Platelets play a crucial role for blood hemostasis, the process that prevents bleeding. In addition, platelets have been demonstrated to promote cancer progression and cancer related complications like metastasis and thrombosis. Platelets can affect cancer related diseases either directly or by interacting with other blood cells or molecules in the circulation of individuals with cancer. The current thesis addresses the role of platelets in tumor progression and tumor-induced systemic effects of cancer, with a special focus on the effects on the vasculature. In the first paper, the role of platelets in tumor progression in histidine-rich glycoprotein (HRG)-deficient mice was addressed. We report that HRG-deficient mice show enhanced tumor growth, epithelial to mesenchymal transition (EMT) and metastasis. The enhanced platelet activity in the absence of HRG is responsible for the accelerated tumor progression. In the second paper, we demonstrate that platelet-derived PDGFB is a central player to keep the tumor vessels functional. Moreover, in a pancreatic neuroendocrine carcinoma model with PDGFB-deficient platelets, spontaneous liver metastasis was enhanced. With this finding we identify a previously unknown role of platelet derived PDGFB. In the third paper, we found that TBK1 mediates platelet-induced EMT by activation of NF-kB signaling, which suggest that TBK1 contributes to tumor invasiveness in mammary epithelial tumors. In the last paper, we report that the vascular function in organs that are neither affected by the primary tumor, nor represent metastatic sites, is impaired in mice with cancer. We show that tumor-induced formation of intravascular neutrophil extracellular traps (NETs), a fibril matrix consisting of neutrophils with externalized DNA and histones, granule proteases and platelets, are responsible for the impaired peripheral vessel function.

Histidine-rich glycoprotein (HRG) is a heparin-binding plasma protein modulating immune, hemostatic and vascular functions. I have studied the antiangiogenic functions of HRG in vitro and in vivo in order to understand the molecular mechanisms of action of HRG as an angiogenesis inhibitor. Angiogenesis is the formation of new blood vessels from the pre-existing vasculature. It is a central rate-limiting step of tumor development and thus a possible target for cancer therapeutics. Previous studies have shown that HRG has antiangiogenic functions in vivo and that the antiangiogenic effects are mediated via the proteolytically released His/Pro-rich domain of HRG. In this thesis we demonstrate that HRG can inhibit endothelial cell migration by interfering with focal adhesion and cytoskeletal turnover. Moreover we have identified the minimal active domain of HRG, a 35 amino acid peptide derived from the histidine- and proline-rich domain of HRG. Analyzing human tumor tissue samples, we have found that a His/Pro-rich fragment of HRG is bound to the vasculature from cancer patients but not to the vasculature from healthy individuals. The fragment is found in association with platelets, and we show that activated platelets can induce a functional microenvironment for the His/Pro-rich fragment. Cancer patients often display an increased coagulation and our data describe a new mechanism to confer specificity of an angiogenesis inhibitor for situations with enhanced platelet activation, as in the tumor. We have further studied the role of HRG in tumor growth by crossing HRG-deficient mice with a transgenic mouse model of pancreatic insulinoma. We show that mice lacking HRG display an elevated “angiogenic switch” and that the total tumor volume is larger in these mice than in wild type mice. HRG is also involved in regulation of platelet function and platelets can stimulate angiogenesis in various ways. We have depleted mice of platelets to study the possible connection between the function of HRG in angiogenesis and platelet regulation. Our data suggest an involvement of platelets in the antiangiogenic activities of HRG.

Recent studies suggest that factor (F) XII plays a key role in thrombus stabilization and growth but is dispensable for hemostasis. We have previously shown that histidine-rich glycoprotein (HRG), a protein present in platelets and plasma, binds FXIIa and inhibits FXII autoactivation and FXIIa-mediated activation of FXI, thereby downregulating thrombosis. HRG binds various ligands, including FXIIa, fibrin(ogen), nucleic acids and polyphosphate (polyP). Studies have shown that polyP, released from activated platelets, and artificial surfaces like catheters, can promote FXII activation. This suggests that HRG can downregulate the activation of the contact system. This thesis aims to determine the potential mechanisms by which HRG modulates platelet function and thrombosis induced by polyP or catheters. We show that HRG binds polyP with high affinity and inhibits the procoagulant, prothrombotic and cardiotoxic effects of polyP via at least two mechanisms. First, HRG binds polyP and neutralizes its procoagulant activities and cytotoxic effects. Second, HRG binds FXIIa and attenuates its capacity to promote autoactivation and activate FXI. Also, we identify that HRG serves as a molecular brake for the contact system by attenuating the procoagulant activity of FXIIa regardless of whether FXII activation is triggered systemically with polyP or occurs locally on the surface of catheters. Our studies have identified HRG as a novel ligand for platelet receptor GPIbα on resting platelets, and upon activation, it competes with fibrinogen for binding to GPIIb/IIIa integrin, thereby inhibiting platelet aggregation. These findings suggest that HRG may modulate coagulation as well as platelet function. Therefore, supplementation with HRG or HRG analogs may serve as a potential therapeutic option to attenuate polyP or catheter-induced thrombosis without perturbing hemostasis. / Dissertation / Doctor of Philosophy (PhD)

Implantation requires complex molecular and cellular events involving coagulation, angiogenesis and immunological processes that need to be well regulated for a pregnancy to establish and progress normally.  The overall aim of this thesis was to study different models associated with atypical angiogenesis, impaired implantation and/or placentation, such as recurrent miscarriage (RM), oocyte donation (OD) and preeclampsia. Histidine-rich glycoprotein (HRG), a serum protein with angiogenic potential has been previously shown to have an impact on implantation and fertility.  In two retrospective case-control studies, women suffering from RM (Study I) and gestational hypertensive disorders (GHD) (Study IV) have been compared to healthy control women, regarding carriership of HRG genotypes (HRG A1042G and C633T SNP, respectively).  According to the findings of this thesis, heterozygous carriers of the HRG A1042G SNP suffer from RM more seldom than homozygous carriers (Study I).  Additionally, the presence of the HRG 633T allele was associated with increased odds of GHD (GHD IV).  Studies II and III comprised a national cohort of relatively young women with optimal health status conceiving singletons with donated oocytes versus autologous oocytes (spontaneously or via IVF).  We explored differences in various obstetric (Study II) and neonatal (Study III) outcomes from the Swedish Medical Birth Register.  Women conceiving with donated oocytes had a higher risk of GHD, induction of labor and cesarean section, as well as postpartum hemorrhage and retained placenta, when compared to autologously conceiving women.  OD infants had higher odds of prematurity and lower birthweight and length when born preterm, compared to neonates from autologous oocytes.  With regard to the indication of OD treatment, higher intervention but neverthelss favourable neonatal outcomes were observed in women with diminished ovarian reserve; the risk of GHD did not differ among OD recipients after adjustment. In conclusion, HRG genetic variation appears to contribute to placental dysfunction disorders.  HRG is potential biomarker that may contribute in the prediction of the individual susceptibility for RM and GHD.  Regarding OD in Sweden, the recipients-despite being of optimal age and health status- need careful preconceptional counselling and closer prenatal monitoring, mainly due to increased prevalence of hypertensive disorders and prematurity.

Chapter: 1 Introduction Genus Salmonella is a Gram-negative rod shaped facultative anaerobic bacteria that can survive inside the host macrophages and cause persistent infection. Salmonella Typhimurium, Salmonella Typhi and Salmonella Enteritidis are the serovars, which belong to the Salmonella enterica species. S. Typhi causes typhoid fever in humans. S. Typhimurium is one of the important causes for food poisoning in humans. It causes typhoid like fever in mice and serves as a good model system to study Salmonella pathogenesis. Salmonella infection occurs via the orofecal route following which it invades the intestinal mucosa through several ways, namely by antigen sampling M cells, CD18+ macrophages present in the intestinal lumen or via a forced entry in the non phagocytic enterocytes. Upon entry Salmonella resides in an intracellular phagosomal compartment called the Salmonella containing vacuole (SCV). The SCV only transiently acquires endocytic markers like TfnR, EEA1, Rab4, Rab5, Rab11 and Rab7. It eventually uncouples from the endocytic pathway to avoid lysosomal fusion and ultimately reaches the golgi apparatus achieving a perinuclear position. The mechanisms by which phagocytes kill the virulent Salmonella are not completely understood, however the role of nicotinamide-adenine dinucleotide phosphate (NADPH) phagocytic oxidase system has been strongly implicated. The generation of reactive oxygen species (ROS) occurs via a membrane-bound flavocytochrome b558, consisting of two phagocytic oxidase components (gp91phox and p22phox) and four cytosolic components, p40phox, p47phox, p67phox, and a GTP-binding Rac protein. Further, professional phagocytes like macrophages generate nitric oxide (NO) that acts as a potent agent to limit the growth of many intracellular pathogens including Salmonella. Chapter:2 Resistance to host Nitrosative stress in Salmonella by quenching L-arginine. Arginine is a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The competition between iNOS and arginase for arginine contributes to the outcome of several parasitic and bacterial infections. Salmonella infection in macrophage cell line RAW264.7 induces iNOS. Because the availability of L-arginine is a major determinant for nitric oxide (NO) synthesis, we hypothesize that in the Salmonella infected macrophages NO production may be regulated by arginase. Here we report for the first time that Salmonella up-regulates arginase II but not arginase I isoform in RAW264.7 macrophages. Blocking arginase increases the substrate L-arginine availability to iNOS for production of more nitric oxide and perhaps peroxynitrite molecules in the infected cells allowing better killing of virulent Salmonella in a NO dependent manner. RAW264.7 macrophages treated with iNOS inhibitor aminoguanidine reverts the attenuation in arginase blocked condition. Further, the NO block created by Salmonella was removed by increasing concentration of L-arginine. In the whole-mice system arginase I, although constitutive, is much more abundant than the inducible arginase II isoform. Inhibition of arginase activity in mice during the course of Salmonella infection reduces the bacterial burden and delays the disease outcome in a NO dependent manner. Chapter:3 Hrg (hydrogen peroxide resistant gene), a LysR type transcriptional regulator confers resistance to oxidative stress in Salmonella LysR type transcriptional regulators are one of the key players that help bacteria adapt to different environments. We have christened STM0952, a putative LysR type transcriptional regulator in Salmonella enterica serovar Typhimurium as the hydrogen peroxide resistance gene (hrg). By generating a knock out of the hrg gene, we demonstrate that the hrg mutant serovar Typhimurium is sensitive to oxidative products of the respiratory burst, specifically to hydrogen peroxide. The hrg mutant is profoundly attenuated in the murine model of infection and shows decreased intracellular proliferation in macrophages. It was also found to induce increased amount of reactive oxygen species and co-localization with gp91phox in the macrophage cell line, when compared to the wild type. An overproducing strain of this gene showed a survival advantage over the wild type Salmonella under hydrogen peroxide induced stress condition. Microarray analysis suggested the presence of a Hrg regulon, which is required for resistance to the toxic oxidative products of the reticulo-endothelial system. Chapter:4 Importance of the host oxidative stress in antigen presentation and its modulation by Salmonella: Role of TLR Synthetic CpG containing oligodeoxynucleotide TLR-9 agonist (CpG ODN) activates innate immunity and can stimulate antigen presentation against numerous intracellular pathogens. We report that Salmonella Typhimurium growth can be inhibited by the CpG ODN treatment in the murine dendritic cells. This inhibitory effect was shown to be mediated by an increased reactive oxygen species (ROS) production. We further show that the CpG ODN treatment of the dendritic cells during Salmonella infection leads to a ROS dependent increased antigen presentation. In addition, TLR-9 signaling inhibitor was able to inhibit the CpG ODN mediated increased antigen presentation, ROS production and pathogen killing. These data indicate that CpG ODN can improve the ability of the murine dendritic cells to contain the growth of the virulent Salmonella through ROS dependent killing and could as well be used as an effective adjuvant in vaccines against Salmonella infection.

In part to search for a possible critical point (CP) in the phase diagram of hot nuclear matter, a beam energy scan was performed at the Relativistic Heavy-Ion Collider at Brookhaven National Laboratory. The Solenoidal Tracker at RHIC (STAR) collected Au+Au data sets at beam energies, √sNN , of 7.7, 11.5, 19.6, 27, 39, 62.4, and 200 GeV. Such a scan produces hot nuclear matter at different locations in the phase diagram. Lattice and phenomenological calculations suggest that the presence of a CP might result in divergences of the thermodynamic susceptibilities and correlation lengths. The statistical moments of the identified-particle multiplicity distributions directly depend on both the thermodynamic susceptibilities and correlation lengths, possibly making the shapes of these multiplicity distributions sensitive tools for the search for the critical point. The statistical moments of the multiplicity distributions of a number of different groups of identified particle species were analyzed. Care was taken to remove a number of experimental artifacts that can modify the shapes of the multiplicity distributions. The observables studied include the lowest four statistical moments (mean, variance, skewness, kurtosis) and some products of these moments. These observables were compared to the predictions from several approaches lacking critical behavior, such as the Hadron Resonance Gas model, mixed events, (negative) binomial, and Poisson statistics. In addition, the data were analyzed after gating on the event-by-event antiproton-to-proton ratio, which is expected to more tightly constrain the event trajectories on the phase diagram.