fibrinopeptide-b and Blood-Coagulation-Disorders

Topics: Animals; Antithrombin III Deficiency; Blood Coagulation; Blood Coagulation Disorders; Diabetes Complications; Estrogens; Female; Fibrinopeptide B; Hemostasis; Humans; Male; Myocardial Infarction; Neoplasms; Partial Thromboplastin Time; Postoperative Complications; Pregnancy; Prospective Studies; Thrombosis

A large number of families have now been described in whom affected individuals have within their plasmas an abnormal species of fibrinogen (factor I). These defects, presumably examples of the phenomenon of allotypy--i.e., the synthesis of variant forms of a normal protein--have been inherited as autosomal dominant characteristics. In the great majority of cases, clotting is abnormally slow when thrombin is added to the abnormal plasma. Sometimes this defect appears to reside in impaired release of fibrinopeptides by thrombin. In other cases, fibrinopeptide release proceeds normally, but aggregation of fibrin monomers is impeded. In the latter instance, aggregation may be abnormally slow or, once it begins, it may proceed at a normal rate. Curiously, a bleeding tendency is more likely to occur in patients in whom fibrinopeptide release is impaired, while dehiscence of operative wounds rarely complicates dysfibrinogenemias associated with impaired aggregation of fibrin monomers; thrombosis has been described in both groups of patients. Most of the reported cases may be distinguished by functional criteria and by the physicochemical behavior and biochemical nature of the abnormal protein. Additonally, one family has been described in which plasma clots abnormally rapidly upon addition of thrombin, and two others in which crosslinking of fibrin by fibrin-stabilizing factor (factor XIII) is defective.

Topics: Afibrinogenemia; Amino Acid Sequence; Blood Coagulation Disorders; Child; Diagnosis, Differential; Female; Fibrinogen; Fibrinopeptide A; Fibrinopeptide B; Genes, Dominant; Humans; Male

Fibrinogen Milano XII was detected in an asymptomatic Italian woman, whose routine coagulation test results revealed a prolonged thrombin time. Fibrinogen levels in functional assays were considerably lower than levels in immunologic assays. Polymerization of purified fibrinogen was strongly impaired in the presence of calcium or ethylenediaminetetraacetic acid (EDTA). Two heterozygous structural defects were detected by DNA analysis: Aalpha R16C and gamma G165R. As seen previously with other heterozygous Aalpha R16C variants, thrombin-catalyzed release of fibrinopeptide A was 50% of normal. Additionally, the release of fibrinopeptide B was delayed. Immunoblotting analysis with antibodies to human serum albumin indicated that albumin is bound to Aalpha 16 C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmin digests of fibrinogen Milano XII in the presence of calcium or EDTA showed both normal and novel D1 and D3 fragments. Further digestion of abnormal D3 fragments by chymotrypsin resulted in degradation products of the same size as the fragments derived from normal fibrinogen. SDS-PAGE analysis under reducing conditions showed no difference between normal fibrinogen and fibrinogen Milano XII or between their plasmic fragments. Circular dichroism analysis revealed a shift in the mean residual ellipticity and a significant reduction of the alpha-helix content in the variant D3 fragment. It is concluded that the Aalpha-chain substitution is mainly responsible for the coagulation abnormalities, whereas the substitution in the gamma-chain induced a conformational change in the D3 fragment.

Topics: Aged; Blood Coagulation Disorders; Calcium; Chymotrypsin; Circular Dichroism; DNA; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Female; Fibrinogen; Fibrinogens, Abnormal; Fibrinolysin; Fibrinopeptide A; Fibrinopeptide B; Heterozygote; Humans; Immunoblotting; Kinetics; Mutation; Peptide Fragments; Protein Conformation; Sequence Analysis, DNA; Serum Albumin; Thrombin; Thrombin Time

Topics: Adult; Blood Coagulation Disorders; Female; Fibrin Fibrinogen Degradation Products; Fibrinopeptide A; Fibrinopeptide B; Humans; Hypothyroidism; Male; Middle Aged; Peptide Fragments

To determine the effects of disseminated intravascular coagulation (DIC) and head injury on posttrauma coagulation and fibrinolysis.. Case-control study.. General ICU (tertiary care center) in a city hospital serving a population of 150 million people.. Forty trauma victims: 15 with DIC; 25 without DIC.. Measurement of six types of coagulation and fibrinolytic molecular markers (fibrinopeptide A, fibrinopeptide B beta 15-42, plasmin antiplasmin complex, D-dimer, tissue plasminogen activator antigen concentration, tissue plasminogen activator activity) immediately after trauma, 3 days later, and 6 days later. Anticoagulant treatment with gabexate mesilate at 1.45 +/- 0.06 mg/kg/hr.. Fibrinopeptide A, fibrinopeptide B beta 15-42, plasmin antiplasmin complex, and D-dimer showed high values immediately after trauma and exceeded normal activity for the first 6 days. When trauma was complicated with DIC, the molecular markers showed significantly higher values than those for non-DIC patients on all days. In the head-injured patients, such effect was not noted. Tissue plasminogen activator antigen concentration and tissue plasminogen activator activity were within a normal physiologic range of variation. By contrast, tissue plasminogen activator antigen concentration increased significantly after trauma in patients with DIC. When anticoagulant treatment was found effective, it caused a reduction in fibrinopeptide A.. a) Fibrinolytic shut-down and its reactivation cannot be confirmed after trauma. b) Head injury does not lead to an increase in posttrauma coagulation or fibrinolytic activity. c) DIC enhances posttrauma coagulation and fibrinolytic activity and plasminogen activator inhibitor activity can be inferred in DIC patients. d) Increase in tissue plasminogen activator antigen concentration without tissue plasminogen activator activation may be a prognostic factor indicative of DIC and its chances of improvement, and fibrinopeptide A as an assessment criterion for the effectiveness of anticoagulant treatment.

Topics: Adult; Antifibrinolytic Agents; Antigen-Antibody Complex; Antigens; Antithrombin III; Biomarkers; Blood Coagulation Disorders; Case-Control Studies; Craniocerebral Trauma; Disseminated Intravascular Coagulation; Female; Fibrin Fibrinogen Degradation Products; Fibrinolysin; Fibrinolysis; Fibrinopeptide A; Fibrinopeptide B; Glasgow Coma Scale; Hospitals, General; Humans; Injury Severity Score; Japan; Male; Middle Aged; Multiple Trauma; Partial Thromboplastin Time; Platelet Count; Predictive Value of Tests; Prothrombin Time; Tissue Plasminogen Activator

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.

Topics: Amino Acid Sequence; Arginine; Blood Coagulation Disorders; Calcium; Child; Chromatography, High Pressure Liquid; Cysteine; Fibrinogens, Abnormal; Fibrinopeptide A; Fibrinopeptide B; Humans; Macromolecular Substances; Male; Molecular Sequence Data; Platelet Aggregation

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.

Topics: Adult; Amino Acid Sequence; Blood Coagulation Disorders; Blood Coagulation Tests; Female; Fibrinogens, Abnormal; Fibrinopeptide A; Fibrinopeptide B; Humans; Leucine; Molecular Sequence Data; Polymers; Proline

An asymptomatic 50-year-old male with a gamma globulin paraprotein was found to have prolonged prothrombin time, activated partial thromboplastin time, and thrombin time but a normal reptilase time. The prolonged clotting times were not the result of a factor deficiency because they were not corrected by the addition of normal plasma. Instead, this patient had an antibody that delayed thrombin-mediated fibrinopeptide B release thereby producing an apparent dysfibrinogenaemia. His isolated IgG prolonged the thrombin clotting time of both normal plasma and fibrinogen. Precincubation of his IgG with fibrinopeptide B, but not with fibrinopeptide A or thrombin, decreased its ability to prolong the thrombin clotting time. The patient's purified IgG but not control IgG delayed thrombin-mediated fibrinopeptide B release from fibrinogen without affecting the release of fibrinopeptide A. These studies define a novel, clinically silent dysfibrinogenaemia due to an antibody that delays thrombin-mediated fibrinopeptide B release from fibrinogen thereby markedly prolonging the clotting times.

Topics: Blood Coagulation Disorders; Fibrinopeptide B; Humans; Immunoglobulin G; Male; Middle Aged; Partial Thromboplastin Time; Prothrombin Time; Thrombin Time; Time Factors

A new case of heterozygous dysfibrinogenemia characterized by the replacement of NH2-terminal amino acid of fibrin beta-chain was found in a 50-year-old man. Despite a prolonged thrombin time, the propositus' fibrinogen had a normal reptilase time with the normal release of fibrinopeptide A. Release of fibrinopeptide B by thrombin was strongly affected, but a very high concentration of thrombin almost completely released fibrinopeptide B with a normal elution pattern on reversed-phase high performance liquid chromatography (HPLC). Lysylendopeptidase-cleavage of purified B beta-chains analyzed on HPLC showed the decrease of one peptide compared with the normal 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 demonstrated that B beta glycine-15, NH2-terminus of the fibrin beta-chain, was replaced by cysteine. These findings will be of particular importance because they strongly support the hypothesis that the NH2-terminal portion of the fibrin beta-chain is involved in the polymerization reaction by thrombin. The propositus' daughter and two sisters had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Ise. During these studies, we found that a very high concentration of thrombin cleaves not only the A alpha Arg19-Val20 bond but also the COOH-terminal region of alpha-chains, which results in the generation of further degraded alpha-chains with apparent molecular weights of 44,000 or less.

Topics: Amino Acid Sequence; Batroxobin; Blood Coagulation Disorders; Blood Coagulation Tests; Chromatography, High Pressure Liquid; Cysteine; Fibrin; Fibrinogens, Abnormal; Fibrinopeptide A; Fibrinopeptide B; Glycine; Humans; Male; Middle Aged; Molecular Sequence Data; Thrombin

Fibrinogen Baltimore I is one of the very first congenital abnormal fibrinogens reported over several decades ago; however, the molecular defect of this dysfibrinogen has eluded identification. In fact, several reports misidentified the functional defect of Baltimore I, which has impaired fibrin monomer polymerization. Reversed-phase high-performance liquid chromatography analysis of lysyl endopeptidase digest of the purified Baltimore I gamma-chain showed an abnormal peptide not found in the co-existing normal gamma-chain of this heterozygote. Amino acid sequencing of this peptide indicated that gamma-chain Gly292 is replaced by valine. This observation was confirmed, and the genetic defect was determined by direct nucleotide sequencing of a polymerase chain reaction product containing codon gamma 292, which is mutated: GGC----GTC. The molecular defect of Fibrinogen Baltimore I lies in a region of the gamma-chain required for fibrin polymerization, suggesting that the integrity of gamma Gly292 is critical for fibrin assembly.

Topics: Adult; Amino Acid Sequence; Base Sequence; Blood Coagulation Disorders; Chromatography, High Pressure Liquid; Codon; Female; Fibrinogens, Abnormal; Fibrinopeptide A; Fibrinopeptide B; Glycine; Humans; Molecular Sequence Data; Mutation; Polymers; Serine Endopeptidases; Valine

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).

Topics: Adolescent; Amino Acids; Blood Coagulation Disorders; Calcium; Electrophoresis, Polyacrylamide Gel; Fibrinogens, Abnormal; Fibrinopeptide A; Fibrinopeptide B; Heterozygote; Humans; Isoelectric Focusing; Kinetics; Male; Pedigree; Platelet Aggregation; Thrombin

Little information is available on the prevalence and etiology of the coagulopathy present in some children with acute leukemia at disease presentation. We studied 102 children with newly diagnosed acute leukemia (50 retrospective: Group A; and 52 prospective: Group B) with prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT), fibrinogen (FIB), and fibrin degradation products (FDP). All patients in Group B also had assessment of thrombin activation by measurement of the crosslinked fibrin fragment, D-dimer, and of primary fibrinolysis with the B beta 1-42 peptide. Additionally, ten patients from Group B had Factors II, V, VII, and X measured, and eight of these patients had measurement of tissue factor from sonicated bone marrow cells. Thirty-two percent of Group A and 40% of Group B had totally normal coagulation studies, whereas 20% of Group A and 10% of Group B had a severe coagulopathy on disease presentation. A high percentage of both groups had elevated PT (Group A, 52%; Group B, 27%) and increased FDP (Group A, 39%; Group B, 25%). In Group B, 38% of the patients had a positive D-dimer, whereas only 4% of this prospective group had an elevated B beta 1-42 peptide (P less than 0.00001). Nine of ten patients with a positive D-dimer had low levels of one or more of the extrinsic pathway factors. Three of four patients with the highest tissue factor levels were of monocytoid leukemia cell type. These data indicate that the coagulopathy associated with acute leukemia of childhood is usually mediated by thrombin activation.

Topics: Adolescent; Blood Coagulation Disorders; Child; Child, Preschool; Fibrin Fibrinogen Degradation Products; Fibrinolysis; Fibrinopeptide B; Humans; Infant; Peptide Fragments; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prevalence; Prospective Studies; Prothrombin Time; Retrospective Studies; Thrombin

To clarify the mechanism which causes the coagulation time of fetal fibrinogen to be longer than that of adult fibrinogen, the N-terminal amino acid residues of fetal fibrinogen were analyzed. The results showed that the amount of A alpha chain's N-terminal alanine residues in fetal fibrinogen was only 54% of that in adult fibrinogen. When the amount of A alpha chain's N-terminal residual alanine in adult fibrinogen was decreased to 57% of the normal level by digestion with aminopeptidase M, the adult fibrinogen yielded a prolonged thrombin time and a retarded release rate for fibrino-peptides A and B, both values approximating to those of fetal fibrinogen. The results suggest that the deficiency in alanine residue at the N-terminus of the A alpha chain is a major cause of the slow coagulation of fetal fibrinogen.

Topics: Alanine; Amino Acids; Aminopeptidases; Blood Coagulation Disorders; CD13 Antigens; Electrophoresis, Polyacrylamide Gel; Fetal Blood; Fibrinogen; Fibrinopeptide A; Fibrinopeptide B; Glycine; Humans; Infant, Newborn; Thrombin Time

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.

Topics: Blood Coagulation Disorders; Fibrinogen; Fibrinogens, Abnormal; Fibrinopeptide A; Fibrinopeptide B; Humans; Male; Middle Aged; Pedigree; Plasminogen; Protein Binding; Thrombophlebitis; Tissue Plasminogen Activator; Whole Blood Coagulation Time

Thrombin preferentially cleaves fibrinopeptides A (FPA) from fibrinogen resulting in the formation of desAA-fibrin from which most of the fibrinopeptides B (FPB) are then released with an enhanced rate. Kinetics of fibrinopeptide release from normal and dysfunctional fibrinogens were investigated in order to further characterize the mechanism of accelerated FPB release during desAA-fibrin polymerization. Dysfunctional fibrinogens London I and Ashford, exhibiting primary polymerization abnormalities (i.e., an abnormality present when all fibrinopeptides have been cleaved), which in the case of fibrinogen London I is believed to be caused by a defect in the D-domain, were shown to exhibit a decreased rate of FPB release compared with normal fibrinogen. While Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, was shown to decrease the rate of FPB release from normal fibrinogen by a factor of 5, normal fragment D1, although inhibiting clot formation of normal fibrinogen, did not influence the acceleration of FPB release. On the other hand, the presence of fragment D1 did not enhance FPB release from fibrinogen London I, suggesting that interaction of D-domains in functional isolation with desAA-fibrin E-domains is not sufficient to enhance FPB release. Although clot formation was inhibited by the concentrations of fragment D1 used, the formation of small desAA-fibrin oligomers was hardly affected. Thus, small fibrin polymers, but not desAA-fibrin monomers, act as optimal substrates for the release of FPB by thrombin.

Topics: Blood Coagulation Disorders; Fibrin; Fibrinogen; Fibrinogens, Abnormal; Fibrinopeptide B; Humans; Kinetics; Nephelometry and Turbidimetry; Polymers; Thrombin

We describe the coagulopathy of a 65-year-old woman with a thrombotic disorder associated with dysfibrinogenemia and lupus anticoagulant (LA). The patient's prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT), and batroxobin time were prolonged and could not be corrected by mixing with equal volumes of normal plasma. Fibrinogen quantitation showed approximately twice as much immunoreactive as thrombin-clottable protein. The batroxobin and thrombin clotting times of the patient's isolated fibrinogen were prolonged and could not be corrected by mixture with normal fibrinogen. Turbidimetrically assessed fibrin monomer aggregation in response to thrombin, ancrod, or batroxobin and fibrin monomer reaggregation experiments disclosed clearly delayed onset and a lower maximum opacity. In other turbidimetric and clotting-time experiments, the patient's fibrinogen displayed a dose-dependent inhibition of the reaggregation of normal fibrin. Fibrinopeptide A and B release rates and sialic acid content were normal. Assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of reduced samples, the subunit structure of the patient's fibrinogen and its fully cross-linked fibrin was normal. The presence of LA was established by two techniques, the blood thromboplastin inhibition test and the platelet neutralization procedure (PNP). A positive PNP could not be produced by mixing afibrinogenemic plasma with the patient's purified fibrinogen. The patient's inactivated serum and her isolated IgG prolonged the PT and PTT of normal plasma but showed no inhibitory effect on the clotting of purified normal fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aged; Blood Coagulation Disorders; Blood Coagulation Factors; Female; Fibrinogen; Fibrinopeptide A; Fibrinopeptide B; Humans; Immunoglobulin G; Lupus Coagulation Inhibitor; Partial Thromboplastin Time; Platelet Aggregation; Prothrombin Time; Thrombophlebitis

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.

Topics: Blood Coagulation Disorders; Chromatography, High Pressure Liquid; Fibrinogen; Fibrinopeptide A; Fibrinopeptide B; Humans

Fibrinogen Aarhus was found in a woman with slightly prolonged whole blood clotting time. The thrombin induced clotting of plasma and purified fibrinogen was much prolonged. Kinetic analysis of FPA and FPB release revealed larger apparent Km and Vmax values for fibrinogen Aarhus than for normal fibrinogen. No clot formation of fibrinogen Aarhus was demonstrated in the presence of Batroxobin and the release of FPA was slower than normal. Upon addition of the clotting enzyme from Agkistrodon contortrix contortrix clotting did occur but the clotting time was much prolonged in comparison with normal fibrinogen. The turbidity of fibrin gels obtained from fibrinogen Aarhus was similar to normal fibrin gels at low thrombin concentrations. Increasing thrombin concentration resulted in appearance of degradation products in the fibrin gels from fibrinogen Aarhus and at the same time a relative increase in turbidity of the gels was observed. Possibly reasons for the slow release of fibrinopeptides, the delayed gelation, and susceptibility to degradation by thrombin are discussed.

Topics: Adult; Amino Acids; Batroxobin; Blood Coagulation Disorders; Blood Coagulation Tests; Crotalid Venoms; Electrophoresis, Polyacrylamide Gel; Female; Fibrinogen; Fibrinogens, Abnormal; Fibrinolysin; Fibrinopeptide A; Fibrinopeptide B; Gels; Humans; Kinetics; Nephelometry and Turbidimetry; Radioimmunoassay; Thrombin

A congenitally abnormal fibrinogen was isolated from blood of a young man with deep-vein thrombosis. Two other affected members of his family had three episodes of severe arterial thrombosis. The fibrinogen showed a delayed clotting by thrombin, but a normal clotting by Arvin, Reptilase, and prothrombin-staphylocoagulase complex. Analysis of the fibrinopeptides A and B by High Performance Liquid Chromatography did not reveal an abnormal peptide structure. The rate of release of A and B peptides by thrombin was strongly delayed, whereas the rate of release of fibrinopeptide A by Arvin appeared to be normal. The fibrin polymerization rate was normal. Interactions between the abnormal fibrinogen, platelets and the fibrinolytic system were also normal. Evidence is presented that the defective interaction between fibrinogen Milano II and thrombin is associated with a defective binding of thrombin to the fibrin moiety of the abnormal fibrinogen.

Topics: Adult; Blood Coagulation Disorders; Fibrin; Fibrinogen; Fibrinogens, Abnormal; Fibrinopeptide A; Fibrinopeptide B; Humans; Male; Pedigree; Platelet Aggregation; Thrombin; Thrombophlebitis; Thrombosis

An abnormal fibrinogen, denoted as "fibrinogen Bergamo I", has been characterized. Its defect consists in an exchange of arginine by cysteine in position 16 of the A alpha-chain, thus corresponding to that found in a number of other fibrinogen variants. The abnormal fibrinopeptide A cannot be split off by thrombin from intact fibrinogen Bergamo I. We describe three different chemical modifications of the cysteine A alpha 16, i.e. aminoethylation, methylation and carboxamidomethylation, and their effects on the susceptibility of fibrinogen Bergamo I towards thrombin attack. S-aminoethylation of the A alpha 16Cys renders the peptide bond A alpha 16-17 cleavable by thrombin. Following methylation or carboxamidomethylation, the A alpha 19-arginyl bond becomes accessible for thrombin. The chemically modified extended fibrinopeptide A can be readily separated from the normal fibrinopeptide A by HPLC. The latter two modifications are suitable alternative procedures for detecting the molecular defect A alpha 16Arg----Cys of fibrinogen.

Topics: Amino Acids; Aziridines; Blood Coagulation Disorders; Blood Coagulation Tests; Cysteine; Female; Fibrinogen; Fibrinogens, Abnormal; Fibrinopeptide A; Fibrinopeptide B; Humans; Male; Mercaptoethanol; Methylation; Thrombin

Topics: Blood Coagulation Disorders; Fibrin; Fibrinogen; Fibrinopeptide A; Fibrinopeptide B; Humans; Polymers; Prothrombin Time; United States; Wound Healing

Fibrinogen Seattle, a clinically silent, slow-clotting dysfibrinogen, releases 50% of the normal amount of fibrinopeptide B as assessed by amino acid analysis. The reduced dysfibrin exhibited equal quantities of chains with B beta- and beta-charge mobility on polyacrylamide gel electrophoresis in 2 M urea at low pH. By these same techniques, the release of fibrinopeptide A was normal. Clots formed by repolymerizing the thrombin and batroxobin dysfibrin monomers showed a maximal turbidity that was lower than normal. Fibrinogen Seattle was indistinguishable from normal fibrinogen by radial immunodiffusion and immunoelectrophoresis. Degradation by plasmin and transamination by factor XIIIa were normal. The characteristics of fibrinopeptide release by fibrinogen Seattle distinguish it from other reported dysfibrinogens.

Topics: Amino Acids; Blood Coagulation Disorders; Female; Fibrinogen; Fibrinogens, Abnormal; Fibrinopeptide B; Humans; Immunodiffusion; Macromolecular Substances; Thrombin; Washington

Fibrinogen Manchester is an abnormal fibrinogen with an impaired release of fibrinopeptide A (FPA) and a polymerization abnormality. In the accompanying article we have identified the amino acid substitution in fibrinogen Manchester as A alpha 16 Arg leads to His. When fibrinogen Manchester was digested with low thrombin concentrations approximately 40-50% of the total FPA content was release at a rate similar to FPA release from normal fibrinogen. The fibrin so formed exhibited an impaired polymerization of monomers. Digestion of fibrinogen Manchester with high concentrations of thrombin for prolonged times released the remaining FPA which had an abnormal retention time when studied by high performance liquid chromatography (HPLC). This fibrinopeptide has been shown previously to contain the A alpha 16 Arg leads to His substitution. fibrin resulting from this exhaustive digestion had normal polymerization of monomers. The normal and substituted FPAs were isolated by HPLC and compared in a double antibody competitive-binding assay for normal FPA. The immunological cross-reactivity of the abnormal peptide was reduced, so that approximately 5 times more abnormal peptide was required on a molar basis to displace labelled normal FPA. Normal intact fibrinogen was 10-fold less reactive (on a half molar basis) than free normal FPA and the crossreactivity of fibrinogen Manchester was measurably less than that of normal fibrinogen. It is concluded that immunological measurement alone of FPA released from abnormal fibrinogens may not give a complete description of the kinetics of peptide release if the amino acid substitution lies within the FPA sequence. The combination of radioimmunoassay and HPLC, however, provides a powerful analytical approach that should be useful in classifying and characterizing abnormal fibrinogens.

Topics: Blood Coagulation Disorders; Chromatography, High Pressure Liquid; Cross Reactions; Female; Fibrin; Fibrinogen; Fibrinogens, Abnormal; Fibrinopeptide A; Fibrinopeptide B; Humans; Male; Thrombin

In a 81 year old health woman, gross abnormalities of fibrin formation led to the discovery of an abnormal fibrinogen named fibrinogen Bondy. Clottability of purified fibrinogen Bondy was only 53% compared to 95-98% for normal fibrinogen. Functional studies revealed (i) delayed coagulation by thrombin and batroxobin (Reptilase), (ii) incomplete release of fibrino-peptides A and B, (iii) poor fibrin monomer aggregation, (iv) delayed fibrin proteolysis by plasmin. Electrophoretic mobility of fibrinogen Bondy, its three chains and the products of fibrin cross-linking, was normal. Fibrinogen NH2-terminal residues of fibrinogen Bondy were found to be normal. The presence of Ala, in addition to Gly and Tyr in the fibrin clot and its supernatant, showed that a part of fibrinogen molecules was not clotted, i.e. either copolymerised with fibrin or remaining in solutions. Gel filtration of the supernatant allowed the separation of both soluble complexes and fibrinogen. This fibrinogen population was shown to be unclottable by thrombin and to inhibit clotting of normal fibrinogen.

Topics: Aged; Blood Coagulation Disorders; Chemical Phenomena; Chemistry, Physical; Chromatography, Gel; Cross-Linking Reagents; Female; Fibrin Fibrinogen Degradation Products; Fibrinogen; Fibrinogens, Abnormal; Fibrinolysin; Fibrinopeptide A; Fibrinopeptide B; Humans; Thrombin Time

The kinetics of the thrombin-catalysed release of fibrino-peptides A and B from human fibrinogen have been investigated and a mechanism correlating the release of fibrinopeptides to fibrin formation is presented. The sequential release of fibrinopeptides results in sequential activation of two sets of polymerisation sites.

Topics: Binding Sites; Blood Coagulation; Blood Coagulation Disorders; Calcium; Fibrin; Fibrinogen; Fibrinopeptide A; Fibrinopeptide B; Humans; Kinetics; Macromolecular Substances; Protein Binding; Thrombin

Topics: Blood Coagulation Disorders; Disulfides; Factor XIII; Fibrinogen; Fibrinopeptide A; Fibrinopeptide B; Humans; Molecular Conformation; Peptide Chain Termination, Translational; Thrombin

A new case of congenital dysfibrinogenemia has been discovered in a 20 year old woman, who suffered from a severe postpartal hemorrhage after the delivery of her first child, followed by episodes of thrombosis. Coagulation studies reveal a prolongation of thrombin time, reptilase time was immeasurable. Thromboplastin time and partial thromboplastin time were slightly prolonged. Low fibrinogen levels were obtained by techniques, which depend on the coagulation velocity following addition of thrombin, while immunological procedures gave slightly diminished values of fibrinogen. Patients's fibrinogen had a moderate inhibitory effect on the fibrin formation in normal plasma. However, inhibitors of the fibrinogen-fibrin conversion could not be detected. Coagulation factors were normal, fibriolysis as well. The cause of the coagulation disorder was found to be a defect of the fibrinogen molecule, leading to an abnormal fibrin polmerization of patient's fibrin monomers. The release of the fibrinopeptides in the paperelectrophoresis was normal. The defect of the fibrinogen molecule did not protect from thrombotic complications. The same defect could be found in the lower scale in patient's father, 4 of her 7 brothers and sisters, and her son.

Topics: Adult; Blood Coagulation Disorders; Blood Coagulation Tests; Female; Fibrinogen; Fibrinolysis; Fibrinopeptide A; Fibrinopeptide B; Hemorrhagic Disorders; Humans