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Genome-Wide Association Study for Circulating Tissue Plasminogen Activator Levels and Functional Follow-Up Implicates Endothelial STXBP5 and STX2

and the Cohorts for Heart and Aging Research in Genome Epidemiology (CHARGE) Consortium Neurology Working Group
and the CARDIoGRAM Consortium
and the CHARGE Consortium Hemostatic Factor Working Group
Originally publishedhttps://doi.org/10.1161/ATVBAHA.113.302088Arteriosclerosis, Thrombosis, and Vascular Biology. 2014;34:1093–1101

Abstract

Objective—

Tissue plasminogen activator (tPA), a serine protease, catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for endogenous fibrinolysis. In some populations, elevated plasma levels of tPA have been associated with myocardial infarction and other cardiovascular diseases. We conducted a meta-analysis of genome-wide association studies to identify novel correlates of circulating levels of tPA.

Approach and Results—

Fourteen cohort studies with tPA measures (N=26 929) contributed to the meta-analysis. Three loci were significantly associated with circulating tPA levels (P<5.0×10−8). The first locus is on 6q24.3, with the lead single nucleotide polymorphism (SNP; rs9399599; P=2.9×10−14) within STXBP5. The second locus is on 8p11.21. The lead SNP (rs3136739; P=1.3×10−9) is intronic to POLB and <200 kb away from the tPA encoding the gene PLAT. We identified a nonsynonymous SNP (rs2020921) in modest linkage disequilibrium with rs3136739 (r2=0.50) within exon 5 of PLAT (P=2.0×10−8). The third locus is on 12q24.33, with the lead SNP (rs7301826; P=1.0×10−9) within intron 7 of STX2. We further found evidence for the association of lead SNPs in STXBP5 and STX2 with expression levels of the respective transcripts. In in vitro cell studies, silencing STXBP5 decreased the release of tPA from vascular endothelial cells, whereas silencing STX2 increased the tPA release. Through an in silico lookup, we found no associations of the 3 lead SNPs with coronary artery disease or stroke.

Conclusions—

We identified 3 loci associated with circulating tPA levels, the PLAT region, STXBP5, and STX2. Our functional studies implicate a novel role for STXBP5 and STX2 in regulating tPA release.

Introduction

Tissue plasminogen activator (tPA) is a glycoprotein produced mainly by vascular endothelial cells that catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for endogenous fibrinolysis and an important regulatory element in thrombosis. Circulating tPA is implicated in the progression and incidence of clinically apparent atherothrombotic cardiovascular diseases (CVDs), such as myocardial infarction and stroke, and is associated, in some studies, with advanced atherosclerosis.111 Recombinant tPA is approved for use in patients with acute myocardial infarction and is the only drug approved by the US Food and Drug Administration for the treatment of acute ischemic stroke.5,7

See accompanying editorial on page 964

The estimated heritability for circulating tPA level is as high as 0.67, based on family and twin studies, providing substantial evidence of genetic influences on circulating levels.1214 Little is known about genetic predictors of circulating tPA. Several genetic polymorphisms within the PLAT gene locus have been identified, including the well-studied 311 bp Alu-repeat insertion/deletion polymorphism (rs4646972).15 In some cohorts, this Alu-repeat polymorphism has been associated with levels of circulating tPA and with CVD risk, although this finding was not seen in all cohorts studied.4,16,17 Circulating levels of tPA are also associated with common polymorphisms in genes contained in the renin–angiotensin and bradykinin systems.18,19

To date, no genome-wide association study (GWAS) on this circulating biomarker has been reported. We conducted a meta-analysis of 14 studies that had both tPA measurement and genome-wide genotype data to identify common variants that are associated with variations in the circulating levels of tPA antigen. Our study included a total of 26 929 participants who were enrolled in 14 cohorts of European ancestry with genome-wide markers. For replication, we evaluated lead single nucleotide polymorphisms (SNPs) in an independent sample. We sought evidence for the biological function for lead SNPs within each locus, using human gene expression databases and RNA silencing studies in endothelial cells. We further sought to identify evidence for a role, if any, of associated genetic variants with thrombosis-related clinical end points, including apparent coronary artery disease (CAD) and stroke.

Materials and Methods

Materials and Methods are available in the online-only Supplement.

Results

Cohort Characteristics

The characteristics of a total of 26 929 participants in the 14 discovery cohorts are summarized in Table I in the online-only Data Supplement. The age ranged from 45.2 to 76.7 years. The percentage of men ranged from 38.5% to 75.3%, except for the largely female Twins UK, in which men comprised 4.8%. The body mass index was similar across the cohorts, with a range of 26.1 to 27.9 kg/m2. The mean tPA level ranged from 5.06 to 11.01 ng/mL.

Results of Primary GWAS

The P values of our discovery meta-analysis for the 2 455 857 meta-analyzed SNPs are presented in Figure 1. A total of 3 loci reached the genome-wide significance threshold of 5×10−8 (Table 1). For the first locus, we identified multiple SNPs (n=61) of genome-wide significance in the 6q24.3 region that harbors the STXBP5 gene.20 The SNP rs9399599 (within intron 26 of STXBP5) has the smallest P value of 2.9×10−14. Allele T (frequency, 0.54) is the risk allele, with an effect size (SE) of 0.032 (0.004). Because the trait was natural logarithm–transformed, this translates to an increase of 1.033 ng/mL of tPA per copy of the risk allele. The regional plot demonstrates that all significant SNPs in the region are in high linkage disequilibrium (LD) with the lead SNP (Figure I in the online-only Data Supplement; plot A). The second locus includes 7 SNPs reaching the genome-wide significance threshold; 6 of these lie within POLB, whereas 1 lies within PLAT, the gene that encodes tPA. The lead SNP (rs3136739; P=1.3×10−9) resides within intron 3 of POLB. The SNP within PLAT is a nonsynonymous SNP (rs2020921; P=2.0×10−8) within exon 5 of PLAT with the minor allele causing a tryptophan to be substituted for an arginine. Based on the 1000 Genomes Project European data, these 2 SNPs are in LD (r2=0.5). After reanalysis of chromosome 8 conditioning on rs3136739, rs2020921 had a P value of 2.1×10−4 and was the only SNP with P<1×10−3 within the 1.6-Mb region containing these 2 SNPs, suggesting there are 2 separate signals.

Table 1. Association Results for 4 SNPs Within the 3 Significant Loci With Circulating Levels of Tissue Plasminogen Activator (tPA) Antigen

SNPChromosomePositionGeneEffect Allele (Frequency)β, SE (log-transformed)GM ratio* (95% CI)P ValuePHet
rs9399599 (lead SNP)6147744992STXBP5 intron 26T → A (0.54)0.032, 0.0041.032 (1.024–1.041)2.9×10−140.61
rs2020921 (nsSNP)842164122PLAT exon 5G → A (0.95)0.067, 0.0121.069 (1.045–1.095)2.0×10−80.15
rs3136739 (lead SNP)842324237POLB intron 3A → G (0.95)0.063, 0.0101.065 (1.043–1.087)1.3×10−90.33
rs7301826 (lead SNP)12129857054STX2 intron 7C → T (0.43)0.027, 0.0041.027 (1.018–1.036)1.0×10−90.49

CI indicates confidence interval; GM, geometric mean; and SNP, single nucleotide polymorphism.

*Per-allele proportionate increase in geometric mean (GM) PA.

P value for heterogeneity test.

Figure 1.

Figure 1. Manhattan plot showing the association P values for the meta-analyzed single nucleotide polymorphisms in the discovery cohorts. x axis organized by chromosome and base pair positions. y axis shows −log10 of association P values. The horizontal dotted line marks the threshold for genome-wide significance (P=5.0×10−8).

The third genome-wide locus includes a total of 33 SNPs lying within STX2 in the 12q24.33 region. The lead SNP (rs7301826; P=1.0×10−9) resides within intron 7 of STX2. Regional plots for these 3 loci are shown in Figure I in the online-only Data Supplement. Summary statistics of the 3 lead SNPs and the cis-acting SNP within PLAT in each individual GWAS are shown in Table III in the online-only Data Supplement. For all 4 SNPs in these 3 loci, there was no evidence for heterogeneity across studies (P>0.05; Table 1). The individual and combined effect of the 3 top SNPs in explaining phenotypic variance was assessed in the largest contributing study (British 1958 Birth Cohort, B58C). The proportion of variance in log-transformed tPA explained by the top 3 loci combined was 0.75%. This comprised 0.29% variance explained by rs9399599 alone, 0.16% variance explained by rs7301826 alone, and 0.28% variance explained by rs3136739 alone.

To test for replication, genotyping was conducted in 4487 participants from the Prevention of Renal and Vascular End-stage Disease study (PREVEND). In the PREVEND replication cohort, none of the 3 SNPs was associated with tPA (P<0.05). The effect sizes were smaller, −0.001, 0.017, 0.002 compared with 0.032, 0.063, 0.027, respectively, for the 3 lead SNPs within STXBP5, POLB-PLAT, and STX2. After combined meta-analysis of these results with data from the 14 discovery cohorts, the P values for association for the 4 genome-wide associated SNPs (rs9399599, rs2020921, rs3136739, and rs7301826) each remained genome-wide significant (P<5.0×10−8).

Association With Gene Expression

All 3 lead SNPs and their proxies were searched against 3 large expression quantitative trait locus (eQTL) sources as described in Materials and Methods in online-only Data Supplement. eQTL results provided expression association evidence for STXBP5 and STX2, but not for chromosome 8 locus (Table 2). SNP rs7739314 (P<3.1×10−12), located ≈500 bp 3′ of STXBP5, was modestly associated with STXBP5 expression in lymphocytes (P<1.6×10−3), CD4+ lymphocytes (P<1.7×10−4), and liver (P<0.03), though this was not the strongest expression SNP (eSNP) for STXBP5 in these respective tissues. Three perfect proxy SNPs (r2=1.0) for the lead STX2 SNP (rs7301826) were strongly associated with the expression of STX2 in a wide range of blood cells and other tissues. In every case, the strongest eSNP for STX2 was the same or a perfect proxy for the strongest SNP associated with circulating tPA levels, indicating a high degree of concordance between eQTL and association signals. tPA SNPs at STX2 and STXBP5 loci were not significantly associated with the expression of any other genes at those loci.

Table 2. Expression Quantitative Trait Locus (eQTL) for 2 Loci With Proximal Gene Expression in Human Cells and Tissues

Index SNPProxy eSNP* (tPA P; r2)eQTL TissueeQTL PStrongest eSNPStrongest eSNP* (eQTL P; tPA P; r2)
rs9399599 in STXBP5rs7739314 (3.1×10−12; 0.97)CD4+ lymphocytesNArs6946251.4×10−4; 0.02; 0.17
Lymphocytes1.6×10−3rs6207153.5×10−4; 2.4×10−8; 0.90
Liver0.03rs17650287.8×10−6; 4.4×10−6; 0.44
rs7301826 in STX2rs10848205 (1.5×10−6; 1.0)Monocytes3.8×10−274Same as proxy
rs10773819 (1.4×10−6; 1.0)Lymphocytes7.5×10−92Same as proxy
Monocytes1.5×10−80rs11063697.1×10−91; 1.5×10−6; 1.0
Peripheral blood mononucleated cells1.4×10−25
Macrophage1.8×10−24rs11063692.3×10−28; 1.5×10−6; 1.0
CD4+ lymphocytes2.9×10−20Same as proxy
Leukocytes2.7×10−10Same as proxy
Liver9.0×10−6Same as proxy
Mammary artery4.9×10−3Same as proxy
rs2001483 (1.8×10−6; 1.0)Liver2.7×10−5Same as proxy

All sentinel SNPs and their proxies were searched against eQTL sources as described in Materials and Methods in the online-only Data Supplement. eSNP indicates expression single nucleotide polymorphism; and tPA, tissue plasminogen activator.

*Proxy SNP is the measured SNP in highest linkage disequilibrium with index SNP linkage disequilibrium measured in correlation R2, based on HapMap2 CEU data.

Strongest eSNP is the one with the best eQTL P value, for the same tissue and transcript as for the lead proxy eSNP.

Results of Gene Silencing for STXBP5 and STX2 in Human Endothelial Cells

The proteins encoded by STX2 and STXBP5 are expressed in 3 types of vascular endothelial cells (human aortic endothelial cells, human umbilical vein endothelial cells, and human dermal microvascular endothelial cells; Figure 2B–2D). Silencing STX2 and STXBP5 decreased the expression of STX2 and STXBP5 proteins, respectively, in each of the 3 endothelial cell types (Figure 2B–2D). Silencing STXBP5 significantly decreased the release of tPA, whereas silencing STX2 significantly increased tPA release, in both resting and histamine-stimulated vascular endothelial cells (Figure 2A). SNP-specific effects were not evaluated in the current experiments.

Figure 2.

Figure 2. Effect of gene silencing on tissue plasminogen activator (tPA) release. Endothelial cells were transfected with oligonucleotides to silence STXBP5 or STX2 and then treated with histamine to induce tPA release. Levels of tPA in the media were measured by ELISA. A, Silencing STXBP5 decreases tPA release, whereas silencing STX2 increases tPA release. B to D, Silencing STX2 or STXBP5 decreased the target protein expression in human umbilical vein endothelial cells (HUVECs; B), human aortic endothelial cells (HAEC; C), and human dermal microvascular endothelial cells (HDMVEC; D). Each panel includes a 3×3 matrix of Western blot images for the 3 proteins (STX2, STXBP5, β-actin) after 3 gene silencing approaches (siControl for control scrambled oligonucleotide, siSTX2 for siRNA directed against STX2, and siSTXBP5 for siRNA directed against STXBP5).

Association With CAD and Stroke

In a recently updated meta-analysis (based on 13 observational cohort studies and 5494 cases of CAD), a 1-SD increase in tPA antigen, adjusted for conventional cardiovascular risk factors, was associated with an odds ratio of incident CAD of 1.13 (95% confidence interval, 1.06–1.21).11 Because the genetic influences we detected on tPA levels together accounted for <1% of phenotypic variance, and individual SNPs were associated with differences in untransformed tPA levels <0.2 SD, comparing homozygotes to heterozygotes, it is inherently unlikely that any of these variants would impact greatly on CAD risk, and an in silico lookup in previously published GWAS meta-analyses confirms this (Table IV in the online-only Data Supplement). The upper confidence limits in this table exclude clinically or epidemiologically important associations of the 3 top SNPs with CVD, defined as either CAD or stroke.

Findings for Previously Implicated Genes

We examined for evidence of association of SNPs within a 20-kb region of the cis locus, PLAT, as well as SNPs within ACE, AGT, AGTR1, BDKRB2, and SERPINE1.21,22 For a total of 204 SNPs, 32 SNPs within ACE, AGT, BGKRB2, PLAT, and SERPINE1 have a P value <0.05 (Table V in the online-only Data Supplement). However, only 3 SNPs in PLAT (lead rs2020921; P=5.1×10−8) and 5 SNPs in SERPINE1 (lead SNP rs2227667; P=2.2×10−5) remained significant after adjusting for the multiple testing (multiple testing threshold P<2.5×10−4). Given the correlation of SNPs within these 2 loci resulting from residual LD, the associations for each of the 8 SNPs within these 2 loci are robust, extending evidence from previous literature for the existence of a genetic association with the plasma levels of tPA.

Discussion

In a large GWAS of >27 000 research participants of European ancestry, we discovered a total of 3 loci that have not been previously reported to be associated with circulating tPA levels at a genome-wide significant threshold. This is the first GWAS that identifies a nonsynonymous SNP within PLAT that reaches genome-wide significant threshold. eQTL examination provided strong functional evidence for associated SNPs in STXBP5 and STX2, and further studies in human endothelial cells directly implicate these 2 genes in the expression, production, and release of tPA protein.

Previous candidate gene studies have not consistently noted the presence of associations between SNPs in the PLAT gene and circulating levels of tPA, and several studies have found no such association.17,23 The current study substantially extends and strengthens the previous hypothesis of a cis association between SNPs in PLAT and circulating levels of tPA by providing evidence for a strong and genome-wide significant association of SNPs within the PLAT locus. We identify an association with a nonsynonymous SNP rs2020921 within PLAT, suggesting a functional variant, and separately with SNPs in POLB, raising the hypothesis of an independent genetic determination of tPA in this locus. These findings suggest that the cis associations are complex and may have been missed because previous mapping studies focused on mapping a narrow genomic region and were conducted in relatively smaller samples. Little is known about the functional consequences of the nonsynonymous PLAT mutation, and prediction software provides conflicting predictions of its effect (PolyPhen-2: neutral; SIFT: deleterious); therefore, future functional experiments are warranted.

The associations of variants within STXBP5 and STX2 with circulating levels of tPA are novel findings. Syntaxins are members of a family of membrane-integrated SNARE (Soluble NSF Attachment Protein Receptor) proteins that participate in exocytosis.24 Syntaxin 4 plays a role in exocytosis of Weibel–Palade bodies in endothelial cells.25 Our functional studies reveal that STXBP5 and STX2 play a role in the endothelial release of tPA. Our cell culture studies strongly support a role for these 2 genes in regulating endothelial cell tPA expression, production, and release. Although these studies provide novel evidence derived from an unbiased GWAS for the role of STXBP5 and STX2 in the regulation of tPA at the endothelial cell level, further studies are clearly warranted to examine how the manipulation of specific SNPs, rather than silencing the whole gene, affects the dynamics of circulating tPA level at cellular and model organism levels.

SNPs in STXBP5 and STX2 loci were also reported to be associated with the circulating levels of von Willebrand factor (vWF) in a recent study by the Cohorts for Heart and Aging Research in Genome Epidemiology (CHARGE) Consortium.26 Based on the 1000 Genomes Project data, there is moderate to strong correlation of the lead SNP associated with vWF26 and the lead SNP we reported here to be associated with tPA for STXBP5 (rs9390459; r2=0.97; D′=1.0) and for STX2 (rs79789987; r2=0.63; D′=1.0). Although tPA and vWF share associations with common variants at STXBP5 and STX2 loci, these relatively weak genetic associations are not a major explanation for the phenotypic correlation between these hemostatic risk factors. Both plasma components were measured in B58C, and a highly significant (P<10–22) correlation (r=0.13) remained between log-transformed tPA and log-transformed vWF levels, after adjustment for the top SNPs at STXBP5 (rs9399599) and STX2 (rs7301826). The association of identical syntaxin-coding genes with various circulating hemostatic factor levels may provide an opportunity for further investigations on these newly identified mechanisms by which these circulating hemostatic factors are implicated in thrombotic cardiovascular and metabolic diseases.

Our study was motivated, in part, to better understand the mechanism by which endogenous tPA may be implicated in clinically apparent CVD outcomes. Our lead SNP rs9399599 in STXBP5 locus is associated with the circulating levels of vWF26 and with the risk of venous thrombosis.27 Although elevated plasma level of vWF is a predictor of venous thrombosis, available evidence suggests that the level of tPA is not associated with venous thrombosis.28 Another nonsynonymous SNP rs1039084 within STXBP5 has a less significant association with vWF (P=1.0×10−9) compared with the lead SNP in our study, but rs1039084 has a stronger association than rs9399599 has with vWF in a subgroup of coronary heart disease patients.29 For the STX2 locus, the SNP rs7978987 has been previously reported to be associated with vWF levels26 and with an increased risk of arterial thrombosis.29 The P value for the association of this SNP with tPA is 4.5×10−9, similar to that of the lead SNP rs7301826 (P=4.1×10−9). Two other SNPs within STX2 (rs1236 and rs11061158) were also previously reported to be associated with coronary heart disease.29 The former is genome-wide significant and the latter is marginally significant in our study (P=1.9×10−9 and 0.048, respectively). None of the 3 lead SNPs in PLAT/POLB, STXBP5, or STX2 was found to be associated with CAD or stroke based on an in silico examination of results from a large sample for CAD and a moderate-sized sample for stroke. However, the key function of tPA in the coagulation system and the importance of coagulation to the cardiovascular system have been well established, and the novel genes identified in our study merit further study of their potential role as intermediaries in the pathophysiology of atherothrombotic CVD.

The replication of previously reported findings for the associations between genetic variation in SERPINE1 and PLAT with circulating tPA levels was able to act as a positive control for this study and extends evidence for the existence of a genetic association in these genes with the plasma levels of tPA.

We note several potential study limitations. First, samples in our study are of European ancestry; therefore, our findings may not be generalizable to populations of different ethnicity. Second, the replication sample size is quite small, with 80% power to detect an association at a 5% significance level. Although not particularly weak, there was still inadequate power to provide strong evidence for the replication of small effects detected in the discovery study. We, therefore, included biological validation that includes in vitro cell studies. Third, because of the low frequency of lead SNPs within the POLB-PLAT locus, we are not able to use a traditional LD mapping approach to refine the association signals. The lead SNP within POLB (rs3136739) is in perfect LD (r2=1) with the nonsynonymous SNP (rs2020921) within exon 5 of PLAT, based on the HapMap2 genotype data. However, rs3136739 has a missing rate of 10% in the HapMap2 data. Even with the most recent 1000 Genomes Project genotype data, however, it remains difficult to fully characterize the haplotype structure for SNPs with low minor allele frequency (≈5%) based on pair-wise LD. Fourth, although it is impossible to rule out that the association of nonsynonymous mutation with circulating tPA levels is simply a confounder introduced by altered antibody binding during the tPA measurement procedure, it is unlikely after investigating all information available to us at this time. Finally, there may be distinct roles for genetic variation in regulating the circulating levels in healthy individuals compared with elevated levels in individuals in whom thrombolytic activity is induced. Although there is no evidence of heterogeneity of effect by cohort, our results cannot exclude the possibility of meaningful differences in the effects of genetic variation on circulating levels in the overall population versus subgroups of individuals with increased thrombolytic activity.

In conclusion, by analyzing a total of 26 929 participants from 14 discovery cohorts across the United States and Europe, we provide genome-wide evidence of the association of SNPs in STXBP5, PLAT, and STX2 loci with the circulating levels of tPA antigen. Although our analyses do not provide evidence for the association of these SNPs with clinically apparent CAD or stroke, we provided functional evidence in endothelial cells for a novel regulatory role of STXBP5 and STX2 on tPA availability. The strong eQTL result for STX2 in a wide range of tissues supports the hypothesis that associated alleles may modulate the tPA levels via a functional effect on gene expression.

Nonstandard Abbreviations and Acronyms

CAD

coronary artery disease

CVD

cardiovascular disease

eQTL

expression quantitative trait locus

eSNP

expression single nucleotide polymorphism

GWAS

genome-wide association study

LD

linkage disequilibrium

SNP

single nucleotide polymorphism

tPA

tissue plasminogen activator

vWF

von Willebrand factor

Acknowledgments

We acknowledge the essential role of the Cohorts for Heart and Aging Research in Genome Epidemiology (CHARGE) Consortium in providing a collaboration protocol and data-sharing resource for this project. We fully acknowledge the thousands of study participants who volunteered their time to help advance science and the scores of research staff and scientists who made this research possible. The Atherosclerosis Risk in Communities (ARIC) study would like to thank the staff and participants of the ARIC study for their important contributions. British 1958 Birth Cohort (B58C) acknowledges the use of phenotype and genotype data from the B58C DNA collection. CROATIA-Vis would like to acknowledge the invaluable contributions of the recruitment team (including those from the Institute of Anthropological Research in Zagreb) in Vis, the administrative teams in Croatia and Edinburgh, and the people of Vis. Genotyping was performed at the Wellcome Trust Clinical Research Facility in Edinburgh. The Framingham Heart Study acknowledges that the analyses presented here reflect intellectual input and resource development from the Framingham Heart Study investigators participating in the SNP Health Association Resource (SHARe) project. The Orkney Complex Disease Study (ORCADES) acknowledges the invaluable contributions of Lorraine Anderson, the research nurses in Orkney, and the administrative team in Edinburgh. Twins UK acknowledges essential contribution of Peter Grant and Angela Carter from the Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, United Kingdom, for measurements of clotting factors phenotypes. F.W. Asselbergs is supported by UCL Hospitals’ National Institute for Health Research Biomedical Research Centre.

Writing Group

J.H., S.T., F.W.A., M.S.-L., D.-A.T., J.E.H., A.D.J., N.L.S., S.M.W., C.H., P.v.d.H., A.H., C.J.L., D.P.S. (cochair), and C.J.O. (cochair).

Footnotes

*These first authors contributed equally.

**These last authors contributed equally.

The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.113.302088/-/DC1.

Correspondence to Christopher J. O’Donnell, MD, MPH, National Heart, Lung, and Blood Institute’s Framingham Heart Study, 73 Mt Wayte St, Second Floor, Framingham, MA 01702. E-mail

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Significance

Tissue plasminogen activator (tPA) catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for endogenous fibrinolysis. In some but not all studies, elevated plasma levels of tPA have been associated with coronary artery disease (CAD) and other cardiovascular diseases. Through a genome-wide association study approach, we provide evidence of the association of genetic variants in STXBP5, PLAT, and STX2 loci with circulating levels of tPA antigen. Although our analyses do not provide supportive evidence for the association of these single nucleotide polymorphisms with clinical CAD or stroke, we provided additional functional evidence for a novel regulatory role of STXBP5 and STX2 on tPA availability in endothelial cells. Results from gene expression studies in various tissues support the hypothesis that associated alleles may modulate circulating tPA levels via a functional effect on gene expression. Our findings provide new insights into tPA biology and avenues for future research for the prevention and treatment of thrombosis.