Natural heterogeneity of α2-antiplasmin: functional and clinical consequences.

Human α2-antiplasmin (α2AP, also called α2-plasmin inhibitor) is the main physiological inhibitor of the fibrinolytic enzyme plasmin. α2AP inhibits plasmin on the fibrin clot or in the circulation by forming plasmin-antiplasmin complexes. Severely reduced α2AP levels in hereditary α2AP deficiency may lead to bleeding symptoms, whereas increased α2AP levels have been associated with increased thrombotic risk. α2AP is a very heterogeneous protein. In the circulation, α2AP undergoes both amino terminal (N-terminal) and carboxyl terminal (C-terminal) proteolytic modifications that significantly modify its activities. About 70% of α2AP 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 α2AP to fibrin and consequently prolonged clot lysis. In approximately 35% of circulating α2AP, 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 α2AP. Without its C terminus, α2AP 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 α2AP 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 α2AP with particular focus on the functional and possible clinical implications.

Fibrinogen γ' increases the sensitivity to activated protein C in normal and factor V Leiden plasma.

Activated protein C (APC) resistance, often associated with the factor V (FV) Leiden mutation, is the most common risk factor for venous thrombosis. We observed increased APC resistance in carriers of fibrinogen γ gene (FGG) haplotype 2, which is associated with reduced levels of the alternatively spliced fibrinogen γ' chain. This finding prompted us to study the effects of fibrinogen and its γ' chain on APC resistance. Fibrinogen, and particularly the γA/γ' isoform, improved the response of plasma to added APC in the thrombin generation-based assay. Similarly, a synthetic peptide mimicking the C-terminus of the fibrinogen γ' chain, which binds thrombin and inhibits its activities, greatly increased the APC sensitivity of normal and FV Leiden plasma, likely due to its ability to inhibit thrombin-mediated activation of FV and FVIII. Although the fibrinogen γ' peptide also inhibited protein C activation by the thrombin/thrombomodulin complex, it still increased the sensitivity of plasma to endogenously formed APC when thrombin generation was measured in the presence of soluble thrombomodulin. We conclude that fibrinogen, and particularly fibrinogen γ', increases plasma APC sensitivity. The fibrinogen γ' peptide might form the basis for pharmacologic interventions to counteract APC resistance.

Proteolytic and genetic variation of the alpha-2-antiplasmin C-terminus in myocardial infarction.

Alpha-2-antiplasmin (α2AP) undergoes both N- and C-terminal cleavages, which significantly modify its activities. Compared with other Ser protease inhibitors (serpins), α2AP contains an ~50-residue-extended C-terminus, which binds plasmin(ogen). We developed 2 new ELISAs to measure the antigen levels of free total α2AP and free C-terminally intact α2AP to investigate whether α2AP antigen levels or α2AP C-terminal cleavage were associated with myocardial infarction (MI) in 320 male MI survivors and 169 age-matched controls. Patients had 15.2% reduced total α2AP antigen levels compared with controls (93.8 vs 110.6 U/dL, P < .001), with a 10.1-fold (95% confidence interval [CI]: 5.5-18.9) increased MI risk for levels in the 1st quartile compared with the 4th quartile. The percentage of C-terminal cleavage did not differ between patients and controls (38.7% and 38.1%, respectively, P = .44). In addition, all individuals were genotyped for the polymorphism Arg407Lys, which is located near the start of the extended C-terminus. Arg407Lys was not associated with α2AP C-terminal cleavage, total α2AP antigen levels, or MI risk (odds ratios compared with Arg/Arg: Arg/Lys 0.74, 95% CI: 0.50-1.10; Lys/Lys 0.77, 95% CI: 0.31-1.92). Our data show that levels of free full-length α2AP were decreased in MI, that the percentage of C-terminally cleaved α2AP was unaltered, and that Arg407Lys did not influence α2AP levels or MI risk.

Polyphosphate modifies the fibrin network and down-regulates fibrinolysis by attenuating binding of tPA and plasminogen to fibrin.

Activated platelets secrete a negatively charged polymer, polyphosphate (polyP). Here, we explore the interactions of polyP with fibrin(ogen) and its effect on fibrin structure and fibrinolysis. Electrophoretic mobility and binding assays indicate that polyP interacts with fibrinogen and soluble fibrin. Clots formed in the presence of polyP exhibited reduced turbidity and permeability indicative of a tighter fibrin network, but these changes were not related to cross-linking or fibrinopeptide release. Microscopy showed a change in fibrin distribution in clots formed with polyP; with formation of tight aggregates of fibrin fibers interspaced with large pores in contrast to homogenous fiber distribution in control clots. Lysis by tissue plasminogen activator (tPA) and plasminogen or plasmin was delayed in clots formed with polyP and depended on both the activator and polyP concentration. Adding polyP to the clot after fibrin formation or to repolymerizing soluble fibrin did not affect lysis, indicating changes induced by polyP occur at the level of conversion of fibrinogen to fibrin. Surface plasmon resonance showed that the presence of polyP reduced the binding of both plasminogen and tPA to partially lysed fibrin surfaces. These data show that polyP directly influences fibrin architecture and attenuates fibrinolysis through reduced binding of fibrinolytic proteins.

The pleiotropic role of the fibrinogen gamma' chain in hemostasis.

A fraction of fibrinogen contains a differently spliced gamma chain called gamma', which presents itself mainly as heterodimer with the common gammaA chain as gammaA/gamma' fibrinogen. The gamma' chain differs from the gammaA chain in its C-terminus and has important functional implications for fibrinogen. The presence of the gamma' chain modulates thrombin and FXIII activity, influences clot architecture, and eliminates a platelet-binding site. Associations of gammaA/gamma' fibrinogen levels with arterial and venous thrombosis have been reported, indicating that the functional effects of gammaA/gamma' fibrinogen may contribute to the pathology of thrombosis. This review summarizes the key biologic aspects of this interesting variant of fibrinogen and discusses inconsistencies in current reports.

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.

On the localization of the cleavage site in human alpha-2-antiplasmin, involved in the generation of the non-plasminogen binding form.

BACKGROUND: Alpha-2-antiplasmin (α2AP) is the main natural inhibitor of plasmin. The C-terminus of α2AP is crucial for the initial interaction with plasmin(ogen) and the rapid inhibitory mechanism. Approximately 35% of circulating α2AP has lost its C-terminus (non-plasminogen binding α2AP/NPB-α2AP) and thereby its rapid inhibitory capacity. The C-terminal cleavage site of α2AP is still unknown. A commercially available monoclonal antibody against α2AP (TC 3AP) detects intact but not NPB-α2AP, suggesting that the cleavage site is located N-terminally from the epitope of TC 3AP. OBJECTIVES: To determine the epitope of TC 3AP and then to localize the C-terminal cleavage site of α2AP. METHODS: For epitope mapping of TC 3AP, commercially available plasma purified α2AP was enzymatically digested with Asp-N, Glu-C, or Lys-N. The resulting peptides were immunoprecipitated using TC 3AP-loaded Dynabeads® Protein G. Bound peptides were eluted and analyzed by liquid chromatography-tandem mass spectometry (LC-MS/MS). To localize the C-terminal cleavage site precisely, α2AP (intact and NPB) was purified from plasma and analyzed by LC-MS/MS after enzymatic digestion with Arg-C. RESULTS: We localized the epitope of TC 3AP between amino acid residues Asp428 and Gly439. LC-MS/MS data from plasma purified α2AP showed that NPB-α2AP results from cleavage at Gln421-Asp422 as preferred site, but also after Leu417, Glu419, Gln420, or Asp422. CONCLUSIONS: The C-terminal cleavage site of human α2AP is located N-terminally from the TC 3AP epitope. Because C-terminal cleavage of α2AP can occur after multiple residues, different proteases may be responsible for the generation of NPB-α2AP.

Fibrinogen gamma gene 3'-end polymorphisms and risk of venous thromboembolism in the African-American and Caucasian population.

Genetic determinants of venous thromboembolism (VTE) in the African-American population are poorly characterised. It was recently shown that fibrinogen gamma gene (FGG) polymorphisms 10034C>T and 9340T>C influence VTE risk in the Caucasian population. In the African-American population these polymorphisms are common, with allele frequencies above 25%. Here we evaluated whether these and other FGG 3'-end polymorphisms were associated with VTE risk in the African-American population and aimed to replicate the association in the Caucasian population. We examined 557 Caucasian patients and 678 Caucasian controls, and 537 African-American patients and 586 African-American controls from the ;Genetic Attributes and Thrombosis Epidemiology' (GATE) study. In the African-American population, 10034C>T and 9340T>C marginally influenced VTE-risk, with a 20% increase in risk for 10034TT carriers and a 20% reduction in risk for 9340CC carriers. In the Caucasian population, 10034TT was associated with a 1.7-fold increase in risk, which increased to 2.1-fold for idiopathic VTE patients. 9340CC significantly reduced VTE risk approximately two-fold. In conclusion, both FGG polymorphisms 10034C>T and 9340T>C influence VTE-risk, with the strongest effects observed in the Caucasian population, confirming previous data on these polymorphisms in this population.

Heritability of clot formation.

The development of occlusive arterial and venous disease is contingent on the formation of a fibrin mesh that occurs following tissue damage and activation of the coagulation system. Clinical evidence indicates that fibrin structure and function are important determinants of cardiovascular risk, and the difference between clots formed from plasma and from purified fibrinogen highlights the importance of plasma factors in determining final clot structure. Twin, family, and case-control studies indicate there is a significant genetic contribution to variance in coagulation and fibrinolytic factors that may influence clot structure. Additionally, studies indicate a smaller but significant genetic contribution to fibrin structure, with a larger component provided by the environmental contribution. Future studies of the influence of post-translational modifications to fibrin(ogen) and other factors involved in clot formation may provide important insights into thrombotic disease mechanisms.

Fibrinogen gamma' in ischemic stroke: a case-control study.

BACKGROUND AND PURPOSE: To determine the contribution of fibrinogen gamma' levels and FGG haplotypes to ischemic stroke. METHODS: Associations between fibrinogen gamma' levels, fibrinogen gamma'/total fibrinogen ratio, and FGG haplotypes with the risk of ischemic stroke were determined in 124 cases and 125 controls. RESULTS: Fibrinogen gamma'/total fibrinogen ratio was higher in patients than in controls during the acute phase of the stroke and lower in the convalescent phase 3 months after the stroke. FGG haplotype 3 (H3) was associated with a reduced risk of ischemic stroke (odds ratio 0.60; 95% CI, 0.38 to 0.94), but not with the fibrinogen gamma'/total fibrinogen ratio. In contrast, FGG-H2 was associated with a decreased fibrinogen gamma'/total fibrinogen ratio, but not with risk of stroke. CONCLUSIONS: Fibrinogen gamma'/total fibrinogen ratio is associated with ischemic stroke, especially in the acute phase of the disease. In addition, FGG-H3 haplotype appears to be protective against ischemic stroke.

The level of circulating fibroblast activation protein correlates with incorporation of alpha-2-antiplasmin into the fibrin clot.

INTRODUCTION: Circulating fibroblast activation protein (cFAP) cleaves alpha-2-antiplasmin (α2AP) N-terminally, converting native Met-α2AP into Asn-α2AP. Previous studies in purified model systems showed that Asn-α2AP is faster incorporated into a fibrin clot by activated factor XIII than Met-α2AP, making the fibrin clot more resistant to fibrinolysis. The objective was to investigate whether cFAP level in plasma associated with the amount of α2AP incorporation into fibrin in a new plasma-based clotting assay. MATERIALS AND METHODS: We included 118 arterial thrombotic patients of the ATTAC study; 59 patients with diabetes mellitus (DM) and 59 age- and sex-matched patients without DM, additionally matched for type of arterial thrombosis (myocardial infarction or ischemic stroke). The percentage of α2AP incorporation was assessed with an α2AP incorporation assay mimicking physiological conditions with endogenous α2AP and physiological cFAP variation. cFAP levels were measured previously by ELISA. RESULTS: We found that on average 32.3 ±â€¯5.1% of α2AP was incorporated into fibrin, with slightly more α2AP incorporation in individuals with DM (33.3 ±â€¯4.9%) compared to individuals without DM (31.4 ±â€¯5.2%, p = 0.047), which validates our assay according to literature. The main finding of this study was that cFAP level positively correlated with α2AP incorporation into the fibrin clot (r = 0.296, p = 0.001). CONCLUSION: The findings of a positive association between cFAP level and α2AP incorporation in a plasma-based system under physiological conditions support the hypothesis that N-terminal cleavage of α2AP leads to faster and more incorporation of α2AP into the fibrin clot, which may be clinically relevant.

Generation and characterization of monoclonal antibodies against the N-terminus of alpha-2-antiplasmin.

Around 70% of circulating alpha-2-antiplasmin (α2AP), the main natural plasmin inhibitor, is N-terminally cleaved between residues Pro12 and Asn13 by antiplasmin-cleaving enzyme. This converts native Met-α2AP into the more potent fibrinolysis inhibitor Asn-α2AP. The Arg6Trp (R6W) polymorphism affects the N-terminal cleavage rate of Met-α2AP in a purified system, with ~8-fold faster conversion of Met(R6)-α2AP than Met(W6)-α2AP. To date, assays to determine N-terminally intact Met-α2AP in plasma have been limited to an ELISA that only measures Met(R6)-α2AP. The aim of this study was to generate and characterize monoclonal antibodies (mAbs) against Met(R6)-α2AP, Met(W6)-α2AP and all α2AP forms (total-α2AP) in order to develop specific Met(R6)-α2AP and Met(W6)-α2AP ELISAs. Recombinant Met(R6)-α2AP, Met(W6)-α2AP and Asn-α2AP were expressed in Drosophila S2 cells. Using hybridoma technology, a panel of 25 mAbs was generated against a mixture of recombinant Met(R6)-α2AP and Met(W6)-α2AP. All mAbs were evaluated for their specific reactivity using the three recombinant α2APs in one-site non-competitive ELISAs. Three mAbs were selected to develop sandwich-type ELISAs. MA-AP37E2 and MA-AP34C4 were selected for their specific reactivity against Met(R6)-α2AP and Met(W6)-α2AP, respectively, and used for coating. MA-AP15D7 was selected for its reactivity against total-α2AP and used for detection. With the novel ELISAs we determined Met(R6)-α2AP and Met(W6)-α2AP levels in plasma samples and we showed that Met(R6)-α2AP was converted faster into Asn-α2AP than Met(W6)-α2AP in a plasma milieu. In conclusion, we developed two specific ELISAs for Met(R6)-α2AP and Met(W6)-α2AP, respectively, in plasma. This will enable us to determine N-terminal heterogeneity of α2AP in plasma samples.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

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.

Circulating fibroblast activation protein activity and antigen levels correlate strongly when measured in liver disease and coronary heart disease.

BACKGROUND AND AIM: Circulating fibroblast activation protein (cFAP) is a constitutively active enzyme expressed by activated fibroblasts that has both dipeptidyl peptidase and endopeptidase activities. We aimed to assess the correlation between cFAP activity and antigen levels and to compare variations in levels. METHODS: In plasma of 465 control individuals, 368 patients with coronary heart disease (CHD) and 102 hepatitis C virus (HCV) infected patients with severe liver disease before and after liver transplant, cFAP activity levels were measured with a newly developed cFAP activity assay. In the same samples, cFAP antigen levels were measured using a commercially available cFAP ELISA. Correlation analyses between activity and antigen levels were performed by calculating Pearson's correlation coefficient (ρ). Additionally, normal ranges, determinants and differences between cohorts and between anticoagulants were investigated. RESULTS: cFAP activity and antigen levels significantly correlated in controls (ρ: 0.660, p<0.001) and in CHD patients (ρ: 0.709, p<0.001). cFAP activity and antigen levels in the HCV cohort were significantly lower in the samples taken after liver transplantation (p<0.001) and normalized toward levels of healthy individuals. Furthermore, cFAP activity and antigen levels were higher in men and significantly associated with body mass index. Also, cFAP activity and antigen levels were higher in EDTA plasma as compared to the levels in citrated plasma from the same healthy individuals. CONCLUSIONS: For analyzing cFAP levels, either activity levels or antigen levels can be measured to investigate differences between individuals. However, it is of importance that blood samples are collected in the same anticoagulant.

Sarcoglycanopathies and the risk of undetected deletion alleles in diagnosis.

We have designed Multiplex Amplifiable Probe Hybridization (MAPH) probes for 28 exons of the sarcoglycan genes SGCA, SGCB, SGCG, and SGCD. The set was used to screen DNA from limb-girdle muscular dystrophy (LGMD) patients for the presence of pathogenic deletion or duplication mutations. An unexpected heterozygous deletion of SGCG exon 7 was detected in a patient from a consanguineous family in which a known c.525delT mutation segregates. The exon 7 deletion was inherited from the father, who was part of the consanguineous c.525delT branch of the family but who did not carry the c.525delT mutation. A similar, homozygous deletion had been identified in two unrelated LGMD patients from southern Italy. The deletion breakpoints were mapped, isolated, and sequenced, and were identical in all cases. Haplotype analysis showed the same alleles segregating with the mutation in all three patients, suggesting a common ancestor. Exonic deletions in sarcoglycanopathies appear to be rare events. However, we recommend screening for exonic deletions/duplications in patients where a mutation has not been identified in both alleles, as well as in seemingly homozygous cases where segregation of the mutations can not be confirmed in the parents.

Plasma levels of soluble fibroblast activation protein in arterial thrombosis: determinants and cleavage of its substrate alpha-2-antiplasmin.

BACKGROUND: Fibroblast activation protein (FAP) is a transmembrane glycoprotein with dipeptidyl-peptidase and endopeptidase activities and circulates in blood in a truncated, soluble form (sFAP). Fibrinolysis inhibitor α2-antiplasmin (α2AP) has been described as a potential in vivo substrate of sFAP. We aimed to investigate sFAP levels and α2AP cleavage in young arterial thrombosis patients and in control individuals, study the correlation between sFAP levels and α2AP cleavage and investigate determinants of these variables. METHODS: sFAP levels and α2AP cleavage were determined by ELISA in the plasma samples of 391 coronary heart disease (CHD) patients, 221 ischemic stroke patients, 51 peripheral arterial disease patients and 501 control individuals. RESULTS: Median sFAP levels were similar in arterial thrombotic patients and in control individuals, but in CHD patients sFAP levels significantly increased with time (number of months) between the event and study inclusion (Spearman's rho: 0.209, p<0.001), indicating reduced sFAP levels at time of event. sFAP levels and percentage α2AP cleavage significantly correlated in controls and in patients. Furthermore, sex, use of oral contraceptives and hyperlipidemia were significant determinants of sFAP levels. CONCLUSIONS: sFAP levels were reduced in the CHD patient population, but only in the first months after the event, indicating that over time sFAP levels may normalize. The significant correlation between sFAP level and α2AP cleavage indicates that in vivo sFAP (at least partly) regulates cleavage of α2AP, irrespective of disease status. Differences in sFAP level due to sex, use of oral contraceptives and hyperlipidemia might suggest hormonal control of sFAP levels.

Genetic variation in the fibrinogen gamma gene increases the risk for deep venous thrombosis by reducing plasma fibrinogen gamma' levels.

We investigated the association between haplotypes of fibrinogen alpha (FGA), beta (FGB), and gamma (FGG), total fibrinogen levels, fibrinogen gamma' (gammaA/gamma' plus gamma'/gamma') 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 gamma' levels and reduced ratios of fibrinogen gamma' to total fibrinogen. Multivariate analysis showed that reduced fibrinogen gamma' levels and elevated total fibrinogen levels were both associated with an increased risk for thrombosis, even after adjustment for FGG-H2. A reduced fibrinogen gamma' to total fibrinogen ratio (less than 0.69) also increased the risk (OR, 2.4; 95% CI, 1.7-3.5). We propose that FGG-H2 influences thrombosis risk through htSNP 10034C/T [rs2066865] by strengthening the consensus of a CstF site and thus favoring the formation of gammaA chain above that of gamma' chain. Fibrinogen gamma' contains a unique high-affinity, nonsubstrate binding site for thrombin, which seems critical for the expression of the antithrombin activity that develops during fibrin formation (antithrombin 1).