Author + information
- Received December 3, 1999
- Revision received April 28, 2000
- Accepted June 26, 2000
- Published online November 1, 2000.
- Przemyslaw M Mrozikiewicz, MD∗,
- Ingolf Cascorbi, MD, PhD∗,
- Sabine Ziemer, MD†,
- Michael Laule, MD‡,
- Christian Meisel, MD∗,
- Verena Stangl, MD‡,
- Wolfgang Rutsch, MD‡,
- Klaus Wernecke, PhD§,
- Gert Baumann, MD‡,
- Ivar Roots, MD∗ and
- Karl Stangl, MD‡,* ()
- ↵*Reprint requests and correspondence: Dr. Karl Stangl, Medizinische Klinik, Kardiologie, Angiologie, Pneumologie, Charité, Campus Mitte, Humboldt Universität zu Berlin, Schumannstrasse 20/21, D-10117 Berlin, Germany
We have focused on the role of coagulation factor VII (FVII) Arg353Gln polymorphism as a risk predictor of complications following percutaneous transluminal coronary angioplasty (PTCA), directional coronary atherectomy (DCA), and stenting.
The FVII Arg353Gln mutation decreases FVII activity, and presence of the Gln353 allele could be protective against thrombus formation during catheter interventions.
A total of 666 consecutive patients with coronary artery disease who had undergone PTCA (n = 280), DCA (n = 104), or stenting (n = 282) were followed up for a 30-day composite end point, which included need for target vessel revascularization, myocardial infarction, and death. The Arg353Gln polymorphism of FVII was determined by PCR/RFLP assay.
Carriers of the Gln353 allele had significantly lower levels of total FVII activity (FVIIc, −20.7%, p < 0.001) and of activated circulating FVII (FVIIa, −32.7%, p = 0.03) compared with Arg353/Arg353. The composite end point occurred in 43 patients: 4 were heterozygous Arg353/Gln353, and 39 were homozygous Arg353/Arg353. The incidence of the composite end point was 2.5% in carriers of the Gln353 allele and 7.7% in Arg353/Arg353 homozygotes (p = 0.013). This corresponds to a 72% risk reduction in carriers of the Gln353 allele (relative risk: 0.28; 95% confidence interval: 0.09–0.81; p = 0.02).
The Gln353 allele of FVII is associated with substantial risk reduction in adverse events that complicate coronary catheter interventions. With the perspective of active site-blocked activated FVII (FVIIai) as conjunctive medication, the results suggest that the FVII genotype should be taken into due consideration in assessment of FVIIai medication and of its dosage.
There is emerging evidence that the extrinsic coagulation pathway plays a major role in the pathophysiology of intravascular thrombus formation after tissue injury (1,2). This pathway is initiated when tissue factor (TF), a membrane-bound glycoprotein receptor, is exposed to flowing blood (3,4). Circulating factor VII (FVII), a single-chain zymogen, is the only known TF ligand (3). Binding to TF leads to an enhanced cleavage of FVII at Arg152 resulting in formation of activated FVII (FVIIa), a two-chain serine protease, which activates coagulation factors X and IX, ultimately leading to thrombin generation (1–5).
Increased FVII activity represents a risk factor for thrombotic events in the course of coronary artery disease (CAD) (6). Several polymorphisms influencing FVII activity have been recently identified. A single G10,976A-point mutation (7) in exon 8 of the FVII gene causes an exchange of arginine to glutamine (Arg353Gln), which in turn results in decreased FVII activity by 20% to 30% (6–9). It has been suggested that the Gln353 allele protects against myocardial infarction (MI) (6). Thus, presence of the Gln353 allele may consequently also be protective in other situations in which thrombus formation is a fundamental pathophysiological mechanism, as it is for adverse events complicating coronary catheter interventions. We, therefore, focused on the role of FVII Arg353Gln polymorphism as a risk predictor of complications following percutaneous transluminal coronary angioplasty (PTCA), directional coronary atherectomy (DCA), and stenting.
Study population and operational definitions
The 666 subjects participating in the present study were consecutive CAD patients receiving coronary catheter interventions at the Charité University Medical Center between October 1995 and January 1997. The design of this study has been described in detail elsewhere (10). Coronary artery disease was defined on the basis of angiographic criteria as stenosis ≥50% in a major coronary artery or major branch, with classification according to the number of affected arteries.
Angiograms were analyzed by quantitative angiography. To ascertain the occurrence of the composite end point (in particular, periprocedural MI), we routinely performed surveillance of post-procedure creatine kinase, MB fraction levels at least twice for all patients. The CK levels greater than twice normal were established as criteria for suspecting MI. In addition, at least one post-procedural electrocardiogram was carried out. In case of MI suspicion on the basis of clinical, enzymatic and/or ECG evidence (11), angiography was again immediately carried out. The final diagnosis of MI was confirmed only in combination with angiographical findings.
The 30-day composite clinical end point encompassed the following: need for target-vessel revascularization (TVR), MI, and death.
Percutaneous transluminal coronary angioplasty was performed by standard techniques. Indications for DCA followed commonly accepted criteria, with target lesions located in the proximal sections of the three major coronary branches. Predilation was used to facilitate passage of the 7F Simpson atherocath. In each pass, 8 to 12 cuts were performed and repeated if necessary. In a manner analogous to the OARS (Optimal Atherectomy Restenosis Study) protocol (12), post-procedural PTCA at low inflation pressures (4 atm) was performed to reduce residual stenosis to a level <20% (i.e., optimal DCA). Elective indications for stent implantation were de novo lesions (lesion length <30 mm; vessel diameters ≥2.75 mm), restenosis, venous-bypass graft lesions, and residual stenosis after PTCA or DCA ≥30%. Bail-out procedures were performed in type-D→F dissections, for flow deterioration of at least 1 thrombolysis in myocardial infarction trial (TIMI) degree after PTCA or DCA, and in cases of acute vessel occlusion (i.e., TIMI flow grades 0 and 1). One coil stent type (Wiktor stent) and four tubular stent types (Palmaz-Schatz, NIR, Multi-link, Micro AVE) were used. The median number of stents/attempted lesion was 2.0 (1.2 to 2.8). Stents were inserted with use of high inflation pressures (>12 atm), and balloons were slightly oversized on the basis of visual estimate of the angiogram.
We administered aspirin and 10,000 U of heparin to all patients before intervention and, as required, repeated doses of 2,500 U heparin to maintain activated clotting time >250 s. Patients undergoing stent deployment received an additional 500 mg of ticlopidine orally after the procedure and for the following four weeks.
All patients provided written consent according to the study protocol approved by the Charité Hospital Ethics Committee.
Genotyping of FVII Arg353Gln, FVII −323 nt 0/10-bp promoter, and glycoprotein IIIa A1/A2 polymorphism; FVII determinations
Genomic DNA was amplified with primers 5′-GGG AGA CTC CCC AAA TAT CAC and 5′-ACG CAG CCT TGG CTT TCT CTC (7). Amplification conditions were 35 cycles for 30 s at 94°C, 30 s at 60°C, and 60 s at 72°C. Amplicons (312 bp) were digested with MspI. Fragments of 206 bp, 67 bp, and 39 bp were detected in presence of the Arg353 allele (G10,976), and 273-bp and 39-bp bands indicated the Gln353 allele (A10,976).
The FVII −323 nt 0/10-bp promoter polymorphism was genotyped according to the procedure described by Marchetti et al. (13). Genotyping of the A1/A2 polymorphism of glycoprotein IIIa (GP IIIa) has been described earlier (10).
For determination of FVIIa and total FVII activity (FVIIc), venous blood samples were collected in tubes with sodium citrate solution between 7 and 8 am, after overnight fasting of at least 10 h. After drawing, the samples were centrifuged for 15 min at a relative centrifugation force of at least 2,500. Total FVII activity was determined with FVII Deficient Plasma (Immuno AG, Vienna, Austria) and thromboplastin (Immunoplastin HIS, Immuno AG, Vienna, Austria) (14). Activated FVII (FVIIa) was determined by a commercial kit (STACLOT VIIa-rTF, Diagnostica Stago, Asnières-sur-Seine, France) (15). Levels of fibrinogen, von Willebrand factor, C-reactive protein C, and plasminogen activator inhibitor were determined by commercial kits.
Frequencies of genotypes were compared by chi-square (χ2) test or the Fisher exact test. Values of FVIIc and FVIIa of different genotypes were compared by Mann-Whitney U-test. In an initial step we made univariate comparison of the genotypes—stratified by device (PTCA, DCA, and stent)—with respect to baseline characteristics, as well as common and procedural risk factors (Table 1). In a second step we developed a logistic regression in which the composite end point was included as dependent variable and the parameters listed in Table 4 were independent variables.
Possible correlations between GPIIIa and FVII genotypes were tested using Pearson’s exact chi-square test. All statistical tests were calculated using SPSS 9.0.
Table 1 depicts demographic and angiographic baseline characteristics for the 666 patients studied according to genotype. The prevalence of common atherogenic and procedural risk factors did not differ between both groups (Table 1). This also held true when both groups were further stratified according to device. Genotype frequencies of Arg353/Arg353 homozygotes, Arg353/Gln353 heterozygotes, and Gln353/Gln353 homozygotes were 76.4% (95% confidence interval [CI], 73.0–79.6%), 22.2% (19.1–25.6%), and 1.4% (0.6%–2.6%), respectively, being in accordance with Hardy-Weinberg equilibrium. The corresponding predicted values were 76.6%, 21.8%, and 1.6%. Allelic frequency of the Gln353 mutation was 12.5% (10.7%–14.4%).
Correlation to FVII −323 nt 0/10-bp promoter and glycoprotein IIIa A1/A2 polymorphism
We further investigated the functionally relevant −323 nt 0/10-bp promoter polymorphism (16) in our intervention group. The genotype frequencies were 0-bp/0-bp 75.7% (predicted: 75.9%), 95% CI: 72.2–78.9%, 0-bp/10-bp 22.9% (predicted: 22.4%), 95% CI: 19.8–26.4%, and 10-bp/10-bp 1.4% (predicted: 1.7%), 95% CI: 0.6%–2.4%. In 96.9% of patients the −323 nt 0/10-bp promoter polymorphism was linked with the Arg353Gln polymorphism, indicating almost complete linkage disequilibrium.
Furthermore, because the glycoprotein IIIa A1/A2 polymorphism has been described as inherited risk factor during stenting (17), we controlled our results for GP IIIa genotypes. In our results we found no significant correlations between FVII and GP IIIa genotypes (p = 0.47).
Arg353Gln polymorphism and FVIIc and FVIIa
To determine FVIIa and FVIIc in a representative sample of the patient population, the total of 666 patients was randomly mixed, and then every second patient, up to number 359, was sampled. Allelic and genotype frequencies of this sample did not differ from those of the total population (data not shown).
As shown in Table 2, FVIIc levels were lower by 20.7% (p < 0.001) and 36.3% (p = 0.04), respectively, lower in heterozygous and homozygous carriers of Gln353, compared with Arg353/Arg353. Similarly, FVIIa was reduced by 30.9% (p = 0.05) and 49.0% (p = 0.12), respectively. In the group reaching the composite end point there were no significant differences in FVIIc and FVIIa levels between homozygous Arg353/Arg353 and carriers of Gln353.
FVII Arg353Gln polymorphism and 30-day composite end point
As Table 3shows, 280 patients underwent PTCA; 104, DCA; and 282, stenting. The composite end point occurred in 43 patients, of which 42 occurred within the first seven days in the hospital. Immediate repeat angiography was performed for these patients. The only case of death was sudden and out-of-hospital, after 14 days.
Of these 43 patients, 4 were heterozygous Arg353/Gln353 and 39 were homozygous Arg353/Arg353. No Gln353/Gln353 homozygote was found in the group with complications; we therefore calculated the combined risk for both Arg353/Gln353 and Gln353/Gln353. The incidence of the composite end point was 2.5% in carriers of the Gln353 allele and 7.7% in Arg353/Arg353 homozygotes (odds ratio [OR], 0.32; 95% CI, 0.08–0.90; p = 0.013). There was also a tendency to Gln353-allele-dependent risk reduction when patients were further subgrouped by device (Table 3). Moreover, comparison for each single end point between carriers of the Gln353 allele and Arg353/Arg353 homozygotes revealed, respectively, significant risk reduction for TVR: 3 (1.9%) versus 27 (5.3%), p = 0.05; trends for MI: 1 (0.6%) versus 11 (2.2%), and death: 0 (0%) versus 1 (0.2%).
To control for potential confounding in the relationship between the Arg353 polymorphism and ischemic complications, we employed logistic regression in the next step, with the composite end point as dependent variable and the risk factors listed in Table 4as independent variables. As shown in Table 4 with a relative risk of 0.28 (95% CI: 0.09–0.81, p = 0.02) for the Arg353 polymorphism, this logistic regression model confirmed the marked risk reduction established in univariate analysis (Table 3). Furthermore, lesion type C proved to be associated with higher risk when compared with types A, B1, and B2, and von Willebrand factor demonstrated a slight association with excess risk (Table 4).
FVII Arg353Gln polymorphism and acute coronary syndromes
In our intervention group the Arg353Gln polymorphism was not associated with protection against acute MI (OR: 1.33; 95% CI: 0.80–2.24; p = 0.28) or unstable angina (OR: 0.89; 95% CI: 0.60–1.32; p = 0.58). We have also examined the question of association in the overall population of the study, with 2,000 cases and controls (10). Here, as well, we determined no associations with the 578 patients with a history of MI (OR: 0.82; 95% CI: 0.61–1.12; p = 0.13), the 91 with acute MI (OR: 1.47; 95% CI: 0.90–2.41; p = 0.14), or the 144 with unstable angina (OR: 1.06; 95% CI: 0.69–1.63; p = 0.74).
The present study provides first evidence that the Gln353 allele of coagulation factor VII is associated with substantial risk reduction (in our findings, by two thirds) following coronary catheter interventions. It was a crucial finding of this study that the risk reduction was fairly consistent across the component end points as well as across the three different types of catheter interventions.
In the large sample size of 666 patients, 43 patients suffered from major complications during the first 30 days and reached the composite end point. This small absolute number of complications may explain why the reduction in risk achieved significance only in the context of our total patient population.
Because we had hypothesized that the Arg353Gln polymorphism associated with a reduction of FVIIa could reduce the vascular events such as thrombosis, we were slightly surprised to find no reduction of risk for acute coronary syndromes or for chronic MI. Conversely, these results of ours did concur with case-control studies with large populations (18) as well as with the results of the prospective Framingham Study (19).
The surprising results for the group as a whole (i.e., the substantial reduction of procedural risk among carriers of the Gln353 allele) must be interpreted with the necessary circumspection. Discussion of the possibility of coincidence in the finding data, or of selection bias in the patient group, is necessary with respect to Gln353 allelic frequency and the complication rate. The following, however, speaks against the possibility of selection bias: the fact that the Gln353 allelic frequency of 12.5% disclosed in our study lies within the reported range for Middle European Caucasians (7,9). Moreover, the prevalence of the composite end point of 6.5% as determined by us corresponds to comparable figures achieved by others with consecutive patients (20). From the standpoint of work being performed today, our DCA frequency of 16% appears to be very high. This level may be justified by considering that our interventions took place basically in 1996, at the beginning of the stent era, and that this percent figure is in line with data published in a comparable registry (20).
During catheter interventions the protective endothelial lining of the arterial wall is disrupted, and such catheter-induced endothelial injury may trigger thrombogenesis. It has been evidenced by our findings (Table 2) that the Gln353 mutation possesses functional importance, suggesting a gene-dose effect that consists in a FVIIc reduction of 21% in heterozygous and of 36% in homozygous Gln353 carriers. An analogous phenomenon could be observed in FVIIa levels, even though the 49% reduction in Gln353 homozygotes did not reach significance. These phenotypical findings accord well with the literature (6–9) and may explain the observed protective effect of the Gln353 allele.
Because a −323 nt 0/10-bp polymorphism in the promoter region has been described as functionally relevant in lowering FVII levels (16), we examined this aspect in our study group. The almost complete disequilibrium between this promoter polymorphism and the Arg353Gln polymorphism determined in our study accords with findings of other groups (21). By contrast, these “within-gene” allelic interactions do not in the clinical context of our study allow differentiation between the effects of each single polymorphism on decreased FVII levels. Instead, we can represent the decreased levels only as cumulative effects.
Although many factors influence FVII concentration and activity, presence of the Gln353 allele is not necessarily associated with lower FVII levels, as demonstrated in the group of Gln353 allele carriers presenting with complications. By contrast to functional variations of FVIIc or FVIIa levels, the FVII genotype is a constant predictor of the lifelong tendency of individual FVII activity.
Our observation (i.e., risk reduction for thrombotic events) is in line with findings by Iacoviello and colleagues (6), who described a protective effect against MI in carriers of the Gln353 allele. By contrast, several studies failed to demonstrate any protection (18,22).
Our results could well have two clinically important implications: first, that FVII polymorphism has an impact on the development of catheter complications and may serve as a risk predictor. Second, with the perspective of active site-blocked activated FVII (FVIIai) (23) as conjunctive medication during catheter interventions, our findings suggest that the FVII genotype should be taken into due consideration in use, dosage, and assessment of FVIIai effectiveness.
This work was supported in part by a grant of the German Federal Ministry of Education, Science, Research, and Technology (No. 01 GG 9845-5).
- coronary artery disease
- directional coronary atherectomy
- factor VII
- activated FVII
- active site-blocked activated FVII
- total FVII activity
- myocardial infarction
- percutaneous transluminal coronary angioplasty
- tissue factor
- thrombolysis in myocardial infarction
- target-vessel revascularization
- Received December 3, 1999.
- Revision received April 28, 2000.
- Accepted June 26, 2000.
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