Combined Use of Markers of Muscle Necrosis and Fibrinogen Conversion in the Early Differentiation of Myocardial Infarction and Unstable Angina

Cardiovascular Res. Inst. Maastricht, Maastricht Univ., P.O. Box 616, 6200 MD Maastricht, The Netherlands

To the Editor:

Intracoronary formation of blood clots on ruptured arteriosclerotic plaques is considered the main cause of acute myocardial infarction (AMI) (1). After such ruptures, exposed tissue factor binds to factor VIIa from plasma, and the resulting tissue factor-factor VIIa complex activates factor X toward factor Xa, the enzyme converting prothrombin to thrombin. By cleavage of fibrinopeptides A and B, thrombin produces desAABBfibrin monomers that polymerize into still-soluble complexes called "thrombus precursor proteins" (TpPs). New antigens formed on these complexes were used for a TpP assay (2).

Because the acute thrombotic event precedes coronary occlusion and muscle necrosis, detection of activated coagulation potentially allows early detection of AMI. Until now, attempts in this field have focused on markers for factor Xa and thrombin activity, such as prothrombin fragment 1.2 and thrombin-antithrombin complexes. However, these markers are not necessarily closely related to the actual formation of fibrin clots (3), especially in chronically hypercoagulable patients, and TpP could perform better in this respect. We therefore studied plasma concentrations of TpP and fibrin monomers (FMs) in patients with suspected AMI. The results were compared with two small cytosolic cardiac marker proteins, myoglobin (Mb) and fatty acid-binding protein (FABP), that are early markers for necrosis(4, 5) and with a highly cardiospecific marker, troponin T (TnT).

TpP was determined with a monoclonal sandwich ELISA provided by American Biogenetic Sciences (2). Intraassay imprecision, estimated on three different days by the 11-fold determination of three pools of citrated plasma with 2.9, 10.1, and 20.7 mg/L of TpP added, was 16%, 11%, and 12%, respectively (CVs). Interassay imprecision, estimated from duplicate measurements on 20 different days of citrated donor plasma and similar plasma with added TpP, was 23% and 30%, respectively, with mean values of 2.5 and 11.6 mg/L. FMs were determined with a sandwich ELISA (Boehringer Mannheim). The assay measures the free amino terminus of fibrin A-chains. Mb was determined with an immunoturbidimetric assay (Hoffmann-La Roche); FABP was determined with a monoclonal sandwich ELISA as described (6), creatine kinase MB isoenzyme (CK-MB) was determined with a microparticle immunoassay (Abbott), and TnT was determined with a one-step sandwich ELISA (Boehringer Mannheim). ROC curves were obtained from double logarithmic plots (7).

Twenty-five patients entering the University Hospital Maastricht with AMI or unstable angina pectoris (UAP) were studied. The final diagnosis of AMI required at least two of the following conditions: (a) typical chest pain, (b) electrocardiogram changes indicating AMI, or (c) a plasma CK-MB concentration exceeding the locally established cutoff value of 8 µg/L. Standard treatment included oral aspirin, intravenous heparin, and elective thrombolytic therapy or percutaneous transluminal coronary angioplasty. As of October 1, 1997, none of the patients had died, and no patient with UAP developed AMI, during the 1.6 y follow-up.

After patients gave informed consent, a blood sample was obtained before medication was given. Clotting was prevented with sodium citrate, and plasma was stored at -80 °C. The median delay between the first symptoms and acquisition of the blood sample was 3 (range, 0.8–5) h in 15 patients with AMI (9 men and 6 women) and 2.6 (0.5–6) h in 10 patients with UAP (6 men and 4 women). The median ages were 71 (35–87) years and 64 (51–78) years, respectively. A tendency toward higher TpP values when delays in hospitalization were shorter (4 mg · L^-1 · h^-1) was noted, but that tendency did not hold for FM. As shown in Fig. 1 , the assay for TnT did not discriminate between AMI and UAP in patients who were hospitalized early. Median TnT values, with first and third quartiles, were 0.12 (0.08–0.14) µg/L in patients with AMI and 0.13 (0.05–0.25) µg/L in patients with UAP. Better discrimination was observed for FM and for TpP, and even better discrimination was observed for Mb and FABP. For clinical application of the low cutoff values shown in Fig. 1 , day-to-day imprecision of the TpP and FABP assays should be reduced.

View larger version (26K): [in this window] [in a new window]   Figure 1. ROC curves for detection of AMI in patients having either AMI or UAP. Lack of discrimination by TnT is apparent from its coincidence with the line of identity. Arrows indicate cutoff values of 43 µg/L, 4.5 µg/L, 4 mg/L, and 2.5 mg/L for Mb, FABP, TpP, and FM, respectively. Corresponding sensitivities (true positives) and specificities (100 - false positives) were 67%, 69%, 62%, and 58% and 80%, 80%, 67%, and 70%, respectively.

In the AMI group, FABP and Mb concentrations were highly correlated (r = 0.90), whereas the correlation between TpP and FM was much weaker (r = 0.49). Interestingly, FABP and TpP were not significantly correlated (r = 0.01). Apparently the two markers identified different patients; this suggests a potential for clinical utility in the combined use of these markers. Indeed, 87% sensitivity with 80% specificity was obtained when either FABP >6 µg/L or TpP >7 mg/L was considered as diagnostic for AMI. Moreover, the performance of such a combined test will be relatively insensitive to delays in hospitalization because the TpP assay will perform better in patients hospitalized earlier, whereas the FABP assay will perform better in patients admitted later(4, 5).

Low TpP values were found in patients with UAP, with the TpP concentration exceeding 6 mg/L (8.1 mg/L) in only one patient. Moreover, this patient was the only one showing a slight CK-MB increase (to 6 µg/L) over the next day, suggesting minor necrosis. Such lack of coagulation activation in UAP patients contrasts with data in the literature (8)(9) and may be explained by two factors: Prothrombin fragment 1.2 and thrombin-antithrombin complexes, as measured in these studies, may correlate poorly with fibrin generation in spite of a hypercoagulable state (3)(9) and minimal myocardial necrosis, as reflected by borderline increases of TnT or of CK-MB, identified UAP patients with no better prognosis than patients with AMI (10). Our use of a CK-MB cutoff of 8 µg/L shifted such patients to the AMI group, which could explain the lack of TpP and FM in the remaining UAP patients.

Acknowledgments

We thank David G. M. Carville, American Biogenetic Sciences, and Norbert Drees, Roche Diagnostic Systems, for providing the TpP and Mb assays.

Footnotes

*Author for correspondence. Fax 31-43-3670916; e-mail w.hermens{at}carim.unimaas.nl

References

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