Nuala A. Booth

Plasminogen activator inhibitor (PAI-1) in plasma and platelets

The distribution of PAI‐1 in the plasma and platelets of normal individuals and of patients with platelet abnormalities was studied. An ELISA, capable of measuring PAI‐1 in plasma at 1.5 ng/ml, and a functional assay of t‐PA inhibition were used to assay platelet‐free plasma (PFP), platelet‐rich plasma in which the platelets were lysed (PRP) and serum. The PAI‐1 concentration of normal PFP was 21.0 ± 7.2 ng/ml (mean ± SD) and those of PRP and serum were 282.6 ± 68.0 and 270.3 ± 71.9 ng/ml. The concentration of PAI‐1 in PRP was proportional to the platelet count with 0.67 ± 0.18 ng/106 platelets. Patients with thrombocy‐topenia had approximately normal PAI‐1 concentrations in PFP; the extremely low concentrations in serum or PRP reflected the platelet count. A patient with grey platelet syndrome showed a comparable pattern, confirming that PAI‐1 occurs in the platelet α‐granules and indicating that the plasma concentration of PAI‐1 is independent of the platelet pool of PAI‐1. The median inhibitory activities towards t‐PA were 1.6, 8.7 and 8.3 units/ml in normal PFP. PRP and serum respectively. PAI‐1 in PFP had a median specific activity (units/mg PAI‐1) about 5‐fold higher than platelet PAI‐1. Plasma and platelets represent two distinct pools of PAI‐1, both of which should be considered in studies on the relationship between circulating PAI‐1 and thrombotic disease.

Candida albicans binds human plasminogen: identification of eight plasminogen-binding proteins

Several microbial pathogens augment their invasive potential by binding and activating human plasminogen to generate the proteolytic enzyme plasmin. Yeast cells and cell wall proteins (CWP) of the human pathogenic fungus Candida albicans bound plasminogen with a Kd of 70 ± 11 nM and 112 ± 20 nM respectively. Bound plasminogen could be activated to plasmin by mammalian plasminogen activators; no C. albicans plasminogen activator was detected. Binding of plasminogen to CWP and whole cells was inhibited by ɛACA, indicating that binding was predominantly to lysine residues. Candida albicans mutant strains defective in protein glycosylation did not show altered plasminogen binding, suggesting that binding was not mediated via a surface lectin. Binding was sensitive to digestion by basic carboxypeptidase, implicating C‐terminal lysine residues in binding. Proteomic analysis identified eight major plasminogen‐binding proteins in isolated CWP. Five of these (phosphoglycerate mutase, alcohol dehydrogenase, thioredoxin peroxidase, catalase, transcription elongation factor) had C‐terminal lysine residues and three (glyceraldehyde‐3‐phosphate dehydrogenase, phosphoglycerate kinase and fructose bisphosphate aldolase) did not. Activation of plasminogen could potentially increase the capacity of this pathogenic fungus for tissue invasion and necrosis. Although surface‐bound plasmin(ogen) degraded fibrin, no direct evidence for a role in invasion of endothelial matrix or in penetration and damage of endothelial cells was found.

Inhibitors of fibrinolysis are elevated in atherosclerotic plaque.

The proteins of the fibrinolytic system have been examined in the human normal and atherosclerotic arterial wall by immunohistochemical techniques and by quantitative immunoassay of extracts. The concentration of plasminogen activator inhibitor-1 (PAI-1) increased significantly during the progression from normal vessels to fatty streaks to the developed atherosclerotic plaque. Staining for PAI-1 was strongly positive, particularly in the areas adjacent to the plaque. In these areas, PAI-1 appeared to colocalized with its binding protein vitronectin. Alpha2-antiplasmin (alpha2-AP) was present in the aorta at even higher concentrations than PAI-1; a small but significant increase was seen in some atherosclerotic compared with normal vessel walls. Tissue plasminogen activator (TPA) showed the opposite trend, being lowest in lesions with plaque. Thus, higher concentrations of the two principal inhibitors of fibrinolysis, PAI-1 and alpha2-AP, together with lower levels of TPA, are characteristic of advanced atheromatous lesions. Alteration in the balance of the fibrinolytic system, favoring its inhibition, may predispose to the development or maintenance of atherosclerotic plaque.

Distribution of plasminogen activator inhibitor (PAI-1) in tissues.

Extracts of human tissue were analysed for plasminogen activator inhibitor (PAI-1) antigen and activity. PAI-1 was localised in tissues by an immunochemical method, using monoclonal antibodies. PAI-1 occurred throughout the body; its concentration and activity differed considerably from organ to organ. Extracts of liver and spleen had the greatest abundance of PAI-1, but the activity of the inhibitor was much higher in liver than in spleen: the liver may be a source of plasma PAI-1. Immunochemical staining for PAI-1 was observed in endothelium, platelets and their precursor cells, the megakaryocytes, and locations central to the process of haemostasis. PAI-1 also occurred in neutrophil polymorphs and macrophages, cells important in inflammatory and immune processes, but not in lymphocytes. Other cell types, in particular, vascular smooth muscle cells and mesangial cells, also stained positively for PAI-1 and such cells seem to represent an important reservoir of PAI-1.

From famine to feast: the role of methylglyoxal production in Escherichia coli

The enzyme methylglyoxal synthase (MGS) was partially purified from Escherichia coli extracts, and the amino‐terminal sequence of candidate proteins was determined, based on the native protein being a tetramer of about 69 kDa. Database analysis identified an open reading frame in the E. coli genome, YccG, corresponding to a protein of 16.9 kDa. When amplified and expressed from a controlled promoter, it yielded extracts that contained high levels of MGS activity. MGS expressed from the trc promoter accumulated to approximately 20% of total cell protein, representing approximately 900‐fold enhanced expression. This caused no detriment during growth on glucose, and the level of methylglyoxal (MG) in the medium rose to only 0.08 mM. High‐level expression of MGS severely compromised growth on xylose, arabinose and glycerol. A mutant lacking MGS was constructed, and it grew normally on a range of carbon sources and on low‐phosphate medium. However, the mutant failed to produce MG during growth on xylose in the presence of cAMP, and growth was inhibited.

The effects of chronic smoking on the fibrinolytic potential of plasma and platelets

We studied tissue‐type plasminogen activator (t‐PA) and plasminogen activator inhibitor type 1 (PAI‐1) in healthy individuals divided by smoking habit into current smokers, former smokers and non‐smokers (who had never smoked). Plasma PAI‐1 antigen was significantly higher in smokers than in non‐smokers with intermediate levels in former smokers. A similar trend was observed for plasma PAI activity but this did not reach statistical significance. Platelet PAI‐1 and plasma t‐PA were not significantly different when comparing the three groups. After venous occlusion t‐PA rose significantly in all groups; no significant change in plasma PAI‐1 was observed. The ratio of t‐PA to PAI‐1 in plasma was similar in non‐smokers and former smokers but lower in smokers, suggesting that there is at least partial restoration of plasma fibrinolytic potential after smoking cessation. Plasma PAI‐1 antigen and PAI activity correlated with estimated pack‐years of cigarettes smoked among smokers and former smokers. When all subjects were studied collectively, plasma PAI‐1 correlated strongly with plasma t‐PA and triglycerides; plasma t‐PA also correlated strongly with triglycerdes.

The antifibrinolytic function of factor XIII is exclusively expressed through α2-antiplasmin cross-linking

Factor XIII (FXIII) generates fibrin-fibrin and fibrin-inhibitor cross-links. Our flow model, which is sensitive to cross-linking, was used to assess the effects of FXIII and the fibrinolytic inhibitor, α₂-antiplasmin (α₂AP) on fibrinolysis. Plasma model thrombi formed from FXIII or α₂AP depleted plasma lysed at strikingly similar rates, 9-fold faster than pooled normal plasma (PNP). In contrast, no change was observed on depletion of PAI-1 or thrombin activatable fibrinolysis inhibitor (TAFI). Inhibition of FXIII did not further enhance lysis of α₂AP depleted thrombi. Addition of PNP to FXIII or α₂AP depleted plasmas normalized lysis. Lysis rate was strongly inversely correlated with total cross-linked α₂AP in plasma thrombi. Reconstitution of FXIII into depleted plasma stabilized plasma thrombi and normalized γ-dimers and α-polymers formation. However, the presence of a neutralizing antibody to α₂AP abolished this stabilization. Our data show that the antifibrinolytic function of FXIII is independent of fibrin-fibrin cross-linking and is expressed exclusively through α₂AP.

Cross-linking of Plasminogen Activator Inhibitor 2 and α2-Antiplasmin to Fibrin(ogen)

In this study, we identified lysine residues in the fibrinogen Aα chain that serve as substrates during transglutaminase (TG)-mediated cross-linking of plasminogen activator inhibitor 2 (PAI-2). Comparisons were made with α2-antiplasmin (α2-AP), which is known to cross-link to lysine 303 of the Aα chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to α2-AP but not for cross-linking to PAI-2. Further evidence that PAI-2 did not cross-link via Lys-303 was the cross-linking of PAI-2 to I-9 and des-αC fibrinogens, which lack 100 and 390 amino acids from the C terminus of the Aα chain, respectively. PAI-2 or α2-AP was cross-linked to fibrinogen and digested with trypsin or endopeptidase Glu-C, and the resulting peptides analyzed by mass spectrometry. Peptides detected were consistent with tissue TG (tTG)-mediated cross-linking of PAI-2 to lysines 148, 176, 183, 457 and factor XIIIa-mediated cross-linking of PAI-2 to lysines 148, 230, and 413 in the Aα chain. α2-AP was cross-linked only to lysine 303. Cross-linking of PAI-2 to fibrinogen did not compete with α2-AP, and the two proteins utilized different lysines in the Aα chain. Therefore, PAI-2 and α2-AP can cross-link simultaneously to the α polymers of a fibrin clot and promote resistance to lysis.

TAFIa, PAI‐1 and α2‐antiplasmin: complementary roles in regulating lysis of thrombi and plasma clots

Summary.  PAI‐1 and α2‐antiplasmin (α2AP) are the principal direct inhibitors of fibrinolytic proteases. Thrombin activatable fibrinolysis inhibitor (TAFI), a plasma procarboxypeptidase activated by thrombin–thrombomodulin to form TAFIa, also regulates fibrinolysis by modulating fibrin. In this study, the relative contributions of PAI‐1, α2AP and TAFIa to inhibition of lysis were assessed. In platelet‐poor plasma clots, α2AP, TAFIa and PAI‐1 all inhibited lysis, as shown by the addition of neutralizing antibodies to α2AP and PAI‐1 ± CPI, a potato carboxypeptidase inhibitor. α2AP played the largest role in regulating plasma clot lysis, but neutralization of inhibitors in combinations was more effective in shortening lysis times, with a maximal effect when all three inhibitors were neutralized. In platelet‐rich clots, a larger contribution of PAI‐1 was evident. Tissue plasminogen activator induced lysis of model thrombi, made from whole blood, was approximately doubled on incorporation of CPI, illustrating a substantial contribution of TAFIa to inhibition of thrombus lysis. Similar increases in thrombus lysis were observed on inclusion of neutralizing antibodies to PAI‐1 and α2AP, with α2AP playing the dominant role. Maximal thrombus lysis occurred upon neutralization of all three inhibitors. These observations suggest that, despite the differences in concentrations and activities of inhibitors, and the different modes of action, the roles of the three are complementary in both plasma clot lysis and thrombus lysis.

Metformin Causes a Reduction in Basal and Post‐venous Occlusion Plasminogen Activator Inhibitor‐1 in Type 2 Diabetic Patients

The effects of metformin on the fibrinolytic system were studied pre‐ and post‐venous occlusion in 38 Type 2 diabetic patients in a double‐blind, placebo‐controlled trial. After a 3‐week run‐in period, 21 patients received metformin and 17 placebo, for 6 weeks. In the metformin‐treated patients basal plasminogen activator inhibitor‐1 antigen (PAI‐1Ag) fell from 57.4 μg l−1 before treatment to 36.1 (p < 0.05) and 41.0 μg l−1 (p < 0.01) after 3 and 6 weeks therapy. In this group post‐venous occlusion PAI‐1Ag also fell after 3 weeks (p < 0.002) and 6 weeks (p < 0.05) treatment. There were no changes in either basal or post‐venous occlusion concentrations of PAI‐1Ag in the placebo treated group. The fall in PAI‐1Ag was not associated with an increase in basal plasminogen activator activity (PAA) which remained unchanged in both groups. Post‐venous occlusion values for PAA in the metformin treated patients were increased at 3 weeks (p < 0.05) although there was no difference at 6 weeks.