Structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex

The serine proteinase alpha-thrombin causes blood clotting through proteolytic cleavage of fibrinogen and protease-activated receptors and amplifies its own generation by activating the essential clotting factors V and VIII. Thrombomodulin, a transmembrane thrombin receptor with six contiguous epidermal growth factor-like domains (TME1-6), profoundly alters the substrate specificity of thrombin from pro- to anticoagulant by activating protein C. Activated protein C then deactivates the coagulation cascade by degrading activated factors V and VIII. The thrombin-thrombomodulin complex inhibits fibrinolysis by activating the procarboxypeptidase thrombin-activatable fibrinolysis inhibitor. Here we present the 2.3 A crystal structure of human alpha-thrombin bound to the smallest thrombomodulin fragment required for full protein-C co-factor activity, TME456. The Y-shaped thrombomodulin fragment binds to thrombin's anion-binding exosite-I, preventing binding of procoagulant substrates. Thrombomodulin binding does not seem to induce marked allosteric structural rearrangements at the thrombin active site. Rather, docking of a protein C model to thrombin-TME456 indicates that TME45 may bind substrates in such a manner that their zymogen-activation cleavage sites are presented optimally to the unaltered thrombin active site.     

PAR3 is a cofactor for PAR4 activation by thrombin

Identification of the mechanisms by which the coagulation protease thrombin activates platelets is critical for understanding haemostasis and thrombosis. Thrombin activates cells at least in part by cleaving protease-activated G-protein-coupled receptors (PARs). PAR3 and PAR4 are thrombin receptors expressed in mouse platelets. Inhibition of thrombin binding to mPAR3 (ref. 4) and knockout of the mPAR3 gene inhibited mouse platelet activation at low but not high concentrations of thrombin. Thus PAR3 is important for thrombin signalling in mouse platelets. Expression of human PAR3 in heterologous expression systems reliably resulted in responsiveness to thrombin. Curiously, despite its importance for the activation of mouse platelets by thrombin, mouse PAR3 (mPAR3) did not lead to thrombin signalling even when overexpressed. We now report that mPAR3 and mPAR4 interact in a novel way: mPAR3 does not itself mediate transmembrane signalling but instead functions as a cofactor for the cleavage and activation of mPAR4 by thrombin. This establishes a paradigm for cofactor-assisted PAR activation and for a G-protein-coupled receptor's acting as an accessory molecule to present ligand to another receptor.     

Role of thrombin signalling in platelets in haemostasis and thrombosis.

Platelets are critical in haemostasis and in arterial thrombosis, which causes heart attacks and other events triggered by abnormal clotting. The coagulation protease thrombin is a potent activator of platelets ex vivo. However, because thrombin also mediates fibrin deposition and because multiple agonists can trigger platelet activation, the relative importance of platelet activation by thrombin in haemostasis and thrombosis is unknown. Thrombin triggers cellular responses at least in part through protease-activated receptors (PARs). Mouse platelets express PAR3 and PAR4 (ref. 9). Here we show that platelets from PAR4-deficient mice failed to change shape, mobilize calcium, secrete ATP or aggregate in response to thrombin. This result demonstrates that PAR signalling is necessary for mouse platelet activation by thrombin and supports the model that mouse PAR3 (mPAR3) does not by itself mediate transmembrane signalling but instead acts as a cofactor for thrombin cleavage and activation of mPAR4 (ref. 10). Importantly, PAR4-deficient mice had markedly prolonged bleeding times and were protected in a model of arteriolar thrombosis. Thus platelet activation by thrombin is necessary for normal haemostasis and may be an important target in the treatment of thrombosis.     

Enhancing protein C interaction with thrombin results in a clot-activated anticoagulant.

Human protein C is a vitamin K-dependent plasma glycoprotein that circulates as an inactive zymogen. At the endothelial cell surface, thrombin in complex with the integral membrane protein thrombomodulin converts protein C to its active form by specific cleavage of an activation peptide. The activated form of protein C has potent anticoagulant activity as a feedback regulator of thrombin generation (reviewed in refs 4-6), and also has profibrinolytic, anti-ischaemic and anti-inflammatory properties. Protein C is effective in the treatment of model and human thrombotic diseases but, except when it has been used to treat genetic or acquired deficiencies and microvascular thrombosis, it is administered as the activated enzyme, which has a short biological half-life. We have altered two putative inhibitory acidic residues near the thrombin cleavage site, which results in a 30-fold increase in substrate utilization by alpha-thrombin. We combined these changes with a genetically altered glycoform to generate a zymogen protein C with a 60-fold increased cleavage rate by free alpha-thrombin, independent of its cofactor thrombomodulin. We show that this 'proform' of protein C, unlike the natural circulating zymogen, can be activated by thrombin generated in clotting human plasma, resulting in an inhibition of further clot formation. Our data therefore show that we have engineered a site-activated agent, which only has anticoagulant activity when significant amounts of thrombin are being generated.     

Structure of the C2 domain of human factor VIII at 1.5 A resolution

Human factor VIII is a plasma glycoprotein that has a critical role in blood coagulation. Factor VIII circulates as a complex with von Willebrand factor. After cleavage by thrombin, factor VIIIa associates with factor IXa at the surface of activated platelets or endothelial cells. This complex activates factor X (refs 6, 7), which in turn converts prothrombin to thrombin in the presence of factor Va (refs 8, 9). The carboxyl-terminal C2 domain of factor VIII contains sites that are essential for its binding to von Willebrand factor and to negatively charged phospholipid surfaces. Here we report the structure of human factor VIII C2 domain at 1.5 A resolution. The structure reveals a beta-sandwich core, from which two beta-turns and a loop display a group of solvent-exposed hydrophobic residues. Behind the hydrophobic surface lies a ring of positively charged residues. This motif suggests a mechanism for membrane binding involving both hydrophobic and electrostatic interactions. The structure explains, in part, mutations in the C2 region of factor VIII that lead to bleeding disorders in haemophilia A.     

人肠激酶轻链基因的克隆及其在大肠杆菌中的表达

研究目的：克隆表达人肠激酶轻链编码基因,以期应用于融合蛋白的切割与纯化．方法：从人的全基因组中扩增出编码人肠激酶轻链的5个外显子基因片段,经过酶切、连接后得到正确编码的人肠激酶轻链完整基因序列;随后,将目的基因片段插入原核表达载体pBV220中,构建成融合型表达载体pBV—EKL,转化大肠杆菌BL21（DE3）,调节温度至42℃诱导重组蛋白表达;通过镍亲和层析纯化得到重组蛋白．结果：所表达的重组蛋白经SDS—PAGE分析,相对表观分子质量约为31kDa,表达量占菌体总可溶蛋白量的46％;通过镍亲和层析纯化得到的重组蛋白经复性后显示出具有催化切割活性．结论：成功构建了能大量表达重组人肠激酶轻链的菌株,为进一步进行重组人肠激酶活性的研究及其在生产和医学方面的应用研究奠定了基础．     

A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain

Signalling through the receptor protein Notch, which is involved in crucial cell-fate decisions during development, requires ligand-induced cleavage of Notch. This cleavage occurs within the predicted transmembrane domain, releasing the Notch intracellular domain (NICD), and is reminiscent of gamma-secretase-mediated cleavage of beta-amyloid precursor protein (APP), a critical event in the pathogenesis of Alzheimer's disease. A deficiency in presenilin-1 (PS1) inhibits processing of APP by gamma-secretase in mammalian cells, and genetic interactions between Notch and PS1 homologues in Caenorhabditis elegans indicate that the presenilins may modulate the Notch signalling pathway. Here we report that, in mammalian cells, PS1 deficiency also reduces the proteolytic release of NICD from a truncated Notch construct, thus identifying the specific biochemical step of the Notch signalling pathway that is affected by PS1. Moreover, several gamma-secretase inhibitors block this same step in Notch processing, indicating that related protease activities are responsible for cleavage within the predicted transmembrane domains of Notch and APP. Thus the targeting of gamma-secretase for the treatment of Alzheimer's disease may risk toxicity caused by reduced Notch signalling.     

A chimaeric receptor allows insulin to stimulate tyrosine kinase activity of epidermal growth factor receptor

The cell surface receptors for insulin and epidermal growth factor (EGF) appear to share a common evolutionary origin, as suggested by structural similarity of cysteine-rich regions in their extracellular domains and a highly conserved tyrosine-specific protein kinase domain. Only minor similarity is found outside this catalytic domain, as expected for receptors that have different ligand specificities and generate different biological signals. The EGF receptor is a single polypeptide chain but the insulin receptor consists of distinct alpha and beta subunits that function as an alpha 2 beta 2 heterotetrameric receptor complex. Provoked by this major structural difference in two receptors that carry out parallel functions, we have designed a chimaeric receptor molecule comprising the extracellular portion of the insulin receptor joined to the transmembrane and intracellular domains of the EGF receptor to investigate whether one ligand will activate the tyrosine kinase domain of the receptor for the other ligand. We show here that the EGF receptor kinase domain of the chimaeric protein, expressed transiently in simian cells, is activated by insulin binding. This strongly suggests that insulin and EGF receptors employ closely related or identical mechanisms for signal transduction across the plasma membrane.     

NMDA application potentiates synaptic transmission in the hippocampus

The NMDA (N-methyl-D-aspartate) class of glutamate receptor plays a critical role in a variety of forms of synaptic plasticity in the vertebrate central nervous system. One extensively studied example of plasticity is long-term potentiation (LTP), a remarkably long-lasting enhancement of synaptic efficiency induced in the hippocampus by brief, high-frequency stimulation of excitatory synapses. LTP is a strong candidate for a cellular mechanism of learning and memory. The site of LTP induction appears to be the postsynaptic cell and induction requires both activation of NMDA receptors by synaptically released glutamate and depolarization of the postsynaptic membrane. It is proposed that this depolarization relieves a voltage-dependent Mg2+ block of the NMDA receptor channel, resulting in increased calcium influx which is the trigger for the induction of LTP. This model predicts that application of a large depolarizing dose of NMDA should be sufficient to evoke LTP. In agreement with a previous study, we have found that NMDA or glutamate application does potentiate synaptic transmission in the hippocampus. This agonist-induced potentiation is, however, decremental and short-lived, unlike LTP. It is occluded shortly after the induction of LTP and a similar short-term potentiation can be evoked by synaptically released glutamate. We thus propose that LTP has two components, a short-term, decremental component which can be mimicked by NMDA receptor activation, and a long-lasting, non-decremental component which, in addition to requiring activation of NMDA receptors, requires stimulation of presynaptic afferents.     

Tissue factor and PAR1 promote microbiota-induced intestinal vascular remodelling

The gut microbiota is a complex ecosystem that has coevolved with host physiology. Colonization of germ-free (GF) mice with a microbiota promotes increased vessel density in the small intestine, but little is known about the mechanisms involved. Tissue factor (TF) is the membrane receptor that initiates the extrinsic coagulation pathway, and it promotes developmental and tumour angiogenesis. Here we show that the gut microbiota promotes TF glycosylation associated with localization of TF on the cell surface, the activation of coagulation proteases, and phosphorylation of the TF cytoplasmic domain in the small intestine. Anti-TF treatment of colonized GF mice decreased microbiota-induced vascular remodelling and expression of the proangiogenic factor angiopoietin-1 (Ang-1) in the small intestine. Mice with a genetic deletion of the TF cytoplasmic domain or with hypomorphic TF (F3) alleles had a decreased intestinal vessel density. Coagulation proteases downstream of TF activate protease-activated receptor (PAR) signalling implicated in angiogenesis. Vessel density and phosphorylation of the cytoplasmic domain of TF were decreased in small intestine from PAR1-deficient (F2r(-/-)) but not PAR2-deficient (F2rl1(-/-)) mice, and inhibition of thrombin showed that thrombin-PAR1 signalling was upstream of TF phosphorylation. Thus, the microbiota-induced extravascular TF-PAR1 signalling loop is a novel pathway that may be modulated to influence vascular remodelling in the small intestine.     

Structural basis of steroid hormone perception by the receptor kinase BRI1

Polyhydroxylated steroids are regulators of body shape and size in higher organisms. In metazoans, intracellular receptors recognize these molecules. Plants, however, perceive steroids at membranes, using the membrane-integral receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1). Here we report the structure of the Arabidopsis thaliana BRI1 ligand-binding domain, determined by X-ray diffraction at 2.5?? resolution. We find a superhelix of 25 twisted leucine-rich repeats (LRRs), an architecture that is strikingly different from the assembly of LRRs in animal Toll-like receptors. A 70-amino-acid island domain between LRRs 21 and 22 folds back into the interior of the superhelix to create a surface pocket for binding the plant hormone brassinolide. Known loss- and gain-of-function mutations map closely to the hormone-binding site. We propose that steroid binding to BRI1 generates a docking platform for a co-receptor that is required for receptor activation. Our findings provide insight into the activation mechanism of this highly expanded family of plant receptors that have essential roles in hormone, developmental and innate immunity signalling.     

Structure of the guide-strand-containing argonaute silencing complex

The slicer activity of the RNA-induced silencing complex is associated with argonaute, the RNase H-like PIWI domain of which catalyses guide-strand-mediated sequence-specific cleavage of target messenger RNA. Here we report on the crystal structure of Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-base DNA guide strand, thereby identifying the nucleic-acid-binding channel positioned between the PAZ- and PIWI-containing lobes, as well as the pivot-like conformational changes associated with complex formation. The bound guide strand is anchored at both of its ends, with the solvent-exposed Watson-Crick edges of stacked bases 2 to 6 positioned for nucleation with the mRNA target, whereas two critically positioned arginines lock bases 10 and 11 at the cleavage site into an unanticipated orthogonal alignment. Biochemical studies indicate that key amino acid residues at the active site and those lining the 5'-phosphate-binding pocket made up of the Mid domain are critical for cleavage activity, whereas alterations of residues lining the 2-nucleotide 3'-end-binding pocket made up of the PAZ domain show little effect.     

Splicing-related catalysis by protein-free snRNAs

Removal of intervening sequences from eukaryotic messenger RNA precursors is carried out by the spliceosome, a complex assembly of five small nuclear RNAs (snRNAs) and a large number of proteins. Although it has been suggested that the spliceosome might be an RNA enzyme, direct evidence for this has been lacking, and the identity of the catalytic domain of the spliceosome is unknown. Here we show that a protein-free complex of two snRNAs, U2 and U6, can bind and position a small RNA containing the sequence of the intron branch site, and activate the branch adenosine to attack a catalytically critical domain of U6 in a reaction that is related to the first step of splicing. Our data provide direct evidence for the catalytic potential of spliceosomal snRNAs.