1. Wolfgang Koenig, MD, FESC, FACC Dept. of Internal Medicine II - Cardiology University of Ulm Medical Center Ulm, Germany CRP, Lp-PLA 2, and Other Serum Markers of Disease and Vulnerability of Disease and Vulnerability The 2 nd Vulnerable Patient Satellite Symposium Towards a National Screening Program New Orleans, LA, March 6 th, 2004 The 2 nd Vulnerable Patient Satellite Symposium Towards a National Screening Program New Orleans, LA, March 6 th, 2004

2. Identity Test Positive Test Negative 0.50.40.30.20.10.0 0.05 0.1 0.15 0.2 0.05 0.1 0.15 0.2 Pre-test Probability of CHD Event in 10 Yrs Post-test Probability of CHD Event in 10 Yrs modified after Greenland et al. Circulation 2001;104:1863-1867 Low-Risk Intermediate-Risk High-Risk Low-Risk Intermediate-Risk High-Risk (~35 % of Pts.) (~40% of Pts.) (~25% of Pts.) <6 (10) % 6 (10) -19 % ≥ 20 % <6 (10) % 6 (10) -19 % ≥ 20 % over 10 years over 10 years CHD Risk Assessment in Asymptomatic Patients: Selective Use of Noninvasive Testing Modification of Probability Estimates of CHD by Non-invasive Testing  Assessment by multivariable statistical models: e.g. statistical models: e.g. Framingham Risk Score or Framingham Risk Score or PROCAM Score PROCAM Score  Clear guidelines for high or low risk subjects, but not so for risk subjects, but not so for those at intermediate risk those at intermediate risk

3. * ESR, erythrocyte sedimentation rate; PAI-1, plasminogen activator inhibitor-1; vWF, von Willebrand factor; CIC, circulating immune complexes; Lp-PLA 2 lipoprotein-associated phospholipase A 2 Acute Phase Reactants Investigated Prospectively in Epidemiological Studies Non-Protein Markers Frequently Studied Proteins Infrequently Studied Proteins Leukocytes C-reactive protein Orosomucoid ESR* Serum amyloid A Alpha 1 -antitrypsin Plasma viscosity FibrinogenHaptoglobin AlbuminCeruloplasmin Plasminogen C3, C4 PAI-1* IgA, G, M, and E vWF* Sialic acid Cytokines (IL-6, 8, 10, 18) CIC* CAMs Lp (a) Lp-PLA 2 *, sPLA 2 -IIA mod. after Koenig & Rosenson. Sem Vasc Med 2002;2:417-24

4. Kuller MRFIT 1996 CHD death Ridker PHS 1997 MI Ridker PHS 1997 Stroke Tracy CHS/RHPP 1997 CHD Ridker PHS 1998,2001 PAD Ridker WHS 1998,2000,2002 CVD Koenig MONICA 1999 CHD Roivainen HELSINKI 2000 CHD Mendall CAERPHILLY 2000 CHD Danesh BRITAIN 2000 CHD Gussekloo LEIDEN 2001 Fatal Stroke Lowe SPEEDWELL 2001 CHD Packard WOSCOPS 2001 CV Events Ridker AFCAPS 2001 CV Events Rost FHS 2001 Stroke Pradhan WHI 2002 MI, CVD death Albert PHS 2002 Sudden Death Sakkinen HHS 2002 MI 0 1.02.03.04.05.06.0 0 1.02.03.04.05.06.0 Relative Risk (upper versus lower quartile) Ridker PM. Circulation 2003;107:363-369 CRP as a Risk Factor for Future CVD – Results from Population-Based Studies

5. Ridker et al. N Engl J Med 2002;347:1557-1565 Relative Risk (RR) of a First Cardiovascular Event, According to CRP and LDL- C at Baseline (WHS)

6. 0 5 10 15 20 25 0-12-45-9  10 <1.0 1.0-3.0 >3.0 CRP mg/L <1.0 1.0-3.0 >3.0 0 1 2 3 <130130-160<160 Multivariable Relative Risk Framingham Estimate of 10-Year Risk (%) LDL Cholesterol (mg/dL) RR of Cardiovascular Disease According to Levels of CRP and the Estimated10-Year Risk and According to Levels of CRP and Categories of LDL-C (WHS) Ridker et al. N Engl J Med 2002;347:1557-1565

7. Class I: Should be performed Class II: Conflicting evidence/opinion a: Weight in favor of usefulness/efficacy a: Weight in favor of usefulness/efficacy b: Usefulness/efficacy less well established b: Usefulness/efficacy less well established Class III: Should not be performed Class I: Should be performed Class II: Conflicting evidence/opinion a: Weight in favor of usefulness/efficacy a: Weight in favor of usefulness/efficacy b: Usefulness/efficacy less well established b: Usefulness/efficacy less well established Class III: Should not be performed AHA/CDC Recommendations for Clinical and Public Health Practice  Of current inflammatory markers identified, hs-CRP has the analyte & assay characteristics most conducive to use in practice (Class IIa, Level of Evidence B)  Other inflammatory markers should not be measured for determination of CV risk in addition to hs-CRP (Class III, Level of Evidence C) Laboratory Tests AHA/CDC Statement. Circulation 2003;107:499–511

8. The Value of CRP in Cardiovascular Risk Prediction: The Rotterdam Study  Nested case-control study (157/500) within a population based cohort study of 7983 men and women >55 years  Multivariable RR (Q4-Q1) for CRP 1.2 (95% CI, 0.6-2.2)  Assessment of Framingham Risk Score w/o and with CRP  Assessment of AUC by ROC analysis Variable AUC (SE) Basic risk * 0.642 (0.026) Risk function 1 † 0.773 (0.021) with CRP with CRP 0.777 (0.021) Risk function 2 ‡ 0.746 (0.021) with CRP with CRP 0.748 (0.021) * Indicated by age, age squared, sex; † Indicated by age, age squared, sex, current smoking, BMI, BP, DM, family hystory of early MI, TC, HDL; ‡ based on the Framingham risk function + LVH BP, DM, family hystory of early MI, TC, HDL; ‡ based on the Framingham risk function + LVH Van der Meer et al. Arch Intern Med 2003;163:1323-1328

9. < 6 6-10 11-14 15-19  20 0 1 2 3 4 5 6 7 8 RR of CHD According to the Estimated 10-Yr Risk Alone and in Combination With CRP: MONICA Augsburg Cohort < 6 6-10 11-14 15-19  20 0 1 2 3 4 5 6 7 8 P=0.19 P=0.28 P=0.02 P=0.03 P=0.14 <1.0 1.0 – 3.0 > 3.0 CRP mg/L 18 32 35 50 56 18 32 35 50 56 Population at risk 809 914 650 526 536 Framingham Estimate of 10-Year Risk (%) Multivariable Relative Risk AIC 2776 AIC 2789 (N=3,435 Men, 45-74 Yrs; 191 Events, FU 6.6 Yrs) Koenig et al. Circulation (in press)

10. FactorEvents/n HR (95%CI) P-value P-value FRS 1 <6 18/809 18/809Ref.Ref. (%) (%)6-19117/2090 2.81 (1.71-4.62) 2.39 (1.45-3.94)  20 56/536 56/536 6.19 (3.64-10.54) <0.0001 4.85 (2.82-8.33) <0.0001 AIC28162797 ∆AIC 19 AUC0.7130.7400.0077 FRS 2 <6 18/809 18/809Ref.Ref. (%) (%) 6-10 6-10 32/914 32/914 1.63 (0.91-2.90) 1.46 (0.82-2.61) 11-14 35/650 35/650 2.70 (1.53-4.77) 2.35 (1.32-4.16) 15-19 50/526 50/526 5.61 (3.27-9.62) 4.50 (2.59-7.80)  20 56/536 56/536 6.21 (3.65-10.57) <0.0001 5.01 (2.91-8.62) <0.0002 AIC27892776 ∆AIC 13 AUC0.7350.7500.0163 AIC, Akaike’s Information Criterion; ΔAIC, AIC (model without CRP) – AIC (model with CRP); AUC, Area under the curve Risk of a First Coronary Event by Cox Model Without and With CRP for the FRS With 3 and 5 Categories Koenig et al. Circulation (in press)

11. MONICA Augsburg Cohort Study: Summary  Elevated CRP concentrations and an elevated TC/HDL-C ratio were both independently related to incident CHD.  The addition of CRP to a prediction model of TC/HDL-C or the FRS resulted in a better fit of the model containing CRP and significantly improved prediction of incident CHD for TC/HDL-C and the calculated FRS.  The latter was particularly true for those at intermediate risk (10-20% over 10 years).  Thus, CRP measurement modulates coronary risk and may therefore modify the physician`s interpretation of the patient`s risk status.  However, these findings have to be replicated in other populations. Koenig et al. Circulation (in press)

12. * ESR, erythrocyte sedimentation rate; PAI-1, plasminogen activator inhibitor-1; vWF, von Willebrand factor; CIC, circulating immune complexes; Lp-PLA 2 lipoprotein-associated phospholipase A 2 Acute Phase Reactants Investigated Prospectively in Epidemiological Studies Non-Protein Markers Frequently Studied Proteins Infrequently Studied Proteins Leukocytes C-reactive protein Orosomucoid ESR* Serum amyloid A Alpha 1 -antitrypsin Plasma viscosity FibrinogenHaptoglobin AlbuminCeruloplasmin Plasminogen C3, C4 PAI-1* IgA, G, M, and E vWF* Sialic acid Cytokines (IL-6, 8, 10, 18) CIC* CAMs Lp (a) Lp-PLA 2 *, sPLA 2 -IIA mod. after Koenig & Rosenson. Sem Vasc Med 2002;2:417-24

13. IL-18 and Risk of CHD*: PRIME Combined Endpoint Coronary Death and MI 235 235 235 235 [pg/mL][pg/mL] Relative Risk (95% CI) 32103210 F F F F B B B B Blankenberg et al. Circulation 2003;108:2453-2459 * In tertiles of IL-18

14.  Lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) Platelet-activating factor acetylhydrolase 50kDa, Ca-insensitive lipase Produced predominantly by macrophages/ monocytes, T-cells, and mast cells monocytes, T-cells, and mast cells Not responsive to IL-1, IL-6, TNF-   Secretory phospholipase A 2 (sPLA 2 ) 14 kDa, Ca-dependent lipase Produced by arterial wall SMC and macrophages Increased by cytokines IL-1, IL-6, TNF-  Phospholipases A 2 and Atherosclerosis

15. Theory: Lp-PLA 2 Promotes Atherogenesis  Generates lyso-PC during oxidation of LDL  Lp-PLA 2 -dependent oxFFA are also bioactive human monocyte chemoattractants  Anti-atherosclerotic effect of inhibitor demonstrated in WHHL rabbit  Plasma levels correlate with CHD in patients?

16. LumenIntimaMediaLDL Oxidized LDL LDL Lp-PLA 2 Lyso-PC+ OxFA OxFA AdhesionMolecules Monocyte PlaqueFormationPlaqueFormation Cytokines MacrophageMacrophage Foam Cell Role of Lp-PLA 2 in Coronary Heart Disease

17. West of Scotland Coronary Prevention Study (WOSCOPS) WOSCOPS Study Design  randomized, double blind, placebo controlled trial  6,595 men, aged 45 to 65  elevated LDL levels (range 174-232 mg/dL or 4.5-6.0 mM)  no previous myocardial infarction (MI)  mean follow-up of 5 years Study Results  treatment with Pravastatin reduced risk of first time heart attack (by 31%) and death (by 22%) Packard et al. N Engl J Med 2000;343:1148-1155

18. Lp-PLA 2 as a Novel Risk Factor in CHD: WOSCOPS Baseline samples (stored @ -70 o C) plasma n=6,595 4.9 years 580 coronary events 1,160 event free 1,160 event free (randomly selected, but age, smoking matched) Cases Controls Samples drawn from freezer Packard et al. N Engl J Med 2000;343:1148-1155

19. CRP, Lp-PLA 2 and CHD Risk: WOSCOPS univariate Inflam. markers All risk factors Relative Risk (confidence interval) Model 1 2 3 CRP 1.27 (1.14 -1.42) 1.21 (1.06 -1.39) 1.13 (0.98 -1.29) WCC 1.22 (1.09 -1.37) 1.15 (1.02 -1.31) 1.10 (0.97 -1.25) Fibrinogen 1.19 (1.07 -1.31) 1.04 (0.92 -1.17) 1.02 (0.90 -1.15) Lp- PLA 2 1.20 (1.08 -1.34) 1.19 (1.07 -1.33) 1.18 (1.05 -1.33) Packard et al. N Engl J Med 2000;343:1148-1155

20. Methods: Patient Population and Assays  12,819 apparently healthy men and women free of CHD at ARIC visit 2  608 individuals with incident CHD between visit 2 and visit 4 (6- to 8-year follow-up), with 740 controls from a cohort random sample  Lp-PLA 2 : diaDexus PLAC™ test (Dada et al. Expert Re Mol Diagn 2002), dual monoclonal Ab immunoassay standardized to recombinant Lp-PLA 2  hs-CRP: Denka Seiken asssay, which has been validated to Dade Behring method (Roberts et al. Clin Chem 2001) Lp-PLA 2 and Risk of CHD: ARIC Ballantyne et al. Circulation 2004;109:837-842

21. Weighted-Adjusted* Means of Risk Factors Variable Cases (n=608) Noncases(n=740) P-value DM 28.7 15.1<0.001 BMI 28.7 28.1 0.014 TC219.7207.2<0.001 TG144.8124.5<0.001 HDL-C 45.6 51.2<0.001 LDL-C145.1131.2<0.001 SBP127.5121.6<0.001 DBP 73.1 72.6 0.350 Lp-PLA 2 404373<0.001 hs-CRP 4.05 3.04<0.001 * Adjusted for age, sex, and race Ballantyne et al. Circulation 2004;109:837-842 Lp-PLA 2 and Risk of CHD: ARIC

22. 2 (310-422 μg/L) 3 (≥422 μg/L) Model 1 † 1.26 (0.94-1.69) 1.78 (1.33-2.38) Model 2 ‡ 1.02 (0.73-1.43) 1.16 (0.82-1.65) Model 2 ‡, LDL-C<130 mg/dL 1.83 (1.11-3.00) 1.99 (1.17-3.38) Model 3 § 1.00 (0.71-1.41) 1.15 (0.81-1.63) Model 3 §, LDL-C<130 mg/dL 1.83 (1.10-3.05) 2.08 (1.20-3.62) CHD HRs (95% CI) by Lp-PLA 2 Tertiles Lp-PLA 2 Tertiles * *Lowest tertile (<310µg/L) is reference; † Adjusted for age, sex, and race; ‡ Also adjusted for smoking status, SBP, LDL-C, HDL-C, and diabetes; § Additionally adjusted for CRP Ballantyne et al. Circulation 2004;109:837-842 Lp-PLA 2 and Risk of CHD: ARIC

23. Weighted-Correlation Between Lp-PLA 2 and Other Risk Factors: ARIC Pearson Correlation Pearson Correlation Risk Factor Coefficient P-Value Total cholesterol 0.23 0.23<0.0001 LDL-C 0.36 0.36<0.0001 HDL-C - 0.33 <0.0001 SBP 0.04 0.04NS DBP - 0.01 NS hs-CRP - 0.05 NS BMI - 0.02 NS Triglycerides 0.13 0.13 0.0006 0.0006 Ballantyne et al. Circulation 2004;109:837-842

24. 0 1 2 3 Association of Lp-PLA2 and hs-CRP with Incident CHD in Patients with Low LDL-C (<130mg/dL) CHD Hazard Ratio Lp-PLA 2 µg/L hs-CRP, mg/L Lp-PLA 2 and Risk of CHD: ARIC Ballantyne et al. Circulation 2004;109:837-842 95% CI 1.47 - 5.94, P=0.002 High (≥ 422) High (≥ 422) Low-Med (< 422) High (>3) Low-Med (≤3) High (>3) Low-Med (≤3) 2.95 1.14 1.00 0.99

25. Lp-PLA 2 and Risk of CHD: MONICA-Augsburg Cohort 1984-98  934 men aged 45-64 years, participating in the first MONICA survey 1984/85  Exclusion of prevalent CHD  Standardized assessment of cardiovascular risk factors  Lp-PLA 2 by diaDexus PLAC ™ test (enzyme immunoassay); CRP by a high-sensitivity immunoradiometric assay (Hutchinson et al. Clin Chem 2000)  Endpoint determination according to the MONICA protocol (fatal and non-fatal MI and SCD) Koenig et al. (AHA 2003)

26. Age-adjusted Baseline Characteristics of 934 Men, Aged 45-64 Years Participating in the MONICA Augsburg Survey 1984/85 With Follow-up 1998 Characteristic Men with event (n=97) Men w/o event (n= 837) P-value BMI (kg/m 2 ) 27.627.6n.s. Total cholesterol (mg/dl) 259.6243.30.001 HDL cholesterol (mg/dl) 46.851.8 0.0037 0.0037 TC/HDL-Ratio § 5.664.83 < 0.0001 Systolic BP (mmHg) 139.0136.6n.s. Diastolic BP (mmHg) 84.984.5n.s. Regular smoker % 53.928.0<0.0001 Physical activity % 25.735.60.05 Diabetes % 8.43.1 0.0096 0.0096 Education (<12 years) % 74.273.7n.s. Lp-PLA 2 (ng/ml) 292.3263.4 0.0013 0.0013 C-reactive protein § (mg/L) 2.491.54 < 0.0001 § geometric means calculated from the log-transformed distribution Koenig et al. (AHA 2003)

27. Correlation Between Lp-PLA 2, CRP and Other Cardiovascular Risk Factors: MONICA Risk factor Spearman correlation coefficient (P-value) Lp-PLA 2 CRP Lp-PLA 2 CRPAge 0.13 (<0.0001) 0.13 (<0.0001) 0.15 (<0.0001) 0.15 (<0.0001) Total-C 0.30 (<0.0001) 0.30 (<0.0001) 0.07 (0.03) 0.07 (0.03) HDL-C 0.11 (0.0006) 0.11 (0.0006) -0.15 (<0.0001) TC/HDL-C 0.07 (0.04) 0.07 (0.04) 0.16 (<0.0001) 0.16 (<0.0001) Systolic BP 0.03 (0.40) 0.03 (0.40) 0.11 (0.0013) 0.11 (0.0013) BMI -0.04 (0.24) 0.21 (<0.0001) 0.21 (<0.0001) CRP 0.07 (0.04) 0.07 (0.04) 1.0 1.0 Koenig et al. (AHA 2003)

28. MONICA: RR of CHD by a 1 SD Increase in CRP or Lp-PLA 2 (separate models) Model RR (95% CI) P-value CRP only Unadjusted 1.57 (1.30-1.90) 0.0001 Adjusted for age, DM, smoking 1.32 (1.07-1.62) 0.008 Multivariable adjustment* 1.28 (1.03-1.60) <0.03 Lp-PLA 2 only Unadjusted 1.37 (1.16-1.62) 0.0002 Adjusted for age, DM, smoking 1.26 (1.05-1.54) 0.01 Multivariable adjustment* 1.23 (1.02-1.47) 0.03 *Age, systolic BP, TC/HDL-ratio, physical activity, BMI, smoking, DM, alcohol intake, education Koenig et al. (AHA 2003)

29. MONICA: RR of CHD by a 1 SD Increase in Lp-PLA 2 or CRP (same model) Model RR (95% CI) P-valueUnadjustedCRP Lp-PLA 2 1.55 (1.28-1.89) 1.35 (1.14-1.60) <0.0001 <0.0001 0.006 0.006 Adjusted for age, DM, smoking CRP Lp-PLA 2 1.31 (1.07-1.62) 1.25 (1.04-1.50) 0.01 0.01 <0.02 <0.02 Multivariable adjustment* CRP Lp-PLA 2 1.27 (1.01-1.59) 1.21 (1.01-1.45) 0.04 0.04 *Age, systolic BP, TC/HDL-ratio, physical activity, BMI, smoking, DM, alcohol intake, education Koenig et al. (AHA 2003)

30. Unadjusted Adjusted for age Multivariable DM, smoking adjustment* DM, smoking adjustment* *Age, systolic BP, TC/HDL-ratio, physical activity, BMI, smoking, DM, alcohol intake, education smoking, DM, alcohol intake, education 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.06.0Ref.Ref.Ref. CRP ≤ 3 mg/L Lp-PLA 2 < 290.8 ng/mL N=447 CRP > 3 mg/L Lp-PLA 2 < 290.8 ng/mL N=176 CRP ≤ 3 mg/L Lp-PLA 2 ≥ 290.8 ng/mL N=203 CRP > 3 mg/L Lp-PLA 2 ≥ 290.8 ng/mL N=108 Additive Effect of CRP and Lp-PLA 2 in Coronary Risk Prediction: MONICA Hazard Ratio (95% CI) Koenig et al. (AHA 2003)

31. Summary and Conclusions  Lp-PLA 2 was the strongest predictor/biomarker of coronary events, and was independent of traditional and emerging risk factors, including CRP in hyperlipidemic individuals (WOSCOPS)  In particular, in individuals with low LDL-C (<130 mg/dL), levels of Lp-PLA 2 were independently associated with incident CHD in multivariable analysis including CRP (ARIC)  Lp-PLA 2 was predictive of coronary events in a population- based sample of initially healthy middle-aged men with moderately elevated total cholesterol levels during long- term FU of 14 years (MONICA cohort)  In addition to CRP, Lp-PLA 2 appears to be a promising marker of atherosclerotic complications and deserves further study

32. Break Diagnosing Risk: CRP and Lp-PLA 2

33. Case-Control Study: Population and Laboratory Methods Patients with clinically stable CAD aged 40-68 years (n=312) Age- and gender- matched voluntary blood donors (n=479) Lp-PLA 2 plasma levels - ELISA, diaDexus Inc. Inflammatory markers Hemostatic markers Complete lipid profile Khuseyinova et al. (unpublished data)

34. CAD patients (n=312) Controls (n=479) CAD patients (n=312) Controls (n=479) Age (yrs) 57.7  7.4 55.8  7.2 Male (%) 85.674.9 Family status married (%) 85.983.9 School education <10 yr (%) 69.258.5 Daily alcohol consumption (%) 29.528.5 Smoked pack years 20.310.8 Current smoker 9.6 9.614.2 BMI (kg/m 2 ) 27.3  3.6 26.3  3.2 History of high blood pressure (%) 57.720.5 History of diabetes (%) 13.52.7 History of hyperlipidemia (%) 67.320.9 Lp-PLA 2 (ng/mL)* 296.1  122.5 266.0  109.8 C-reactive protein (mg/L) † 1.7 (0.7-3.8) 1.2 (0.5-2.6) Demographic Characteristics of Patients with Coronary Artery Disease (CAD) and Controls *mean  SD; † median and their interquartile ranges BMI = body mass index BMI = body mass index Khuseyinova et al. (submitted)

35. Distribution of Lipid Variables, Markers of Coagulation, Fibrinolysis and Inflammation (I) CAD patientsControlsP-value Lp-PLA 2 [ng/mL]* 296.1  122.5266.0  109.8 <0.0001 Total cholesterol [mmol/L]* 5.05  1.065.27  0.85 0.0002 HDL cholesterol [mmol/L]* 1.09  0.271.33  0.34 0.0001 LDL cholesterol [mmol/L]* 3.10  0.753.19  0.79 0.11 Apolipoprotein A1 [mg/dL]* 129  21.5145  23.0 0.0001 Apolipoprotein A2 [mg/dL]* 45  7.749  15.6 0.0001 Apolipoprotein B [mg/dL]* 107  27.5103  23.6 0.09 Apolipoprotein C2 [mg/dL]* 4.4  2.754.1  2.94 0.10 Apolipoprotein C3 [mg/dL]* 15.6  6.6614.8  4.21 0.11 Apolipoprotein E [mg/dL]* 9.4  4.929.0  2.58 0.91 Lipoprotein (a) [mg/dL] † 14.8 (5.4-47.1)9.7 (3.5-25.3)<0.0001 *mean  SD † median and their interquartile ranges Khuseyinova et al. (submitted)

36. CAD patients Controls P-value Leukocytes [10 3 /µL]* 6.9  1.815.8  1.53 0.0001 C-reactive protein [mg/L] † 1.7 (0.7-3.8)1.2 (0.5-2.6)0.0001 Serum amyloid A [mg/L] † 3.1 (1.9-4.9)2.6 (1.7-4.1)0.002 Interleukin-6 [pg/mL] † 2.20 (1.53-3.95)1.34 (0.92-2.04)0.0001 TNF-α [pg/mL] † 256 (202-359)184 (130-262)0.0001 sICAM-1 [ng/mL]* 518  166488  141 0.009 Fibrinogen [g/L]* 2.82  0.632.52  0.61 0.0001 Plasma viscosity [m Pas]* 1.22  0.071.19  0.05 0.0001 Plasminogen [%]* 113.6  17.4114.1  16.8 0.43 PAI-1 activity [U/mL] † 11.8 (7.4-19.3)8.2 (4.1-13.7)0.0001 D-Dimers [ng/mL] † 11.2 (0-28.9)2.8 (0-15.1)<0.001 Von Willebrand factor [activity %] † 144 (110-181)134 (99-162)0.0001 sCD14 [µg/mL] † 4.07 (3.36-4.81)4.06 (3.38-4.84)0.51 *mean  SD † median and their interquartile ranges Khuseyinova et al. (submitted) Distribution of Lipid Variables, Markers of Coagulation, Fibrinolysis and Inflammation (II)

37. VariablesCasesP-valueControlsP-valueAge-0.0020.960.170.0001 TC0.29<0.00010.100.03 HDL-C-0.150.008-0.050.24 LDL-C0.33<0.00010.140.002 Apo B 0.27<0.00010.100.03 CRP (log) 0.130.020.050.31 ICAM-10.150.0090.0450.33 Plasma viscosity 0.140.010.060.20 D-Dimer (log) 0.110.05-0.070.11 vWF0.080.120.18<0.0001 Plasminogen-0.110.04-0.040.35 Spearman Rank Correlation Coefficients Between Traditional Risk Factors, Lipid Variables, Systemic Inflammatory and Hemostatic Markers, and Lp-PLA 2 No significant effect for BMI, smoking, leukocytes, fibrinogen, IL-6, TNF- , PAI-1 activity, sCD14, Apo A1, Apo A2, Apo C2, Apo C3, Apo E Khuseyinova et al. (submitted)

38. Odds Ratios (OR) of CAD Associated With Lp-PLA 2 Levels After Various Adjustments Lp-PLA 2 Distribution (ng/mL) Lp-PLA 2 Distribution (ng/mL) Q1 Q2 Q1 Q2 (  188.2) (>188.2-249.2 ) (  188.2) (>188.2-249.2 )Q3 (>249.2-323.5 ) Q4 (>323.5 ) Model 1* OR (95 % CI) 1 Ref 0.98 (0.63-1.52) 1.23 (0.80-1.88) 1.61 (1.07-2.44) Model 2 † OR (95 % CI) 1 Ref 0.91 (0.54-1.52) 1.36 (0.84-2.21) 1.72 (1.07-2.76) Model 3 ‡ OR (95 % CI) 1 Ref 0.93 (0.55-1.56) 1.40 (0.85-2.29) 1.84 (1.12-2.99) Model 4 § OR (95 % CI) 1 Ref 1.03 (0.58-1.83) 1.74 (1.01-3.01) 2.04 (1.19-3.48) Model 5 ¶ OR (95 % CI) 1 Ref 0.98 (0.55-1.76) 1.65 (0.94-2.91) 1.84 (1.05-3.12) * Adjustment for age and gender (matching variables) † Adjustment for matching variables and for BMI, smoking status, alcohol intake, school education years, hypertension, diabetes hypertension, diabetes ‡ Model 2 and additional adjustment for TC and HDL cholesterol § Model 3 and additional adjustment for statin intake ¶ Additionally adjusted for CRP, fibrinogen, vWF, sICAM-1, plasma viscosity, plasminogen, D-Dimer Q = quartile Khuseyinova et al. (submitted)

39. Atherogenic Activities of Lyso-PC Endothelial cells Promotes endothelial dysfunction, upregulates adhesion molecules Monocytes Chemoattractant, stimulates cytokine production Macrophages Stimulates proliferation, inhibits migration, cytotoxic T-lymphocytes Chemoattractant, upregulates cytokine & CD40 ligand expression Smooth muscle cells Cytotoxic, upregulates growth factor expression