Edward L. G. Pryzdial

Antibacterial activity, inflammatory response, coagulation and cytotoxicity effects of silver nanoparticles

The incorporation of nanoparticles (NPs) in industrial and biomedical applications has increased significantly in recent years, yet their hazardous and toxic effects have not been studied extensively. Here, we studied the effects of 24 nm silver NPs (AgNPs) on a panel of bacteria isolated from medical devices used in a hospital intensive care unit. The cytotoxic effects were evaluated in macrophages and the expression of the inflammatory cytokines IL-6, IL-10 and TNF-α were quantified. The effects of NPs on coagulation were tested in vitro in plasma-based assays. We demonstrated that 24 nm AgNPs were effective in suppressing the growth of clinically relevant bacteria with moderate to high levels of antibiotic resistance. The NPs had a moderate inhibitory effect when coagulation was initiated through the intrinsic pathway. However, these NPs are cytotoxic to macrophages and are able to elicit an inflammatory response. Thus, beneficial and potential harmful effects of 24 nm AgNPs on biomedical devices must be weighed in further studies in vivo. From the Clinical Editor: The authors of this study demonstrate that gallic acid reduced 24 nm Ag NPs are effective in suppressing growth of clinically relevant antibiotic resistant bacteria. However, these NPs also exhibit cytotoxic properties to macrophages and may trigger an inflammatory response. Thus, the balance of beneficial and potential harmful effects must be weighed carefully in further studies.

Thrombin generates previously unidentified C5 products that support the terminal complement activation pathway

The coagulation and complement pathways simultaneously promote homeostasis in response to injury but cause tissue damage when unregulated. Mechanisms by which they cooperate are poorly understood. To delineate their interactions, we studied the effects of thrombin and C5 convertase on C5 in purified and plasma-based systems, measuring release of the anaphylatoxin C5a, and generation of C5b, the initial component of the lytic membrane attack complex. Thrombin cleaved C5 poorly at R751, yielding minimal C5a and C5b. However, thrombin efficiently cleaved C5 at a newly identified, highly conserved R947 site, generating previously undescribed intermediates C5(T) and C5b(T). Tissue factor-induced clotting of plasma led to proteolysis of C5 at a thrombin-sensitive site corresponding to R947 and not R751. Combined treatment of C5 with thrombin and C5 convertase yielded C5a and C5b(T), the latter forming a C5b(T)-9 membrane attack complex with significantly more lytic activity than with C5b-9. Our findings provide a new paradigm for complement activation, in which thrombin and C5 convertase are invariant partners, enhancing the terminal pathway via the generation of newly uncovered C5 intermediates. Delineating the molecular links between coagulation and complement will provide new therapeutic targets for diseases associated with excess fibrin deposition and complement activation.

Dengue virus binding and replication by platelets

Dengue virus (DENV) infection causes ∼200 million cases of severe flulike illness annually, escalating to life-threatening hemorrhagic fever or shock syndrome in ∼500,000. Although thrombocytopenia is typical of both mild and severe diseases, the mechanism triggering platelet reduction is incompletely understood. As a probable initiating event, direct purified DENV-platelet binding was followed in the current study by quantitative reverse transcription-polymerase chain reaction and confirmed antigenically. Approximately 800 viruses specifically bound per platelet at 37°C. Fewer sites were observed at 25°C, the blood bank storage temperature (∼350 sites), or 4°C, known to attenuate virus cell entry (∼200 sites). Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and heparan sulfate proteoglycan were implicated as coreceptors because only the combination of anti-DC-SIGN and low-molecular-weight heparin prevented binding. Interestingly, at 37°C and 25°C, platelets replicated the positive sense single-stranded RNA genome of DENV by up to ∼4-fold over 7 days. Further time course experiments demonstrated production of viral NS1 protein, which is known to be highly antigenic in patient serum. The infectivity of DENV intrinsically decayed in vitro, which was moderated by platelet-mediated generation of viable progeny. This was shown using a transcription inhibitor and confirmed by freeze-denatured platelets being incapable of replicating the DENV genome. For the first time, these data demonstrate that platelets directly bind DENV saturably and produce infectious virus. Thus, expression of antigen encoded by DENV is a novel consideration in the pathogen-induced thrombocytopenia mechanism. These results furthermore draw attention to the possibility that platelets may produce permissive RNA viruses in addition to DENV.

Thrombin induces endothelial cell-surface exposure of the plasminogen receptor annexin 2

Cell-surface annexin 2 (A2) and its ligand p11 have been implicated in fibrinolysis because of their ability to accelerate tissue plasminogen activator (tPA)-mediated activation of plasminogen to plasmin. Because thrombin is a potent cell modulator obligately produced at the site of clot formation, we hypothesized that the amount of cell-surface A2 and p11 might be altered by thrombin with consequent effects on plasmin generation. In support of this hypothesis, immunofluorescence microscopy and hydrophilic biotinylation experiments showed that both A2 and p11 were significantly increased on the surface of human umbilical vein endothelial cells (HUVECs) treated with thrombin (0.8-8 nM) for 5 minutes followed by 1 hour at 37°C. Intracellular immunofluorescence microscopy and immunoblot analyses of whole cell extracts revealed increased p11 but unchanged A2 in response to thrombin, suggesting that transbilayer trafficking of A2 might be controlled by p11. The thrombin receptor-activating peptide (TRAP) similarly affected cells, demonstrating that cell signaling at least involved the type-1 protease activated receptor (PAR-1). An effect on the fibrinolysis pathway after treatment of HUVECs with thrombin was shown by increased fluorescein-labeled plasminogen binding to cells, which was inhibited by an antibody specific for p11. This was confirmed by observing that thrombin pretreatment of HUVECs increased biotin-modified plasminogen binding. Utilizing a chromogenic assay, pretreatment of HUVECs by thrombin further enhanced activation of the Glu and Lys forms of plasminogen by tPA. These data suggest a novel mechanism that links the coagulation and fibrinolysis pathways by thrombin-mediated feedback.

Plasmin Converts Factor X from Coagulation Zymogen to Fibrinolysis Cofactor

Known anticoagulant pathways have been shown to exclusively inhibit blood coagulation cofactors and enzymes. In the current work, we first investigated the possibility of a novel anticoagulant mechanism that functions at the level of zymogen inactivation. Utilizing both clotting and chromogenic assays, the fibrinolysis protease plasmin was found to irreversibly inhibit the pivotal function of factor X (FX) in coagulation. This was due to cleavage at several sites, the location of which were altered by association of FX with procoagulant phospholipid (proPL). The final products were ∼28 and ∼47 kDa for proPL-bound and unbound FX, respectively, which did not have analogues when activated FX (FXa) was cleaved instead. We next investigated whether the FX derivatives could interact with the plasmin precursor plasminogen, and we found that plasmin exposed a binding site only on proPL-bound FX. The highest apparent affinity was for the 28-kDa fragment, which was identified as the light subunit disulfide linked to a small fragment of the heavy subunit (Met-296 to ∼Lys-330). After cleavage by plasmin, proPL-bound FX furthermore was observed to accelerate plasmin generation by tissue plasminogen activator. Thus, a feedback mechanism localized by proPL is suggested in which plasmin simultaneously inhibits FX clotting function and converts proPL-bound FX into a fibrinolysis cofactor. These data also provide the first evidence for an anticoagulant mechanism aimed directly at the zymogen FX.

Thrombin enhances herpes simplex virus infection of cells involving protease-activated receptor 1.

Summary.  Background: We have previously shown that the surface of purified herpes family viruses can initiate thrombin production by expressing host‐encoded and virus‐encoded procoagulant factors. These enable the virus to bypass the normal cell‐regulated mechanisms for initiating coagulation, and provide a link between infection and vascular disease. Objective: In the current study we investigated why these viruses may have evolved to generate thrombin. Methods: Using cytolytic viral plaque assays, the current study examines the effect of thrombin on human umbilical vein endothelial cell (HUVEC) or human foreskin fibroblast (HFF) infection by purified herpes simplex virus type 1 (HSV1) and type 2 (HSV2). Results: Demonstrating that the availability of thrombin is an advantage to the virus, purified thrombin added to serum‐free inoculation media resulted in up to a 3‐fold enhancement of infection depending on the virus strain and cell type. The effect of thrombin on HUVEC infection was generally greater than its effect on HFF. To illustrate the involvement of thrombin produced during inoculation, hirudin was shown to inhibit the infection of each HSV strain, but only when serum containing clotting factors for thrombin production was present in media. The involvement of protease‐activated receptor 1 (PAR1) was supported using PAR1‐activating peptides in place of thrombin and PAR1‐specific antibodies to inhibit the effects of thrombin. Conclusion: These data show that HSV1 and HSV2 initiate thrombin production to increase the susceptibility of cells to infection through a mechanism involving PAR1‐mediated cell modulation.

Tissue factor and glycoprotein C on herpes simplex virus type 1 are protease-activated receptor 2 cofactors that enhance infection

The coagulation system provides physiologic host defense, but it can also be exploited by pathogens for infection. On the HSV1 surface, host-cell-derived tissue factor (TF) and virus-encoded glycoprotein C (gC) can stimulate protease activated receptor 1 (PAR1)-enhanced infection by triggering thrombin production. Using novel engineered HSV1 variants deficient in either TF and/or gC, in the present study, we show that activated coagulation factors X (FXa) or VII (FVIIa) directly affect HSV1 infection of human umbilical vein endothelial cells in a manner that is dependent on viral TF and gC. The combination of FXa and FVIIa maximally enhanced infection for TF(+)/gC(+) HSV1 and receptor desensitization and Ab inhibition demonstrated that both proteases act on PAR2. Inhibitory TF Abs showed that the required TF source was viral. Individually, TF or gC partly enhanced the effect of FXa, but not FVIIa, revealing gC as a novel PAR2 cofactor for FVIIa. In sharp contrast, thrombin enhanced infection via PAR1 independently of viral TF and gC. Thrombin combined with FXa/FVIIa enhanced infection, suggesting that PAR1 and PAR2 are independently involved in virus propagation. These results show that HSV1 surface cofactors promote cellular PAR2-mediated infection, indicating a novel mode by which pathogens exploit the initiation phase of the host hemostatic system.

Involvement of the contact phase and intrinsic pathway in herpes simplex virus‐initiated plasma coagulation

Summary.  Background: A hemostatic response to vascular injury is initiated by the extrinsic pathway of coagulation and amplified by the intrinsic pathway. We previously reported that purified herpes simplex virus type‐1 (HSV1) has constitutive extrinsic pathway tissue factor (TF) and anionic phospholipid on its surface derived from the host cell, and can consequently bypass strict cellular control of coagulation. Objective: The current work addresses the hypothesis that HSV1‐induced plasma coagulation also involves intrinsic pathway, factor VIII (FVIII), and upstream contact activation pathway, factor XII (FXII). Results: HSV1‐initiated clotting was accelerated when purified FVIII was added to FVIII‐deficient plasma and in normal plasma attenuated by an inhibitory anti‐FVIII antibody (Ab). High HSV1 concentrations predictably reduced the effect of FVIII due to the availability of excess viral TF. To further define TF‐independent clotting mechanisms initiated by HSV1, the extrinsic pathway was disabled using factor VII‐deficient plasma. The intrinsic pathway is triggered by activation of FXII associated with surface‐bound kallikrein, which subsequently activates factor XI. Here we found that an inhibitor of activated FXII, corn trypsin inhibitor, and anti‐FXII, anti‐kallikrein and anti‐FXI Abs inhibited HSV1‐initiated clotting. HSV1‐enhanced activation of purified FXII was confirmed by Western blot, but required prekallikrein. Conclusion: The current work shows that HSV1 can trigger and amplify coagulation through the contact phase and intrinsic pathway, and suggests an additional mechanism that may contribute to vascular pathology.

Enhanced fibrinolysis by proteolysed coagulation factor Xa.

We previously showed that coagulation factor Xa (FXa) enhances activation of the fibrinolysis zymogen plasminogen to plasmin by tissue plasminogen activator (tPA). Implying that proteolytic modulation occurs in situ, intact FXa (FXaalpha) must be sequentially cleaved by plasmin or autoproteolysis, producing FXabeta and Xa33/13, which acquire necessary plasminogen binding sites. The implicit function of Xa33/13 in plasmin generation has not been demonstrated, nor has FXaalpha/beta or Xa33/13 been studied in clot lysis experiments. We now report that purified Xa33/13 increases tPA-dependent plasmin generation by at least 10-fold. Western blots confirmed that in situ conversion of FXaalpha/beta to Xa33/13 correlated to enhanced plasmin generation. Chemical modification of the FXaalpha active site resulted in the proteolytic generation of a product distinct from Xa33/13 and inhibited the enhancement of plasminogen activation. Identical modification of Xa33/13 had no effect on tPA cofactor function. Due to its overwhelming concentration in the clot, fibrin is the accepted tPA cofactor. Nevertheless, at the functional level of tPA that circulates in plasma, FXaalpha/beta or Xa33/13 greatly reduced purified fibrin lysis times by as much as 7-fold. This effect was attenuated at high levels of tPA, suggesting a role when intrinsic plasmin generation is relatively low. FXaalpha/beta or Xa33/13 did not alter the apparent size of fibrin degradation products, but accelerated the initial cleavage of fibrin to fragment X, which is known to optimize the tPA cofactor activity of fibrin. Thus, coagulation FXaalpha undergoes proteolytic modulation to enhance fibrinolysis, possibly by priming the tPA cofactor function of fibrin.

The Procoagulant Envelope Virus Surface: Contribution to Enhanced Infection

Many virus types are covered by a lipid bilayer. This structure called an envelope, is derived from the host cell and includes host- and virus-encoded proteins. Because envelope components first interact with the host, it is the trigger for infection, immunity and pathology. The roles of especially host-derived constituents are poorly understood. Focusing on herpes simplex type 1 (HSV1) as a model, we have shown that the envelope acquires the physiological initiators of coagulation from the host cell; tissue factor (TF) and procoagulant phospholipid (proPL). Unlike resting cells, where TF and proPL accessibility is carefully restricted, their expression is constitutive on the purified virus enabling factor VIIa (FVIIa)-dependant factor Xa (FXa) and thrombin generation. Interestingly, HSV1-encoded glycoprotein C (gC) on the virus enhances FXa production. In addition to coagulation proteases, HSV1 also facilitates fibrinolytic plasmin generation. HSV1 TF and gC combine to optimally enhance cultured cell infection when both FVIIa and FXa are available through protease activated receptor (PAR) 2. Plasmin also increases infection through PAR2, whereas thrombin provides an additive effect via PAR1. Thus, depending on the host cell, TF and proPL may be a general feature of enveloped viruses, enabling coagulation protease activation and PAR-mediated effects on infection.