Mapping of the fibrinogen-binding site on the staphylocoagulase C-terminal repeat region.

Fibrin (Fbn) deposits are a hallmark of staphylocoagulase (SC)-positive endocarditis. Binding of the N terminus of Staphylococcus aureus SC to host prothrombin triggers formation of an active SC·prothrombin∗ complex that cleaves host fibrinogen to Fbn. In addition, the C-terminal domain of the prototypical SC contains one pseudorepeat (PR) and seven repeats (R1 → R7) that bind fibrinogen/Fbn fragment D (frag D) by a mechanism that is unclear. Here, we define affinities and stoichiometries of frag D binding to C-terminal SC constructs, using fluorescence equilibrium binding, NMR titration, alanine scanning, and native PAGE. We found that constructs containing the PR and single repeats bound frag D with KD ∼50 to 130 nM and a 1:1 stoichiometry, indicating a conserved binding site bridging the PR and each repeat. NMR titration of PR-R7 with frag D revealed that residues 22 to 49, bridging PR and R7, constituted the minimal peptide (MP) for binding, corroborated by alanine scanning, and binding of labeled MP to frag D. MP alignment with the PR-R and inter-repeat junctions identified critical conserved residues. Full-length PR-(R1 → R7) bound frag D with KD ∼20 nM and a stoichiometry of 1:5, whereas constructs containing the PR and various three repeats competed with PR-(R1 → R7) for frag D binding, with a 1:3 stoichiometry. These findings are consistent with binding at PR-R and R-R junctions with modest inter-repeat sequence variability. CD of PR-R7 and PR-(R1 → R7) suggested a disordered flexible structure, allowing binding of multiple fibrin(ogen) molecules. Taken together, these results provide insights into pathogen localization on host fibrin networks.

Multimodal imaging of bacterial-host interface in mice and piglets with Staphylococcus aureus endocarditis.

Acute bacterial endocarditis is a rapid, difficult to manage, and frequently lethal disease. Potent antibiotics often cannot efficiently kill Staphylococcus aureus that colonizes the heart's valves. S. aureus relies on virulence factors to evade therapeutics and the host's immune response, usurping the host's clotting system by activating circulating prothrombin with staphylocoagulase and von Willebrand factor-binding protein. An insoluble fibrin barrier then forms around the bacterial colony, shielding the pathogen from immune cell clearance. Targeting virulence factors may provide previously unidentified avenues to better diagnose and treat endocarditis. To tap into this unused therapeutic opportunity, we codeveloped therapeutics and multimodal molecular imaging to probe the host-pathogen interface. We introduced and validated a family of small-molecule optical and positron emission tomography (PET) reporters targeting active thrombin in the fibrin-rich environment of bacterial colonies. The imaging agents, based on the clinical thrombin inhibitor dabigatran, are bound to heart valve vegetations in mice. Using optical imaging, we monitored therapy with antibodies neutralizing staphylocoagulase and von Willebrand factor-binding protein in mice with S. aureus endocarditis. This treatment deactivated bacterial defenses against innate immune cells, decreased in vivo imaging signal, and improved survival. Aortic or tricuspid S. aureus endocarditis in piglets was also successfully imaged with clinical PET/magnetic resonance imaging. Our data map a route toward adjuvant immunotherapy for endocarditis and provide efficient tools to monitor this drug class for infectious diseases.

Specificity and affinity of the N-terminal residues in staphylocoagulase in binding to prothrombin.

In Staphylococcus aureus-caused endocarditis, the pathogen secretes staphylocoagulase (SC), thereby activating human prothrombin (ProT) and evading immune clearance. A previous structural comparison of the SC(1-325) fragment bound to thrombin and its inactive precursor prethrombin 2 has indicated that SC activates ProT by inserting its N-terminal dipeptide Ile1-Val2 into the ProT Ile16 pocket, forming a salt bridge with ProT's Asp194, thereby stabilizing the active conformation. We hypothesized that these N-terminal SC residues modulate ProT binding and activation. Here, we generated labeled SC(1-246) as a probe for competitively defining the affinities of N-terminal SC(1-246) variants preselected by modeling. Using ProT(R155Q,R271Q,R284Q) (ProTQQQ), a variant refractory to prothrombinase- or thrombin-mediated cleavage, we observed variant affinities between ∼1 and 650 nm and activation potencies ranging from 1.8-fold that of WT SC(1-246) to complete loss of function. Substrate binding to ProTQQQ caused allosteric tightening of the affinity of most SC(1-246) variants, consistent with zymogen activation through occupation of the specificity pocket. Conservative changes at positions 1 and 2 were well-tolerated, with Val1-Val2, Ile1-Ala2, and Leu1-Val2 variants exhibiting ProTQQQ affinity and activation potency comparable with WT SC(1-246). Weaker binding variants typically had reduced activation rates, although at near-saturating ProTQQQ levels, several variants exhibited limiting rates similar to or higher than that of WT SC(1-246). The Ile16 pocket in ProTQQQ appears to favor nonpolar, nonaromatic residues at SC positions 1 and 2. Our results suggest that SC variants other than WT Ile1-Val2-Thr3 might emerge with similar ProT-activating efficiency.

Protein Z-dependent protease inhibitor (ZPI) is a physiologically significant inhibitor of prothrombinase function.

Current thought holds that factor Xa (FXa) bound in the prothrombinase complex is resistant to regulation by protein protease inhibitors during prothrombin activation. Here we provide evidence that, contrary to this view, the FXa-specific serpin inhibitor, protein Z-dependent protease inhibitor (ZPI), complexed with its cofactor, protein Z (PZ), functions as a physiologically significant inhibitor of prothrombinase-bound FXa during prothrombin activation. Kinetics studies showed that the rapid rate of inhibition of FXa by the ZPI-PZ complex on procoagulant membrane vesicles (ka(app) ∼107 m-1 s-1) was decreased ∼10-fold when FXa was bound to FVa in prothrombinase and a further ∼3-4-fold when plasma levels of S195A prothrombin were present (ka(app) 2 × 105 m-1 s-1). Nevertheless, the ZPI-PZ complex produced a major inhibition of thrombin generation during prothrombinase-catalyzed activation of prothrombin under physiologically relevant conditions. The importance of ZPI-PZ complex anticoagulant regulation of FXa both before and after incorporation into prothrombinase was supported by thrombin generation assays in plasma. These showed enhanced thrombin generation when the inhibitor was neutralized with a PZ-specific antibody and decreased thrombin generation when exogenous ZPI-PZ complex was added whether prothrombin was activated directly by FXa or through extrinsic or intrinsic pathway activators. Moreover, the PZ antibody enhanced thrombin generation both in the absence and presence of activated protein C (APC) anticoagulant activity. Taken together, these results suggest an important anticoagulant role for the ZPI-PZ complex in regulating both free FXa generated in the initiation phase of coagulation as well as prothrombinase-bound FXa in the propagation phase that complement prothrombinase regulation by APC.

Platelet heterogeneity in activation-induced glycoprotein shedding: functional effects.

The platelet receptors glycoprotein Ibα (GPIbα) and GPVI are known to be cleaved by members of a disintegrin and metalloprotease (ADAM) family (ADAM10 and ADAM17), but the mechanisms and consequences of this shedding are not well understood. Our results revealed that (1) glycoprotein shedding is confined to distinct platelet populations showing near-complete shedding, (2) the heterogeneity between (non)shed platelets is independent of agonist type but coincides with exposure of phosphatidylserine (PS), and (3) distinct pathways of shedding are induced by elevated Ca2+, low Ca2+ protein kinase C (PKC), or apoptotic activation. Furthermore, we found that receptor shedding reduces binding of von Willebrand factor, enhances binding of coagulation factors, and augments fibrin formation. In response to Ca2+-increasing agents, shedding of GPIbα was abolished by ADAM10/17 inhibition but not by blockage of calpain. Stimulation of PKC induced shedding of only GPIbα, which was annulled by kinase inhibition. The proapoptotic agent ABT-737 induced shedding, which was caspase dependent. In Scott syndrome platelets that are deficient in Ca2+-dependent PS exposure, shedding occurred normally, indicating that PS exposure is not a prerequisite for ADAM activity. In whole-blood thrombus formation, ADAM-dependent glycoprotein shedding enhanced thrombin generation and fibrin formation. Together, these findings indicate that 2 major activation pathways can evoke ADAM-mediated glycoprotein shedding in distinct platelet populations and that shedding modulates platelet function from less adhesive to more procoagulant.

Межсекторальные сети охраны психического здоровья в Бельгии: пример успешной реформы системы охраны психического здоровья посредством реорганизации модели предоставления услуг: информационный обзор по передовому опыту

Бельгия сделала выбор в пользу проведения общенациональной реформы сектора охраны психического здоровья в ответ на необходимость улучшения ориентированных на население подходов к оказанию медицинской помощи людям с психическими расстройствами, что явилось императивом обеспечения ее эффективности. Реформа была направлена на укрепление предложения услуг на общинном уровне и сокращение числа больничных коек в психиатрических больницах; она способствовала улучшению интеграции медицинской помощи, улучшению социальной реабилитации и выздоровления пользователей медицинскими услугами, включая улучшение качества жизни пациентов и лиц, осуществляющих уход за ними. Центральное место в реформе занимают межсекторальные сети охраны психического здоровья, предлагающие услуги по индивидуальной поддержке, профилактику, стационарную и амбулаторную психиатрическую помощь, первичную медико-санитарную помощь, дневной уход, а также профессионально-техническую, жилищную и социальную помощь. Общенациональная реформа согласуется с Комплексным планом действий ВОЗ в области психического здоровья на 2013–2020 гг., в котором предлагается осуществить переход от институционального медицинского обслуживания к созданию системы медицинского обслуживания в области психического здоровья на общинном уровне. Реформа способствовала улучшению долгосрочной стабильности состояния пациентов и сокращению срока их пребывания в больнице. Она также способствовала значительному сокращению числа больничных коек в психиатрических больницах в связи с расширением индивидуальной поддержки людей с психическими заболеваниями на первичном уровне.

Multisectoral mental health networks in Belgium: an example of successful mental health reform through service delivery redesign: good practice brief

Belgium has opted for a nationwide reform of the mental health sector in response to the need for improved people-centred approaches for people with mental health conditions, an efficiency imperative. The reform aimed to strengthen the community-based supply of care and to reduce the number of psychiatric hospital beds; it improved care integration, social rehabilitation and service users’ recovery, including users’ and carers’ quality of life. Central to the reform are multisectoral mental health care networks offering outreach services, prevention, in-and outpatient mental health services, primary care, day care, and vocational, housing and social care services. The nationwide reform is in line with WHO’s Mental Health Action Plan 2013–2020, calling for a shift from institutional care to community services. The reform contributed to improving the long-term health of patients and reducing hospital stays. It also significantly reduced the number of psychiatric hospital beds in favour of outreach services to people with mental health conditions.

Carbon and amide detect backbone assignment methods of a novel repeat protein from the staphylocoagulase in S. aureus.

The C-terminal repeat domain of staphylocoagulase that is secreted by the S. aureus is believed to play an important role interacting with fibrinogen and promotes blood clotting. To study this interaction by NMR, full assignment of each amide residue in the HSQC spectrum was required. Despite of the short sequence of the repeat construct, the HSQC spectrum contained a substantial amount of overlapped and exchange broadened resonances, indicating little secondary or tertiary structure. This caused severe problems while using the conventional, amide based NMR method for the backbone assignment. With the growing interest in small apparently disordered proteins, these issues are being faced more frequently. An alternative strategy to improve the backbone assignment capability involved carbon direct detection methods. Circumventing the amide proton detection offers a larger signal dispersion and more uniform signal intensity. For peptides with higher concentrations and in combination with the cold carbon channels of new cryoprobes, higher fields, and sufficiently long relaxation times, the disadvantage of the lower sensitivity of the 13C nucleus can be overcome. Another advantage of this method is the assignment of the proline backbone residues. Complete assignment with the carbon-detected strategy was achieved with a set of only two 3D, one 2D, and a HNCO measurement, which was necessary to translate the information to the HSQC spectrum.

Complete genome of Staphylococcus aureus Tager 104 provides evidence of its relation to modern systemic hospital-acquired strains.

BACKGROUND: Staphylococcus aureus (S. aureus) infections range in severity due to expression of certain virulence factors encoded on mobile genetic elements (MGE). As such, characterization of these MGE, as well as single nucleotide polymorphisms, is of high clinical and microbiological importance. To understand the evolution of these dangerous pathogens, it is paramount to define reference strains that may predate MGE acquisition. One such candidate is S. aureus Tager 104, a previously uncharacterized strain isolated from a patient with impetigo in 1947. RESULTS: We show here that S. aureus Tager 104 can survive in the bloodstream and infect naïve organs. We also demonstrate a procedure to construct and validate the assembly of S. aureus genomes, using Tager 104 as a proof-of-concept. In so doing, we bridged confounding gap regions that limited our initial attempts to close this 2.82 Mb genome, through integration of data from Illumina Nextera paired-end, PacBio RS, and Lucigen NxSeq mate-pair libraries. Furthermore, we provide independent confirmation of our segmental arrangement of the Tager 104 genome by the sole use of Lucigen NxSeq libraries filled by paired-end MiSeq reads and alignment with SPAdes software. Genomic analysis of Tager 104 revealed limited MGE, and a νSaß island configuration that is reminiscent of other hospital acquired S. aureus genomes. CONCLUSIONS: Tager 104 represents an early-branching ancestor of certain hospital-acquired strains. Combined with its earlier isolation date and limited content of MGE, Tager 104 can serve as a viable reference for future comparative genome studies.

In Vivo Tracking of Streptococcal Infections of Subcutaneous Origin in a Murine Model.

PURPOSE: Generation of plasmin in vivo by Streptococcus pyogenes is thought to localize the active protease complexes to the pathogen surface to aid in tissue dissemination. Here, we chose to follow cutaneous streptococcal infections by the use of non-invasive bioluminescence imaging to determine if this pathogen can be followed by this approach and the extent of bacterial spread in the absence of canonical plasminogen activation by streptokinase. PROCEDURES: Mice were injected subcutaneously with either bioluminescent strains of streptococci, namely Xen20 and Xen10 or S. pyogenes ALAB49. Bioluminescence imaging was performed daily and results were correlated with microbiological and histological analyses. RESULTS: Comparative analysis of chronologic non-invasive datasets indicated that Xen20 did not disseminate from the initial infection site. Contrary to this, microbiological and histological analyses of Xen20 mice for total bacterial burden indicated sepsis and widespread pathogen involvement. CONCLUSIONS: The use of bioluminescence in microbe-based studies requires genomic and pathologic characterization to correlate imaging results with underlying pathology.

Rapid binding of plasminogen to streptokinase in a catalytic complex reveals a three-step mechanism.

Rapid kinetics demonstrate a three-step pathway of streptokinase (SK) binding to plasminogen (Pg), the zymogen of plasmin (Pm). Formation of a fluorescently silent encounter complex is followed by two conformational tightening steps reported by fluorescence quenches. Forward reactions were defined by time courses of biphasic quenching during complex formation between SK or its COOH-terminal Lys(414) deletion mutant (SKΔK414) and active site-labeled [Lys]Pg ([5-(acetamido)fluorescein]-D-Phe-Phe-Arg-[Lys]Pg ([5F]FFR-[Lys]Pg)) and by the SK dependences of the quench rates. Active site-blocked Pm rapidly displaced [5F]FFR-[Lys]Pg from the complex. The encounter and final SK ·[5F]FFR-[Lys]Pg complexes were weakened similarly by SK Lys(414) deletion and blocking of lysine-binding sites (LBSs) on Pg kringles with 6-aminohexanoic acid or benzamidine. Forward and reverse rates for both tightening steps were unaffected by 6-aminohexanoic acid, whereas benzamidine released constraints on the first conformational tightening. This indicated that binding of SK Lys(414) to Pg kringle 4 plays a role in recognition of Pg by SK. The substantially lower affinity of the final SK · Pg complex compared with SK · Pm is characterized by a ∼ 25-fold weaker encounter complex and ∼ 40-fold faster off-rates for the second conformational step. The results suggest that effective Pg encounter requires SK Lys(414) engagement and significant non-LBS interactions with the protease domain, whereas Pm binding additionally requires contributions of other lysines. This difference may be responsible for the lower affinity of the SK · Pg complex and the expression of a weaker "pro"-exosite for binding of a second Pg in the substrate mode compared with SK · Pm.

Targeting inflammation in cancer-related-fatigue: a rationale for mistletoe therapy as supportive care in colorectal cancer patients.

BACKGROUND: Cancer-related fatigue (CRF) affects a majority of patients (pts) with symptoms lasting up to several years after finishing therapy. These symptoms lead to decreased health related quality of life. Fatigue during treatment for colorectal cancer is common, but poorly understood and can affect compliance with post-surgical cancer therapy. We examined the fatigue levels during first-line chemo- or radio-chemotherapy protocols, which were supported by a pharmaceutical mistletoe preparation (Iscador(®)Qu) (181patients). We compared the outcome to a parallel control group (143 patients), which did not receive this supportive care treatment. METHODS: The medical records of 324 patients with non-metastasized colorectal cancer (UICC stage I-III), which were obtained from hospitals and resident physicians, were assessed. The documented treatment decision by chemo- or radio-chemotherapy supported by mistletoe interventions was followed for a median treatment period of 8.6 months. During the post-surgical treatment period the patients were diagnosed twice for the presence of fatigue symptoms by structural interviews carried out by physicians. RESULTS: At the end of the median treatment period, 16/181 patients (8.8%) were diagnosed with CRF in the supportive care group and 86/143 (60.1%) in the chemo- or radio-chemotherapy group without supportive mistletoe medication. Multivariable-adjusted ORs provided evidence for a chance to improve CRF by supportive mistletoe medication compared to chemo- or radio-chemotherapy alone over the time of treatment. The OR = 10.651 (95% CI 5.09-22.28; p < 0.001) declined from the first visit to OR = 0.054 (95 CI 0.02-0.13; p < 0.001) at the end of therapy. Furthermore, 14 confounding factors for risk assessment of CRF were compared by means of forest plots. It turned out that the hospital versus office-based treatment and the co-morbidity/inflammation represent independent but important determinants for fatigue levels. CONCLUSION: The clinically used mistletoe medication (Iscador(®)Qu) is the first candidate to be included in a supportive care modus into chemo- or chemo-radiotherapy protocols for colorectal patients to improve CRF without discernable toxicities.

Full time course kinetics of the streptokinase-plasminogen activation pathway.

Our previously hypothesized mechanism for the pathway of plasminogen (Pg) activation by streptokinase (SK) was tested by the use of full time course kinetics. Three discontinuous chromogenic substrate initial rate assays were developed with different quenching conditions that enabled quantitation of the time courses of Pg depletion, plasmin (Pm) formation, transient formation of the conformationally activated SK·Pg* catalytic complex intermediate, formation of the SK·Pm catalytic complex, and the free concentrations of Pg, Pm, and SK. Analysis of full time courses of Pg activation by five concentrations of SK along with activity-based titrations of SK·Pg* and SK·Pm formation yielded rate and dissociation constants within 2-fold of those determined previously by continuous measurement of parabolic chromogenic substrate hydrolysis and fluorescence-based equilibrium binding. The results obtained with orthogonal assays provide independent support for a mechanism in which the conformationally activated SK·Pg* complex catalyzes an initial cycle of Pg proteolytic conversion to Pm that acts as a trigger. Higher affinity binding of the formed Pm to SK outcompetes Pg binding, terminating the trigger cycle and initiating the bullet catalytic cycle by the SK·Pm complex that converts the residual Pg into Pm. The new assays can be adapted to quantitate SK-Pg activation in the context of SK- or Pg-directed inhibitors, effectors, and SK allelic variants. To support this, we show for the first time with an assay specific for SK·Pg* that fibrinogen forms a ternary SK·Pg*·fibrinogen complex, which assembles with 200-fold enhanced SK·Pg* affinity, signaled by a perturbation of the SK·Pg* active site.

Effect of zymogen domains and active site occupation on activation of prothrombin by von Willebrand factor-binding protein.

Prothrombin is conformationally activated by von Willebrand factor-binding protein (vWbp) from Staphylococcus aureus through insertion of the NH(2)-terminal residues of vWbp into the prothrombin catalytic domain. The rate of prothrombin activation by vWbp(1-263) is controlled by a hysteretic kinetic mechanism initiated by substrate binding. The present study evaluates activation of prothrombin by full-length vWbp(1-474) through activity progress curve analysis. Additional interactions from the COOH-terminal half of vWbp(1-474) strengthened the initial binding of vWbp to prothrombin, resulting in higher activity and an ∼100-fold enhancement in affinity. The affinities of vWbp(1-263) or vWbp(1-474) were compared by equilibrium binding to the prothrombin derivatives prethrombin 1, prethrombin 2, thrombin, meizothrombin, and meizothrombin(des-fragment 1) and their corresponding active site-blocked analogs. Loss of fragment 1 in prethrombin 1 enhanced affinity for both vWbp(1-263) and vWbp(1-474), with a 30-45% increase in Gibbs free energy, implicating a regulatory role for fragment 1 in the activation mechanism. Active site labeling of all prothrombin derivatives with D-Phe-Pro-Arg-chloromethyl ketone, analogous to irreversible binding of a substrate, decreased their K(D) values for vWbp into the subnanomolar range, reflecting the dependence of the activating conformational change on substrate binding. The results suggest a role for prothrombin domains in the pathophysiological activation of prothrombin by vWbp, and may reveal a function for autocatalysis of the vWbp·prothrombin complexes during initiation of blood coagulation.

Development of coagulation factor probes for the identification of procoagulant circulating tumor cells.

Metastatic cancer is associated with a hypercoagulable state, and pathological venous thromboembolic disease is a significant source of morbidity and the second leading cause of death in patients with cancer. Here we aimed to develop a novel labeling strategy to detect and quantify procoagulant circulating tumor cells (CTCs) from patients with metastatic cancer. We hypothesize that the enumeration of procoagulant CTCs may be prognostic for the development of venous thrombosis in patients with cancer. Our approach is based on the observation that cancer cells are capable of initiating and facilitating cell-mediated coagulation in vitro, whereby activated coagulation factor complexes assemble upon cancer cell membrane surfaces. Binding of fluorescently labeled, active site-inhibited coagulation factors VIIa, Xa, and IIa to the metastatic breast cancer cell line, MDA-MB-231, non-metastatic colorectal cell line, SW480, or metastatic colorectal cell line, SW620, was characterized in a purified system, in anticoagulated blood and plasma, and in plasma under conditions of coagulation. We conclude that a CTC labeling strategy that utilizes coagulation factor-based fluorescent probes may provide a functional assessment of the procoagulant potential of CTCs, and that this strategy is amenable to current CTC detection platforms.

Notecarin D binds human factor V and factor Va with high affinity in the absence of membranes.

Notecarin D (NotD) is a prothrombin (ProT) activator in the venom of the tiger snake, Notechis scutatus, and a factor Xa (FXa) homolog. NotD binds specifically to the FXa binding site expressed on factor V (FV) upon activation to factor Va (FVa) by thrombin. NotD active site-labeled with 5-fluorescein ([5F]FFR-NotD) binds FV and FVa with remarkably high affinity in the absence of phospholipids (K(D) 12 and ≤ 0.01 nm, respectively). In the presence of membranes, the affinity of [5F]FFR-NotD for FVa is similar, but increased ∼55-fold for FV. Binding of FXa active site-labeled with Oregon Green to FV and FVa in the presence of phospholipids is ∼5,000- and ∼80-fold weaker than [5F]FFR-NotD, respectively. NotD reports FVa and not FV binding by a 3-fold increase in tripeptide substrate hydrolysis, demonstrating allosteric regulation by FVa. The NotD·FVa·membrane complex activates ProT with K(m)((app)) similar to prothrombinase, and ∼85-fold weaker without membranes. Active site-blocked NotD exhibits potent anticoagulant activity in plasma thrombin generation assays, representing inhibition of productive prothrombinase assembly and possible disruption of FXa inhibition by the tissue factor pathway inhibitor. The results show that high affinity binding of NotD to FVa is membrane-independent, unlike the strict membrane dependence of FXa for high affinity FVa binding.

In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation.

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, the bacteria initiate the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin could be used to detect S. aureus in endocarditic vegetations via noninvasive fluorescence or positron emission tomography (PET) imaging. These prothrombin derivatives bound 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.

Structural basis of thrombin-mediated factor V activation: the Glu666-Glu672 sequence is critical for processing at the heavy chain-B domain junction.

Thrombin-catalyzed activation of coagulation factor V (FV) is an essential positive feedback reaction within the blood clotting system. Efficient processing at the N- (Arg(709)-Ser(710)) and C-terminal activation cleavage sites (Arg(1545)-Ser(1546)) requires initial substrate interactions with 2 clusters of positively charged residues on the proteinase surface, exosites I and II. We addressed the mechanism of activation of human factor V (FV) using peptides that cover the entire acidic regions preceding these cleavage sites, FV (657-709)/ (FVa2) and FV(1481-1545)/(FVa3). FVa2 appears to interact mostly with exosite I, while both exosites are involved in interactions with the C-terminal linker. The 1.7-Å crystal structure of irreversibly inhibited thrombin bound to FVa2 unambiguously reveals docking of FV residues Glu(666)-Glu(672) to exosite I. These findings were confirmed in a second, medium-resolution structure of FVa2 bound to the benzamidine-inhibited proteinase. Our results suggest that the acidic A2-B domain linker is involved in major interactions with thrombin during cofactor activation, with its more N-terminal hirudin-like sequence playing a critical role. Modeling experiments indicate that FVa2, and likely also FVa3, wrap around thrombin in productive thrombin·FV complexes that cover a large surface of the activator to engage the active site.

Engineering streptokinase for generation of active site-labeled plasminogen analogs.

We previously demonstrated that streptokinase (SK) can be used to generate active site-labeled fluorescent analogs of plasminogen (Pg) by virtue of its nonproteolytic activation of the zymogen. The method is versatile and allows stoichiometric and active site-specific incorporation of any one of many molecular probes. The limitation of the labeling approach is that it is both time-consuming and low yield. Here we demonstrate an improved method for the preparation of labeled Pg analogs by the use of an engineered SK mutant fusion protein with both COOH- and NH(2)-terminal His(6) tags. The NH(2)-terminal tag is followed by a tobacco etch virus proteinase cleavage site to ensure that the SK Ile(1) residue, essential for conformational activation of Pg, is preserved. The SK COOH-terminal Lys(414) residue and residues Arg253-Leu260 in the SK ß-domain were deleted to prevent cleavage by plasmin (Pm) and to disable Pg substrate binding to the SK·Pg(∗)/Pm catalytic complexes, respectively. Near elimination of Pm generation with the SKΔ(R253-L260)ΔK414-His(6) mutant increased the yield of labeled Pg 2.6-fold and reduced the time required more than 2-fold. The versatility of the labeling method was extended to the application of Pg labeled with a near-infrared probe to quantitate Pg receptors on immune cells by flow cytometry.

Active site-labeled prothrombin inhibits prothrombinase in vitro and thrombosis in vivo.

Mouse and human prothrombin (ProT) active site specifically labeled with D-Phe-Pro-Arg-CH(2)Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating the requirement for all three ProT domains. Kinetics of inhibition of ProT activation by the inactive ProT(S195A) mutant were compatible with competitive inhibition as an alternate nonproductive substrate, although FPR-ProT deviated from this mechanism, implicating a more complex process. FPR-ProT exhibited ∼10-fold more potent anticoagulant activity compared with ProT(S195A) as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProT(S195A), the pathway of FPR-ProT cleavage by prothrombinase was redirected from meizothrombin toward formation of the FPR-prethrombin 2 (Pre 2)·F1.2 inhibitory intermediate. Localization of ProT labeled with Alexa Fluor® 660 tethered through FPR-CH(2)Cl ([AF660]FPR-ProT) during laser-induced thrombus formation in vivo in murine arterioles was examined in real time wide-field and confocal fluorescence microscopy. [AF660]FPR-ProT bound rapidly to the vessel wall at the site of injury, preceding platelet accumulation, and subsequently to the thrombus proximal, but not distal, to the vessel wall. [AF660]FPR-ProT inhibited thrombus growth, whereas [AF660]FPR-Pre 1, lacking the F1 membrane-binding domain did not bind or inhibit. Labeled F1.2 localized similarly to [AF660]FPR-ProT, indicating binding to phosphatidylserine-rich membranes, but did not inhibit thrombosis. The studies provide new insight into the mechanism of ProT activation in vivo and in vitro, and the properties of a unique exosite-directed prothrombinase inhibitor.