Temporary Rise in Blood Thrombogenicity in Patients with Acute Myocardial Infarction

Objective  Although blood thrombogenicity seems to be one of the determinant factors for the development of acute myocardial infarction (MI), it has not been dealt with in-depth. This study aimed to investigate blood thrombogenicity and its change in acute MI patients. Methods and Results  We designed a prospective, observational study that included 51 acute MI patients and 83 stable coronary artery disease (CAD) patients who underwent cardiac catheterization, comparing thrombogenicity of the whole blood between: (1) acute MI patients and stable CAD patients; and (2) acute and chronic phase in MI patients. Blood thrombogenicity was evaluated by the Total Thrombus-Formation Analysis System (T-TAS) using the area under the flow pressure curve (AUC 30 ) for the AR-chip. Acute MI patients had significantly higher AUC 30 than stable CAD patients (median [interquartile range], 1,771 [1,585–1,884] vs. 1,677 [1,527–1,756], p  = 0.010). Multivariate regression analysis identified acute MI with initial TIMI flow grade 0/1 as an independent determinant of high AUC 30 ( β  = 0.211, p  = 0.013). In acute MI patients, AUC 30 decreased significantly from acute to chronic phase (1,859 [1,550–2,008] to 1,521 [1,328–1,745], p  = 0.001). Conclusion  Blood thrombogenicity was significantly higher in acute MI patients than in stable CAD patients. Acute MI with initial TIMI flow grade 0/1 was significantly associated with high blood thrombogenicity by multivariate analysis. In acute MI patients, blood thrombogenicity was temporarily higher in acute phase than in chronic phase.


Introduction
Acute myocardial infarction (MI) is caused by plaque disruption and subsequent thrombotic coronary occlusion. However, plaque disruptions and mural thrombus formation are often detected in the coronary arteries without causing any coronary event, that is, silent plaque disruptions. 1,2 The determinant factors for mural thrombus at disrupted plaque to become occlusive have been unknown.
Blood thrombogenicity is one of the well-known determinant factors of thrombus formation. However, it is not always stable, due to which change is likely to influence the Keywords ► blood thrombogenicity ► acute myocardial infarction ► coronary artery disease ► T-TAS

Abstract
Objective Although blood thrombogenicity seems to be one of the determinant factors for the development of acute myocardial infarction (MI), it has not been dealt with in-depth. This study aimed to investigate blood thrombogenicity and its change in acute MI patients.

Methods and Results
We designed a prospective, observational study that included 51 acute MI patients and 83 stable coronary artery disease (CAD) patients who underwent cardiac catheterization, comparing thrombogenicity of the whole blood between: (1) acute MI patients and stable CAD patients; and (2) acute and chronic phase in MI patients. Blood thrombogenicity was evaluated by the Total Thrombus-Formation Analysis System (T-TAS) using the area under the flow pressure curve (AUC 30 ) for the AR-chip. Acute MI patients had significantly higher AUC 30  Conclusion Blood thrombogenicity was significantly higher in acute MI patients than in stable CAD patients. Acute MI with initial TIMI flow grade 0/1 was significantly associated with high blood thrombogenicity by multivariate analysis. In acute MI patients, blood thrombogenicity was temporarily higher in acute phase than in chronic phase. development of acute MI. In fact, blood thrombogenicity has been reported to vary under some factors 3-5 that may trigger the onset of MI. 6,7 Although a hypothesis that a temporary rise in blood thrombogenicity triggers thrombotic coronary occlusion after plaque disruption has been presented, 8 it appears not to be adequately established. Previous studies investigated thrombogenic components of blood in MI patients, such as platelet function or coagulation factors activities, and suggested that blood thrombogenicity was temporarily increased in the acute phase of MI patients 9-14 ; however, these thrombogenic components only partially reflect thrombogenicity of whole blood, which in acute MI patients has not been adequately elucidated.
The present study therefore aimed to test the following pre-specified hypotheses: (1) that thrombogenicity of whole blood is higher in the patients with acute MI than in those with stable coronary artery disease (CAD); and (2) that thrombogenicity of whole blood in MI patients is temporarily high in the acute phase. We used the Total Thrombus-Formation Analysis System (T-TAS; Fujimori Kogyo, Tokyo, Japan) to evaluate thrombogenicity of whole blood under flow conditions. 15

Patients and Study Design
We designed a prospective, single-center, observational study, in which we enrolled patients with acute MI or stable CAD who received left heart catheterization from August 2018 to September 2020, comparing thrombogenicity of the whole blood between: (1) acute MI patients and stable CAD patients; and (2) acute and chronic phase in MI patients. Acute MI was defined as type 1 MI according to the fourth universal definition of MI. 16 Stable CAD was defined as asymptomatic or symptomatic CAD with stable symptoms after initial diagnosis or revascularization. Patients using anticoagulants or extracorporeal membrane oxygenation with heparin coating before collection of blood samples were excluded because they were known to affect the measurement by T-TAS. 15

Antithrombotic Therapy and Catheterization
Antiplatelet therapy was performed with aspirin (100 mg/d) and/or prasugrel (3.75 mg/d) or clopidogrel (75 mg/d) as a maintenance dose for patients who had received percutaneous coronary intervention (PCI). In cases of primary PCI, a loading dose of aspirin (200 mg) and prasugrel (20 mg), or clopidogrel (300 mg) was administered to each naïve patient. Intravenous unfractionated heparin (100 U/kg) was administered at the beginning of catheterization, and an additional dose was repeated to maintain an activated clotting time of !250 s during the procedure. GP-IIb/IIIa inhibitors were not used because they were not approved in Japan. Catheterization was performed via radial, brachial, or femoral artery approach using a 6-Fr or 7-Fr sheath and catheters. Coronary angiography was recorded by Artis zee biplane (Siemens Healthineers AG, Erlangen, Germany).

Collection of Blood Samples
In stable CAD patients and in acute phase MI patients, blood samples were collected before the administration of unfractionated heparin. The administration timing of antiplatelet agents was left to the discretion of attending physician. In MI patients, blood samples were collected again at discharge as the chronic phase. The blood sample was collected into plastic tubes containing 3.2% sodium citrate (Terumo, Tokyo, Japan). It was allowed to stand for 1 to 3 hours, of which 480 µL was mixed with 20 µL of 0.3 mol/L CaCl 2 containing 1.25 mg/mL of corn trypsin inhibitor immediately before measurement.

Measurement of Thrombogenicity of Whole Blood
Thrombogenicity of whole blood was determined with T-TAS using the AR chip. The AR chip can assess the thrombogenicity of the whole blood associated with both platelets and coagulation systems. The AR chip contains a capillary channel that is 15 mm long, 300 µm in width, and 80 µm in depth, coated with type I collagen and tissue thromboplastin. The blood sample is perfused through the capillary by the precision pump with a shear rate of 600 s À1 (a flow rate of 10 µL/min). After the perfusion of blood initiated, collagen and tissue thromboplastin activate platelets and the extrinsic coagulation pathway. Depending on the formation of thrombus, the capillary is gradually occluded, and the flow pressure monitored by the sensor is increased. The area under the flow pressure curve for the first 30 minutes at a flow rate of 10 µL/min (AUC 30 ) was used to evaluate blood thrombogenicity. 15 Prior literatures reported the coefficients of variation for AUC 30 in AR-chip analysis as 1.2 to 5.0%. 17,18 Statistical Analysis Continuous variables were expressed as median (interquartile range [IQR]) and were compared by Mann-Whitney Utest or Kruskal-Wallis test. Categorical variables were expressed as absolute numbers (percentage) and were compared by the Chi-square test or Fisher's exact test. Wilcoxon signed-rank test was used to compare AUC 30 between acute and chronic phases in MI patients. Spearman rank correlation was used to determine the relationship between AUC 30 and each variable. Multivariate linear regression analysis was performed to elucidate the factors associated with AUC 30 , including platelet count, P2Y12 antagonists use, angiotensinconverting enzyme inhibitors/angiotensin II receptor blockers use, beta-blockers use, and acute MI (model 1) or acute MI with TIMI flow grade 0/1 (model 2). Variables with p <0.10 by univariate analysis were used for these models. All statistical analysis was regarded as significant when p-value was <0.05.

Study Patients
Included in the analysis were 51 patients with acute MI and 83 patients with stable CAD. Patients' characteristics are presented in ►Table 1. Platelet count was higher in the acute MI patients than in stable CAD patients, whereas antiplatelet agents were used more frequently in stable CAD patients than in acute MI patients. There was no significant difference in AUC 30

Comparison between the Acute and Chronic Phases in Acute MI Patients
Both of acute phase and chronic phase T-TAS measurements were available in 13 patients. Baseline characteristics are presented in ►Table 3. The measurement interval between acute Fig. 1

Discussion
The present study demonstrated that thrombogenicity of whole blood was significantly higher in acute MI patients, especially in those with TIMI flow grade 0/1, than in stable CAD patients. Furthermore, blood thrombogenicity decreased significantly from acute to chronic phase among acute MI patients, suggesting that thrombogenicity was temporarily high in the acute phase. Several studies have demonstrated the association between the onset of MI and coagulation factors. The increased levels of fibrinogen and von Willebrand factor antigen were independent predictors of subsequent acute coronary syndromes in a prospective cohort study with angina pectoris patients. 9 The increased factor VII, IX, and XI activity, and the decreased levels of antithrombin III and protein C were detected in the acute phase of MI patients. [10][11][12] Moreover, the activation of factor XI and increased serum levels of fibrinopeptide A were temporarily observed in the acute phase of MI patients. 13,14 On the other hand, factor X did not differ between acute MI and stable CAD patients, 11 and factor V and XII were rather reduced in the acute phase of MI patients. 10,12 Therefore, in those prior studies, thrombogenicity of whole blood cannot be judged from the findings on the individual coagulation factors. The importance and novelty of our study are that whole blood samples under flow conditions mimicking in vivo atherosclerotic lesion were used to investigate the status of blood thrombogenicity in acute MI patients. The present study demonstrated that blood thrombogenicity is temporarily high in the acute phase of MI patients, supporting the hypothesis that a temporary increase in blood thrombogenicity is involved in the development of acute MI. We found that MI patients with initial TIMI flow grade 0/1 was independently associated with high AUC 30 . This finding may be pathophysiologically plausible. High blood thrombogenicity may cause occlusive large and firm thrombus formation leading to TIMI flow grade 0/1; however, relatively low blood thrombogenicity may cause the formation of relatively fragile thrombus leading to spontaneous recanalization with the higher TIMI flow grade, which results in the stronger relationship between AUC 30 and TIMI 0/1 acute MI rather than with acute MI in general.
The present study showed that platelet count was also independently associated with AUC 30 , which was consistent with previous studies. 19,20 It is controversial whether platelet count is a risk of MI, as some population-based cohort studies have reported high platelet count as a risk of cardiovascular death and cardiovascular disease 21,22 but others have reported that it is not. 23,24 However, since acute MI patients had higher platelet count than stable CAD patients and platelet count was independently associated with high blood thrombogenicity in the present study, platelet count would be a risk of MI.
The measurement of blood thrombogenicity by T-TAS using AR chip is known to be affected by anticoagulants, GPIIb/IIIa antagonists, and GPIbα antagonists 15,20 but not by aspirin and P2Y12 antagonists, i.e., clopidogrel, prasugrel, and ticagrelor. 17,19,25,26 The reason for this has been mentioned in prior literature that P2Y12 inhibitors may have no effect in AR chip, in which thrombus formation is more dependent upon fibrin formation, as platelets play little in thrombus formation at low shear rates. 17 AR chip measures the formation of occlusive thrombus in a capillary channel coated with type I collagen and tissue thromboplastin, mimicking the formation of occlusive thrombus in the atherosclerotic artery. Aspirin and P2Y12 inhibitors have an antithrombotic effect through GPIIb/IIIa, which mainly contributes to platelet instability. 27 Although platelets are generally important for the arterial thrombus formation, thrombus formed at the culprit lesion of MI is mixed thrombus with high fibrin content, 28,29 suggesting that coagulation system plays an important role in the formation of occlusive thrombus. We therefore believe that the measurement using T-TAS with AR-chip would be appropriate for assessing blood thrombogenicity related to the development of acute MI.
Epidemiologic studies have shown that various factors such as temperature, exercise, and mental stress trigger the onset of MI 6,7 and that those factors increase platelet and coagulation activities. [3][4][5] Our present study added important evidence for the association between the increase in blood thrombogenicity and acute MI onset. However, further studies are still needed to clarify the mechanisms that contribute to the formation of occlusive thrombus at the thrombogenic vessel wall by which acute MI is caused. The hypothesis 30 that a temporary increase in blood thrombogenicity is involved in the development of acute MI should also be tested in those studies.
Although AR-chip mimics the formation of occlusive thrombus in the atherosclerotic artery, nobody knows if it is the ideal model for acute MI culprit lesion. The dynamic circadian, daily, or monthly changes of blood thrombogenicity and its determinant factors has not yet been clarified.

Conclusion
Blood thrombogenicity was significantly higher in acute MI patients than in stable CAD patients. Acute MI with initial TIMI flow grade 0/1 was significantly and independently associated with high blood thrombogenicity by multivariate analysis. Furthermore, in acute MI patients, blood thrombogenicity was temporarily higher in acute phase than in chronic phase.
What Is Known about This Topic?
• Blood thrombogenicity seems to be one of the determinant factors for mural thrombus at disrupted coronary plaques to become occlusive. • Acute myocardial infarction is associated with a hypercoagulable state with increased fibrinogen, von Willebrand factor antigen, and factor VII, IX, and XI activity, and decreased antithrombin III and protein C.
What Does This Paper Add?
• Thrombogenicity of whole blood measured by T-TAS was significantly higher in the patients with acute myocardial infarction than in those with stable coronary artery disease. • In acute myocardial infarction patients, thrombogenicity of the whole blood was temporarily higher in acute phase than in chronic phase.