Aspirin resistance assessment in acute myocardial infarction patients

¶ … Aspirin for Acute Myocardial Infarction Patients

Item

Organization of the Study

IV ASPIRIN FOR ACUTE MYOCARDIAL INFARCTION PATIENTS

IV ASPIRIN FOR ACUTE MYOCARDIAL INFARCTION PATIENTS

Aspirin (ASA) has been known to influence the bleeding time of individuals who take aspirin and it this is presumably due to the inhibition of prostaglandis biosynthesis and the resultant platelet secretion time. All observations in man indicate that long-term ingestion of aspirin is associated with impaired platelet function and prolongation of the bleeding time. Patients with inadequate platelet inhibition by aspirin, which is commonly referred to as aspirin resistance, may have an increased risk of suffering cardiovascular events. Because of this, identification of these patients with aspirin resistance by measuring platelet function is of great interest. This work reports a study that used the VerifyNow Assay to formulate IV aspirin for administration to patients with Acute Myocardial Infarction.

IV ASPIRIN FOR ACUTE MYOCARDIAL INFARCTION PATIENTS

CHAPTER ONE

INTRODUCTION

Aspirin (ASA) has been known to influence the bleeding time of individuals who take aspirin and it this is presumably due to the inhibition of prostaglandis biosynthesis and the resultant platelet secretion time. All observations in man indicate that long-term ingestion of aspirin is associated with impaired platelet function and prolongation of the bleeding time. Patients with inadequate platelet inhibition by aspirin, which is commonly referred to as aspirin resistance, may have an increased risk of suffering cardiovascular events. Because of this, identification of these patients with aspirin resistance by measuring platelet function is of great interest.

As the platelets adhere, they undergo secretion and recruitment of other platelets to build a platelet plug. This is termed 'platelet aggregation' and 'platelet secretion'. Low platelet response to aspirin has been consistently reported to be associated with a high incidence of vascular events. The ability of platelets to aggregate in response to an agonist is determined in clinicopathological studies by using the principle of light transmission.

In vitro platelet aggregation studies the Born method has traditionally been used and is the method of choice in assessing platelet function. This method has been used to assess aspirin sensitivity in several studies. Another method that has been used extensively is measurement of the urinary TXA2 metabolite 11-dehyro-TXss using both radioimmunoassay (RIA) and enzyme immunoassay (EIA). A revision of EIA is commercially available under the marketed name of 'Aspirin Works' (Creative Chemical Concepts Inc.).

Two automated point-of-care (POC) Platelet function assays have also been studied: the Platelet Function Analyzer 100 (PFA-100) (Dade Behring Inc.) and the verify Now Aspirin Assay (Accumetrics Inc.). Platelet aggregation is also determine dosing an optical platelet aggregometer (OPA) such as the PAP4 (Bio Data Corp) and Model 700 whole blood optical Lumi Aggregometer (Chronolog Inc.) This method relies on the concept that the platelet aggregations in a uniform solution of platelet-rich plasma (PRP) decreases the turbidity of the solution as the platelets cross-link and clump together. The amount of platelet aggregation is directly related to the amount of light that is allowed to be transmitted through the solution. Results area expressed in units of percentage of light transmission on a scale of 0-100%.

Like the PFA-100 the VerifyNow is a whole blood (WB) point-of-care device that conveniently measures platelet aggregation using different easy-to-load cartridges for different applications. Unlike the PFA-100 the VerifyNow is designed only to detect platelet dysfunctions or the result of exposure to antiplatelet agents such as aspirin, clopidogrel and glycoprotection II-IIIa receptor antagonists (eg absiximab). Platelet aggregation detection is based on the agglutination of platelets on fibringogen-coated beads detected by an optical turbidity method.

Anticoagulant therapy was routine management of patients with myocardial infarction in the 1950s however, this management "fell into disrepute because of the fear of bleeding complications and doubt about its effectiveness." (Stein, 1998, p.639) Stein states that the primary objectives of anticoagulant treatment in patients with myocardial infarction are: (1) to improve survival; (2) to prevent recurrent infarction, mural thrombosis and systemic embolism; and (3) to prevent the complication of venous thromboembolism. (p.639)

The use of "long-term anticoagulant therapy for improving survival and preventing recurrent infarction has been controversial." (Stein, p.639) Stein reports that the risk of complications from bleeding associated with long-term anticoagulant therapy in patients with prosthetic heart valves has been reported to range form 1% to 40%. Major bleeding ranged from 0% to 7% and fatal bleeding from 0% to 4.1%. (Stein, 1998, p. 640) When aspirin is added to warfarin the risk of bleeding increases yet, when aspirin in low doses are used (100 mg/day) the increase is due to bleeding episodes of a minor nature without major bleeding increases.

STATEMENT OF THE PROBLEM

Aspirin has been found by many studies across many decades to be effective in treating patients with heart disease including myocardial infarction however, the problem is that many patients cannot successful take aspirin due to complicating factors including conditions that result in bleeding being triggered by aspirin.

PURPOSE OF THE STUDY

The hydrolysis of aspirin markedly reduced its antiplatelet activity. This has prevented the development of an effective IV formulation of aspirin that would improve the treatment of acute coronary syndromes. This study reports the development of a formulation that is believed to have the capacity to resist hydrolysis and preserve platelet inhibition.

ORGANIZATION OF THE STUDY

The following Chapter or Chapter 2 will contain a literature review in this area of study. Chapter three will contain the methods of the research as well as the research summary and conclusion.

TERMS & DEFINITIONS

CI confidence interval

COX cyclo-oxygenase

CRP C-reactive protein

CRT cardiac resynchronization therapy

ECG electrocardiographic/electrocardiogram

GP glycoprotein h hour

HDL high-density lipoprotein

INR international normalized ratio

i.v. intravenous

LDL low-density lipoprotein

LMWH low-molecular-weight heparin

LV left ventricular min minute

MBG myocardial blush grade

MRI magnetic resonance imaging

NSAID non-steroidal anti-inflammatory drug

NYHA New York Heart Association

OR odds ratio

PCI percutaneous coronary intervention

PDA personal digital assistant

PET positron emission tomography

s seconds

s.c. subcutaneous

SCD sudden cardiac death

SPECT single-photon emission computed tomography

STEMI acute ST-segment elevation myocardial infarction

TIMI thrombolysis in myocardial infarction

IV ASPIRIN FOR ACUTE MYOCARDIAL INFARCTION PATIENTS

CHAPTER TWO

LITERATURE REVIEW

Myocardial infarction is stated in the work of Van de Werf, et al. (2008) to be defined "from a number of different perspectives related to clinical, electrocardiographic (ECG) biochemical and pathological characteristics." (p.2912) The present guidelines are stated to pertain to patients "presenting with ischaemic symptoms and persistent ST-segment elevation on the ECG (STEMI). The great majority of these patients will show a typical rise of biomarkers of myocardial necrosis and progress to Q-wave myocardial infarction." (Van de Werf, et al., 2008, p.2913)

According to Van de Werf, et al. The majority of cases of STEMI are caused "by an occlusion of a major coronary artery. Coronary occlusion and reduction in coronary blood flow are usually due to physical disruption of an atherosclerotic plaque with subsequent formation of an occluding thrombus. Concomitant coronary vasoconstriction and microembolization may be involved to some extent." (Van de Werf, et al., 2008, p.2913)

It is less common however there is the possibility of the formation of a thrombus due to a "superficial erosion of the endothelial surface. The risk of plaque disruption depends on plaque composition and vulnerability (plaque type) and degree of stenosis (plaque size). As many as three-quarters of all infarct-related thrombi appear to evolve over plaques causing only mild to moderate stenosis." (Van de Werf, et al., 2008, p.2913)

It is related that portions of the coronary arterial tree that look normal according to angiographic criteria have been found to "...harbor a substantial burden of atherosclerosis. In particular, plaques with substantial outward remodeling, or 'compensatory enlargement', can have thin, fibrous caps and large lipid pools without encroachment of the lumen. However, severe stenoses are as likely to undergo plaque events leading to infarction as mild ones" (Van de Werf, et al., 2008, p.2913)

Van de Werf et al. state that there is often up to a two-week delay between the plaque rupturing and the clinical consequences. Playing a key role in the instability of plaque is the factor of inflammation which also affects the pathogenesis of acute coronary syndromes. Van de Werf, et al. report that increased sympathetic stimulation activities and those associated with vasoconstriction including stress either physical or emotional in nature are known to cause plaque disruption and coronary thrombosis. Coronary artery occlusion causes myocardial necrosis which develops approximately fifteen to thirty minutes of severe ischaemia and "...progresses from the subendocardium to the subepicardium in a time-dependent fashion ('the wavefront phenomenon'). Reperfusion, including recruitment of collaterals, may save myocardium at risk from undergoing necrosis, and subcritical but persistent forward flow may extend the time window for achieving myocardial salvage. The thrombotic response to plaque disruption is dynamic: thrombosis and clot lysis, often associated with vasospasm, occur simultaneously, and may cause intermittent flow obstruction and distal embolization." (Van de Werf, et al., 2008. p.2914)

The work of Chan, et al. (nd) entitled: "Reappraisal of Long-Term Tolerance of Aspirin in Patients After Myocardial Infarction" states that aspirin has "by virtue of an irreversible inhibition of platelet cyclooxygenase leading to diminished platelet aggregation...has found wide applications in the prophylaxis of cardiac and cerebrovascular catastrophes." (p.566) In fact, it is reported that aspirin alone resulted in a reduction in post myocardial infarct (MI) mortality "at five weeks by 23% in the Second International Study of Infarct Survival (ISIS-2)." (Chan et al., nd, p.566)

Chan et al. states that MI prophylaxis with aspirin "can be associated with a reduction of up to 15% total deaths and 30% of reinfarction rates." (nd, p.566) Chan et al. notes that there is wide recognition of "...drug-induced acute and chronic gastroduodenal complications" from aspirin and state that "the magnitude of drug related adverse events is important in determining the proportion of patients who can sustain the proven prophylactic benefit. Variations in methodology in epidemiological studies or other therapeutic randomized studies often result in widely differing estimates of such magnitude." (Chan et al., nd, p. 566) Chan et al. report a study and the results of a retrospective survey into the tolerance long-term of low dosage of aspirin utilized as a secondary prophylaxis following myocardial infarction.

Figure 1

Gastrointestinal Intolerance Due to Aspirin in Prospective Randomized Trials

Source: Chan et al. (nd)

Figure 2

Comparison of Risk Factors in Patients with (Group B) or without (Group A) Adverse Upper Gastrointestinal symptoms or events

Source: Chan et al. (nd)

Figure 3

Comparison of Risk Factors in Patients with (Group B) or without (Group A) adverse GI Symptoms of events -- excluding all patients on antacids of ulcer healing drugs

Source: Chan et al. (nd)

Figure 4

Outcome of Patients with Upper GI Symptoms or Events While Taking Long-Term Aspirin

Source: Chan et al. (nd)

Figure 5

Dosages and Formulations of Aspirin and Pattern of Co-Prescription with Antacids or Ulcer Healing Drugs

Source: Chan et al. (nd)

Chan et al. reports analysis of the casenotes of patients who were admitted to the Coronary Care Unit, United Christian Hospital between the first of April 1991 and June 30th 1995. Survey participants were stated to have had:

(1) A definite diagnosis of acute myocardial infarction after admission;

(2) been discharged with aspirin as long-term secondary prevention; and (3) subsequent clinical follow-up for three months or more. (Chan et al., nd, p. 567)

Excluded from the study were patients who:

(1) had died within the same admission;

(2) those with follow-up shorter than three months; and (3) those who were transferred to other cardiac centers for subsequent follow-up. (Chan, et al., nd, p.567)

Chan et al. reports that data on patient characteristics including age, gender, smoking, alcohol consumption, concurrent illnesses, and history of peptic ulcer and/or gastrointestinal hemorrhage were extracted from in -- and out-patient records. Furthermore "the reason of aspirin withdrawal, co-prescription with antacids, gastric cytoprotective or anti-ulcer drug and results of upper gastrointestinal investigation were also noted." (Chan et al., nd, p.567) The study reported by Chan et al. (nd) states that the proportion of patients both with and without side effects of a gastrointestinal nature were compared in regards to common risk factors. Group A is stated to have been comprised by patients who did not "complain of any dyspeptic symptoms and had no upper GI events diagnosed throughout the follow-up period; and group B. consisted of patients who either had symptoms of dyspepsia with or without subsequent positive investigations or had a diagnosis of peptic ulceration, unexplained anemia, acute GI hemorrhage or perforation. Chi-square test or two tailed Fisher exact test was used to detect significance for categorical variables. A p-value of < 0.05 was considered significant." (nd, p.568)

Results of the study reported by Chan et al. (nd) state that 173 patients met the criteria for analysis and was comprised of 113 males and 60 females with a mean age of 66.5±SD 9.8 yrs (range 35-93 ). Chan et al. states:

"Thirty-eight of the 173 (22%) patients continued to smoke. 8 patients (4.6%) continued to consume excessive alcohol. Coexisting renal dysfunction with serum creatinine exceeding 150 umol/l were found in 19 patients (11%). Nine patients (5.2%) had a past history of peptic ulcer with or without hemorrhage. Concurrent use of non-steroid anti-inflammatory drugs (NSAID), corticosteroid and oral anticoagulant were found in 4 (2.3%), O (0%), and 3 (1.7%) patients, respectively." (nd, p. 568)

Chan et al. (nd) state that aspirin has become "an integral component of everyday cardiology practice. Its efficacy in the treatment and prophylaxis of cerebrovascular and cardiovascular disease is well established. The inherent risk of associated gastroduodenal complications requires a careful weighing of risks vs. benefits of the therapy. A previously proposed theory of gastric adaptation with prolonged administration has generated controversies. Whether this adaptation occurs in a clinically predictable or protective manner remains largely unknown." (p. 569)

Chan et al. states that the "...magnitude of treatment risk can be derived from case-control studies or large randomized studies. The former may be subject to biases in questioning and in control selection whilst the latter may have strict exclusion criteria that underestimate risks in GI adverse events. Comparing the magnitude of risks or adverse events in randomized trials also poses difficulties because of different dose regimes and varying length of follow up." (nd, p. 570)

The reported incidence of adverse gastrointestinal events was stated to differ widely and that this was not surprising to researchers in the study. Chan et al. reports that one of every seven patients who they had intended to maintain on long-term low dose aspirin following a myocardial infarct had the drug withdrawn and 4/5 of the reasons are stated to be due to GI adverse symptoms or events. While it is understood that lower doses of aspirin are likely to be safer it is also understood that no dose of aspirin leaves the patient free of these types of risks. It is related that renal impairment or failure was more common in the group where gastrointestinal adverse symptoms and/or events took place while taking aspirin which supports the hypothesis that the "intrinsic bleeding tendency due to uremia might be aggravated with the use of aspirin." (Chan et al., nd, p. 539)

Chan et al. states that while a great proportion of the patients (127 of 173) were co-prescribed antacids with aspirin that it has not yet been proven to have "prophylactic value against ulcer and other complications." (Chan et al., nd, p. 539)

Key findings stated by Chan et al. include those as follows:

(1) Aspirin has been proven to be effective and widely prescribed for secondary prevention following myocardial infarction;

(2) Chan et al. (nd) reports their studying show one in seven patients were withdrawn from aspirin on long-term follow-up;

(3) The primary reason of aspirin cessation was gastrointestinal adverse effects;

(4) Renal impairment was associated with a higher risk of gastrointestinal adverse effects;

(5) The potential side effects of aspirin should be carefully monitored in every patient. (Chan et al., nd)

The work entitled: "Thrombolysis for Acute Myocardial Infarction: Drug...: The Aspirin Factor" states that in the nine major trials of thrombolytic agents vs. standard treatment for EMI, which established the role of thrombolytic therapy "all patients received aspirin in 4 trials and half of the patients took it in ISIS-2, where aspirin allocation was random. Of all randomized trials of anticoagulants for AMI since the 1970s, only ISIS-2 looked at aspirin and thrombolysis independently and in combination.

Aspirin or another antiplatelet drug alone provides an unknown portion of the overall reduction in mortality when combined with thrombolytic agents, but aspirin causes almost none of the toxicity or expense. Given these comparisons, evidence of an additional survival benefit of thrombolysis above that of aspirin depends entirely on ISIS-2 and whether ISIS-2 data is consistent with findings of the other trials." (Cundiff, 2002)

It is stated that ISIS-2 "randomized 17,187 patients with suspected AMI, comparing aspirin, SK, both, and neither. This study reported that aspirin decreased the odds of death in 5 weeks in the aspirin arm by 23% (24 per 1000 patient deaths prevented), SK reduced it by 25% (28 per 1000 patient deaths prevented), and both together by 42% (52 per 1000 patient deaths prevented). The relative efficacy of aspirin and thrombolysis on AMI mortality in ISIS-2 differs considerably from those of the meta-analyses of the aspirin and thrombolysis trials. The mortality reduction in the meta-analysis of aspirin vs. placebo studies (approximately 20,000 patients studied; survival benefit = 29% [about 40/1000 deaths prevented] exceeds that of the meta-analysis of thrombolysis trials (58,511 patients studied; survival benefit = 18% [about 20/1000 deaths prevented]).(Cundiff, 2002)

The omission of the ISIS-2 from the meta-analysis provides for a survival benefit of approximately 13% or approximately 15/1000 deaths prevented. The question addressed is what thrombolytics do to AMI mortality compared with aspirin. IN comparison of the mortality of the fibrinolysis studies that did not include routine use of aspirin vs. The trails in which all patients were given aspirin results in showing a different in efficacy of thrombolysis that is stated to be striking. ISIS-2 and the studies that were not inclusive of routine aspirin "account for virtually all of the meta-analysis' statistical significant concerning a survival advantage with fibrinolysis in AMI. The 21,144 patients randomized in studies including routine aspirin demonstrate no significant survival advantage with fibrinolysis (P = .14)." (Cundiff, 2002)

The work of Borna, et al. (2005) entitled: "Resistance to Aspirin is Increased by ST-elevation Myocardial Infarction and Correlates with Adenosin Diphosphate Levels" state that in order to be fully activated "platelets are dependent on two positive feedback loops; the formation of thromboxane A2 by cyclooxygenase in the platelets and the release of ADP from dense platelet granules. Thromboxane A2 and ADP then activates specific receptors on the extracellular side of the platelet membrane. Therapeutic intervention aimed at the first positive feedback loop by inhibiting cyclooxygenase with aspirin is highly efficient in reducing death and cardiovascular events by approximately 25%." (p1.)

It is stated by Borna, et al., (2005) that ADP may be "...even more important as evidenced by the CAPRIE-study, in which the ADP receptor antagonist clopidogrel was more beneficial than aspirin in reducing cardiovascular events. Furthermore, the CURE and CREDO studies have established clopidogrel in combination with aspirin as a valuable treatment for acute coronary syndromes." (p.1)

Approximately 9% to 45% of patients are found to have aspirin resistance and there is little known in relation to the clinical consequences of resistance to aspirin although two recent studies have indicated that aspirin resistance might be associated with an increase in the number of cardiovascular events. Assessment of platelet activation is difficult at best and laboratory testing currently available is not sufficiently reliable or is complicated and not eligible for routine use in clinical studies.

Borna et al. (2005) reports the use of a "...novel platelet function test, PFA-100. PFA-100 is an ex-vivo assay of shear stress induced platelet adhesion and aggregation in whole blood. It simulates an injured blood vessel by a collagen-coated membrane together with either epinephrine or ADP. It has been found to be a sensitive test of aspirin resistance. Aspirin resistance has previously been studied in healthy controls and in stable patients with a previous myocardial infarction.

Borna et al. reports the evaluation of the effect of aspirin on platelet function in patients with acute coronary syndromes with the stated hypothesis that "increased levels of ADP in patients with acute coronary syndromes could contribute to aspirin resistance." (p.1) Borna et al. (2005) reports that 135 patients were enrolled from among those admitted to the emergency ward for chest pain at Lung University Hospital between 2001 and 2003. Only patients with chest pain in the last hour prior to admittance were eligible for the study. The study defined patients as either being aspirin users or patients not taking aspirin in the past three weeks and not having received aspirin while be transported to the hospital.

Those who were defined as users of aspirin were patients who had been taking aspirin daily for at least one week prior to hospital admittance. The majority of patients were taking aspirin at a dosage of 75 mg one time a day but there were a few patients in this study that were on 320 mg one time a day although these were less than 10% of the total. Borna, et al. (2005) states exclusion criteria to include: "...ingestion of clopidogrel, dipyridamole, nonsteroidal anti-inflammatory drugs, heparin, low molecular heparin, warfarin, receiving bolus dose of aspirin on their way to hospital, platelet count

Three prespecified subgroups were compared upon the basis of diagnosis at discharge: (1) chest pain with no sign of cardiac disease (NCD); (2) Non-ST elevation myocardial infarction (NSTEMI); and (3) STEMI. Borna et al. (2005) states that the NCD group presented no recent ECG changes and normal values of TNT. Exercise test where appropriate before discharge were negative. A minor number of patients in the NSTEMI and STEMI group were on beta-blocker, ACE-inhibitors and Ca-channel blockers. However, the groups were to small for subgroup analysis." (p.1)

Borna et al. states that the PFA-100 system has been described in the work of Kundu et al. And it is stated that the PFA-100 utilizes a disposal test cartridge that simulates an injured blood vessel. The PFA-100 simulates primary homeostasis by flowing whole blood at a high shear rate through an aperture (147 ?m diameter) cut into a collagen-coated membrane coated with either ADP (50 ?g) or epinephrine (10 ?g), where it comes into contact with the membrane surface and aggregate. A platelet plug forms, with occlusion of the aperture and cessation of blood flow. The closure time reflects platelet function in the sample evaluated. Shorter closure times indicate increased platelet aggregation. Testing was done in whole blood from antecubital vein samples anticoagulated with 3.8% sodium citrate. Samples were obtained at admission in 135 patients. PFA-100 tests were performed within 30 min after blood sampling, within 1 hour after admission to hospital. The epinephrine-collagen cartridge (EPI-COLL) is sensitive to aspirin and can be used for the detection of aspirin resistance. The ADP-collagen cartridge (ADP-COLL) is only weakly sensitive to aspirin. Aspirin resistance was defined as normal EPI-COLL closure times (

Nucleotides were measured in 64 patients including 16 patients with STEMI, 16 patients with NSTEMI and 32 patients with NCD. It is reported that ADP was measured with "...HPLC from antecubital vein samples. Sampling was done at admission. 5 ml blood was added to tubes containing citrate and immediately centrifuged for 10 min at 1200 G, 4°C. Platelet contamination was excluded by Burker chamber examination. The plasma was aspirated and mixed with an equal amount of 10% TCA to precipitate all proteins and inactivate ectonucleotideases. After centrifugation the protein free supernatant was frozen at -80°. Samples were sent on dry ice by courier, to Department of Medicine, University of North Carolina, School of Medicine, Chapel Hill, USA for analysis. Samples were thawed at room temperature and TCA was extracted three times with six volumes of ethyl ether. Ethyl ether was removed by gassing N2, and the resulting samples diluted in the corresponding nucleotide assay buffer as indicated below." (Borna, et al., 2005)

Borna et al. states that the Luciferin-luciferase assay involved extracts being diluted 1:20 in HEPES-buffered Hanks Balanced Salt Solution 9 HBSS, pH [HIDDEN] and a 30 ul sample being added to a test tube and the volume adjusted to 300 ul with JPLC-grade H20. "The luciferin-luciferase reaction mix (100 ?l) was added to tubes with a built-in injector into the light protected chamber of an Auto-Lumat LB953 luminometer. Luminescence was subsequently recorded during 10 seconds and compared against an ATP standard curve performed in parallel. Luminescence was linear with slope of one between 0.1 and 1000 nM ATP." (Borna, et al., 2005, p.1) Borna et al. (2005) states that they adopted and "slightly modified a derivatization protocol originally described by Levitt and co-workers. Sample extracts were incubated for 30 min at 72°C in the presence of 1.0 M. chloroacetaldehyde and 25 mM Na2HPO4 (pH 4.0) in a 200-?l final volume. Samples were transferred to ice, alkalinized with 50 ?l 0.5 M. NH4HCO3, and analyzed by HPLC within 24 h. Identification and quantification of ethenylated species were performed with an automated Waters HPLC apparatus equipped with a fluorescence detector. Derivatized samples were transferred to 0.7 ml plastic shell vials and kept at 4°C in the sample injector rack. A 100 ?l sample aliquot was injected into a 250-mm, 10 ?m Hamilton PRP-X100 anion exchange column. The mobile phase (2 ml/min, 30% methanol) developed linearly from 0.250 to 0.275 M. NH4HCO3 (pH 8.5) during the first 8 min, remaining isocratic at 0.275 M. NH4HCO3for additional 4 min. The column was subsequently rinsed for 3 min with 0.425 M. NH4HCO3 in 30% methanol, and re-equilibrated to the initial conditions for 15 min. Elution times (in min) were: -ADO, 3.2; -AMP, 5.9; -ADP, 7.6, and ?-ATP, 9.4." (2005, p.1) Borna et al. go on to explain that [C]glucose-1P (300 McI/mmol) and molecular biology grade ATP and UTP were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). ADP, AMP, and adenosine were from Roche Molecular Biochemicals (Indianapolis, IN). Etheno-adenyl standards were from Sigma (St. Louis, MO). Firefly luciferase and luciferin were purchased from PharMingen International (San Diego, CA). Other chemicals were of the highest purity available." (2005, p.1) The GraphPad Prism 3.02 software was used for calculations and statistics and values are presented as mean ± SD. Statistical significance was accepted when P < 0.05 (two-tailed test). For continuous variables Kruskal-Wallis test followed by Dunnett multiple comparisons test was used. Spearman's rank correlation coefficient test was used for regression analysis. Categorical variables were compared using the Fisher exact test."

Findings stated by Borna et al. (2005) state that in patients without aspirin therapy there was "...an increased platelet activation in the STEMI group compared to NCD in the EPI-coll. NCD: 139 ± 44, NSTEMI: 121 ± 23 (n.s.), STEMI: 99 ± 28 sec (p < 0.001, mean ± SD). These differences were also seen in patients on aspirin. NCD: 280 ± 41, NSTEMI: 243 ± 72 (n.s.), STEMI: 116 ± 56 sec (p < 0.001). Lower values indicate increased platelet activation. Similar results were observed with ADP-COLL measurements." (p.1) It is related that patients with chronic aspirin treatment for at least one week prior to admission "significantly reduced platelet aggregation in NCD and NSTEMI as seen by increased PFA-100 times for EPI-COLL, but only in NCD for ADP-COLL. However, aspirin had no effect in either EPI-COLL or ADP-COLL in patients with STEMI.Aspirin resistance defined as

Plasma levels of ADP were stated to be "markedly increased in STEMI but not in NSTEMI, compared to NCD." (Borna et al., 2005, p.1) There were stated to be similar findings for other purines. ADP levels are stated to have "correlated with increased platelet activity measured with ADP-COLL in the whole material." (Borna, et al., 2005, p.1) Borna et al. states that similar results were witnessed for total purines and that there was a non-significant trend for EPI-COLL to correlate with both ADP and total purines in patients with aspirin treatment." (2005, p.1)

Aspirin resistant patients are reported to have had higher ADP levels when compared to the aspirin responders and increased levels of total purines. There was no significant correlation with age for purine measurements or with diabetes, haemoglobin, CKMB or troponin T. levels. Borna et al. (2005) state that platelet contamination "was excluded by cell counting" and there were no correlations witnessed between platelet counts and purine levels. Furthermore it is reported that nucleotide turnover is "fast in whole blood due to ectonucleotidases. ATP degradation was evaluated both in samples with endogenous ATP and in samples were ATP had been added. The degradation was rapid in samples where ATP was added with a T. 1/2 of 5.2 min. Endogenous ATP levels had a slower degradation rate, with a T. 1/2 of approximately 30 min. Baseline levels of adenosine were markedly lower than those of its nucleotides and barely detectable, most likely due to both rapid uptake into the red blood cells and degradation. This was because we have not included adenosine deaminase and nucleoside transport inhibitors in the perfused solution. The magnitude of changes in adenosine levels could therefore not be studied but this was not the aim of the study." (Borna, et al., 2005, p.1)

Borna et al. state that confirming previous studies they found that "platelets are activated in acute coronary syndromes. Furthermore, for the first time we could demonstrate a rise in systemic levels of ADP and a decreased platelet inhibitory effect of aspirin in patients with STEMI. It is possible that the raised ADP level contributes to the increased platelet activity and the reduced effect of aspirin." (2005, p.1)

According to Borna et al. evidence is growing that many patients do not derive benefit from therapy involving standard doses of aspirin although aspirin resistance is a term that is poorly defined and used to describe a number of conditions including the inability of aspirin to protect individuals from cardiovascular events, to inhibit platelet aggregation measured with a number of different methods and to inhibit thromboxane A2 formation. We found aspirin resistance levels in controls (NCD) of 9.7%, which is in agreement with a previous study that found 9.5% aspirin resistance using PFA-100. It is also in agreement or even lower than other methods that found frequencies of 9 -- 45%." (2005, p.1)

Borna et al. state that it is interesting that they found "...increased frequency of aspirin resistance in acute coronary syndromes rising from 9.7% in controls to 26.0% in NSTEMI (n.s.) and 83.3% in STEMI (p < 0,001). In fact, we could not see any significant effect of aspirin on platelet aggregation measured with PFA-100 in acute STEMI. Thus, in the situations where the patients need the platelet inhibitory effect of aspirin the most, the aspirin resistance is most frequent." (Borna, et al., 2005, p.1)

The causative factors of aspirin resistance are stated to remain unclear although Altman et al. (2004) provided a description for several mechanisms that may possibly be the cause for aspirin resistance as well as for the difficulty in interpreting and making comparison of the results of different studies. Borna et al. states that the possibilities of "..inadequate doses of aspirin and thromboxane independent platelet aggregation mechanisms have been discussed. In some patients aspirin resistance could simply reflect non-compliance with drug therapy.

Weber et al., (2002) suggested several possible types of aspirin resistance where one is linked to inability to inhibit thromboxane formation. Another possible mechanism is classified as "pseudo resistance" since aspirin exerted the expected pharmacodynamic effect of inhibiting thromboxane formation but platelet aggregation was not inhibited. Hillarp et al., (2003) found that in a series of 200 aspirin treated patients, none was found to have unblocked cyclooxygenase activity. Aspirin resistance despite blocked cyclooxygenase activity has been suggested to be explained by increased platelet sensitivity to ADP and collagen." (Borna et al., 2005, p.1)

The study conducted and reported by Borna et al. (2005) shows that aspirin resistance increases in STEMI and that this is likely due to a general activation of the platelets since platelet aggregation was also noted to increase in patients who were not treated with aspirin. The important positive feedback systems such as ADP and thromboxane could explain the increased aspirin resistance and as well it could be explained by "an increase in vW factor that are released from the endothelial cells under high shear stress." (Borna, et al., 2005, p.1)

Borna et al. (2005) state that several studies have demonstrated that sufficient inhibition of platelet aggregation with the 75 mg dose of aspirin however a recent randomized study involving sixty patients with stable coronary artery disease demonstrated that the effect of aspirin "was dose-dependent and the conclusion was that doses of less than100 mg of aspirin was less effective inhibiting platelet aggregation than doses greater than 100 mg. This could indicate that higher doses of aspirin could be necessary to inhibit increased activity in the thromboxane positive feedback system, but for the time being there is no clear evidence saying that higher doses of aspirin improve clinical outcome." (Borna et al., 2005, p.1)

Aspirin resistance however is stated to possibly be dependent on "increased activity of the ADP positive feedback system. ADP is released by activated platelets, but also in the heart from cardiac myocytes during ischemia, or from endothelial cells, red blood cells and sympathetic nerves. Erythrocytes are known to contain large amounts of ADP, which may increase the platelet activity and modulate the effect of aspirin. It is known that nucleotides are released from numerous cells during stress and exercise.. The control group in this study was therefore chosen to present pain and stress and it is our belief that this control group is more relevant for comparison than unstressed healthy individuals to prove ischemia induced increases in ADP levels." (Borna, et al., 2005, p.1)

Borna et al. states that they have for the first time demonstrated that "...in man that ADP levels are increased during myocardial infarction. It is possible that increased ADP levels during acute myocardial ischemia could contribute to the increased frequency of aspirin resistance. There are at least two subtypes of ADP receptors on the platelet. The P2Y12 receptor is linked via Gi protein to adenylate cyclase. The P2Y12 receptor stimulates platelet aggregation and has a high expression in platelets. Clopidogrel blocks the P2Y12 receptor irreversibly and the value of this treatment has been established by the CURE and CAPRIE studies. P2Y1 is a Gq protein linked ADP receptor expressed in platelets that mediates shape change. P2Y12 and thromboxane receptors act via different intracellular second messenger mechanisms, cAMP and inositol triphosphate (IP3), respectively. This explains the additive clinical effect of clopidogrel when it is combined with aspirin. However, the P2Y1 receptor acts via the same second messenger system as thromboxane (IP3). Thus, high levels of ADP could replace thromboxane as stimulator of IP3 by activation of P2Y1 receptors. If ADP contributes to aspirin resistance, it may not be sufficient to block the P2Y12 receptor. It is possible that P2Y1 antagonists also will be necessary to achieve inhibition of both the important intracellular second messenger systems in the platelet." (p.1)

Borna et al. states that previous studies have demonstrated that patients who are aspirin resistant have higher incidences of cardiovascular events. Several studies have shown a higher incidence of cardiovascular events in patients reported to be aspirin resistant. The findings of Gum et al. were that "...aspirin resistance was associated with an increased risk of death, myocardial infarction or cerebrovascular accident compared to aspirin sensitive patients. (Borna et al., p.1) Borna et al. states that the work of Grotemeyer and colleagues provided a description in 1993 of a "...40% risk for major events (stroke, myocardial infarction or vascular death) for aspirin resistant patients compared to a 4% risk in the aspirin responder group." (p.1)

The conclusions stated by Borna et al. include that platelets are activated and resistance to aspirin is more frequent in STEMI and that this is likely do to a general activation of platelets. Further stated is that ADP levels are "...increased in STEMI and correlates with platelet activation. Increased levels of ADP could be one reason for increased platelet activity and aspirin resistance." (Borna et al., 2005, p.1) The following illustration have been adapted from the work of Borna et al. (2005).

Figure 6

a) Closure time measurements (epi-collagen) in NCD (no sign of cardiac disease), NSTEMI (non ST elevation myocardial infarction) and STEMI (ST elevation myocardial infarction) groups. (b) Closure time measurements (ADP-collagen) in the NCD, NSTEMI and STEMI groups. *** p < 0.001, compared to NCD. Lower values indicate increased platelet activation. (Borna et al., 2005)

Figure 7

Figure 8

(a) Effect of chronic aspirin treatment in the NCD (no sign of cardiac disease), NSTEMI (non ST elevation myocardial infarction) and STEMI (ST elevation myocardial infarction) groups measured as closure time with the EPI-collagen cartridge. (b) Effects of aspirin in the NCD, NSTEMI and STEMI groups measured as closure time with the ADP-collagen cartridge. White bars: no aspirin treatment, black bars: aspirin treated patients. Values are expressed as mean values ± SD, *** p < 0.001, * p < 0.05, n.s. = not significant, compared to NCD. Lower values indicate increased platelet activation.

Figure 9

Plasma concentrations of extracellular purines in NCD (no sign of cardiac disease), NSTEMI (non ST elevation myocardial infarction) and STEMI (ST elevation myocardial infarction) groups expressed as mean values ± SD. * p < 0.05, ** p < 0.01, compared to NCD.

Figure 10

a) Correlation between extracellular total purine levels and platelet activity measured as closure time with the ADP-COLL cartridge. (b) Correlation between extracellular ADP levels and platelet activity measured as closure time with the ADP-COLL cartridge.

The work of Sakethou, et al. (1997) entitled: "Emergency Department Use of Aspirin in Patients with Possible Acute Myocardial Infarction" states that there have been efforts made to improve the suboptimal use of aspirin after hospitalization of patients with acute myocardial infarction. The study reported by Sakethou et al. (1997) reports the analysis of admissions to the emergency departments of four hospitals, all of which are affiliated with Brown University. Each of these hospitals were representative of a different patient population and different cardiovascular service level.

The study conducted is stated to demonstrate that "aspirin is underscrutinized in patients who are admitted with suspected myocardial infarction and related syndromes that prove to be acute myocardial infarctions at discharge. Patients who did receive aspirin in the emergency department received the aspirin only following a delay of a substantial nature and findings state that of the patients who were documented at discharge as having acute myocardial infarction, only 45% of these received aspirin in the emergency department. Aspirin use was found to be most likely when there was an initial suspicion of myocardial infarction.

Sakethou et al. (1997) state that an approach that is of a rigid nature and that "restricts aspirin use to patients who are admitted with the strongest evidence of acute myocardial infarction deprives many others of the benefits of early aspirin therapy." (p.128) Sakethou et al. additionally state that 1994 guidelines recommend the immediate administration of 160 mg of aspirin to patients "in whom myocardial infarction is suspected" and state that recently this recommendation was reaffirmed with strong clinical evidence supporting the efficacy of aspirin. The ISIS-2 (Second International Study of Infarct Survival) trail involving approximately 17,000 patients who were suspected of having myocardial infarction found a 23% in patient mortality rate among patients who received aspirin with streptokinase. Aspirin use is reported to have been influenced by clinical trials.

Sakethou et al. report that aspirin use before myocardial infarction increased between 1987 and 1990 from 16% to 24%. Aspirin use after myocardial infarction increased from 39% to 72%. Sakethou et al. (2000) state that other studies of aspirin therapy have concluded that "practitioners who refer patients for hospital admission underutilize aspirin therapy." (1997, p.43)

The replacement of aspirin in the treatment of myocardial infarction is examined in an article published in the European Heart Journal (2000) which states that acute myocardial infarction is the result of a thrombotic coronary occlusion secondary to vulnerable plague rupture. Plaque vulnerability depends on the size and consistency of the atheromatous core, the fibrous cap thickness and the existence of ongoing inflammation with activated macrophagues and T-lympocytes." (Sakethou et al., 1997, p.430)

There are stated to be four major groups of antiplatelet compounds as follows:

(1) drugs that inhibit cyclooxygenase (aspirin is a reference standard of this group, in which triflusal is also included);

(2) agents that interfere with ADP-mediated platelet reactions;

(3) Thrombin inhibitors such as hirudin; and (4) GPIIb/IIIa reception antagonists such as abciximab. (Sakethou et al., 1997, p.431)

The claim of Sakethou et al., (2000) is affirmed in this European Heart Journal article that the ISIS 2 made provision of evidence that is conclusive relating to aspirin benefits in myocardial infarction and that patients receiving low aspirin dosage when compared with the effects of a placebo were found to have a 33% "reduction in risk of cardiovascular mortality at 5 weeks and a 50% reduction non-fatal reinfarction." (p.2000)

Anti-Platelet Effects of Aspirin

Platelets are small cells found in the blood and which have a key role in clotting of blood. When clots are formed inappropriately inside the blood vessels these clots can be transferred to the heart of brain. A blockage in the heart or brain results in localized ischaemia and the result is a heart attack or stroke respectively. Aspirin in low dosage impacts the function of platelets by inhibiting platelet cyclooxygenase and ultimately preventing the aggregating agent thromboxane A2 from forming. This is an irreversible effect which lasts the platelet lifetime however, unless there are repeated dosages of aspirin the new platelets are not affected significantly. The first randomized controlled trial of aspirin for the purpose of preventing vascular events was published in 1974 and stated findings of a "non-significant reduction in all cause mortality by aspirin of 24%." (DISPRIN, 2009)

In 1994 the Anti-platelet Trailists Collaboration meta analysis represented the culmination of many studies having been conducted and was a combination of findings from 145 randomized controlled trials with a total of 102,459 patients and 10,943 outcome events. This study established that aspirin conclusively brought about a reduction in non-fatal myocardial infarction by c. 34% and all cause mortality by ca. 16%." (DISPRIN, 2009) There is stated to have been no evidence of any significant differences in the benefit proportions following various previous clinical events and this included "unstable angina, myocardial infarction, stroke, transient ischaemic attack, angioplasty, valve replacement and peripheral vascular disease. Nor was there any significant heterogeneity in the reduction achieved with different groups of individuals including males/females, diabetic/non-diabetic, hypertensive/non-hypertensive and older/younger patients." (DISPRIN, 2009)

It is important that aspirin be administered within four hours of an ischaemic event because if the aspirin is given later it is less effective The United Kingdom states recommendations that doctors, paramedics and ambulance staff all should carry aspirin and provide it as a treatment for patients in which there is a suspicion of myocardial infarction unless there is an indication that the use of aspirin is contradictory for a specific patient. Studies conducted have demonstrated a total platelet inhibition in participants in the study in approximately four to five minutes following chewing 300mg of soluble aspirin.

Studies have shown complete platelet inhibition in volunteers only 15 minutes after chewing 300mg of soluble aspirin. However any aspirin as long as it is chewed and swallowed will be effective.

Aspirin has different formulation which are of a wide variation throughout the world and include aspirin that is "...plain, soluble, mouth dispersible, microencapsulated, buffered and enteric coated forms are all available in low dose. Recent evidence suggests however that there is little difference between formulations in terms of the likelihood of a serious of gastrointestinal bleed." (DISPRIN, 2009) It is stated that "the risk of a bleed which would necessitate hospitalization as a result of low dose aspirin use is very low at around one incident per 1000 patients per year. This is contrasted with the 30-40 patients with a recent myocardial infarct given low dose aspirin who will avoid a vascular event for every 1000 patients treated per year. An accurate estimate of the percentage of patients who suffer dyspeptic symptoms as a result of low dose aspirin usage is not available but many of these patients may tolerate enteric coated formulations." (DISPRIN, 2009)

or the very few myocardial infarction patients with contradiction absolutely to the use of aspirin there are a number of effective alternatives although they are more expensive. It is stated that a patient when treated with "an acute, evolving myocardial infarction...all formulations of aspirin when chewed and swallowed will affect platelets." (DISPRIN, 2009) There is however, evidence that 300mg soluble aspirin will within fifteen minutes of ingestion inactivate platelets because they are so rapidly absorbed. Therefore, a soluble form of aspirin is recommended as one with an enteric coating will be of little effect in the situation that is acute.

The treatment costs of aspirin prophylaxis are stated to be minimal when compared to the amount needed to prevent one death in the first 12 months following an infarct is estimated at $130.00 per life saved. In comparison, a product introduced recently that is an antagonizer of ADP-induced platelet aggregation indicates that the cost is approximately $100,000 per life saved. The cost-effectiveness of aspirin use in this situation is clear.

It is reported that allergic reactions to aspirin doses do occur and one of these reactions is known as Kounis Syndrome which is the result of inflammatory mediators being released during mast cell degranualation.

The work of Van de Werf (2008) states that adjunctive antithrombotic treatment and devices include Aspirin, NSAID, and COX-2 Inhibitors and that aspirin should be given to all patients with a STEMI immediately following the diagnosis. It is stated that there are "...few contraindications to the use of aspirin, but it should not be given to those with a known hypersensitivity, active gastrointestinal bleeding, known clotting disorders, or severe hepatic disease. Aspirin may occasionally trigger bronchospasm in asthmatic patients. Aspirin should be started at a dose of 150 -- 325 mg in a chewable form (enteric-coated aspirin should not be given because of slow onset of action)." (p. 1)

Van de Werf states that there is an alternative approach when oral ingestion is impossible and that is the I.V. administration of aspirin at a does of 250-500 mg, although no specific data are available on the relative merits of this strategy." (Van de Werf, 2008, p. 1) Van de Werf additionally states that NSAIDS (other than aspirin) and selection cyclo-oxygenase (COX-2) inhibitors 'have been demonstrated to increase the risk of death, reinfarction, cardiac rupture, and other complications in STEMI patients: discontinuation of these drugs is indicated at the time of STEMI." (2008, p. 1) The following figure states recommendations for antithromboic treatment without reperfusion therapy.

Figure 11

Antithrombotic Treatment without Reperfusion Therapy

Figure 12

Contraindications to Fibrinolytic Therapy

Figure 13

Doses of Antiplatelet Co-Therapies

The work of Roberts, McLeod, Cossum and Vial (1984) entitled: "Inhibition of Platelet Function by a Controlled Release Acetysalicylic Acid Formulation -- Single and Chronic Dosing" states that the extent to which a controlled release acetylsalicylic acid (ASA) formulation inhibited platelet function has been evaluated in single and chronic dosing studies." (p.1)

It is reported that in the single dose study, the platelet inhibitory effect of the controlled release formulation was compared with that of an equivalent dose of soluble ASA and an equimolar dose of sodium salicylate (SA). In the chronic dosing study, ASA dose-response curves for platelet function, including cycloosygenase activity were determined for various doses (20-1300 mg) of the controlled release (enteric coated pellets) ASA formulation taken by volunteers daily for one week." (Roberts, McLeod, Cossum and Vial, 1984)

It is stated that platelet function was assessed by the "...degree of inhibition of aggregation for several aggregating agents, and the degree of inhibition of activity of platelet cyclooxygenase quantified by the estimation of malondialdehyde (MDA) production. Plasma ASA and SA concentrations were also determined in each study. The controlled release product inhibited platelet function to the same extent as an equimolar dose of soluble ASA, but did so with much lower and sometimes undetectable peak systemic plasma ASA concentrations. SA, the direct metabolite of aspirin, did not have any effect on platelet function. The ASA dose-platelet function response curves obtained from chronic dosing with the controlled release formulation appeared to be similar to those reported previously for the soluble product. The inhibition of platelet function appeared to be unrelated to plasma ASA concentrations." (Roberts, McLeod, Cossum and Vial, 1984)

The work of McConnell (2002) entitled: "Aspirin Protection Reaffirmed for High Risk Myocardial Infarction or Stroke" states that aspirin or other antiplatelet drugs "protect patients who are at high risk of serious vascular events and should be considered routinely for all such patients." (McConnell, 2002, p.1) McConnell states that this has been demonstrated in a meta-analysis of 287 trials including more than 200,000 patients in which compared was an antiplatelet drug with a control or different antiplatelet drugs. McConnell states that "...Antiplatelet therapy reduced the risk of any serious vascular event by about one quarter; risk of non-fatal heart attack was reduced by one third, non-fatal stroke by one quarter, and vascular death by one sixth. Low dose aspirin (75-150 mg daily) seemed to be as effective as higher doses for long-term use." (2002, p.1)

McConnell states that the reports notes that "...previous meta-analyses of randomized trials have shown that antiplatelet therapy prevents serious vascular events, arterial occlusion, and venous thromboembolism among a wide range of patients at high risk of occlusive vascular events. The proportional reduction in serious vascular events (non-fatal myocardial infarction, non-fatal stroke, or death from a vascular cause) was about one quarter in a wide range of high risk patients, irrespective of why the risk was high and irrespective of age, sex, blood pressure, or history of diabetes." (2002, p.1) McConnell notes that the previous meta-analyses "...left some important clinical questions unanswered," the report continues. For instance, although long-term antiplatelet therapy was shown to be of substantial benefit after ischaemic stroke, it was not known whether antiplatelet drugs were of net benefit as an immediate treatment in the acute phase of such strokes." (2002, p.1)

McConnell states as well that there was reported to be some uncertainty "...about whether antiplatelet therapy was of net benefit in patients with chronic conditions such as atrial fibrillation, stable angina, and atherosclerotic peripheral arterial disease that had been less extensively studied. Daily doses of at least 75 mg of aspirin had been shown to be effective in long-term use, but theoretical advantages had been proposed for lower doses." (2002, p.l) It is noted that it has been pointed out by researchers that "...large amounts of information have become available from trials in patients having coronary artery procedures and in patients with acute stroke, stable angina, atrial fibrillation, peripheral arterial disease, and diabetes mellitus. Consequently, this analysis extends the direct evidence of benefit from antiplatelet therapy to a much wider range of patients at high risk of occlusive vascular disease." (2002, p.1)

Antiplatelet therapy is noted to have reduced the risk for serious vascular events "...by about one quarter, not just among patients with unstable angina, acute myocardial infarction, stroke, or transient ischaemic attacks but also among other patients with coronary or peripheral arterial disease and those at high risk of embolism." (McConnell, 2002, p.1)

Furthermore, it is reported that "Overall mortality was also significantly reduced in these high risk patients, and, compared with these benefits, the absolute risk of fatal and major non-fatal bleeds was small." (McConnell, 2002, p.1) Results in the meta-analysis are reported to have reinforced the "value of ensuring that antiplatelet therapy with 75-150 mg aspirin daily (or some other effective antiplatelet regimen) is considered routinely for all such patients at high or intermediate risk of occlusive vascular events (more than about 2% a year), irrespective of whether they have already had a major vascular event. Adding a second antiplatelet drug to aspirin may produce additional benefits in some clinical circumstances, but more research into this strategy is needed." (McConnell, 2002, p.1)

McConnell concludes by stating that "An unanswered question, however, is whether it is possible to identify particular groups of apparently healthy people who may be at increased risk of myocardial infarction or stroke and for whom the benefits of daily aspirin outweigh the hazards. This is currently being investigated in an analysis of primary prevention trials." (2002, p.1) The findings reported include that for most healthy individuals, and for who the "...risk of a vascular event is likely to be substantially less than 1% a year, daily aspirin may well be appropriate." (McConnell, 2002, p.1)

VerifyNow Assay

The work of Matthew J. Price entitled: "Accumetrics VerifyNow System" reports use of the Accumetrics VerifyNow System. The following figure which has been adapted from the work of Price displays the VerifyNow assay device and shows the different parts of this device.

Figure 14

VerifyNow Assay Device

Source: Price (nd)

The principle of the use of the VerifyNow platelet function assay is shown in the following figure.

Figure 15

Principle of the Use of the VerifyNow Platelet Function Assay

Source: Price (nd)

The following shows the increase in light transmittance with agglutination of beads.

Figure 16

Increase in Light Transmittance with Agglutination of Beads

Source: Price (nd)

The following illustration displays aspirin resistance by VerifyNow Assay and how this is associated with Peri-Procedural Myonecrosis after elective PCI.

Figure 17

Source: Price (nd)

The VerifyNow P2Y12 Assay reporting of results is shown in the following example.

Figure 18

Source: Price (nd)

The following illustration shows the measured effect of different loading doses of Clopidogrel in Healthy Volunteers using the VerifyNow P2Y12 Assay

Figure 19

Source: Price (nd)

The following example shows GRAVITAS or 'Gauging Responsiveness with a VerifyNow Assay -- impact on Thrombosis and Safety.

Figure 20

Source: Price (nd)

The VerifyNow assay is stated to be a "true point-of-care assay that can measure patient response to aspirin, clopidogrel and GP IIb/IIa inhibitors." (Price, nd) Aspirin non-responsiveness as measured by the VerifyNow assay is stated to be "associated with post-PCI myocardial infarction." (Price, nd) The GRAVITAS study is stated to be geared toward determining:

(1) whether increasing the maintenance dose overcomes non-responsiveness; and (2) whether tailoring the dose based on the VerifyNow assay improves clinical outcomes.

The work of Hillman (2009) entitled: "Platelet Aspirin Resistance Detection and Validation" states that it was concluded by Tantry et al. that the occurrence of aspirin resistance "in published reports is overestimated, implying that this was due to the use of assays that did not measure cyclooxygenase-1 (COX-1) activity. Although these investigators reported an incidence of aspirin resistance of _0.4%, several reviews estimate, based on a large number of studies, that the incidence of aspirin resistance is between 5% and 45%." (Hillman, 2009) There are stated to be serious limitations in the Tantry et al. study that "call into question the generalizability of their findings." (Hillman, 2009) Stated first is that the research population of Tantry et al. was comprised mainly of patients with stable angina. It is stated that a number of studies have demonstrated that "...aspirin resistance is more prevalent in patients with acute coronary syndromes than in patients with stable angina, and thus their study may underestimate the prevalence of aspirin resistance in those at greatest risk." (Hillman, 2009)

Stated secondly is that through "...employing a dose of 325 mg of aspirin, their data may not be applicable to the vast majority of patients who currently are treated with 81 mg per day." (Hillman, 2009) Third stated is that because their definition of aspirin resistance "...has not been validated against clinical outcomes, it is arbitrary and thus must be viewed as speculative. Fourth, the thrombelastograph (TEG) assay they use has not undergone extensive critical evaluation." (Hillman, 2009)

Furthermore it is stated that Tantry et al. furthermore do not "...accurately portray the VerifyNow -- ASA System (Ultegra System, Accumetrics, San Diego, California), which employs arachidonic acid (AA) as the agonist and thus does assess COX-1 activity. When compared to light transmission aggregometry (LTA) using AA, the VerifyNow assay demonstrated 91.4% sensitivity and 100% specificity. Although a previous generation of the VerifyNow device did use propyl gallate (PG) as the agonist, Schwartz et al. demonstrated that PG detects aspirin-induced inhibition of platelets with 100% sensitivity and differentiates the degree of aspirin induced inhibition of platelet function produced by single doses of 81 mg and 325 mg, and hence is a specific activator of the aspirin-inhibited COX-1 pathway. Most importantly, results of the VerifyNow assay have been correlated to clinical outcome in several studies, including one published in JACC (4 -- 6)." (Hillman, 2009)

Hillman reports that there have been several studies including that reported by Lee et al. with VerifyNow that have documented that "...demonstrated that aspirin resistance decreases with increasing dose of aspirin, from 81 mg to 325 mg." (2009) It is further stated by Hillman (2009) that it was found by Maree et al. through using "...AA-induced LTA as well as serum thromboxane measurements, the gold standard, that "many patients who are prescribed low-dose aspirin (81 mg) have persistent uninhibited platelet COX activity." (Hillman, 2009) The work of Serebruany et al. have demonstrated that "...major bleeding increases from 1% to 3% as aspirin dose is increased from 81 mg to 325 mg Thus, there is a graded effect in both safety and efficacy of aspirin that should be considered." (Hillman, 2009)

Further contributing to the incidents reported of aspirin resistance in the "real world of clinical practice" are factors of noncompliance and drug interactions with nonsteroidal anti-inflammatory drugs such as Ibuprofen. (Hillman, 2009) It was concluded in the work of Cotter et al. that "..."nonadherence is a significant mediator of poor outcome . . . And it is important to evaluate whether or not patients are taking their medications in clinical settings." Thus, assays that ensure an antiplatelet effect may have great clinical value even in patient populations in whom, as reported by Tantry et al. biochemical aspirin resistance is uncommon." (Hillman, 2009) Schneider stated that "...initial techniques used to assess the effect of glycoprotein IIb/IIIa inhibitors on platelet function were inadequate. The original VerifyNow IIb/IIIa assay was used in the AU-Assessing Ultegra (GOLD) study which served to definitively establish the correlation between platelet inhibition and clinical outcomes." (Hillman, 2009)

The VerifyNow Aspirin Assay is inclusive of all needed reagents to make the provision of results following analysis for a specific assay. The VerifyNow Aspirin Assay includes the following components:

(1) Finger Grip: Each assay device has a handle with which the user can insert or remove the assay device from the assay device port.

(2) Staging Well: The blood sample is held in this area for temperature stabilization prior to analysis.

(3) Assay Device Spot Code: Each assay device is labeled with a unique spot code. Upon insertion of the assay device into the assay device port, the instrument automatically scans the spot code and determines which assay is being performed, records the lot number, and verifies that the assay device has not expired.

(4) Sample Well: The sample well is the area of the assay device where the blood collection tube is inserted. The well contains a Blood Collection Tube Spike for sampling directly from a closed blood collection tube. A protective sheath is removed from the assay device spike before use.

(5) Humidity Indicator: Each assay device has a humidity sensor to determine whether the assay device has been exposed to excess humidity. (Biomedicine, 2009)

(6) Mixing Chamber/Detection Wells: The wells contain all necessary reagents for the specific assays.

According to the article published in the Biomedicine online journal aspirin "...is the medication most commonly prescribed for the prevention of life threatening vascular events such as heart attacks and strokes. However, a number of studies have shown that as many as 30% of people taking daily aspirin therapy to prevent a recurrent vascular event may not be receiving aspirin's full benefits. These people are at greater risk for a heart attack or stroke. The VerifyNow Aspirin Assay is a qualitative test to aid in the detection of platelet dysfunction due to aspirin (ASA) ingestion in citrated whole blood for the point of care or laboratory setting." (Biomedicine, 2009)

The Biomedicine Journal article states that the VerifyNow System "...is a turbidimetric-based optical detection system which measures platelet induced aggregation as an increase in light transmittance. The system consists of an instrument, a disposable assay device and controls. The assay device contains reagents based on microbead agglutination technology. The assay device contains a lyophilized preparation of human fibrinogen-coated beads, platelet agonist, preservative and buffer. The patient sample is 3.2% citrated whole blood, which is automatically dispensed from the blood collection tube into the assay device by the instrument, with no blood handling required by the user." (Biomedicine, 2009)

The work of Roglieri, Hill, and Warburton (1996) entitled: "Aspirin After Myocardial Infarction: Patient Experience in a Managed Care Setting" states that myocardial infarction (MI) "...remains the leading cause of death among American men and women, accounting for almost 500,000 death annually." (p.S42) Reported is a study conducted to determine the treatment post-MI and it is stated in the findings that 88% of all patients were prescribed aspirin post-MI. These findings are shown in the following table.

Figure 21

Source: Roglieri, Hill, and Warburton (1996)

It is stated that of patients who were prescribed aspirin, that 93.5% were taking aspirin at the time of the interview. Compliance is stated to have been significantly varied by age but no differences between genders were noted in terms of compliance. These findings are shown in the following figure.

Figure 22

Source: Roglieri, Hill, and Warburton (1996)

IV ASPIRIN FOR ACUTE MYOCARDIAL INFARCTION PATIENTS

CHAPTER THREE

METHDOLOGY, RESULTS, SUMMARY & CONCLUSION

The purpose of this study was to evaluate effects of a new formulation of ASA for IV use on the aggregability of platelets. Pharmaceutical aggregability was evaluated using platelet induced aggregation measure as an increase in light transmittance. The results area reported as values of aspirin response units (ARU). These measurements were made using the Accumetrics 'VerifyNow' Aspirin Assay System.

METHODOLOGY

Blood was obtained from 2 volunteer donors in a 3.2% sodium citrate using a 21-guage needle. The donors stated that they had not taken any aspirin, NSAID, or other drug that would affect platelet function within the past ten days. The first 2 ml was drawn into a discard rube and not used for the aspiring assay. The remainder was pooled, mixed by gentle inversion, and allocated into 2 m. portions into tubes manufactured for use with the VerifyNow assay.

Formulation solutions of aspirin were prepared (see table 1) by the procedures described below. Solutions containing a concentration of 325 mg/ml of aspirin in 40% ethanol, 58% propylene glycol (or PEG 300 and 2% NN-dimethlacetamide (NNDMA) at pH 6.8 of 162.5 mg/ml and 81.25 mg/ml were also prepared. All of these formulation solutions were clear, colorless without any turbidity and showed acceptable stability (presence of at least 98% of aspirin) when stored at RT and 40 degrees Celsius for a period of up to six months. A2ul were incubated at room temperature for 30 minutes prior to the performance of the assay. Following incubation, the tubes were inserted four to five times and placed in the device. The results of this assay are reported in table 2.

RESULTS OF STUDY

Table 1

Formulation Solutions of Aspirin

Solution

Description

1

100 mg ASA/ml in NaOH2 Freshly prepared

2

100 mg ASA/ml 1 NaOH after storage for 1 month

3

2% NNDMA in 40% ethanol and 58% propylene glycol pH6.8

4

325 mg ASA/ml as shown in #3 above, freshly prepared

5

325 mg ASA/ml as shown in #3 above, after storage for 1 month at 40 degrees Celsius

6

325 mg ASA/ml as shown in #3 above after storage for 6 months at 40 degrees Celsius

Table 2

Results of VerifyNow Assays

Solution #

Sample 1 (ARU)

Sample 2 (ARU)

1

2

3

4

5

6

* Test performed at Accumetrics, San Diego, CA

Additionally, HPLC assay was performed using solutions listed in Table 1. The analytical results are summarized in table 3. Solutions #1 and 2 indicated hydrolysis of aspirin to salicylic acid, while solutions # 4, 5, and 6 did not contain salicylic acid as the hydrolysis product.

Table 3

HPLC Results of Aspirin Formulation Solutions

Solution #

Retention of Time (mins) of Aspirin

% Aspirin (as the unhydrolyzed product)

Figure #

1

1.32

96.23

1

2

1.47

43.29 (salicylic acid, 56.68%)

2

3

1.83

99.22

3

4

1.58

98.84

4

5

1

98.6

5

HPLC was performed using Thermo-Electron (Spectra-Physics, P2000) instrument and Luna 5 u:, C8(2) 100A. 100 X 4.6 mm (Phenomenex, CA) column. The solvent system was NaH2HP04 (0.025m):NaH2HPO4) 0.025m: MeOH (25:25:50v/v) and pH was adjusted to 3.2 with phosphoric acid (or with conc. HCI. The flow rate was 1 ml/min and UV detector was set to 230 nm.

Solutions containing 325 ASA mg/ml (#4,5,6) produced inhibition of platelet activity consistent with the effects of aspirin reported and indicate that these solutions did not contain salicylic as the hydrolyzed product. The HPLC results of Solutions containing 100mg ASA/ml in an aqueous solution reported decreased platelet activity consistent with aspirin response indicating that these solutions (#1,2) contained salicylic acid as the hydrolyzed product. The formulation containing the 2% diluent (solution #3) did not contain ASA and did not appear to affect the performance of assay results, indicating that this solvent did not cause any hindrance or change in the assay. A quality of ul of ASA of 325 mg/ml solution that was used in this assay corresponds to 0.325 mg ASA/ml of blood. This amount closely resembles the amount (0.1-0.3 mg/ml) obtained from the effective dose and level serum concentration obtained in pharmacokinetic studies with aspirin.

ASA in 2% NNDMA results in a stable formulation of ASA for IV injection that does not hydrolyze and retains the anti-plate activity of aspirin. The aspirin formulation described in these studies that contained up to 325 mg/ml in 2% NNDMA 40% ethanol, 58% propyleneglycol (or PEG 300) at pH6.8 is suitable for parenteral administration and the formulation demonstrates stability.

Hydrolysis was determined by HPLC and platelet function evaluated 'ex vivo' using the 'Verify Now' assay developed by Accumetrics. Six solutions were prepared

(1) ASA in aqueous solution of 1N NaOH,

(2) ASA solution stored for a month;

(3) new formulation vehicle;

(4) ASA in new formulation freshly prepared;

(5) ASA in new formulation stored at 40 degrees Celsius for 1 month; and (6) ASA new formulation stored at 40 degrees Celsius for 6 months.

HPLC was preformed using a solvent system NaH2PO4, NaH2PO4, MeOH with pH adjusted to 3.2 and UV detector set at 230mm. The 'Verify Now' assay required 2ul of whole blood incubated at room temperature for 30 minutes and assays repeated twice in duplicate.

SUMMARY OF RESULTS

ASA in an aqueous solution showed 57% hydrolysis, but was 99% non-hydrolyzed in the new formulation at all time points studies (freshly prepared, 1 month and 6 months at 40 degree Celsius. Vehicle solution had no effect. Platelet function showed almost no inhibition from aqueous samples but showed a 37.37 and 39% platelet inhibition for fresh, 1 and 6-month storage samples. Vehicle solution had no effect on platelet function. Solutions containing 325 ASA mg/ml or numbers 4, 5, and 6, produced inhibition of platelet activity consistent with the effects of aspirin reported and this indicates that these solutions did not contain salicylic acid as the hydrolyzed product. The HPLC results of Solutions containing 100 mg ASA/ml in an aqueous solution reported decreased platelet activity consistent with aspirin response indicating that these solutions (#1,2) contained salicylic acid as the hydrolyzed product. The formulation containing the 2% diluent (solution #4) did not contain ASA and did not appear to affect the performance of assay results, indicating that this solvent did not cause any hindrance or change in the assay. A quantity of ul of ASA of 325 mg/ml solution that was used in these assays corresponds to 0.325 mg ASA/ml of blood. This amount closely resembles the amount (O.1-0.3 mg/ml) obtained form the effective dose and level serum concentration obtained in pharmacokinetic studies with aspirin. ASA in 2% NNDMA results in a stable formulation of ASA for IV injection that does not hydrolyze and retains the anti-platelet activity of aspirin. The aspirin formulation described in this study that contained up to 325 mg/ml in 2% NNDMA, 40% ethanol, 58% popyleneglycol (or PEG 300) at pH6.8 is suitable for parenteral administration and the formulation demonstrates stability.

CONCLUSIONS OF THE STUDY

As noted in the literature review in this study the work of Van de Werf, et al. (2008) entitled: "Management of Acute Myocardial Infarction in Patient Presenting with Persistent ST-segment Elevation" states that aspirin "...Aspirin should be given to all patients with a STEMI as soon as possible after the diagnosis is deemed probable. There are few contraindications to the use of aspirin, but it should not be given to those with a known hypersensitivity, active gastrointestinal bleeding, known clotting disorders, or severe hepatic disease. Aspirin may occasionally trigger bronchospasm in asthmatic patients." (2008) Van de Werf, et al. further relates that aspirin should be given at the beginning does of 150-325 mg however, it should be given in a chewable form as enteric coated aspirin will be slow in onset. Van de Werf states that an alternative approach is administering aspirin through an IV at a does of 250-500 mg "although no specific data are available on the relative merits of this strategy." (2008) Aspirin is critically important for patients with acute myocardial infarction however, due to the contradictions of aspirin in some patients there are those patients who have in the past gone without the much needed aspirin. The present study has discovered that there is a way to mitigate these dangers and risks.

This study has found that aqueous solutions of ASA hydrolyzes rapidly and this correlates with a diminished 'ex vivo' ASA effect on platelet function. A new formulation of ASA for IV administration shows no hydrolysis at one and six months of storage at forty degrees Celsius and this lack of hydrolysis correlates with 'ex vivo' measured platelet inhibition by ASA. Availability of an IV formulation of ASA may be useful in the treatment of acute coronary syndromes and will remain stable if stored over time. This study has successfully formulated a method by which patients with acute myocardial infarction may be administered aspirin and the dangers and risks of the contradictions of the use of aspirin mitigated.