Peptides as inhibitors of thrombin coagulation activity and of thrombocyte aggregation]

The analysis of literature and our own data of regulatory peptides influence on the blood coagulation system is presenting. Various natural and synthetic peptides inhibit the activity of thrombin and platelet aggregation. Direct specific inhibitors of thrombin are peptides developed on the base of D-Phe-Pro-Arg sequence. Strong specific inhibitors of the prothrombinase complex factor Xa were isolated from tissues and saliva of the blood-sucking organisms. These inhibitors decrease thrombin generation at the early stage of blood coagulation cascade Anticoagulating peptides from the tick Ornithodoros moubata tissue (TAP), the recombinant rTAP from the saliva glands of tick Ornithodoros savignyi and peptide with even greater anticoagulating activity from saliva glands of fly Glossina morsitans morsitans were isolated and characterised. For complete and reliable suppression of thrombus formation simultaneous administration of thrombin and platelet aggregation inhibitors is necessary. Main terminal stage of platelet aggregation is the interaction of receptor GP IIb/IIIa with adhesive fibrinogen sequence Arg-Pro-Asp (RGD). Peptides derived on the base of this sequence compete with fibrinogen in reaction with platelet receptors. A lot of corresponding peptidomimetics were synthesised, e.g. MK-852, RO-44 and particularly effective compound integrelin. Many direct platelet aggregation inhibitors were found in snake venoms. Recombinant peptide TAP mentioned above exerts both antithrombin and antiaggregation activity. Peptides and peptide mimetics of this type rapidly and irreversibly bound with receptor GP IIb/IIIa. They have short half life time in the blood plasma. Their preference in comparison with other drugs is particularly rapid and strong action. In our experiments it was demonstrated, that simple proline-containing peptides Pro-Gly, Trp-Pro, Pro-Gly-Pro (putative fragments of collagen and elastin) possesses significant antithrombotic and anticoagulant potential in vitro and in in vivo. Perhaps these peptides are members on intrinsic complex of haemostasis regulators.     

Effect of neonatal androgenization on positive feedback in female mice

Exposure of female mice to androgens within 5 days of birth impairs fertility. Such treatment in rats results in a post-pubertal acyclic state of persistent vaginal cornification and in an inability, when ovariectomized, to show normal positive feedback on luteinizing hormone (LH) release in response to steroid challenge. In the present study, we explored whether neonatally androgenized mice demonstrate positive feedback. Female mice were administered 100 micrograms of testosterone propionate (TP) on either Day 1 (TP1) or Day 5 (TP5) after birth, or vehicle on Day 1 (SO1). Androgen-treated mice had a statistically significant advance in onset of vaginal opening as compared with vehicle-treated mice. All mice that received TP entered constant vaginal estrus, whereas those given vehicle showed variable cytology. All mice were ovariectomized at 7 wk of age and received Silastic capsules containing a priming dose of 17 beta-estradiol. When all mice were challenged 1 wk later with sequential administration of estradiol benzoate and progesterone, a significant increase in plasma LH level was present only in the vehicle-treated mice. We conclude that neonatal androgenization defeminizes the neuroendocrine mechanisms controlling gonadotropin release.     

Rewiring mitogen-activated protein kinase cascade by positive feedback confers potato blight resistance

Late blight, caused by the notorious pathogen Phytophthora infestans, is a devastating disease of potato (Solanum tuberosum) and tomato (Solanum lycopersicum), and during the 1840s caused the Irish potato famine and over one million fatalities. Currently, grown potato cultivars lack adequate blight tolerance. Earlier cultivars bred for resistance used disease resistance genes that confer immunity only to some strains of the pathogen harboring corresponding avirulence gene. Specific resistance gene-mediated immunity and chemical controls are rapidly overcome in the field when new pathogen races arise through mutation, recombination, or migration from elsewhere. A mitogen-activated protein kinase (MAPK) cascade plays a pivotal role in plant innate immunity. Here we show that the transgenic potato plants that carry a constitutively active form of MAPK kinase driven by a pathogen-inducible promoter of potato showed high resistance to early blight pathogen Alternaria solani as well as P. infestans. The pathogen attack provoked defense-related MAPK activation followed by induction of NADPH oxidase gene expression, which is implicated in reactive oxygen species production, and resulted in hypersensitive response-like phenotype. We propose that enhancing disease resistance through altered regulation of plant defense mechanisms should be more durable and publicly acceptable than engineering overexpression of antimicrobial proteins.     

Effect of thrombomodulin on specific thrombin functions: fibrinogen coagulation and thrombocyte aggregation

The effect of thrombomodulin (TM), prepared from rabbit lungs, on fibrinogen clotting and platelets aggregation by alpha-thrombin has been investigated. It has been established that TM caused a dose-dependent decrease in fibrinogen-clotting activity of thrombin (Ki = 14.7 +/- 1.24 nM). TM was shown to reduce thrombin-induced platelet aggregation but not to alter ADP-induced one. It was found that the kinetic parameters for hydrolysis of synthetic substrates by alpha-thrombin were not altered by TM.     

Oscillations in MAPK cascade triggered by two distinct designs of coupled positive and negative feedback loops

ABSTRACT: BACKGROUND: Feedback loops, both positive and negative are embedded in the Mitogen Activated Protein Kinase (MAPK) cascade. In the three layer MAPK cascade, both feedback loops originate from the terminal layer and their sites of action are either of the two upstream layers. Recent studies have shown that the cascade uses coupled positive and negative feedback loops in generating oscillations. Two plausible designs of coupled positive and negative feedback loops can be elucidated from the literature; in one design the positive feedback precedes the negative feedback in the direction of signal flow and vice-versa in another. But it remains unexplored how the two designs contribute towards triggering oscillations in MAPK cascade. Thus it is also not known how amplitude, frequency, robustness or nature (analogous/digital) of the oscillations would be shaped by these two designs. RESULTS: We built two models of MAPK cascade that exhibited oscillations as function of two underlying designs of coupled positive and negative feedback loops. Frequency, amplitude and nature (digital/analogous) of oscillations were found to be differentially determined by each design. It was observed that the positive feedback emerging from an oscillating MAPK cascade and functional in an external signal processing module can trigger oscillations in the target module, provided that the target module satisfy certain parametric requirements. The augmentation of the two models was done to incorporate the nuclear-cytoplasmic shuttling of cascade components followed by induction of a nuclear phosphatase. It revealed that the fate of oscillations in the MAPK cascade is governed by the feedback designs. Oscillations were unaffected due to nuclear compartmentalization owing to one design but were completely abolished in the other case. CONCLUSION: The MAPK cascade can utilize two distinct designs of coupled positive and negative feedback loops to trigger oscillations. The amplitude, frequency and robustness of the oscillations in presence or absence of nuclear compartmentalization were differentially determined by two designs of coupled positive and negative feedback loops. A positive feedback from an oscillating MAPK cascade was shown to induce oscillations in an external signal processing module, uncovering a novel regulatory aspect of MAPK signal processing.     

Development of inhibitors in the ubiquitination cascade

The ubiquitin proteasome system (UPS) is essential in regulating myriad aspects of protein functions. It is therefore a fundamentally important regulatory mechanism that impacts most if not all aspects of cellular processes. Indeed, malfunction of UPS components is implicated in human diseases such as neurodegenerative and immunological disorders and many cancers. The success of proteasome inhibitors in cancer therapy suggests that modulating enzymes in the ubiquitination cascade would be clinically important for therapeutic benefits. In this review, we summarize advances in developing inhibitors of a variety of UPS components. In particular, we highlight recent work done on the protein engineering of ubiquitin as modulators of the UPS, a novel approach that may shed light on innovative drug discovery in the future.     

Structural basis for inhibition promiscuity of dual specific thrombin and factor Xa blood coagulation inhibitors

BACKGROUND: A major current focus of pharmaceutical research is the development of selective inhibitors of the blood coagulation enzymes thrombin or factor Xa to be used as orally bioavailable anticoagulant drugs in thromboembolic disorders and in the prevention of venous and arterial thrombosis. Simultaneous direct inhibition of thrombin and factor Xa by synthetic proteinase inhibitors as a novel approach to antithrombotic therapy could result in potent anticoagulants with improved pharmacological properties. RESULTS: The binding mode of such dual specific inhibitors of thrombin and factor Xa was determined for the first time by comparative crystallography using human alpha-thrombin, human des-Gla (1--44) factor Xa and bovine trypsin as the ligand receptors. The benzamidine-based inhibitors utilize two different conformations for the interaction with thrombin and factor Xa/trypsin, which are evoked by the steric requirements of the topologically different S2 subsites of the enzymes. Compared to the unliganded forms of the proteinases, ligand binding induces conformational adjustments of thrombin and factor Xa active site residues indicative of a pronounced induced fit mechanism. CONCLUSION: The structural data reveal the molecular basis for a desired unselective inhibition of the two key components of the blood coagulation cascade. The 4-(1-methyl-benzimidazole-2-yl)-methylamino-benzamidine moieties of the inhibitors are able to fill both the small solvent accessible as well as the larger hydrophobic S2 pockets of factor Xa and thrombin, respectively. Distal fragments of the inhibitors are identified which fit into both the cation hole/aromatic box of factor Xa and the hydrophobic aryl binding site of thrombin. Thus, binding constants in the medium-to-low nanomolar range are obtained against both enzymes.     

Blood flow controls coagulation onset via the positive feedback of factor VII activation by factor Xa

Background  

Blood coagulation is a complex network of biochemical reactions, which is peculiar in that it is time- and space-dependent, and has to function in the presence of rapid flow. Recent experimental reports suggest that flow plays a significant role in its regulation. The objective of this study was to use systems biology techniques to investigate this regulation and to identify mechanisms creating a flow-dependent switch in the coagulation onset.     

Introduction to the blood coagulation cascade and cloning of blood coagulation factors

The coagulation cascade that occurs in mammalian plasma involves a large number of plasma proteins that participate in a stepwise manner and eventually give rise to the formation of thrombin. This enzyme then converts fibrinogen to an insoluble fibrin clot. This series of reactions involves a number of glycoproteins that particupate as enzymes as well as cofactors. These proteins that circulate in the blood in a precursor or zymogen form are multifunctional proteins that share many common segments or domains. One group includes the vitamin K-dependent glycoproteins (prothrombin, factor IX, factor X, and protein C) that show considerable homology in both their amino acid sequences and their gene structures. The proteins that participate in the contact or early phase of the blood coagulation cascade include plasma prekallikrein, factor XII, and factor IX. The amino-terminal regions of both factor XI and plasma prekallikrein contain four tandem repeats of about 90 amino acids, and these tandem repeats show considerable amino acid sequence homology. Factor XII contains four different domains in the amino-terminai region of the protein, including a kringle structure, two growth factor domains, and type I and type II finger domains. The finger domains were first identified in fibronectin. The carboxyl-terminal portion of plasma prekallikrein, factor XII, and factor XI contains the serine or protease portion of the molecule. These various plasma proteins that share common domains appear to have evolved by gene shuffling that may have, in some cases, involved introns.     

Mathematical modeling identifies inhibitors of apoptosis as mediators of positive feedback and bistability

The intrinsic, or mitochondrial, pathway of caspase activation is essential for apoptosis induction by various stimuli including cytotoxic stress. It depends on the cellular context, whether cytochrome c released from mitochondria induces caspase activation gradually or in an all-or-none fashion, and whether caspase activation irreversibly commits cells to apoptosis. By analyzing a quantitative kinetic model, we show that inhibition of caspase-3 (Casp3) and Casp9 by inhibitors of apoptosis (IAPs) results in an implicit positive feedback, since cleaved Casp3 augments its own activation by sequestering IAPs away from Casp9. We demonstrate that this positive feedback brings about bistability (i.e., all-or-none behaviour), and that it cooperates with Casp3-mediated feedback cleavage of Casp9 to generate irreversibility in caspase activation. Our calculations also unravel how cell-specific protein expression brings about the observed qualitative differences in caspase activation (gradual versus all-or-none and reversible versus irreversible). Finally, known regulators of the pathway are shown to efficiently shift the apoptotic threshold stimulus, suggesting that the bistable caspase cascade computes multiple inputs into an all-or-none caspase output. As cellular inhibitory proteins (e.g., IAPs) frequently inhibit consecutive intermediates in cellular signaling cascades (e.g., Casp3 and Casp9), the feedback mechanism described in this paper is likely to be a widespread principle on how cells achieve ultrasensitivity, bistability, and irreversibility.     

Effect of thrombomodulin on the kinetics of the interaction of thrombin with substrates and inhibitors.

Thrombomodulin decreased by 20-30% the Michaelis constant of two tripeptidyl p-nitroanilide substrates of thrombin. Thrombomodulin increased the rate of inactivation of thrombin by two peptidyl chloromethane inhibitors by a similar amount. This effect appeared to be due to a decrease in the dissociation constants of the inhibitors. An improved method for the separation of fibrinopeptides A and B by h.p.l.c. was developed, and this method was used to study the effect of thrombomodulin on the thrombin-catalysed cleavage of fibrinogen. In this reaction, thrombomodulin was a competitive inhibitor with respect to the A alpha-chain of fibrinogen. The release of fibrinopeptide B was also inhibited by thrombomodulin. Analysis of the inhibition caused by thrombomodulin with respect to fibrinopeptides A and B yielded the same dissociation constant for the thrombin-thrombomodulin complex. In the presence of thrombomodulin, the rate of inactivation of thrombin by antithrombin III was stimulated 4-fold. This stimulation showed saturation kinetics with respect to thrombomodulin. Thrombomodulin was found to compete with hirudin for a binding site on thrombin. As a result of this competition, hirudin became a slow-binding inhibitor of thrombin at high thrombomodulin concentrations. Estimates of the dissociation constant for thrombomodulin were obtained in several of the above experiments, and the weighted mean value was 0.7 nM.     

Pharmacology of benzamidine-type thrombin inhibitors

Findings on the pharmacology of derivatives of benzamidine characterized as potent competitive inhibitors of the clotting enzyme thrombin were presented including data on their toxicity. Bis-benzamidines and amidinophenylalanine amides were shown to exert pharmacodynamic effects in intact animals and isolated preparations (hypotension; influence on smooth muscle reactions to serotonin and histamine). Studies with 14C-4-amidinophenylpyruvic acid and 3H-N alpha-tosyl-(3-amidino)-phenylalanine piperidide on the pharmacokinetics in rabbits showed that benzamidine derivatives are suited, in principle, for use as anticoagulants in vivo. Pharmacokinetic properties of individual thrombin inhibitors have to be taken into account in order to reach and maintain adequate plasma levels. The antithrombotic effect of benzamidine-type thrombin inhibitors in animals experiments is directly related to their antithrombin activity.     

Interconnections between autophagy and the coagulation cascade in hepatocellular carcinoma

Autophagy has an important role in tumor biology of hepatocellular carcinoma (HCC). Recent studies demonstrated that tissue factor (TF) combined with coagulation factor VII (FVII) has a pathological role by activating a G-protein-coupled receptor called protease-activated receptor 2 (PAR2) for tumor growth. The present study aimed to investigate the interactions of autophagy and the coagulation cascade in HCC. Seventy HCC patients who underwent curative liver resection were recruited. Immunohistochemical staining and western blotting were performed to determine TF, FVII, PAR2 and light chain 3 (LC3A/B) expressions in tumors and their contiguous normal regions. We found that the levels of autophagic marker LC3A/B-II and coagulation proteins (TF, FVII and PAR2) were inversely correlated in human HCC tissues. Treatments with TF, FVII or PAR2 agonist downregulated LC3A/B-II with an increased level of mTOR in Hep3B cells; in contrast, knockdown of TF, FVII or PAR2 increased LC3A/B. Furthermore, mTOR silencing restored the impaired expression of LC3A/B-II in TF-, FVII- or PAR2-treated Hep3B cells and activated autophagy. Last, as an in vivo correlate, we administered TF, FVII or PAR2 agonist in a NOD/severe combined immunodeficiency xenograft model and showed decreased LC3A/B protein levels in HepG2 tumors with treatments. Overall, our present study demonstrated that TF, FVII and PAR2 regulated autophagy mainly via mTOR signaling. The interaction of coagulation and autophagic pathways may provide potential targets for further therapeutic application in HCC.     

Mechanism of action of heparin through thrombin on blood coagulation.

It has been suggested that heparin can affect blood coagulation through thrombin, i.e. the binding of heparin to thrombin induces a conformational change in the enzyme, facilitating a complex formation between thrombin and antithrombin (Machovich, T., Blaskó, Gy. and Pálos, L. (1975) Biochim. Biophys. Acta 379, 193-200). This hypothesis seems to have been proved. Modification of arginine residues in thrombin did not result in decreased thrombin activity and decreased sensitivity to antithrombin, whereas the heparin sensitivity of the enzyme and the thrombin-antithrombin reaction were diminished.     

Assessment of coagulation cascade during air microembolization of the lung

Experiments were performed to determine whether activation of the coagulation cascade was required for pulmonary vascular permeability to increase during microembolization of the lung. For 30-45 min air microemboli were intravenously infused (0.05-0.10 ml X kg-1 X min-1) into awake sheep with chronic lung-lymph fistulas and anesthetized mongrel dogs. During embolization the pulmonary arterial pressure increased, and O2 partial pressure (PaO2) fell by more than 20 Torr (P less than 0.01). Subsequently lymph flow nearly tripled without a change in the lymph-to-plasma protein concentration ratio. Partial thromboplastin and prothrombin times, biological activity of antithrombin III, and circulating concentration of 125I-labeled dog or sheep fibrinogen did not change during or following air infusion. In two additional sheep an intravenous infusion of thrombin at 0.6 U X kg-1 X min-1 for 15 min resulted in a 20% decrease in 125I-labeled sheep fibrinogen concentration without a change in pulmonary arterial pressure or PaO2. We conclude that air microembolization can increase permeability to water and protein without a detectable activation of the coagulation cascade in the sheep or dog.     

Collagen-induced platelet shape change is not affected by positive feedback pathway inhibitors and cAMP-elevating agents

Shape change is the earliest response of platelets to stimuli; it is mainly dependent upon Ca(2+)/calmodulin interaction subsequent to Ca(2+) mobilization and is mediated by myosin light chain kinase (MLCK) activation. It has been recently suggested that collagen itself is not able to elicit platelet shape change in the absence of ADP and thromboxane A(2) costimulation but is capable of inducing MLCK activation. Since we hypothesize that the morphological changes of the few platelets that adhere to collagen might not be revealed by turbidimetry, the aim of this study was to assess platelet shape change using transmission electron microscopy, in the absence of the amplificatory feedback pathways of ADP and thromboxane A(2). Our results demonstrated that only the platelets in contact with insoluble collagen fibers underwent a typical shape change, whereas those further away remained quiescent. Moreover, since cAMP enhances Ca(2+) mobilization in response to collagen, in the present study, we also investigated whether cAMP is involved in the inhibition of collagen-induced platelet shape change and MLC phosphorylation. Platelets were thus treated with iloprost (28 nm) prior to stimulation. Electron microscopy studies demonstrated that iloprost did not modify collagen-induced shape change, whereas immunoblotting studies showed a slight inhibition of MLC phosphorylation in the presence of enhanced cAMP levels. We can thus conclude that collagen is able to cause platelet shape change through activation of Ca(2+)/calmodulin-dependent MLCK, without the involvement of amplificatory pathways. Enhanced cytosolic cAMP levels do not inhibit collagen-induced platelet shape change but exert a weak inhibitory action on MLCK.