M.P.M. de Maat

C-reactive protein: A cardiovascular risk factor report on the CRP hot-topic workshop october 1, 1997

On October 1, 1997, a 1 day hot-topic workshop on C-reactive protein (CRP) was organized in Leiden, the Netherlands, aiming at further evaluating the importance of inflammation as a critical mechanism in cardiovascular disease. C-reactive protein (CRP) is an acute phase protein that is associated with risk of cardiovascular events in healthy individuals and patients with angina pectoris. At the workshop, several investigators who are the pioneers in studies on CRP as a cardiovascular risk factor presented the latest data and their views on the topic. The discussions were designed to evaluate where we stand at present, and what should be done in the near future to promote evidence-based diagnostics and evidence-based intervention/ prevention strategies. The discussion focussed on the association between CRP and cardiovascular disease (CVD), a possible causal role of CRP in CVD, the effect of drugs on CRP levels, the role of infectious agents in the relation between CRP and CVD, and the measurement of low levels of CRP. Although many questions remained, the meeting resulted in a better insight into the role of CRP in cardiovascular disease and a basis for further studies in this area.

Insulin resistance and changes in haemostatic variables

Abstract The insulin resistance syndrome is briefly introduced and the relationship with cardio-vascular diseases is summarized. In view of the relationship with cardio-vascular disease the question is addressed as to how and whether insulin resistance translates into changes in haemostasic variables. Earlier studies focused on a possible direct relationship between hyperinsulinaemia and elevations in circulating plasminogen activator inhibitor 1 (PAI-1). Recent data on obese middle aged men and obese patients with type II diabetes indicate however that elevations in PAI-1 are not related directly to insulin levels but to insulin resistance. In accordance, infusions of insulin have not been found to elicit an increase in circulating PAI-1. New data (presented in this manuscript) on a small group of obese middle aged men and obese patients with type II diabetes mellitus show no relationship between insulin resistance and concentrations of circulating fibrinogen, plasminogen, von Willebrand factor, or histidine-rich glycoprotein. In addition, PAI-1 levels were found to correlate with tissue-type plasminogen activator (t-PA) antigen levels, and t-PA antigen with insulin resistance, indicating that the mechanism of effect of increased insulin resistance in haemostasis most likely involves the endothelium which produces both PAW and t-PA. It is concluded that insulin resistance has a rather specific effect on the endothelium involving strongly the haemostasis variables t-PA and PAI-1, but not von Willebrand factor.

Isolation and storage of DNA for population studies

For genetic population studies, human genomic DNA is commonly isolated from peripheral blood. A fast, non-invasive DNA sampling method is developed involving oral samples taken with cotton swabs. In addition various procedures were compared for isolation of DNA from different sources: whole blood or buffy-coats stored at -20°C for 5-10 years or buccal cells collected freshly with the non-invasive method. The differences in these procedures, which do not contain a phenol-extraction, are based on the use of either 1) high concentration ammonium acetate followed by DNA precipitation or 2) high concentration potassium acetate, followed by chloroform extraction and normal DNA precipitation.

The effect of ticlopidine upon plasma fibrinogen levels in patients undergoing suprapubic prostatectomy

The mechanisms of the antithrombotic effect of the platelet aggregation inhibiting agent Ticlopidine might include a decrease of the plasma fibrinogen level. The effect of ticlopidine on increased fibrinogen synthesis following trauma, such as surgery, is however not known. 46 patients who underwent suprapubic prostatectomy were randomized to receive either (group A) Ticlopidine (2 x 250 mg daily) from the second preoperative day until the seventh postoperative day or (group B) placebo up till the day of surgery and further acenocoumarol against post-operatively and observed that the level and in particular the rise of the plasma fibrinogen concentration was not different in the two groups. It is concluded that compared with the standard treatment in group B ticlopidine does not influence trauma-induced fibrinogen increase.

Plasma factor VII levels are determined by polymorphisms in the factor VII gene

Summary Elevated factor VII coagulant activity (FVII:C) is associated with increased risk of cardiovascular disease. FVII:C and FVII protein concentration (FVII:Ag) are influenced by environmental factors such as diet and by genetic factors. We examined the effects of FVII polymorphisms on 1) fasting and non-fasting values of FVII:C, FVII:Ag and activated FVII (FVIIa) and 2) the relation between total triglycerides and FVII measures. Fortyfour males (age 30–60 y) participated in the study, and had blood drawn in the fasting state. A subgroup (n=19) was also studied in the non-fasting state after consumption of two high-fat meals. FVII:C, FVII:Ag, FVIIa and total triglycerides were measured as well as genotypes of the promoter, HVR4 and Msp1 polymorphisms of the FVII gene. There was a marked effect of the promoter and Msp1 polymorphism on fasting FVII:C, FVII:Ag and FVIIa, and of the Msp1 polymorphism on the fasting FVIIa/FVIIag ratio, with about 15–40% lower levels in heterozygotes. The Msp1 polymorphism also had an effect on non-fasting FVII:C. A significant correlation between fasting total triglycerides and FVII measures was only seen in individuals homozygous for the common Msp1 and promoter alleles.

Correlations between plasma levels of fibrin(ogen) derivatives as quantified by different assays based on monoclonal antibodies

New plasma assays for fibrin(ogen) degradation products have become available which are based upon monoclonal antibodies and can be performed in plasma. In this study we have evaluated four of such specific enzyme immuno assays i.e.: for the total of degradation products of fibrin and of fibrinogen (TDP), fibrin degradation products (D-dimer and FbDP) and fibrinogen degradation products (FgDP) in patients suspected of having deep venous thrombosis of the leg (DVT) and patients with cirrhosis of the liver. DVT was assessed by impedance plethysmography (IPG). In each of the (sub) groups of patients, a very good correlation (0.90 less than r less than 0.98) was observed between the actually measured TDP values and the calculated sum of the separately measured FbDP and FgDP levels. Only 2% (5 patients) of the cases showed a discrepancy of more than a factor two between the found TDP values and the calculated sum of the measured FbDP and FgDP levels. About 90% of the fibrin degradation products were crosslinked. FbDP levels correlated well with the FgDP levels (0.72 less than r less than 0.94) and D-dimer levels (0.82 less than r less than 0.91) in both patients with DVT and cirrhotics. In those patients also a good correlation (0.67 less than r less than 0.83) was observed between FgDP and D-dimer levels, but not in patients suspected of having DVT but with a normal IPG test result. Secondary fibrinolysis appeared to be accompanied by fibrinogenolysis.

Deep-vein thrombosis is not associated with the P/S186 polymorphism of histidine-rich glycoprotein

Background: In several studies, higher plasma levels of histidine-rich glycoprotein (HRG) have been observed in patients with venous thrombosis than in healthy subjects. Apart from environmental factors, such as the use of oral contraceptives, the plasma HRG levels are mainly determined genetically. The most important genetic determinant is P/S186 polymorphisms in exon 5 of the HRG gene which is associated with 40% higher HRG levels. In this study we investigated the relationship between the HRG P/S 186 polymorphism and venous thrombosis. Methods and Results: DNA was available from 466 patients and 471 controls of the Leiden Thrombophilia Study (LETS), a population-based case- control study on venous thrombosis. Both in patients and controls, the genotype distribution of the P/S186 polymorphism was not different from that predicted by the Hardy-Weinberg equilibrium. No association between the genotypes of the P/S186 polymorphism and deep-vein thrombosis was found (PS 186 genotype: OR: 0.97 (CI:0.24,1.70) SS 186 genotype: OR: 1.12 (CI:0.21,2.04), PP 186 is the reference category). Conclusion: The results of this study suggest that the HRG P/S 186 polymorphism is not associated with first venous thrombotic events.

Analysis of activation markers of coagulation, fibrinolysis and inflammation in unstable angina by probit transformation

In order to investigate the role of the hemostatic, fibrinolytic and inflammatory system in unstable angina, we assessed the levels of thrombin-antithrombin III (TAT), Prothrombin Fragment 1+2 (F1+2), plasmin-antiplasmin (PAP) and D-Dimer (DD) and of C-reactive protein (CRP) in 51 patients admitted to our CCU with severe unstable angina. Thirty-one pts had a complicated in hospital course (G1), levels of CRP and PAP were significantly higher in this group than in patients with non complicated in-hospital course (G2). Using the probit transformation we assessed the following cut-off point between G1 and G2: 2.5 μg/1 for TAT, 0.9 nmol/ml for F1+2, 13 μg/l for DD, 500 μg/l for PAP, and 3.5 mg/l for CRP. Our study provides further evidence of the importance of markers of inflammation and fibrinolysis in unstable angina, and demonstrates the utility of data analysis by probit transformation.

40. Modulation of plasma fibrinogen levels by ticlopidine in healthy volunteers and patients with stable angina pectoris

Elevated plasma fibrinogen levels represent an increased risk for cardiac events. Ticlopidine is a drug that inhibits the ADP-induced aggregation of blood platelets and it also has been described that ticlopidine can decrease the plasma fibrinogen level in patients with vascular diseases. The mechanism of this decrease has not yet been elucidated and therefore mechanisms that are known to affect fibrinogen levels were studied, viz. the acute phase reaction, total fibrin and fibrinogen degradation (TDP) levels and the fibrinogen G/A4 -gene polymorphism. The fibrinogen lowering effect of ticlopidine was studied in 26 healthy volunteers and in 26 patients with stable angina pectoris in a double blind, randomized cross-over study versus placebo. Functional plasma fibrinogen levels were measured with the Clauss assay and antigen levels with an enzyme immunoassay. C-reactive protein (CRP) and TOP levels were measured with an enzyme immuno assay. In the healthy volunteers the mean (SD) baseline level of functional plasma fibrinogen was 2.35 g/L (SD 0.35) and for fibrinogen antigen this was 2.49 g/L (SD 0.63). The geometrical mean (central 95% range) of CRP as 0.21 mg/L (0.02-2.36) and for TOP this was 0.18 ng/mL (0.03-1.00). After 4 weeks of ticlopidine administration, the functional fibrinogen levels had decreased with 0.20 g/L (9%, p=0.005 using the paired Student t-test) whereas the fibrinogen antigen levels, the CRP and the TOP levels had not significantly changed. In the stable angina pectoris patient the mean (SD) baseline levels of functional plasma fibrinogen were 3.44 g/L (SD 0.61) and of fibrinogen antigen they were 2.60 g/L (SD 0.49). The geometrical mean (central 95% range) of CRP was 1.45 mg/L (0.15-14.44) and for TOP this was 0.28 ng/mL (0.05-1.62). These baseline fibrinogen, CRP and TOP levels were significantly higher than in the volunteer group. After four weeks ticlopidine administration the functional fibrinogen levels had decreased with 0.39 g/L (11%, p<0.005), whereas the fibrinogen antigen, the CRP and the TDP levels had not significantly changed. The functional and antigen levels of fibrinogen, CRP and TDP did not change significantly during the placebo period in the volunteers or the patients. Neither in the volunteers nor in the patients was the effect of ticlopidine on the fibrinogen levels associated with the fibrinogen G/A~455 genotype. Therefore, the fibrinogen lowering effect of ticlopidine is likely to be a modulation of the functionality of the molecule and unlikely to be modulated by the acute phase reaction, TDP-levels or the fibrinogen β-gene polymorphisms.

Modulation of plasma fibrinogen levels by medication

Elevated plasma fibrinogen levels represent an increased risk for cardiac events. This has enhanced the interest in identifying agents that can normalize elevated plasma fibrinogen levels. Agents that have this capacity are the lipid lowering fibric acid derivatives (e.g. ciprofibrate) and the platelet aggregation inhibitor ticlopidine. Fibrinogen is a very heterogenous molecule that has a number of different functions. Fibrinogen is an important protein in the coagulation process. Besides that, the concentration of fibrinogen is the main determinant of the plasma viscosity. The functionality of fibrinogen is partly determined by the molecular weight (MW) form of fibrinogen: the high MW (HMW) form clots faster than the low and low (LMW and LMW) forms. The mechanism of the fibrinogen decrease by fibrates and ticlopidine has not yet been elucidated and, therefore, we studied the effects of treatment with these drugs on functional fibrinogen, fibrinogen antigen and (HMW+LMW) levels. The effect of ticlopidine was studied in 26 healthy volunteers and in 26 patients with stable angina pectoris. The effect of ciprofibrate was studied in 51 hypercholesterolemic patients. In the healthy volunteers, the mean (SD) baseline level of functional fibrinogen was 2.35 g/L (SD 0.35), for fibrinogen antigen this was 2.49 g/L (SD 0.63), and for (HMW+LMW) fibrinogen 1.97 g/L (SD 0.53). After 4 weeks of ticlopidine administration, the functional fibrinogen level had decreased with 0.20 g/L (9%, p=0.005 using the paired Student t-tesl), whereas the fibrinogen antigen levels had not significantly changed. In the stable angina patients, the mean (SD) baseline levels of functional plasma fibrinogen was 3.44 g/L (SD 0.61), fibrinogen antigen 2.60 g/L (SD 0.49) and for (HMW+LMW) fibrinogen 2.70 g/L (SD 0.60). These baseline fibrinogen levels were significantly higher than in the volunteer group. After four weeks ticlopidine administration the functional fibrinogen levels had decreased with 0.39 g/L (11%, p<0.005), whereas the fibrinogen antigen and (HMW+LMW) fibrinogen levels had not significantly changed. In the hypercholesterolemic group, the mean (SD) baseline level of functional fibrinogen was 3.38 g/L (SD0.61), for (HMW+LMW) fibrinogen this was 3.04 (SD 0.80). After 12 weeks of treatment with ciprofibrate, the functional fibrinogen levels were significantly decreased to 2.42 g/L (SD 0.42) (p<0.0005), whereas the (HMW+LMW) fibrinogen levels had not significantly changed. Therefore, the fibrinogen lowering effect of ticlopidine and ciprofibrate are likely to be a modulation of the functionality of the molecule.