Plasminogen activator inhibitor-1 regulates the vascular expression of vitronectin.

Essentials Vitronectin (VN) is produced by smooth muscle cells (SMCs) and promotes neointima formation. We studied the regulation of vascular VN expression by plasminogen activator inhibitor-1 (PAI-1). PAI-1 stimulates VN gene expression in SMCs by binding LDL receptor-related protein 1. Stimulation of VN gene expression may be a mechanism by which PAI-1 controls vascular remodeling. SUMMARY: Background Increased expression of vitronectin (VN) by smooth muscle cells (SMCs) promotes neointima formation after vascular injury, and may contribute to chronic vascular diseases, such as atherosclerosis. However, the molecular regulation of vascular VN expression is poorly defined. Given the overlapping expression profiles and functions of VN and plasminogen activator inhibitor (PAI)-1, we hypothesized that PAI-1 regulates vascular VN expression. Objectives To determine whether PAI-1 regulates VN expression in SMCs and in vivo. Methods The effects of genetic alterations in PAI-1 expression, pharmacologic PAI-1 inhibition and recombinant PAI-1 on SMC VN expression were studied, and vascular VN expression in wild-type (WT) and PAI-1-deficient mice was assessed. Results VN expression was significantly lower in PAI-1-deficient SMCs and significantly increased in PAI-1-overexpressing SMCs. PAI-1 small interfering RNA and pharmacologic PAI-1 inhibition significantly decreased SMC VN expression. Recombinant PAI-1 stimulated VN expression by binding LDL receptor-related protein-1 (LRP1), but another LRP1 ligand, α2 -macroglobulin, did not. As compared with WT controls, carotid artery VN expression was significantly lower in PAI-1-deficient mice and significantly higher in PAI-1-transgenic mice. In a vein graft (VG) model of intimal hyperplasia, VN expression was significantly attenuated in PAI-1-deficient VGs as compared with WT controls. The plasma VN concentration was significantly decreased in PAI-1-deficient mice versus WT controls at 4 weeks, but not at 5 days or 8 weeks, after surgery. Conclusions PAI-1 stimulates SMC VN expression by binding LRP1, and controls vascular VN expression in vivo. Autocrine regulation of vascular VN expression by PAI-1 may play important roles in vascular homeostasis and pathologic vascular remodeling.

Recombinant soluble apyrase APT102 inhibits thrombosis and intimal hyperplasia in vein grafts without adversely affecting hemostasis or re-endothelialization.

Essentials New strategies are needed to inhibit thrombosis and intimal hyperplasia (IH) in vein grafts (VG). We studied effects of apyrase (APT102) on VGs and smooth muscle and endothelial cells (SMC/EC). APT102 inhibited thrombosis, SMC migration, and IH without impairing hemostasis or EC recovery. Apyrase APT102 is a single-drug approach to inhibit multiple processes that cause VG failure. SUMMARY: Background Occlusion of vein grafts (VGs) after bypass surgery, owing to thrombosis and intimal hyperplasia (IH), is a major clinical problem. Apyrases are enzymes that scavenge extracellular ATP and ADP, and promote adenosine formation at sites of vascular injury, and hence have the potential to inhibit VG pathology. Objectives To examine the effects of recombinant soluble human apyrase, APT102, on platelets, smooth muscle cells (SMCs) and endothelial cells (ECs) in vitro, and on thrombosis and IH in murine VGs. Methods SMC and EC proliferation and migration were studied in vitro. Inferior vena cava segments from donor mice were grafted into carotid arteries of recipient mice. Results APT102 potently inhibited ADP-induced platelet aggregation and VG thrombosis, but it did not impair surgical hemostasis. APT102 did not directly inhibit SMC or EC proliferation, but significantly attenuated the effects of ATP on SMC and EC proliferation. APT102 significantly inhibited SMC migration, but did not inhibit EC migration, which may be mediated, at least in part, by inhibition of SMC, but not EC, migration by adenosine. At 4 weeks after surgery, there was significantly less IH in VGs of APT102-treated mice than in control VGs. APT102 significantly inhibited cell proliferation in VGs, but did not inhibit re-endothelialization. Conclusions Systemic administration of a recombinant human apyrase inhibits thrombosis and IH in VGs without increasing bleeding or compromising re-endothelialization. These results suggest that APT102 has the potential to become a novel, single-drug treatment strategy to prevent multiple pathologic processes that drive early adverse remodeling and occlusion of VGs.

C-reactive protein induces expression of tissue factor and plasminogen activator inhibitor-1 and promotes fibrin accumulation in vein grafts.

BACKGROUND: C-reactive protein (CRP) promotes tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) expression in vitro, and an elevated plasma CRP concentration is associated with an increased risk of vein graft (VG) thrombosis after coronary artery bypass surgery. However, little is known about the effects of CRP on VG TF and PAI-1 expression in vivo, or on VG thrombosis. OBJECTIVES: We studied transgenic (Tg) mice expressing human CRP in a VG model to explore in vivo cause-and-effect relationships between CRP and TF, PAI-1, and VG thrombosis. METHODS: Vein segments from wild-type (WT) and CRP-Tg donors were transplanted into carotid arteries of WT and CRP-Tg recipients. VGs were analyzed 1-4 weeks later. RESULTS: Human CRP accumulated in VGs during the first 4 weeks after surgery, but appeared to originate exclusively from systemic sources, rather than local production. Human CRP significantly increased TF gene expression, protein concentration and activity in VGs. Human CRP also increased PAI-1 concentrations in VGs, although only in vascular endothelial cells. Human CRP stimulated macrophage migration, invasion into VGs, and TF expression. Fibrin deposition was significantly greater in VGs of CRP-Tg mice than in WT controls. CONCLUSIONS: CRP accumulates in VGs early after surgery, originating from systemic sources rather than local synthesis. Human CRP promotes TF and PAI-1 expression in VGs, although with different expression patterns. Human CRP stimulates macrophage invasion and fibrin deposition within VGs. These results suggest that CRP induces pathologic changes in VGs that contribute to early VG occlusion.

Plasminogen activator inhibitor-1 and vitronectin expression level and stoichiometry regulate vascular smooth muscle cell migration through physiological collagen matrices.

BACKGROUND: Vascular smooth muscle cell (VSMC) migration is a critical process in arterial remodeling. Purified plasminogen activator inhibitor-1 (PAI-1) is reported to both promote and inhibit VSMC migration on two-dimensional (D) surfaces. OBJECTIVE: To determine the effects of PAI-1 and vitronectin (VN) expressed by VSMC themselves on migration through physiological collagen matrices. METHODS: We studied migration of wild-type (WT), PAI-1-deficient, VN-deficient, PAI-1/VN doubly-deficient (DKO) and PAI-1-transgenic (Tg) VSMC through three-D collagen gels. RESULTS: WT VSMC migrated significantly slower than PAI-1- and VN-deficient VSMC, but significantly faster than DKO VSMC. Experiments with recombinant PAI-1 suggested that basal VSMC PAI-1 expression inhibits migration by binding VN, which is secreted by VSMC and binds collagen. However, PAI-1-over-expressing Tg VSMC migrated faster than WT VSMC. Reconstitution experiments with recombinant PAI-1 mutants suggested that the pro-migratory effect of PAI-1 over-expression required its anti-plasminogen activator (PA) and LDL receptor-related protein (LRP) binding functions, but not VN binding. While promoting VSMC migration in the absence of PAI-1, VN inhibited the pro-migratory effect of active PAI-1. CONCLUSIONS: In isolation, VN and PAI-1 are each pro-migratory. However, via formation of a high-affinity, non-motogenic complex, PAI-1 and VN each buffers the other's pro-migratory effect. The level of PAI-1 expression by VSMC and the concentration of VN in extracellular matrix are critical determinants of whether PAI-1 and VN promote or inhibit migration. These findings help to rectify previously conflicting reports and suggest that PAI-1/VN stoichiometry plays an important role in VSMC migration and vascular remodeling.

Plasminogen activator inhibitor type 1 enhances neointima formation after oxidative vascular injury in atherosclerosis-prone mice.

BACKGROUND: Plasminogen activator inhibitor type 1 (PAI-1) inhibits neointima formation after vascular injury. Hyperlipidemia modulates the expression of multiple genes, however, and the effects of PAI-1 on the arterial response to injury under hyperlipidemic conditions are unknown. The purpose of this study was to examine the impact of PAI-1 on intimal hyperplasia and other vascular changes that develop after arterial injury in apolipoprotein E-deficient (apoE(-/-)) mice. METHODS AND RESULTS: Ferric chloride injury of the midportion of the common carotid arteries of apoE(-/-) mice (n=22) induced formation of a neointima that contained smooth muscle cells, foam cells, neutral lipid, tissue factor, and von Willebrand factor. Interactions between vascular injury and apolipoprotein E deficiency were strongly synergistic; either stimulus alone was insufficient to induce significant neointima formation. Mean intima/media ratios were significantly greater (P<0.03) in apoE(-/-), PAI-1(+/+) mice (5.6+/-1.8, n=12) than in apoE(-/-), PAI-1(-/-) mice (1.2+/-0.55, n=12), as were the percentages of bromodeoxyuridine-positive cells in the intima and media (P<0.03). Transiently occlusive (<48 hours) and nonocclusive mural thrombi persisted longer in apoE(-/-), PAI-1(+/+) mice than in apoE(-/-), PAI-1(-/-) mice. CONCLUSIONS: In atherosclerosis-prone mice, PAI-1 promotes neointima formation after oxidative vascular injury. The apparent hyperlipidemia-dependent effect of PAI-1 may be mediated by its capacity to inhibit the clearance of platelet-fibrin thrombi that can deliver growth factors to the blood vessel wall or be incorporated into developing vascular lesions. Alternatively, hyperlipidemia may alter the pattern of gene expression in the blood vessel wall to enhance potential effects of PAI-1 on antiproliferative processes, such as transforming growth factor-beta activation and apoptosis.

Streptokinase triggers conformational activation of plasminogen through specific interactions of the amino-terminal sequence and stabilizes the active zymogen conformation.

Cleavage of Arg(561)-Val(562) in plasminogen (Pg) generates plasmin (Pm) through a classical activation mechanism triggered by an insertion of the new amino terminus into a binding pocket in the Pg catalytic domain. Streptokinase (SK) circumvents this process and activates Pg through a unique nonproteolytic mechanism postulated to be initiated by the intrusion of Ile(1) of SK in place of Val(562). This hypothesis was evaluated in equilibrium binding and kinetic studies of Pg activation with an SK mutant lacking Ile(1) (SK(2--414)). SK(2--414) retained the affinity of native SK for fluorescein-labeled [Lys]Pg and [Lys]Pm but induced no detectable conformational activation of Pg. The activity of SK(2--414) was partially restored by the peptides SK(1--2), SK(1--5), SK(1--10), and SK(1--15), whereas Pg(562--569) peptides were much less effective. Active site-specific fluorescence labeling demonstrated directly that the active catalytic site was formed on the Pg zymogen by the combination of SK(1--10) and SK(2--414), whereas sequence-scrambled SK(1-10) was inactive. The characterization of SK(1--10) containing single Ala substitutions demonstrated the sequence specificity of the interaction. SK(1--10) did not restore activity to the further truncated mutant SK(55-414), which was correlated with the loss of binding affinity of SK(55--414) for labeled [Lys]Pm but not for [Lys]Pg. The studies support a mechanism for conformational activation in which the insertion of Ile(1) of SK into the Pg amino-terminal binding cleft occurs through sequence-specific interactions of the first 10 SK residues. This event and the preferentially higher affinity of SK(2--414) for the activated proteinase domain of Pm are thought to function cooperatively to trigger the conformational change and stabilize the active zymogen conformation.

An analysis of mechanisms underlying the antifibrinolytic properties of radiographic contrast agents.

BACKGROUND: Radiographic contrast agents inhibit fibrinolysis, although by poorly defined pathways. The purpose of this study was to define specific mechanisms by which contrast agents inhibit clot lysis. METHODS AND RESULTS: Diatrizoate (high osmolar ionic agent), ioxaglate (low osmolar ionic), and ioversol (nonionic) were studied in vitro. Diatrizoate inhibited clot lysis by 81.3+/-0.6% vs. control (p<0.001). Ioxaglate inhibited clot lysis by 41.7+/-11.9%, which was of borderline significance (p=0.07). Ioversol did not significantly inhibit clot lysis (14.9+/-11.5% decrease vs. control; p>0.3). Inhibition of fibrinolysis was not explained by the high osmolarities of contrast agents, by their iodine content, or by their effects on the amidolytic activities of t-PA, urokinase, or plasmin. However, plasminogen activation by t-PA, urokinase, or streptokinase was significantly inhibited by contrast agents. Diatrizoate, ioxaglate, and ioversol inhibited plasminogen binding to plasma clots by 51+/-4% (p<0.001), 30.1+/-4% (p<0.01), and 19.4+/-7% (p=0.07), respectively. Plasma clots formed in the presence of contrast agents were resistant to lysis by plasmin. Diatrizoate produced the most potent effect, inhibiting clot lysis by 40+/-5.7% (p<0.03). Contrast agents did not inhibit plasminogen binding to fibrin or plasmin-mediated fibrinolysis if they were added after clot formation. Contrast agents altered clot turbidity, an index of fibrin structure, if present during clot formation, but not if added to preformed clots. Contrast agents did not affect plasminogen activator inhibitor-1 or alpha(2)-antiplasmin function. CONCLUSIONS: Contrast agents inhibit clot lysis by inhibiting plasminogen activation and by disrupting interactions of plasminogen and plasmin with fibrin by altering fibrin structure. Significant variation in antifibrinolytic properties exists between different contrast agents.

Streptokinase binds to human plasmin with high affinity, perturbs the plasmin active site, and induces expression of a substrate recognition exosite for plasminogen.

Binding of streptokinase (SK) to plasminogen (Pg) conformationally activates the zymogen and converts both Pg and plasmin (Pm) into specific Pg activators. The interaction of SK with Pm and its relationship to the mechanism of Pg activation were evaluated in equilibrium binding studies with active site-labeled fluorescent Pm derivatives and in kinetic studies of SK-induced changes in the catalytic specificity of Pm. SK bound to fluorescein-labeled and native Pm with dissociation constants of 11 +/- 2 pm and 12 +/- 4 pm, which represented a 1,000-10,000-fold higher affinity than determined for Pg. Stoichiometric binding of SK to native Pm was followed by generation of a two-fragment form of SK cleaved at Lys(59) (SK'), which exhibited an indistinguishable affinity for labeled Pm, while a truncated, SK(55-414) species had a 120-360-fold reduced affinity. Binding of SK to native Pm was accompanied by a >50-fold enhancement in specificity for activation of Pg, which was paralleled by a surprising 2.6-10-fold loss of specificity of Pm for 8 of 11 tripeptide-pNA substrates. Further studies with Pm labeled at the active site with 2-anilinonaphthalene-6-sulfonic acid demonstrated directly that binding of SK to Pm resulted in expression of a new substrate binding exosite for Pg on the SK.Pm complex. It is concluded that SK activates Pg in part by preferential binding to the active zymogen conformation. High affinity binding of SK to Pm enhances Pg substrate specificity principally through emergence of a substrate recognition exosite.

Plasminogen activator inhibitor-1 is a major determinant of arterial thrombolysis resistance.

BACKGROUND: Platelet-rich thrombi are resistant to lysis by tissue plasminogen activator (tPA). Plasminogen activator inhibitor-1 (PAI-1), a rapid inhibitor of tPA, may contribute to arterial thrombolysis resistance. However, few data are available regarding the effect of PAI-1 on arterial thrombolysis in animals. We used a murine carotid injury model to test the hypothesis that PAI-1 inhibits thrombolysis mediated by pharmacological concentrations of tPA. METHODS AND RESULTS: Platelet-rich thrombi were induced in wild-type mice (PAI-1 +/+; n=11) and PAI-1-deficient mice (PAI-1 -/-; n=11) with ferric chloride. Baseline carotid blood flows and mean occlusion times did not differ between PAI-1 +/+ and PAI-1 -/- mice. Clot lysis was induced by infusion of heparin (200 U/kg bolus, 70 U. kg-1. h-1 drip), human plasminogen (50 mg/kg), and tPA at 20 (n=10) or 100 (n=12) microg. kg-1. min-1. Mean plasma tPA antigens were 2.7 microg/mL (tPA infusion, 20 microg. kg-1. min-1) and 5.5 microg/mL (tPA infusion, 100 microg. kg-1. min-1), with no significant differences between PAI-1 +/+ mice and PAI-1 -/- mice. Reperfusion after tPA 20 microg. kg-1. min-1 occurred in 1 of 5 PAI-1 +/+ mice versus 5 of 5 PAI-1 -/- mice (P=0.0006). Reperfusion occurred in all mice that received tPA 100 microg. kg-1. min-1, but reperfusion times were significantly shorter in PAI-1 -/- mice (17. 8+/-2.6 minutes, n=6) than in PAI-1 +/+ mice (35.7+/-5.1 minute, n=6; P=0.01). Histological analyses confirmed that carotid thrombi were platelet rich and that PAI-1 was distributed uniformly throughout thrombi from PAI-1 +/+ mice. Lysates of PAI-1 +/+ platelets inhibited human tPA, whereas PAI-1 -/- platelet lysates did not. CONCLUSIONS: PAI-1 is a major determinant of the resistance of platelet-rich arterial thrombi to lysis by pharmacological concentrations of tPA. Strategies to inhibit or resist PAI-1 may enhance thrombolysis.

Vitronectin inhibits the thrombotic response to arterial injury in mice.

Vitronectin (VN) binds to plasminogen activator inhibitor-1 (PAI-1) and integrins and may play an important role in the vascular response to injury by regulating fibrinolysis and cell migration. However, the role of VN in the earliest response to vascular injury, thrombosis, is not well characterized. The purpose of this study was to test the hypothesis that variation in vitronectin expression alters the thrombotic response to arterial injury in mice. Ferric chloride (FeCl3) injury was used to induce platelet-rich thrombi in mouse carotid arteries. Wild-type (VN +/+, n = 14) and VN-deficient (VN -/-, n = 15) mice, matched for age and gender, were studied. Time to occlusion after FeCl3 injury was determined by application of a Doppler flowprobe to the carotid artery. Occlusion times of VN -/- mice were significantly shorter than those of VN +/+ mice (6.0 +/- 1.2 minutes v 17.8 +/- 2.3 minutes, respectively, P < .001). Histologic analysis of injured arterial segments showed that thrombi from VN +/+ and VN -/- mice consisted of dense platelet aggregates. In vitro studies of murine VN +/+ and VN -/- platelets showed no significant differences in ADP-induced aggregation, but a trend towards increased thrombin-induced aggregation in VN -/- platelets. Purified, denatured VN inhibited thrombin-induced platelet aggregation, whereas native VN did not. Thrombin times of plasma from VN -/- mice (20.5 +/- 2.1 seconds, n = 4) were significantly shorter than those of VN +/+ mice (34.2 +/- 6.7 seconds, n = 4, P < .01), and the addition of purified VN to VN -/- plasma prolonged the thrombin time into the normal range, suggesting that VN inhibits thrombin-fibrinogen interactions. PAI-1-deficient mice (n = 6) did not demonstrate significantly enhanced arterial thrombosis compared with wild-type mice (n = 6), excluding a potential indirect antithrombin function of VN mediated by interactions with PAI-1 as an explanation for the accelerated thrombosis observed in VN -/- mice. These results suggest that vitronectin plays a previously unappreciated antithrombotic role at sites of arterial injury and that this activity may be mediated, at least in part, by inhibiting platelet-platelet interactions and/or thrombin procoagulant activity.

Effects of amiodarone on tumor necrosis factor-alpha levels in congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy.

Tumor necrosis factor-alpha (TNF-alpha) has been implicated in the pathogenesis of congestive heart failure and may be associated with an increase in mortality. A recent in vitro study showed that amiodarone decreases TNF-alpha production by human blood mononuclear cells in response to lipopolysaccharide. However, no previous clinical studies have determined the effect of chronic amiodarone therapy on TNF-alpha levels. Thus, the purpose of this study was to determine whether amiodarone affects TNF-alpha levels in patients with ischemic and nonischemic cardiomyopathy. TNF-alpha levels were analyzed by an enzyme-linked immunoassay using plasma samples at baseline, 1, and 2 years of follow-up in New York Heart Association class III patients (n = 40 in each of the placebo and amiodarone groups, mean ejection fraction 0.25+/-0.09) who were randomized in the Congestive Heart Failure-Survival Trial of Antiarrhythmic Therapy, a multicenter, double-blind, placebo-controlled study in which the effect of amiodarone on survival was investigated. TNF-alpha levels were elevated in both groups of patients at baseline, 6.6+/-3.1 and 7.7+/-5.3 pg/ml in the amiodarone and placebo groups, respectively (p = 0.3). There were no significant differences in demographic or clinical variables between the 2 groups. Amiodarone treatment was associated with a significant increase in TNF-alpha levels in patients with ischemic cardiomyopathy, 12.7+/-12.5 and 6.8+/-3.7 pg/ml in the amiodarone and placebo groups, respectively (p = 0.03) at 1 year. No change in TNF-alpha levels was observed in patients with nonischemic cardiomyopathy. In contrast to the in vitro data, amiodarone treatment is associated with an increase in TNF-alpha levels in patients with ischemic cardiomyopathy. This increase is not associated with an adverse effect on survival.

Acute myocardial infarction in two adolescent males.

Acute myocardial infarction in previously healthy children is rare in the absence of congenital anomalies. We describe two cases of acute anterior myocardial infarction in adolescent males with no congenital heart disease, without prior history of or risk factors for coronary heart disease, and with no history of drug abuse. These cases illustrate that myocardial infarction in the absence of systemic illness or coronary anomalies can occur in an adolescent population.

Effects of radiographic contrast agents on thrombin formation and activity.

Clinical trials suggest that the risk of thrombosis during coronary angioplasty is lower with ionic contrast agents than with nonionic contrast agents. However, the molecular mechanisms underlying this effect are unknown. This study examined the effects of contrast agents on thrombin formation and its interaction with substrates, inhibitors, and ligands to define potential mechanisms by which contrast agents affect thrombus formation. Two ionic agents, diatrizoate and ioxaglate, and one nonionic agent, ioversol, were studied. Ionic agents inhibited factor X activation by the tissue factor-factor VIIa complex more potently than ioversol (53 +/- 3.7, 43.0 +/- 1.9, and 26.5 +/- 2.4% inhibition by diatrizoate, ioxaglate, and ioversol, respectively, at concentrations of 5%). Ionic contrast agents were potent inhibitors of prothrombinase function, inhibiting thrombin formation by >75% at contrast concentrations of 0.6% (p <0.005). Ioversol inhibited prothrombinase to a significantly lesser extent than ionic agents. Clotting assays suggested that ioxaglate was the most potent inhibitor of thrombin generation in plasma despite having the least effect on fibrin polymerization. Contrast agents inhibited binding of thrombin to fibrin, with ionic agents producing a more potent effect than ioversol (p <0.02). However, contrast agents did not inhibit thrombin-mediated platelet activation, had only a minor effect on inhibition of thrombin by antithrombin III, and did not affect thrombin-hirudin interactions. In summary, these studies identify specific mechanisms by which radiographic contrast agents inhibit thrombin formation and function -- i.e. inhibition of tissue factor-dependent factor Xa generation, inhibition of the prothrombinase complex, and inhibition of thrombin binding to fibrin. These findings may help to explain the reduced risk of thrombosis during coronary angioplasty associated with ionic contrast agents.

Functional analysis of the amino- and carboxyl-termini of streptokinase.

Streptokinase (SK) is a 414 amino acid bacterial protein that activates human plasminogen. Streptokinase fragments derived from the central portion of the protein bind plasminogen, but are inactive, indicating that the amino- and/or carboxyl-termini are required for normal plasminogen activator activity. To better define the function of the N- and C-termini of SK we generated and characterized 21 N-terminal and 20 C-terminal deletion mutants. All mutants lacking > or = 18 N-terminal or > or = 51 C-terminal amino acids exhibited markedly reduced plasminogen activator activity, while mutants lacking < or = 12 N-terminal or < or = 40 C-terminal residues were fully active. The decrease in SK activity with N-terminal deletion appeared to result not from loss of plasminogen binding capacity, but rather from increased susceptibility of deletion mutants to degradation by plasmin. Point mutations at positions 13 (SK V13D) or 20 (SK V20D) produced functional abnormalities similar to those observed in N-terminal deletion mutants, with SK V13D exhibiting delayed amidolytic activity and SK V20D exhibiting only 1% plasminogen activator activity and marked sensitivity to degradation by plasmin. C-terminal deletion mutants lacking > or = 51 amino acids also bound plasminogen, but did not induce significant amidolytic activity in plasminogen or activator activity in plasmin. Prevention of cleavage at position 59 of SK had no effect on plasminogen activator activity, suggesting that the rapid hydrolysis of this bond that occurs after SK-plasminogen complex formation is not required for normal function of the N-terminus. These results suggest that residues within or near positions 13-20 of SK are important determinants of its capacity to generate amidolytic activity and are a critical determinant of the stability of SK, while residues within or near position 364-374 are required for generating amidolytic activity and for conferring plasminogen activator activity to plasmin(ogen). These results also suggest that SK fragments significantly smaller than SK 13-374 are unlikely to be effective thrombolytic agents.

Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice.

BACKGROUND: Platelet-rich arterial thrombi are resistant to lysis by plasminogen activators. However, the mechanisms underlying thrombolysis resistance are poorly defined. Plasminogen activator inhibitor-1 (PAI-1), which is present in plasma, platelets, and vascular endothelium, may be an important determinant of the resistance of arterial thrombi to lysis. However, in vitro studies examining the regulation of platelet-rich clot lysis by PAI-1 have yielded inconsistent results. METHODS AND RESULTS: We developed a murine arterial injury model and applied it to wild-type (PAI-1 [+/+]) and PAI-1-deficient (PAI-1 [-/-]) animals. FeCl3 was used to induce carotid artery thrombosis. Thrombi consisted predominantly of dense platelet aggregates, consistent with the histology of thrombi in large-animal arterial injury models and human acute coronary syndromes. To examine the role of PAI-1 in regulating endogenous clearance of platelet-rich arterial thrombi, thrombi were induced in 22 PAI-1 (+/+) mice 14 PAI-1 (-/-) mice. Twenty-four hours later, the amount of residual thrombus was determined by histological analysis of multiple transverse sections of each artery. Residual thrombus was detected in 55 of 85 sections (64.7%) obtained from PAI-1 (+/+) mice compared with 19 of 56 sections (33.9%) from PAI-1 (-/-) mice (P=.009). Computer-assisted planimetry analysis revealed that mean thrombus cross-sectional area was 0.033+/-0.0271 mm2 in PAI-1 (+/+) mice versus 0.016+/-0.015 mm2 in PAI-1 (-/-) mice (P=.048). CONCLUSIONS: PAI-1 is an important determinant of thrombolysis at sites of arterial injury. Application of this model to other genetically altered mice should prove useful for studying the molecular determinants of arterial thrombosis and thrombolysis.

Human plasminogen activator inhibitor-1 (PAI-1) deficiency: characterization of a large kindred with a null mutation in the PAI-1 gene.

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue- and urokinase-type plasminogen activators, is considered a critical regulator of the fibrinolytic system. We previously reported a child with abnormal bleeding and complete PAI-1 deficiency caused by a frame-shift mutation in exon 4 of the PAI-1 gene. The purpose of this study was to provide genetic and clinical data on the extended pedigree of the original proband to better define the phenotype associated with PAI-1 deficiency. Allele-specific oligonucleotide hybridization was used to genotype individuals, and serum PAI-1 antigen was measured by enzyme-linked immunosorbent assay. By this approach we have identified 19 individuals who are heterozygous for the PAI-1 null allele and 7 homozygous individuals with complete PAI-1 deficiency. Clinical manifestations of PAI-1 deficiency were restricted to abnormal bleeding, which was observed only after trauma or surgery in homozygous affected individuals. A spectrum of bleeding patterns was observed, including intracranial and joint bleeding after mild trauma, delayed surgical bleeding, severe menstrual bleeding, and frequent bruising. Fibrinolysis inhibitors, including epsilon-aminocaproic acid and tranexamic acid, were effective in treating and preventing bleeding episodes. Other than abnormal bleeding, no significant developmental or other abnormalities were observed in homozygous PAI-1-deficient individuals. Heterozygous PAI-1 deficiency was not associated with abnormal bleeding, even after trauma or surgery. These observations define the clinical spectrum of PAI-1 deficiency and provide additional evidence to support the hypothesis that the primary function of plasminogen activator inhibitor-1 in vivo is to regulate vascular fibrinolysis.

High concentrations of active plasminogen activator inhibitor-1 in porcine coronary artery thrombi.

Addition of exogenous plasminogen activator inhibitor-1 (PAI-1) to fibrin clots inhibits fibrinolysis in vivo. However, it is unknown whether the localized concentrations of active PAI-1 necessary to produce this antifibrinolytic effect can be recruited to acute arterial thrombi by endogenous mechanisms. We measured PAI-1 activity and antigen in porcine coronary artery thrombi that formed in response to acute vascular injury. Mean PAI-1 activity in thrombi (n = 5) was 36 +/- 5.1 micrograms/mL, which is > 2000 times its concentration in normal porcine plasma. The presence of markedly elevated concentrations of active PAI-1 in thrombi was confirmed by an immunoactivity assay and by demonstrating formation of sodium dodecyl sulfate-stable complexes after addition of 125I-urokinase to thrombus extracts. Comparative analysis of PAI-1 antigen by Western blotting and urokinase inhibition assay suggested that approximately one third of thrombus-associated PAI-1 was active. Histological examination of coronary thrombi revealed that they consisted predominantly of dense aggregates of platelets with interspersed islands of fibrin, which closely resemble the histological appearance of thrombi in patients with myocardial infarction and unstable angina pectoris. Washed porcine platelets prepared from peripheral blood contained sufficient PAI-1 antigen and activity to account for the concentrations observed in coronary artery thrombi. However, the specific activity of human platelet PAI-1 was lower than that of porcine platelet PAI-1 (2% versus 50% active, respectively), and human platelets inhibited in vitro fibrinolysis to a lesser extent than did porcine platelets. These results indicate that active PAI-1 accumulates in porcine coronary artery thrombi in concentrations markedly higher than those present in plasma and that PAI-1 may be an important determinant of the known resistance of platelet-rich thrombi to lysis by tissue-type plasminogen activator. These studies also underscore the importance of considering possible species differences in protein function when comparing animal models of thrombosis to acute coronary thrombosis in humans.

Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene.

Impaired fibrinolytic activity within the lung is a common manifestation of acute and chronic inflammatory lung diseases. Because the fibrinolytic system is active during repair processes that restore injured tissues to normal, reduced fibrinolytic activity may contribute to the subsequent development of pulmonary fibrosis. To examine the relationship between the fibrinolytic system and pulmonary fibrosis, lung inflammation was induced by bleomycin in transgenic mice that either overexpressed or were completely deficient in murine plasminogen activator inhibitor-1 (PAI-1). 2 wk after 0.075 U of bleomycin, the lungs of transgenic mice overexpressing PAI-1 contained significantly more hydroxyproline (118 +/- 8 micrograms) than littermate controls (70.5 +/- 8 micrograms, P < 0.005). 3 wk after administration of a higher dose of bleomycin (0.15 U), the lung hydroxyproline content of mice completely deficient in PAI-1 (49 +/- 8 micrograms) was not significantly different (P = 0.63) than that of control animals receiving saline (37 +/- 1 micrograms), while hydroxyproline content was significantly increased in heterozygote (77 +/- 12 micrograms, P = 0.06) and wild-type (124 +/- 19 micrograms, P < 0.001) littermates. These data demonstrate a direct correlation between the genetically determined level of PAI-1 expression and the extent of collagen accumulation that follows inflammatory lung injury. These results strongly support the hypothesis that alterations in fibrinolytic activity influence the extent of pulmonary fibrosis that occurs after inflammatory injury.