Effect of Mean Platelet Volume on Postintervention Coronary
Blood Flow in Patients with Chronic Stable Angina Pectoris

Platelets play a crucial role in the pathophysiology, morbidity and mortality of coronary artery disease (CAD). It has been shown that platelet size, measured as mean platelet volume (MPV), correlates with their reactivity.¹ MPV showing platelet size reflects platelet function and activity. Platelet production and stimulation indirectly elevates values, which can result in cardiovascular disease, as larger platelets are more reactive than normal-sized ones.^2,3 Change in platelet behavior, such as increased platelet aggregability, has proved to be an independent risk factor for cardiovascular events.⁴
The thrombolysis in myocardial infarction (TIMI) frame count (TFC) is a simple clinical tool for assessing quantitative indexes of coronary blood flow (CBF). TFC is objective, quantitative, reproducible and sensitive to changes in coronary flow.⁵ To our knowledge, there are no reports regarding the effect of MPV on CBF in patients with stable CAD undergoing percutaneous coronary intervention (PCI). In our study we sought to determine whether MPV, measured on admission, could be used in determining the risk of decreased CBF in stable CAD patients treated with PCI.

Methods
Study population. A total of 66 consecutive eligible patients (mean age 58 ± 5 years, 74% male) with a diagnosis of stable CAD and who were hospitalized for elective PCI were prospectively enrolled in our study. PCI was performed based upon ongoing symptoms (refractory angina) despite optimal medical therapy. Chronic stable angina was defined as exertional angina with objective ischemic evidence on thallium scintigraphy or exercise testing over the previous 6 months. Hypertension was defined as blood pressure 3 140/90 mmHg, or treatment with antihypertensive medication, presence of diabetes mellitus (fasting blood glucose > 125 mg/dL), or being on antidiabetic drugs, current cigarette smoking, hypercholesterolemia (low-density lipoprotein level > 130 mg/dL), or treatment with a statin, and a family history of CAD (if patients had a first-degree male relative > 55 years of age or a female relative > 65 years of age with CAD). Body mass index was also measured. Exclusion criteria included acute coronary syndromes, severe hepatic or renal dysfunction, diabetes mellitus, thrombocytopenia, oral anticoagulation and anti-inflammatory drug use, and complex coronary lesions (total occlusions, highly calcified lesion, left main coronary artery lesion, restenosis and vein graft lesion). All patients gave informed consent, and the study protocol was approved by the institution’s ethics committee.
Adjunctive pharmacotherapy. The study called for all patients to receive 100 mg of aspirin daily before the intervention and unfractionated heparin during PCI. Clopidogrel was administered (300 mg loading dose at least 6 hours preprocedure, and 75 mg daily thereafter) for a period of 4 weeks. A glycoprotein (GP) IIb/IIIa inhibitor was administered at the discretion of the operator. An unfractionated heparin bolus of 100 IU/kg (50–70 IU/kg if a GP IIb/IIIa receptor inhibitor was administered) was given to all patients, and a periprocedural activated clotting time (ACT) was measured. Unfractionated heparin dosage was adjusted under ACT guidance (ACT in the range of 250–300 seconds, or 200–250 seconds if a GP IIb/IIIa receptor inhibitor was given).
Angiographic analysis and PCI procedure. Selective coronary angiographic examinations were performed by the standard Judkins technique (Philips DCI, Eindhoven, The Netherlands). The coronary diameters and stenosis percentages were measured by computerized quantitative angiography in a biplane mode. A luminal narrowing > 50% in a major vessel (left anterior descending artery, left circumflex artery, right coronary artery) or in a major side branch (first or second diagonal or marginal) was defined as significant stenosis. Angiographic assessment was always performed by two independent angiographers blinded to the MPV results.
Coronary angioplasty was performed in the usual manner with a femoral approach using standard 7 Fr guiding catheters. Only bare-metal stents with or without balloon predilatation were used in this study. Stent diameter and length were also recorded. PCI was considered successful if the final percent diameter stenosis was < 50%, with TIMI grade 3 flow in the absence of recurrent ischemia, myocardial infarction, need for bailout stenting or urgent coronary bypass surgery during hospitalization, or death. Digital angiograms were than analyzed by two independent, experienced interventional cardiologists, blinded to the MPV results. All angiograms were assessed with respect to TIMI flow grade and TFC for the treated vessels at baseline and after PCI.
TIMI flow grades. TIMI flow grades were described previously⁶: Grade 0: no perfusion (no antegrade flow beyond the point of occlusion); Grade 1: penetration without perfusion (contrast material passes beyond the area of obstruction, but fails to opacify the entire coronary bed distal to the obstruction for the duration of the cineangiographic filming sequence); Grade 2: partial perfusion (contrast material passes across the obstruction and opacifies the coronary artery distal to the obstruction, however, the rate of entry of contrast material into the vessel distal to the obstruction or its rate of clearance from the distal bed (or both) is perceptibly slower than its flow into or clearance from comparable areas not perfused by the previously occluded vessel (for example, the opposite coronary artery or the coronary bed proximal to the obstruction); and Grade 3: complete perfusion (antegrade flow into the bed distal to the obstruction occurs as promptly as antegrade flow into the bed proximal to the obstruction, and clearance of contrast material from the involved bed is as rapid as clearance from an uninvolved bed in the same vessel or the opposite artery).
Determination of TIMI frame count. Coronary flow rates of all subjects were documented by TFC for each major coronary artery included in the study according to the method first described by Gibson et al.⁵ The number of cineangiographic frames (recorded at 30 frames per second) required for the leading edge of the column of radiographic contrast to reach a predetermined landmark is determined. The first frame is defined as the frame in which concentrated dye occupies the full width of the proximal coronary artery lumen, touching both borders of the lumen, with forward motion down the artery. The final frame is designated when the leading edge of the contrast column initially arrives at the distal landmark. In the left anterior descending coronary artery (LAD), the landmark used is the most distal branch nearest the apex of the left ventricle, commonly referred to as the “pitchfork” or “whale’s tail.” The LAD is usually longer than the other major coronary arteries and the TFC for this vessel is often higher. To obtain a corrected TFC (CTFC) for the LAD in our study, the TFC was divided by 1.7. The right coronary artery (RCA) distal landmark is the first branch of the posterolateral artery after the origin of the posterior descending artery, regardless of the size of this branch. The branch of the left circumflex coronary artery (LCX) that encompassed the greatest total distance traveled by contrast was used to define the distal landmark of the LCX. The TFC in the LAD and LCX was assessed in a right anterior oblique projection with caudal angulation, and in the RCA in a left anterior oblique projection with cranial angulation. Intraobserver and interobserver variability for CTFC was 1.1 ± 0.6 and 2.3 ± 0.8 frames, respectively.
Blood collection and measurement of MPV. Blood samples for MPV estimation and platelet count obtained on admission were measured on the day of the scheduled PCI. A fasting blood sample was obtained from all patients in a sitting position. Blood samples were taken into standardized tubes containing dipotassium ethylenedinitro tetra-acetic acid (EDTA). All measurements were performed 30 minutes after blood collection using a Sysmex system (Roche Diagnostics, Germany). The assessment of MPV was made without clopidogrel, heparin or GP IIb/IIIa inhibitor. The range of expected values for MPV in our laboratory is 7.8–11 fL for a platelet count of 150–450 (x 10/L). Total cholesterol, triglyceride, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels were also measured by an auto-analyzer (Olympus AU 5200, Japan).
Statistical analysis. Statistical analysis was performed using the SPSS 10.0 statistical package (SPSS, Inc., Chicago, Illinois). Continuous variables were expressed as mean ± standard deviation and categorical variables as percentages. While the chi-square test or Fisher’s exact test was used for categorical values between the two groups, the Student’s t-test or the Mann-Whitney U-test was utilized to compare the continuous variables between groups. Correlation analysis was performed by using Pearson’s correlation coefficients. The multivariable logistic regression test was used to define independent factors influencing CTFC after PCI. A p-value < 0.05 was considered statistically significant.

Results

The characteristics of the study population are listed in Table 1. Final TIMI 3 flow was achieved in all patients without complications. Target ACT levels during PCI were achieved in all patients. An intracoronary vasodilator agent was not used. General characteristics of patient groups according to admission MPV levels are shown in Table 2. PCI properties, except for LAD intervention, were similar in the two groups (Table 2). The number of LAD interventions was higher in patients with a normal MPV than in patients with a high MPV (p = 0.02). Patients with a high MPV had significantly higher CTFC than those with a normal MPV (24 ± 3 vs. 17 ± 5; p = 0.001) (Figure 1). The MPV was correlated strongly with post-PCI CTFC (R = 0.625; p = 0.0001) (Figure 2). The higher the MPV value, the greater the number of cine frames needed to reach the distal part of the target vessel after PCI. Platelet count was found lower in patients with a high MPV than in those with a normal MPV (Table 2). No significant differences were found between the two groups for any other analyzed variables. Multiple logistic regression analysis showed that only the MPV was an independent predictor of post-PCI CTFC after adjustment for baseline characteristics (OR 1.9, 95% CI 1.2–2.3; p = 0.001). There were no significant correlations between post-PCI CTFC and hemodynamic variables (systolic and diastolic blood pressure) and clinical variables (age, sex, body surface area, hypertension, hypercholesterolemia and smoking).

Discussion
The results of the present study suggest that an increased MPV may contribute to the reduction in CBF after PCI in patients with stable CAD. Several experimental studies have indicated that platelet size and function correlate since large platelets are hemostatically more reactive than platelets of normal size. Large platelets contain more dense α-granules,⁷ produce more prothrombotic factors like thromboxane B2,³ and release more serotonin and b-thromboglobulin than smaller platelets.^8,9 Moreover, large platelets express more GP Ib and IIb/IIIa receptors¹⁰ and exhibit an enhanced in vitro aggregability after ADP challenge.¹¹ MPV increases in patients with acute myocardial infarction,¹² stroke,¹³ stable CAD,² and diabetes mellitus,¹⁴ and has been shown to be a predictive marker for future adverse coronary events after survived myocardial infarction.¹⁵ Also, in a recently published study, it was found that the MPV was higher in patients with acute coronary syndrome than in patients with stable CAD, and was significantly higher in patients with non-ST-segment elevation myocardial infarction than in patients with unstable angina pectoris.¹⁶
No-reflow phenomenon, defined as inadequate myocardial perfusion of the adequately dilated target vessel without evidence of angiographic mechanical obstruction, has been observed in 0.6–2% of all PCIs.^17,18 The noreflow phenomenon is a well-recognized negative prognostic indicator after angioplasty in patients with acute coronary syndromes.¹⁹ Despite the achievement of optimal epicardial coronary artery patency, 30–40% of patients exhibit the phenomenon of distal no-reflow after primary PCI, which negates the advantages of coronary recanalization.²⁰ No-reflow also occurs in stable lesions. The mechanisms underlying no-reflow are complex and only partially understood.²¹ Microvascular obstruction due to neutrophil and platelet plugging, intense vasoconstriction, local platelet activation and external microvascular compression caused by myocardial edema are potential causes.19 Furthermore, in patients treated with primary PCI, distal embolization is likely to play a contributory role.²² Although we did not observe no-reflow in our study population with stable CAD, we did observe diminished flow, which usually indicates “low-reflow”.²³
In experimental studies with electron microscopy, detection of intraluminal platelets and fibrin-rich thrombus in a microvascular bed of no-reflow areas supported the idea that intravascular occlusion with fibrin and platelets might contribute to the no-reflow phenomenon.^24,25 Animal studies provide supportive evidence for the role of platelet-dependent microembolization via the release of products such as thromboxane A2, serotonin, and adenosine from granules contained in platelet aggregates.²⁶
Conventional TIMI flow grading is a predictor of cardiac outcome after acute myocardial infarction and PCI, but it has several limitations.^27,28 CTFC, another approach to grade flow impairment, is an objective, quantitative, reproducible and sensitive index of CBF.⁵ As indicated in our study, TIMI flow may appear normal visually, but may correlate to abnormal CTFC. CTFC has been proposed to have incremental prognostic accuracy in predicting survival outcome with reperfusion therapy.²⁹ This measurement has been significantly correlated with flow velocity measured with the FloWire^® (Volcano Corp., Rancho Cordova, California) by several investigators during baseline conditions or hyperemia.^30,31 Thus, CTFC may be an index of microvascular behavior, which reflects coronary vascular resistance.³²
Higher CTFC values (slower flow) after PCI have also been found to be associated with poor clinical outcome.³³ In the present study, we demonstrated that low-reflow occurs significantly more frequently in patients with high baseline values of MPV. Moreover, MPV correlated with CBF expressed as a continuous variable (CTFC), which further strengthens the findings of this study. We assume that the presence of larger, more reactive platelets or platelet aggregates may be associated with intravascular plugging on both epicardial and tissue levels of the culprit vessel, thus resulting in low-reflow (higher CTFC) after PCI. A higher MPV may correspond with the increased number of both platelet-leukocyte and plateletplatelet aggregates.³⁴
Study limitations. There are several limitations of this study. First of all, apart from the limited number of patients enrolled in our study, previous studies have reported that MPV increases in a time-dependent manner when EDTA is used as an anticoagulant.³⁵ However, a recently published study proved that when the measurement is performed within 2 hours after venipuncture, the anticoagulation with EDTA accounts for less than a 0.5 fL increase in MPV.³⁶ To minimize the effect of EDTA on platelet size in the present study, all samples were processed early (at 30 minutes) after blood collection. Another constraint was the fact that CTFC, used to assess coronary flow, was not compatible with other methods such as Doppler flow wire studies, nuclear scintigraphic imaging, myocardial contrast echocardiography and magnetic resonance imaging. Another major limitation was the impossibility of patient follow up, which could have informed us concerning the impact of MPV on restenosis and long-term clinical outcomes. While the MPV was studied as a surrogate parameter for increased platelet activity, no functional tests were performed. A better knowledge of the control mechanisms governing the size of platelets might help to understand the exact role of the MPV in diminished coronary flow following PCI.

Conclusions
Results from the present study suggest that MPV may be considered as a useful, independent, hematological marker allowing for early and easy identification of patients with stable CAD who are at a higher risk of post-PCI low-reflow. Measuring the MPV may also carry further practical, therapeutic implications. Large platelets express more GP Ib and IIb/IIIa receptors and exhibit an enhanced in vitro aggregability after ADP challenge.^10,11 Patients with a high MPV might benefit from GP IIb/IIIa receptor blocker administration. Also, diminished coronary flow may contribute to restenosis, stent thrombosis and other clinical adverse events. Further studies are warranted to explore these issues.

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