Structural insights into the catalytic mechanism of granzyme B upon substrate and inhibitor binding.

Human granzyme B (hGzmB), which is present in various immune cells, has attracted much attention due to its role in various pathophysiological conditions. The hGzmB activity is triggered at a catalytic triad (His59, Asp103, Ser198), cleaving its specific substrates. To date, the drug design strategy against hGzmB mainly targets the catalytic triad, which causes the non-specificity problem of inhibitors due to the highly conserved active site in serine proteases. In the present work, microsecond classical molecular dynamics simulations are devoted to exploring the structural dynamics of the hGzmB catalytic cycle in the presence of Ac-IEPD-AMC, a known substrate (active hGzmB), and Ac-IEPD-CHO, a known inhibitor (inactive hGzmB). By comparing active and inactive forms of hGzmB in the six different stages of the hGzmB catalytic cycle, we revealed, for the very first time, an additional network of interactions involving Arg216, a residue located outside the conventional binding site. Upon activation, the His59∙∙∙Asp103 hydrogen bond is broken due to the formation of the Asp103∙∙∙Arg216 salt bridge, expanding the active site to facilitate the substrate-binding. On the contrary, the binding of inhibitor Ac-IEPD-CHO to hGzmB prevents the Arg216-mediated interactions within the catalytic triad, thus preventing hGzmB activity. In silico Arg216Ala mutation confirms the role of Arg216 in enzyme activity, as the substrate Ac-IEPD-AMC failed to bind to the mutated hGzmB. Importantly, as Arg216 is not conserved amongst the various granzymes, the current findings can be a major step to guide the design of hGzmB specific therapeutics.

Intracellular and Extracellular Roles of Granzyme K.

Granzymes are a family of serine proteases stored in granules inside cytotoxic cells of the immune system. Granzyme K (GrK) has been only limitedly characterized and knowledge on its molecular functions is emerging. Traditionally GrK is described as a granule-secreted, pro-apoptotic serine protease. However, accumulating evidence is redefining the functions of GrK by the discovery of novel intracellular (e.g. cytotoxicity, inhibition of viral replication) and extracellular roles (e.g. endothelial activation and modulation of a pro-inflammatory immune cytokine response). Moreover, elevated GrK levels are associated with disease, including viral and bacterial infections, airway inflammation and thermal injury. This review aims to summarize and discuss the current knowledge of i) intracellular and extracellular GrK activity, ii) cytotoxic and non-cytotoxic GrK functioning, iii) the role of GrK in disease, and iv) GrK as a potential therapeutic target.

Granzyme A inhibition reduces inflammation and increases survival during abdominal sepsis.

Aims: Peritonitis is one of the most common causes of sepsis, a serious syndrome characterized by a dysregulated systemic inflammatory response. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed by NK and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role and the therapeutic potential of GzmA in the pathogenesis of peritoneal sepsis. Methods: The level of extracellular GzmA as well as GzmA activity were analyzed in serum from healthy volunteers and patients with confirmed peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (WT) and GzmA-/- mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics alone or in combination serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days, levels of some proinflammatory cytokines were measured in serum and bacterial load and diversity was analyzed in blood and spleen at different times. Results: Clinically, elevated GzmA was observed in serum from patients with abdominal sepsis suggesting that GzmA plays an important role in this pathology. In the CLP model GzmA deficient mice, or WT mice treated with an extracellular GzmA inhibitor, showed increased survival, which correlated with a reduction in proinflammatory markers in both serum and peritoneal lavage fluid. GzmA deficiency did not influence bacterial load in blood and spleen and GzmA did not affect bacterial replication in macrophages in vitro, indicating that GzmA has no role in bacterial control. Analysis of GzmA in lymphoid cells following CLP showed that it was mainly expressed by NK cells. Mechanistically, we found that extracellular active GzmA acts as a proinflammatory mediator in macrophages by inducing the TLR4-dependent expression of IL-6 and TNFα. Conclusions: Our findings implicate GzmA as a key regulator of the inflammatory response during abdominal sepsis and provide solid evidences about its therapeutic potential for the treatment of this severe pathology.

Granzyme B inhibition reduces disease severity in autoimmune blistering diseases.

Pemphigoid diseases refer to a group of severe autoimmune skin blistering diseases characterized by subepidermal blistering and loss of dermal-epidermal adhesion induced by autoantibody and immune cell infiltrate at the dermal-epidermal junction and upper dermis. Here, we explore the role of the immune cell-secreted serine protease, granzyme B, in pemphigoid disease pathogenesis using three independent murine models. In all models, granzyme B knockout or topical pharmacological inhibition significantly reduces total blistering area compared to controls. In vivo and in vitro studies show that granzyme B contributes to blistering by degrading key anchoring proteins in the dermal-epidermal junction that are necessary for dermal-epidermal adhesion. Further, granzyme B mediates IL-8/macrophage inflammatory protein-2 secretion, lesional neutrophil infiltration, and lesional neutrophil elastase activity. Clinically, granzyme B is elevated and abundant in human pemphigoid disease blister fluids and lesional skin. Collectively, granzyme B is a potential therapeutic target in pemphigoid diseases.

Granzyme B Inhibition by Tofacitinib Blocks the Pathology Induced by CD8 T Cells in Cutaneous Leishmaniasis.

In cutaneous leishmaniasis, the immune response is not only protective but also mediates immunopathology. We previously found that cytolytic CD8 T cells promote inflammatory responses that are difficult to treat with conventional therapies that target the parasite. Therefore, we hypothesized that inhibiting CD8 T-cell cytotoxicity would reduce disease severity in patients. IL-15 is a potential target for such a treatment because it is highly expressed in human patients with cutaneous leishmaniasis lesions and promotes granzyme B‒dependent CD8 T-cell cytotoxicity. Here we tested whether tofacitinib, which inhibits IL-15 signaling by blocking Jak3, might decrease CD8-dependent pathology. We found that tofacitinib reduced the expression of granzyme B by CD8 T cells in vitro and in vivo systemic and topical treatment, with tofacitinib protecting mice from developing severe cutaneous leishmaniasis lesions. Importantly, tofacitinib treatment did not alter T helper type 1 responses or parasite control. Collectively, our results suggest that host-directed therapies do not need to be limited to autoimmune disorders and that topical tofacitinib application should be considered a strategy for the treatment of cutaneous leishmaniasis disease in combination with antiparasitic drugs.

Extracellular Granzyme A Promotes Colorectal Cancer Development by Enhancing Gut Inflammation.

If not properly regulated, the inflammatory immune response can promote carcinogenesis, as evident in colorectal cancer (CRC). Aiming to gain mechanistic insight into the link between inflammation and CRC, we perform transcriptomics analysis of human CRC, identifying a strong correlation between expression of the serine protease granzyme A (GzmA) and inflammation. In a dextran sodium sulfate and azoxymethane (DSS/AOM) mouse model, deficiency and pharmacological inhibition of extracellular GzmA both attenuate gut inflammation and prevent CRC development, including the initial steps of cell transformation and epithelial-to-mesenchymal transition. Mechanistically, extracellular GzmA induces NF-κB-dependent IL-6 production in macrophages, which in turn promotes STAT3 activation in cultured CRC cells. Accordingly, colon tissues from DSS/AOM-treated, GzmA-deficient animals present reduced levels of pSTAT3. By identifying GzmA as a proinflammatory protease that promotes CRC development, these findings provide information on mechanisms that link immune cell infiltration to cancer progression and present GzmA as a therapeutic target for CRC.

Inhibition of SHP-1 Expands the Repertoire of Antitumor T Cells Available to Respond to Immune Checkpoint Blockade.

The presence and activity of CD8+ T cells within the tumor microenvironment are essential for the control of tumor growth. Utilizing B16-F10 melanoma tumors that express altered peptide ligands of chicken ovalbumin, OVA257-264, we measured high- and low-affinity OVA-specific responses following adoptive transfer of OT-I CD8+ T cell into mice subsequently challenged with tumors. T-cell receptor (TCR) affinity positively correlated with the frequency of OT-I tumor-infiltrating lymphocytes (TIL). Differences in TCR affinity inversely corresponded to in vivo tumor growth rate. Blockade of the PD-1 and CTLA-4 checkpoints preferentially increased the frequency and antitumor function of TIL responding to high-affinity antigens, while failing to enhance the antitumor activity of low-affinity T cells. To determine whether lowering the TCR activation threshold could enhance the breadth and magnitude of the antitumor T-cell response, we inhibited Src homology region 2 domain-containing phosphatase 1 (SHP-1) in OT-I T cells prior to tumor antigen exposure. SHP-1 knockdown increased the cytokine-producing potential of high- and low-affinity T cells but failed to enhance control of tumor growth. In contrast, when SHP-1 knockdown of OT-I T cells was combined with immunotherapy, we observed a significant and long-lasting suppression of tumor growth mediated by low-affinity T cells. We conclude that lowering the TCR activation threshold by targeting SHP-1 expands the repertoire of T cells available to respond to conventional checkpoint blockade, leading to enhanced control of tumor growth.

Improved Antitumor Efficacy of Chimeric Antigen Receptor T Cells that Secrete Single-Domain Antibody Fragments.

Chimeric antigen receptor (CAR) T-cell therapy is effective in the treatment of cancers of hematopoietic origin. In the immunosuppressive solid tumor environment, CAR T cells encounter obstacles that compromise their efficacy. We developed a strategy to address these barriers by having CAR T cells secrete single-domain antibody fragments [variable heavy domain of heavy chain antibodies (VHH) or nanobodies] that can modify the intratumoral immune landscape and thus support CAR T-cell function in immunocompetent animals. VHHs are small in size and able to avoid domain swapping when multiple nanobodies are expressed simultaneously-features that can endow CAR T cells with desirable properties. The secretion of an anti-CD47 VHH by CAR T cells improves engagement of the innate immune system, enables epitope spreading, and can enhance the antitumor response. CAR T cells that secrete anti-PD-L1 or anti-CTLA-4 nanobodies show improved persistence and demonstrate the versatility of this approach. Furthermore, local delivery of secreted anti-CD47 VHH-Fc fusions by CAR T cells at the tumor site limits their systemic toxicity. CAR T cells can be further engineered to simultaneously secrete multiple modalities, allowing for even greater tailoring of the antitumor immune response.

Granzyme B-expressing treg cells are enriched in colorectal cancer and present the potential to eliminate autologous T conventional cells.

In addition to expressing inhibitory cytokines and suppressive molecules, Treg cells could downplay inflammation by releasing cytotoxic molecules and eliminating proinflammatory immune cells. Colorectal cancer (CRC) is a common malignancy that has led to many cancer-related deaths. In this study, we investigated the cytotoxic aspect of Treg cells in CRC patients. Data showed that tumor-infiltrating FOXP3+ Treg cells expressed granzyme B immediately following resection, indicating that granzyme B-expressing Treg cells were present directly ex vivo. In the tumor-associated lymph nodes (LNs) and circulating lymphocytes, however, granzyme B-expressing Treg cells were only scarcely found. We then attempted to stimulate granzyme B expression in circulating Treg cells. Granzyme B upregulation in Treg cells could not be activated by standard T cell receptor (TCR) activation through anti-CD3/CD28 and IL-2 but required stimulation with bacterial products, such as with heat-killed Staphylococcus aureus. Interestingly, granzyme B expression was highly concentrated in TIM-3+ Treg cells, a Treg subset previously shown to be enriched in the tumor microenvironment and presented increased suppressive capacity. These TIM-3+ Treg cells presented higher cytolytic capacity toward autologous T conventional cells than the TIM-3- Treg cells, in a manner that was dependent on granzyme B but not TIM-3. Overall, we found that granzyme B-expressing Treg cells were enriched in the tumors from CRC patients and had the potential to eliminate autologous T conventional cells.

Granzyme B as a therapeutic target for wound healing.

Introduction: Granzyme B is a serine protease traditionally understood as having a role in immune-mediated cytotoxicity. Over the past decade, this dogma has been challenged, with a new appreciation that granzyme B can exert alternative extracellular roles detrimental to wound closure and remodeling. Granzyme B is elevated in response to tissue injury, chronic inflammation and/or autoimmune skin diseases, resulting in impaired wound healing. Areas covered: This review provides a historical background of granzyme B and a description of how it is regulated. Details are provided on the role of granzyme B in apoptosis as well as newly identified extracellular roles, focusing on those affecting wound healing, including on inflammation, dermal-epidermal junction separation, re-epithelialization, scarring and fibrosis, and autoimmunity. Finally, the use of pharmacological granzyme B inhibitors as potential therapeutic options for wound treatment is discussed. Expert opinion: Endogenous extracellular granzyme B inhibitors have not been identified in human bio-fluids, thus in chronic wound environments granzyme B appears to remain uncontrolled and unregulated. In response, targeted granzyme B inhibitors have been developed for therapeutic applications in wounds. Animal studies trialing inhibitors of granzyme B show improved healing outcomes, and may therefore provide a novel therapeutic approach for wound treatment.

Topical small molecule granzyme B inhibitor improves remodeling in a murine model of impaired burn wound healing.

Granzyme B (GzmB) is a serine protease that has long been thought to function exclusively in lymphocyte-mediated apoptosis. In recent years, this paradigm has been revisited due to the recognition that GzmB accumulates in the extracellular milieu in many autoimmune and chronic inflammatory disorders, and contributes to impaired tissue remodeling due to the cleavage of extracellular matrix proteins. Knockout studies suggest that GzmB-mediated cleavage of decorin (DCN) contributes to impaired collagen fibrillogenesis and remodeling. As DCN is anti-fibrotic and contributes to reduced hypertrophic scarring, GzmB-induced DCN cleavage could play a role in wound healing following burn injury. In the present study, a novel, gel-formulated, first-in-class small-molecule inhibitor of GzmB, VTI-1002, was assessed in a murine model of impaired, diabetic burn wound healing. VTI-1002 exhibited high specificity, potency, and target selectivity. Gel-formulated VTI-1002 was able to penetrate the stratum corneum and was retained in the skin with minimal systemic absorption. Daily topical administration of VTI-1002 gel for 30 days following thermal injury showed significantly accelerated wound closure, increased DCN protein levels, and collagen organization that was translated into significantly increased wound tensile strength compared to controls. Overall, VTI-1002 gel was well-tolerated in vivo and no adverse events were observed. Topical application of VTI-1002 represents a novel therapeutic approach for the treatment of cutaneous burn wounds.

Inhibition of granzyme B activity blocks inflammation induced by lipopolysaccharide through regulation of endoplasmic reticulum stress signaling in NK92 cells.

Granzyme B (GrB) is a serine protease that is expressed in the lytic granules of natural killer (NK) cells and cytotoxic T lymphocytes (CTL), and which has been widely reported to serve a crucial role for target cell apoptosis. GrB may serve a non­cytotoxic role in inflammation, but the evidence remains unclear. The present study aimed to establish an inflammatory cell model by using NK92 cells stimulated with lipopolysaccharide (LPS) to investigate whether GrB was involved in the development of inflammation. The extracellular levels of tumor necrosis factor­α (TNF­α), interleukin­1ß (IL­1ß) and GrB were examined by ELISA, and it was demonstrated that LPS treatment increased the extracellular levels of TNF­α, IL­1ß and GrB, and these increased expression levels were inhibited by pretreatment with the GrB inhibitor serpin A3N (SA3N). The protein expression levels of glucose­regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), nuclear factor­κB (NF­κB), inhibitor of NF­κB (IκBα) and GrB were examined by western blot analysis. The results demonstrated that LPS stimulation increased the expression levels of GRP78, CHOP, NF­κB and GrB, and decreased the expression of IκBα, and these changes were inhibited by SA3N, which indicated that inhibition of GrB activity may suppress endoplasmic reticulum (ER) stress signaling. Therefore, it was suggested that GrB may be a potential pro­inflammatory factor, and inhibition of GrB activity may aid the prevention of the development of inflammation by suppressing ER stress signaling.

GZMB gene silencing confers protection against synovial tissue hyperplasia and articular cartilage tissue injury in rheumatoid arthritis through the MAPK signaling pathway.

INTRODUCTION: Rheumatoid arthritis (RA) represents the most commonly occurring inflammatory type of arthritis and is a major cause of disability. Reports have placed emphasis on the potential of, granzyme B (GZMB) as a potentially valuable prognostic marker in early RA, the mechanism of which still remains largely unclear. Thus, the aim of the current study was to investigate the effects GZMB gene silencing influences synovial tissue hyperplasia and articular cartilage tissue injury of RA through the regulation of the MAPK signaling pathway. METHODS: Following the successful establishment of the collagen-induced animal model of RA in rats, a five-grade scoring method was applied to evaluate the swelling degree measurement of the rats for model identification. The various rat responses to GZMB shRNA and U-46619 (activator of the MAPK signaling pathway) were subsequently detected. The general status of rats was observed and recorded, with their weight and ankle diameter kept accurate record of. ELISA was employed to detect the levels of inflammatory cytokines, while RT-qPCR and Western blotting techniques were applied to determine the expressions of GZMB and pathway-related genes and proteins. RESULTS: GZMB gene silencing was observed to aid in the maintenance of rat weight increases, while acting to reduce the degree of ankle swelling, while hypertrophy of the synovial tissue and the injury of the articular cartilage tissue were not obvious. GZMB gene silencing was shown to decrease inflammatory cytokine levels, as well as decreased bcl-2, Cyclin D1, VEGF and bFGF while increasing caspase 3. Notably, GZMB gene silencing suppressed the activation of the MAPK signaling pathway by reducing the phosphorylation extent of ERK and MEK. CONCLUSION: Taken together, the key findings of the present study ultimately suggest that GZMB gene silencing acts to inhibit MAPK signaling pathway through regulating the expressions of inflammatory factors, factors correlated with apoptosis (bcl-2 and caspase), as well as factors associated with angiogenesis (VEGF and bFGF), thus relieving synovial tissue hyperplasia and articular cartilage tissue injury brought about by RA. The GZMB gene could well be a new therapeutic target for RA treatment.

A pro-survival role for the intracellular granzyme B inhibitor Serpinb9 in natural killer cells during poxvirus infection.

Intracellular serpins are proposed to inactivate proteases released from lysosome-related organelles into the host cell interior, preventing cell death. Serpinb9 opposes the immune cytotoxic protease, granzyme B, and in a number of settings protects cells against granzyme B-mediated cell death. Using a knockout mouse line engineered to express green fluorescent protein under the serpbinb9 promoter, we demonstrate that serpinb9 is vital for host survival during Ectromelia virus infection by maintaining both mature natural killer NK) cells, and activated CD8+ T cells. Serpinb9 expression parallels granzyme B expression within both populations during infection. Maturing serpinb9-null NK cells exhibit higher levels of granzyme B-mediated apoptosis during infection; hence there are fewer mature NK cells, and these cells also have lower cytotoxic potential. Thus the serpinb9-granzyme B axis is important for homeostasis of both major cytotoxic effector cell populations.

The Untold Story of Granzymes in Oncoimmunology: Novel Opportunities with Old Acquaintances.

For more than 20 years perforin and granzymes (GZMs) have been recognized as key cell death executors of cytotoxic T (Tc) and natural killer (NK) cells during cancer immunosurveillance. In immune surveillance, perforin and GZMB, the most potent cytotoxic molecules, act mainly as antitumoral and anti-infectious factors. However, when expressed by immune regulatory cells they may contribute to immune evasion of specific cancer types. By contrast, the other major granzyme, GZMA, seems not to play a major role in Tc/NK cell-mediated cytotoxicity, but acts as a proinflammatory cytokine that might contribute to cancer development. Members of the GZM family also regulate other biological processes unrelated to cell death, such as angiogenesis, vascular integrity, extracellular matrix remodeling, and barrier function, all of which contribute to cancer initiation and progression. Thus, a new paradigm is emerging in the field of oncoimmunology. Can GZMs act as protumoral factors under some circumstances? We review the diverse roles of GZMs in cancer progression, and new therapeutic opportunities emerging from targeting these protumoral roles.

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis.

We have found that granzyme B (GB)-induced apoptosis also requires reactive oxygen species resulting from the alteration of mitochondrial complex I. How GB, which does not possess a mitochondrial targeting sequence, enter this organelle is unknown. We show that GB enters the mitochondria independently of the translocase of the outer mitochondrial membrane complex, but requires instead Sam50, the central subunit of the sorting and assembly machinery that integrates outer membrane ß-barrel proteins. Moreover, GB breaches the inner membrane through Tim22, the metabolite carrier translocase pore, in a mitochondrial heat-shock protein 70 (mtHsp70)-dependent manner. Granzyme A (GA) and caspase-3 use a similar route to the mitochondria. Finally, preventing GB from entering the mitochondria either by mutating lysine 243 and arginine 244 or depleting Sam50 renders cells more resistant to GB-mediated reactive oxygen species and cell death. Similarly, Sam50 depletion protects cells from GA-, GM- and caspase-3-mediated cell death. Therefore, cytotoxic molecules enter the mitochondria to induce efficiently cell death through a noncanonical Sam50-, Tim22- and mtHsp70-dependent import pathway.

Histone H4 is cleaved by granzyme A during staurosporine-induced cell death in B-lymphoid Raji cells.

Granzyme A (GzmA) was first identified as a cytotoxic T lymphocyte protease protein with limited tissue expression. A number of cellular proteins are known to be cleaved by GzmA, and its function is to induce apoptosis. Histones H1, H2B, and H3 were identified as GzmA substrates during apoptotic cell death. Here, we demonstrated that histone H4 was cleaved by GzmA during staurosporine-induced cell death; however, in the presence of caspase inhibitors, staurosporine-treated Raji cells underwent necroptosis instead of apoptosis. Furthermore, histone H4 cleavage was blocked by the GzmA inhibitor nafamostat mesylate and by GzmA knockdown using siRNA. These results suggest that histone H4 is a novel substrate for GzmA in staurosporine-induced cells. [BMB Reports 2016; 49(10): 560-565].

The analysis of estrogen receptor-α positive breast cancer stem-like cells unveils a high expression of the serpin proteinase inhibitor PI-9: Possible regulatory mechanisms.

Breast cancer stem cells seem to play important roles in breast tumor recurrence and endocrine therapy resistance, although the underlying mechanisms have not been well established. Moreover, in some tumor systems the immunosurveillance failure against cancer cells has been related to the presence of the granzyme B inhibitor PI-9. This study explored the status of PI-9 in tumorspheres isolated from estrogen receptor-α positive (ERα+) breast cancer MCF7 cells. Studies were performed in tertiary tumorspheres which possess high levels of stemness markers (Nanog, Oct3/4 and Sox2) and self-renewal ability. The exposure to estrogens (17-ß estradiol and genistein) increased the number and sizes of tumorspheres, promoting cell proliferation as demonstrated by the increase in the proliferating cell nuclear antigen (PCNA). The study of the three isoforms (66, 46 and 36 kDa) of ERα disclosed that tertiary tumorspheres exhibit a marked increase in ERα36, while the level of ERα66, which is highly expressed in MCF7 cells, declines. Although it is known that PI-9 is a transcriptional target of ERα66, surprisingly in tertiary tumorspheres, despite the reduced level of ERα66, the protein and mRNA content of PI-9 is higher than in MCF7 cells. Treatment with estrogens further increased PI-9 level while decreased that of ERα66 isoform thus excluding the involvement of this receptor isoform in the event. Moreover, our studies also provided evidence that tertiary tumorspheres express elevated levels of CXCR4 and phospho-p38, suggesting that the high PI-9 content might be ascribed to the activation of the proliferative CXCR4/phospho-p38 axis. Taken together, these events could supply a selective advantage to breast cancer stem cells by interfering with immunosurveillance systems and open up the avenue to new possible targets for breast cancer treatment.

Extracellular granzyme K mediates endothelial activation through the cleavage of protease-activated receptor-1.

Granzymes are a family of serine proteases that were once thought to function exclusively as mediators of cytotoxic lymphocyte-induced target cell death. However, non-apoptotic roles for granzymes, including granzyme K (GzK), have been proposed. As recent studies have observed elevated levels of GzK in the plasma of patients diagnosed with clinical sepsis, we hypothesized that extracellular GzK induces a proinflammatory response in endothelial cells. In the present study, extracellular GzK proteolytically activated protease-activated receptor-1 leading to increased interleukin 6 and monocyte chemotactic protein 1 production in endothelial cells. Enhanced expression of intercellular adhesion molecule 1 along with an increased capacity for adherence of THP-1 cells was also observed. Characterization of downstream pathways implicated the mitogen-activated protein kinase p38 pathway for intercellular adhesion molecule 1 expression, and both the p38 and the extracellular signal-regulated protein kinases 1 and 2 pathways in cytokine production. GzK also increased tumour necrosis factor α-induced inflammatory adhesion molecule expression. Furthermore, the physiological inhibitor of GzK, inter-α-inhibitor protein, significantly inhibited GzK activity in vitro. In summary, extracellular GzK promotes a proinflammatory response in endothelial cells.