Many bacterial pathogens secrete proteins that activate host trypsinogen-like enzyme precursors, most notably the proenzymes of the blood coagulation and fibrinolysis systems. Staphylococcus aureus, an important human pathogen implicated in sepsis and endocarditis, secretes the cofactor staphylocoagulase, which activates prothrombin, without the usual proteolytic cleavages, to directly initiate blood clotting. Here we present the 2.2 A crystal structures of human alpha-thrombin and prethrombin-2 bound to a fully active staphylocoagulase variant. The cofactor consists of two domains, each with three-helix bundles; this is a novel fold that is distinct from known serine proteinase activators, particularly the streptococcal plasminogen activator streptokinase. The staphylocoagulase fold is conserved in other bacterial plasma-protein-binding factors and extracellular-matrix-binding factors. Kinetic studies confirm the importance of isoleucine 1 and valine 2 at the amino terminus of staphylocoagulase for zymogen activation. In addition to making contacts with the 148 loop and (pro)exosite I of prethrombin-2, staphylocoagulase inserts its N-terminal peptide into the activation pocket of bound prethrombin-2, allosterically inducing functional catalytic machinery. These investigations demonstrate unambiguously the validity of the zymogen-activation mechanism known as 'molecular sexuality'.

Objective-Platelets play a dual role in thrombosis by forming aggregates and stimulating coagulation. We investigated the commitment of platelets to these separate functions during collagen-induced thrombus formation in vitro and in vivo. Methods and Results-High-resolution 2-photon fluorescence microscopy revealed that in thrombus formation under flow, fibrin(ogen)-binding platelets assembled into separate aggregates, whereas distinct patches of nonaggregated platelets exposed phosphatidylserine. The latter platelet population had inactivated ␣IIb␤3 integrins and displayed increased binding of coagulation factors. Coated platelets, expressing serotonin binding sites, were not identified as a separate population. Thrombin generation and coagulation favored the transformation to phosphatidylserine-exposing platelets with inactivated integrins and reduced adhesion. Prolonged tyrosine phosphorylation in vitro resulted in secondary downregulation of active ␣IIb␤3. Conclusions-These results lead to a new spatial model of thrombus formation, in which aggregated platelets ensure thrombus stability, whereas distinct patches of nonaggregated platelets effectuate procoagulant activity and generate thrombin and fibrin. Herein, the hemostatic activity of a developing thrombus is determined by the balance in formation of proaggregatory and procoagulant platelets. This balance is influenced by antiplatelet and anticoagulant medication. Key Words: aggregation Ⅲ coagulation Ⅲ microdomains Ⅲ platelets Ⅲ integrin activation A ctivated platelets have a dual role in hemostasis and thrombosis. They form the building blocks of a thrombus and provide the membrane surface for coagulation factor activation, which results in thrombin and fibrin formation. 1 Once formed, thrombin greatly enhances platelet activation and aggregation. Given the strong interdependency of thrombin generation and platelet activation, it is intuitively assumed that those platelets that participate in aggregate formation are also involved in the coagulation process, but this has not been investigated.The mechanism(s) by which platelets contribute to thrombin generation and coagulation have been investigated for decades. Kinetic evidence shows that collagen/thrombin-activated platelets expose phosphatidylserine (PS) at their outer surface and then bind Gla domain-containing coagulation factors, mediating factor Xa and thrombin generation. 2 However, even after activation with strong agonists, only a subpopulation of the platelets tends to expose PS. 3 Conversely, it has been argued that PS exposure alone is insufficient to explain the procoagulant contribution of platelets. 4,5 There is also evidence that subfractions of activated platelets have different roles in the coagulation process. Several reports indicate an imperfect relation of PS exposure and the binding of coagulation factors Va, VIIIa, IXa, and Xa to the platelet surface. 6 -8 Another report characterizes a subfraction of activated platelets according to their so-called SCIP morphology (for susta...

The binding of signal recognition particle (SRP) to ribosome-bound signal sequences has been characterized directly and quantitatively using fluorescence spectroscopy. A fluorescent probe was incorporated cotranslationally into the signal sequence of a ribosome⅐nascent chain complex (RNC), and upon titration with SRP, a large and saturable increase in fluorescence intensity was observed. Spectral analyses of SRP and RNC association as a function of concentration allowed us to measure, at equilibrium, K d values of 0.05-0.38 nM for SRP⅐RNC complexes with different signal sequences. Competitive binding experiments with nonfluorescent RNC species revealed that the nascent chain probe did not alter SRP affinity and that SRP has significant affinity for both nontranslating ribosomes (K d ‫؍‬ 71 nM) and RNCs that lack an exposed signal sequence (K d ‫؍‬ 8 nM). SRP can therefore distinguish between translating and nontranslating ribosomes. The very high signal sequence-dependent SRP⅐RNC affinity did not decrease as the nascent chain lengthened. Thus, the inhibition of SRPdependent targeting of RNCs to the endoplasmic reticulum membrane observed with long nascent chains does not result from reduced SRP binding to the signal sequence, as widely thought, but rather from a subsequent step, presumably nascent chain interference of SRP⅐RNC association with the SRP receptor and/or translocon.In mammalian cells, ribosomes are found both in the cytoplasm and at the membrane of the endoplasmic reticulum (ER).1 These two classes of ribosome differ in the nature of their translation products, with membrane-bound ribosomes synthesizing secretory or membrane proteins that are being translocated across or integrated into the ER membrane cotranslationally. The structural feature that distinguishes the cytoplasmic ribosomes from the membrane-bound ribosomes is the presence in the latter of a nascent chain that contains a signal sequence. When a nascent chain signal sequence emerges from the ribosome, it is recognized and bound by a ribonucleoprotein termed the signal recognition particle (SRP) (for review, see Ref. 1). The binding of SRP to the signal sequence-containing ribosome-nascent chain complex (RNC) temporarily prevents it from synthesizing protein.The resulting "elongation-arrested complex" then diffuses to the ER membrane where a GTP-dependent interaction with the SRP receptor initiates a series of events which includes the binding of the RNC to the site of cotranslational translocation and integration at the ER membrane (the translocon), the release of the signal sequence from the SRP, the release of SRP and the SRP receptor from the translocon, and the resumption of protein synthesis by the ribosome (for review, see Refs. 1-3). After targeting is complete, nascent chain translocation or integration then proceeds at the translocon (for review, see Ref. 4). SRP therefore has a critical regulatory role in the cell because it is responsible both for the proper trafficking of newly synthesized proteins and for the conversion of ...

Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where (together with fibrin and platelets) it initiates the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin detected S. aureus in endocarditic vegetations via noninvasive fluorescence or PET imaging. These prothrombin derivatives bound to staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor.

To cite this article: Bock PE, Panizzi P, Verhamme IMA. Exosites in the substrate specificity of blood coagulation reactions. J Thromb Haemost 2007; 5 (Suppl. 1): 81-94.Summary. The specificity of blood coagulation proteinases for substrate, inhibitor, and effector recognition is mediated by exosites on the surfaces of the catalytic domains, physically separated from the catalytic site. Some thrombin ligands bind specifically to either exosite I or II, while others engage both exosites. The involvement of different, overlapping constellations of exosite residues enables binding of structurally diverse ligands. The flexibility of the thrombin structure is central to the mechanism of complex formation and the specificity of exosite interactions. Encounter complex formation is driven by electrostatic ligand-exosite interactions, followed by conformational rearrangement to a stable complex. Exosites on some zymogens are in low affinity proexosite states and are expressed concomitant with catalytic site activation. The requirement for exosite expression controls the specificity of assembly of catalytic complexes on the coagulation pathway, such as the membrane-bound factor Xa•factor Va (prothrombinase) complex, and prevents premature assembly. Substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed by a conformational change to engage the FXa catalytic site. Prothrombin and its activation intermediates bind prothrombinase in two alternative conformations determined by the zymogen to proteinase transition that are hypothesized to involve prothrombin (pro)exosite I interactions with FVa, which underpin the sequential activation pathway. The role of exosites as the major source of substrate specificity has stimulated development of exosite-targeted anticoagulants for treatment of thrombosis.

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Binding of streptokinase (SK) to plasminogen (Pg) conformationally activates the zymogen and converts both Pg and plasmin (Pm) into specific Pg activators. The interaction of SK with Pm and its relationship to the mechanism of Pg activation were evaluated in equilibrium binding studies with active site-labeled fluorescent Pm derivatives and in kinetic studies of SK-induced changes in the catalytic specificity of Pm. SK bound to fluorescein-labeled and native Pm with dissociation constants of 11 ؎ 2 pM and 12 ؎ 4 pM, which represented a 1,000 -10,000-fold higher affinity than determined for Pg. Stoichiometric binding of SK to native Pm was followed by generation of a two-fragment form of SK cleaved at Lys 59 (SK ), which exhibited an indistinguishable affinity for labeled Pm, while a truncated, SK 55-414 species had a 120 -360-fold reduced affinity. Binding of SK to native Pm was accompanied by a >50-fold enhancement in specificity for activation of Pg, which was paralleled by a surprising 2.6 -10-fold loss of specificity of Pm for 8 of 11 tripeptide-pNA substrates. Streptokinase (SK), 1 a 47,000 molecular weight protein from Streptococcus equisimilis, is used as a thrombolytic drug to activate plasminogen (Pg) into plasmin (Pm), the serine proteinase responsible for dissolution of fibrin clots (1, 2). SK possesses no intrinsic enzyme activity, but binds specifically to Pg and Pm, converting both the zymogen and proteinase into Pg activators. SK binding to Pg results in the conformational expression of an active catalytic site on the zymogen that cleaves specifically the Arg 561 -Val 562 activation bond in the catalytic domain of Pg to form Pm (3-7). SK also binds to Pm, transforming the substrate specificity of the proteinase from one which is incapable of Pg activation into a specific activator (7-9), and decreasing greatly the reactivity of Pm toward its physiological serpin inhibitor, ␣ 2 -antiplasmin (10). The mechanism of conformational activation of Pg and the origin of the dramatic macromolecular substrate specificity change that Pm exhibits upon SK binding are unknown. The x-ray crystal structure of SK bound to the catalytic domain of Pm shows that SK consists of three, similarly-folded globular domains (11). SK surrounds the catalytic site, the three domains forming a "three-sided crater" with the active site of Pm at the bottom (11). The structure suggests that SK may induce a conformational change affecting the specificity of the catalytic site and/or participate directly in the binding of Pg as a substrate (11). However, the contribution of these two mechanisms to the change in Pm specificity has not been established. Structurefunction correlation studies with recombinant or proteolytic SK derivatives support a direct role for SK in recognition of Pg as a substrate (12)(13)(14). SK has been reported to cause modest changes in the kinetic constants for Pm with two peptide chromogenic substrates, suggesting that SK binding may also cause a change in catalytic specificity (9,15,16).Quantitative equilibr...

Thrombin and factor Xa, two important pro-coagulant proteinases, can be regulated through direct and indirect inhibition mechanisms. Recently, we designed sulfated dehydropolymers (DHPs) of 4-hydroxycinnamic acids that displayed interesting anticoagulant properties (Monien, B. H., Henry, B. L., Raghuraman, A., Hindle, M., and Desai, U. R. (2006) Bioorg. Med. Chem. 14, 7988 -7998). To better understand their mechanism of action, we studied the direct inhibition of thrombin, factor Xa, factor IXa, and factor VIIa by CDSO3, FDSO3, and SDSO3, three analogs of sulfated DHPs. All three sulfated DHPs displayed a 2-3-fold preference for direct inhibition of thrombin over factor Xa, whereas this preference for inhibiting thrombin over factor IXa and factor VIIa increased to 17-300-fold, suggesting a high level of selectivity. Competitive binding studies with a thrombin-specific chromogenic substrate, a fluorescein-labeled hirudin peptide, bovine heparin, enoxaparin, and a heparin octasaccharide suggest that CDSO3 preferentially binds in or near anion-binding exosite II of thrombin. Studies of the hydrolysis of H-D-hexahydrotyrosol-Ala-Arg-p-nitroanilide indicate that CDSO3 inhibits thrombin through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. Overall, designed sulfated DHPs appear to be the first molecules that bind primarily in the region defined by exosite II and allosterically induce thrombin inhibition. The molecules are radically different in structure from all the current clinically used anticoagulants and thus represent a novel class of potent dual thrombin and factor Xa inhibitors.The coagulation cascade is composed of two intertwined pathways, called the extrinsic and the intrinsic pathways, that operate in a highly complex, but tightly regulated, manner to bring about controlled formation of the fibrin polymer. Several enzymes participate in this process, including factor IXa and factor VIIa, which belong to the intrinsic and extrinsic pathways, respectively, and thrombin and factor Xa, which belong to the common pathway (1, 2). The cascade is regulated by several proteins present naturally in the plasma, of which antithrombin is a major regulator (3, 4).Antithrombin, a member of the serpin (serine proteinase inhibitor) family of proteins, primarily inhibits thrombin, factor Xa, and factor IXa and also possibly inhibits several other enzymes to a lesser extent. Yet antithrombin is a rather poor inhibitor of these pro-coagulant enzymes and requires the presence of heparin to exhibit its anticoagulant potential (3, 4). Heparin is a highly sulfated polysaccharide that greatly enhances the rate of antithrombin inhibition of thrombin, factor Xa, and factor IXa under physiological conditions (5). This acceleration is the primary reason for the continued use of heparin as an effective anticoagulant for the past 8 decades. Yet heparin suffers from several limitations, including enhanced risk for bleeding, variable patient response, heparin-induced thrombocytopenia, and the ...