Gizella Haramura

Severe bleeding complications caused by an autoantibody against the B subunit of plasma factor XIII: a novel form of acquired factor XIII deficiency

Acquired factor XIII (FXIII) deficiency due to autoantibody against FXIII is a very rare severe hemorrhagic diathesis. Antibodies directed against the A subunit of FXIII, which interfere with different functions of FXIII, have been described. Here, for the first time, we report an autoantibody against the B subunit of FXIII (FXIII-B) that caused life-threatening bleeding in a patient with systemic lupus erythematosus. FXIII activity, FXIII-A(2)B(2) complex, and individual FXIII subunits were undetectable in the plasma, whereas platelet FXIII activity and antigen were normal. Neither FXIII activation nor its activity was inhibited by the antibody, which bound to structural epitope(s) on both free and complexed FXIII-B. The autoantibody highly accelerated the elimination of FXIII from the circulation. FXIII supplementation combined with immunosuppressive therapy, plasmapheresis, immunoglobulin, and anti-CD20 treatment resulted in the patients recovery. FXIII levels returned to around 20% at discharge and after gradual increase the levels stabilized above 50%.

Anti-factor V auto-antibody in the plasma and platelets of a patient with repeated gastrointestinal bleeding

Summary.  Development of autoantibody against coagulation factor V (FV) is a rare clinical condition with hemorrhagic complications of varying severity. The aim of this study was to establish the pathomechanism of an acquired FV deficiency and characterize the FV inhibitor responsible for the clinical symptoms. A 78‐year‐old female was admitted to hospital with severe gastrointestinal bleeding. General clotting tests and determination of clotting factors were performed by standard methods. FV antigen and FV containing immune complexes were measured by ELISA. The FV molecule was investigated by Western blotting and by sequencing the f5 gene. The binding of patients IgG to FV and activated FV (FVa) was demonstrated in an ELISA system and its effect on the procoagulant activity of FVa was tested in clotting tests and in a chromogenic prothrombinase assay. Localization of the epitope for the antibody was performed by blocking ELISA. FV activity was severely suppressed both in plasma and platelets. FV antigen levels were normal by ELISA using polyclonal anti‐FV antibody or monoclonal antibody against the connecting region of FV, but depressed when HV1 monoclonal antibody against the C2 domain in the FV light‐chain was used as capture antibody. The FV molecule was found intact. An IgG reacting with both FV and FVa was present in the patients plasma and its binding to FV was inhibited by HV1 antibody. FV‐containing immune complexes were detected in the patients plasma and platelet lysate. The patients IgG inhibited the procoagulant function of FVa. An anti‐FV IgG was present in the patients plasma and platelets. The autoantibody reacted with an epitope in the C2 domain of FV light chain and neutralized the procoagulant function of FVa.

Down-regulation of activated factor XIII by polymorphonuclear granulocyte proteases within fibrin clot

Activated clotting factors are down-regulated by two major mechanisms which involve protease inhibitors or proteolytic degradation. To date, no down-regulating mechanism for activated factor XIII (FXIIIa) has been demonstrated. As the hemostatic plug contains polymorphonuclear granulocytes (PMNs) rich in proteolytic enzymes, we tested if these proteases are released in fibrin clots, and become involved in the down-regulation of FXIIIa. The supernatant of stimulated granulocytes proteolytically degraded and inactivated FXIIIa. In the fibrin clot formed from fibrinogen solution elastase, cathepsin G and matrix metalloprotease-9 (MMP-9) were released from granulocytes without any external stimulus. PMN proteases released in fibrin clot exerted a fibrinolytic effect and almost completely degraded both FXIII subunits. The elastase inhibitor, ONO 5046, partially inhibited the proteolytic degradation of FXIII in PMN-supplemented fibrin clots. Cathepsin G and MMP-9 inhibitors provided less protection; in these cases intermediate split products accumulated. The proteolytic degradation of FXIII by PMNs was also significant when the clot was made from whole plasma. The main plasma protease inhibitor, alpha1-antitrypsin, provided only partial protection. In the fibrin clot which contained alpha1-antitrypsin FXIIIa was degraded by PMN proteases significantly faster than cross-linked fibrin. The results suggest that the degradation of FXIII subunits by the concerted action of PMN proteases released within the clot represents a novel mechanism for the down-regulation of FXIIIa.

The pool of fatty acids covalently bound to platelet proteins by thioester linkages can be altered by exogenously supplied fatty acids

The goals of this investigation were, first, to develop a chemical strategy to identify and quantitate the mass of fatty acid which is covalently bound to proteins by thioester linkage in unactivated platelets, and, second, to determine whether exogeneously added fatty acids can alter the fatty acid composition of thioester bound fatty acids. Studies with radio-labeled fatty acids cannot identify and quantitate the actual fatty acids bound to proteins because they permit analysis of only the radiolabeled fatty acids added and their metabolites. Therefore, in the absence of metabolic labeling by radiolabeled fatty acids, we isolated the thioester-linked fatty acids from platelet proteins using hydroxylamine at neutral pH to form fatty acid hydroxamates. The hydroxamates were subsequently converted to fatty acid methyl esters by acid methanolysis for quantitation by gas chromatography-mass spectrometry. Using platelet specimens from 14 subjects, 74% of the fatty acid recovered from the unactivated platelet proteins as thioester linked was palmitate. Importantly, however, 22% was stearic acid, and oleate was 4% of the total thioester bound fatty acid. There was minimal variability (2.6-fold at maximum) between the subjects in the amount of the thioester-linked palmitate and thioester-linked stearate. However, there was substantial variability (>100-fold at maximum) between subjects in the amount of thioester-linked oleate. We also demonstrated that incubation of platelets with exogenous fatty acids can alter the profile of fatty acids bound to platelet proteins by thioester linkages. Incubation of platelets with 100 µM palmitate for 3 h increased the amount of thioester-linked palmitate by up to 26%, and incubation of platelets with 100 µM stearate increased the amount of thioester-linked stearate up to 30%. In support of the observation that radiolabeled fatty acids other than palmitate were shown to be capable of binding to platelet proteins by thioester linkage, our results indicate that the fatty acids actually bound to unactivated platelet proteins include a significant amount of stearate, and variable amounts of oleate, as well as palmitate. In addition, the data show that palmitate and stearate can be increased, as a percentage of total protein-bound fatty acid, by incubation with exogenous palmitate and stearate, respectively.

Cleavage of factor XIII by human neutrophil elastase results in a novel active truncated form of factor XIII A subunit

The first step in the activation of plasma factor XIII (FXIII) is the cleavage of R37-G38 bond in FXIII-A subunit (FXIII-A) by thrombin, which makes the subsequent formation of an active transglutaminase possible. No active truncated form of FXIII-A, other than G38-FXIII-A, has been identified. In contrast to thrombin, which has a preference toward arginine residues, human neutrophil elastase (HNE) cleaves peptide bonds at small side-chain aliphatic amino acids, preferably at valine. As there are several valine residues close to the thrombin cleavage-site, we tested if an active truncated FXIII-A was formed during fragmentation of FXIII by HNE. It was demonstrated by Western blotting and transglutaminase assay that HNE induced a limited cleavage of FXIII-A resulting in the activation of both plasma and cellular FXIII; the maximal transglutaminase activities were 52.5% and 67.4% of thrombin-activated FXIII, respectively. After the relatively rapid activation a much slower inactivation occurred. HNE-activated FXIII cross-linked fibrin gamma- and alpha-chains in the clot formed by batroxobin moojeni. MALDI-TOF analysis of the cleaved fragments and N-terminal Edman degradation of the truncated protein identified V39-N40 as the primary cleavage-site and N40-FXIII-A as the active form. No primary cleavage occurred at V34, V35, V47, V50 residues. FXIII-A V34L polymorphism, which increases the rate of FXIII-A cleavage by thrombin, was without effect on FXIII activation by HNE. Molecular modeling located the primary HNE cleavage-site in the middle of the flexible and accessible Q32-L45 loop and showed that other neighboring valine residues were in less favorable position.

Molecular mechanism of the interaction between activated factor XIII and its glutamine donor peptide substrate

Summary.  Background: Activated factor XIII (FXIII), a dimer of truncated A‐subunits (FXIII‐A2*), is a transglutaminase that crosslinks primary amines to peptide‐bound glutamine residues. Because in the few natural substrates of FXIII‐A2* no consensus sequence could be identified around the reactive glutamine, studying the interaction between individual substrates and FXIII‐A2* is of primary importance. Most of the α2‐plasmin inhibitor (α2PI) molecules become truncated by a plasma protease, and the truncated isoform (N1‐α2PI) is an important substrate of FXIII‐A2*. The crosslinking of N1‐α2PI to fibrin plays a major role in protecting fibrin from fibrinolysis. Methods: We studied the interaction of FXIII‐A2* with its dodecapeptide glutamine donor substrate, N1‐α2PI(1–12), the sequence of which corresponds to the N‐terminal sequence of N1‐α2PI. Kinetic parameters for N1‐α2PI(1–12) and for its truncated or synthetic mutants were determined by a spectrophotometric assay. The interaction of N1‐α2PI(1–12) with FXIII‐A2* was investigated by proton nuclear magnetic resonance (NMR) and saturating transfer difference (STD) NMR. Results and Conclusions: Kinetic experiments with peptides in which the Asn1 residue was either truncated or replaced by alanine and proton NMR analysis of the FXIII‐A2*–N1‐α2PI(1–12) complex demonstrated that Asn1 is essential for effective enzyme–substrate interaction. Experiments with C‐terminally truncated peptides proved that amino acids 7–12 are essential for the interaction of N1‐α2PI(1–12) with the enzyme, and suggested the existence of a secondary binding site on FXIII‐A2*. Hydrophobic residues, particularly Leu10 and the C‐terminal Lys12, seemed to be especially important in this respect, and direct interaction between hydrophobic C‐terminal residues and FXIII‐A2* was demonstrated by STD NMR.

Factor XDebrecen: Gly204Arg mutation in factor X causes the synthesis of a non-secretable protein and severe factor X deficiency

Inherited factor X deficiency is a rare coagulopathy with severe bleeding symptoms in homozygous patients. Several mutations in the F10 gene have been described, the Gly204Arg mutation causes structural changes in the molecule and a secretion defect due to retention at the trans Golgi-late endosome level. Due to a homozygous Gly204Arg mutation in the factor X (FX) gene no detectable FX antigen was found in the plasma of a one-year old patient with severe bleeding diathesis. The amino acid replacement destabilized the disulfide bond that holds the two FX chains together, decreasing the interaction between the Cys201-Cys206 loop region and the region connecting the EGF2 and serine protease domains. Both Gly204 FX and Arg204 FX were synthesized in transfected cells, but only the wild type protein became secreted. The mutant protein was diverted from the normal secretory pathway and retained at the trans Golgi-late endosome level.

Molecular characterization of p.Asp77Gly and the novel p.Ala163Val and p.Ala163Glu mutations causing protein C deficiency

INTRODUCTION Protein C (PC) is a major anticoagulant and numerous distinct mutations in its coding gene result in quantitative or qualitative PC deficiency with high thrombosis risk. Homozygous or compound heterozygous PC deficiency usually leads to life-threatening thrombosis in neonates. PATIENTS AND METHODS The molecular consequences of 3 different missense mutations of two patients have been investigated. The first patient suffered from neonatal purpura fulminans and was a compound heterozygote for p.Asp77Gly and p.Ala163Glu mutations. The second patient had severe deep venous thrombosis in young adulthood and carried the p.Ala163Val mutation. The fate of mutant proteins expressed in HEK cells was monitored by ELISA, by Western blotting, by investigation of polyubiquitination and by functional assays. Their intracellular localization was examined by immunostaining and confocal laser scanning microscopy. Molecular modeling and dynamics simulations were also carried out. RESULTS AND CONCLUSIONS The 163Val and 163Glu mutants had undetectable levels in the culture media, showed intracellular co-localization with the 26S proteasome and were polyubiquitinated. The 77Gly mutant was secreted to the media showing similar activity as the wild type. There was no difference among intracellular PC levels of wild type and mutant proteins. The 163Val and 163Glu mutations caused significant changes in the relative positions of the EGF2 domains suggesting misfolding with the consequence of secretion defect. No major structural alteration was observed in case of 77Gly mutant; it might influence the stability of protein complexes in which PC participates and may have an impact on the clearance of PC requiring further research.

Thrombomodulin-dependent effect of factor V Leiden mutation on the cross-linking of α2-plasmin inhibitor to fibrin and its consequences on fibrinolysis

INTRODUCTION It has been shown that thrombomodulin (TM) considerably delays factor XIII (FXIII) activation and this effect is abrogated by Factor V Leiden (FV(Leiden)) mutation. The aim of the study was to explore the effect of TM on the cross-linking of α(2)-plasmin inhibitor (α(2)-PI) to fibrin in plasma samples of different FV genotypes and how this effect is related to the impaired fibrinolysis of FV(Leiden) carriers. METHODS In the plasma samples of fifteen individuals with different FV genotypes and in FV deficient plasma supplemented with wild type FV or FV(Leiden) coagulation was initiated by recombinant human tissue factor and phospholipids with or without recombinant human TM (rhTM). In the recovered clots the extent of α(2)-PI-fibrin cross-linking was evaluated by Western blotting and quantitative densitometry. The effect of rhTM on tissue plasminogen activator (tPA) induced clot lysis was measured by turbidimetric method. RESULTS rhTM significantly delayed the formation of α(2)-PI-fibrin α-chain heterodimers/oligomers in plasma samples containing wild type FV. This effect of rhTM was impaired in the presence of FV(Leiden). rhTM delayed tPA-induced clot lysis and this effect of rhTM was more pronounced in plasma containing FV(Leiden). When TAFIa was inhibited by potato carboxypeptidase inhibitor, rhTM accelerated clot lysis in the presence of wild type FV, which is explained by the delayed α(2)-PI-fibrin cross-linking. This effect of rhTM did not prevail in the presence of FV(Leiden). CONCLUSION FV(Leiden) abrogates the delaying effect of rhTM on α(2)-PI-fibrin cross-linking, which contributes to the impaired fibrinolysis observed in FV(Leiden) carriers.

Variant type Glanzmann thrombasthenia caused by homozygous p.724R>X mutation in β3 integrin

a Clinical Research Center, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary b Thrombosis, Haemostasis and Vascular Biology Research Group of the Hungarian Academy of Sciences, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary c Pal Heim Childrens Hospital, Budapest, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary d Department of Obstetrics and Gynecology, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary e Clinical Biochemistry and Molecular Pathology Department, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary