Frits Haverkate

Reduced response to activated protein C is associated with increased risk for cerebrovascular disease

Resistance to activated protein C (APC) is a recently described coagulation abnormality [1] that is associated with increased risk for venous thromboembolism [2, 3]. The balance of procoagulant and anticoagulant factors undoubtedly plays a critical role in determining the risk for coronary [4, 5] and cerebral thromboembolism [6, 7]. Therefore, some studies [8, 9] have suggested that resistance to APC may also be associated with increased risk for arterial disease. Other studies [10-13], however, have not found evidence to support this conclusion. An extremely low response to APC is often caused by the single-base, Arg 506 to Gln mutation of the factor V gene [14, 15]. This mutation affects the site of cleavage of activated factor V by APC, rendering it relatively resistant to inactivation; the resistance, in turn, leads to increased thrombotic tendency. Accordingly, a functional test for response to APC is used to screen for the factor V mutation. Patients with values below an arbitrarily chosen point are considered potential carriers of the mutation. However, a low response to APC can be caused by other factors; not all patients with low values are carriers of the factor V mutation [16]. We studied whether response to APC is associated with arterial disease by comparing levels of response to APC and prevalence of the factor V Leiden mutation in patients with and without a history of stroke, transient ischemic attack, or myocardial infarction. We also examined other determinants of the level of response to APC. Methods Population We did a casecontrol study of participants in the Rotterdam Study, which is a prospective study of 7983 men and women 55 years of age and older. The rationale and design of the Rotterdam Study have been described elsewhere [17]. Between March 1990 and July 1993, all men and women 55 years of age and older living in Ommoord, a district of Rotterdam, the Netherlands, were invited to participate (n = 10 275). The overall response rate was 78%. The study was approved by the ethics committee of Erasmus University, and written informed consent was obtained from all participants. Selection Patients with a history of myocardial infarction (n = 115) were selected if they had an infarction shown by electrocardiography. Selection was made using the diagnostic classification system of the Modular Electrocardiogram Analysis System (MEANS) [18, 19], independent of a history of chest pain. Patients with a definite or probable history of transient ischemic attack (n = 55) were selected if they had a positive medical history of transient ischemic attack. Four screening questions were asked about temporary visual, locomotor, sensory, or speech disturbances; when responses were affirmative, a detailed history of symptoms was obtained. Symptoms were classified by a neurologist as indicating that the patient had definitely, had probably, or had not had a transient ischemic attack, using methods described elsewhere [20]. Patients with a history of stroke (n = 62) were selected by the question, Did you ever suffer from stroke, diagnosed by a physician? Five patients had a history of both transient ischemic attack and stroke. Therefore, 112 patients were classified as having cerebrovascular disease, which was defined as a stroke, a transient ischemic attack, or both. Controls (n = 222) were selected from among those persons with a normal electrocardiogram, an ankle-to-arm systolic pressure ratio greater than 0.9 (the ankle-to-arm systolic pressure ratio is the ratio of the systolic blood pressure at the posterior tibial artery to the systolic blood pressure at the arm), and no arterial disease (that is, no history of myocardial infarction, stroke, or transient ischemic attack) [21]. Controls were matched in 5-year age strata to persons who had had myocardial infarction. Patients using anticoagulant drugs were excluded. Measurements Information on current health status, medical history, drug use, and smoking was obtained by using a questionnaire. We measured height and weight and calculated body mass index. We measured blood pressure at the right upper arm while patients were seated by using a random-zero sphygmomanometer, and we used the average of two measurements obtained on one occasion. The electrocardiogram was coded using the MEANS computerized coding system [18, 19]. The methods we used for blood sampling and storage have been described elsewhere [22]. Blood was collected in tubes containing 0.129 mol/L sodium citrate. Platelet-poor plasma was obtained by two-stage centrifugation: Samples were centrifuged at 1600 g and 4 C for 10 minutes; after the plasma midlayer was carefully transferred, a second centrifugation was done at 10 000 g and 4 C for 10 minutes. Plasma was immediately frozen in liquid nitrogen and stored at 80C for a mean of 2 years. Plasma from 30 healthy volunteers was centrifuged for 30 minutes at 2000 g and 4 C and was pooled to serve as reference plasma for the test of response to APC. The response to APC for the reference plasma was 3.27. The response of the plasma-activated partial thromboplastin time to APC was determined using the Coatest APC resistance test of Chromogenix (kit 0548-51, Molndal, Sweden) and is expressed as the ratio of the activated partial thromboplastin time with the addition of APC to the activated partial thromboplastin time without the addition of APC. Serum total and high-density lipoprotein (HDL) cholesterol levels were measured with an automated enzymatic procedure. Whole blood that was collected and stored at baseline was thawed for DNA extraction. Genotype assay using polymerase chain reaction was done by laboratory personnel who were blinded to case or control status. The Arg 506 to Gln mutation was detected by amplification of a 220-base pair fragment of exon 10-intron 10 of the factor V gene, followed by digestion with the restriction enzyme Mnl I. The primers and conditions that we used have been described elsewhere [14, 23]. Statistical Analysis We calculated means and proportions for potential determinants for five categories of response to APC and adjusted for a history of myocardial infarction or cerebrovascular disease (two dummy variables in the regression model) using linear regression analysis. Logistic regression was used to assess the association of response to APC and the factor V Leiden mutation with cerebrovascular disease and myocardial infarction. Odds ratios with corresponding 95% CIs estimated from the logistic model were used as the measure of association. With myocardial infarction or cerebrovascular disease as the outcome variable, we compared levels of response to APC and genotypes of the factor V mutation adjusted for age and sex. By adding current smoking, total cholesterol level, and activated partial thromboplastin time as covariates in the logistic regression model, we evaluated whether these potentially confounding factors affected the estimates of the odds ratios. In addition, logistic regression was used to explore the association of disease status with response to APC as a continuous variable. Information on factor V mutation was missing for five participants for whom no blood cells were available. In the regression models with factor V as a confounder, the indicator method for missing data was used [24]. The results were similar to analyses done without these participants. Results Response to Activated Protein C The response to APC ranged from 1.5 to 9.5. Mean responses (SD) were 4.3 1.1 among controls, 3.9 1.0 among patients with a history of cerebrovascular disease, and 4.3 1.1 among patients with a history of myocardial infarction. Mean response to APC was 2.5 0.6 in participants with the factor V mutation and 4.3 1.0 in those without the mutation. The response to APC was higher in men (n = 202; response, 4.5 [CI, 4.4 to 4.7]) than in women (n = 247; response, 3.9 [CI, 3.8 to 4.1]). Several cardiovascular risk factors were compared across five levels of response for men and women (Table 1). In men, response to APC decreased with increasing age by 0.18 (CI, 0.01 to 0.35) per decade. In women, a trend of 0.08 (CI, 0.06 to 0.23) per decade was seen toward an increase in response to APC with advancing age. Men who smoked had a mean response that was 0.47 (CI, 0.16 to 0.78) higher than that of men who did not smoke. Response to APC of women who smoked did not differ from that of women who did not. Increased cholesterol levels were associated with a decreased response to APC in men but not in women. In men, an increase in cholesterol level of 1 mmol/L was associated with a decrease in response to APC of 0.14 (CI, 0.01 to 0.27). Table 1. Cardiovascular Risk Factors in Response to Activated Protein C* The odds ratio of cerebrovascular disease (stroke and transient ischemic attack) increased gradually with decreasing response to APC (odds ratio per 1-unit decrease, 1.43 [CI, 1.12 to 1.81]) after adjustment for age and sex. Separate analyses for stroke (odds ratio, 1.32 [CI, 0.99 to 1.77]) and transient ischemic attack (odds ratio, 1.56 [CI, 1.14 to 2.14]) showed no material difference in their relation to decreasing response to APC. The odds ratios for cerebrovascular disease according to varying levels of response to APC are presented in Table 2. Adjustment for presence of the factor V mutant allele did not substantially change the results; the adjusted odds ratio of cerebrovascular disease for each 1-unit decrease in response to APC was 1.43 (CI, 1.12 to 1.81). Table 2. Prevalence of Cerebrovascular Disease and Myocardial Infarction by Levels of Response to Activated Protein C* Response to APC was not associated with myocardial infarction; the odds ratio for response to APC as a continuous variable was 1.10 (CI, 0.89 to 1.37) (Table 2). Factor V Mutation Heterozygosity for the factor V mutation was present in 5% of controls (11 of 222), 6% of patients with cerebrovascular disease (6 of 107), and 4% of patients with myocar

Tissue plasminogen activator and risk of myocardial infarction : The Rotterdam study

BACKGROUND Impaired fibrinolytic capacity, as assessed by euglobulin clot lysis time or plasma concentration of fibrinolytic parameters, has been associated with an increased risk of myocardial infarction (MI). We studied the association of a polymorphism in the gene for TPA and of plasma concentrations of TPA (antigen and activity) with the prevalence of MI. METHODS AND RESULTS A case-control study was performed. Subjects with a history of MI (n = 121) and controls (n = 250) were drawn from the Rotterdam Study, a population-based cohort study of 7983 subjects > or = 55 years old. We determined TPA antigen and activity in plasma and genotyped all subjects for the Alu repeat insertion/deletion polymorphism in intron h in the TPA gene. Homozygosity for the insertion was associated with twice as many cases of MI as was homozygosity for the deletion (odds ratio, 2.24; 95% CI, 1.11-4.50). TPA antigen was positively associated with the risk of MI; compared with that in the lowest quartile, the relative risks (odds ratio) in the second, third, and upper quartiles were 1.7 (CI, 0.9-3.3), 2.3 (1.2-4.4), and 2.0 (1.0-3.8), respectively. When adjusted for body mass index, HDL and total cholesterol, systolic and diastolic blood pressures, and current smoking, the risk associated with TPA antigen concentration was attenuated. Increased concentrations of TPA activity tended to be associated with an increased risk of MI. CONCLUSIONS This study provides evidence for an independent association of the insertion allele of the insertion/deletion polymorphism in the TPA gene with nonfatal MI. Increased TPA antigen is associated with an increased risk of MI; however, this association was not independent of cardiovascular disease risk factors.

Elevated Plasma Fibrinogen Cause or Consequence of Cardiovascular Disease

An association between increased plasma fibrinogen and an increased risk for myocardial infarction (MI) is well established, but the nature of this association is subject to debate. Our aim was to shed light on the potentially causal nature of this association. We examined whether increased plasma fibrinogen, due to a condition that is independent of cardiovascular events, also increases the risk for MI. A case-control study was performed in 139 subjects with a history of MI and 287 control subjects selected from the Rotterdam Study, a population-based cohort of 7983 subjects aged 55 years and older. The genotype of the -455G/A polymorphism in the fibrinogen beta-gene was determined by polymerase chain reaction. Functional plasma fibrinogen levels were determined according to von Clauss. The plasma level of fibrinogen was significantly higher in subjects with one or two A alleles compared with subjects with the GG genotype: 3.8 (95% confidence interval [CI], 3.6 to 3.9) g/L and 3.6 (3.5 to 3.7) g/L, respectively. With increasing plasma fibrinogen level, the risk for MI increased gradually; a rise in fibrinogen of 1 g/L was associated with a 45% increased risk (odds ratio adjusted for age, sex, and smoking, 1.45; 95% CI, 1.12 to 1.88). There was no association between the genotype of the -455G/A polymorphism and the risk for MI. The -455G/A polymorphism is therefore associated with increased plasma fibrinogen levels but not with an increased risk for MI. These findings indicate that an increased plasma fibrinogen level due to this genetic factor does not increase the risk for MI.

A model for the harmonisation of test results of different quantitative D-dimer methods.

The numerical test results of different D-dimer assays vary widely. Because of the complexity of the analyte of target as well as the variability in specificity of different D-dimer assays, only harmonisation of the test results seems to be feasible. The use of a single conversion factor does not take into account for several methods the lack of commutability between test results and consensus values at different D-dimer levels. This is probably related to the mutually different response of methods to high and low levels. We therefore designed a harmonisation model based on the transformation of a method-specific regression line to a reference regression line. We used the data for the measurement of a set of plasma samples with different D-dimer levels by 353 different laboratories using 7 of the most frequently used quantitative D-dimer methods. For each method we calculated the method-specific consensus value for each sample. The overall median value was also estimated. Per method linear regression was applied throughout the method-specific consensus values using the amount of patient pooled plasma added to the different plasma samples as the independent variable. The line through the overall median values of all 7 methods was used as the reference line. Harmonisation between the methods was obtained by transformation of the method-specific regression line to the reference line. This harmonisation resulted in a reduction of the variability between the method-specific consensus values from about 75% to about 5.5%. Clinical validation of this concept had shown significant improvement of the test result comparability. We conclude that this model is a feasible approach in the harmonisation of D-dimer methods. If the harmonisation procedure is included in the calibration procedure by the manufacturers, customers will automatically obtain harmonised test results.

The Dutch Vascular Factors in Dementia Study: rationale and design

Abstract Dementia is a rapidly increasing health problem in the industrialized countries. With the ageing of the population the number of demented persons increases both in relative and absolute terms. Obviously, there is a need for prevention and intervention strategies. We describe the methods and baseline findings of a large study aimed at identifying potentially modifiable vascular, thrombogenic, and metabolic determinants of dementia. The study population consists of subjects 55 years of age or older. Since the vascular wall of the cerebral vessels is different from that of the coronary or peripheral vessels, we formed three subgroups in which vascular risk factors for dementia are studied. Subjects with stroke were distinguished from subjects with coronary or peripheral artery disease, and from subjects without stroke or coronary or peripheral artery disease. To obtain a large enough number of subjects with stroke, cases and controls from a stroke registry were combined with cases and controls of a population-based study from the same region. For the diagnosis of dementia the DSM-III-R criteria were used. Extensive information on cardiovascular risk factors was collected, including indicators of atherosclerosis. Blood and urine were sampled to study platelet function and thrombogenic and metabolic factors. The study population consists of 7,466 subjects, of whom 300 were recruited from a hospital-based stroke registry. Coronary or peripheral artery disease was present in 956 subjects and stroke in 617. Dementia was present in 434 (5.8%) of all subjects. The prevalence of dementia was 3.0, 24.0, and 4.4% in subjects with a history of coronary or peripheral artery disease, a history of stroke, and subjects without a history of coronary or peripheral artery disease or stroke, respectively. The study will allow us to investigate the role of vascular factors in dementia, irrespective of its cause.

The long-term within- and between-laboratory variability for assay of antithrombin, and proteins C and S: results derived from the external quality assessment program for thrombophilia screening of the ECAT Foundation.

Summary.  A stable laboratory performance is important for comparability and transferability of laboratory data both within and between laboratories. The lack of a reference system within hemostasis hampers laboratories in establishing their laboratory performance over a prolonged period of time. Therefore, based on data from an external quality assessment program, we evaluated the between laboratory variation (CVBETWEEN) and the long‐term within‐laboratory variation (LCVa) for antithrombin, and proteins C and S. We evaluated the CVBETWEEN for the period 1996–2001, including the results of 64–240 laboratories from 23 different surveys (protein S activity 15 surveys). We observed a quite high CVBETWEEN and a broad range for each analyte. The CVBETWEEN was significantly higher for antithrombin and protein S for samples with low levels similar to heterozygous deficiencies. We also evaluated the LCVa, including the results of 136 laboratories. The lowest LCVa[median and 95% content interval (CI)] was observed for antithrombin (7.6%; 3.6–35.5%), intermediate values for protein C activity and antigen (8.6%; 3.5–25.3% and 10.8%; 4.8–33.1%, respectively) and highest values for the protein S variables (13.4%; 6.4–50.6% for total protein S antigen, 14.1%; 6.5–79.1% for free protein S antigen and 17.2%; 7.2–84.3% for protein S activity). We concluded that the main reason for the high CVBETWEEN is the long‐term within‐laboratory variability. Application of linear regression on data of an external quality assessment program is a useful model to demonstrate per analyte per laboratory the long‐term variability (LCVa). It is concluded that improvement of the long‐term within‐laboratory test performance is the first priority in hemostasis to yield important improvements in the comparability and transferability of laboratory data.

The performance of quantitative D-dimer assays in laboratory routine

Assessment of D-dimer in plasma is routinely used for the exclusion of venous thrombosis and the monitoring of hypercoagulability. Little information is available about the performance of D-dimer assays in clinical laboratories examined by external quality assessment schemes. We obtained results from 423 laboratories measuring plasma pools from patients with elevated D-dimer levels mixed with human normal plasma. The results from five samples were reported containing D-dimer from the lower normal range up to a 20-fold increased level. In addition, information about the assignment of a cut-off point and the medical need to apply these assays was obtained by standardized questionnaire. Participants reported results and additional information from 20 different assays. Lack of standardization regarding the calibration concepts obstructs comparability of results. Results in one sample varied up to 20-fold between the assays applied. In addition, a high variability was reported around the cut-off values introduced for the exclusion of venous thrombosis and pulmonary embolism. As a consequence, generally accepted cut-off values cannot be established. For cut-off assignment, 62% of participants used the kit insert but also 14% used local validation. In conclusion, standardization or at least harmonization of D-dimer assays is necessary to ensure comparability of D-dimer plasma levels measured in clinical routine.

Coagulation and Fibrinolysis Markers and Risk of Dementia

We performed a cross-sectional case-control study among 277 subjects with dementia and 298 control subjects drawn from participants of the Rotterdam Study, a population-based cohort study among subjects aged 55 years or over, and from participants of the Rotterdam Stroke Databank, a hospital-based stroke registry, with the objective to evaluate the association of indicators of coagulability, fibrinogen, prothrombin fragments 1+2, thrombin-antithrombin complex (TAT), and indicators of fibrinolysis, plasmin-inhibitor complex, D-dimer and tissue-type plasminogen activator (t-PA) with dementia. Increased levels of TAT, D-dimer and t-PA activity were associated with an increased risk of dementia. Additional stratified analyses indicated that an increased TAT level was the primary factor related to dementia. The present study provides evidence that predominantly increased thrombin generation is associated with dementia.

Performance goals for the laboratory testing of antithrombin, protein C and protein S.

To achieve a reliable analytical quality for both monitoring and diagnostic testing, laboratories need to fulfil the widely accepted analytical performance goals based on the biological variation of the analytes of testing. Not only is the short-term analytical performance, which regularly is assessed by internal quality control procedures, of importance, but also the long-term analytical performance. To assess the long-term analytical performance, data obtained from an external quality assessment programme can be used. In this study we have used the evaluation model designed by the ECAT Foundation for the assessment of the longterm analytical performance, including imprecision, bias and total analytical error. The model was applied to the data from 136 different laboratories for the assay of antithrombin (activity), protein C (activity and antigen) and protein S (activity, total and free antigen). The imprecision (median; range), reflected by the long-term analytical coefficient of variation (LCV (A) ), was the lowest for antithrombin (7.6%; 2.6 - 43.8%) and the highest for protein S activity (17.2%; 4.3 - 88.6%). For bias and total error the same pattern was observed (antithrombin: 3.8%; 0.3 - 17.1% and 9.1%; 3.4 - 34.3%, respectively; protein S activity: 12.8%; 3.1 - 34.8% and 24.5%; 9.9 - 87.0%, respectively). For the majority of the laboratories (70 - 85%) the imprecision contributes considerably more to the total error than the bias. However the effect of the bias on the analytical quality is not negligible. Assays for antithrombin, protein C and protein S are mainly used for diagnostic testing. About 70 - 100% of the laboratories can fulfil the desirable performance goal for imprecision. The desirable performance goal for bias was reached by 50 - 95% of the laboratories. In all cases the highest numbers of laboratories fulfilling performance goals was obtained for the protein C variables. To improve the analytical quality in assays of antithrombin, protein C and protein S it is highly recommended that primarily imprecision (non-systematic failures) be suppressed. However the effect of the bias (systematic failures) on the analytical quality should not be neglected. A useful tool for determining the imprecision (LCV (A) ) and bias is the long-term analytical performance evaluation model as used by the ECAT Foundation.

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.