Detection of tissue factor antigen and coagulation activity in coronary artery thrombi isolated from patients with ST-segment elevation acute myocardial infarction

Abstract

Introduction: Although ruptured atherosclerotic plaques have been extensively analyzed, the composition of thrombi causing arterial occlusion in patients with ST-segment elevation acute myocardial infarction has been less thoroughly investigated. We sought to investigate whether coagulant active tissue factor can be retrieved in thrombi of patients with STEMI undergoing primary percutaneous coronary intervention.

Methods: Nineteen patients with ST-segment elevation acute myocardial infarction referred for primary percutaneous coronary intervention were enrolled in this study. Coronary thrombi aspirated from coronary arteries were routinely processed for paraffin embedding and histological evaluation (4 patients) or immediately snap frozen for evaluation of tissue factor activity using a modified aPTT test (15 patients). Immunoprecipitation followed by immunoblotting was also performed in 12 patients.

Results: Thrombi aspirated from coronary arteries showed large and irregular areas of tissue factor staining within platelet aggregates, and in close contact with inflammatory cells. Some platelet aggregates stained positive for tissue factor, whereas others did not. Monocytes consistently stained strongly for tissue factor, neutrophils had a more variable and irregular tissue factor staining, and red blood cells did not demonstrate staining for tissue factor. Median clotting time of plasma samples containing homogenized thrombi incubated with a monoclonal antibody that specifically inhibits tissue factor-mediated coagulation activity (mAb 5G9) were significantly longer than their respective controls (88.9 seconds versus 76.5 seconds, respectively; p<0.001). Tissue factor was also identified by immunoprecipitation in 10 patients, with significant variability among band intensities.

Conclusions: Active tissue factor is present in coronary artery thrombi of patients with ST-segment elevation acute myocardial infarction, suggesting that it contributes to activate the coagulation cascade ensuing in coronary thrombosis.

Figure 1. Histological assessment of thrombi aspirated from coronary arteries of patients with ST-segment elevation acute myocardial infarction.

(A) Hematoxylin and eosin staining shows platelet aggregates (*) intermingled in a fibrin network (°) with red and white blood cells. Original magnification 100x. (B) and (C) Immunohistochemistry of coronary thrombi prepared with anti-TF CD142 antibody showing large and irregular areas of tissue factor staining (brown colour) within platelet aggregates and white blood cells. Some portion of the platelet aggregates stained intensely (*), whereas other lightly (++) with anti-TF CD142 antibody. B: original magnification 200x; C: original magnification 400x. (D) Detail of a coronary thrombus showing tissue factor staining in close contact with platelet aggregates (*). Some areas of these platelet aggregates do not display tissue factor staining (++). Monocyte are strongly positive for tissue factor, granulocytes display a weak and irregular staining, and red cells are negative. Original magnification 400x. (E) Negative control for TF performed with immunoperoxidase staining on sections not incubated with the primary antibody against TF. Magnification 100x. (F) Negative control using an isotype-matched irrelevant primary antibody (mouse IgG anti-epithelial membrane antigen).

Figure 2. Immunohistochemistry of coronary thrombi.

(A,B and C) Immunohistochemistry prepared with 10H10 showing large and irregular areas of tissue factor staining (in brown) within platelet aggregates and inflammatory cells. Some platelet aggregates stained positive for tissue factor (*), whereas other did not (++). Original magnification is ×200. (D) Detail of a coronary thrombus prepared with 10H10. Monocytes are strongly positive for tissue factor, granulocytes display a weak and irregular staining, and red cells are negative. Original magnification is ×400.

Figure 3. Clotting times measured with the modified aPTT test.

(A) Clotting times of plasma samples incubated with buffer, 5G9, 10H10 or IgG. (B) Clotting times of plasma samples containing homogenized thrombi incubated with or without 5G9. (C) Pairwise comparison of clotting times of plasma samples containing homogenized thrombi incubated with or without 5G9. (D) Differences in clotting times between plasma samples containing homogeneized thrombi incubated with or without 5G9.

Figure 4. Immunoblotting analysis.

(A) Immunoblotting of thrombi harvested from 12 patients showing the presence of various amount of tissue factor antigen (47 kD). (B) Band intensity of the immunoblot expressed in arbitrary unit (AU).