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Jerzy Mosiewicz - One of the best experts on this subject based on the ideXlab platform.

D C Rijken - One of the best experts on this subject based on the ideXlab platform.

  • On the localization of the cleavage site in human Alpha‐2‐antiplasmin, involved in the generation of the non‐plasminogen binding form
    'Wiley', 2020
    Co-Authors: Abdul S, D C Rijken, Dekkers Dhw, Ariëns Ras, Leebeek Fwg, Su ,de Willige
    Abstract:

    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

  • plasma levels of soluble fibroblast activation protein in arterial thrombosis determinants and cleavage of its substrate Alpha 2 antiplasmin
    International Journal of Cardiology, 2015
    Co-Authors: Shirley Uitte De Willige, Joyce J M C Malfliet, Jaap W Deckers, Diederik W J Dippel, Frank W G Leebeek, D C Rijken
    Abstract:

    Abstract 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 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.

  • increased n terminal cleavage of Alpha 2 antiplasmin in patients with liver cirrhosis
    Journal of Thrombosis and Haemostasis, 2013
    Co-Authors: Uitte S De Willige, Joyce J M C Malfliet, Frank W G Leebeek, Harry L A Janssen, D C Rijken
    Abstract:

    Summary 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.

Shirley Uitte De Willige - One of the best experts on this subject based on the ideXlab platform.

  • the level of circulating fibroblast activation protein correlates with incorporation of Alpha 2 antiplasmin into the fibrin clot
    Thrombosis Research, 2018
    Co-Authors: Shirley Uitte De Willige, Joyce J M C Malfliet, Frank W G Leebeek, Shiraazkhan Abdul, Dingeman C. Rijken
    Abstract:

    Abstract 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 ± 5.1% of α2AP was incorporated into fibrin, with slightly more α2AP incorporation in individuals with DM (33.3 ± 4.9%) compared to individuals without DM (31.4 ± 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
    2018
    Co-Authors: Shiraazkhan Abdul, Joyce J M C Malfliet, Frank W G Leebeek, Miet Peeters, Els Brouwers, Paul J. Declerck, Dingeman C. Rijken, Shirley Uitte De Willige
    Abstract:

    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.

  • plasma levels of soluble fibroblast activation protein in arterial thrombosis determinants and cleavage of its substrate Alpha 2 antiplasmin
    International Journal of Cardiology, 2015
    Co-Authors: Shirley Uitte De Willige, Joyce J M C Malfliet, Jaap W Deckers, Diederik W J Dippel, Frank W G Leebeek, D C Rijken
    Abstract:

    Abstract 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 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.

  • proteolytic and genetic variation of the Alpha 2 antiplasmin c terminus in myocardial infarction
    Blood, 2011
    Co-Authors: Shirley Uitte De Willige, Megan Miedzak, Angela M Carter, Ton Lisman, Frits R Rosendaal, Peter J Grant, Helen Philippou, Robert A S Ariens
    Abstract:

    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.

Maciej Zakrzewski - One of the best experts on this subject based on the ideXlab platform.

H. Roger Lijnen - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of the Murine Plasma Fibrinolytic System
    European Journal of Biochemistry, 1994
    Co-Authors: H. Roger Lijnen, Berthe Van Hoef, Veerle Beelen, Desire Collen
    Abstract:

    The main components of the murine plasma fibrinolytic system, including fibrinogen, plasminogen, Alpha 2-Antiplasmin, tissue-type plasminogen activator and plasminogen activator inhibitor-1, were purified to homogeneity and their interactions were quantitated and compared with those of the human counterparts. Initial activation rates of murine and human plasminogen by autologous tissue-type plasminogen activator were comparable (catalytic efficiencies, k2/Km, of 0.4 and 0.6 mM-1 s-1, respectively), but murine plasminogen appeared to be resistant to activation by human tissue-type plasminogen activator (k2/Km = 0.01 mM-1 s-1). Plasminogen activation by tissue-type plasminogen activator was stimulated 100- and 160-fold in autologous murine and human systems, respectively, with saturating concentrations of 0.45 and 0.32 microM, respectively, of CNBr-digested fibrinogen. Nearly quantitative binding (85-90%) of tissue-type plasminogen activator to fibrin was observed both in autologous and heterologous systems. Murine and human plasmin were very rapidly inhibited by autologous and heterologous Alpha 2-Antiplasmin (second-order inhibition rate constants, k1,app, of 2.1-2.3 x 10(7) M-1 s-1) and murine and human tissue-type plasminogen activator were very rapidly inhibited by autologous or heterologous plasminogen activator inhibitor-1 (k1,app of 1.8-4.9 x 10(7) M-1 s-1). Two-chain murine tissue-type plasminogen activator (added at a concentration of 1 microgram/ml) was inhibited in normal or plasminogen activator inhibitor-1-deficient murine plasma with half-lives of 6.5 min and 4.2 min, respectively, as compared to 80 min for human tissue-type plasminogen activator, suggesting that murine plasma contains proteinase inhibitors other than plasminogen activator inhibitor-1 which efficiently inhibit autologous tissue-type plasminogen activator. Clot lysis experiments in autologous plasma revealed that the murine plasma fibrinolytic system is more resistant to activation than the human system (20-30% clot lysis in 2 h with 100 nM tissue-type plasminogen activator in the murine system, as compared to 50% clot lysis in 2 h with 3.5 nM tissue-type plasminogen activator in the human system). Several mechanisms appear to be involved in this relative resistance observed in the murine system, including resistance of murine plasminogen to quantitative activation and short plasma half-life of murine tissue-type plasminogen activator. Thus, although these quantitative interactions between purified components of the murine fibrinolytic system appear to be comparable to those between the human counterparts, murine plasma clots are > 30-fold more resistant to lysis with autologous tissue-type plasminogen activator than human plasma clots.

  • interaction between staphylokinase plasmin ogen and Alpha 2 antiplasmin recycling of staphylokinase after neutralization of the plasmin staphylokinase complex by Alpha 2 antiplasmin
    Journal of Biological Chemistry, 1993
    Co-Authors: Karen Silence, Desire Collen, H. Roger Lijnen
    Abstract:

    Abstract Although the plasminogen activating equimolar complex of staphylokinase (STA) with human plasmin is very rapidly inhibited by Alpha 2-Antiplasmin, STA is a potent fibrinolytic agent in a human plasma milieu which contains 1 microM Alpha 2-Antiplasmin. In the present study, it was found that the complex of plasmin with recombinant STA (STAR), after neutralization with Alpha 2-Antiplasmin, retained the full plasminogen activating potential of STAR when added to a plasminogen solution (93 +/- 5% residual activity). When added to human plasma containing a 125I-fibrin-labeled plasma clot, equi-effective concentrations (causing 50% lysis in 2 h) were 17 +/- 3.0, 13 +/- 1.0, and 20 +/- 1.0 nM for STAR, equimolar plasmin-STAR mixtures, and plasmin-STAR mixtures neutralized by Alpha 2-Antiplasmin, respectively. Gel filtration of mixtures of plasmin(ogen) and STAR revealed elution as plasmin-STAR complex (Mr approximately 100,000), whereas after addition of Alpha 2-Antiplasmin, STAR eluted with an apparent Mr of 20,000. When mixtures of plasmin and STAR were adsorbed to lysine-Sepharose, STAR adsorbed quantitatively (96 +/- 1%) to the gel, whereas it was nearly quantitatively recovered in the unbound fraction (92 +/- 4%) after addition of Alpha 2-Antiplasmin to the mixture. Scatchard analysis of the binding of STAR to plasmin-Sepharose yielded a dissociation constant of 55 nM, whereas no specific binding of STAR to plasmin-Alpha 2-Antiplasmin-Sepharose could be demonstrated. These findings indicate that, both in purified systems and in a human plasma milieu containing a 125I-fibrin-labeled plasma clot, neutralization of the plasmin-STAR complex by Alpha 2-Antiplasmin results in dissociation of functionally active STAR from the complex and recycling of STAR to other plasminogen molecules. This dissociation-recycling process may explain the high fibrinolytic potency of STAR in a plasma milieu in the presence of high concentrations of Alpha 2-Antiplasmin.