Alpha-Chain cross-linking in fibrin(ogen) Marburg

The fibrinogen structural variant, Marburg (A alpha 1-460B beta gamma)2, is comprised of normal B beta and gamma chains but contains severely truncated A alpha chains that are missing approximately one half of their factor XIIIa cross-linking domain. Immunochemical studies of fibrin(ogen) Marburg were conducted to characterize the degree to which deletion of a defined A alpha-chain segment, A alpha 461-610, can affect the process of fibrin stabilization, ie, the factor XIIIa- mediated covalent interaction that occurs between alpha chains of neighboring fibrin molecules and between alpha chains and alpha 2 antiplasmin (alpha 2PI). The ability of Marburg (and control) alpha chains to serve as a substrate for factor XIIIa and undergo cross- linking was examined in an in vitro plasma clotting system. The capacity for alpha-chain cross-linking was evaluated both as the covalent incorporation of the small synthetic peptide, NQEQVSPLTLLK (which represents the first 12 amino acids of alpha 2PI and includes the factor XIIIa-sensitive glutamine residue responsible for the cross- linking of alpha 2PI to fibrin), and as the appearance of native (ie, natural), high-molecular-weight, cross-linked alpha-chain species. Antibodies specific for the (A)alpha and gamma/gamma-gamma chains of fibrin(ogen) and for the peptide and its parent protein, alpha 2PI (68 kD), were used as immunoblotting probes to visualize the various cross- linked products formed during in vitro clotting. Recalcification of Marburg plasma in the presence of increasing concentrations of peptide resulted in the formation of peptide-decorated Marburg alpha-chain monomers. Their size at the highest peptide concentration examined indicated the incorporation of a maximum of 3 to 4 mol of peptide per mole of alpha-chain. In the absence of alpha 2PI 1-12 peptide, the alpha chains of Marburg fibrin cross-linked to form oligomers and polymers, as well as heterodimers that included alpha 2PI. Both the peptide-decorated monomers and the native cross-linked alpha-chain species of Marburg fibrin were smaller than their control plasma counterparts, consistent with the truncated structure of the parent Marburg A alpha chain. Collectively, the findings indicate that, although deletion of the A alpha chain region no. 461-610 in fibrinogen Marburg prevents formation of an extensive alpha polymer network (presumably due to the absence of critical COOH-terminal lysine residues), it does not interfere with initial events in the fibrin stabilization process, namely, factor XIII binding and the ability of alpha chains to undergo limited cross-linking to one another and to alpha 2PI.     

Severe hypodysfibrinogenemia in compound heterozygotes of the fibrinogen AalphaIVS4 + 1G>T mutation and an AalphaGln328 truncation (fibrinogen Keokuk)

Two siblings with hypofibrinogenemia have lifelong trauma-related bleeding. Recently, the brother experienced recurrent thrombosis after cryoprecipitate infusions following surgery. The sister had 6 miscarriages. Plasma clots in each were resistant to compression and fibrinolysis and were soluble in 5 M urea. Examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed only the presence of crosslinked gamma-gamma fibrin chain dimers without high polymers of alpha n. Fibrin clots contained an abnormal 35-kDa constituent recognized by an antibody to the mature fibrinogen Aalpha-chain residues 241-476 but not by antibodies to Aalpha219-348 or Aalpha349-406. DNA analysis revealed a heterozygous CAA-->TAA mutation at the codon for amino acid 328 of the Aalpha gene in these siblings and 2 asymptomatic family members. The Gln328stop mutation (fibrinogen Keokuk) predicted a 46% truncation and the production of a 35-kDa Aalpha chain. Analysis of purified fibrinogen revealed expression of the abnormal Aalpha chain in 4 family members but found no normal fibrinogen in the 2 hypofibrinogenemic patients. This paradox was resolved when they and their asymptomatic mother were found to be heterozygous for a second Aalpha mutation, a GT-->TT splice site mutation in intron 4 (IVS4 + 1 G> T). However, compound heterozygosity for both mutations was required for the expression of severe hypodysfibrinogenemia and for clinical symptoms.     

A bleeding disorder due to deficiency of alpha 2-antiplasmin

A deficiency of alpha 2-antiplasmin has been identified in a female patient with severe and frequent bleeding episodes. Routine coagulation and platelet assays of the patient's plasma were within normal limits. However, abnormally rapid whole blood or dilute plasma clot lysis times and an abnormal FXIII test in which clots were lysed in the presence of urea or saline suggested an abnormal fibrinolytic system. Analysis of alpha 2-antiplasmin levels by radioimmunoassay revealed less than 1.0 microgram/ml alpha 2-antiplasmin. Functional assays indicated an alpha 2-antiplasmin level less than or equal to 10% of normal. Addition of purified alpha 2-antiplasmin to the patient's plasma restored its ability to inhibit plasmin in in vitro assays, and mixtures of patient plasma with normal plasma did not interfere with the antiplasmin activity of the normal plasma. Whereas normal platelets contain 68 ng alpha 2-antiplasmin/10(9) platelets, platelets from the patient contained 30% of the normal level of antigen. Analysis of alpha 2- antiplasmin functional and antigenic levels in the plasma of both parents and four siblings of the propositus provided evidence consistent with an autosomal mechanism of inheritance of alpha 2- antiplasmin deficiency. One sibling appeared to be homozygous and three siblings and the parents were heterozygous for the deficiency. Two heterozygotes had positive bleeding histories. The association of a bleeding disorder with a deficiency of alpha 2-antiplasmin emphasizes that lack of regulation of the fibrinolytic system can result in a hemostatic dysfunction.     

Fibrinogen Kanazawa: a congenital dysfibrinogenaemia with delayed polymerization having a replacement of proline-18 by leucine in the A alpha-chain

Congenital dysfibrinogenaemia was found in a 39-year-old female and her two children. The proposita, apparently heterozygous for this abnormality, had no episode of bleeding or thrombosis. The abnormal fibrinogen showed normal release of fibrinopeptides A and B but impaired polymerization of the fibrin monomer. Amino acid sequence analysis of the whole A alpha-chain isolated from fibrinogen Kanazawa showed a substitution of Leu for Pro at position 18 in the A alpha-chain. This substitution was corroborated by the analysis of the amino acid sequence which demonstrated the lysyl endopeptidase peptides derived from the A alpha-chain of fibrinogen Kanazawa. The minimal genetic exchange responsible for this substitution was a C----T transition in the middle position of the Pro codon. We conclude that Pro-18 in the A alpha-chain is crucial for the polymerization of the fibrin monomer.     

Fibrinogen Kyoto II, a new congenitally abnormal molecule, characterized by the replacement of A alpha proline-18 by leucine

A new case of heterozygous dysfibrinogenemia characterized by an amino acid replacement in the NH2-terminal region of the fibrin alpha-chain was found in a 27-year-old woman with a bleeding problem. Her one-stage prothrombin time and activated partial thromboplastin time were slightly prolonged, and the purified fibrinogen from this patient had a markedly prolonged thrombin or reptilase time. Release of fibrinopeptides A and B was normal, but the polymerization of fibrin monomers was impaired. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fibrinogen under the reduced condition showed no abnormalities in the apparent molecular weights of its three chains. Reverse-phase high performance liquid chromatography (HPLC) of the lysylendopeptidase-cleaved purified A alpha-chains showed a decrease in one peptide compared with the normal amount and the appearance of an abnormal peptide peak. These peptides were treated with thrombin and further separated on HPLC. Amino acid sequence analysis of the abnormal peptide indicated that A alpha proline-18, the second residue from the NH2-terminus of the fibrin alpha-chain, was replaced by leucine. The synthetic peptide Gly-Pro-Arg-Pro inhibited both thrombin- and reptilase-induced fibrin aggregation, but Gly-Leu-Arg-Pro showed little or no inhibition under the same conditions. The discovery of this abnormal fibrinogen supports the findings that A alpha proline-18 is important as part of the polymerization site in the NH2-terminus of the fibrin alpha-chain. The propositus' mother had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Kyoto II.     

Fibrinogen Milano. VI: A heterozygous dysfibrinogenemia (A alpha 16 Arg----His) with bleeding tendency

Congenital heterozygous dysfibrinogenemia was diagnosed in a young woman with bleeding tendency. 3 other asymptomatic members of her family (mother and the 2 sisters) had abnormal fibrinogen. The proposita's plasma exhibited prolonged thrombin and reptilase times. Plasma fibrinogen concentration determined by functional assay was 0.3 g/l, whereas immunologic assay revealed normal fibrinogen levels. Turbidity curves, representing the rate of thrombin-induced fibrin formation, were markedly delayed both in the presence and absence of Ca2+. Isoelectric focusing and SDS electrophoresis of reduced fibrinogen showed normal charge and size of the subunit chains. Release of fibrinopeptide B by thrombin was normal, whereas HPLC elution diagrams of fibrinopeptide A showed an abnormal peak A* with a slightly shorter retention time than the normal fibrinopeptide A. The amino acid analysis showed that the arginine in peak A* is replaced by histidine (A alpha 16 Arg----His).     

Fibrinogen Matsumoto II: γ308Asn → Lys (AAT → AAG) mutation associated with bleeding tendency

Fibrinogen Matsumoto II is a hereditary dysfibrinogenaemia identified in a woman with Basedow's disease and a bleeding tendency. Coagulation tests of the patient's plasma revealed a prolonged thrombin time and a decreased fibrinogen level determined by functional method. Release of fibrinopeptide A and B was normal, whereas fibrin monomer polymerization was delayed. Fibrinogen γ-chain gene of the propositus was heterozygous for a missense mutation that resulted in Asn → Lys substitution at codon 308. Though the same amino acid substitution was also attributed to fibrinogen Kyoto I and Bicetre II, fibrinogen Matsumoto II showed different clinical manifestations from them.     

Fibrinogen Seoul (FGG Ala341Asp): a novel mutation associated with hypodysfibrinogenemia.

Dysfibrinogenemia is a coagulation disorder caused by a variety of structural abnormalities in the fibrinogen molecule that result in fibrinogen function. The molecular basis of hypodysfibrinogenemia was investigated in a 66-year-old woman with peripheral artery obstructive disease and in her family members. Plasma level of functional fibrinogen determined using the Clauss method was lower (75 mg/dL; normal, 140-460 mg/dL) than that measured with immunologic nephelometric assay (137 mg/dL; normal, 180-400 mg/dL). Similar results were also observed in two family members through two generations. DNA was extracted from whole blood, and the coding regions and intron/exon boundaries of gamma chain gene (FGG) were amplified. A novel (Fibrinogen Seoul) heterozygous FGG mutation (GCT->GAT, Ala341Asp) was identified in all three affected family members. Thrombin-catalyzed polymerization was found to be defective on the analysis of purified fibinogen from the propositus. Molecular modeling also showed a conformational change of fibrinogen structure.     

Congenital hypodysfibrinogenemia associated with a novel deletion of three residues (γAla289_Asp291del) in fibrinogen

Hypodysfibrinogenemia is the least frequently reported congenital fibrinogen disorder, characterized by both quantity and quality defects of fibrinogen. In this study, we investigated the molecular basis of hypodysfibrinogenemia in a Chinese family. Functional fibrinogen was measured by Clauss method, and the antigenic fibrinogen was measured by immunoturbidimetry assay. All the exons and exon–intron boundaries of fibrinogen genes (FGA, FGB and FGG) were analysed by direct DNA sequencing. To further evaluate its molecular and functional characterizations, fibrinogen was purified from the plasma of propositus, then SDS-PAGE, fibrin polymerization, clot lysis, and electron microscopy scanning were all performed. The propositus showed a slight decrease of immunologic fibrinogen (1.52 g/L) but dramatically reduced functional fibrinogen (0.3 g/L). DNA sequencing revealed a novel heterozygous CCTTTGATG deletion in the exon 8 of FGG, leading to the deletion of Ala289, Phe290, and Asp291 in fibrinogen γ-chain. The polymerization of the fibrinogen from the propositus was markedly impaired, with prolonged lag period and decreased final turbidity. The fibrinogen clottability showed a reduced fraction of participating clot formation. While the clot lysis showed normal. Scanning electron microscopy revealed that the fibers of the propositus were thicker than normal, with larger pores and curlier meshworks. We conclude that γAla289_Asp291del is responsible for the hypodysfibrinogenemia in this case.     

A novel nonsense mutation in the FGA gene in a Chinese family with congenital afibrinogenaemia.

Congenital afibrinogenaemia is a rare autosomal recessive coagulation disorder. Here we describe the genetic defect in the fibrinogen A alpha-chain underlying afibrinogenaemia in a Chinese family. The proposita had a life-long bleeding tendency, both her parents and paternal grandparents had a consanguineous marriage. The blood-clotting indices of the proposita and her father were prolonged, and their functional and immunologic fibrinogen was absent. To identify the mutations of fibrinogen genes in this family, all the exons and exon-intron boundaries of the three fibrinogen genes (FGA, FGB, FGG) were amplified by polymerase chain reaction, and direct sequencing of polymerase chain reaction products was performed, then the restriction endonuclease (RsaI) analysis was used to confirm the mutation. A homozygous C --> T mutation was found at nucleotide 3108 in exon 4 of the FGA gene of the proposita and her father; it is a null mutation predicting to produce severely truncated A alpha-chains because of the presence of premature termination at the Gln 150 codon (or truncated at the 131 residues according to the mature A alpha-chain). Her mother and some other family members were heterozygous. The g.3108C --> T (Gln150 --> stop) nonsense mutation in the FGA gene is a novel genetic defect of congenital afibrinogenaemia that, to our knowledge, has not been described previously.     

Afibrinogenemia resulting from homozygous nonsense mutation in A alpha chain gene associated with multiple thrombotic episodes.

Congenital afibrinogenemia is a rare disorder characterized by the absence in circulating fibrinogen, a hexamer composed of two sets of three polypeptides (Aalpha, Bbeta and gamma). Although predisposition to thrombosis is a well known feature of dysfibrinogenemia, the relatively frequent thrombotic manifestations seen in congenital afibrinogenemia are puzzling. We herein report a mutational analysis of a young afibrinogenemic man from Turkey with multiple thrombo-embolic events involving both arteries and veins. Purified DNAs of the propositus was used for amplification by polymerase chain reaction of all the exons of the A subunit gene with primers allowing the analysis of the intron-exon boundaries. Analysis of the genes coding for the three fibrinogen chains of the propositus found a homozygous G to A transition in the exon 5 of the A alpha chain gene (g.g4277a; access number gi458553). The TGG to TGA codon change predicts a homozygous W315X in the A alpha chain (p.W334X when referring to the translation initiation codon). Both parents and his brother were found to carry this heterozygous mutation. This is the first report of a patient homozygous for this rare mutation associated with afibrinogenemia. Our patient was free of known risk factors as well as diseases associated with thrombosis including atherosclerosis, vasculitis, Buerger's disease, and it seems therefore probable that afibrinogenemia itself might have contributed to both arterial and venous thrombosis.     

Fibrinogen Kaiserslautern (γ 380 Lys to Asn): a new glycosylated fibrinogen variant with delayed polymerization

An adult woman diagnosed with cerebral thrombosis following a caesarean section was found to have severely prolonged thrombin and reptilase times. Five other family members also had prolonged, but variable, thrombin and reptilase times. Analysis of purified fibrinogen on reducing SDS-PAGE revealed an additional band, in all family members, which migrated immediately below the normal Bβ band. Western blotting indicated that this band was a gamma chain and endoglycosidase-F digestion established that it contained an additional oligosaccharide side chain. Partial acid hydrolysis localized the new oligosaccharide to the C-terminus of the gamma chain. Amplification of this region by PCR and subsequent DNA sequencing demonstrated a single base substitution altering the normal 380 Lys (AAG) codon to Asn (AAT), producing a new Asn-Lys-Thr glycosylation site. The propositus and one other family member were homozygous for this mutation but the remaining four family members were heterozygous. The polymerization of purified fibrin monomers from the propositus was grossly abnormal; however, the polymerization curve was almost normalized by the removal of terminal sialic acid residues. This suggests that the polymerization defect was primarily caused by additional negatively charged sialic acid residues present on the new oligosaccharide. Further analysis of the D domain of purified fibrinogen established that calcium binding to the high affinity site remained unaffected by the bulky carbohydrate side chain or negatively charged sialic acid residues.     

Fibrinogen Geneva, a new case of A alpha 16 Arg----Cys dysfibrinogenaemia

A new congenital variant of fibrinogen, from which only half the normal amount of fibrinopeptide A can be released by thrombin, was found in three members of a family having no major bleeding or thrombotic tendency. Following carboxamidomethylation of the reduced fibrinogen chains, an abnormal peptide was cleaved by thrombin from the amino terminus of the A alpha-chain (A* alpha 1-19) and isolated by reversed phase high-performance liquid chromatography. Amino acid analysis indicated the presence of carboxymethyl cysteine in this A alpha-chain fragment which in normal fibrinogen is devoid of cysteine. We conclude that fibrinogen Geneva is another fibrinogen variant with the substitution A alpha 16 Arg----Cys.     

Direct analysis of plasma fibrinogen-derived fibrinopeptides by high-performance liquid chromatography: investigation of nine congenital fibrinogen abnormalities

A simple method has been developed for the rapid analysis of fibrinopeptides contained on fibrinogen in small anticoagulated plasma samples. Following incubation with thrombin the plasma is diluted, boiled and then studied by high performance liquid chromatography (HPLC). The three forms of FPA (AP, A, AY) and two forms of FPB (B, des Arg B) can be identified and quantified in samples of less than 200 microliters. Additionally, the FPB peak height can be used to measure the plasma fibrinogen level. This method has been used to screen plasma samples with abnormal clotting times for possible congenital fibrinogen abnormalities. Results of the study of nine unrelated cases are presented. Four cases of congenital dysfibrinogenaemia were diagnosed directly from HPLC analysis alone. Fibrinogen Sheffield and Paris VI were identified as A alpha Arg 16----His substitutions and fibrinogens London VI and Madrid II were found to be heterozygous for an unknown substitution preventing thrombin cleavage at A alpha Arg 16. A case of dysfibrinogenaemia (fibrinogen Ashford) with a normal fibrinopeptide release stoichiometry was confirmed to have a primary polymerization abnormality using purified fibrin monomers. Similarly, a case of hypodysfibrinogenaemia (fibrinogen London V) had normal fibrinopeptides and a fibrin polymerization abnormality. In one case of hypofibrinogenaemia and two cases of afibrinogenaemia, no fibrinopeptide or functional abnormalities could be definitely established. This rapid and simple method of fibrinopeptide analysis is recommended for screening of plasma samples taken from patients suspected of having abnormalities of fibrinogen synthesis.     

Fibrinogen Ledyard (A alpha Arg16----Cys): biochemical and physiologic characterization

Fibrinogen Ledyard was discovered in a 10-year-old boy with a mild bleeding history. His father had the same defect and a bleeding history after surgery. Both patients were heterozygous. The plasma fibrinogen concentration was normal immunologically (335 mg/dL) and very low functionally (52 mg/dL). Purified fibrinogen Ledyard had a prolonged polymerization, which was somewhat corrected by addition of Ca2+ ions. High performance liquid chromatography (HPLC) analyses of the fibrinopeptides released by thrombin showed 1 mol of fibrinopeptide A (FPA) and 2 mol of fibrinopeptide B (FPB) released per mole of fibrinogen Ledyard. Steady-state kinetic parameters were evaluated for release of FPA by thrombin. When the concentration of fibrinogen Ledyard was corrected to 50% of total protein, because only 50% of fibrinogen Ledyard can release FPA, the kinetic constants were similar to those of control fibrinogen (Km = 7.5 mumol/L for A alpha chain, kcat = 54 s-1). This finding indicates that the cleavage site of the A alpha chain in these abnormal molecules may not interact with the catalytic site of thrombin. The three chains of fibrinogen Ledyard were isolated on reverse-phase C4-HPLC. The sequence of the amino terminus of A alpha chain showed that Arg in position 16 was replaced by Cys in the abnormal molecules. Approximately half of fibrinogen Ledyard (52%) was clotted by reptilase, suggesting that fibrinogen Ledyard may consist of 50% normal homodimers (A alpha Arg16 . A alpha Arg16) and 50% abnormal homodimers (A alpha Cys16 . A alpha Cys16). Abnormal molecules could form disulfide bond between the A alpha Cys16 residues. Thus, the abnormal molecules have a different structure that does not bind to thrombin. Probably the abnormality of polymerization of fibrinogen Ledyard results from the interaction of the abnormal molecules with normal fibrin monomers, so that the growth of fibrin protofibrils is inhibited. This abnormal fibrinogen supports adenosine diphosphate-induced platelet aggregation in a normal manner.     

Biophysical characterization of fibrinogen Caracas I with an Aalpha-chain truncation at Aalpha-466 Ser: identification of the mutation and biophysical characterization of properties of clots from plasma and purified fibrinogen.

Fibrinogen Caracas I is a dysfibrinogenemia with a mild bleeding tendency; a novel nonsense mutation, in the gene coding the Aalpha-chain, identified in this study as G4731T, giving rise to a new stop codon at Aalpha-Glu 467. Fibrinogen from two family members, the mother and sister of the propositus, both heterozygous for the mutation were studied, analyzing clots made from both plasma and purified fibrinogen. Clot structure and properties were characterized by turbidity, permeation, scanning electron microscopy and rheological studies. Permeation through Caracas I plasma clots was decreased, consistent with the decreased final turbidity. As shown by scanning electron microscopy, plasma clots from the patients were composed of very thin fibers, with increased fibrin density and reduced pore size. Viscoelastic measurements revealed that fibrinogen Caracas I plasma clots were much stiffer and less subject to compaction. These results demonstrate a key role of the carboxyl-terminal alpha chains of fibrin in lateral aggregation during polymerization and reinforce the utility of studying plasma clots. It is important to point out that the biophysical studies with fibrinogen purified by two different methods yielded contradictory results, which can be accounted for by selective purification of certain molecular species as seen by two-dimensional electrophoresis.     

The C–T substitution in the distal CACCC box of the β-globin gene promoter is a common cause of silent β thalassaemia in the Italian population

This paper describes four families of Italian descent in each of which the propositus had the clinical phenotype of thalassaemia intermedia, resulting from the compound heterozygous state for high HbA2 beta thalassaemia and type I silent beta thalassaemia. Direct sequencing on amplified DNA and/or oligonucleotide analysis detected, in all families but one, the compound heterozygous state for codon 39 nonsense mutation and the C-T substitution at position -101 in the distal CACCC box of the beta-globin gene promoter (beta th-101). Members of these families who are heterozygous for high HbA2 beta thalassaemia showed the codon 39 nonsense mutation, while those with the clinical phenotype of silent beta thalassaemia had the beta th-101 mutation. In the remaining family, the propositus and one of his siblings had the compound heterozygous state for a molecularly undefined high HbA2 beta thalassaemia and the beta th-101 mutation in combination with the triple alpha globin gene arrangement. These patients showed a more severe thalassaemia intermedia like clinical phenotype as compared to those with the same beta-globin genotype and a normal alpha-globin gene arrangement. In the families investigated the beta th-101 was always associated with haplotype I. A group of patients with thalassaemia intermedia from Southern Italy, either homozygous or heterozygous for haplotype I and in whom previous studies had failed to define the mutation in one of the beta thalassaemia globin genes, were screened by oligonucleotide analysis for the presence of the beta th-101. Three out of nine were positive. These results indicate that the beta th-101 mutation is a common cause of the type I silent beta thalassaemia phenotype in the Southern Italian population.     

Prothrombin Habana: a new dysfunctional molecule of human prothrombin associated with a true prothrombin deficiency

A Cuban family with a new congenital dysprothrombinaemia is described. The propositus was a 5-year-old female who presented with umbilical bleeding after birth followed by easy bruising and bleeding tendency throughout her life. The main laboratory features of the defect included prolongation of prothrombin time and partial thromboplastin time. Prothrombin activity was less than 10% in several one- and two-stage systems. However, the staphylocoagulase-complexed prothrombin level and immunologic methods yielded levels of about 50%. The migration of the abnormal prothrombin was more anodic in single and bidimensional immunoelectrophoresis system and did not change by the addition of calcium. Family studies revealed that the father had approximately 50% prothrombin activity and antigen, whereas the mother had 45% prothrombin activity but about 100% prothrombin antigen. We suggest that the propositus is heterozygous for an abnormal prothrombin and heterozygous for true prothrombin deficiency.     

Occurrence of the αI 22 Arg→His (CGT→CAT) spectrin mutation in Tunisia: potential association with severe elliptopoikilocytosis

A category of spectrin alpha I domain variants are manifested by the increase of the alpha I 74 kDa fragment at the expense of the parent 80 kDa fragment following partial tryptic digestion. We describe a particular case of alpha I/74 abnormality in a Tunisian family. The propositus was severely ill and had an elliptopoikilocytosis. To the contrary, his father, who carried the same alpha I/74 variant, displayed no clinical signs and a few elliptocytes. The increase of the alpha I 74 kDa fragment was more pronounced in the propositus than in his father. Unexpectedly, the spectrin content was reduced to similar (and limited) extents in both of them, and the father displayed nearly as pronounced an increase of the spectrin dimer percentage as the propositus following low ionic strength extraction. In vitro spectrin dimer reconstitution experiments indicated that the primary mutation was located in the alpha-chain itself (not in the beta-chain as is the case in some alpha I/74 mutants). Following polymerase chain reaction (PCR) amplification, cloning and sequencing of exon 2 of spectrin alpha-gene in the father, we found the G----A substitution at position 2 of codon 22 (CGT----CAT; Arg----His). This mutation has been recently discovered in a family of French descent. Dot blot hybridization confirmed that the substitution was transmitted with the alpha I/74 abnormality. As previously shown, the enhancement of its expression level in the propositus, with respect to the father, was accounted for by the presence of a factor carried by the alpha-spectrin allele in trans and transmitted by the mother.     

Early cross-linked fibrin in human plasma contains alpha-polymers with intact fibrinopeptide A.

alpha-polymer formation, as opposed to gamma-chain dimerization has been considered a relatively late event in factor XIII-induced fibrin stabilization. Recently it has been shown, however, that plasma from healthy individuals and from patients with fibrinaemia contains small amounts of soluble fibrin/fibrinogen oligomers interlinked through dimerized gamma-chains as well as cross-linked alpha-chains. The present work was carried out to see if these early alpha-chain polymers also arise during coagulation of plasma in vitro. Plasma samples from healthy individuals, prepared by immediate centrifugation of blood collected without anticoagulant, were allowed to clot spontaneously for varying periods. The plasma clots were solubilized in SDS-urea-mercaptoethanol and samples were subjected to SDS-PAGE and Western blotting using polyclonal antibodies to human fibrinogen, or monoclonal antibodies specific either for A alpha/alpha-chains, for fibrinopeptide A-containing chains, for the N-terminus of the fibrin beta-chain or for the gamma-chains. Fibrin/fibrinogen oligomers were seen to form long before visible gelation of plasma. These oligomers were cross-linked through gamma-chain dimerization, but also through A alpha- or alpha-chain polymerization. The number and amount of alpha-polymers containing A alpha-chains increased immediately after clot formation, but these disappeared about 20 min later, due to complete removal of fibrinopeptide A (FPA) by thrombin. It is concluded that alpha-polymer formation is a very early event during plasma coagulation in vitro, and that both A alpha- and alpha-chains are involved.