Effect on Atherogenesis
Observations in Individuais With a Hereditary Bleeding Tendency
A Srdmek, MD, J H C Reiber, PhD, W B J Gemts, MD, F R Rosendaal, MD
Background—Hemostasis affects ischemic cardiovascular disease through its role m formation of occludmg artenal
thrombi Several studies suggest that hemostasis also might play a role m atherogenesis We mvestigated whether mdividuals with an mhented bleedmg tendency are protected agamst development of atherosclerosis
Methods and Results—A total of 76 mdividuals with an mhented bleedmg tendency (hemophiha and von Willebrand
disease) and 142 healthy controls were included m the present study Early atherosclerotic vessel-wall changes were quantified by measurement of mtima-media thickness m the carotid and femoral artenes by B-mode ultrasonography To validate mtima-media thickness measurements, measuiements also were performed in 77 mdividuals with clinically proven atherosclerosis and m 34 healthy, age-matched controls A large difference m mtima-media thickness was found between mdividuals with proven atherosclerosis and healthy controls, in particular for the femoral artery (difference for carotid artery, 0 16 mm, femoral artery, 0 53 mm) Companson between patients with a bleedmg tendency and healthy controls showed only mimmally reduced mtima-media m femoral artery m mdividuals with a bleedmg tendency (adjusted difference, —0078 mm, 95% CI, — 0 1 7 to 0018 mm) Subgroup analysis revealed that m subjects with modeiate to severe hemophiha, vessel walls were thmnest (adjusted difference, — 0 1 0 mm, 95% CI, — 0 2 7 to 0061 mm)
Conclusions—Hypocoagulability caused by hemophiha or von Willebrand disease has at most a hmited effect on
atherogenesis (Circulation. 2001; 104:762-767.)
Key Words: atherosclerosis · coagulation · carotid artenes · femoral artenes · ultrasonics
R
esults of studies concernmg the role of hemostasis in ischemic cardiovascular disease mdicate that hyperco-agulabihty mcreases nsk of ischemic cardiovascular disease, whereas hypocoagulabihty decreases that nsk In many stud-ies, high fibrmogen levels proved to be a nsk factor for myocardial infarction and stroke ' 6 Results for otherclottmg-factor levels, such äs factor VII, are less consistent In these studies, clottmg-factor levels were m the normal or subnormal ränge Patients with a hereditary deficiency of clottmg factors VIII and IX (hemophiha A and B, respec-tively) have considerable protection agamst myocardial m-farctions 7 8 Furthermore, increased nsk of myocardial
mfarc-tions was found among carriers of a common prothrombm vanant (20210 G to A) that is associated with mcreased prothrombm levels 9 10 Overall results of these studies
indi-cate that coagulabihty plays a role in ischemic heart disease, particularly when extremes of coagulabihty are considered
Myocardial infarction is the result of 2 processes slow and chromc development of an atherosclerotic plaque and acute fonnation of an occludmg thrombus at the site of the
plaque " l2 Obviously, hemostasis plays an important role in
formation of occludmg artenal thrombi However, results of a number of studies suggest that coagulabihty also might play a role m atherogenesis Among patients who suffer from severe penpheral artenal disease, hereditary thrombophiha (caused by protem S deficiency) was more prevalent than m the general population 13 Furthermore, expenmental animal
studies showed that pigs with complete deficiency of von Willebrand factor were protected agamst spontaneous devel-opment of atherosclerotic plaques l 4 1 5
In the present study, we exammed whether hypocoagula-bihty protects agamst development of atherosclerotic plaques Early atherosclerotic vessel wall changes were quantified by measurmg mtima-media thickness in superficial artenes by ultrasonography Measurements were performed m patients with a hereditary bleedmg tendency caused by a clottmg-factor deficiency and m healthy controls for companson Individuais with a bleedmg tendency were patients with hemophiha A or B or who suffered from mild von Willebrand disease Because hemophihacs are almost exclusively men,
Rcccivcd Fcbruary 2, 2001, revision rcccivcd June 5, 2001, acccpted June 7, 2001
From the Dcpartmcnts of Climcal Epidemiology ( A S , F R R ) , and Radiology (J H C R ) and the Hemostasis and Thrombosis Research Center (F R R ) , Leiden Umvcrsity Mcdical Center, Leiden, and the Department of Hcmatology (W B J G ), Leyenburg Hospital, The Hague, the Ncthcrlands
Correspondcnce to Prof Dr F R Rosendaal, Department of Climcal Epidemiology, CO-P46, Leiden Umvcrsity Mcdical Center, PO Box 9600, 2300 RC Leiden, Ncthcrlands E-mail f r rosendaal@lumc nl
© 2001 Amcncan Hcart Association, Ine
Circulation is available at http //www.circulationaha org
Ciirolid iirlery Femoral artery Carotid artery Femoral artery
Figure 1. Sohematic visual presentation of the measurement
areas in the carotid (left) and femoral (nght) artenes Areas in the far wall at which measurements of mtima-media thickness were performed are mdicated by a thick gray hne BIF mdicates carotid bifurcation, CCA, common carotid artery, CFA, common femoral artery, DFA, deep femoral artery, and SFA, superficial femoral artery
the present study was restncted to men Established nsk factors of atherogenesis were determmed to adjust for differ-ences among groups
Methods
Study Design
To vahdate the methodology, measurements were performed m 77 men who had undergone coronary bypass surgery and in 34 healthy, age-matched men To determme the role of hypocoagulability m atherogenesis, 76 men with congemtal bleedmg tendency caused by a deficiency of clottmg factors VIII or IX or von Willebrand factor were mcluded All male patients &30 years of age who were treated m the 2 participatmg centers (Leiden and Den Haag) were ehgible and asked to participate The group consisted of 17 patients with heterozygous von Willebrand disease (mean factor VIII antigen, 45 lU/dL, ränge, 18 to 97 lU/dL, mean von Willebrand factor antigen, 36 lU/dL, ränge, 12 to 65 lU/dL) and 34 with mild (>5 lU/dL), 5 with moderate (l to 5 lU/dL), and 20 with severe (<1 lU/dL) hemophilia (52 with hemophüia A and 7 with hemophiha B) For companson, 108 mdividuals were mcluded who had received tem-poranly prophylactic anticoagulant treatment because of an orthope-dic condition such äs surgery or fractures The 34 healthy mdividuals from the vahdation study were added, which resulted m a total of 142 healthy male controls Atthe time of study, none of the controls was usmg anticoagulant therapy The present study was approved by the Medical Ethics Committee of Leiden Umversity Medical Center, and all subjects gave mformed consent
Ultrasound
We used an Aloka SSD 1200 ultrasound machine with a 7 5-MHz linear transducer An elaborate descnption of the technique and image analysis is given elsewhere l6 In bnef, 2 frozen Images of the mtima-media complex in the common carotid artery of both sides (4 images total) were saved on an S-VHS videotape Images were frozen dunng the R phase of the heart cycle by use of an ECG tnggermg module attached to the ultrasound machine Similarly, 4 Images m the caiotid bifurcation and common femoral artery were recorded At a later stage, Images were digitized m a personal Computer and saved on a recordable CD All Images for each mdividual were saved under a smgle, randomly allocated identifica-tion number to ensure matching of the subject's identity dunng later measurements Images were magmfied 4 times to visuahze clearly the mtima-media complex m the far wall äs has been defmed by Pignoh et al 17 In every image, mtima-media thickness of the far wall was measured 6 times at constant mtervals and averaged by use of dedicated Software ls Figure l is a schematic that shows areas m the carotid and femoral artenes at which measurements were performed Mean mtima-media thickness m carotid artery was calculated by averagmg measurements of Images of the common carotid artery and carotid bifurcation Mean thickness in the femoral artery was based on
1 60-1 40 · 1 20 100 080 ·
I
I 16 102 . 0 88 -L- im1
,<1 665 143HtaUtiy Controls CABG Ilcallhy controls (AB(,
Figure 2. Visual companson of mean intima-media thickness m
carotid and femoral artenes between coronary bypass patients and healthy, age-matched controls Mean and 95% confidence mtervals of intima-media thickness (m mm) are mdicated by —
measurements from Images of the common femoral artery Image acquisition and measurements were performed by l mdividual (A §) Measurement of Risk Factors
Information about use of medication, smoking habits, and presence of cardiovascular disease (myocardial mfarction and stroke) before the age of 65 years for first-degree relatives was obtamed by use of a standardized questionnaire Smoking was categonzed äs number of pack-years an mdividual had smoked Body-fat distnbution was determmed by measurement of waist-to-hip ratio For each subject, blood pressure was measured 3 times by use of a Hawksley random-zero mercury sphygmomanometei while the subject was m a supme position and after £20 mmutes of rest From each mdividual, we drew fastmg blood samples (serum and plasma) Fractions for VLDL, LDL, and HOL determmations were obtamed by ultracentnfugation of serum samples Total serum cholesterol, HDL, LDL, VLDL, and tnglycende concentrations were determmed by use of an enzymatic colonmetnc method
Statistical Analysis
Vessel-wall thickness of patients and controls was compared by Student's t lest for contmuous variables and by )? lest for discrete variables, after log-transformation when appropriate Association of all determmants studied with intima-media thickness was quantified by linear regression For umvanate regression analysis, mcludmg blood pressure, we excluded 27 mdividuals who used antihyperten-sive drugs, 7 mdividuals from the regression analysis of lipids were excluded because they used lipid-lowermg drugs Multivanate re-gression analysis was used to correct for differences m exposure to estabhshed nsk factors
Results
Patients with coronary bypasses had substantially thicker vessel walls both m the carotid and femoral artenes than healthy, age-matched controls (Figure 2) For the carotid artery, mean difference was 0 16 mm, for the femoral artery, 053 mm
TABLE 1. General Characteristics of Patients With a Clotting Disorder and Healthy Individuais Variables Mean age y Smoking, pack y Waist-to-hip ratio Blood pressure, mm Hg Systolic Diastolic
Total cholesterol, mmol/L VLDL, mmol/L LDL, mmol/L HOL, mmol/L Triglycendes, mmol/L Positive family history % Cholesterol-lowermg drugs, % Antihypertension drugs, % Hypertension, % Diabetes, % Bleedmg Tendency (n=76) 488 1612 097 1179 766 5 2 090 3 3 0 9 4 1 2 19(25) 4(5) 12(16) 14(18) 3(4) Healthy Controls (n =142) 51 5 1347 097 1190 770 5 4 089 3 3 098 1 1 30(21) 3(2) 15(11) 24(17) 2(1) 95% Cl Of Difference -62-07 -15-32* -0017-0015 -3 0-1 0* -3 37-2 61 -04-02 -1 12-308* -0 2-0 2 -012-0045 -0 20-0 25* -8-16 -2-8 -4-15 -9-12 -2-7
Hypertension was defmed äs systolic blood pressure &90 mm Hg, or use of antihypertension drugs
*After logarrthmic transformation
£160 mm Hg, diastohc blood pressure
For the intima-media thickness m the carotid artery, we observed no difference between healthy controls and patients with a bleeding tendency (Figure 3) Figure 4 shows results for the femoral artery Mean mtima-media was shghtly lower for patients with a bleeding tendency, this effect appeared restncted to those with hemophiha (mean difference after loganthmic transformation, 0 11 mm, 95% CI, -00028 to 022)
In umvanate linear regression analysis, most of the estab-lished nsk factors for atherosclerosis showed a clear relation to intima-media thickness m both artenes (Table 2) Except for HOL, variables showed a more-pronounced effect on thickness in the femoral artery than m the carotid artery This apphed particularly for smokmg (coefficient, 0 022 versus 0 068), total cholesterol (coefficient, 0 035 versus 0 11), LDL (coefficient, 0 035 versus 0 14), and presence of
hypercho-Carotid artery 1 0 0 -ε ~ί 0 9 0 -l - » „ s . •ϊυ £ ϊ; 080· Ü + 07V « 0 70 · | 06V η An ·
Ι
Ο 80 -ρ Ι) Μ V7, J-076 -1-072 -Τ 0861
080 076 071Ϊ
1.1 SO 076 07J 1-0691
086 078 0 7 2 MI wllb hl»d ng lcnd«nc) ModcrNlc Mi Mild wtrr hcnwphll!* hcnraphllll ftndVWDFigure 3. Visual companson of mean intima-media thickness in
carotid artery between healthy mdividuals and subgroups of mdividuals with a bleeding tendency Mean and 95% confidence mtervals of the intima-media thickness (m mm) are indicated by — VWD mdicates von Willebrand disease
lesterolemia (coefficient, 0 070 versus 0 23) Umvanate re-gression analysis of intima-media thickness between controls and mdividuals with a clotting disorder showed results similar to those shown m Figures 3 and 4 no difference m mtima-media thickness of the carotid artery but a shghtly thinner femoral mtima-media m mdividuals with a bleeding tendency (-0091 mm, 95% CI, -020 to 0022 mm), particularly m those with hemophiha (-011, 95% CI, -0 23 to 0016 mm)
Adjustment for estabhshed nsk factors attenuated differ-ences between controls and patients, but intima-media re-mamed thinner among patients with a coagulation disorder compared with controls (Table 3) This occurrence was true especially m the companson between patients suffenng from moderate to severe hemophiha and controls (adjusted differ-ence, -0 10 mm, 95% CI, -0 27 to 0 061)
Discussion
In the present study, we exammed whether hypocoagulabihty results in less atherosclerotic vessel wall changes than normal coagulation As a model for hypocoagulabihty, we selected patients suffermg from a hereditary deficiency of clotting factor VIII or IX or von Willebrand factor We observed no clmically relevant effect of decreased coagulability on atherogenesis
We used B-mode ultrasonography to quantify early athero-sclerotic vessel wall changes In a recent study, we showed that the ultrasound technique used m the present study is accurate and reliable for determmation of mtima-media thick-ness m the carotid and femoral artenes 16 Numerous studies
TABLE 2. Univariate Regression Analysis of Intima-Media Thickness in Carotid and Femoral Arteries
Artery Carotid Variables Age (10 y) Smoking (1 0 pack-y) Waist-to-hip ratio Blood pressure (10 mm Hg) Systolic Diastolic
Total cholesterol (mmol/L) VLDL (mmol/L) LDL (mmol/L) HOL (mmol/L) Tnglycendes (mmol/L) Positive family history Hypertension Hypercholesterolemia All with bleedmg tendency Hemophilia
Moderate to severe hemophilia Mild hemophilia and VWD
VWD Coefficient 010 0022 1 05 0046 0038 0035 0065 0035 -0057 0036 0054 020 0070 -0 0093 -0013 00060 -0017 00043 95% Cl 0087-012 00071-0039 0 63-1 48 0 033-0 060 0014-0061 0011-0059 0015-012 0 0039-0 065 -015-0033 0 0080-0 059 -00064-012 013-026 0015-013 -0 063-0 044 -0 073-0 046 -0 078-0 90 -0 077-0 043 -0 089-0 098 Femoral Coefficient 016 0068 160 0085 0092 011 013 014 0018 0070 011 028 023 -0091 -011 -0097 -0088 -0033 95% Cl 012-019 0035-010 0 67-2 51 0055-011 0042-014 0067-016 0 026-0 23 0 075-0 20 -017-020 0018-012 -0016-024 014-042 012-035 -0 20-0 022 -023-0016 -0 28-0 084 -0 22-0 043 -025-018
VWD mdicates von Willebrand disease Hypertension was defmed äs systolic blood pressure >160 mm Hg, diastohc blood pressure >90 mm Hg, or use of antinypertension drugs Hypercho-lesterolemia was defmed äs total cholesterol >6 or use of cholesterol-lowermg drugs Regression Coefficient determmed for subgroups with a bleedmg tendency mdicates difference in mtima-media thickness (mm) between subgroups and controls (le mtima-media thickness of subgroup minus mtima media thickness of controls)
young mdividuals 19-22 Intima-media thickness m the carotid
and femoral artenes is a good mdicator of presence of atherosclerosis in coronary and other penpheral artenes 23 24
We also found a clear effect of established nsk factors on intima-media thickness Furthermore, we found a large dif-ference m mtima-media thickness between patients with clmically proven coronary atherosclerosis and healthy controls
Numerous studies have been performed about the role of coagulation and clottmg factor levels m ischermc
cardiovas-Femoral artery 1" 09* · ΪΓ 090 ·
IG °"·
ΐ ·*· 0 7S · | 070 · JÜ 065 ntn-Ι
«Μ Ο«3 γ υ > 2 OJjl 078 Ι Τ Τ ° 7* 4- 0 74 0711 1.068 F 0 8 7 ill.S4 06Ϊ -097 • 080 •^ 06ο •064Modcralc and Mild
u»re honopl Illi
hemophila «nd VHD
Figure 4. Visual companson of mean intima-media thickness m
femoral artery between healthy mdividuals and subgroups of indi-viduals with a bleedmg tendency Mean and 95% confidence mter-vals of the intima-media thickness (in mm) are mdicated by —
cular disease In particular, fibrmogen emerged äs a nsk
factor from these studies ' 6 25 However, m these studies,
climcal end pomts of cardiovascular disease such äs myocar-dial mfarction or stroke were used Therefore, these studies do not clanfy whether coagulation plays a role only in artenal thrombosis or also m atherogenesis Several studies used carotid intima-media thickness measurements äs a Surrogate end pomt of ischermc cardiovascular disease Results of these studies show no consistent results with respect to association of clottmg factor levels with early atherosclerotic chang-es 19 26~28 A possible explanation for this inconsistency is that
clottmg-factor levels in mdividuals studied were mamly m the normal to subnormal ränge In the present study, we maximized contrast by including patients with hereditary low clottmg-factor levels Because patients were selected on the basis of a genetic trait with high penetrance, selection and confoundmg are unhkely
Our results suggest that highly decreased clottmg-factor levels may have a small protective effect agamst atheroscle-rosis m the femoral artery and no effect m the carotid artery Previous studies have suggested that different nsk factor patterns exist for the carotid and femoral artenes 29 30 This
TABLE 3. Comparison of Intima-Media Thickness in Carotid and Femoral Arteries of Subgroups of Patients With a Bleeding Disorder to Group of Controls After Multivariate Regression Analysis
Artery
Subgroup
All with bleedmg tendency Hemophilia
Moderate to severe hemophilia Mild hemophilia and VWD
VWD Coefficient 00091 00047 -0016 -0017 0028 Carotid 95% Cl -0 030-0 048 -0040-0049 -0081-0048 -0 077-0 043 -0044-0099 Femoral Coefficient -0078 -0087 -010 -0088 -0068 95% Cl -017-0018 -0 20-0 020 -0 27-0 061 -0 22-0 043 -025-011 VWD mdicates von Willebrand disease Variables adjusted for in the regression analysis were age, smokmg habits, waist-to-hip ratio, systohc blood pressure, total cholesterol VLDL LDL, HOL, tnglycendes, family history of cardiovascular disease, use of cholesterol-lowermg drugs, and use of antihypertension drugs Regression coetficient determmed for subgroups with a bleedmg tendency mdicates adjusted difference m mtima-media thickness (mm) between subgroup and controls
plausible, explanation is that measurements in the femoral artery are more sensitive than those m the carotid artery for detection of early atherosclerotic changes In the companson between patients with proven coronary atherosclerosis and healthy controls, we found a larger difference m mtima-media thickness m the femoral artery than in the carotid artery This finding suggests that minor changes are detected better m the femoral than carotid artery
Our results indicate minor effects on atherosclerosis in individuals with extreme hypocoagulabihty In a recently published study, presence of advanced atherosclerotic lesions m the carotid artery between patients suffermg from hemo-philia A and von Willebrand disease were compared with healthy controls 32 Results suggested considerable protection
m the patients with a clotting-factor deficiency, even in those suffermg from mild von Willebrand disease However, m that study, the selection procedure and a general charactenstic of the study groups (ie, age) were not documented Furthermore, only crude data were presented and no adjustments were performed In expenmental animal studies with pigs, protec-tion agamst atherosclerosis was found in pigs with severe von Willebrand disease, whereas no protection was found in pigs suffermg from the mild type of the disease 1433 These results
are not in disagreement with our results, because we mcluded only subjects with mild von Willebrand disease (albeit m only a small number of patients)
We studied hereditary deficiencies of clottmg factor VIII and IX and von Willebrand factor äs a model for hypocoagu-labihty of blood Deficiencies of these clottmg factor levels will result in less formation of thrombin and fibrin (impaired secondary hemostasis) or less platelet aggregation (impaired pnmary hemostasis) The present model is vahd only for the hypotheses m which mcorporation of mural thrombi in the vessel wall, mitogen, and other atherosclerotic effects of an mcreased thrombin and fibrm production are a part of the pathway to atherosclerosis Some studies suggest that fibnn-ogen also might have an effect on atherfibnn-ogenesis through its effect on the viscosity of blood In recent studies, viscosity of blood emerged äs an important nsk factor for atherosclerotic disease 34 35
In the present study, patients with a bleedmg tendency were treated temporanly to correct the clottmg disorder durmg hemorrhages Some patients received prophylactic treatment to avoid hemorrhages Durmg penods of prophy-laxis and treatment of hemorrhages, the coagulation levels of these patients were mcreased A study concernmg treatment of hemophilia in the Netherlands36 showed that =e50% of
patients who suffered from severe hemophilia (m particular, younger individuals) receive prophylactic treatment Prophy-lactic dosage level is determmed so that mean clotting-factor VIII or IX level will be mcreased by 0 05 to 0 l lU/mL Thus, durmg prophylactic treatment, seventy of hemophilia is "converted" from severe to mild Patients with mild hemo-philia usually receive no prophylactic treatment Durmg bleedmgs, treatment is aimed at a higher mcrease m clottmg factors However, treatment m these cases is given only for a penod of several days The same study showed that patients with moderate or severe hemophilia expenence a bleedmg episode every 3 to 4 weeks, whereas those with mild hemophilia rarely bleed spontaneously Therefore, treatment has only a hmited effect on overall coagulability Even m case of prophylactic treatment, a high contrast remams in hfetime coagulability between individuals with a severe bleedmg disorder and individuals with normal coagulation
In conclusion, results of the present study suggest a mimmal protective effect of extreme hypocoagulabihty on atherogenesis Our results indicate that the protection agamst myocardial mfarctions that has been reported m hemophilia patients probably is pnmanly caused by a decreased tendency to form occludmg artenal thrombi Because we compared early atherosclerotic changes between individuals with a high contrast m coagulability, the protective effect is neghgible m individuals with coagulation in the normal or subnormal ränge
Acknowledgments
The present study was funded by the Netherlands Heart Foundation
(No 93 110) We express our gratitude to all individuals who
participated m this study WethankM Horstmanshoff for secretanal
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