Components Of Hemostatic System Research Articles

Crosstalk between hemostasis and immunity in cancer pathogenesis

Studies spanning the last 3 decades have fundamentally altered our understanding of the interplay between the hemostatic and immune systems. A plethora of studies have revealed that there is bidirectional crosstalk between these two systems at multiple levels that likely evolved as a means to coordinate the host response to numerous challenges, including trauma, infection, and thermal or chemical injury. Such challenges require reestablishment of vascular integrity, the clearance of pathogens, and removal of cellular and external debris. Clearly, bidirectional coordination of hemostasis and immunity would be beneficial in such contexts. Many types of malignancies take advantage of the interplay between hemostasis and immunity, co-opting these mechanisms to promote tumorigenesis, the formation of a supportive stroma, and metastasis to distant organs. Three important “bridges” that mechanistically link the hemostatic system to immune functions that have been shown to play a key role in cancer biology include the platelet/fibrinogen axis, protease activated receptor-1 (PAR-1) and protease activated receptor-2 (PAR-2). These hemostatic system components have been shown to regulate a variety of immune functions that support tumorigenesis in the context of inflammation-driven malignancy, metastasis, and escape from adaptive antitumor immunity. Understanding the mechanisms coupling these bridges between hemostasis and immunity, as well as others, could provide novel targets for the prevention and treatment of a variety of cancers.

Use of thromboelastography in children on extracorporeal membrane oxygenation

IntroductionExtracorporeal membrane oxygenation (ECMO) profoundly impacts inflammatory and coagulation pathways, and strict monitoring is essential to guide therapeutic anticoagulation. Thromboelastography (TEG) offers a global evaluation of whole blood hemostatic system components and may be a valuable measurement of hemostatic function in these patients. There is a paucity of data correlating TEG parameters with standard measures of coagulation in heparinized pediatric patients. MethodsChildren on ECMO during a 10-year period were retrospectively reviewed. Standard measures of coagulation were matched to TEGs drawn within 30 min of each other. ResultsOut of 296 unique patients with 331 ECMO runs, 74.3% (n = 246) had at least one set of matched laboratory samples for a total of 2502 matched samples. The aPTT correlated with R-time (p<0.001). Platelets and fibrinogen correlated with α-angle (p<0.001). Fibrinogen (p<0.001) and platelets (p<0.001) were each associated with maximum amplitude (MA). 158 (47.7%) patients had at least one bleeding complication, and 100 (30.2%) had at least one thrombotic complication. Interestingly, a decreasing MA was associated with increased thrombotic complications (p<0.001). DiscussionTEG correlated well with traditional measures of hemostasis in pediatric ECMO patients. However, there was not a clear benefit of the TEG over these other measures Level of evidenceIII.

The thrombin–inflammation axis in cancer progression

The last half century of cancer research has seen an explosion in our understanding of the complex interplay between cancer cells and host-derived factors critical for cancer progression. Two important host-derived arms that are part of this complex interplay are the inflammatory immune compartment and the hemostatic system. Chronic pathological inflammation is a major factor in the development of multiple common malignancies, including adenocarcinomas of the colon, pancreas, prostate and breast. Hemostatic system components have also been shown to promote cancer progression in multiple contexts. What is only recently been recognized is the link between inflammation and hemostasis in cancer progression. The hemostatic and inflammatory innate immune systems co-evolved to deal with many of the same challenges, including trauma, infections, and thermal/chemical injuries. Their co-evolution necessarily led to bidirectional cross-talk whereby inflammatory cells can activate and alter hemostasis, and hemostatic system components serve as important regulators of inflammatory processes. This cross-talk is critical for the maintenance of vascular integrity, host defense, and wound healing. However, in the context of malignancy, the interplay of these integrated host systems has the capacity to promote multiple stages of malignancy, including tumorigenesis, tumor growth and metastatic dissemination. This review focuses on the interplay of inflammatory cells with the thrombin-fibrinogen axis and protease-activated receptor-1 in cancer pathobiology. Dissecting the mechanisms by which the inflammatory and hemostatic systems cooperatively promote cancer progression will fill in critical knowledge gaps in our understanding of malignancy, and also likely reveal novel therapeutic targets to treat and/or prevent cancer.

A study of prevalence of polymorphic variants of genes of blood coagulation factors in onco-logical patients

Aim. To study the prevalence of carriage of polymorphic allele variants of genes of blood coagulation factors in oncological patients.&#x0D; Materials and Methods. 213 Patients with morphologically confirmed oncological diseases were examined. Samples of genomic DNA of peripheral blood of the patients were examined. Using polymerase chain reaction (PCR), polymorphic sites of genes of hemostatic system were studied in real time: F2 (G20210А, rs1799963), F5 (G1691A, rs6025), F7 (G10976A, rs6046), F13 (G226A, rs5985), FGB G(-455)A (rs1800790), ITGA2-2 (C807T, rs1126643), ITGB3-b (Т1565С, rs5918), PAI-1 4G(-675)5G, rs1799889).&#x0D; Results. The prevalence of carriage of alternative allele of F2 (G20210А) polymorphic locus in the studied group was 1.6%, of F5 (G1691A) 3.5%, of F7 (G10976A) 13.4%, of F13 (G226A) 28.2%, of FGB G(-455)A 24.9%, of ITGA2-2 (C807T) 41.5%, of ITGB3-b (Т1565С) 15.5%, of PAI-1 4G(-675)5G 56.6%. A statistically significant increase in the frequency of risk alleles of F5 G1691A (р=0.0169), F13 G226A (р=0.0007), FGB G(-455)A (р0.0001) and ITGA2-2 C807T (р=0.0201) polymorphic loci was found in oncological patients as compared to the general population. In the same loci, except ITGA2-2 (C807T), statistically significant differences in the frequency of alternative alleles were found in different localizations of the oncological process. In 92.0% of patients, SNR combination was determined in different components of hemostatic system.&#x0D; Conclusion. Taking into account a high frequency of identification of risk alleles in all components of hemostatic system, it is reasonable to carry out additional research to determine the necessity of addition of antiaggregants to antithrombotic therapy in oncological patients.

Effects of cerium oxide nanoparticles on hemostasis: Coagulation, platelets, and vascular endothelial cells.

Cerium oxide nanoparticles (nanoceria [NC]) have attracted much attention in biomedicine due to their surface composition that confers interesting redox activities and regenerative properties. Studies have demonstrated that the application of NPs in biomedicine can influence components of hemostatic system, inducing blood clotting, alterations of blood cells, and endothelial cell functions. NC were tested in vitro to assess their hemocompatibility and anticoagulant, anti-inflammatory, and anti-senescence activity in human endothelial cells. Hemocompatibility has been evaluated in vitro looking at the impact of NC on coagulation times, fibrinogen, and platelet aggregation. The effect of NC on vascular endothelial cells were assayed by testing cell viability, antioxidant activity, anticoagulant (tissue factor [TF]-mRNA expression) and anti-inflammatory properties (VCAM-1 exposure, cytokine release), and senescence (telomere shortening). NC did not show significant effects on coagulation process, hemolysis, or platelet aggregation. In endothelial cells, NC did not affect cell viability, reduced oxidative stress, inhibited mRNA-TF expression, VCAM-1 expression, and cytokine release. Moreover, NC reduce telomere shortening, possibly counteracting premature senescence. The hemocompatibility combined with anticoagulant and anti-inflammatory phenotype and the ability of counteract the premature senescence in vascular cells make NC a promising therapeutic tool in oxidative stress-related conditions. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019.

Co-localization of Coagulation Factor X and its Inhibitory System, PZ/ZPI, in Human Endometrial Cancer Tissue.

Hemostatic system components contribute to cancer progression independently from their roles in hemostasis. It has been shown that protein Z (PZ)/protein Z-dependent protease inhibitor (ZPI) inhibit coagulation factor X (FX). The aim of the study was to analyze the expression of PZ/ZPI in relation to the main coagulation factor - FX in human endometrial cancer tissue. Immunohistochemical analysis was performed on 21 endometrial cancer specimens employing antibodies against ZPI, PZ and FX. Endometrial cancer cells showed a strong expression of ZPI and PZ and medium expression of FX. Normal endometrial tissue showed no expression of ZPI, PZ or FX. Strong expression of PZ and ZPI in endometrial cancer cells suggests a role of these proteins in endometrial cancer.

Mechanisms coupling thrombin to metastasis and tumorigenesis

The association of malignancy and thrombophilia is bidirectional, as evidenced by four decades of studies in animal models showing that hemostatic system components support cancer progression. Consistent with this view, clinical studies have suggested that anticoagulants not only limit thromboembolic complications associated with cancer, but also improve survival by impeding cancer progression, and may even prevent the development of cancer. In order to fully capitalize on this association, a detailed understanding of the mechanisms coupling hemostatic factors to cancer pathogenesis is required. Multiple studies have shown that thrombin-mediated procoagulant functions strongly promote metastatic potential. In particular, the platelet/fibrin(ogen) axis has been shown to protect newly formed micrometastases from innate immune surveillance, contribute to creation of a metastatic niche by recruitment of prometastatic inflammatory cells, and promote the epithelial to mesenchymal transition of metastatic cells. Thrombin-mediated functions have also been shown to support tumor growth in some contexts, and have even been linked to tumorigenesis in the setting of inflammation-driven colon cancer. Here, local thrombin-mediated extravascular fibrin deposition, and specifically fibrin-αMβ2 integrin interaction, push intestinal inflammatory cells toward a pro-tumorigenic phenotype, resulting in the elaboration of key cytokines and growth factors that support the proliferation and survival of transformed intestinal epithelial cells. These studies reveal that hemostatic factors can serve as a bridge between pathological inflammation and the development of cancer. As a large proportion of cancers are caused by pathological inflammation, these studies suggest that therapies targeting the nexus between hemostasis and inflammation could be used to prevent cancer development.

Thromboelastography as a novel viscoelastic method for hemostasis monitoring: Its methodology, applications, and constraints

Thromboelastography (TEG) is a novel viscoelastic method which provides a comprehensive assessment of hemostasis from clot initiation and development to fibrinolysis involving both cellular and plasmatic components of hemostatic system. Apart from surgery its role is expanding into medical specialties with increasing integration into laboratory settings. TEG complements the conventional coagulation tests in assessment of bleeding disorders. Further hemotherapy based on TEG results has been shown to reduce transfusion requirements in varied clinical settings besides helping in identifying coagulopathies in patients with major bleedings. This review article addresses briefly the methodology, clinical applications, interpretation of TEG results including authors' own experience of TEG in different clinical scenarios and limitations of TEG. Overall, this technique seems to be helpful for evaluation of hypercoagulable state and in detecting fibrinolysis which are difficult to be detected with conventional coagulation tests. Kaolin activated citrated blood samples analyzed within 30-60 min of sampling can provide reliable results in a laboratory setting. However, multiple assays using different activators or modifiers may be required for accurate results in selected cases. The operator should be aware about the various preanalytical and analytical variables which can affect the results including limitations of this technique. The tracing of the thromboelastography should be interpreted cautiously taking into consideration the clinical picture of the patient and results of other laboratory tests. With improved model and availability of more assays it is hoped that TEG and other such hemostasis analyzers would bring in the paradigm shift in the hemostasis monitoring and treatment of patients in future.

Protease-activated receptors - biology and role in cancer.

The fact that blood coagulation disorders may accompany malignant disease is well established. However, many studies have shown that components of the haemostatic system may also elicit signaling leading to cancer developement and progression. The potential mechanism by which coagulation factors play a role in cancer invasion is not completely understood, but one hypothesis is that protease-activated receptors (PARs) play a prominent role. PARs are transmembrane G-protein-coupled receptors (GPCRs) that are activated by a unique proteolytic mechanism. They have important functions in haemostasis and inflammation but may also be implicated in cancer cell progression. Thrombin, tissue factor (TF) and matrix metalloproteinases (MMPs) are the main activators of these receptors. The mechanism of persistent activation of PARs was also described in cancer cells. Here, we discuss the physiological and pathological role of PARs with a particular focus on PARs' contribution to cancer biology. We also present therapeutic options tailored specifically to inhibition of PAR-induced signalling in cancer patients.

Active nano/microbilayer hemostatic agents for diabetic rat bleeding model.

Patients with diabetes mellitus have an increased cardiovascular risk due to the abnormality of hemostatic system components. Therefore, hemostasis is an important concept when considering that diabetics are under risk due to potential bleeding complications during surgical operation. The aim of our study was to examine the efficiency of a fabricated nano/microbilayer hemostatic dressing for bleeding control in diabetic patients. For this purpose, we prepared a nano/microbilayer hemostatic dressing that has a porous sublayer, including chitosan (CTS), bacterial cellulose (BC) as basement and active agents in coagulation cascade, such as vitamin K (Vit K), protamine sulfate (PS), and kaolin (Kao) as a filler and an upper layer consisting of silk fibroin (SF) or SF/phosphatidylcholine (PC) blend to achieve complete hemostasis in diabetic rats. Coagulative performances of the prepared hemostatic dressings were examined by the determination of bleeding time, blood loss, and mortality rate through diabetic rat femoral artery injury model. The percent of diabetic rat blood absorption was found to be 247±5% for gauze as opposed to 2214±56% for SF-coated PS/BC/CTS. Vit K-reinforced within 138s and SF-coated BC/CTS hemostatic dressings within 144s showed a rapid coagulation time. In vivo coagulation studies demonstrated that hemostatic agent-reinforced BC/CTS hemostatic dressing, especially PS/BC/CTS showed a significant hemostatic plug formation. This study suggests that the high positive charge and porosity give to these hemostatic agents reinforced hemostatic dressings the ability to rapidly swell and to promote the accumulation of red blood cells and platelets through electrostatic interactions. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1573-1585, 2017.

Fibrinogen Is at the Interface of Host Defense and Pathogen Virulence in Staphylococcus aureus Infection.

Fibrinogen not only plays a pivotal role in hemostasis but also serves key roles in antimicrobial host defense. As a rapidly assembled provisional matrix protein, fibrin(ogen) can function as an early line of host protection by limiting bacterial growth, suppressing dissemination of microbes to distant sites, and mediating host bacterial killing. Fibrinogen-mediated host antimicrobial activity occurs predominantly through two general mechanisms, namely, fibrin matrices functioning as a protective barrier and fibrin(ogen) directly or indirectly driving host protective immune function. The potential of fibrin to limit bacterial infection and disease has been countered by numerous bacterial species evolving and maintaining virulence factors that engage hemostatic system components within vertebrate hosts. Bacterial factors have been isolated that simply bind fibrinogen or fibrin, promote fibrin polymer formation, or promote fibrin dissolution. Staphylococcus aureus is an opportunistic gram-positive bacterium, the causative agent of a wide range of human infectious diseases, and a prime example of a pathogen exquisitely sensitive to host fibrinogen. Indeed, current data suggest fibrinogen serves as a context-dependent determinant of host defense or pathogen virulence in Staphylococcus infection whose ultimate contribution is dictated by the expression of S. aureus virulence factors, the path of infection, and the tissue microenvironment.

ФАКТОРИ РИЗИКУ ГЕМОСТАТИЧНОГО ДИСБАЛАНСУ У ХВОРИХ НА ЗАПАЛЬНІ ЗАХВОРЮВАННЯ ТА НОВОУТВОРЕННЯ ЛОР-ОРГАНІВ

To undertake a comparative study of some components of haemostatic system in patients with inflammatory diseases and tumors of URT for clarification of their role in pathologic process and for reveal of their informative value for evaluation of the pathologic process' flow. The indices of prothrombine pool, content of tissue activator of plasminogen, inhibitor of plasminogen's acnivator PAI-1, and von Willebrandt factor were tested in groups of patients with inflammatory diseases and tumors of URT-organs in contrast of the same ones in the group healthy donors as well as dependently to the kind and stage of disease. The informative value of all the tested indices as well as their dependence of the kind and the stage of disease were proved. Their differences from the norm prove for deep alterations in haemostatic system's functioning at considered diseases. Obtained data seems to be promising for complex evaluation of the state of patients with inflammatory diseases and tumors of URT-organs.

The role of coagulation/fibrinolysis during Streptococcus pyogenes infection.

The hemostatic system comprises platelet aggregation, coagulation and fibrinolysis and is a host defense mechanism that protects the integrity of the vascular system after tissue injury. During bacterial infections, the coagulation system cooperates with the inflammatory system to eliminate the invading pathogens. However, pathogenic bacteria have frequently evolved mechanisms to exploit the hemostatic system components for their own benefit. Streptococcus pyogenes, also known as Group A Streptococcus, provides a remarkable example of the extraordinary capacity of pathogens to exploit the host hemostatic system to support microbial survival and dissemination. The coagulation cascade comprises the contact system (also known as the intrinsic pathway) and the tissue factor pathway (also known as the extrinsic pathway), both leading to fibrin formation. During the early phase of S. pyogenes infection, the activation of the contact system eventually leads to bacterial entrapment within a fibrin clot, where S. pyogenes is immobilized and killed. However, entrapped S. pyogenes can circumvent the antimicrobial effect of the clot by sequestering host plasminogen on the bacterial cell surface that, after conversion into its active proteolytic form, plasmin, degrades the fibrin network and facilitates the liberation of S. pyogenes from the clot. Furthermore, the surface-localized fibrinolytic activity also cleaves a variety of extracellular matrix proteins, thereby enabling S. pyogenes to migrate across barriers and disseminate within the host. This review summarizes the knowledge gained during the last two decades on the role of coagulation/fibrinolysis in host defense against S. pyogenes as well as the strategies developed by this pathogen to evade and exploit these host mechanisms for its own benefit.

Protein Z/protein Z-dependent protease inhibitor system in loco in human gastric cancer

In gastric cancer, hemostatic system components contribute to cancer progression, as activation of factor X (FX) was observed. The protein Z (PZ)/protein Z-dependent protease inhibitor (ZPI) complex inhibits factor Xa proteolytic activity. The purpose of this study was to determine the distribution of ZPI and PZ in relation to FX, and prothrombin fragment (F1 + 2), a standard marker for blood coagulation activation, in human gastric cancer tissue. ABC procedures and a double staining method employed polyclonal antibodies against PZ, FX, and F1 + 2 and a monoclonal antibody against ZPI. In situ hybridization (ISH) methods employed biotin-labeled 25-nucleotide single-stranded DNA probes directed to either PZ or ZPI mRNAs. FX and components of PZ/ZPI coagulation inhibitory system were observed in cancer cells. F1 + 2 was observed in gastric cancer cells as well. Double staining studies revealed FX/PZ, FX/ZPI, and PZ/ZPI co-localization on gastric cancer cells. ISH studies demonstrated the presence of PZ mRNA and ZPI mRNA in gastric cancer cells indicating induced synthesis of these proteins. The co-localization of PZ/ZPI and FX in gastric cancer cells indicates in loco that these proteins may play a role in anticoagulant events at the tumor tissue.

Thrombin Supports Colitis and Colitis-Associated Cancer Pathogenesis

Abstract 2237Chronic inflammation has been recognized as a major factor in the development and progression of multiple cancers. A prime example of this is the strong association between colitis and colon cancer. However, the specific factors that regulate disadvantageous immune processes in the context of inflammation-associated cancers remain poorly defined. Growing evidence suggests that hemostatic system components, traditionally associated with the maintenance of vascular integrity and prevention of blood loss, also directly regulate inflammatory processes. Furthermore, thrombin and several thrombin targets (e.g., PARs, fibrinogen, factor XIII) have been shown to regulate tumor cell proliferation and apoptosis, support metastasis, and protect tumor cells from innate immune surveillance mechanisms in other experimental contexts. A logical extension of these findings is the hypothesis that thrombin, as a master regulator of both inflammatory processes and tumor cell biology, is a major determinant of the progression of inflammation-driven cancers such as colitis-associated colon cancer (CAC). To test this hypothesis, we induced CAC in mice carrying prothrombin levels 50% of normal (fII+/−) and WT mice in parallel using an established two step protocol consisting of azoxymethane (AOM) and dextran sodium sulfate (DSS) exposure. The modest diminution in prothrombin levels imposed by the fII+/− genotype resulted in a dramatic diminution in the number of colonic adenomas formed after AOM/DSS challenge relative to WT mice. In order to determine if the diminution in adenoma formation observed in fII+/− mice was coupled to thrombin function, wildtype mice challenged with AOM/DSS were treated with daily i.p. injections of hirudin, a direct thrombin inhibitor, or saline carrier. Similar to the finding in mice with a genetically-imposed diminution in circulating prothrombin, hirudin treatment significantly blunted adenoma formation. To determine if reduction of thrombin generation improved the inflammation preceding the development of colonic adenomas, we used a novel, highly-specific factor XI antisense oligonucleotide “gapmer” (ISIS Pharmaceuticals) to inhibit hepatic factor XI synthesis prior to DSS challenge. Gapmer-mediated diminution of fXI levels to ∼15% of normal resulted in a dramatic improvement in colitis related symptoms. Gapmer-treated mice had less intestinal bleeding and weight loss associated with DSS challenge relative to mice treated with a control oligonucleotide. Consistent with these gross observations, microscopic analyses of colonic tissue showed that fXI gapmer treatment significantly limited mucosal ulceration. Factor XI gapmer treatment also significantly diminished local levels of several inflammatory cytokines known to play a role in colon cancer progression (i.e., IL-6, IL-1β, IL-12). These results demonstrate that thrombin is a crucial driver of the pathogenesis of colitis-associated colon cancer and suggest that therapies directed at thrombin or thrombin generation could treat or prevent inflammation-driven colon cancer. As pathological inflammation has been estimated to account for as many as 1 in 5 cancer-related deaths, thrombin-directed therapies could have broad applicability to multiple malignancies. Disclosures:Mullins:Baxter: Consultancy. Monia:Isis Pharmaceuticals: Employment. MacLeod:Isis Pharmaceuticals: Employment. Revenko:Isis Pharmaceuticals: Employment. Palumbo:Novo Nordisk: Research Funding.

Thrombin Control Mechanisms and Thrombin Targets in Cancer Biology

Abstract SCI-17 A link between hemostasis and cancer has been recognized for more than a century, but the last decade has seen substantial strides in understanding the mechanisms by which hemostatic system components actively contribute to the malignant phenotype. The expression of procoagulants, such as tissue factor (TF), by tumor cells has been shown to be a poor prognostic factor in clinical studies and a crucial determinant of metastasis in animal models. While TF expressed by tumor cells likely plays a multifaceted role in cancer biology, a substantial body of evidence indicates that tumor cell-associated and circulating hemostatic system components (e.g., prothrombin, fibrinogen, platelets) play a cooperative role in supporting metastasis. The capacity of tumor cells to generate thrombin has been proposed to support metastasis through several mechanisms, including tumor cell proliferation, stable adhesion, regulation of apoptosis, and escape from innate immune surveillance mechanisms. More recently, the fundamental importance of endothelial regulators of thrombin activity in metastasis was established through studies of tumor dissemination in mice expressing mutant forms of thrombomodulin (TM). Mice expressing a TM derivative with reduced thrombin affinity (TMPro) exhibited a profoundly prometastatic phenotype relative to wild-type (WT) mice. The TMPro mutation was shown to support metastasis by promoting the survival of tumor cell emboli newly localized to the lung. The impact of the TMPro mutation on metastasis was dependent on tumor cell-associated tissue factor, prothrombin, thrombin function, and platelets. In contrast, mice expressing a mutant form of TM lacking the lectin-like domain (TMLed) that were shown previously to have altered immune function but normal thrombin affinity, exhibited metastatic potential comparable to wild-type mice. These studies further highlight the importance of the hemostatic system in metastasis and demonstrate that apart from tumor cell-associated and circulating procoagulants, TM-mediated regulation of hemostatic function strongly influences tumor cell metastatic success. In addition, recent studies of inflammation-driven cancer have revealed that the role of hemostatic factors in tumor biology is not limited to later phases of malignant progression, such as metastasis. Fibrin(ogen)-mediated regulation of leukocyte function was shown to support tumor development and tumor proliferation in a murine model of inflammation-driven colon cancer. Recent advances in our understanding of the role of hemostatic factors in cancer biology demonstrate that this system of proteins can be important in multiple phases of malignant progression, and underscore the potential utility of targeting selected coagulation factors as a novel adjunct therapy in the treatment of cancer. Disclosures: Palumbo: Novo Nordisk Corporation: Research Funding.

Co-localization of Protein Z, Protein Z-Dependent protease inhibitor and coagulation factor X in human colon cancer tissue: Implications for coagulation regulation on tumor cells

IntroductionSeveral hemostatic system components, including factor X (FX), contribute to cancer progression. The Protein Z (PZ)/protein Z-dependent protease inhibitor (ZPI) complex directly inhibits factor Xa proteolytic activity. The aim of this study was to determine the antigenic distribution of ZPI and PZ, in relation to FX, as well as indicators of blood coagulation activation (F1+2 and fibrin) in human colon cancer tissue. Materials & methodsStudies were performed on human colon cancer fragments. Immunohistochemical (IHC) ABC procedures and double staining method employed polyclonal antibodies against PZ, FX, F1+2 and monoclonal antibodies against ZPI and fibrin. In-situ hybridization (ISH) methods employed biotin-labeled 25-nucleotide single-stranded DNA probes directed to either FX, PZ or ZPI mRNAs. ResultsExpression of FX, PZ and ZPI in association with colon cancer cells was observed by IHC. Moreover, the presence of both F1+2 and fibrin in association with colon cancer cells was found, which indicates that blood coagulation activation proceeds extravascularly at the tumor site. Furthermore, expression of FX and PZ was visualized in association with endothelial cells. In turn, colon cancer-associated macrophages were characterized by FX , PZ and ZPI presence. The double staining studies revealed strong FX/PZ, FX/ZPI, as well as PZ/ZPI co-localization on colon cancer cells. ISH studies revealed the presence of FX mRNA, PZ mRNA and ZPI mRNA in colon cancer cells indicating induced synthesis of these proteins. ConclusionsThe localization of PZ/ZPI and FX in colon cancer cells indicates that PZ/ZPI may contribute to anticoagulant events at the tumor site. Strong co-localization of PZ/ZPI and FX in cancer cells, and the presence of the mRNAs encoding the proteins, suggests their role in the tumor's biology. However, the presence of F1+2 and fibrin at the colon cancer site also suggests that the regulation of FXa by the PZ/ZPI complex at this site is incomplete.

Protein Z is present in human breast cancer tissue

Thromboembolic complications are common findings in breast cancer patients [1, 2]. Hemostatic system components play a role in tumor progression, independent of their role in hemostasis [1, 2]. Since the report of multifactorial activation of factor X in cancer patients [1], inhibitors which control the activity of this factor have attracted attention. Protein Z (PZ), a vitamin K-dependent plasma nonpeptidase glycoprotein [3, 4], is involved in one mechanism responsible for the direct inhibition of factor Xa. PZ circulates in plasma complexed with PZ-dependent protease inhibitor (ZPI) and enhances the rate of ZPImediated factor Xa inhibition [5, 6]. The presence of ZPI in breast cancer tissue was previously reported [7], while information on PZ in tumor malignant tissue is lacking. The purpose of the present study was to evaluate the localization of PZ (both protein and mRNA) at the primary site of human breast cancer. Tumor fragments were obtained during the surgical treatment of 13 previously untreated breast cancer patients (clinical stage T1-2N0M0). Studies were performed on 4 cases of G2 and 9 cases of G3 invasive ductal carcinomas. Immunohistochemical studies (IHC) were performed (Vectastain Kits, Vector Laboratories, Burlingame, CA, USA) [7] employing a polyclonal antibody against homogeneous, plasma-derived human PZ (prepared in rabbits, and purified from immune sera by protein A-Sepharose chromatography). The antibody did not cross-react with all other purified human plasma vitamin K-dependent proteins including prothrombin, protein C, protein S, factor VII, factor X, and factor IX by ELISA. Controls consisted of omission of the primary antibody from the procedure. The control fragments of normal breast tissues obtained from neoplasm-free surgical margins were processed simultaneously as well. Antigen staining was detected by the dark brown reaction product. A semiquantitative analysis of PZ IHC expression in cancer cells (based on both the percentage of cancer cells with PZ positive staining and the intensity of staining) was performed [7]. Immunoreactive score (IRS) values of 1–4 were interpreted as weak, 5–8 as medium, and 9–12 as strong PZ expression. In situ hybridization (ISH) method employed a biotin-labeled 25-nucleotide single-stranded DNA probe (probe sequence: 50 Biotin-CGTCATACCGCATGTGCA CATGGAC-Biotin 30) specific for PZ mRNA. The probe was synthesized by Sigma-Aldrich, Poznan, Poland. The ISH protocol of R&D Systems (R&D Systems, Minneapolis, MN, USA) was followed. Hybrids were detected with rabbit anti-biotin monoclonal antibodies according to the IHC ABC technique associated with ImmunoMax amplification technique described in detail elsewhere [7]. Negative controls included hybridization without addition of the molecular probe and incubation of slides in a RNase A solution (R&D Systems, Minneapolis, MN, USA) before hybridization. Reaction results appeared as dark brown staining. E. Sierko M. Z. Wojtukiewicz (&) Department of Oncology, Medical University, 12 Ogrodowa St, Bialystok, Poland e-mail: mzwojtukiewicz@gmail.com

Host Fibrinogen and the S. Aureus-Encoded Procoagulant Vwbp Are Context-Dependent Determinants of Bacterial Virulence.

AbstractAbstract 1152Microbial pathogens frequently express bacterial products that are specifically designed to engage hemostatic system components in their vertebrate hosts. Staphylococcus aureus has evolved a particularly impressive repertoire of factors to control fibrin deposition, dissolution, and binding, including two bacterial products that form proteolytically active procoagulant complexes with prothrombin, staphylocoagulase (Coa) and von Willebrand factor binding protein (vWbp). These findings support the working hypothesis that hemostatic factors, in general, and fibrinogen, in particular, are likely to be important determinants of S. aureus virulence/host defense. To directly explore the role of fibrin(ogen) in host inflammatory/antimicrobial processes, a comparative analysis of bacterial clearance was done in control and fibrinogen-deficient mice in the context of S. aureus peritonitis. Control mice challenged with 109 CFU of S. aureus were found to efficiently clear S. aureus within the peritoneal cavity and eliminated ∼99.5% of bacterial CFUs within 20 minutes, whereas fibrin(ogen)-deficient mice exhibited little capacity to clear the microbe, even after several hours. Consistent with these findings, fibrinogen-deficient mice challenged with S. aureus peritonitis also exhibited a poor survival profile relative to control animals. More detailed studies to define the mechanism(s) underlying the rapid, fibrin(ogen)-dependent clearance of S. aureus have thus far excluded a critical contribution of host T cells, B cells, neutrophils, immunglobulins, complement, toll-like receptor signaling pathways, and the bacterial fibrinogen receptor clumping factor A (ClfA). However, mice carrying low levels of circulating prothrombin or expressing a mutant form of fibrinogen that cannot be converted to fibrin, exhibited a profound defect in S. aureus clearance following i.p. infection, suggesting a central role for fibrin polymer in the implementation of an effective antimicrobial program. Although fibrin formation is necessary, it is not sufficient for efficient bacterial clearance; Fibγ390-396A mice retaining full clotting function, but lacking the leukocyte integrin Mac-1 binding motif, also exhibit an impediment in bacterial clearance relative to wild-type mice. Complementary studies of S. aureus mutants deficient in bacterial procoagulants indicate that vWbp, but not Coa, is a fundamental determinant of bacterial clearance in this peritonitis model. Remarkably, bacterial procoagulant vWbp is distinctly counter-productive to the microbe in the context of peritonitis and supports the rapid, fibrin(ogen)-dependent clearance of intraperitoneal S. aureus. Based on findings indicating that host fibrinogen and bacterial factors that engage fibrin(ogen) support S. aureus virulence in other infection settings, the present findings suggest that host hemostatic factors and the bacterial procoagulant vWbp are likely to be context-dependent determinants of bacterial virulence. A better understanding of the interactions between bacterial proteins and host hemostatic factors, as well as interactions between the host hemostatic and inflammatory/immune systems, may well reveal novel therapeutic approaches for limiting microbial infections and sepsis. Disclosures:No relevant conflicts of interest to declare.

Host Prothrombin and Fibrinogen Support S. Aureus Virulence In the Context of Acute Bacteremia

AbstractAbstract 821Staphylococcus aureus is a common and re-emerging gram-positive bacteria that is a leading cause of both community and hospital-acquired infections. Multiple bacteria-derived products appear to drive pathogenicity, but some of the most potent virulence factors identified are those that engage host hemostatic system components. To directly investigate the hypothesis that host prothrombin and key thrombin substrates significantly contribute to S. aureus virulence in the context of acute bacteremia, we evaluated the survival profile of mice with either genetically-imposed deficiencies or functional alterations in prothrombin and thrombin targets following intravenous infection with S. aureus. Mice expressing 10% of normal levels of prothrombin (termed fIIflox/- mice) were found to have a major survival advantage following intravenous infection with ∼2.5 × 108 colony forming units (cfu) of wild-type Newman-strain S. aureus. This difference in survival did not appear to be tied to platelet function as mice that were either pharmacologically depleted of platelets or deficient in the platelet integrin receptor subunit αIIb did not display a survival advantage when similarly challenged with intravenous S. aureus. Fibrinogen was found to be at least one thrombin substrate critical to supporting S. aureus virulence, as the genetic elimination of fibrinogen resulted in dramatically prolonged survival following acute S. aureus bacteremia relative to fibrinogen-sufficient animals. One mechanism driving this prolongation in survival was linked to S. aureus binding of host fibrinogen via the bacterial fibrinogen receptor, clumping factor A (ClfA). Mice expressing a mutant form of fibrinogen that retains normal clotting function but lacks the binding motif for ClfA located on the fibrinogen g chain (termed FibγΔ5) exhibited a profound survival advantage to wild-type animals over a wide bacterial dose range (e.g., LD50 to an LD100 for control animals). Additional studies revealed that the survival advantage in FibγΔ5 mice correlated with reduced bacterial burdens in the hearts and kidneys, diminished histological evidence of cardiac damage, and significantly lower levels of plasma markers of cardiac damage (i.e., lactose dehydrogenase and cardiac troponin I) relative to infected control mice. To determine whether the observed survival advantages for fIIflox/- and FibγΔ5 mice were strictly linked to ClfA, identical survival studies were performed with ClfA-deficient S. aureus. Consistent with the concept that ClfA is a potent virulence factor, mice of each genotype survived longer after infection with ClfA-deficient S. aureus than with the same dose of ClfA-positive bacteria. However, the survival advantage observed in FibγΔ5 mice over wild-type animals was largely, but not entirely eliminated when challenged with i.v. ClfA-deficient S. aureus. The fact that FibγΔ5 mice continued to display a modest, but significant, survival advantage relative to control animals when infected with bacteria lacking ClfA suggests that the loss of C-terminal residues of the gamma chain provides an additional, but still unidentified, benefit to the host. These data indicate that the host prothrombin and fibrinogen as well as the bacterial fibrinogen receptor ClfA are, cooperatively, major determinants of S. aureus virulence in the context of bacteremia, and that strategies designed to disrupt S. aureus engagement of these proteins may limit disease progression and improve host survival in S. aureus bacteremia/sepsis. Disclosures:No relevant conflicts of interest to declare.