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PubMed2004

Crystal structure of thrombin bound to heparin.

Thrombin is the final protease in the blood coagulation cascade and serves both pro- and anticoagulant functions through the cleavage of several targets. The ability of thrombin to specifically recognize a wide range of substrates derives from interactions that occur outside of the active site of thrombin. Thrombin possesses two anion binding exosites, which mediate many of its interactions with cofactors and substrates, and although many structures of thrombin have been solved, few such interactions have been described in molecular detail. Glycosaminoglycan binding to exosite II of thrombin plays a major role in switching off the procoagulant functions of thrombin by mediating its irreversible inhibition by circulating serpins and by its binding to the endothelial cell surface receptor thrombomodulin. Here we report the 1.85-A structure of human alpha-thrombin bound to a heparin fragment of eight monosaccharide units in length. The asymmetric unit is composed of two thrombin dimers, each sharing a single heparin octasaccharide chain. The observed interactions are fully consistent with previous mutagenesis studies and illustrate on a molecular level the cofactor interaction that is critical for the restriction of clotting to the site of blood vessel injury.

Anions↗

PubMed2005

Efficient tyrosine phosphorylation of proteins after activation of platelets with thrombin depends on intact glycoprotein Ib.

The role of platelet glycoprotein Ib as a thrombin receptor has been often a subject of controversy. We have investigated the role of the thrombin receptors, GPIb and protease-activated receptor (PAR)-1. Tyrosine phosphorylation in whole platelet lysates and in cytoskeletal extracts was evaluated after activation with thrombin and with the thrombin receptor-activating peptide (TRAP). Different experimental approaches were applied including: (i) congenital deficiency of platelet GPIb (Bernard Soulier syndrome, BSS), (ii) antibody to GPIb (AP1), (iii) selective protease cleavage (metalloprotease), and (iv) antibody to (PAR)-1. After activation of control platelets with thrombin or TRAP, multiple proteins became tyrosine phosphorylated in platelet lysates and some of them associated with the cytoskeletal fraction. These effects were absent in BSS platelets. Presence of AP1 or metalloprotease treatment showed an inhibitory effect when platelets were activated with a low concentration of thrombin or TRAP. Blockade of PAR-1 with a specific antibody, SPAN 12, inhibited platelet response to both agonists. This study reinforces the hypothesis that GPIb is the high-affinity receptor for thrombin. The signaling mechanisms occurring through tyrosine phosphorylation of proteins triggered by thrombin seem to be dependent on intact GPIb. Moreover, our results indicate that both receptors, GPIb and PAR-1, are necessary to achieve a full platelet response to thrombin.

Bernard-Soulier Syndrome↗

PubMed2004

Thrombin and its protease-activated receptor-1 (PAR1) participate in the endothelial-mesenchymal transdifferentiation process.

The serine protease thrombin, independently of its participation in hemostasis and thrombosis, has been involved in tissue repair and remodeling, embryogenesis, angiogenesis, and development and progression of atherosclerosis. Many of these functions appear to be mediated by specific thrombin receptors, particularly the protease-activated receptor-1 (PAR1). In this study, we investigated whether both thrombin and PAR1 were present in the aortic wall of chicken embryos at days 11 and 12 of development. We found that PAR1 was limited to some cells of the intimal thickening and the inner media, whereas thrombin appeared distributed across the aortic wall. We also investigated whether PAR1 was present during endothelial-mesenchymal transdifferentiation (EMT) in vitro. A moderate immunoreactivity was detected in the monolayer of endothelial cells. In contrast, a strong cytoplasmic immunoreactivity was observed in the detaching and migrating cells and those that had acquired mesenchymal characteristics. This PAR1 expression was confirmed by flow cytometry. In this study, the addition of thrombin to arrested endothelial cell cultures was assessed. We found that thrombin stimulated endothelial cell spreading and migration, as no migrating cells were observed in serum-free medium (SFM) condition. Immunolocalization of PAR1 in the thrombin-treated cultures showed strong cytoplasmic immunoreactivity in the monolayers and in spreading and migrating cells, whereas in the SFM condition undetectable PAR1 immunoreactivity was observed. Flow cytometry of these cultures revealed an elevated expression of PAR1 in the presence of thrombin, in contrast to that detected in SFM and complete medium. These data indicate that both thrombin and PAR1 are involved in the remodeling of the aortic wall and intimal thickening formation, and in the endothelial-mesenchymal transdifferentiation process.

Animals↗

PubMed1998

Regulation of the actin cytoskeleton by thrombin in human endothelial cells: role of Rho proteins in endothelial barrier function.

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.

Actins↗

PubMed1997

The thrombin E192Q-BPTI complex reveals gross structural rearrangements: implications for the interaction with antithrombin and thrombomodulin.

Previous crystal structures of thrombin indicate that the 60-insertion loop is a rigid moiety that partially occludes the active site, suggesting that this structural feature plays a decisive role in restricting thrombin's specificity. This restricted specificity is typified by the experimental observation that thrombin is not inhibited by micromolar concentrations of basic pancreatic trypsin inhibitor (BPTI). Surprisingly, a single atom mutation in thrombin (E192Q) results in a 10(-8) M affinity for BPTI. The crystal structure of human thrombin mutant E192Q has been solved in complex with BPTI at 2.3 A resolution. Binding of the Kunitz inhibitor is accompanied by gross structural rearrangements in thrombin. In particular, thrombin's 60-loop is found in a significantly different conformation. Concomitant reorganization of other surface loops that surround the active site, i.e. the 37-loop, the 148-loop and the 99-loop, is observed. Thrombin can therefore undergo major structural reorganization upon strong ligand binding. Implications for the interaction of thrombin with antithrombin and thrombomodulin are discussed.

Antithrombin III↗

PubMed1997

Thrombin stimulates production of transforming growth factor-beta by cultured human mesangial cells.

Fibrin formation within the glomeruli occurs in various forms of human and experimental glomerulonephritis and it may play an important role in progressive glomerular injury. Transforming growth factor-beta (TGF-beta) has been shown to participate in the glomerular accumulation of extracellular matrix in glomerulonephritis. We investigated whether thrombin, an important coagulation factor, could modulate the production of TGF-beta by cultured human mesangial cells (HMC). TGF-beta levels in the culture supernatants were measured by ELISA using a specific antibody. The TGF-beta concentration was significantly increased by incubation of HMC with thrombin in a time-dependent manner. The stimulating effect of thrombin on TGF-beta was inhibited by addition of hirudin (a natural thrombin inhibitor) and argatroban (a synthetic thrombin inhibitor). In addition DFP-inactivated thrombin, which has no enzymatic activity, did not stimulate TGF-beta production. A protein kinase C inhibitor (H7) and a tyrosine kinase inhibitor (herbimycin A) also inhibited thrombin induced TGF-beta production. These findings suggested that thrombin may modulate the synthesis of TGF-beta via protein kinase C- and tyrosine kinase-dependent mechanisms in cultured HMC. Thus thrombin may participate in the accumulation of extracellular matrix in glomeruli through the augmentation of TGF-beta production.

Cells, Cultured↗

PubMed2004

Novel role of microtubules in thrombin-induced endothelial barrier dysfunction.

Disturbances in endothelial cell (EC) barrier regulation are critically dependent upon rearrangements of EC actin cytoskeleton. However, the role of microtubule (MT) network in the regulation of EC permeability is not well understood. We examined involvement of MT remodeling in thrombin-induced EC permeability and explored MT regulation by heterotrimeric G12/13 proteins and by small GTPase Rho. Thrombin induced phosphorylation of MT regulatory protein tau at Ser409 and Ser262 and peripheral MT disassembly, which was linked to increased EC permeability. MT stabilization by taxol attenuated thrombin-induced permeability, actin remodeling, and paracellular gap formation and diminished thrombin-induced activation of Rho and Rho-kinase. Expression of activated Galpha12/13 subunits involved in thrombin-mediated signaling or their effector p115RhoGEF involved in Rho activation caused MT disassembly, whereas p115RhoGEF-specific negative regulator RGS preserved MT from thrombin-induced disassembly. Consistent with these results, expression of activated RhoA and Rho-kinase induced MT disassembly. Conversely, thrombin-induced disassembly of peripheral MT network was attenuated by expression of dominant negative RhoA and Rho-kinase mutants or by pharmacological inhibition of Rho-kinase. Collectively, our data demonstrate for the first time a critical involvement of MT disassembly in thrombin-induced EC barrier dysfunction and indicate G-protein-dependent mechanisms of thrombin-induced MT alteration.

Cell Membrane Permeability↗

PubMed1994

Regulation of thrombin-induced endothelial cell activation by bacterial toxins.

It has previously been shown that thrombin effects on endothelial cells can be mediated via G-proteins, which couple the thrombin receptor to several key physiological responses. As G-proteins are known targets of bacterial toxins, specific toxins were used to further characterize G-protein involvement in thrombin activation of bovine pulmonary arterial endothelial cells (BPAEC) and human umbilical vein endothelial cells (HUVEC). Homogenates were exposed to several bacterial toxins in the presence of 32P-NAD and ADP ribosylation of proteins determined by autoradiography of SDS-PAGE gels. Major substrates were a 40 kDa protein for pertussis toxin, 39, 45 and 52 kDa proteins (Gs) for cholera toxin, a 21 kDa protein for botulinum toxin C, and a 43 kDa protein (actin) for botulinum toxin C2a. The increase in either HUVEC or BPAEC PGI2 release induced by thrombin was not altered by pretreatment with any toxin. However, 1 h treatment of BPAEC monolayers with 1 microgram/ml pertussis toxin resulted in dramatic barrier dysfunction, which was synergistic with the albumin permeability induced by 1 microM thrombin. In contrast, pretreatment with 1 microgram/ml cholera toxin completely prevented the thrombin-induced barrier dysfunction. Moreover, contraction and gap formation due to thrombin challenge, observed by phase contrast microscopy, was greatly augmented by pertussis toxin and prevented by cholera toxin. Whereas 5 micrograms/ml botulinum toxin C did not affect either basal or thrombin-induced barrier dysfunction, botulinum toxin C2a increased basal BPAEC permeability over four-fold. Thus, bacterial toxins have specific and divergent effects on thrombin-induced endothelial cell responses. Botulinum toxin C2a appears to interact directly with actin to produce barrier dysfunction. In contrast, cholera toxin promotes barrier function via its known effects on Gs, stimulating adenylate cyclase and increasing cAMP. Because cholera toxin and pertussis toxin (via inhibition of G(i)) both increase cAMP, yet have opposing effects on barrier function, the present results suggest that pertussis toxin produces barrier dysfunction via ADP ribosylation of a novel G-protein other than G(i) or via a novel action of G(i).

Actins↗

PubMed1998

The inactivation of single-chain urokinase-type plasminogen activator by thrombin in a plasma milieu: effect of thrombomodulin.

Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a virtually inactive two-chain form (tcu-PA/T), a process which may contribute to the maintenance of a fresh blood clot. We have examined the inactivation of scu-PA by thrombin in a plasma milieu to get more insight in the physiological relevance of this phenomenon. Citrated pooled normal plasma was treated with thrombin in the absence and presence of thrombomodulin. After an incubation period of 30 min the concentrations of scu-PA and tcu-PA/T were measured using specific bioimmunoassays. The inactivation of scu-PA in citrated plasma was found to be stimulated fourfold by thrombomodulin. Kinetic experiments showed that the inactivation of scu-PA by thrombin in the absence and presence of thrombomodulin occurred rapidly and declined within 1 min as a result of rapid inhibition by antithrombin III (ATIII) and other possible inhibitors. Calcium had no direct effect on the inactivation of scu-PA by exogenously added thrombin in the absence and presence of thrombomodulin. However, recalcification of plasma induced significant inactivation of scu-PA in plasma as a result of endogenous thrombin generation through the contact activation system. This calcium-induced inactivation of scu-PA was completely abolished in the presence of thrombomodulin, most likely as a result of activation of protein C by the complex formed between thrombomodulin and endogenously generated thrombin. Thrombomodulin thus appeared to play a dual role both by stimulating the inactivation of scu-PA by thrombin, and by inhibiting calcium-induced inactivation of scu-PA in plasma. In the plasma from a patient heterozygous for protein C deficiency, thrombomodulin could not prevent calcium-induced generation of tcu-PA/T, whereas the stimulating effect of thrombomodulin predominated instead. This result implied that disturbance of the protein C pathway may lead to the inactivation of substantial amounts of scu-PA in plasma under (patho)physiological circumstances and may provide an additional explanation for the association found between thromboembolism and deficiencies in the protein C pathway. This study shows that the amount of scu-PA that is inactivated in plasma depends mainly on the generation of thrombin and on thrombomodulin. We conclude that the inhibition of scu-PA-induced fibrinolysis appears to be regulated by activation of the coagulation system, providing a link between coagulation and fibrinolysis.

Fibrinolysis↗

PubMed2001

In vitro interaction of C1-inhibitor with thrombin.

Previous observations of increased generation of thrombin during acute attacks of angioedema in plasma of patients with C1-inhibitor (C1-INH) deficiency prompted us to evaluate the interaction of C1-INH with thrombin in both purified systems and human plasma. For this purpose, we used several methods: (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis; (2) enzyme-linked immunosorbent assays to measure complexes between C1-INH and thrombin and inactivated C1-INH; and (3) kinetic studies using a chromogenic assay. We found that the interaction of purified C1-INH with thrombin is associated with the formation of bimolecular complexes of molecular weight (Mr) 130 000 and 120 000 as well as with the appearance of a cleaved form of C1-INH of Mr 97 000. The kinetic studies of inhibition of thrombin by C1-INH showed an average second-order rate constant of 19/s per mol/l, which was significantly increased in the presence of heparin. The addition of thrombin to human plasma was not associated with detectable C1-INH-thrombin complex formation or with cleavage of C1-INH. In conclusion, our data demonstrate that C1-INH upon interaction with thrombin, in part, forms enzyme-inhibitor complexes and, in part, is cleaved. The low second-order rate constant and the lack of a significant interaction in plasma suggest that the inhibition of thrombin by C1-INH has a minor role in circulating blood; however, its role might be important at the endothelial surface, where high concentrations of glycosaminoglycans occur.

Complement C1 Inactivator Proteins↗

PubMed2004

Comparison of the effect of fondaparinux and enoxaparin on thrombin generation during in-vitro clotting of whole blood and platelet-rich plasma.

Fondaparinux, a selective antithrombin-dependent inhibitor of activated factor X (FXa), is effective in the prevention and treatment of deep vein thrombosis and seems to be superior to enoxaparin. However, the exact mechanism of fondaparinux antithrombotic action is still unclear. We compared the effect of clinically relevant concentrations of fondaparinux and enoxaparin on the initiation and propagation phase of prothrombin activation and on the endogenous thrombin potential (ETP). Coagulation was triggered either in whole blood or in platelet-rich plasma (PRP) by recalcification in the presence of diluted thromboplastin. Prothrombin activation in whole blood was assessed with an original method by measuring the kinetics of prothrombin F1+2 formation using an enzyme-linked immunosorbent assay. We also assessed the maximum concentration of thrombin (Cmax) and the ETP in PRP using the Thrombogram-Thrombinoscope assay. Concentrations of fondaparinux achieved in prophylaxis (0.11-0.28 anti-FXa IU/ml) prolonged the initiation phase and reduced the velocity of the propagation phase of F1+2 formation. Concentrations of enoxaparin achieved in prophylaxis (0.1-0.25 anti-FXa IU/ml) did not significantly modify these parameters. Concentrations of fondaparinux equal to or higher than 0.57 anti-FXa IU/ml significantly reduced the Cmax of F1+2 or thrombin as well as the ETP. At fondaparinux concentrations equal to or higher than 0.91 anti-FXa IU/ml, a maximum 60% inhibition of thrombin generation was observed. In the presence of enoxaparin concentrations equal to or higher than 0.8 anti-FXa IU/ml, the inhibition of thrombin generation was higher than 80%. Fondaparinux prolonged the initiation phase, decreased the velocity of the propagation phase of thrombin generation and partially reduced the total amount of generated thrombin. The inhibitory effect of fondaparinux on the initiation and propagation phase of thrombin generation seems to be responsible for its antithrombotic action. The more profound inhibition of thrombin generation induced by enoxaparin is due to its supplementary anti-activated factor II activity.

Anticoagulants↗

PubMed1999

Effects of alpha-thrombin on superoxide dismutase levels in human cerebral microvascular endothelial cells.

BACKGROUND: Sequelae of traumatic brain injury include generation of oxygen-free radicals and fibrin deposition, which worsen the initial injury. Superoxide dismutases (SODs) scavenge and bind to the free-radical superoxide anion (O2-), potentially defending against oxidative stress. In the present study, we investigated the production of SOD within human cerebral microvascular endothelial (HCME) cells after exposure to alpha-thrombin, hypothesizing that manganese SOD (MnSOD) expression is increased. Our aims were to determine whether alterations in SOD are observed at the mRNA level, to examine whether a particular species is preferentially expressed, and to determine the requirement of the active site of alpha-thrombin. METHODS: HCME cells were characterized and grown to confluence. Control cells and cells exposed to 10 nmol/L alpha-thrombin were harvested for mRNA isolation using reverse transcriptase-polymerase chain reaction. Quantitation of mRNA production determined the levels of copper-zinc SOD and MnSOD. Active site blocked alpha-thrombin was used as a negative control and determined the specificity of the response. RESULTS: The cells in culture were identified as endothelial after fulfilling criteria, such as positive immunocytochemical staining for factor VIII/von Willebrand factor antigen and binding of Ulex europaeus agglutinin-1 lectin. Levels of MnSOD mRNA were elevated at all time points in response to alpha-thrombin, whereas the cytosolic form was undetectable. HCME cells that were exposed to active site-blocked alpha-thrombin produced mRNA levels of MnSOD that were increased above those of controls, but this increase was half that of mRNA levels of MnSOD produced by HCME cells that were exposed to alpha-thrombin. CONCLUSION: Our study showed for the first time that alpha-thrombin partially modulates SOD in HCME cells, causing a preferential increase in MnSOD. Further investigation into secondary brain injury will provide insights into the role of alpha-thrombin in the mechanism of free radical-induced alterations, potentially improving the outcome of patients with head injury.

Cells, Cultured↗

PubMed2006

Thrombin and prothrombin are expressed by neurons and glial cells and accumulate in neurofibrillary tangles in Alzheimer disease brain.

Thrombin is a serine protease that is generated by proteolytic cleavage of its precursor, prothrombin. We previously showed that thrombin proteolyses the microtubule-associated protein tau and that phosphorylation of tau inhibits this process. To characterize further the role of thrombin in the brain, we investigated prothrombin and thrombin expression in cultured brain cells and in brains of control, Alzheimer disease (AD) and parkinsonism-dementia complex of Guam (PDCG). We show by reverse transcriptase-polymerase chain reaction that prothrombin mRNA is expressed in brain tissues, neuroblastoma cells, and cultured human astrocytes, oligodendrocytes, and microglial cells. We also show by immunohistochemistry that the proteins prothrombin and thrombin are present in brain using specific monoclonal and polyclonal antibodies for both proteins. All antibodies stained residual serum in blood vessels, as well as normal pyramidal neurons and their processes, and some astrocytes. Additionally, in AD and PDCG cases, all antibodies stained extra- and intracellular neurofibrillary tangles (NFTs), senile plaques, and reactive microglial cells. The ubiquitous expression of prothrombin and thrombin in brain cells suggests that thrombin plays an important physiological role in normal brain. The accumulation of thrombin and prothrombin in NFTs supports the hypothesis that thrombin may be involved in tau proteolysis and that failure to metabolize tau may lead to its aggregation in neurodegenerative diseases.

Aged↗

PubMed1999

Structures of thrombin retro-inhibited with SEL2711 and SEL2770 as they relate to factor Xa binding.

Most thrombin active-site inhibitors form a short antiparallel beta-strand with residues Ser214-Gly216. However, the Selectide Corp. inhibitors SEL2711 and SEL2770 bind to thrombin in a retro fashion, making a parallel beta-strand with Ser214-Gly216 similar to other retro-binding inhibitors. The crystallographic structures of thrombin-hirugen complexed with SEL2711 and SEL2770, which are isostructural with the binary thrombin-hirugen complex, have been determined and refined in the 9.0-2.1 A resolution range to final R values of 16.5 and 16.7%, respectively. The structures of the SEL2711 and SEL2770 complexes contain 131 and 104 water molecules, respectively, both of which correspond to occupancies of greater than 0.5. The L-4-amidinophenylalanyl residues of SEL2711 and SEL2770 are fixed at the S1 specificity site, utilizing favorable ionic and hydrogen-bonding interactions between the N atoms of the amidino group and the side-chain O atoms of Asp189. The Glu192 residue of thrombin adopts an extended conformation, which allows the L-cyclohexylglycyl residue in the P2 retro-binding position of the inhibitors to occupy a similar site to the P3 aspartate in thrombin platelet-receptor peptides bound to thrombin. The N-terminal acetyl group of both inhibitors is located in the S2 subsite, while the L-3-pyridyl-(3-methyl)-alanyl of SEL2711 and the L-(N,N-dimethyl)lysine of SEL2770 occupy the S3 D-Phe subsite of D-PheProArg chloromethyl ketone (PPACK) in the thrombin-PPACK complex. The two C-terminal residues of SEL2711 (leucine and proline) point into the solvent and have no electron density in the thrombin complex. Those of SEL2770 are also positioned into the solvent, but surprisingly produce weak electron density with high B values ( = 50 A2). Since the Selectide inhibitors are about 10(4) times more specific for factor Xa, modeling retro-binding to the latter suggests that the selectivity can be a consequence of interactions of the inhibitors in the S3-S4 binding subsites of factor Xa.

Anticoagulants↗

PubMed1985

The importance of thrombin inhibition for the expression of the anticoagulant activities of heparin, dermatan sulphate, low molecular weight heparin and pentosan polysulphate.

The effects of standard heparin, three low molecular weight derivatives of heparin, dermatan sulphate and pentosan polysulphate on the intrinsic coagulation pathway were compared in order to evaluate the contributions of the anti-factor Xa and anti-thrombin activities to their anticoagulant activities. The anticoagulant potency was measured by the ability of each sulphated polysaccharide to inhibit the generation of thrombin activity in plasma. Similarly, the ability of the six sulphated polysaccharides to enhance the rates of inactivation either factor Xa or thrombin in defibrinated plasma containing calcium chloride and cephalin were also determined. Standard heparin was the only sulphated polysaccharide that could equally inhibit thrombin generation and enhance the inactivation of factor Xa and thrombin by plasma. Dermatan sulphate and pentosan polysulphate were more effective as inhibitors of thrombin generation than potentiators of factor Xa inactivation. The two smallest derivatives of heparin, which had high anti-factor Xa (but low antithrombin) activity, were the poorest inhibitors of thrombin generation. Our results therefore suggest that only sulphated polysaccharides that enhance the inactivation of thrombin by plasma and/or inhibit the generation of thrombin activity in plasma are good anticoagulants. These two activities of sulphated polysaccharides appear to be good predictors of the relative antithrombotic potency in vivo.

Anticoagulants↗

PubMed1995

Altered platelet phospholipid-dependent thrombin generation in thrombocytopenia and thrombocytosis.

Platelet phospholipid dependent thrombin generation was determined in 20 patients with thrombocytosis, 18 with thrombocytopenia, and 25 normal controls, using a quantitative chromogenic assay. Platelets from patients with myeloproliferative disorders displayed normal lag times to 20 nM thrombin concentration but increased thrombin potentials, even when corrected for platelet size. Platelets from patients with reactive thrombocytosis supported normal thrombin generation. ITP platelets were large, with a proportionate increase in thrombin potentials, but very short lag times to 20 nM thrombin concentration. Following marrow ablation there was a progressive loss of activation-induced enhancement of thrombin generation. Platelets from patients with idiopathic aplastic anaemia supported normal thrombin generation. These findings indicate that platelet phospholipid-dependent thrombin generation is altered in many patients with a variety of quantitative platelet disorders, and this may be an important determinant of the clinical expression of these disorders.

Anemia, Aplastic↗

PubMed1985

Thrombin specificity. Selective cleavage of antibody light chains at the joints of variable with joining regions and joining with constant regions.

Selective cleavage of polypeptides by alpha-thrombin can be reasonably predicted [Chang, J.Y. (1985) Eur. J. Biochem. 151,217-224]. This knowledge was applied to the selective cleavage of antibody light chains with the aim of producing intact fragments of both variable region and constant region. (a) Mouse kappa light chains 10K26 and 10K44 from anti-(azobenzene arsonate) antibodies contain 20 Arg/Lys-Xaa bonds. Only two of them, one ProArg-Thr bond located at the joint of the variable region with the joining peptide and one ValLys-Ser bond located near the carboxyl-terminal end of the constant region, were selectively cleaved by alpha-thrombin. The ProArg-Thr bond has a 50% cleavage time of about 10 min under the designated conditions, whereas the ValLys-Ser has a 50% cleavage time approx. 9-10 h. A single selective cleavage at the joining position of the variable region and joining peptide can be achieved by short-time thrombin digestion. Fragments containing intact variable region (1-96) and intact joining peptide-constant region (97-214) obtained from both denatured and native light chains of 10K26 can be separated by gel filtration. (b) lambda light chains from both human and mouse all begin with the GlnProLys-(Ala/Ser) structure (positions 108-111) at their constant regions. This ProLys-Ala/Ser bond is also susceptible to specific thrombin cleavage. Four human lambda chain (KERN, NEI, NEW, VOR) and one mouse lambda chain (RPC20) were shown to be selectively cleaved by thrombin at these ProLys-Ala/Ser bonds. For human lambda chains, the 50% cleavage time at this ProLys-Ala bond was approx. 3-4 h under the designated conditions. Six additional thrombin specific cleavages were also detected within the variable regions of NEI, VOR and RPC-20. (c) Heparin inhibits thrombin cleavage of Arg/Lys-Xaa bonds located near the center of the antibody light chain, but slightly activates thrombin cleavage of those located near the amino or carboxyl-terminal ends of the protein. The significance of these findings is threefold. (a) It demonstrates that selective cleavage of large polypeptides by alpha-thrombin can also be reasonably predicted. (b) It provides a useful method for light chain fragmentation which can greatly facilitate amino acid sequencing of antibodies. (c) It serves to generate fragments containing intact variable regions and constant regions from antibody light chains of human and mouse. Such fragments may be useful for chemical semisynthesis of a human-mouse light chain chimeras.

Amino Acid Sequence↗

PubMed1991

Ca2+ ionophore A23187 and thrombin inhibit the pertussis-toxin-induced ADP-ribosylation of the alpha-subunit of the inhibitory guanine-nucleotide-binding protein and other proteins in human platelets.

Inhibitory guanine-nucleotide-binding proteins (Gi proteins) are substrates for pertussis toxin and the decreased pertussis-toxin-dependent ADP ribosylation of Gi proteins upon prior specific hormonal stimulation of cells is thought to reflect the receptor-mediated activation of Gi proteins, leading to their subsequent dissociation into alpha i and beta/gamma subunits. In the present study, the effect of various platelet stimuli on the subsequent pertussis-toxin-dependent ADP ribosylation of the alpha subunit of Gi (Gi alpha) in saponized platelets and platelet membranes were studied. Stimulation of intact platelets with the Ca(2+)-ionophore A23187 or thrombin, but not phorbol 12,13-dibutyrate, decreased the subsequent pertussis-toxin-dependent ADP ribosylation of Gi alpha in saponin-permeabilized platelets in a time-dependent and dose-dependent manner. Thrombin was more effective than A23187. Parallel measurements of Ca2+ mobilization and pertussis-toxin-dependent ADP ribosylation of Gi alpha in platelets showed that Ca2+ mobilization could only partly account for the decrease in pertussis-toxin-dependent ADP ribosylation in platelets stimulated by thrombin. When the ADP-ribosylation reaction was carried out in platelet membranes, a decrease in ADP ribosylation was still observed after stimulation of platelets with thrombin, but not with A23187. In addition to Gi alpha, two other proteins were found to be ADP ribosylated by pertussis toxin; their ADP ribosylation was also decreased after A23187 and thrombin stimulation of platelets. The results indicate that Ca2+ mobilization can decrease the pertussis-toxin-dependent ADP ribosylation of Gi alpha in saponized platelets; the decrease of pertussis-toxin-dependent ADP ribosylation of Gi alpha after thrombin stimulation of platelets can only, in part, be explained by Ca2+ mobilization and involves additional mechanisms; the decrease in pertussis-toxin-dependent ADP ribosylation after A23187 and thrombin stimulation is not confined to G1 alpha and involves other proteins. We conclude that the decrease in pertussis-toxin-dependent ADP ribosylation of Gi in thrombin-stimulated platelets might not be solely caused by a specific structural change, such as dissociation of Gi. It is likely that A23187 and thrombin stimulation of platelets generates substances which interfere with the ADP-ribosylating activity of pertussis toxin.

Adenosine Diphosphate Ribose↗