We investigated the association between haplotypes of fibrinogen alpha (FGA), beta (FGB), and gamma (FGG), total fibrinogen levels, fibrinogen ␥ (␥A/␥ plus ␥/␥) levels, and risk for deep venous thrombosis. In a population-based case-control study, the Leiden Thrombophilia Study, we typed 15 haplotype-tagging single nucleotide polymorphisms (htSNPs) in this gene cluster. None of these haplotypes was associated with total fibrinogen levels. In each gene, one haplotype increased the thrombosis risk approximately 2-fold. After adjustment for linkage disequilibrium between the genes, only FGG-H2 homozygosity remained associated with risk (odds ratio [OR], 2.4; 95% confidence interval [95% CI], 1.5-3.9). FGG-H2 was also associated with reduced fibrinogen ␥ levels and reduced ratios of fibrinogen ␥ to total fibrinogen. Multivariate analysis showed that reduced fibrinogen ␥ levels and elevated total fibrinogen levels were both associated with an increased risk for thrombosis, even after adjustment for FGG-H2. A reduced fibrinogen ␥ to total fibrinogen ratio (less than 0.69) also increased the risk (OR, 2.4; 95% CI, 1.7-3.5)

Summary. 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.

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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.

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. Objective:  We aimed to investigate the molecular mechanism underpinning the influence of γ′ on fibrin polymerization, structure and viscoelasticity. Methods:  γA/γA and γA/γ′ fibrinogens were separated using anion exchange chromatography. Cross‐linking was controlled with purified FXIIIa and a synthetic inhibitor. Fibrin polymerization was analyzed by turbidity and gel‐point time was measured using a coagulometer. We used atomic force microscopy (AFM) to image protofibril formation while final clot structure was assessed by confocal and scanning electron microscopy. Clot viscoelasticity was measured using a magnetic microrheometer. Results:  γA/γ′ fibrin formed shorter oligomers by AFM than γA/γA, which in addition gelled earlier. γA/γ′ clots displayed a non‐homogenous arrangement of thin fibers compared with the uniform arrangements of thick fibers for γA/γA clots. These differences in clot structure were not due to thrombin inhibition as demonstrated in clots made with reptilase. Non‐cross‐linked γA/γA fibrin was approximately 2.7 × stiffer than γA/γ′. Cross‐linking by FXIIIa increased the stiffness of both fibrin variants; however, the difference in stiffness increased to approximately 4.6 × (γA/γA vs. γA/γ′). Conclusions:  Fibrinogen γ′ is associated with the formation of mechanically weaker, non‐uniform clots composed of thin fibers. This is caused by direct disruption of protofibril formation by γ′.

Human a2-antiplasmin (a2AP, also called a2-plasmin inhibitor) is the main physiological inhibitor of the fibrinolytic enzyme plasmin. a2AP inhibits plasmin on the fibrin clot or in the circulation by forming plasmin-antiplasmin complexes. Severely reduced a2AP levels in hereditary a2AP deficiency may lead to bleeding symptoms, whereas increased a2AP levels have been associated with increased thrombotic risk. a2AP is a very heterogeneous protein. In the circulation, a2AP undergoes both amino terminal (N-terminal) and carboxyl terminal (C-terminal) proteolytic modifications that significantly modify its activities. About 70% of a2AP is cleaved at the N terminus by antiplasmin-cleaving enzyme (or soluble fibroblast activation protein), resulting in a 12-amino-acid residue shorter form. The glutamine residue that serves as a substrate for activated factor XIII becomes more efficient after removal of the N terminus, leading to faster crosslinking of a2AP to fibrin and consequently prolonged clot lysis. In approximately 35% of circulating a2AP, the C terminus is absent. This C terminus contains the binding site for plasmin(ogen), the key component necessary for the rapid and efficient inhibitory mechanism of a2AP. Without its C terminus, a2AP can no longer bind to the lysine binding sites of plasmin(ogen) and is only a kinetically slow plasmin inhibitor. Thus, proteolytic modifications of the N and C termini of a2AP constitute major regulatory mechanisms for the inhibitory function of the protein and may therefore have clinical consequences. This review presents recent findings regarding the main aspects of the natural heterogeneity of a2AP with particular focus on the functional and possible clinical implications. (Blood. 2016; 127(5):538-545) Introduction a2-antiplasmin (a2AP, also called a2-plasmin inhibitor) is a key player in the fibrinolytic system (Figure 1). The fibrinolytic system is crucial for dissolving fibrin clots, facilitating tissue repair, and preventing clots from occluding vessels.1 Recent clinical studies have shown that reduced fibrinolysis (eg, due to an increase in a2AP level) is associated with an increase in both venous and arterial thrombotic risk. 2In contrast, an increase in fibrinolysis due to a2AP deficiency is associated with hemophilia-like bleeding symptoms, which typically occur after initial hemostasis as a result of the premature dissolution of fibrin. The phenotype of a2AP deficiency is heterogeneous. Complete congenital a2AP deficiency leads to severe bleeding with hemophilialike bleeding symptoms such as joint bleeding, whereas heterozygous a2AP-deficient patients typically have mild or no bleeding symptoms. 3,4 In addition, acquired a2AP deficiency may also occur in liver disease 5 and amyloidosis 6 or during fibrinolytic therapy. 7The main enzyme of the fibrinolytic system is the serine protease plasmin, which is predominantly responsible for the degradation of fibrin into rapidly cleared soluble fibrin degradation products (Figure 1). The inactive proenzyme plasminogen ca...

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To cite this article: Rijken DC, Abdul S, Malfliet JJMC, Leebeek FWG, Uitte de Willige S. Compaction of fibrin clots reveals the antifibrinolytic effect of factor XIII. J Thromb Haemost 2016; 14: 1453-61. Essentials• Factor XIIIa inhibits fibrinolysis by forming fibrinfibrin 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 a2-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 a 2 -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 a 2 -antiplasmin to fibrin. The FXIIIa inhibitor reduced the amount of a 2 -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 a 2 -antiplasmin to fibrin by FXIIIa, which prevents the plasmin inhibitor from being fully expelled from the clot during compaction/retraction.