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Kinetic studies on the effect of heparin and fibrin on plasminogen activators.

ABSTRACT: 1. Possible interactions between fibrin(ogen) and heparin in the control of plasminogen activation were studied in model systems using the thrombolytic agents tissue-type plasminogen activator (t-PA), urokinase and streptokinase.plasminogen activator complex and the substrates Glu- and Lys-plasminogen. 2. Both t-PA and urokinase activities were promoted by heparin and by pentosan polysulphate, but not by chondroitin sulphate or hyaluronic acid. The effect was on Km. 3. In the presence of soluble fibrin (and its mimic, CNBr-digested fibrinogen) the effect of heparin on t-PA was attenuated, although not abolished. In studies using a monoclonal antibody and 6-aminohexanoic acid, it was found that heparin and fibrin did not seem to share a binding site on t-PA. 4. The activity of t-PA B-chain was unaffected by heparin, so the binding site is located on the A-chain of t-PA (and urokinase). 5. Fibrin potentiated the activity of heparin on urokinase. The activity of streptokinase.plasminogen was unaffected by heparin whether or not fibrin was present. 6. If these influences of heparin and fibrin also occur in vivo, then, in the presence of heparin, the relative fibrin enhancement of t-PA will be diminished and the likelihood of systemic activation by t-PA is increased.

SUBMITTER: Fears R

PROVIDER: S-EPMC1148668 | biostudies-other | 1988 Jan

REPOSITORIES: biostudies-other

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Kinetic studies on the interaction of streptokinase and other plasminogen activators with plasminogen and fibrin.

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Crystal Structure of the Michaelis Complex between Tissue-type Plasminogen Activator and Plasminogen Activators Inhibitor-1.

Project description:Thrombosis is a leading cause of death worldwide. Recombinant tissue-type plasminogen activator (tPA) is the Food and Drug Administration-approved thrombolytic drug. tPA is rapidly inactivated by endogenous plasminogen activator inhibitor-1 (PAI-1). Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort. Precise details, with atomic resolution, of the molecular interactions between tPA and PAI-1 remain unknown despite previous extensive studies. Here, we report the crystal structure of the tPA·PAI-1 Michaelis complex, which shows significant differences from the structure of its urokinase-type plasminogen activator analogue, the uPA·PAI-1 Michaelis complex. The PAI-1 reactive center loop adopts a unique kinked conformation. The structure provides detailed interactions between tPA 37- and 60-loops with PAI-1. On the tPA side, the S2 and S1β pockets open up to accommodate PAI-1. This study provides structural basis to understand the specificity of PAI-1 and to design newer generation of thrombolytic agents with reduced PAI-1 inactivation.

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Project description:Accumulation and deposition of Aβ is one of the main neuropathological hallmarks of Alzheimer's disease (AD) and impaired Aβ degradation may be one mechanism of accumulation. Plasmin is the key protease of the plasminogen system and can cleave Aβ. Plasmin is activated from plasminogen by tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). The activators are regulated by inhibitors which include plasminogen activator inhibitor-1 (PAI-1) and neuroserpin. Plasmin is also regulated by inhibitors including α2-antiplasmin and α2-macroglobulin. Here, we investigate the mRNA levels of the activators and inhibitors of the plasminogen system and the protein levels of tPA, neuroserpin and α2-antiplasmin in post-mortem AD and control brain tissue. Distribution of the activators and inhibitors in human brain sections was assessed by immunoperoxidase staining. mRNA measurements were made in 20 AD and 20 control brains by real-time PCR. In an expanded cohort of 38 AD and 38 control brains tPA, neuroserpin and α2-antiplasmin protein levels were measured by ELISA. The activators and inhibitors were present mainly in neurons and α2-antiplasmin was also associated with Aβ plaques in AD brain tissue. tPA, uPA, PAI-1 and α2-antiplasmin mRNA were all significantly increased in AD compared to controls, as were tPA and α2-antiplasmin protein, whereas neuroserpin mRNA and protein were significantly reduced. α2-macroglobulin mRNA was not significantly altered in AD. The increases in tPA, uPA, PAI-1 and α2-antiplasmin may counteract each other so that plasmin activity is not significantly altered in AD, but increased tPA may also affect synaptic plasticity, excitotoxic neuronal death and apoptosis.

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Fibrin-Enhanced Canonical Wnt Signaling Directs Plasminogen Expression in Cementoblasts.

Project description:Cementum is a mineralized layer on the tooth's root surface and facilitates the biomechanical anchoring of fibrous connective tissues as a part of tooth-supportive complexes. Previously, we observed that OCCM30 cementoblasts cultured on fibrin matrices underwent apoptosis due to fibrin degradation through the expression of proteases. Here, we demonstrated that OCCM30 on fibrin matrices (OCCM30-fibrin) enhanced canonical Wnt signaling, which directed to plasminogen expression. The OCCM30-fibrin showed higher levels of Wnt3a expression, nuclear translocation of ?-catenin, and T-cell factor (TCF) optimal motif (TOP) reporter activity than the cells on tissue culture dishes (OCCM30-TCD), indicating that the OCCM30-fibrin enhanced canonical Wnt/?-catenin signaling. Also, OCCM30-fibrin expressed biomineralization-associated markers at higher levels than OCCM30-TCD, of which levels were further increased with LiCl, a Wnt signaling activator. The OCCM30 cementoblasts simultaneously showed that high levels of plasminogen, a critical component of fibrinolysis, were expressed in the OCCM30-fibrin. Activation of canonical Wnt signaling with LiCl treatment or with forced lymphoid enhancer factor 1 (LEF1)-expression increased the expression of plasminogen. On the contrary, the inhibition of canonical Wnt signaling with siRNAs against Wnt3a or ?-catenin abrogated fibrin-enhanced plasminogen expression. Furthermore, there are three conserved putative response elements for the LEF1/?-catenin complex in the plasminogen proximal promoter regions (-900 to +54). Site-directed mutations and chromatin immunoprecipitation indicated that canonical Wnt signaling directed plasminogen expression. Taken together, this study suggests that fibrin-based materials can modulate functional periodontal formations in controlling cementoblast differentiation and fibrin degradation.

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Purification of plasminogen activators from human seminal plasma.

Project description:Two plasminogen activators (1 and 2) were isolated from human seminal plasma by hiigh-speed centrifugation, Sephadex-gel filtration and ion-exchange chromatography. The activators were shown to be homogeneous by polyacrylamide-disc -gel electrophoresis at pH 8.3 and 4.5, and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weights of activators 1 and 2 were estimated as 69 000 and 74 000. Their amino acid compositions are very similar, both being high in aspartic acid, glutamic acid, serine, glycine and leucine, and low in methionine, tryptophan, tyrosine, isoleucine and histidine. Activators 1 and 2 each possess 16 cysteine residues. Both activators have isoelectric points of approx. 7.0, are stable over a wide pH range at temperatures up to 60 degrees C, but lose activity at higher temperatures, particularly under very basic or acidic conditions. They are not inhibited by EDTA, Mg2+ and Ca2+ at 10 mM concentrations, but their activity decreases on addition of 10 mM-cysteine or Fe2+ and 6-aminohexanoate or sera from pregnant women. The precipitin band formed between urokinase and its antiserum is continuous with the precipitin bands formed between the seminal plasminogen activators and the urokinase antiserum. Antisera to urokinase inhibit both the activity of urokinase and the seminal plasminogen activators.

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