Plasma levels of plasminogen activator inhibitor-1 and bleeding phenotype in patients with von Willebrand disease.

INTRODUCTION: von Willebrand disease (VWD) is the most common inherited bleeding disorder. In VWD patients, large variations in bleeding tendency are observed, which cannot be completely explained by the variation in von Willebrand factor levels or activities. Thus, there must be additional factors, for instance, changes in fibrinolysis that have an effect on the variation in bleeding tendency in VWD patients. AIM: To investigate whether plasminogen activator inhibitor-1 (PAI-1) level influences the variation in bleeding tendency in VWD patients. METHODS: PAI-1 antigen levels were measured in the plasma of 633 patients with moderate or severe VWD who participated in the 'Willebrand in the Netherlands' (WiN) study, a nationwide multicentre cross-sectional study. Bleeding severity was assessed using the Tosetto bleeding score. RESULTS: PAI-1 levels increased with age (Spearman's rho: 0.225, P < 0.001) and were higher in men (23 [IQR 12-60] vs. 20 [IQR 10-44] ng mL-1 in women, P = 0.039), whereas the bleeding score was higher in women (11 [IQR 7-17] vs. 9 [IQR 5-14] ng mL-1 in men, P = 0.002). After adjustment for age and sex by stratification, PAI-1 level and bleeding score were negatively correlated (Spearman's rho: -0.170, P = 0.017) in the group of 196 young (age ≤ 45 year) female VWD patients, accounting for 31% of our study population. CONCLUSION: In young female VWD patients, we observed that low PAI-1 levels were associated with a higher bleeding score, which may partly explain the observed variability in bleeding phenotype in VWD patients.

Compaction of fibrin clots reveals the antifibrinolytic effect of factor XIII.

UNLABELLED: 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.

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

BACKGROUND: 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, Bß, 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. OBJECTIVE: To determine whether γA/γ' fibrinogen is prothrombotic in vivo. METHODS: We separated γA/γA and γA/γ' fibrinogen from human plasma-purified fibrinogen and determined the effects on in vitro plasma clot formation and on in vivo thrombus formation and circulating thrombin-antithrombin complexes in mice. RESULTS AND CONCLUSIONS: Both γA/γA and γA/γ' fibrinogen were cleaved by murine and human thrombin and were incorporated into murine and human clots. When γA/γA or γA/γ' was spiked into plasma, γA/γA increased the fibrin formation rate to a greater extent than γA/γ'. In mice, compared to controls, γA/γA infusion shortened the time to carotid artery occlusion, whereas γA/γ' infusion did not. Additionally, γA/γ' infusion led to lower levels of plasma thrombin-antithrombin complexes following arterial injury, whereas γA/γA infusion did not. These data suggest that γA/γ' binds thrombin in vivo and decreases prothrombotic activity. Together, these findings indicate that elevated levels of γA/γA fibrinogen promote arterial thrombosis in vivo, whereas γA/γ' does not.

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

BACKGROUND: 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. OBJECTIVES: We developed two novel enzyme-linked immunosorbent assays (ELISAs) to determine the N-terminal variation of α2AP to test the hypothesis that liver cirrhosis, characterized by increased expression of FAP/APCE, results in increased N-terminal cleavage of α2AP. PATIENTS/METHODS: α2AP and FAP/APCE antigen levels were measured in the plasma samples of 75 patients with cirrhosis with different severities and 30 healthy control individuals. The percentage of N-terminal cleavage of α2AP was calculated. RESULTS: Compared with levels (median [interquartile range]) in control individuals, total PB-α2AP levels and Met-PB-α2AP levels were reduced in cirrhosis patients (27.3 [21.4-41.3] µg mL(-1) vs. 56.2 [49.6-62.8] µg mL(-1) , P < 0.001, and 2.7 [1.7-5.5] µg mL(-1) vs. 12.1 [11.0-15.3] µg mL(-1) , P < 0.001, respectively). Interestingly, the percentage of N-terminal cleavage was increased in the patients (87.8 [85.0-91.6]% vs. 77.2 [72.2-79.8]% in controls, P < 0.001), as well as the plasma FAP/APCE levels (166 [60-550] ng mL(-1) in patients vs. 107 [67-157] ng mL(-1) in controls, P < 0.001). Additionally, all variables significantly correlated with the severity of disease. CONCLUSIONS: Using our novel ELISAs we found increased N-terminal cleavage of α2AP in liver cirrhosis patients, which correlated with the severity of disease and is likely to have reflected the increased FAP/APCE levels in these patients.

Inhibition of thrombin-mediated factor V activation contributes to the anticoagulant activity of fibrinogen γ'.

BACKGROUND: 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. OBJECTIVES: To investigate the overall anticoagulant activity of fibrinogen and particularly of fibrinogen γ' in plasma, and to verify whether the fibrinogen γ' carboxyl-terminal peptide affects thrombin-mediated FV activation. METHODS: Thrombin generation was measured by calibrated automated thrombography in whole and defibrinated plasma and in plasma supplemented with the (sulfated) fibrinogen γ' carboxyl-terminal peptide (0-500 µmol L(-1) ). The effect of the peptide on thrombin-mediated FV activation was studied in model systems and in plasma. RESULTS: Total fibrinogen prolonged the lag time of thrombin generation at low tissue factor (TF) concentrations. The fibrinogen γ' peptide dose-dependently prolonged the lag time and decreased the peak height of thrombin generation at low TF, whereas a scrambled control peptide was ineffective. These effects persisted in the presence of an anti-FVIII antibody, suggesting that the peptide may also inhibit thrombin-mediated activation of FV. This was confirmed in model systems and in plasma. CONCLUSIONS: Total fibrinogen and the fibrinogen γ' peptide have an overall anticoagulant effect on thrombin generation determined at low TF. Inhibition of thrombin-mediated FV activation by the fibrinogen γ' peptide is a novel mechanism of the anticoagulant activity of fibrinogen γ'.

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.

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

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'.

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.

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

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.

Polymorphism 10034C>T is located in a region regulating polyadenylation of FGG transcripts and influences the fibrinogen gamma'/gammaA mRNA ratio.

BACKGROUND: Fibrinogen gamma haplotype 2 (FGG-H2) is associated with reduced fibrinogen gamma' levels and fibrinogen gamma'/total fibrinogen ratios and with an increased deep-venous thrombosis (DVT) risk. Two FGG-H2 tagging single nucleotide polymorphisms (SNPs), 9615C>T and 10034C>T, are located in the region of alternative FGG pre-mRNA processing. 10034C>T is located in a GT-rich downstream sequence element (DSE) that comprises a putative cleavage stimulation factor (CstF) binding site. OBJECTIVES: To investigate the functionality of SNPs 9615C>T and 10034C>T, and the importance of the DSE containing 10034C>T. METHODS: Different minigene constructs containing FGG exon 9, intron 9, exon 10 and the 3' region were transiently transfected into HepG2 cells and quantitative real-time polymerase chain reaction was used to measure relative polyadenylation (pA) signal usage (pA1/pA2 ratio). RESULTS: Compared with the reference construct CC (9615C-10034C; FGG-H1; pA1/pA2 ratio set at 100%), the pA1/pA2 ratio of construct TT (9615T-10034T; FGG-H2) was 1.4-fold decreased (71.5%, P = 0.015). The pA1/pA2 ratio of construct CT (9615C-10034T) was almost 1.2-fold decreased (85.3%, P = 0.001), whereas the pA1/pA2 ratio of construct TC (9615T-10034C) did not differ significantly from the reference construct (101.6%, P = 0.890). Functionality of the putative CstF binding site was confirmed using constructs in which this site was deleted or its sequence altered by point mutations. CONCLUSIONS: SNP 10034C>T is located in a GT-rich DSE involved in regulating the usage of the pA2 signal of FGG, which may represent a CstF binding site. We propose that the 10034C>T change is the functional variation in FGG-H2 that is responsible for the reduction in the fibrinogen gamma'/total fibrinogen ratio and the increased DVT risk.

Plasma fibrinogen gamma' chain content in the thrombotic microangiopathy syndrome.

BACKGROUND: Human fibrinogen gamma chain variants, termed gamma' chains, contain a unique 20-residue sequence after gamma chain residue 407 that ends at gamma'427, and is designated gamma'(427L). Full-length (FL) gamma'(427L) chains are constituents of a fibrin-dependent thrombin inhibitory system known as antithrombin I, whereas a gamma' chain processed in vivo, termed gamma'(423P), lacks the C-terminal tetrapeptide EDDL, and does not bind thrombin. Together, the gamma'(423P) and gamma'(427L) chains comprise the total plasma fibrinogen gamma' chain content. OBJECTIVES: Lowered plasma gamma' chain content (i.e. gamma' chain-containing fibrinogen/total fibrinogen ratio) has been shown to correlate with susceptibility to venous thrombosis, thus prompting this study on the total and FL gamma' chain content in 45 subjects with thrombotic microangiopathy (TMA), a disorder characterized by microvascular thrombosis. METHODS: We measured by enzyme-linked immunosorbent assay the total gamma' chain-containing fibrinogen/total fibrinogen (Total gamma'-fgn/Total fgn) ratio and the FL gamma' chain-containing fibrinogen/total fibrinogen (FL gamma'-fgn/Total fgn) ratio in these plasmas and in healthy subjects (n = 87). RESULTS: In healthy subjects, the mean Total gamma'-fgn/Total fgn ratio was 0.127, whereas the FL gamma'-fgn/Total fgn ratio was somewhat lower at 0.099 (P < 0.0001), a difference reflecting the presence of gamma'(423P) chains. In TMA plasmas, both the Total gamma'-fgn and FL gamma'-fgn/Total fgn ratios (0.099 and 0.084, respectively) were lower than those of their healthy subject counterparts (P < 0.0001). CONCLUSIONS: These findings in TMA suggest that reductions in the gamma' chain content indicate reduced antithrombin I activity that may contribute to microvascular thrombosis in TMA.

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.