Impaired plasminogen binding in patients with venous thromboembolism: Association with protein carbonylation.

INTRODUCTION: Venous thromboembolism (VTE) is associated with hypofibrinolysis. Its mechanisms are poorly understood. We investigated plasminogen-fibrin interaction and its association with fibrinolytic capacity and protein oxidation/carbonylation in VTE patients. MATERIALS AND METHODS: Plasma-purified plasminogen conversion to plasmin and surface plasmon resonance employed for plasminogen-fibrin interactions were individually evaluated in all healthy controls and non-anticoagulated patients following VTE, 10-23months since the event. We also assessed plasma fibrin clot permeability (Ks), clot lysis time (LT), activators and inhibitors of fibrinolysis together with oxidation/carbonylation markers. RESULTS: VTE patients had impaired plasminogen binding to fibrin (apparent Kd, +290%, p=0.002), reduced rate of plasmin generation (-4.7%, p=0.001), and longer LT (+18.6%, p<0.001) compared with controls. Fibrinogen and Ks were similar in both groups. Apparent Kd correlated with LT (r=0.43, p=0.037), tissue plasminogen activator-plasminogen activator inhibitor 1 (tPA-PAI-1) complexes (r=0.63, p=0.012), and active PAI-1 (r=0.49, p=0.03). Compared with controls, VTE patients had higher thiobarbituric acid reactive substances (TBARS), total protein carbonyl content (PC), and lower total antioxidant capacity (all p<0.001), that all were associated with LT (r=0.61, r=0.56, and r=-0.47, respectively, all p<0.05). Impaired plasminogen binding to fibrin reflected by apparent Kd positively correlated with TBARS (r=0.48, p=0.032) and PC (r=0.54, p=0.013) in the whole group. CONCLUSIONS: Plasminogen-fibrin interactions are altered in young and middle-aged VTE patients, without known thrombophilias, except increased factor VIII. The mechanisms underlying these phenomena remain to be established.

Characterization of the interactions between human high-molecular-mass kininogen and cell wall proteins of pathogenic yeasts Candida tropicalis.

Candida tropicalis is one of the most frequent causes of serious disseminated candidiasis in human patients infected by non-albicans Candida species, but still relatively little is known about its virulence mechanisms. In our current study, the interactions between the cell surface of this species and a multifunctional human protein - high-molecular-mass kininogen (HK), an important component of the plasma contact system involved in the development of the inflammatory state - were characterized at the molecular level. The quick release of biologically active kinins from candidal cell wall-adsorbed HK was presented and the HK-binding ability was assigned to several cell wall-associated proteins. The predicted hyphally regulated cell wall protein (Hyr) and some housekeeping enzymes exposed at the cell surface (known as "moonlighting proteins") were found to be the major HK binders. Accordingly, after purification of selected proteins, the dissociation constants of the complexes of HK with Hyr, enolase, and phosphoglycerate mutase were determined using surface plasmon resonance measurements, yielding the values of 2.20 × 10(-7) M, 1.42 × 10(-7) M, and 5.81 × 10(-7) M, respectively. Therefore, in this work, for the first time, the interactions between C. tropicalis cell wall proteins and HK were characterized in molecular terms. Our findings may be useful for designing more effective prevention and treatment approaches against infections caused by this dangerous fungal pathogen.

Kinetic and thermodynamic characterization of the interactions between the components of human plasma kinin-forming system and isolated and purified cell wall proteins of Candida albicans.

Cell wall proteins of Candida albicans, besides their best known role in the adhesion of this fungal pathogen to host's tissues, also bind some soluble proteins, present in body fluids and involved in maintaining the biochemical homeostasis of the human organism. In particular, three plasma factors - high-molecular-mass kininogen (HK), factor XII (FXII) and prekallikrein (PPK) - have been shown to adhere to candidal cells. These proteins are involved in the surface-contact-catalyzed production of bradykinin-related peptides (kinins) that contribute to inflammatory states associated with microbial infections. We recently identified several proteins, associated with the candidal cell walls, and probably involved in the binding of HK. In our present study, a list of potential FXII- and PPK-binding proteins was proposed, using an affinity selection (on agarose-coupled FXII or PPK) from a whole mixture of ß-1,3-glucanase-extrated cell wall-associated proteins and the mass-spectrometry protein identification. Five of these fungal proteins, including agglutinin-like sequence protein 3 (Als3), triosephosphate isomerase 1 (Tpi1), enolase 1 (Eno1), phosphoglycerate mutase 1 (Gpm1) and glucose-6-phosphate isomerase 1 (Gpi1), were purified and characterized in terms of affinities to the human contact factors, using the surface plasmon resonance measurements. Except Gpm1 that bound only PPK, and Als3 that exhibited an affinity to HK and FXII, the other isolated proteins interacted with all three contact factors. The determined dissociation constants for the identified protein complexes were of 10(-7) M order, and the association rate constants were in a range of 10(4)-10(5) M(-1)s(-1). The identified fungal pathogen-host protein interactions are potential targets for novel anticandidal therapeutic approaches.