Thrombomodulin tightens the thrombin active site loops to promote protein C activation

Thrombomodulin (TM) forms a 1:1 complex with thrombin. Whereas thrombin alone cleaves fibrinogen to make the fibrin clot, the thrombin-TM complex cleaves protein C to initiate the anticoagulant pathway. Crystallographic investigations of the complex between thrombin and TMEGF456 did not show any changes in the thrombin active site. Therefore, research has focused recently on how TM may provide a docking site for the protein C substrate. Previous work, however, showed that when the thrombin active site was occupied with substrate analogues labeled with fluorophores, the fluorophores responded differently to active (TMEGF1-6) versus inactive (TMEGF56) fragments of TM. To investigate this further, we have carried out amide H/(2)H exchange experiments on thrombin in the presence of active (TMEGF45) and inactive (TMEGF56) fragments of TM. Both on-exchange and off-exchange experiments show changes in the thrombin active site loops, some of which are observed only when the active TM fragment is bound. These results are consistent with the previously observed fluorescence changes and point to a mechanism by which TM changes the thrombin substrate specificity in favor of protein C cleavage.     

Receptors that activate platelets.

This review highlights the increasing knowledge of the biochemistry, pathology, and cell and molecular biology of platelet receptors. A receptor for ADP has been identified using the affinity label FSBA as aggregin, a 100-kDa membrane protein responsible for shape change, aggregation, and exposure of fibrinogen binding sites. A variety of putative receptors for collagen have been described, with GPIa/IIa and GPIV receiving the most attention recently. A thromboxane A2 receptor has been identified using receptor antagonists and photoaffinity labels. The alpha 2-adrenergic receptor has been cloned and expressed. The platelet thrombin receptor has been tentatively identified as GPIb. Following binding of thrombin to this receptor, activation of calpain occurs, with cleavage of aggregin leading to exposure of GPIIb/III alpha and platelet aggregation. Isolation, expression, or both of the ADP, collagen, and thrombin receptors as single gene products of the human platelet responsible for activation, and more complete understanding of stimulus-response coupling, should allow for greater specificity of drugs with selective therapeutic actions.     

Thrombomodulin lacking the cytoplasmic domain efficiently internalizes thrombin via nonclathrin-coated,pit-mediated endocytosis

The plasminogen activation (PA) system of human Co 115 colon carcinoma cells was investigated. Analysis at the levels of protein and mRNA of cultured cells and of histozymography of tumor xenografts in nude mice showed that Co 115 cells produce only tissue type PA (t-PA) and no urokinase (u-PA). Also, mRNA for the u-PA receptor and for PA inhibitorr type 2 (PAI-2), but not for PAI-I, were detected. We developed a quantitative degradation assay using glutaraldehyde-immobilized¹²⁵I-Iaminin to investigate the capacity of Co 115 cells to degrade laminin. Laminin degradation by Co 115 cells was completely inhibited by 100 μg/ml of polyclonal anti-t-PA IgG, by the plasmin inhibitors aprotinin (100μg/ml) or ε-aminocaproic acid (EACA; at 0.3 M), but not by antibodies against u-PA or u-PAR nor by nonimmune IgG. Cycloheximide-treated Co 115 cells were unable to degrade laminin but increased laminin degradation induced by conditioned medium of Co 115 cells or recombinant t-PA. No potentiation was observed when Co 115 cells and laminin were kept separated by Transwell™ inserts. Our results suggest that Co 115 human colon carcinoma cells degrade laminin by potentiating t-PA-mediated plasminogen activaton at the cells surface which requires close contact between tumor cells and laminin substrate. © 1994 Wiley-Liss, Inc.     

Repeated stress alters the ability of nicotine to activate the hypothalamic-pituitary-adrenal axis

Acute nicotine administration has been shown to activate the hypothalamic-pituitary-adrenal (HPA) axis and stimulate secretion of adrenocorticotrophic hormone (ACTH), corticosterone/cortisol and beta-endorphin (beta-END) in both rodents and humans, raising the possibility that activation of the HPA axis by nicotine may mediate some of the effects of nicotine. Since stress can increase the risk of drug use and abuse, we hypothesized that repeated stress would increase the ability of nicotine to stimulate the secretion of HPA hormones. To test our hypothesis, mice were exposed to repeated stress (swimming in 15 degrees C water for 3 min/day for 5 days) and killed 15 min after injection of saline or nicotine (0.1 mg/kg, s.c.). Repeated exposure to stress increased the ability of nicotine to stimulate plasma ACTH (p<0.05) and beta-END (p<0.05), but not corticosterone secretion. In contrast, repeated exposure to stress increased the post-saline injection levels of corticosterone (p<0.05), but not ACTH and beta-END. The present results suggest that chronic stress leads to an enhanced sensitivity of some components of the HPA axis to a subsequent nicotine challenge.     

Identification of the phosphotyrosine proteome from thrombin activated platelets

Signalling cascades are regulated both positively and negatively by tyrosine phosphorylation. Integrin mediated platelet adhesion triggers signal transduction cascades involving translocation of proteins and tyrosine phosphorylation events, ultimately causing large signalling complexes to be assembled. In resting platelets, a small number of phosphorylated proteins are evident with molecular mass of 50-62 kDa and 120-130 kDa. In thrombin activated human platelets, however, there is a large increase in the number of tyrosine phosphorylated signalling proteins detected. These proteins include pCas (130 kDa), FAK (125 kDa), PI(3)k (85 kDa) and src (85 kDa). However, it is unlikely that this list of proteins represents all the dynamic changes that occur after platelet activation and it is understood that more proteins remain unidentified. In this study, we propose a method for the isolation of the phosphotyrosine proteome from both resting and thrombin activated human platelets. All the dynamic phosphotyrosine events that occur in the platelet after thrombin activation were isolated by immunoprecipitation, using the monoclonal antibody 4G10, allowing us to obtain higher concentrations of relatively low abundant proteins. The resulting phosphotyrosine proteomes were separated by two-dimensional gel electrophoresis. Sixty-seven proteins were reproducibly found to be unique in the thrombin activated platelet proteome when compared to resting platelets. We have positively identified ten of these proteins by Western blotting and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and these include FAK, Syk, ALK-4, P2X6 and MAPK kinase kinase. This proteomics approach to understanding the signalling events following platelet activation may elucidate potential drug targets for the treatment of coronary thrombosis.     

Benzylisoquinoline compounds inhibit the ability of calmodulin to activate cyclic nucleotide phosphodiesterase

Benzylisoquinoline compounds antagonised the ability of calmodulin (CaM) to stimulate the activity of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaM-PDE). This 'anti-CaM' activity was related to the hydrophobicity of the non-polar terminal region of the antagonist molecule. Antagonistic potency increased with the increase of hydrophobicity; the anti-CaM activity did not change when the polar terminus was a tertiary amine or quarternary amine. The anti-CaM potency was greater for bisbenzylisoquinoline compounds than for monobenzylisoquinoline compounds. Among the bisbenzylisoquinoline compounds anti-CaM pathway was: D3 greater than D2 berbamine greater than daurisoline greater than dauricine. Compound D3, which exhibited an IC50 value of 2.8 microM, was one of the most potent calmodulin antagonists, among benzylisoquinoline compounds, so far reported.     

Thrombomodulin changes the molecular surface of interaction and the rate of complex formation between thrombin and protein C

The interaction of thrombin with protein C triggers a key down-regulatory process of the coagulation cascade. Using a panel of 77 Ala mutants, we have mapped the epitope of thrombin recognizing protein C in the absence or presence of the cofactor thrombomodulin. Residues around the Na(+) site (Thr-172, Lys-224, Tyr-225, and Gly-226), the aryl binding site (Tyr-60a), the primary specificity pocket (Asp-189), and the oxyanion hole (Gly-193) hold most of the favorable contributions to protein C recognition by thrombin, whereas a patch of residues in the 30-loop (Arg-35 and Pro-37) and 60-loop (Phe-60h) regions produces unfavorable contributions to binding. The shape of the epitope changes drastically in the presence of thrombomodulin. The unfavorable contributions to binding disappear and the number of residues promoting the thrombin-protein C interaction is reduced to Tyr-60a and Asp-189. Kinetic studies of protein C activation as a function of temperature reveal that thrombomodulin increases >1,000-fold the rate of diffusion of protein C into the thrombin active site and lowers the activation barrier for this process by 4 kcal/mol. We propose that the mechanism of thrombomodulin action is to kinetically facilitate the productive encounter of thrombin and protein C and to allosterically change the conformation of the activation peptide of protein C for optimal presentation to the thrombin active site.     

Thrombomodulin enhances the reactivity of thrombin with protein C inhibitor by providing both a binding site for the serpin and allosterically modulating the activity of thrombin

Thrombomodulin (TM), or its epidermal growth factor-like domains 456 (TM456), enhances the catalytic efficiency of thrombin toward both protein C and protein C inhibitor (PCI) by 2-3 orders of magnitude. Structural and mutagenesis data have indicated that the interaction of basic residues of the heparin-binding exosite of protein C with the acidic residues of TM4 is partially responsible for the efficient activation of the substrate by the thrombin-TM456 complex. Similar to protein C, PCI has a basic exosite (H-helix) that constitutes the heparin-binding site of the serpin. To determine whether TM accelerates the reactivity of thrombin with PCI by providing a binding site for the H-helix of the serpin, an antithrombin (AT) mutant was constructed in which the H-helix of the serpin was replaced with the same region of PCI (AT-PCIH-helix). Unlike PCI, the H-helix of AT is negatively charged. It was discovered that TM456 slightly (<2-fold) impaired the reactivity of AT with thrombin; however, it enhanced the reactivity of AT-PCIH-helix with the protease by an order of magnitude. Further studies revealed that the substitution of Arg35 of thrombin with an Ala also resulted in an order of magnitude enhancement in reactivity of the protease with both PCI and AT-PCIH-helix independent of TM. We conclude that TM enhances the reactivity of PCI with thrombin by providing both a binding site for the serpin and a conformational modulation of the extended binding pocket of thrombin.     

Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets

The activation of human platelets by alpha-thrombin is mediated at least in part by cleavage of protease-activated G-protein-coupled receptors, PAR-1 and PAR-4. Platelet glycoprotein Ibalpha also has a high affinity binding site for alpha-thrombin, and this interaction contributes to platelet activation through a still unknown mechanism. In the present study the hypothesis that GpIbalpha may contribute to platelet activation by modulating the hydrolysis of PAR-1 on the platelet membrane was investigated. Gel-filtered platelets from normal individuals were stimulated by alpha-thrombin, and the kinetics of PAR-1 hydrolysis by enzyme was followed with flow cytometry using an anti-PAR-1 monoclonal antibody (SPAN 12) that recognizes only intact PAR-1 molecules. This strategy allowed measurement of the apparent k(cat)/K(m) value for thrombin hydrolysis of PAR-1 on intact platelets, which was equal to 1.5 +/- 0.1 x 10(7) m(-1) sec(-1). The hydrolysis rate of PAR-1 by thrombin was measured under conditions in which thrombin binding to GpIb was inhibited by different strategies, with the following results. 1) Elimination of GpIbalpha on platelet membranes by mocarhagin treatment reduced the k(cat)/K(m) value by about 6-fold. 2) A monoclonal anti-GpIb antibody reduced the apparent k(cat)/K(m) value by about 5-fold. 3) An oligonucleotide DNA aptamer, HD22, which binds to the thrombin heparin-binding site (HBS) and inhibits thrombin interaction with GpIbalpha, reduced the apparent k(cat)/K(m) value by about 5-fold. 4) Displacement of alpha-thrombin from the binding site on GpIb using PPACK-thrombin reduced the apparent k(cat)/K(m) value by about 5-fold, and 5) mutation at the HBS of thrombin (R98A) caused a 5-fold reduction of the apparent k(cat)/K(m) value of PAR-1 hydrolysis. Altogether these results show that thrombin interaction with GpIb enhances the specificity of thrombin cleavage of PAR-1 on intact platelets, suggesting that GpIb may function as a "cofactor" for PAR-1 activation by thrombin.     

Effect of thrombin on the radioactive nucleotides of human washed platelets

Radioactive ATP and ADP were found in platelets after incubation of human platelet-rich plasma with either [8-(14)C]adenosine or [8-(14)C]ADP. Treatment of the labelled and washed platelets with thrombin indicated that, though considerable amounts of ATP and ADP were released to the supernatant, radioactive ATP and ADP remained predominantly in the cellular fraction. Breakdown of radioactive ATP took place to form mainly IMP and hypoxanthine, the latter compound appearing in the supernatant. The results indicate the presence of at least two pools of nucleotide in platelets. Evidence is given that the two pools contain approximately the same amounts of ATP plus ADP, and that the ratio of ATP to ADP in the pool released to the supernatant by the action of thrombin is about 0.7-0.8.     

Characteristics of a thrombin inhibitor secreted by activated platelets

A 77-kDa complex of thrombin and a protein secreted by activated platelets had little if any thrombin amidolytic activity, indicating that the secreted protein is an inhibitor. The molecular weight of the inhibitor before reaction with thrombin was approximately 50,000. The apparent second-order rate constant for complex formation was estimated to be 1.3 x 10(6) M-1 s-1 (mean of four measurements); it was not affected by heparin or heparinase. These properties distinguish this inhibitor from other protease inhibitors secreted by platelets. The inhibitor reacted with trypsin and possibly with urokinase but not with factor Xa.     

Mechanisms of the ability of insulin to activate the glucose-transport system in rat adipocytes.

Isolated rat adipocytes were used to assess the mechanisms of the ability of insulin to accelerate glucose transport. Glucose transport was determined by measuring the initial rates of 2-deoxyglucose uptake, and at 24 degrees C insulin increased the Vmax. of transport from 7.3 +/- 1 to 23.1 +/- 2 nmol/min per 10(6) cells, but the Km value remained unchanged (2.5, cf. 2.4 mM). When the Vmax. of basal and insulin-stimulated transport was measured as a function of temperature (15-37 degrees C), parallel Arrhenius plots were obtained yielding equal activation energies of approx. 59kJ/mol. Since both processes have equal activation energies the data indicate that insulin increases Vmax. by increasing the number of available carriers rather than enhancing intrinsic activity of already functioning carriers. Since the ability of insulin to activate glucose transport did not decrease with temperature (whereas plasma-membrane fluidity declines), it is suggested that lateral diffusion of insulin receptors within the plasma-membrane bilayer is not a rat-determining step in insulin action.     

High affinity binding of thrombin to platelets. Inhibition by tetranitromethane and heparin

[¹²⁵I] iodo-α-thrombin has been modified at the macromolecular substrate binding site in order to study the importance of this region in the platelet-thrombin interaction. Modification was effected by the nitration of tyrosine residues with tetranitromethane. This chemical modification abolished the ability of the enzyme to bind with a high affinity to the platelet surface but did not significantly alter low affinity binding. The presence of heparin was also found to inhibit high affinity binding. These results indicate that the high affinity binding site interacts with the fibrinogen binding region of the thrombin molecule and suggests that there are two distinct classes of binding sites for thrombin on the platelet membrane.     

Preparation and application of a photoreactive thrombin analogue: binding to human platelets

alpha-Thrombin has previously been shown to bind to specific, saturable glycoproteins on the platelet surface. Modification of the thrombin active site with tosyllysyl chloromethyl ketone (TosLysCH2Cl) does not alter thrombin's binding characteristics. Interaction of alpha-thrombin with high-affinity binding sites (KD = 10(-9) M) initiates the platelet response which involves proteolytic hydrolysis of this glycoprotein. Although TosLysCH2Cl--thrombin binds to and competes for the same sites as alpha-thrombin, it cannot induce platelet stimulation because it is enzymatically inactive. In this study, we describe the preparation and application of photoreactive tritium-labeled thrombin analogues. The alpha-thrombin derivative retains its platelet-stimulating and enzymatic activities and, upon photoactivation, covalently binds to specific platelet membrane components. When freshly washed human platelets are exposed to less than saturation doses (less than or equal to 2 nM) of the thrombin derivatives in the dark and photoactivated, a single labeled complex is detected. The same experiment with greater than saturating doses (greater than or equal to 20 nM) of the thrombin derivative yields a similar complex as well as two additional ones. Molecular weight estimates of these thrombin-bound complexes were obtained by gel filtration and NaDodSO4--polyacrylamide gel electrophoresis. The low dose (high affinity) complex with TosLysCH2Cl--thrombin has an approximate molecular weight of 200 000, while that with active alpha-thrombin is smaller, approximately 120 000, due to enzymatic cleavage. The additional complexes detected with the high thrombin dose had estimated molecular weights of 400 000 and 46 000, respectively, and appeared to be the same for TosLysCH2Cl--thrombin and for the alpha-thrombin coupled platelets. These isolated complexes appear to correspond to the two previously detected populations of thrombin binding sites on the platelet.     

Preliminary studies on the ability of Drosophila microsomal preparations to activate mutagens and carcinogens

Subcellular fractions from Drosophila melanogaster, known to have several xenobiotic-metabolizing enzymatic activities, were investigated with respect to their ability to biotransform compounds that require metabolic activation before exerting mutagenic effects. Nitrofurazone, dimethylnitrosamine, cyclophosphamide and 2-acetylaminofluorene were activated to mutagens upon incubation with Drosophila microsomes or 20000 x g supernatant: mutagenicity was observed in Chinese hamster ovary cells, Escherichia coli strains 343/113/R-9 and 343/113/uvrB, and Salmonella typhimurium TA1538. Under the conditions used, microsomal preparations of Drosophila were not able to activate benzo[a]pyrene to a mutagen for Salmonella typhimurium TA98. The spectrum of mutagenic effects observed shows some correlation with the known mutagenicity of these compounds in vivo in Drosophila melanogaster. Drosophila microsomes appeared to be at least as active as rat-liver microsomes when compared in this type of mutagenicity testing.     

Cation gradient dependence of the steps in thrombin stimulation of human platelets

Thrombin causes a dose-dependent depolarization of the transmembrane potential of normal human platelets which can be continuously measured by the fluorescent probe, 3,3'-dipropylthiodicarbocyanine, whose distribution across the plasma membrane has been shown to be dependent upon the membrane potential. The dose-dependent depolarization of the platelet's negative membrane potential by thrombin is in large part due to a rapid uptake of sodium. Both the membrane potential change and the rapid sodium influx can be inhibited by a fast acting analog of amiloride, a sodium channel blocker, while valinomycin, a potassium ionophore, has no effect on the potential change nor on the sodium uptake, suggesting that the transmembrane potassium gradient is not important in the thrombin-induced depolarization. Neither the secretion of serotonin nor that of lysosomal enzymes nor the secondary release of the fluorescent probe which correlates with the lysosomal enzyme secretion occur if treatment with valinomycin precedes activation by thrombin. It is thus apparent that: 1) the change in the membrane potential induced by thrombin is directly dependent upon the transmembrane sodium gradient and is primarily due to a dose-dependent sodium uptake by the platelets; and 2) the thrombin-induced secretory processes are dependent upon maintenance of the transmembrane potassium gradients.     

The signal transduction induced by thrombin in human platelets

The stimulation of human platelets by thrombin leads to the activation of phospholipases C and A2, protein kinases, formation of 3-inositol phospholipids and mobilization of Ca2+. These biochemical reactions closely parallel platelet shape change, granular secretion and aggregation. The membrane-bound transducers for the thrombin receptor seem to be the heterotrimeric G protein Gi2 and the ras-related G protein rap 1-b. Phosphorylation of rap 1-b by the action of the cyclic AMP-dependent protein kinase seems to uncouple the thrombin receptor from phospholipases. This causes inhibition of the formation of second messenger molecules and the onset of physiological responses.