SummaryRecently, we reported that an Alu insertion polymorphism of the tissue-type plasminogen activator (t-PA) gene is associated with vascular t-PA release rates in man. In the current study we searched the t-PA gene for putative functional genetic variants in linkage disequilibrium (LD) with this polymorphism. Healthy individuals with different Alu genotypes and contrasting t-PA release rates were studied. Regulatory and coding regions of the t-PA gene were sequenced. Eight singlenucleotide polymorphisms (SNPs) were identified. Three of these were in significant LD with the Alu polymorphism and consequently associated with t-PA release rates; one in the far upstream enhancer, one in exon 6, and one in intron 10. The enhancer SNP resides within a GC box. Electrophoretic mobility shift assay (EMSA) revealed a reduced binding affinity of Sp1 to the T allele, which is the allele associated with a low t-PA release rate. Variations in exon 6 and intron 10 were silent and without apparent effect on splicing, respectively.

Abstract-We have observed marked interindividual differences in release rates of tissue-type plasminogen activator (t-PA) among healthy subjects. The objective of the current study was to test the hypothesis that there is an association between a genetic variation at the t-PA locus and the in vivo release rate of t-PA. Fifty-one healthy males were studied at rest in the morning and 27 of these were also subjected to a mental stress test. Net release rates of total t-PA across the forearm vascular bed were calculated as the product of the venoarterial concentration gradient and forearm plasma flow. Zygosity for an Alu-repeat polymorphism in intron 8 of the t-PA gene was determined by a polymerase chain reaction. Basal t-PA release rates differed markedly by genotype (ANOVA, PϽ0.05); subjects homozygous for the insertion had a significantly higher release rate (mean 10.9 ng ⅐ min Ϫ1 ⅐ L Ϫ1 , nϭ19) than both heterozygotes (4., nϭ26) and subjects homozygous for the deletion (0.9 ng ⅐ min Ϫ1 ⅐ L Ϫ1 , nϭ6). After 2 minutes of mental stress release rates had increased approximately 2-fold in all groups. Arterial and venous plasma levels of t-PA were unrelated to genotype. In conclusion, the current results provide the first evidence of an association between a common genetic variation at the t-PA locus and interindividual differences in net release rates of t-PA in vivo. The relationship is not reflected by circulating steady-state plasma levels and can thus not be disclosed by conventional venous plasma sampling. 1 Common genetic variations (polymorphisms) at the fibrinogen, factor VII, and plasminogen activator inhibitor type 1 (PAI-1) loci have been associated with interindividual differences in the basal steady-state plasma level of the respective proteins. [1][2][3][4] In addition, responses to environmental factors have been shown to differ by genotype. 1A polymorphism has been identified at the tissue-type plasminogen activator (t-PA) locus on chromosome 8, which consists of the presence or absence of a 311 bp Alu sequence in intron 8. 5 The Alu insertion probably arose early in human evolution, and a number of populations have been found to be dimorphic for its presence or absence. 6,7 A similar polymorphism in the angiotensin converting enzyme (ACE) gene has been shown to explain approximately 50% of the variability of plasma ACE levels between individuals. 8 In contrast, it was recently reported that t-PA genotypes did not correlate with basal plasma levels of t-PA. 9 -11 However, in view of the complex regulation of the steady-state plasma level of t-PA, this observation does not exclude the possibility of an association between genotype and local t-PA release rates. The systemic concentration of t-PA not only is dependent both on secretion and clearance but also on its rate and degree of complex-formation with PAI-1. 12 This is due to the fact that the t-PA/PAI-1 complex is cleared at a slower rate than free t-PA. 13 It follows that an increased plasma concentration of PAI-1 will be paralleled by an increas...

Free, biologically active tissue-type plasminogen activator (tPA) is the main initiator of intravascular fibrinolysis, but little is known about the regulation of active tPA on the organ level. The aim was to investigate if the local availability of active tPA on the organ level depends on the local release rate of tPA or the arterial input of tPA and plasminogen activator inhibitor type 1 (PAI-1). Also, we wanted to evaluate if plasma levels predict capacity for endothelial release of fibrinolytic proteins. Invasive perfused-forearm studies were performed in 96 healthy subjects. Local release rates of fibrinolytic proteins were assessed at baseline and during endothelial stimulation. Stimulation by methacholine and desmopressin induced a 6-and 12-fold increase in total tPA release rates, respectively. With increasing local release rates of tPA a gradually closer correlation emerged between the total tPA secretion and the forearm output of active tPA (from r ¼ 0.102, ns to r ¼ 0.85, P < 0.0001). Forearm availability of active tPA was not related to arterial input of either tPA or PAI-1. Release rates and plasma levels of tPA were not correlated. Baseline release rates of active tPA increased to noon. The major determinant for the local availability of active tPA is the capacity of the endothelium to release tPA rather than the arterial input of PAI-1 or tPA. Despite a molar excess of PAI-1, the majority of tPA released during stimulation does not undergo local inactivation. The capacity to release tPA locally cannot be predicted from its plasma concentration.

We recently showed that muscarinic receptor stimulation causes a marked increase in the net release of tissue-type plasminogen activator (TPA) antigen and activity across the human forearm in vivo, in conjunction with endothelium-dependent vasodilation. Because hypertension has been associated with endothelial dysfunction, the aim of the study was to compare forearm TPA release and vasodilation in response to muscarinic stimulation in normotensive (NC) and borderline hypertensive (BH) subjects. The study was performed in 10 apparently healthy young men with BH and 10 male NC subjects. Methacholine (MCh: 0.1, 0.8, and 4.0 micrograms/min) and sodium mitroprusside (SNP: 0.5, 2.5, and 10 micrograms/min) were administered in randomized order as double-blind, stepwise, intrabrachial artery infusions. Forearm blood flow was assessed by plethysmography. Net release/uptake was calculated as the product of the arteriovenous concentration gradient and forearm plasma flow. Vasodilator responses to MCh were similar in both groups (P = NS), whereas the decrease in forearm vascular resistance in response to SNP was somewhat less in BH subjects (P = .005). At rest, both groups showed a significant arteriovenous gradient and net release of TPA antigen across the forearm (P < .05 throughout). However, in contrast to the significant net increment in TPA activity across the forearm in the NC group (P < .018), BH subjects had no basal forearm increment in TPA activity (NC vs BH, P = .006). Arterial and venous plasma levels of plasminogen activator inhibitor 1 (PAI-1) antigen and activity were higher in BH subjects (P < or = .05 throughout), who in contrast to NC subjects, also had a significant forearm net release of PAI-1 antigen (P = .006). Across the whole group, there was a significant inverse relation between arterial PAI-1 antigen levels and increment in TPA activity across the forearm (r = -.57, P = .008) but no relation to TPA antigen release. In response to MCh infusion, both the net release of TPA antigen and increment in TPA activity increased markedly and to similar extents in both groups (P < .01 throughout). SNP infusion had no effect on either TPA antigen release or increment in TPA activity in the NC group but elicited a significant net release of TPA antigen and increase in TPA activity in the BH group (P < .05). Both circulating levels and local release of PAI-1 antigen were significantly correlated to fasting plasma insulin. Endothelium-dependent vasodilation and endothelial TPA release in response to muscarinic receptor stimulation were preserved in BH subjects. At rest, BH subjects had higher circulating PAI-1 antigen levels and a corresponding decrease in circulating levels and local increment of TPA activity. In contrast to NC subjects, BH subjects responded with a TPA release also in response to increased flow, which may indicate an enhanced endothelial cell responsiveness to fluid shear stress.

Systemic administration of desmopressin (DDAVP) induces increased plasma levels of tissue-type plasminogen activator (t-PA), coagulation factor VIII, and von Willebrand factor (vWF). However, the mechanisms behind these responses are not known. We tested the hypothesis that DDAVP acts as a local stimulator of acute endothelial release of t-PA and vWF independently of central pathways. Healthy, young, nonsmoking male volunteers were studied. In a first study (n = 7), DDAVP and placebo were administered as randomized single-blind stepwise intrabrachial artery infusions (0.7, 7.0, and 70 ng/min). In a another subset of subjects (n = 4), a constant-rate DDAVP infusion of 70 ng/min was administered for 20 minutes in the brachial artery of the nondominant arm with the dominant arm as control. To rule out that the observed t-PA release was flow-dependent, 4 additional subjects received stepwise intra-arterial infusions of both DDAVP (7.0, 21, and 70 ng/min) and sodium nitroprusside (SNP; 0.5, 2.5, and 10 μg/min). Brachial venoarterial plasma concentration gradients and forearm plasma flow were used to determine net release/uptake rates of t-PA and vWF. At baseline, the average net release rate of t-PA was 6.7 ng/min across the whole forearm vascular bed, whereas there was no detectable basal release of vWF. Stepwise infusion of DDAVP induced a massive regulated release of t-PA with a peak after 15 minutes on the highest dose-step (ANOVA; P < .0001). The average maximum net release rate was 178 ng/min, and the total amount of t-PA released was, on the average, 3,000 ng. The majority was released in its active form. Constant-rate DDAVP infusion again markedly increased t-PA release in the infusion arm but had no effect whatsoever in the control arm. In contrast, DDAVP did not stimulate a local release of vWF in either study. Central hemodynamics were unchanged during infusions despite a local vasodilatory response with DDAVP. Endothelium-independent flow stimulation by SNP did not elicit any local t-PA release. We conclude that DDAVP induces a massive acute flow-independent release of t-PA, without the simultaneous release of vWF, in the human forearm vascular bed. The lack of a t-PA response in the control arm, as well as the unaltered central hemodynamics with DDAVP, confirms that the observed regulated t-PA release is local and independent of central mechanisms.