium
• Incubate for 5 minutes at
37oC in aggregometer
• Add 0.05 ml of 20%
trichloracetic acid
• Shake suspension for 30
minutes at 4oC, then
centrifuge
• Add 0.03 ml of thrombin
• Assess aggregation through
changes in turbidity within
sample suspensions
containing thrombin,
comparing them with blank
suspension
• Transfer platelet samples to
suspension medium
• Incubate for 30 minutes
• Place in ice bath
• Centrifuge at 12,000g
• Place pellets in 0.1 ml of
distilled water, boil for 30
minutes
• Centrifuge
• Measure quantities of
ADP/ATP through
luciferin/luciferase
luminomentric process
Previous research suggests that established correlations exist between:
1.) platelet aggregation and treatment with aspirin and clopidogrel, and 2.)
intraocular pressures and platelet aggregation. Based upon such
precedents, it is expected that the mediation of aspirin and clopidogrel will
correlate negatively with intraocular pressure.
Literature Cited
Angiolillo, Dominick J., Fernandez-Ortiz, Antonio, Bernardo, Esther, Ramirez, Celia, Sabate, Manel, Jimenez-Quevedo, Pilar, Hernandez, Rosana,
Moreno, Raul, Escaned, Javier, Alfonso, Fernando, Banuelos, Camino, Costa, Marco A., Bass Theodore A., and Macaya, Carlos. 2005. Patient function
profiles in patients with type 2 diabetes and coronary artery disease on combined aspirin and clopidogrel treatment. Diabetes 54: 2430-2435.
Guan, Kit, Hudson, Chris, Wong, Tien, Kisilevsky, Mila, Nrusimhadevara, Ravi K., Lam, Wai Ching, Mandelcorn, Mark, Devenyi, Robert G., and Flanagan,
John, G. 2006. Retinal hemodynamics in early diabetic macular edema. Diabetes 55: 813-818.
Guidry, Clyde, Feist, Richard, Morris, Robert, and Hardwick, Charles W. 2004. Changes in IGF activities in human diabetic vitreous. Diabetes 53: 24282435.
Michno, Anna, Bielarczyck, Hanna, Pawelczyk, Tadeusz, Jankowska-Kulawy, Agnieszka, Klimaszewska, Joanna, and Szutowicz, Andrzej. 2007. Alterations
of adenine nucleotide metabolism and function of blood platelets in patients with diabetes. Diabetes 56: 462-467.
Full Study n=120
• Groups will be designated as the pilot study, consisting of 40 individuals per group
• Protocol within the pilot study will be repeated
• Each of the 120 patients will have their intraocular pressures measured through
Goldmann applanation tonometry
• A retinal specialist will be employed to assess the observed pressures
• Data obtained from subjects’ initial blood samples will be compared to the treated blood
samples
• Intraocular pressures will be compared among all groups (Utilizing ANOVA)
Acknowledgements
I would like to personally thank and offer my sincere gratitude to Dr. Ronald
Kaltreider and Dr. Bruce Smith for their continuous guidance and support
throughout the duration of this research.