Determining Correlations between Intraocular Pressure and Platelet Aggregation in Patients Suffering from Type 2 Diabetes

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.

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).

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.

Expected ResultsProposal Summary 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.

Platelet 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

Measurement of Platelet Aggregation

• 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

ADP/ATP Isolation

• 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

Research Design

Commence Pilot Study n=30

• 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

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)

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.

Literature CitedAngiolillo, 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: 2428-2435.

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.

AcknowledgementsI 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.

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.

Joshua Clineburg, Department of Biology, York College of Pennsylvania

Objective/Hypotheses 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

Platelet Aggregation vs Intraocular Pressure

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Platelet Aggregation vs Intraocular Pressurewhen Mediated with Aspirin and Clopidogrel

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Figure 1a

Figure 1b