Plasminogen activator inhibitor with very long half-life (VLHL PAI-1) can reduce bleeding in PAI-1-deficient patients.

This review summarizes our current knowledge of plasminogen activator inhibitor (PAI-1) deficiency and proposes some novel treatments for this condition. PAI-1 is a fast acting inhibitor of tissue and urokinase plasminogen activators (tPA and uPA). PAI-1 controls/slows clot lysis triggered by tPA activated plasminogen. PAI-1 deficiency was once considered to be an extremely rare disorder characterized by frequent and prolonged bleeding episodes. PAI-1 deficiency is now thought to be more frequent than initially reported and is known to be caused by mutations in the PAI-1 gene that produce a dysfunctional PAI-1 protein or slow the secretion of PAI-1 into the circulation. PAI-1 deficiency is characterized by hyperfibrinolysis that results in frequent bleeding episodes. Patients with this condition form normal blood clots that are quickly lysed by unopposed tPA-activated plasmin. Spontaneous bleeding is rare in PAI-1 deficient patients, but moderate hemorrhaging of the knees, elbows, nose, and gums can be triggered by mild trauma. Additionally, prolonged bleeding after surgery is common and menstrual bleeding may be severe. Moderate PAI-1 deficiency is associated with a lifelong bleeding tendency, but severe deficiencies can be life-threatening. The diagnosis of this disorder remains challenging due to the lack of a clear definition of PAI-1 deficiency as well as a lack of standardized tests. Patients with mild PAI-1 deficiency may be treated with antifibrinolytic agents (ε-aminocaproic acid or tranexamic acid); however, not all patients respond well to these treatments. These patients may be treated with wild-type PAI-1; however, this molecule quickly converts into its inactive form. We propose to use PAI-1 with an extended half-life to treat these patients.

Remarkable extension of PAI-1 half-life surprisingly brings no changes to its structure.

Plasminogen activator inhibitor type 1 (PAI-1) is a serpin protein, a natural inhibitor of urokinase (uPA) and tissue plasminogen activators (tPA). By inhibiting uPA it can block growth of the cancer tumors by suppressing angiogenesis, while when acting on tPA in the blood it can avert conversion of plasminogen to plasmin preventing lysis of the clot. Furthermore, blocking PAI-1 activity can protect against thrombosis. Thus PAI-1 makes great impact on human homeostasis and is desirable for clinical application. Wild-type PAI-1 (wt-PAI-1) has a short span of activity with a t1/2 of ~2 h, being spontaneously converted into a latent form. An enormous effort has been made to create a more stable molecule with >600 PAI-1 variants constructed to study its structure-function relationship. In the present study, we evaluate the structure of the active recombinant VLHL-PAI-1 (very long half life, active >700 h) which is glycosylated similarly to wt-PAI-1 at N232 and N288, with the extended reactive center loop, intact engineered -S-S-bridge (Q174C, G323C) that precludes latency without affecting structure, and can be controlled by a reducing agent to terminate activity at will. We have already proven its usefulness to control cancer in human cancer cells, as well as preventing clot lysis in human whole blood and plasma and in a mouse model. Our results demonstrate the potential therapeutic applications (topical or systemic) of this protein in the treatment of cancer, for the trauma patients to ward off an excessive blood loss, or for people with the PAI-1 deficiency, especially during surgery.

Structure-activity relationships of new N-acylanthranilic acid derivatives as plasminogen activator inhibitor-1 inhibitors.

Novel anthranilic acid derivatives having substituted N-acyl side chains were designed and synthesized for evaluation as plasminogen activator inhibitor-1 (PAI-1) inhibitors. Compounds with a 4-diphenylmethyl-1-piperazinyl moiety on the acyl side chains in general exhibited potent in vitro PAI-1 inhibitory activity and good pharmacokinetic profiles after oral administration in rats. Compound 16f (TM5275) was identified as a promising candidate for further pharmacological evaluation.

A PAI-1 mutant, PAI-1R, slows progression of diabetic nephropathy.

Plasminogen activator inhibitor-1 (PAI-1) has been implicated in renal fibrosis. In vitro, PAI-1 inhibits plasmin generation, and this decreases mesangial extracellular matrix turnover. PAI-1R, a mutant PAI-1, increases glomerular plasmin generation, reverses PAI-1 inhibition of matrix degradation, and reduces disease in experimental glomerulonephritis. This study sought to determine whether short-term administration of PAI-1R could slow the progression of glomerulosclerosis in the db/db mouse, a model of type 2 diabetes in which mesangial matrix accumulation is evident by 20 wk of age. Untreated uninephrectomized db/db mice developed progressive albuminuria and mesangial matrix expansion between weeks 20 and 22, associated with increased renal mRNA encoding alpha1(I) and (IV) collagens and fibronectin. Treatment with PAI-1R prevented these changes without affecting body weight, blood glucose, glycosylated hemoglobin, creatinine, or creatinine clearance; therefore, PAI-1R may prevent progression of glomerulosclerosis in type 2 diabetes.

Comparison of the effects of pioglitazone and voglibose on circulating total and high-molecular-weight adiponectin, and on two fibrinolysis inhibitors, in patients with Type 2 diabetes.

BACKGROUND: To investigate short-term effects of pioglitazone and voglibose on serum concentrations of both total and high-molecular-weight (HMW) adiponectin measured with a novel sandwich enzyme-linked immunosorbent assay (ELISA) ,and on plasma fibrinolysis indicators, in Type 2 diabetic patients with inadequate glycaemic control on sulphonylureas. METHODS: Thirty-four diabetic patients were randomized to receive pioglitazone or voglibose treatment for 12 weeks, after which serum HMW adiponectin was measured. Plasma plasminogen activator inhibitor (PAI) 1 and thrombin-activatable fibrinolysis inhibitor (TAFI), a recently identified inhibitor of fibrinolysis, were measured as fibrinolysis inhibitors. RESULTS: At baseline, serum HMW adiponectin correlated negatively with plasma TAFI in all patients with Type 2 diabetes (r = -0.367, P = 0.0423). Both groups showed similar improvements in glycaemic control. Serum total and HMW adiponectin increased in patients treated with pioglitazone, but did not change in patients treated with voglibose. The HMW : total adiponectin ratio increased significantly after treatment with pioglitazone (P = 0.0004). The change in HbA(1c) correlated negatively with changes in serum HMW adiponectin in patients treated with pioglitazone (r = -0.694, P = 0.0034). Plasma PAI-1 and TAFI did not change with pioglitazone treatment. CONCLUSION: Increased serum HMW adiponectin may contribute to the improvement in glycaemic control after pioglitazone treatment. Plasma PAI-1 and TAFI were unchanged by either drug.

The plasma levels of prostanoids and plasminogen activator inhibitor-1 in primary and secondary thrombocytosis.

An elevated platelet count is a common finding in both hospitalized and ambulatory patients. Thrombosis and bleeding complications are more frequently observed in patients with clonal thrombocytosis than secondary thrombocytosis. The aim of this study was to investigate the behaviors of plasminogen activator inhibitor type 1 (PAI-1), the inhibitor of fibrinolysis; and thromboxane A2 and 6-keto-PGF1 alpha, the products of endoperoxides, in 16 patients affected with clonal thrombocytemia as compared with 16 patients with reactive thrombocytosis and 15 normal controls. In the clonal thrombocytemia group, plasma levels of PAI-1 antigen and activity were significantly higher than both reactive thrombocytosis and control group. Plasma levels of 6-keto-PGF1alpha were significantly higher in the clonal thrombocytemia group than the other two groups and also higher in the reactive thrombocytosis group than the control group, which was also significant. This study confirms that arachidonate metabolism is frequently deranged in patients with thrombocytosis and hypofibrinolysis due to increased PAI-1 plasma levels as shown in the clonal thrombocytosis group. This may explain the thrombotic tendency in myeloproliferative disorders.

An experimental multiple-organ model for the study of regional net release/uptake rates of tissue-type plasminogen activator in the intact pig.

Experimental data indicate large between-organs variations in rates of synthesis of tissue-type plasminogen activator (t-PA), which may reflect important differences in the capacity for constitutive and stimulated t-PA release from the vascular endothelium. In this report we describe a new multiple-organ experimental in vivo model for simultaneous determinations of net release/uptake rates of t-PA across the coronary, splanchnic, pulmonary, and hepatic vascular beds. In eleven intact anesthetized pigs, blood samples were obtained simultaneously from the proximal aorta, coronary sinus, pulmonary artery, and portal and hepatic veins. Plasma flows were monitored separately for each vascular region. Total plasma t-PA was determined by ELISA with a porcine t-PA standard. Regional net release/uptake rates were defined as the product of arteriovenous concentration gradients and local plasma flows. The net release of t-PA across the splanchnic vascular bed was very high, with a mean output of 1,919 ng total t-PA x min(-1) (corresponding to 90 ng per min and 100 g tissue). The net coronary t-PA release was 68 ng x min(-1) (30 ng x min(-1) X 100 g(-1)). Pulmonary net fluxes of t-PA were variable without any significant net t-PA release. The net hepatic uptake rate was 4,855 ng x min(-1) (436 ng x min(-1) x 100 g(-1)). Net trans-organ changes of active t-PA mirrored those of total t-PA. The results demonstrate marked regional differences in net release rates of t-PA in vivo. The experimental model we present offers new possibilities for evaluation of regional secretion patterns in the intact animal.

Clearance of tissue plasminogen activator (TPA) and TPA/plasminogen activator inhibitor type 1 (PAI-1) complex: relationship to elevated TPA antigen in patients with high PAI-1 activity levels.

BACKGROUND: To evaluate the effect of plasminogen activator inhibitor type 1 (PAI-1) levels on the clearance of total tissue plasminogen activator (TPA) antigen, we studied the clearance of active TPA and TPA/PAI-1 complex in subjects with low (181+/-109 pmol/L; n=7) and high (1166+/-322 pmol/L; n=4) baseline active PAI-1. METHODS AND RESULTS: A 5-microg/kg bolus of TPA was infused over a 15-second period followed by measurement of TPA activity, TPA antigen, TPA/PAI-1, TPA/C1 inhibitor, PAI-1 activity, and PAI-1 antigen over a 4-hour period. alpha-Phase clearance of total TPA antigen was faster in subjects with low PAI-1 (t(1/2) of 3.5+/-0.7 minutes) versus high PAI-1 (t(1/2) of 5.3+/-0.9 minutes) (P=.006). Clearance of all factors was best fit by a two-compartment pharmacokinetic model based on a computer-simulated human circulatory system. The average hepatic clearance fraction in the two-compartment model was greater for active TPA (89+/-10%, t(1/2) of 2.4+/-0.3 minutes) than for TPA/PAI-1 complex (48+/-17%, t(1/2) of 5.0+/-1.8 minutes) (P=.0006). CONCLUSIONS: Plasma clearance of active TPA was faster than clearance of TPA/PAI-1 complex. High levels of active PAI-1 converted more TPA into TPA/PAI-1 complex, effectively slowing the clearance of total TPA antigen and explaining in part why high levels of PAI-1 activity are associated with increases in total TPA antigen.

Efficiency of Ginkgo biloba extract (EGb 761) in antioxidant protection against myocardial ischemia and reperfusion injury.

The cardio-protective mechanisms of EGb 761, an extract of Ginkgo biloba leaves, on myocardial ischemia-reperfusion injury were investigated using rabbits subjected to 30 minutes of regional cardiac ischemia and 120 min of reperfusion under anesthesia. Compared to the saline perfused group, EGb 761 treatment (10 mg/kg, injected into the coronary artery) significantly inhibited the increase in lipid peroxidation and maintained total and CuZn-SOD levels in both plasma and tissue during and at the end of reperfusion. Both the decrease in tissue type plasminogen activator (t-PA) and the increase in plasminogen activator inhibitor-1 (PAI-1) caused by ischemia-reperfusion were also significantly suppressed by EGb 761 treatment. Furthermore, the ultrastructure of the myocytes of the EGb 761 treated heart was slightly damaged after ischemia-reperfusion, while the control ischemic-reperfused hearts demonstrated severe histological damages such as swelling and vacuolization of the mitochondria. These results suggest that EGb 761 protects hearts by its antioxidant properties and by its ability to adjust fibrinolytic activity.

Recombinant plasminogen activator inhibitor type 1: a review of structural, functional, and biological aspects.

Plasminogen activator inhibitor type 1 (PAI-1), a member of the serpin family of serine protease inhibitors, inhibits both tissue-type plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA). High PAI-1 levels are associated with an increased risk of thromboembolic disease while PAI-1 deficiency may represent an inherited autosomal recessive bleeding disorder. This review describes the biochemistry of PAI-1 including its purification, conversion between active and latent forms, and interaction with its target serine proteases and its protein cofactor, vitronectin. In addition, an overview of animal studies with PAI-1 is presented to examine its role in regulating fibrinolysis in vivo. For this review, particular emphasis is placed on studies with a recombinant form of bacterially expressed PAI-1 (rPAI-1), which shares many features in common with the active form of native PAI-1.