Norma R. Moore

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.

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.

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.

The platelet and plasma pools of plasminogen activator inhibitor (PAI-1) vary independently in disease

Summary. The relative importance and behaviour of plasma and platelet plasminogen activator inhibitor (PAI‐1) in disease has not hitherto been examined. In this study the concentration of PAI‐1 in the plasma and platelets of patients with a variety of disorders was examined using a specific ELISA and a functional assay. Mean plasma PAI‐1 was elevated in groups of patients with diabetes mellitus, hypertension, alcoholic cirrhosis, angina and myocardial infarction. Plasma PAI‐1 was raised in the post‐operative phase and the PAI‐1 released after surgery was not derived from platelets. In all groups PAI‐1 in the platelet pool reflected the platelet count, except in type II diabetes mellitus and chronic renal failure, where a reduced quantity of PAI‐1 antigen per platelet was found. In severe chronic renal failure, abnormal platelets and diminished platelet PAI‐1 may contribute to the haemorrhagic tendency sometimes seen in this disorder.

Regulation of plasminogen activation by TGF-β in cultured human retinal endothelial cells

BACKGROUND/AIMS Regulation of plasmin mediated extracellular matrix degradation by vascular endothelial cells is important in the development of angiogenesis. The aim was to determine whether transforming growth factor β (TGF-β) affected the regulation of components of the plasminogen system by human retinal endothelial cells, in order to define more clearly the role of TGF-β in retinal angiogenesis in the context of diabetes mellitus. METHODS Human retinal endothelial cells (HREC) were isolated from donor eyes and used between passages 4–8. The cells were cultured in medium supplemented with 2, 5, 15, or 25 mM glucose, plus or minus TGF-β (1 ng/ml). The concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and plasminogen activator inhibitor type 1 (PAI-1) in cell conditioned medium were determined by ELISA and the level of PAI-1 mRNA was determined using northern hybridisation. Cell associated plasminogen activity was determined using a clot lysis assay and a chromogenic assay. RESULTS Under basal conditions (5 mM glucose), HREC produced PAI-1, t-PA, and trace amounts of u-PA. Cell surface plasminogen activation observed by lysis of fibrin or by cleavage of chromogenic substrate, was mediated by t-PA. Glucose at varying concentrations (2–25 mM) had no significant effect on t-PA mediated clot lysis. In contrast, treatment with TGF-β resulted in increased synthesis of PAI-1 protein and mRNA. The increased expression of the PAI-1 mRNAs by TGF-β did not occur uniformly, the 2.3 kb mRNA transcript was preferentially increased in comparison with the 3.2 kb mRNA (p<0.05). CONCLUSIONS These data demonstrate that TGF-β increases PAI-1 and decreases cell associated lysis. This is sufficient to decrease the normal lytic potential of HREC.

The C-terminus of α2-antiplasmin interacts with endothelial cells

The serpin, α2‐antiplasmin (α2AP), has an extended C‐terminus relative to other inhibitors. This 51‐residue region contains an RGD sequence; such sequences constitute a key recognition sequence for cell adhesion, mediated through integrins. In the present study, this sequence was expressed in Escherichia coli and its binding to endothelial cells and whether binding depends on the RGD sequence was investigated. Binding to the surface of human umbilical vein endothelial cells (HUVEC‐C) was observed by flow cytometry and immunohistochemistry. Binding studies on immobilised cells showed specific and RGD‐dependent binding of the peptides to HUVEC‐C. The binding of the wild‐type peptide to the HUVEC‐C was significantly higher than that of a mutant peptide, in which RGD was replaced by SAA (P < 0·05, n = 4). Similarly, ethylenediaminetetraacetic acid decreased the binding of the wild‐type peptide (P < 0·05, n = 4). The binding was competed out by full‐length α2AP, fibronectin and anti‐α5β1. This is the first evidence of binding of the C‐terminus of α2AP to endothelial cells via its RGD sequence, with most but not all of the binding being integrin‐mediated. We speculate that this interaction with α2AP may potentially play a role in the control of cellular fibrinolysis by regulating local plasmin activity on cell surfaces.

Circulating tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in proliferative diabetic retinopathy: a pilot study

Abstract Several haemostatic abnormalities are associated with proliferative diabetic retinopathy. While abnormalities in plasma fibrinolytic activity have been described in diabetic retinopathy, platelets (a rich source of plasminogen activator inhibitor type 1, PAI-1) have received little attention. As a result, little is known about the fibrinolytic potential of circulating whole blood in diabetic retinopathy. The concentrations of tissue-type plasminogen activator (t-PA) and of its fast-acting inhibitor. PAI-1 were measured in plasma from eight patients with type 1 diabetes complicated by proliferative retinopathy, and from eight patients with type 1 diabetes and background or no retinopathy, matched for age, sex and duration of diabetes. The concentration of PAI-1 in platelets was also measured. The ratio of t-PTA to PAI-1 in plasma was significantly higher in patients with proliverative retinopathy than in those without (0.66 vs. 0.37, p < 0.02). The average quantitiy of PAI-1 per patelet was significantly lower in the group with proliferative retinopathy (0.33 vs. 0.50 ng/106 platelets, p < 0.02). These data suggest that among patients with type 1 diabetes, total circulating fibrinolytic potential is higher in those with proliferative retinopathy.

Does chronic smoking influence fibrinolytic potential in type 1 diabetes mellitus

Tissue‐type plasminogen activator (t‐PA) and plasminogen activator inhibitor (PAI‐1) were studied in 18 smokers and 18 closely matched non‐smokers, all of whom had Type 1 diabetes mellitus (DM). None of the patients had advanced complications of diabetes. The t‐PA and PAI‐1 antigen levels were measured in plasma before and after venous occlusion, and were normal in Type 1 diabetes regardless of smoking status. Platelet PAI‐1 levels were also measured and were found to be normal both in smokers and non‐smokers. In smokers with Type 1 DM, plasma PAI‐1 was significantly correlated with triglycerides. The normal fibrinolytic potential found in smokers with diabetes contrasts starkly with the significantly elevated plasma PAI‐1 reported in smokers without diabetes. In smokers, triglycerides may effect low levels of PAI‐1 release into plasma; this process may be significantly augmented in the presence of smoking‐induced insulin resistance. The lack of endogenous insulin release in Type 1 diabetes may obviate the characteristic rise in plasma PAI‐1 found in smokers who do not have diabetes.