Role of thrombin receptor in breast cancer invasiveness. (1/5662)

Invasion, the ability of an epithelial cancer cell to detach from and move through a basement membrane, is a central process in tumour metastasis. Two components of invasion are proteolysis of extracellular matrix and cellular movement through it. A potential promoter of these two processes is thrombin, the serine proteinase derived from the ubiquitous plasma protein prothrombin. Thrombin promotes the invasion of MDA-MB231 breast tumour cells (a highly aggressive cell line) in an in vitro assay. Invasion by MDA-MB436 and MCF-7 cells, less aggressive cell lines, is not promoted by thrombin. Thrombin, added to the cells, is a stimulator of cellular movement; fibroblast-conditioned medium is the chemotaxin. Thrombin-promoted invasion is inhibited by hirudin. Stimulation of invasion is a receptor-mediated process that is mimicked by a thrombin receptor-activating peptide. Thrombin has no effect on chemotaxis in vitro. Thrombin receptor is detectable on the surface of MDA-MB231 cells, but not on the other two cell lines. Introduction of oestrogen receptors into MDA-MB231 cells by transfection with pHEO had no effect on thrombin receptor expression, in the presence or absence of oestradiol. This paper demonstrates that thrombin increases invasion by the aggressive breast cancer cell line MDA-MB231 by a thrombin receptor-dependent mechanism.  (+info)

Activation of stimulus-specific serine esterases (proteases) in the initiation of platelet secretion. I. Demonstration with organophosphorus inhibitors. (2/5662)

The effect of organophosphorus inhibitors of serine esterases (proteases) on secretion from washed rabbit platelets was examined. Five noncytotoxic stimuli were employed: collagen, thrombin, heterologous anti-platelet antibody (in the absence of complement), rabbit C3 bound to zymosan, and platelet activating factor derived from antigen-stimulated, IgE-sensitized rabbit basophils. Diisoprophyl phosphofluoridate, three series of p-nitrophenyl ethyl phosphonates, and a series of cyclohexyl phenylalkylphosphonofluridates were all found to be inhibitory to the platelet secretion. These are irreversible inhibitors of serine proteases but in this system were only inhibitory if added to the platelets concurrently with the stimuli. Pretreatment of either the platelets or the stimuli with the inhibitors followed by washing, was without effect on the subsequent reaction. This suggested the involvement of stimulus-activatable serine proteases in the secretory process. The concept was supported by finding that nonphosphorylating phosphonates or hydrolyzed phosphonates or phosphonofluoridates were without inhibitory action. The effect of a series of phosphonates or phosphonoflouridates in inhibiting each stimulus exhibited a unique activity-structure profile. The demonstration of such unique profiles with four series of inhibitors for each of the five stimuli was interpreted as demonstrating that a specific activatable serine protease was involved in the platelet secretory response to each stimulus.  (+info)

Exosites 1 and 2 are essential for protection of fibrin-bound thrombin from heparin-catalyzed inhibition by antithrombin and heparin cofactor II. (3/5662)

Assembly of ternary thrombin-heparin-fibrin complexes, formed when fibrin binds to exosite 1 on thrombin and fibrin-bound heparin binds to exosite 2, produces a 58- and 247-fold reduction in the heparin-catalyzed rate of thrombin inhibition by antithrombin and heparin cofactor II, respectively. The greater reduction for heparin cofactor II reflects its requirement for access to exosite 1 during the inhibitory process. Protection from inhibition by antithrombin and heparin cofactor II requires ligation of both exosites 1 and 2 because minimal protection is seen when exosite 1 variants (gamma-thrombin and thrombin Quick 1) or an exosite 2 variant (Arg93 --> Ala, Arg97 --> Ala, and Arg101 --> Ala thrombin) is substituted for thrombin. Likewise, the rate of thrombin inhibition by the heparin-independent inhibitor, alpha1-antitrypsin Met358 --> Arg, is decreased less than 2-fold in the presence of soluble fibrin and heparin. In contrast, thrombin is protected from inhibition by a covalent antithrombin-heparin complex, suggesting that access of heparin to exosite 2 of thrombin is hampered when ternary complex formation occurs. These results reveal the importance of exosites 1 and 2 of thrombin in assembly of the ternary complex and the subsequent protection of thrombin from inhibition by heparin-catalyzed inhibitors.  (+info)

Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets. (4/5662)

Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the alpha-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Galphaq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72(syk) and stimulation of pp60(c-src) as well as in phosphorylation of myosin light chain (MLC) in Galphaq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Galphaq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase-mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase-dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change.  (+info)

Regulation of the activities of thrombin and plasmin by cholesterol sulfate as a physiological inhibitor in human plasma. (5/5662)

Thrombin and plasmin, both of which are serine proteases in the plasma of vertebrates, play essential roles in blood clotting and fibrinolysis, respectively, and regulation of their activities is important to suppress the excessive reactions within the vascular network and to prevent tissue injury. Along with the peptidic inhibitors belonging to the serpin family, we found that cholesterol sulfate (CS), which is present at the concentration of 2.0+/-1.2 nmol/ml in human plasma, was a potent inhibitor of both plasma thrombin and plasmin. Thrombin, as determined both using a chromogenic substrate and the natural substrate, fibrinogen, was inactivated upon reaction with CS in a dose-dependent manner, but not in the presence of the structurally related steroid sulfates, I3SO3-GalCer and II3NAalpha-LacCer, suggesting that both the sulfate group and the hydrophobic side chain of CS are necessary for the inhibitory activity of CS. Preincubation of thrombin with CS at 37 degrees C for 10 min was required to achieve maximum inhibition, and virtually complete inhibition was achieved at a molar ratio of CS to thrombin of 18:1. CS-treated thrombin had the same Km and a lower Vmax than the original enzyme, and a higher molecular weight. The molecular weight and activity of the original enzyme were not observed on the attempted separation of the CS-treated enzyme by gel permeation chromatography and native PAGE, indicating that the inactivation of thrombin by CS is irreversible. In contrast, CS was readily liberated from the enzyme by SDS-PAGE, suggesting that hydrophobic interactions are involved in the CS-mediated inactivation of thrombin. When acidic lipids were reacted with thrombin after dissolving them in DMSO, I3SO3-GalCer, steroid sulfates and II3NAalpha-LacCer, as well as CS, but not SDS and sodium taurocholate, exhibited inhibitory activity, probably due to micellar formation facilitating interaction between thrombin and negatively charged lipids. On the other hand, plasmin, as determined using a chromogenic substrate, was more susceptible to acidic lipids than thrombin. CS, I3SO3-GalCer and II3NAalpha-LacCer, all of which are present in serum, inhibited the activity of plasmin in aqueous media, as well as in DMSO-mediated lipid solutions. Thus, acidic lipids in plasma were demonstrated to possess regulatory activity as endogenous detergents toward both enzymes for blood clotting and fibrinolysis.  (+info)

Unexpected crucial role of residue 225 in serine proteases. (6/5662)

Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively found in vertebrates, whereas proteases with P225 (like degradative enzymes) are present from bacteria to human. Saturation mutagenesis of Y225 in thrombin shows that residue 225 affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe, all residues are associated with comparable or greatly reduced catalytic activity relative to Pro. The crystal structures of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically influences the shape of the water channel around the primary specificity site. The activity profiles obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe documented in the vertebrates occurred through Ser and was driven by a significant gain (up to 50-fold) in catalytic activity. In fact, Ser and Phe are documented in 4% of serine proteases, which together with Pro and Tyr account for almost the entire distribution of residues at position 225. The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activity and specificity are more complex than currently believed. These findings have broad implications in the rational design of enzymes with enhanced catalytic properties.  (+info)

Injury-induced gelatinase and thrombin-like activities in regenerating and nonregenerating nervous systems. (7/5662)

It is now widely accepted that injured nerves, like any other injured tissue, need assistance from their extracellular milieu in order to heal. We compared the postinjury activities of thrombin and gelatinases, two types of proteolytic activities known to be critically involved in tissue healing, in nonregenerative (rat optic nerve) and regenerative (fish optic nerve and rat sciatic nerve) neural tissue. Unlike gelatinases, whose induction pattern was comparable in all three nerves, thrombin-like activity differed clearly between regenerating and nonregenerating nervous systems. Postinjury levels of this latter activity seem to dictate whether it will display beneficial or detrimental effects on the capacity of the tissue for repair. The results of this study further highlight the fact that tissue repair and nerve regeneration are closely linked and that substances that are not unique to the nervous system, but participate in wound healing in general, are also crucial for regeneration or its failure in the nervous system.  (+info)

Platelet aggregation and incident ischaemic heart disease in the Caerphilly cohort. (8/5662)

BACKGROUND: Platelets are involved in myocardial infarction but evidence of prediction of infarction by measures of platelet function are sparce. METHODS: Platelet aggregation to thrombin and to ADP in platelet rich plasma was recorded for 2176 men aged 49-65 years in the Caerphilly cohort study. RESULTS: Results from 364 men were excluded, 80 of whom had not fasted before venepuncture; most of the others were excluded because antiplatelet medication had been taken shortly before the platelet tests. During the five years following the platelet tests 113 ischaemic heart disease (IHD) events which fulfilled the World Health Organisation criteria were identified--42 fatal and 71 non-fatal. No measure of platelet aggregation was found to be significantly predictive of incident IHD. The possibility that platelet function is predictive for only a limited time after it is characterised, and that prediction falls off with time, was tested. When IHD events are grouped by their time of occurrence after aggregation had been measured, the test results show a gradient suggestive of prediction of early IHD events. Thus, 24% of the men who had an event within 500 days of the test had had a high secondary response to ADP while only 12% of those whose IHD event had been 1000 or more days after the test had shown a high platelet response at baseline. The trend in these proportions is not significant. CONCLUSIONS: Platelet aggregation to thrombin and ADP in platelet rich plasma was recorded in the Caerphilly cohort study. No measure of aggregation was found to be predictive of IHD.  (+info)