S. Uitte De Willige

Haplotypes of the EPCR gene, plasma sEPCR levels and the risk of deep venous thrombosis.

Background: Binding of protein C (PC) to the endothelial cell PC receptor (EPCR) stimulates PC activation by increasing the affinity of PC for the thrombin‐thrombomodulin complex. A soluble form of this receptor (sEPCR) circulates in plasma and inhibits both PC activation and APC anticoagulant activity. Objectives: The aim of this study was to investigate whether variations in the EPCR gene or plasma sEPCR levels are risk factors for deep venous thrombosis (DVT). Patients/methods: In a large case‐control study, the Leiden Thrombophilia Study (LETS), sEPCR levels were measured by ELISA. All subjects were genotyped for three haplotype‐tagging SNPs, enabling us to detect all four common haplotypes of the EPCR gene. Results: The distribution of sEPCR levels in the control population was trimodal and was genetically controlled by haplotype 3 (H3). This haplotype explained 86.5% of the variation in sEPCR levels. Carriers of two H3 alleles had higher sEPCR levels (439 ng mL−1) than carriers of one H3 allele (258 ng mL−1), which had higher levels than non‐H3 carriers (94 ng mL−1). Haplotype 4 was associated with a slightly increased risk (OR = 1.4, 95%CI:1.0–2.2). The risk of subjects with sEPCR levels in the top quartile (≥ 137 ng mL−1) was increased compared to that of subjects in the first quartile (< 81 ng mL−1), but since there was no dose–response effect, it is most likely that low sEPCR levels reduce the risk of DVT. Conclusions: Our data do not suggest a strong association between EPCR haplotypes and thrombosis risk, but low sEPCR levels appear to reduce the risk of DVT.

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.

Evidence that fibrinogen γ′ directly interferes with protofibril growth: implications for fibrin structure and clot stiffness

Summary.  Background:  Fibrinogen contains an alternatively spliced γ‐chain (γ′), which mainly exists as a heterodimer with the common γA‐chain (γA/γ′). Fibrinogen γ′ has been reported to inhibit thrombin and modulate fibrin structure, but the underlying mechanisms are unknown.

Inhibition of thrombin‐mediated factor V activation contributes to the anticoagulant activity of fibrinogen γ′

Besides its role in blood clotting, fibrinogen exerts a poorly understood anticoagulant function by binding thrombin and modulating its activity. In particular, the γA/γ′ fibrinogen isoform binds with high affinity to thrombin exosite II through the anionic carboxyl‐terminal end of the γ′ chain. This interaction down‐regulates thrombin‐mediated factor VIII (FVIII) activation, but its effect on FV activation is unknown.

Haplotypes of the fibrinogen gamma gene do not affect the risk of myocardial infarction

As the precursor of fibrin, fibrinogen plays an important role in hemostasis [1]. Of all the components of the coagulation system, elevated plasma fibrinogen levels have most consistently been shown to be associated with occlusive vascular disorders [2–5]. Several polymorphisms in the fibrinogen genes (FGA, FGB, FGG) have been reported to be associated with fibrinogen levels. Most studies focused on FGB polymorphisms. However, results are not consistent and none of these polymorphisms has been found to be associated with an increased risk of venous [6,7] or arterial thrombosis [7]. Recently, we reported that a specific haplotype (H2) of the fibrinogen gamma gene (FGG) was associated with a 2.4-fold increased risk of deep venous thrombosis [95% confidence intervals (CI): 1.5–3.9] [6]. In the same study, we found that another haplotype (FGG-H3) was associated with a slight reduction in risk [odds ratio (OR) 1⁄4 0.8, 95% CI: 0.6–1.0]. None of these haplotypes was associated with total fibrinogen levels. However, the FGG-H2 haplotype was associated with reduced levels of fibrinogen c¢, a product of alternative splicing of the FGG gene. In a recent report, Mannila et al. [8] studied the effect of haplotypes across the fibrinogen gene cluster on the risk of myocardial infarction (MI). They determined the *216C > T polymorphism, which is identical to FGG-H2 tagging SNP 10034 C/T [rs2066865]. In their study, the FGGH2 haplotype was not associated with the risk ofMI. However, they found a significant difference in the frequency distribution of the minor allele of the 1299 + 79T > C polymorphism between patients and controls (0.294 vs. 0.342, P 1⁄4 0.04), which suggests that this haplotype might be protective against the development of MI. This polymorphism is identical to FGG-H3 tagging SNP 9340 T/C [rs1049636]. The aim of the present study was to investigate the effect of the four most common haplotypes of the FGG gene on the risk of MI. For this study, a large population-based case–control study, Study of Myocardial Infarctions Leiden (SMILE) was used. Full details of the SMILE study have been described Table 1 Variant allele and haplotype group frequencies for VKORC1 and CYP2C9 in selected populations (actual SNP locations in parentheses). Haplotype groups A and B are based on classifications fromReider et al. [1] where haplotype A represents individuals at risk for excessive anticoagulation with standard warfarin dosing, and haplotype B represents individuals at risk for subtherapeutic anticoagulation from standard warfarin dosing. Overall low dose group defines individuals with at least one haplotype A and/or at least one CYP2C9 variant allele. Combined haplotype A and CYP2C9 group defines individuals with at least one haplotype A in combination with at least one CYP2C9 variant allele. The functional significance of haplotypes not in group A or group B (other) is currently unknown. IVS is standard nomenclature for intronic sequence

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

Elevated plasma fibrinogen is associated 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′). Epidemiological studies have detected elevated γA/γ′ fibrinogen in patients with arterial thrombosis, suggesting that 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.

Selectin haplotypes and the risk of venous thrombosis: influence of linkage disequilibrium with the factor V Leiden mutation

Summary.  Background: Selectins (E‐, L‐ and P‐selectin) and their most important counter‐receptor P‐selectin glycoprotein ligand (SELPLG) facilitate the interaction of platelets, leukocytes and endothelial cells at inflammatory sites. Selectin polymorphisms/haplotypes have been associated with cardiovascular disease. Objectives: We investigated the association between haplotypes (H) of these four genes and deep venous thrombosis (DVT) risk. We additionally explored the effect of linkage disequilibrium (LD) with the nearby Factor V Leiden mutation (FVL). Furthermore, interactions between SELPLG polymorphisms and selectin polymorphisms were investigated. Patients/methods: Leiden Thrombophilia Study (LETS) subjects were genotyped for 24 polymorphisms by TaqMan or PCR–RFLP, detecting all common haplotypes in four blocks. P‐selectin was analyzed in two blocks, upstream (SELPup) and downstream (SELPdown) of the recombination hotspot. Results: In E‐ and L‐selectin, none of the haplotypes was associated with DVT risk. In SELPup, H2‐carriers had a 1.3‐fold increased risk (95% CI, 1.0–1.7), whereas H4‐carriers had a 1.4‐fold decreased risk (95% CI, 0.5–1.0). In SELPdown, H2‐carriers had a 1.3‐fold increased risk (95% CI, 1.0–1.7). Because of LD with FVL, we subsequently excluded all FVL‐carriers and all risks disappeared. Mutual adjustment within a logistic regression model resulted in disappearance of the risks for the SELP haplotypes, whereas FVL risk remained. Conclusions: After adjustment for LD with FVL, none of the selectin haplotypes was associated with DVT risk, showing that the increased risks of the selectin haplotypes were a reflection of the effect of FVL on thrombosis risk.

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.

Increased N-terminal cleavage of alpha-2-antiplasmin in patients with liver cirrhosis

The activity of alpha‐2‐antiplasmin (α2AP), the main fibrinolytic inhibitor, is modified by N‐ and C‐terminal proteolytic cleavages. C‐terminal cleavage converts plasminogen‐binding α2AP (PB‐α2AP) into a non‐plasminogen‐binding derivative. N‐terminal cleavage by antiplasmin‐cleaving enzyme (APCE), a soluble, circulating derivative of fibroblast activation protein (FAP), turns native Met‐α2AP into Asn‐α2AP, which is more quickly crosslinked into fibrin.

Binding of carboxypeptidase N to fibrinogen and fibrin

The ultimate step in the blood coagulation cascade is the formation of fibrin. Several proteins are known to bind to fibrin and may thereby change clot properties or clot function. Our previous studies identified carboxypeptidase N (CPN) as a novel plasma clot component. CPN cleaves C-terminal lysine and arginine residues from several proteins. The activity of CPN is increased upon its proteolysis by several proteases. The aim of this study is to investigate the presence of CPN in a plasma clot in more detail. Plasma clots were formed by adding thrombin, CaCl(2) and aprotinin to citrated plasma. Unbound proteins were washed away and non-covalently bound proteins were extracted and analyzed with 2D gel electrophoresis and mass spectrometry. The identification of CPN as a fibrin clot-bound protein was verified using Western blotting. Clot-bound CPN consisted of the same molecular forms as CPN in plasma and its content was approximately 30 ng/ml plasma clot. Using surface plasmon resonance we showed that CPN can bind to fibrinogen as well as to fibrin. In conclusion, CPN binds to fibrinogen and is present in a fibrin clot prepared from plasma. Because CPN binds to a fibrin clot, there could be a possible role for CPN as a fibrinolysis inhibitor.