Novel genetic risk factors for venous thrombosis; a haplotype-based candidate gene approach

Venous thrombosis (VT) is a common disease, which occurs mostly in the deep veins of the leg. VT clusters within families and is a multicausal disease, in which both genetic and environmental factors interact in the onset of the disease. The aim of the study described in this thesis was to find new genetic risk factors for VT. To identify these new genetic risk factors, we used a haplotype-based candidate gene approach. The main hypothesis of this approach was that relatively common functional variants exist that are the product of unique mutational events in a founder haplotype and that the frequency of such haplotypes will be increased in a patient population. In this thesis, candidate genes were selected on the basis of theoretical knowledge of the proteins encoded by the genes. The candidate gene investigated are the Endothelial cell Protein C Receptor (EPCR) (Chapter 2), Fibrinogen (Chapter 3), and the genes of the selectin family (E-selectin (SELE), L-selectin (SELL) and P-selectin (SELP)) and their most important counter-receptor P-selectin ligand (PSGL-1). The results described in this thesis may contribute to a better understanding of the etiology of VT.Venous thrombosis (VT) is a common disease, which occurs mostly in the deep veins of the leg. VT clusters within families and is a multicausal disease, in which both genetic and environmental factors interact in the onset of the disease. The aim of the study described in this thesis was to find new genetic risk factors for VT. To identify these new genetic risk factors, we used a haplotype-based candidate gene approach. The main hypothesis of this approach was that relatively common functional variants exist that are the product of unique mutational events in a founder haplotype and that the frequency of such haplotypes will be increased in a patient population. In this thesis, candidate genes were selected on the basis of theoretical knowledge of the proteins encoded by the genes. The candidate gene investigated are the Endothelial cell Protein C Receptor (EPCR) (Chapter 2), Fibrinogen (Chapter 3), and the genes of the selectin family (E-selectin (SELE), L-selectin (SELL) and P-selectin (SELP)) and their most important counter-receptor P-selectin ligand (PSGL-1). The results described in this thesis may contribute to a better understanding of the etiology of VT.Dutch Heart Foundation, Dr. Ir. Van der Laar Stichting, J.E. Jurriaanse Stichting, EurogentecUBL - phd migration 201

Inhibition of Fibrinolysis by Coagulation Factor XIII

The inhibitory effect of coagulation factor XIII (FXIII) on fibrinolysis has been studied for at least 50 years. Our insight into the underlying mechanisms has improved considerably, aided in particular by the discovery that activated FXIII cross-links α2-antiplasmin (α2AP) to fibrin. In this review, the most important effects of different cross-linking reactions on fibrinolysis are summarized. A distinction is made between fibrin-fibrin cross-links studied in purified systems and fibrin-α2AP cross-links studied in plasma or whole blood systems. While the formation of γ chain dimers in fibrin does not affect clot lysis, the formation of α chain polymers has a weak inhibitory effect. Only strong cross-linking of fibrin, associated with high molecular weight α chain polymers and/or γ chain multimers, results in a moderate inhibition fibrinolysis. The formation of fibrin-α2AP cross-links has only a weak effect on clot lysis, but this effect becomes strong when clot retraction occurs. Under these conditions, FXIII prevents α2AP being expelled from the clot and makes the clot relatively resistant to degradation by plasmin

α−α Cross-Links Increase Fibrin Fiber Elasticity and Stiffness

Fibrin fibers, which are ∼100 nm in diameter, are the major structural component of a blood clot. The mechanical properties of single fibrin fibers determine the behavior of a blood clot and, thus, have a critical influence on heart attacks, strokes, and embolisms. Cross-linking is thought to fortify blood clots; though, the role of α–α cross-links in fibrin fiber assembly and their effect on the mechanical properties of single fibrin fibers are poorly understood. To address this knowledge gap, we used a combined fluorescence and atomic force microscope technique to determine the stiffness (modulus), extensibility, and elasticity of individual, uncross-linked, exclusively α–α cross-linked (γQ398N/Q399N/K406R fibrinogen variant), and completely cross-linked fibrin fibers. Exclusive α–α cross-linking results in 2.5× stiffer and 1.5× more elastic fibers, whereas full cross-linking results in 3.75× stiffer, 1.2× more elastic, but 1.2× less extensible fibers, as compared to uncross-linked fibers. On the basis of these results and data from the literature, we propose a model in which the α-C region plays a significant role in inter- and intralinking of fibrin molecules and protofibrils, endowing fibrin fibers with increased stiffness and elasticity

Compaction of fibrin clots reveals the antifibrinolytic effect of factor XIII

Essentials Factor XIIIa inhibits fibrinolysis by forming fibrin-fibrin and fibrin-inhibitor cross-links. Conflicting studies about magnitude and mechanisms of inhibition have been reported. Factor XIIIa most strongly inhibits lysis of mechanically compacted or retracted plasma clots. Cross-links of α2-antiplasmin to fibrin prevent the inhibitor from being expelled from the clot. Summary: Background Although insights into the underlying mechanisms of the effect of factor XIII on fibrinolysis have improved considerably in the last few decades, in particular with the discovery that activated FXIII (FXIIIa) cross-links α2-antiplasmin to fibrin, the topic remains a matter of debate. Objective To elucidate the mechanisms of the antifibrinolytic effect of FXIII. Methods and Results Platelet-poor plasma clot lysis, induced by the addition of tissue-type plasminogen activator, was measured in the presence or absence of a specific FXIIIa inhibitor. Both in a turbidity assay and in a fluorescence assay, the FXIIIa inhibitor had only a small inhibitory effect: 1.6-fold less tissue-type plasminogen activator was required for 50% clot lysis in the presence of the FXIIIa inhibitor. However, when the plasma clot was compacted by centrifugation, the FXIIIa inhibitor had a strong inhibitory effect, with 7.7-fold less tissue-type plasminogen activator being required for 50% clot lysis in the presence of the FXIIIa inhibitor. In both experiments, the effects of the FXIIIa inhibitor were entirely dependent on the cross-linking of α2-antiplasmin to fibrin. The FXIIIa inhibitor reduced the amount of α2-antiplasmin present in the compacted clots from approximately 30% to < 4%. The results were confirmed with experiments in which compaction was achieved by platelet-mediated clot retraction. Conclusions Compaction or retraction of fibrin clots reveals the strong antifibrinolytic effect of FXIII. This is explained by the cross-linking of α2-antiplasmin to fibrin by FXIIIa, which prevents the plasmin inhibitor from being fully expelled from the clot during compaction/retraction

Generalizing Terwilliger's likelihood approach: a new score statistic to test for genetic association

<p>Abstract</p> <p>Background:</p> <p>In this paper, we propose a one degree of freedom test for association between a candidate gene and a binary trait. This method is a generalization of Terwilliger's likelihood ratio statistic and is especially powerful for the situation of one associated haplotype. As an alternative to the likelihood ratio statistic, we derive a score statistic, which has a tractable expression. For haplotype analysis, we assume that phase is known.</p> <p>Results:</p> <p>By means of a simulation study, we compare the performance of the score statistic to Pearson's chi-square statistic and the likelihood ratio statistic proposed by Terwilliger. We illustrate the method on three candidate genes studied in the Leiden Thrombophilia Study.</p> <p>Conclusion:</p> <p>We conclude that the statistic follows a chi square distribution under the null hypothesis and that the score statistic is more powerful than Terwilliger's likelihood ratio statistic when the associated haplotype has frequency between 0.1 and 0.4 and has a small impact on the studied disorder. With regard to Pearson's chi-square statistic, the score statistic has more power when the associated haplotype has frequency above 0.2 and the number of variants is above five.</p

Fibrin clot structure remains unaffected in young, healthy individuals after transient exposure to diesel exhaust

Exposure to urban particulate matter has been associated with an increased risk of cardiovascular disease and thrombosis. We studied the effects of transient exposure to diesel particles on fibrin clot structure of 16 healthy individuals (age 21- 44). The subjects were randomly exposed to diesel exhaust and filtered air on two separate occasions. Blood samples were collected before exposure, and 2 and 6 hours after exposure. There were no significant changes on clot permeability, maximum turbidity, lag time, fibre diameter, fibre density and fibrinogen level between samples taken after diesel exhaust exposure and samples taken after filtered air exposure. These data show that there are no prothrombotic changes in fibrin clot structure in young, healthy individuals exposed to diesel exhaust

The fibrinogen γA/γ′ isoform does not promote acute arterial thrombosis in mice

Elevated plasma fibrinogen associates with arterial thrombosis in humans and promotes thrombosis in mice by increasing fibrin formation and thrombus fibrin content. Fibrinogen is composed of six polypeptide chains: (Aα, Bβ, and γ)2. Alternative splicing of the γ chain leads to a dominant form (γA/γA) and a minor species (γA/γ’). Epidemiologic studies have detected elevated γA/γ’ fibrinogen in patients with arterial thrombosis, suggesting this isoform promotes thrombosis. However, in vitro data show that γA/γ’ is anticoagulant due to its ability to sequester thrombin, and suggest its expression is upregulated in response to inflammatory processes