Fibrinogen Barcelona I. Congenital dysfibrinogenemia characterized by defective release of fibrinopeptide A and fibrinogen degradation products

A congenital dysfibrinogenemia, fibrinogen Barcelona I, was detected in a 28 year-old woman with no prior history of bleeding. The thrombin induced clotting of plasma and purified fibrinogen was much prolonged. Fibrin monomer aggregation was impaired. The abnormal fibrinogen polymerized in the presence of calcium and can be further cross-linked by factor XIIIa. The turbidity of fibrin gels obtained from fibrinogen Barcelona was much lower than normal fibrinogen. The kinetic constant Km for fibrinogen Barcelona plus normal fibrinogen gelation was similar to normal fibrinogen gelation. The release rate of fibrinopeptide A by thrombin was slower than that of normal fibrinogen. However, two mol of fibrinopeptide A was released per mol of fibrinogen in 30 min. SDS-PAGE of abnormal and normal fibrinogens and of reduced fibrinogens showed identical patterns. Sialic acid content was markedly decreased in fibrinogen Barcelona. Plasmin digestion of two fibrinogens showed identical patterns in SDS-PAGE as regards X fragment formation. The kinetics of fibrinogen degradation showed a decrease in the formation rate of D and E fragments. The fact that the patient was in threat of abortion and developing a haemorrhagic syndrome may indicate that the defect in the fibrinogen was important in the pathogenesis of haemorrhage in this patient.     

Fibrinogen Kawaguchi: an abnormal fibrinogen characterized by defective release of fibrinopeptide A

A congenital dysfibrinogenemia was found in a 32-year-old asymptomatic female and her immediate family. The propositus, apparently a heterozygote for the abnormality, characteristically showed defective release of fibrinopeptide A from half of her fibrinogen molecules. No fibrinopeptide A was cleaved off from the isolated abnormal molecule by thrombin or snake venoms (Reptilase and Ancrod) as evidenced by radioimmunoassay, high performance liquid chromatography and determination of the NH2-terminal amino acids. The abnormal fibrinogen formed a solid gel solely by the release of fibrinopeptide B upon incubation with thrombin. We provisionally designate this abnormal fibrinogen as "Fibrinogen Kawaguchi", although possible identity with other abnormal fibrinogens is not excluded.     

Fibrinogen petoskey: Identification of a new dysfibrinogenemia characterized by altered release of fibrinopeptide A

A new congenital dysfibrinogenemia, designated fibrinogen Petoskey, which was traced through four generations of a Michigan family, was found to exhibit an abnormally slow rate of release of fibrino-peptide A upon treatment with thrombin and batroxobin. Batroxobin only partially hydrolyzed and polymerized the fibrinogen from affected individuals, suggesting that these patients had both normal and abnormal fibrinogen in their circulation and that batroxobin was not capable of releasing fibrinopeptide A from the abnormal fibrinogen. Polymerization of fibrin monomers from fibrinogen Petoskey and plasmin mediated digestion of fibrinogen Petoskey were normal. The Factor XIIIa-catalyzed cross-linking of fibrinogen Petoskey was slightly delayed at low (but not at high) concentrations of thrombin. This delayed cross-linking appeared to be a secondary effect of the lower rate of release of fibrinopeptide A.     

Specificity of antisera to human fibrinopeptide A used in clinical fibrinopeptide A assays.

Distinction between fibrinopeptide A (FPA) and larger polypeptides containing the FPA sequence is critical for the interpretation of clinical results with FPA immunoassay methods. Therefore, the immunochemical reactivity of 14 rabbit anti-FPA sera with six different FPA containing antigens was studied in detail. Antigens tested included: fibrinogen; fragment E of fibrinogen; the amino-terminal disulfide knot of fibrinogen; Aalpha 1(Ala)-51(Met); Aalpha 1(Ala)-23(Arg); and, FPA. Synthetic partial sequences of FPA were also tested. The 14 FPA-specific antisera were divided into 3 distinct categories with: I, FPA immunoreactivity of larger polypeptides containing FPA approximately 1/100 of FPA on a molar basis, II, FPA immunoreactivity of the larger polypeptides intermediate between I and III; and III, FPA immunoreactivity of the larger polypeptides approximately equal to that of FPA on a molar basis. The antigenic determinants of a category I antiserum (R 2) are included in Aalpha 7(Asp)-16(Arg) with Asp(7), Phe(8) and Arg(16) being essential. When attached to FPA, the sequence Gly(17)-Arg(23) decreases the immunoreactivity of FPA with category I antisera 100-fold. The practical consequence of these findings is that, when category III antisera are employed, both FPA and larger FPA-containing polypeptides are equally immunoreactive. Since thrombin treatment of the larger polypeptides does not alter their immunoreactivity, category III antisera cannot discriminate between FPA and the larger polypeptides. On the other hand, with category I antisera, although the immunoreactivity of FPA itself is unaltered by thrombin treatment, larger polypeptides [e.g., Aalpha 1(Ala)-23tArg)] show a 100-fold increase in immunoreactivity following thrombin treatment and thus can readily be identified and separately quantitated. It is concluded that antisera with the specificity of category I are essential for the specific and accurate measurement of FPA, and for its distinction from larger FPA-containing polypeptides, in clinical plasma samples.     

Fibrinogen Magdeburg I: a novel variant of human fibrinogen with an amino acid exchange in the fibrinopeptide A (Aalpha 9, Leu-->Pro)

INTRODUCTION: The exchange of Aalpha 16, Arg for Cys or His is the most common molecular defect in dysfibrinogenemia directly affecting the thrombin cleavage site involved in fibrinopeptide A (FPA) release. Other amino acid exchanges within the fibrinopeptide A have been only rarely reported. MATERIALS AND METHODS: In clinically asymptomatic dysfibrinogenemic patients with low functional plasma fibrinogen (Fg) levels and prolonged thrombin time but normal or slightly prolonged batroxobin (reptilase) time, mutation analysis was carried out by direct sequencing of the coding regions of the three fibrinogen genes. Isolated fibrinogen was functionally characterized for thrombin- or batroxobin-induced fibrinopeptide release and fibrin formation. Fibrinogen and fibrinopeptides were structurally studied by electrophoretic techniques or high-performance liquid chromatography. RESULTS AND CONCLUSIONS: Molecular analysis revealed heterozygosity for a novel missense mutation T1182C in the FGA gene causing the amino acid exchange Aalpha 9, Leu-->Pro. Fibrin generation induced by thrombin was moderately impaired, whereas batroxobin-induced fibrin formation was almost normal. Release of the abnormal fibrinopeptide A by thrombin was delayed but fibrin monomer aggregation was almost normal. Cleavage of Aalpha chains by batroxobin was only slightly delayed. Fibrinopeptides A of the patient fibrinogen did not show any gross abnormality in chromatographic behaviour. This new molecular variant designated fibrinogen Magdeburg I supports the view that amino acid residue Leu-9 in the Aalpha chain as part of a small hydrophobic cluster is involved in the interaction with an apolar binding site of thrombin, thus adding to our understanding of the thrombin-fibrinogen interaction crucial in coagulation.     

Fibrin polymerization and release of fibrinopeptide B by thrombin

Although it is well known that thrombin releases fibrinopeptide A (FPA) more rapidly than fibrinopeptide (FPB) from fibrinogen different opinions have been expressed as to whether fibrin I (FPA release) and II (FPB release) are sequential or simultaneous. Evidence has been presented that FPB release depends on the polymerization of fibrin and the finding that the tetrapeptide gly-pro-arg-pro inhibits fibrin formation provides a new tool to investigate the effect of polymerization. The radioimmunoassay technique which permits the initial rates of the release reactions to be accurately measured at low concentrations of reactants was used to investigate FPB release. In the absence of gly-pro-arg-pro the FPB release pattern showed three phases - an initial slow and a second more rapid phase both of which were at a constant rate and a final phase with decreasing rate as the substrate concentration fell. Gly-pro-arg-pro at appropriate concentrations inhibited fibrin polymerization as indicated by optical density and light scattering techniques as well as by gel filtration on Sephadex G-200. FPA release and the initial rate of FPB release were unaffected. The second phase of rapid FPB release was abolished indicating that this increased rate is entirely dependent on the polymerization of fibrin I. Under one set of specified conditions the initial release rates of FPB by thrombin from different substrates were 15.8 pmol/min (fibrinogen), 14.0 pmol/min (fibrinogen plus gly-pro-arg-pro), 27.4 pmol/min (fibrin I monomer) and 164.4 pmol/min (fibrin I polymer). The data indicate that fibrin I and II formation are not sequential but simultaneous and that it is the more rapid formation of fibrin I which results in the appearance of a sequential reaction. It is suggested that a number of other reactions in the hemostatic system including fibrinolysis which appear to be sequential are also simultaneous.     

A thrombin assay based upon the release of fibrinopeptide A from fibrinogen: definition of a new thrombin unit

An assay for thrombin is presented wherein thrombin-catalyzed hydrolysis at Arg-A alpha-16 to release fibrinopeptide A (FPA) from fibrinogen is measured using high-performance liquid chromatography (HPLC). In this assay one thrombin unit (TU) is defined as that amount of thrombin that will release half of the FPA in one min from one ml of a solution of greater than 90% clottable normal human fibrinogen (less than or equal to 0.35 microM) at 37 degrees C, pH 7.4, /2 0.15. One TU is equivalent to approximately 0.1 NIH unit of thrombin and approximately 1 pmol of pure human thrombin. At 37 degrees C, pH 7.4, and plasma levels of fibrinogen of 3 mg/ml, one TU will catalyze the release of 3.6 nmol FPA min-1. Variability in fibrinogen samples which produce dramatic differences in clotting time assays with the same sample of thrombin, produce little or no variation in the catalytic assay for TU. The assay for TU obviates the need for maintenance of a thrombin reference standard.     

Fibrinogen naples I (B beta A68T) nonsubstrate thrombin-binding capacities.

Fibrinogen Naples I (Bbeta A68T) is characterized by defective thrombin binding and fibrinopeptide cleavage at the fibrinogen substrate site in the E domain. We evaluated the fibrinogen of three homozygotic members of this kindred (II.1, II.2, II.3) who have displayed thrombophilic phenotypes and two heterozygotic subjects (I.1, I.2) who were asymptomatic. Electron microscopy of Naples I fibrin networks showed relatively wide fiber bundles, probably due to slowed fibrin assembly secondary to delayed fibrinopeptide release. We evaluated 125I-thrombin binding to the fibrin from subjects I.1, I.2, II.1, and II.2 by Scatchard analysis with emphasis on the high-affinity site in the D domain of fibrin(ogen) molecules containing a gamma chain variant termed gamma'. Homozygotic subjects II.1 and II.2 showed virtually absent low-affinity binding, consistent with the Bbeta A68T mutation, whereas heterozygotes I.1 and I.2 showed only moderately reduced low-affinity binding. The homozygotes also showed impaired high-affinity thrombin binding, whereas that of the heterozygotes was nearly the same as normal. Genomic sequencing of the gamma' coding sequence (I.2, II.2), ELISA measurements of two gamma' chain epitopes (L2B, gamma'409-412, and IF10, gamma'417-427) (I.2, II.1, II.2, II.3), and mass spectrometry of Naples I fibrinogen (II.2) showed no differences from normal, thus indicating that there were no abnormal structural modifications of the gamma' chain residues in Naples I fibrinogen. However, thrombin reportedly utilizes both of its available exosites for binding to high- and low-affinity sites on normal fibrin, suggesting that binding is cooperative. Thus, reduced high-affinity thrombin binding to homozygotic Naples I fibrin may be related to the absence of low-affinity binding sites.     

Radioimmunoassay of fibrinopeptide A-clinical applications.

The radioimmunoassay technique developed by Nossel et al. for measuring fibrinopeptide A released by thrombin action on fibrinogen was used for assessing the degree of intravascular coagulation in patients. Plasma fibrinopeptide A values in 36 normal persons were below 2 ng/ml. Elevated levels were often found in cases of malignancies, burns and fractures, whereas uremic patients usually had normal values. Samples could be stored for several weeks without significant loss of immunoreactivity.     

Synthesis and radioimmunoassay of canine fibrinopeptide A

Canine fibrinopeptide A, the 16 residue NH₂-terminal segment of the Aα chain of canine fibrinogen released by thrombin proteolysis and its NH₂-tyrosyl analogue, were synthesized by stepwise solid-phase syntheses. Following cleavage, the synthetic peptides were purified by ion-exchange chromatography, and characterized by amino acid analysis, thin-layer chromatography, high-voltage paper electrophoresis and Edman degradation analysis. Rabbits immunized with carbodiimide-coupled synthetic canine fibrinopeptide A-bovine albumin conjugates produced antibodies which bound up to 70% of available counts using ¹²⁵I-N-tyrosyl canine fibrinopeptide A as tracer. Quantitative displacement of bound tracer could be effected by both synthetic canine fibrinopeptide A and clot supernates prepared from purified canine fibrinogen. Fifty percent displacement of radiolabelled tracer could be achieved with 1.1 picomole of peptide antigen. Antiserum to canine fibrinopeptide A was about 50-fold less sensitive on a molar basis to canine fibrinogen as compared to the free peptide in solution. This suggests that antigenic determinants on the canine fibrinopeptide are altered by attachment of the peptide to its parent molecule. The molar reactivity of human fibrinopeptide A was about 25% in comparison with canine fibrinopeptide A, indicating that both the human and canine peptides share some antigenic determinants. Using this assay, fibrinopeptide A immunoreactivity was measured in canine plasma following removal of fibrinogen by ethanol precipitation and dialysis. The mean fibrinopeptide A level 4 normal dogs (18 determinations) was found to be 1.5±0.98 pmols/ml anticoagulated plasma.     

The Zurich Study: XXIII. Epidemiology of headache syndromes in the Zurich cohort study of young adults

This study examines the 1 year prevalence rates of headache syndromes in an epidemiologic cohort study of young adults ages 29–30 in Zurich, Switzerland. The 1 year prevalence rates of headache subtypes were 3.3% for migraine with aura and 21.3% of migraine without aura as defined by the International Headache Society (IHS) criteria. The demographic distribution, clinical features, sequelae, and treatment patterns of subjects with specifc headache subtypes are described. The rates of migraine are compared to those of other community samples that have employed the IHS criteria for headache subtypes. Subjects with migraine reported pervasive impairment in nearly every life role including occupation, leisure, and social relationships. Despite the substantial degree of impairment in occupational and social functioning that was associated with migraine, an extremely low proportion of subjects had received professional treatment for headache. These results suggest that a concerted effort should be directed towards education regarding the classification of headache and the availability of efficacious treatment for migraine.     

Fibrinogen Nijmegen: congenital dysfibrinogenemia associated with impaired t-PA mediated plasminogen activation and decreased binding of t-PA

Congenital dysfibrinogenemia was found in a patient with venous thrombosis. Blood clot lysis was prolonged and suggested an impairment of fibrinolysis. We investigated whether this was related to the fibrinogen abnormality. Fibrinopeptide release was normal but fibrin polymerization was defective in the patient. The stimulating effect of the patient's fibrin on t-PA mediated plasminogen activation was impaired. This could not be attributed to defective binding of plasminogen. However, the binding of t-PA to the patient's fibrin was about 16% less than to normal fibrin. A variant t-PA (G K1 K2 P), which contained only one of the two fibrin binding sites, i.e. the kringle-2 domain, was bound to the abnormal fibrin for only 50% of normal. We conclude that the prolongation of blood clot lysis and the impaired stimulation of t-PA mediated plasminogen activation are related to the defective binding of the kringle-2 domain of t-PA onto the fibrin moiety of the abnormal fibrinogen. The impairment of fibrinolysis might explain the occurrence of thrombosis in the patient.     

Elevated fibrinopeptide A (FPA) in patients with Lesch-Nyhan syndrome.

The detection of elevated fibrinopeptide A (FPA) level in a patient with the Lesch-Nyhan syndrome complicated with cerebral infarction prompted us to examine FPA level in 3 other patients with the syndrome. FPA level significantly increased in all patients. Fibrinopeptide B beta 15-42 (FPB beta 15-42) level was increased in two, and both beta-thromboglobulin (beta TG) and platelet factor 4 (PF4) levels were elevated in one patient. These results suggest coagulation abnormalities in patients with Lesch-Nyhan syndrome.     

The stability of fibrinopeptide B immunoreactivity in blood.

The stability of fibrinopeptide B immunoreactivity as measured by radioimmunoassay was studied in buffer solutions, normal human plasma and blood. Fibrinopeptide B immunoreactivity was stable in buffer, but rapid temperature-dependent loss of immunoreactivity occurred in plasma and whole blood. Carboxypeptidase B (porcine pancreas) inactivated fibrinopeptide B immunoreactivity at a rate of 3.1 × 10^?10 mol/ml/min/unit at pH 7.4, 0.15 M Tris-NaCl and 24°. The loss of immunoreactivity was inhibited with 0.01 M o-phenanthroline. O-phenanthroline (0.01 M) completely inhibited the loss of fibrinopeptide B immunoreactivity in blood and other carboxypeptidase inhibitors including EDTA, hippuryl-L-arginine and hippuryl-L-lysine slowed the rate loss of immunoreactivity. Proof that the loss of immunoreactivity resulted from loss of Arginine (Bβ 14) was obtained by demonstrating that Bβ 1–13 immunoreactivity was stable in whole blood. The results of these studies indicate that the loss of fibrinopeptide B immunoreactivity in blood results from the cleavage of Arginine (Bβ 14) by carboxypeptidase B, that the enzyme is effectively inhibited by 0.01 M o-phenanthroline and that Arginine (Bβ 14) is a critical component of one set of antigenic determinants of fibrinopeptide B.     

Turnover of fibrinopeptide A (FPA) in rabbits

FPA disappeared from the circulating blood along a double-exponential decay curve consisting of an initial phase (t 1/2 = 1.8 min) and a late phase (t 1/2 = 34.7 min). The rapid decrease in blood FPA was due to the large extravascular space, the size of which was estimated to be about 5 times larger than that of intravascular space. The actual amounts of 125I-FPA distributed to the organs and tissues were generally quite low. However, in the case of the urine, the injected amount of FPA was excreted at the rate of 50% per hour. Thus, the urinary FPA levels may reflect the occurrence of intravascular coagulation.     

A radioimmunoassay technique for the rapid measurement of human fibrinopeptide A

Fibrinopeptide A (FPA) has been measured in human plasma by a radioimmunoassay (RIA) modified as to give results available 28 or 8 hours after venipuncture. Rabbit antibodies were elicited against synthetic FPA coupled to thyroglobulin. The tracer was prepared by coupling FPA to desaminotyrosine and radioiodinated using the chloramine-T method. The labeled peptide was purified using a two-step method, which prevented label damage for 10 weeks. The shortening of the procedure was mainly achieved by reduction of the FPA extraction from plasma using a new equilibrium dialyser. The recovery of added FPA was 52 – 54%, the detection limit of the method 0.54 ng/ml. A further substantial shortening can be achieved by reduction of the incubation time from 24 to 21/2 hours, the results obtained with these two incubation times showing an excellent correlation when FPA is elevated above 2 ng/ml. In 24 normal individuals the FPA level was 1.01 ng/ml with an SD of 0.45. Grossly elevated levels were found in patients with various diseases in which the fibrinogen-fibrin conversion is accelerated. In patients with acute thrombosis, the FPA levels were markedly reduced 10 min after injection of 5000 U of Heparin. Infusions of the snake venom enzyme Defibrase in one patient produced high levels of FPA, associated with hypofibrinogenemia and negative ethanol gelation test. The rapid FPA assay is thus of particular interest in those clinical emergencies associated with a low plasma fibrinogen and a negative ethanol test.