Heightened Thrombin Formation but Normal Plasma Levels of Activated Factor VII in Patients With Acute Coronary Syndromes
Abstract Plaque rupture with the exposure of a tissue factor–rich procoagulant surface is considered the common pathogenetic mechanism of unstable angina and myocardial infarction. Activated factor VII, the key enzyme for initiating blood coagulation under resting conditions, is increased in pathological situations associated with tissue factor exposure. We measured the plasma levels of activated factor VII and studied their relation with signs of coagulation enzyme activity in patients with acute coronary syndromes. The plasma levels of activated factor VII, prothrombin fragment 1+2, and fibrinopeptide A were measured on admission in consecutive patients presenting with acute myocardial infarction (n=28), unstable angina (n=32), and stable angina (n=17) and in age- and sex-matched healthy individuals (n=33). Plasma determinations of the same markers were also repeated at 15 days and 3 and 6 months. On admission, the patients with unstable angina or myocardial infarction had significantly higher plasma levels of prothrombin fragment 1+2 (P<.0001) and fibrinopeptide A (P<.0001) than those with stable angina or healthy individuals, whereas no differences were detected in the plasma levels of activated factor VII. During follow-up there was a significant decrease in the plasma levels of fibrinopeptide A both in patients with unstable angina (P<.001) and in those with myocardial infarction (P<.001), whereas no changes in plasma prothrombin fragment 1+2 or activated factor VII levels were observed. Hence, in the acute and chronic phases of myocardial infarction and unstable angina, heightened coagulation enzyme activity is not accompanied by an increase in activated factor VII.
- Received March 27, 1995.
- Accepted June 23, 1995.
Plaque rupture or fissuring is considered the key event in the pathogenesis of unstable angina and myocardial infarction (MI).1 The exposure of blood to a procoagulant surface triggers thrombin generation, platelet aggregation, and fibrin deposition, which leads to the subsequent thrombus formation precipitating the acute coronary event. Tissue factor, a transmembrane protein not normally in contact with blood, is considered the major initiator of the coagulation process.2 3 When small quantities of tissue factor come in contact with blood after vessel injury they form a complex with activated factor VII that initiates the activation of factors IX and X and eventually leads to thrombin generation.3
Factor VII is a vitamin K–dependent glycoprotein that is present in plasma either as a single-chain zymogen or as a double-chain enzymatically active form.3 Factor VII is converted to activated factor VII by a variety of plasma proteases including activated factors X, IX, and XII and thrombin. Besides activating factors IX and X, the tissue factor–activated factor VII complex also dramatically increases the conversion of factor VII to activated factor VII through an autoactivation process.4 5
A new, simple, and specific method for quantifying the enzymatic activity of factor VII in plasma6 should make it possible to establish whether the activation of the tissue factor coagulation pathway that occurs in acute coronary syndromes is mirrored by an increase in the plasma levels of activated factor VII (the main enzyme of the pathway) or whether high activated factor VII activity occurs and develops locally (at the cellular level in fissured plaques) with little change in its plasma levels. The present study was designed to investigate the relation between activated factor VII and the presence of heightened coagulation enzyme activity as detected by markers of thrombin generation and activity in patients presenting in the acute phase of MI and unstable angina. All the patients were reinvestigated after 15 days and 3 and 6 months to assess changes in the activation markers during follow-up.
The study populations consisted of patients with unstable angina and patients with acute MI who were consecutively admitted to the Division of Cardiology, Ca’ Granda Niguarda Hospital, Milan, Italy, from February 1993 through March 1994.
Inclusion Criteria and Patient Subgroups
The patients were prospectively assigned to diagnostic subgroups (or excluded from the study) by the physician in charge of the coronary care unit. A log of all hospital admissions was kept during the recruitment phase.
Unstable angina was defined as chest pain occurring at rest within the previous 48 hours accompanied by transient electrocardiographic ischemic changes (ie, ST segment elevation or depression ≥1 mm 0.08 second after the J-point or the pseudonormalization of previously negative T waves) and serum levels of creatine kinase MB fraction of less than twice the upper limit of normal.
Acute MI was defined as chest pain at rest lasting more than 30 minutes accompanied by ST segment elevation evolving into pathological Q wave or T wave inversion and confirmed by an increase in the MB fraction of creatine kinase of more than twice the upper limit of normal.
Patients with comorbid conditions known to alter coagulation enzyme activity or decrease the clearance of activation fragments and those who were taking drugs that affect hemostatic mechanism function were deemed ineligible for the study. Among the eligible patients with unstable angina, 10 patients were excluded because they had one of the following: concomitant peripheral vascular disorders or valvular heart disease (4); coronary artery bypass surgery, angioplasty, or acute MI in the preceding 6 months (5); or severely limited venous access (1). Among the eligible patients with MI, 7 were excluded because of incorrect diagnosis (1), difficult venous access (1), peripheral vascular disease (2), or malignancy (3).
After their inclusion in the study the patients had venous blood samples taken for baseline biochemical and coagulation analyses. Blood withdrawal on admission was performed before any invasive procedure and the start of any anticoagulant therapy, including aspirin. The patients then received standard routine medical therapy that included nitrates, β-blockers, calcium antagonists, and for patients with MI, thrombolytic therapy. All the patients were also put on aspirin. Subsequent blood withdrawal was performed after 15 days and 3 and 6 months in all but the 6 patients with unstable angina and 9 patients with MI who received chronic anticoagulation with Coumadin.
For control subjects we evaluated patients with stable angina or healthy individuals matched for age and sex. Patients with stable angina but no prior history or findings of MI, unstable angina, coronary revascularization, silent ischemia, or peripheral vascular disease were selected from a pool of individuals hospitalized for elective cardiac catheterization. Stable angina was defined as a history of chest pain induced by exercise or usual daily activity for more than 6 months with the development of at least 1-mm ST segment depression during the exercise test and with significant coronary artery disease at angiography. Healthy, nonsmoking individuals matched with the study population for age and sex were selected at random from the hospital personnel and their relatives. Blood samples for biochemical and coagulation analyses were taken from stable angina patients during hospitalization for cardiac catheterization and from the healthy individuals during a morning visit to the hospital under fasting conditions. In both control groups, blood withdrawal was also repeated after 15 days and 3 and 6 months.
Measurement of Coagulation System Activation
Venipunctures were performed atraumatically by specially trained investigators (P.A.M., L.O., and M.B.) by means of 19-gauge butterfly infusion sets and a two-syringe technique. The first 4 mL of blood was used for the measurement of blood lipids. The samples for the activated factor VII and prothrombin fragment 1+2 (F1+2) assays were collected directly into vacutainers (refrigerated for F1+2 or held at room temperature for activated factor VII) containing sodium citrate at a final concentration of 3.8% (wt/vol). Samples for the fibrinopeptide A (FPA) assay were collected into refrigerated vacutainers with a special anticoagulant provided by the manufacturer of the assay (Diagnostica Stago); the ratio of anticoagulant to blood was 1:9 (vol/vol). All blood samples were immediately centrifuged at 2500g for 25 minutes at 4°C; the plasmas were frozen on dry ice and stored at −80°C until analyzed (within 2 months).
All samples were analyzed without any knowledge by the technician of the clinical data. F1+2 was measured by the commercial F1+2 enzyme-linked immunosorbent assay (Behringwerke). Since the calibration curve was linear only up to 2 nmol/L, those samples with F1+2 levels of more than 2 nmol/L were diluted with phosphate-buffered saline (phosphate 0.04 mol/L and saline 0.1 mol/L, pH 7.4) to obtain absorbance with the linear part of the calibration curve. This technique has an intra-assay coefficient of variation of 5%. The plasma concentrations of FPA were determined in duplicate by enzyme-linked immunosorbent assay (Diagnostica Stago) in plasma extracted twice with bentonite to remove fibrinogen. This technique has an intra-assay coefficient of variation of 5.4%. Activated factor VII, the enzymatic form of factor VII, was measured by means of a one-stage, prothrombin time–based assay that used a truncated soluble form of recombinant tissue factor (kindly supplied by Dr Yale Nemerson, Mount Sinai Hospital Medical School, New York, NY) that upon relipidation reacts with the activated but not the zymogen form of factor VII.7 The standard used for activated factor VII was recombinant factor VIIa from Novo Nordisk. Factor VII–deficient plasma was obtained from a congenitally deficient patient with plasma factor VII levels of less than 2 U/dL (antigen and coagulant activity). This technique has an intra-assay coefficient of variation of 7.2%.
The study was approved by the Institutional Review Board of the Ca’ Granda Niguarda Hospital, Milan, Italy, and informed consent was obtained from all subjects.
Descriptive statistics include means and standard deviations or medians and interquartile ranges as appropriate. Baseline characteristics were compared by ANOVA for continuous data and by the χ2 test for categorical data. Given that the plasma levels of coagulation activation markers were not normally distributed, the Kruskal-Wallis one-way ANOVA was used to test the difference between groups; subsequent comparisons were made by using the Mann-Whitney U test with downward adjustment of the α level to compensate for multiple comparisons. Repeated measures were compared by means of the Friedman test, and subsequent pairwise comparisons were made by using the Wilcoxon signed-rank test. Correlation between activation peptides and activated factor VII was performed by calculating the Spearman’s rank correlation coefficient. Two-tailed probability values of below .05 were regarded as significant.
We investigated 32 patients with unstable angina and 28 patients with acute MI. Seventeen patients with chronic stable angina and 33 healthy individuals were also evaluated and served as control subjects. The baseline characteristics of the four study groups are shown in Table 1⇓. No differences were detected between the groups in plasma levels of cholesterol, triglycerides, HDL, and lipoprotein(a).
Coagulation Activation Markers in the Acute Phase
The plasma levels of the coagulation activation markers in the different groups are reported in Table 2⇓ and represented in the Figure⇓. On admission, plasma concentrations of F1+2 and FPA (Figure⇓, top and middle, respectively) were higher in the patients with unstable angina (P=.0001) or acute MI (P=.0001) than in those with stable angina or in healthy individuals, whereas no differences in the plasma levels of activated factor VII were observed between the different groups (Figure⇓, bottom).
A significant correlation between activated factor VII and F1+2 was observed in healthy volunteers and in the patients with stable angina (r=.39, P<.01), whereas no correlation was found between activated factor VII and F1+2 in patients with unstable angina or acute MI. A lack of correlation was also observed between activated factor VII and FPA in all the study groups.
Coagulation Activation Markers During Follow-up
Plasma F1+2 levels in patients with unstable angina or acute MI as well as in subjects with stable angina and healthy control subjects did not change during follow-up (Table 2⇑). Plasma F1+2 levels were significantly higher in patients with unstable angina or MI than in stable patients and healthy volunteers at 15 days (P=.04) and at 3 (P=.003) and 6 (P=.0006) months.
Plasma FPA levels decreased significantly in patients with unstable angina (P=.0001) and acute MI (P=.0018) during follow-up (Table 2⇑); compared with admission levels, plasma levels of FPA were significantly lower at 3 (P<.005) and 6 (P<.005) months. Plasma FPA concentrations did not significantly change in the patients with stable angina and in healthy control subjects at the different time points. At 15 days plasma FPA levels were higher (P=.01) in patients with unstable angina or MI than in stable patients and healthy volunteers. At both 3 and 6 months no differences were observed in plasma FPA levels between the study groups.
No changes were observed in the plasma levels of activated factor VII at the different times in the patients with unstable angina or MI nor in the stable patients and healthy volunteers (Table 2⇑). No differences between the different groups were detected in the plasma levels of activated factor VII at any time.
Tissue factor is a membrane-bound glycoprotein that functions in the extrinsic pathway of blood coagulation by acting as a cofactor of activated factor VII. Tissue factor binds to activated factor VII, and the resulting complex acts as a catalyst for the conversion of factor X to activated factor X and factor IX to activated factor IX, leading to the formation of thrombin. The presence of tissue factor protein and tissue factor synthesizing cells has been demonstrated in atherosclerotic plaques.8 In addition to its activation of the coagulation cascade, we hypothesized that in patients with an acute coronary syndrome, the contact between tissue factor contained in the atherosclerotic plaques and blood would lead to an increased conversion of factor VII to activated factor VII and that this enzyme might be measurable in plasma in larger amounts. According to this hypothesis, there should also be a correlation between the plasma levels of activated factor VII and those of the markers of coagulation enzyme activities, which measure the degree of in vivo thrombin generation and activity, such as F1+2 and FPA. In this study we addressed this issue in patients with acute coronary syndromes by using recently developed assays that allow the precise biochemical characterization of the degree of in vivo activation of the hemostatic mechanism9 10 as well as the direct measurement of activated factor VII.6 7
Our data show that in the acute phase of unstable angina or MI there is an activation of the coagulation system that is indicated by an increase in the plasma levels of the markers of thrombin generation and activity but that this activation is not associated with an increase in the plasma levels of activated factor VII. A significant correlation between activated factor VII and thrombin generation could be found in the control populations, in agreement with the fact that under normal conditions baseline hemostatic activity is maintained through a tissue factor–dependent mechanism,11 but this correlation was not observed in the patients with acute coronary syndromes. At the follow-up evaluations the patients with acute coronary syndromes showed persistently elevated plasma levels of F1+2, whereas there was a normalization of plasma FPA levels. This condition, which has been defined as a hypercoagulable state12 and has been described in other patients with unstable angina and MI,13 was not associated with any change in the plasma levels of activated factor VII.
The reasons for the discrepancy between the signs of heightened thrombin generation and activity and the absence of any increase in the plasma levels of activated factor VII remain speculative. One possible explanation may be related to the fact that, although more activated factor VII is produced as a consequence of tissue factor exposure, it is not released into the plasma, but due to its high affinity for tissue factor, it exerts its activity locally, thus preventing any increase in plasma levels from being observed. Activated factor VII is quickly inactivated by the complex tissue-factor pathway inhibitor–activated factor X,14 and this may also account for the failure to detect increased levels in plasma.
We gratefully acknowledge the technical assistance of Alessandra Spinola and Bianca Bottasso.
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