Pre-eclampsia is a major cause of maternal and perinatal morbidity and mortality worldwide. A predictor of pre-eclampsia would enable intervention, close surveillance and timely delivery, and thereby reduce the negative consequences of the disorder. The overall aim of this thesis was to study potential predictors of pre-eclampsia by biochemical and epidemiological methods. Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2) are regulators of angiogenesis, which is important for placental development. In a prospective and longitudinal study of a low-risk population the Ang-1/Ang-2 ratio was evaluated. The Ang-1/Ang-2 ratio increased during pregnancy in all women but at gestational week 25 and 28 the ratios were significantly lower in women who later developed pre-eclampsia. The relevance of Histidine-rich glycoprotein (HRG), a protein with angiogenic properties, was furthermore evaluated. HRG levels decreased in all women, with significantly lower levels at gestational week 10, 25 and 28 in women who later developed pre-eclampsia. Thus both Ang-1/Ang-2 ratio and HRG may predict pre-eclampsia. To evaluate the predictive value of HRG in combination with uterine artery Doppler early in pregnancy a study was performed in a high-risk population. The results revealed that the combination was better able to predict preterm pre-eclampsia than each marker individually, with a sensitivity of 91% at a specificity of 62%.  A possible association between hyperemesis gravidarum and pre-eclampsia, as well as other placental dysfunctional disorders, was investigated. Hyperemesis gravidarum may be caused by high levels of human chorionic gonadotrophin (hCG) and increased levels of hCG in the second trimester is associated with later development of pre-eclampsia. A cohort of all pregnancies in the Swedish medical birth register between 1997 and 2009 was studied. After adjustment for confounding factors an association between hyperemesis gravidarum in the second trimester and preterm pre-eclampsia, placental abruption and infants born small for gestational age was demonstrated. In conclusion, the ratio of Ang-1/Ang-2 as well as HRG in plasma may be potential predictors of pre-eclampsia. Combination with uterine artery Doppler further increases the predictive value of HRG for preterm pre-eclampsia. Hyperemesis gravidarum in the second trimester may be considered as a clinical risk predictor of pre-eclampsia and other placental dysfunctional disorders.

Recent studies suggest that factor (F) XII, which is dispensable for hemostasis, is important for thrombus stabilization and growth. Therefore, FXIIa inhibition may attenuate thrombosis without disrupting hemostasis. FXII activation is stimulated by polyanions such as polyphosphates released from activated platelets, and nucleic acids released by cells. Previously, we showed that histidine-rich glycoprotein (HRG) binds FXIIa with high affinity, inhibits FXII autoactivation and FXIIa-mediated activation of FXI, and attenuates ferric chloride-induced arterial thrombosis in mice. Thus, HRG has the capacity to downregulate the contact pathway in vitro and in vivo. This thesis aimed to identify the complementary binding domains of HRG and FXIIa, and to further explore the anticoagulants effects of HRG on FXIIa-mediated contact activation. We hypothesized that FXIIa binds to the zinc-binding histidine-rich region (HRR) of HRG and that HRG binds to the non-catalytic heavy chain of FXIIa to exert its anticoagulant activities on FXIIa-mediated contact activation. We have localized the complementary binding sites of HRG and FXIIa to be within the HRR domain of HRG and NH2-FNII-EGF1 (NFE) domains of FXIIa. Moreover, we show that the HRR binds to short chain polyphosphate with high affinity, suggesting a dynamic complex between HRG, FXIIa, and polyphosphate (polyP) on activated platelets. We provide evidence for two potential mechanisms through which HRG modulates the contact system. These include by 1) inhibiting FXIIa activity and 2) attenuating the procoagulant effect of polyanions, such as polyP on FXIIa-mediated reactions. Indeed, we show that the interaction of HRG with FXIIa and polyphosphate is predominantly mediated by the HRR domain and that HRR analogs have the capacity to recapitulate the anticoagulant effects of HRG in purified and plasma systems. Therefore, by modulating FXIIa-mediated contact pathway reactions, like HRG, HRR analogs may attenuate thrombosis without disrupting hemostasis. / Thesis / Doctor of Philosophy (Medical Science)

<p>Heparan sulfate (HS) is an unbranched and negatively charged polysaccharide of the glycosaminoglycan family, based on the repeated (GlcNAcα1-4GlcAβ1-4)disaccharide structure. The HS backbone is modified by epimerization and sulfation in various positions. HS chains are composed of <i>N</i>-sulfated (NS) domains – predominant locations for further modification steps –, the poorly modified <i>N</i>-acetylated (NA) domains and the alternating NA/NS-domains. HS is present at the cell surface and in the extra-cellular matrix and interacts at these sites with various proteins involved in numerous biological processes, such as angiogenesis. Both pro- and anti-angiogenic proteins can interact with HS and this study was focused on how HS binds to the anti-angiogenic proteins endostatin (ES) and histidine-rich glycoprotein (HRGP) and to pro-angiogenic fibroblast growth factors (FGFs).</p><p>Here we show that ES recognizes NS-domains in HS spaced by NA-disaccharides, and that binding to ES is abolish through cleavage at these NA-disaccharides. HRGP335, a peptide derived from the His/Pro-rich domain of HRGP is shown to bind to heparin and HS to the same extent as full-size HRGP, in a Zn²⁺-dependent manner. Moreover, the ability of HRGP to inhibit endothelial cell migration is located to the same region of the protein. We analyzed HS structure in respect to binding to HRGP335 and FGF-2, and show that the ability of HS to bind to those proteins depends on chain length and composition. Finally, the role of HS in FGF–HS–FGF receptor ternary complexes is evaluated using biosynthetic analogs of NS-domains. For stabilization of such complexes the overall sulfation degree of HS seems to play a more pronounced role than the exact distribution of sulfate groups.</p><p>The results presented in this thesis contribute to a greater understanding of the role of HS in angiogenesis and may provide valuable information for the development of cures against angiogenesis-related disorders.</p>

Heparan sulfate (HS) is an unbranched and negatively charged polysaccharide of the glycosaminoglycan family, based on the repeated (GlcNAcα1-4GlcAβ1-4) disaccharide structure. The HS backbone is modified by epimerization and sulfation in various positions. HS chains are composed of N-sulfated (NS) domains – predominant locations for further modification steps –, the poorly modified N-acetylated (NA) domains and the alternating NA/NS-domains. HS is present at the cell surface and in the extra-cellular matrix and interacts at these sites with various proteins involved in numerous biological processes, such as angiogenesis. Both pro- and anti-angiogenic proteins can interact with HS and this study was focused on how HS binds to the anti-angiogenic proteins endostatin (ES) and histidine-rich glycoprotein (HRGP) and to pro-angiogenic fibroblast growth factors (FGFs). Here we show that ES recognizes NS-domains in HS spaced by NA-disaccharides, and that binding to ES is abolish through cleavage at these NA-disaccharides. HRGP335, a peptide derived from the His/Pro-rich domain of HRGP is shown to bind to heparin and HS to the same extent as full-size HRGP, in a Zn2+-dependent manner. Moreover, the ability of HRGP to inhibit endothelial cell migration is located to the same region of the protein. We analyzed HS structure in respect to binding to HRGP335 and FGF-2, and show that the ability of HS to bind to those proteins depends on chain length and composition. Finally, the role of HS in FGF–HS–FGF receptor ternary complexes is evaluated using biosynthetic analogs of NS-domains. For stabilization of such complexes the overall sulfation degree of HS seems to play a more pronounced role than the exact distribution of sulfate groups. The results presented in this thesis contribute to a greater understanding of the role of HS in angiogenesis and may provide valuable information for the development of cures against angiogenesis-related disorders.

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)

Histidine-rich glycoprotein (HRG) is a protein present in plasma at ~ 2 μM, but whose physiologic function is unclear. HRG is a multi-domain protein that contains a unique histidine-rich core that interacts with zinc and hydrogen ions to modulate ligand binding. Due to its modular structure and capacity to sense local changes in zinc and pH, HRG binds several ligands including complement proteins, phospholipids, DNA, fibrin(ogen), heparin, factor (F) XIIa and plasmin. Thus, it is hypothesized that HRG functions as an accessory or adapter protein that bridges different ligands together. Despite the array of ligands and potential involvement in immunity, angiogenesis, coagulation and fibrinolysis, no clear role for HRG has emerged. Congenital HRG deficiency in humans has been associated with a variable phenotype; some investigators report increased susceptibility to thrombosis while others do not. However, studies in HRG-deficient mice reveal that HRG attenuates coagulation. Coagulation is initiated via the intrinsic (or contact) and extrinsic (or tissue factor) pathways and culminates in the generation of thrombin. Thrombin catalyzes the conversion of fibrinogen into a fibrin meshwork that reinforces the platelet plug at sites of vascular injury. There are two circulating isoforms of fibrinogen that differ with respect to their γ-chains. Bulk fibrinogen is composed of a pair of γA-chains, and is designated γA/γA-fibrinogen, whereas a minor variant contains a γA-chain and a γʹ-chain, and is designated γA/γʹ-fibrinogen. The γʹ-chain contains an anionic 20-amino acid residue extension at its COOH-terminus, which provides an accessory binding site for thrombin. Thrombin possesses an anion binding pocket termed exosite II that flanks the active site and mediates its interaction with the γʹ-chain of fibrinogen. Exosite II is an evolutionary feature that is unique to thrombin, as this region is not observed on the prototypic serine protease trypsin or on other defibrinogenating enzymes from snake venom such as batroxobin. Although the physiologic function of the thrombin-γʹ-chain interaction is unclear, it is proposed that this interaction modulates thrombin’s activity when it is bound to fibrin clots. Consistent with this, we show that γA/γʹ-fibrin attenuates thrombin’s capacity to promote clot expansion compared with thrombin bound to γA/γA-fibrin clots, thereby demonstrating that γA/γʹ-fibrin attenuates thrombin’s activity. In the presence of physiologic concentrations of zinc, HRG binds the γʹ-chain of fibrino(gen) and competes with thrombin for binding, thereby suggesting that HRG is a unique modulator of thrombin activity on fibrin clots. Platelets store zinc and HRG in their α-granules and release both components when they undergo activation at sites of injury, which localizes HRG in the vicinity of fibrin-bound thrombin. Consistent with the role of HRG in modulating coagulation, we also show that HRG attenuates contact activation of coagulation, but has no impact on clotting initiated by the extrinsic pathway. The intrinsic pathway is initiated when FXII is activated by polyanions such as RNA and DNA, which are released into the blood after cellular activation, injury or death. FXIIa activates FXI, thereby propagating coagulation and leading to thrombin generation and fibrin formation. Recently, studies using rodent, rabbit and non-human primate models of thrombosis have shown that knock down of FXII or FXI with antisense oligonucleotides or blocking FXIIa or FXIa activity with inhibitors attenuates thrombosis, while having a minimal impact on hemostasis. With increasing evidence that the intrinsic pathway plays an important role in thrombosis, FXII and FXI have emerged as prominent targets for new anticoagulants. However, little is known about how the intrinsic pathway is regulated, so as to prevent uncontrolled clotting. HRG attenuates the intrinsic pathway by binding both FXIIa and the contact activators, RNA and DNA. By binding nucleic acids, HRG is localized to the site of contact activation, where it is poised to inhibit FXIIa. HRG binds to an allosteric region on FXIIa and attenuates its capacity to feedback activate FXII and to activate FXI, thereby inhibiting the initiating steps of contact activation. In addition, HRG attenuates the cofactor role of RNA and DNA in thrombin activation of FXI, which is an important feedback step. With the capacity to modulate multiple steps in the intrinsic pathway, HRG likely serves as a dynamic regulator of contact activation. We tested our hypothesis that HRG is a novel inhibitor of the intrinsic pathway in a murine model of FeCl3-induced arterial injury. HRG-deficient mice exhibit accelerated thrombosis compared with wild type controls, an effect that was abolished by repletion with human HRG. Therefore, these studies indicate that HRG deficiency induces a prothrombotic phenotype. Consistent with the role of HRG as a modulator of the intrinsic pathway, we show that thrombosis after the FeCl3-induced arterial injury is attenuated by administration of RNase, but not DNase, or by knock down of FXII, but not FVII. Therefore, these studies show that thrombosis in this model is induced by RNA and occurs in a FXII-dependent manner. Furthermore, blood loss after tail tip amputation is similar in HRG-deficient and wild type mice, demonstrating that HRG does not modulate hemostasis. Therefore, these studies suggest that HRG is a dynamic regulator of the intrinsic pathway, and acts as a molecular brake to limit procoagulant stimuli. The observations that HRG binds fibrin(ogen), FXIIa and nucleic acids and modulates the thrombin-γʹ-interaction and intrinsic pathway of coagulation, suggest that HRG is a key regulator of coagulation. HRG, the contact system and fibrin are also important in the innate immune response, demonstrating that the interaction of HRG with these factors may provide a unique link between coagulation and immunity. Since immune cells and the coagulation system contribute to both deep vein thrombosis and sepsis, further characterization of the role of HRG in these conditions will contribute to a better understanding of the pathophysiological role of HRG, and may identify novel therapeutic directions. / Thesis / 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.

<p>Angiogenesis, de novo synthesis of blood vessels from the pre-existing vasculature, is required both during embryonic development and in pathophysiological conditions. In particular, tumor growth needs new capillary vessels in order to both deliver oxygen and nutrients and to remove toxin and metabolites. Growth of most solid tumors would be restricted to a microscopic size in the absence of neovascularization. Angiogenesis ensues as a result of a shift in the balance between pro- and anti-angiogenic molecules.</p><p>Histidine-rich glycoprotein (HRGP) is a heparin-binding plasma protein. We showed that HRGP inhibits endothelial cell migration and adhesion to vitronectin. As a consequence, HRGP attenuates growth and vascularization of mouse model tumors. The anti-angiogenic effect of HRGP is mediated by the central histidine/proline (His/Pro)-rich domain, which must be released from the parent molecule to exert its effect. A 35-amino acid residue peptide denoted HRGP330, derived from the His/Pro-rich domain, was identified as a minimal active anti-angiogenic domain of HRGP. HRGP330 induces disruption of molecular interactions required for cell motility, such as the integrin-linked kinase/paxillin complex. Moreover, HRGP330 inhibits VEGF-induced tyrosine phosphorylation of α-actinin, a focal adhesion kinase (FAK) substrate. Consequently, the motility of endothelial cells is arrested. By use of a signal transduction antibody array, we identified FAK, paxillin and growth factor receptor-bound 2 (Grb2) as tyrosine phosphorylated in HRGP330-treated cells. We confirmed that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures. A critical role of FAK in HRGP-inhibition of angiogenesis was validated using a FAK inhibitor, geldanamycin, which allowed rescue of endothelial cell actin rearrangement.</p><p>We identified another potential mechanism in the HRGP/HRGP330 anti-angiogenic effects, exerted through regulation of tumor-associated macrophages (TAMs). HRGP/HRGP330 treatment led to reduced TAM infiltration, which in turn caused a marked decrease in VEGF and MMP-9 levels in the tumor. </p><p>Taken together, our present studies show that HRGP/HRGP330 target endothelial cell adhesion, migration, focal adhesions, and furthermore, that HRGP is involved in regulation of macrophage infiltration.</p>

Angiogenesis, de novo synthesis of blood vessels from the pre-existing vasculature, is required both during embryonic development and in pathophysiological conditions. In particular, tumor growth needs new capillary vessels in order to both deliver oxygen and nutrients and to remove toxin and metabolites. Growth of most solid tumors would be restricted to a microscopic size in the absence of neovascularization. Angiogenesis ensues as a result of a shift in the balance between pro- and anti-angiogenic molecules. Histidine-rich glycoprotein (HRGP) is a heparin-binding plasma protein. We showed that HRGP inhibits endothelial cell migration and adhesion to vitronectin. As a consequence, HRGP attenuates growth and vascularization of mouse model tumors. The anti-angiogenic effect of HRGP is mediated by the central histidine/proline (His/Pro)-rich domain, which must be released from the parent molecule to exert its effect. A 35-amino acid residue peptide denoted HRGP330, derived from the His/Pro-rich domain, was identified as a minimal active anti-angiogenic domain of HRGP. HRGP330 induces disruption of molecular interactions required for cell motility, such as the integrin-linked kinase/paxillin complex. Moreover, HRGP330 inhibits VEGF-induced tyrosine phosphorylation of α-actinin, a focal adhesion kinase (FAK) substrate. Consequently, the motility of endothelial cells is arrested. By use of a signal transduction antibody array, we identified FAK, paxillin and growth factor receptor-bound 2 (Grb2) as tyrosine phosphorylated in HRGP330-treated cells. We confirmed that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures. A critical role of FAK in HRGP-inhibition of angiogenesis was validated using a FAK inhibitor, geldanamycin, which allowed rescue of endothelial cell actin rearrangement. We identified another potential mechanism in the HRGP/HRGP330 anti-angiogenic effects, exerted through regulation of tumor-associated macrophages (TAMs). HRGP/HRGP330 treatment led to reduced TAM infiltration, which in turn caused a marked decrease in VEGF and MMP-9 levels in the tumor. Taken together, our present studies show that HRGP/HRGP330 target endothelial cell adhesion, migration, focal adhesions, and furthermore, that HRGP is involved in regulation of macrophage infiltration.