Joan H. Sobel

Identification of the alpha chain lysine donor sites involved in factor XIIIa fibrin cross-linking.

Biochemical studies of fibrin cross-linking were conducted to identify the specific Aα chain lysine residues that potentially serve as Factor XIIIa amine donor substrates during α polymer formation. A previously characterized Factor XIIIa fibrin lysine labeling system was employed to localize sites of donor activity based on their covalent incorporation of a synthetic peptide acceptor substrate analog modelled after the NH2-terminal cross-linking domain of α2 antiplasmin. Peptide-decorated fibrin was prepared using purified fibrinogen as the starting material. Cyanogen bromide digestion, immunoaffinity chromatography, high pressure liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (anti-peptide) methodologies were employed to isolate purified CNBr fibrin fragments whose structures included the acceptor probe in cross-linked form and, therefore, represented regions of (amine) donor activity. Five α chain CNBr fragments (within Aα 208-610) and one γ chain CNBr fragment (γ 385-411) were the only portions of fibrin found associated with the acceptor peptide, based on collective sequencing, mass, and compositional data. Trypsin digestion, HPLC, and enzyme-linked immunosorbent assay (anti-peptide) methodologies were used to isolate smaller derivatives whose structures included an α chain tryptic cleavage product (the donor arm) cross-linked to the trypsin-resistant synthetic peptide (the acceptor arm). Biochemical characterization and quantitative peptide recovery data revealed that 12 of the 23 potential lysine donor residues within α 208-610 had incorporated the peptide probe, whereas γ chain donor activity was due solely to peptide cross-linking at (γ) Lys406; the α chain lysines, Lys556 and Lys580, accounted for 50% of the total α chain donor cross-linking activity observed, with Lys539, Lys508, Lys418, and Lys448 contributing an additional 28% and Lys601, Lys606, Lys427, Lys429, Lys208, Lys224, and/or Lys219 responsible for the remaining proportion (2-5%, each). The collective findings extend current models proposed for the mechanism of α polymer formation, raise questions concerning the physiological role of multiple α chain donor sites, and, most importantly, provide specific information that should facilitate future efforts to identify the respective lysine and glutamine partners involved in native fibrin α chain cross-linking.

Immunochemical resemblance between human leukemia and hen egg-white lysozyme and their reduced carboxymethyl derivatives

Abstract The immunological resemblance between hen egg and human leukemia lysozymes and their reduced, carboxymethyl derivatives has been examined by radioimmunoassay, immuno-diffusion and micro-complement fixation. No crossreactions were observed between the native proteins despite evidence that their primary structures are identical in 77 of 129 amino-acid positions and that they are also quite similar in three-dimensional structure. In contrast, when the reduced, carboxymethyl derivatives of these lysozymes were examined by the same immunological techniques, it was possible to show that they would crossreact with antisera prepared against either denatured lysozyme. Control experiments using performic acid-oxidized lysozymes demonstrated that these crossreactions were not solely due to antibodies directed against the reduced and alkylated cystine residues. These results indicate that immunological studies using antisera directed against proteins whose native structures have been disrupted may reveal sequence homologies which are otherwise undetected when cross-reactivity between the native molecules is examined. It is concluded that failure to demonstrate cross-reactivity between two similar proteins does not necessarily rule out the possibility that their amino-acid sequences exhibit a high degree of homology. When immunological evidence for such homologous relationships is being sought, it might be advantageous to include experiments with antisera to reduced, carboxymethyl or performic acid-oxidized protein derivatives.

Alpha chain crosslinking of human fibrin: Purification and radioimmunoassay development for two Aα chain regions involved in crosslinking

Abstract Rapid and efficient purification methods that include hydrophobic chromatography on Phenyl Sepharose CL-4B have been developed for the peptides that span residues 241–476 (CNBr VIII), and 518–584 (CNBr X) in the Aα chain of human fibrinogen. Amino acid analysis, NH 2 -terminal sequence determination, and SDS-PAGE indicated that greater than 95% purity of each peptide was achieved. Sensitive and specific radioimmunoassays capable of detecting antigen in the range of 0.1–10.0 pmol/ml have also been developed for CNBr VIII and CNBr X. These assays have been characterized and successfully applied in studies designed to localize the two COOH-terminal Aα chain regions in column effluents of CNBr-digested fibrinogen and crosslinked fibrin. When these immunoassays were used to study purified preparations of a high molecular weight, crosslink-containing CNBr derivative of α polymer, the data provided immunologic confirmation for the involvement of CNBr VIII and CNBr X in α chain crosslinking.

Comparative Structural and Functional Features of the Human Fibrinogen αC Domain and the Isolated αC Fragment CHARACTERIZATION USING MONOCLONAL ANTIBODIES TO DEFINED COOH-TERMINAL Aα CHAIN REGIONS

The αC domain of fibrinogen (Aα-(220-610)) plays a central role in maintaining hemostasis by serving as a substrate for factor XIIIa and plasmin. Monoclonal antibodies that recognize eight distinct epitopes within the COOH-terminal two-thirds of the Aα chain were employed as structural probes to: 1) isolate the human αC domain, 2) compare the topography of the eight epitopes within the αC domain of intact fibrinogen and in purified αC fragments, and 3) explore the degree to which the αC domains role as a factor XIIIa substrate in intact fibrinogen is preserved within the structure of isolated αC fragments. Five antibodies were raised against small, synthetic peptide immunogens (Aα-(220-230), Aα-(425-442), Aα-(487-498), and Aα-(603-610)), and three were generated against larger cyanogen bromide (A)α chain derivatives with each epitope subsequently localized to discrete Aα chain sequences (Aα-(259-276), Aα-(529-539), and Aα-(563-578)). Human αC preparations were isolated from mild plasmin digests of fibrinogen by successive chromatography on concanavalin A-Sepharose, anti-Aα-(425-442)-Sepharose, and Superdex-75 fast protein liquid chromatography. Immunochemical characterization indicated that the NH2-terminal residue of αC fragments was either Aα-220 or Aα-231 and that, although the extreme COOH-terminal region, Aα-(603-610), was absent, all molecules were intact at least through Aα-(563-578). Solution phase competitive assays indicated that the release of the αC domain from intact fibrinogen was associated with several conformational changes, e.g. in the vicinity of Aα-(220-230), Aα-(259-276), Aα-(487-498), and Aα-(529-539), but that the relative accessibility of other localized structures remained unchanged, e.g. Aα-(425-442) and Aα-(563-578). Immunoblotting analysis of αC cross-linking in vitro revealed that isolated αC fragments could serve as a substrate for factor XIIIa. Immunoblotting studies of the Aα chain proteolysis that occurs during thrombolytic therapy indicated that αC fragments, similar in size and epitope content to those isolated from purified fibrinogen, were released in vivo early during fibrinolytic system activation. The collective findings provide new information about the fine structure of the fibrinogen αC domain and its functional implications and also draw attention to the as yet unexplored role of αC fragments in the pathophysiology of thrombosis and hemostasis.

Fibrinogen Proteolysis and Coagulation System Activation during Thrombolytic Therapy

Thrombolytic treatment of patients with acute myocardial infarction is now well accepted as the treatment of choice in most settings (24). At this time most data has been accumulated with the use of streptokinase, and this drug has been convincingly demonstrated to reduce mortality (5). Reperfusion is achieved in 45–75% of patients, with direct intracoronary administration being slightly more effective than administration by the intravenous route (25). Some problems remain, bleeding is significant and rethrombosis continues to be an obstacle (7).

Immunochemical Studies of Aα Chain Crosslinking

Nearly two decades ago our colleague, the late Dr. Hymie Nossel, considered the possibility that radioimmunoassays might have as much application in the field of coagulation as they had been shown to have in the field of endocrinology. He selected fibrinopeptide A as a likely candidate for measurement (1) and initiated a new era in the quantitation of peptides or proteins that are biochemical markers for various aspects of the coagulation process.