Role of γ' fibrinogen in Cardiovascular Disease: Implications and Limitations

2. Introduction • High fibrinogen is a risk factor for CVDMeta-analyses show a two times higher risk for each 1 g/L increase • Fibrinogen is heterogeneous with different isoforms≈ 90% contain only A chains • ≈ 10% contain a ' chain

3. Introduction (cont) • ' Fibrinogen has an altered ' chain • Arises from alternative mRNA processing • Contains a high affinity thrombin binding site • Results in altered clot ultrastructure • Results in a decreased rate of fibrinolysis

4. Introduction (cont) • Increased ' fibrinogen is independently associated with CVD and stroke in case/control studies • Coronary artery disease: n = 133, OR = 7.16 (95% CI = 1.82 - 27.7) • per A/’ fibrinogen quartile (Lovely RS, Falls LA, Al-Mondhiry HA, Chambers CE, Sexton GJ, Ni H, Farrell DH. Thromb Haemost 2002:88:26-31) • Myocardial infarction: n = 774, OR = 1.24 (95% CI = 1.01 - 1.52). (Mannila MN, Lovely RS, Kazmierczak SC, Eriksson P, Samnegård A, Farrell DH, et al. J Thromb Haemost 2007;5:766-73) • Stroke: n = 249, elevated in cases vs. controls (0.42 mg/ml vs. 0.34 mg/ml). (Cheung EY, Uitte de Willige S, Vos HL, Leebeek FW, • Dippel DW, Bertina RM, de Maat MP. Stroke 2008;39:1033-5)

5. Question • Is ' fibrinogen simply a surrogate marker • for total fibrinogen?

6. Results • Assay precision • Respective within-run, run-to-run, and total imprecision estimates: • 13.4%, 28.6%, and 29.1% at 0.127 g/L of ' fibrinogen • 4.8%, 11.2%, and 11.6% at 0.416 g/L of ' fibrinogen

7. Results (cont) • Analytical specificity • ' fibrinogen shows 99.9% sequence homology with A/A fibrinogen • Serial dilutions of anti-' monoclonal antibody 2.G2.H9 showed no measurable activity against A/A fibrinogen

8. Figure 1. Analytical specificity of 2.G2.H9 towards ' fibrinogen. The analytical specificity of anti-' fibrinogen monoclonal antibody 2.G2.H9 and its ability to differentiate ' fibrinogen from fibrinogen lacking ' chains was determined by incubating the indicated dilutions of the antibody with A/A or A/' fibrinogen in microtiter wells. Goat anti-mouse IgG/HRP conjugate was added for detection, followed by O-phenylenediamine, and the absorbance was quantitated at 450 nm.

9. Figure 3. Limit of quantification for the ' fibrinogen ELISA. The limit of quantification of the assay for measurement of ' fibrinogen concentrations in plasma was determined using 8 separate pools of patient plasma. The limit of quantification for the assay was 0.10 g/L, defined as that concentration of ' fibrinogen giving a within-run CV of 20% or less.

10. Question • What are some possible limitations of using • long-term frozen banked plasma for assessing reference intervals and diagnostic decision thresholds? • The Framingham Offspring cohort samples were collected during the 7th examination cycle from 1998-2001 and stored frozen until analysis.

11. Results (cont) • ' fibrinogen concentrations in humans • Central 95th percentile limits (median value) determined from various studies • Framingham offspring controls (n = 2879): 0.088–0.551 g/L (0.234 g/L) • Healthy blood donors (n = 120): 0.115–0.460 g/L (0.281 g/L) • Control subjects from study on CAD (see ref. 7) (n = 42): 0.125–0.676 g/L (0.242 g/L)

12. Figure 4. ' fibrinogen concentrations in healthy individuals from the Framingham Offspring Study. ' fibrinogen was measured in 2879 participants from the Framingham Offspring Study with no previous history of cardiovascular disease. The reference interval of ' fibrinogen varied nearly 40-fold, from a low of 0.037 g/L to a high of 1.443 g/L. The 2.5th and 97.5th percentile limits for ' fibrinogen were 0.088 to 0.551 g/L. The median concentration was 0.234 g/L and the mean concentration was 0.255  0.119 g/L (SD).

13. Figure 5. Receiver-operating characteristic curve for ' fibrinogen in CAD patients. ' fibrinogen concentrations were measured in a prior study in 133 patients referred for elective diagnostic cardiac catheterization. The receiver-operating characteristic curve of ' fibrinogen concentrations in CAD cases and controls showed an area under the curve of 0.76. A maximum diagnostic accuracy of 0.78 was found at a decision threshold of 0.30 g/L.

14. Table 1. Association of ' Fibrinogen with Traditional Cardiovascular Risk Factors. aResults are unadjusted mean (1 standard deviation) or %. bP values assess the significance of the difference in mean/% across fibrinogen tertiles and are age and sex-adjusted. (P value for age is sex-adjusted only, and P value for sex is age-adjusted only.)

15. Association of ' Fibrinogen with Traditional Cardiovascular Risk Factors • Factors showing significant positive association • Age, gender, BMI, smoking, diabetes, glucose, and triglycerides • Factors showing significant inverse correlation • HDL cholesterol • ' Fibrinogen does not show significant association with systolic blood pressure and total cholesterol • Total fibrinogen does show a significant association with systolic blood pressure and total cholesterol. • Addition of ' fibrinogen to total cholesterol and hsCRP may provide additional predictive value for assessment of the risk of CVD

16. Question • What future studies should be performed to further investigate the association between ' fibrinogen and cardiovascular disease risk?

17. Conclusions • The ' fibrinogen ELISA has within-run CVs of 13.4% at 0.127 g/L and 4.8% at 0.416 g/L • ROC curve analysis of results from patients with coronary artery disease yielded an area under the curve of 0.76, with a diagnostic accuracy of 0.78 at a decision threshold of 0.30 g/L • ' fibrinogen shows excellent prognostic utility for cardiovascular risk analysis