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J H Sobel

Publications and source records attributed to J H Sobel.

At least 19 recordsLinked to original sources
PubMed2000

Soluble fibrin species in arterial thrombi.

The aim of this study was to characterize soluble fibrin(ogen) species in human, arterial, in-vivo-formed thrombi, using the immunoblotting technique. Specimens were collected via intra-arterial catheters in six patients scheduled for catheter-directed thrombolysis. Unreduced and reduced samples of the supernatants from the arterial thrombi-derived specimens were electrophoresed on polyacrylamide gels and immunoblotted, using specific mono- and polyclonal anti-fibrin(ogen) antibodies. The reduced samples disclosed substantial amounts of high molecular weight material, consistent with alpha-chain polymers and gammagamma-dimers, as well as lower molecular weight material, such as alpha-, beta- and gamma-chains. No fibrinogen with intact fibrinopeptide A was detectable, and des-AABB fibrin represented a major fibrin derivative in the soluble part of the arterial thrombi. The alpha-chains were C-terminally degraded, most of them distal to position 259. In conclusion, we have demonstrated the presence of cross-linked fibrin derivatives in soluble, arterial thrombus-related material, without signs of fibrinogen-fibrin hybrids. The fibrin derivatives were C-terminally degraded, thus representing X-oligomeric material, most probably originating from plasmin degradation of insoluble thrombus fibrin. The present study supports the hypothesis of a dynamic equilibrium between clotting and lysis in thrombi.

Aged↗
PubMed1998

Antipeptide monoclonal antibodies to defined fibrinogen Aalpha chain regions: anti-Aalpha 487-498, a structural probe for fibrinogenolysis.

The fibrinogen alphaC domain (Aalpha 220-610) is one of the earliest targets attacked by plasmin following fibrinolytic system activation. Monoclonal antibodies (MoAbs) to defined sequences within the alphaC domain provide the opportunity to explore the structure-function relationships involved in plasmin's interaction with its Aalpha chain substrate at greater resolution and can serve as reagents with potential clinical use for detecting fibrinogenolysis in vivo. The MoAb F-104 was raised against a multiple antigenic peptide derivative modelled after the hydrophilic 12-residue sequence corresponding to Aalpha 487-498 within the alphaC domain. A sensitive solution phase competitive enzyme-linked immunosorbent assay (ELISA) was developed for MoAb F-104 that can be applied for the direct measurement of intact fibrinogen (purified or plasma; ED50% approximately 5 pmol Aalpha chain equivalents/mL), with negligible cross-reactive interference from peptide cleavage products released by plasmin from the COOH-terminal end of the Aalpha chain (<3%). Immunoblotting and ELISA studies to characterize the fate of the F-104 epitope during fibrinogenolysis in vitro indicated a rapid loss of fibrinogen-associated immunoreactivity that reflected the heterogeneity of plasmin cleavage sites within the alphaC domain; cleavage at the 493-494 arg-his bond destroyed the F-104 epitope, while cleavage at other sites released it in an altered, inaccessible, conformation within the structure of 35- to 40-kD and 17.5- to 18-kD Aalpha chain degradation products. Application of the F-104 ELISA to monitor the course of Aalpha chain proteolysis in a small study population of patients undergoing thrombolytic therapy for myocardial infarction (n = 14) showed that the loss of fibrinogen-associated F-104 immunoreactivity was a very early marker (within 15 to 30 minutes) of in vivo fibrinogenolysis. Additional data obtained suggest that MoAb F-104 may have promise as a reagent for evaluating the creation of an effective lytic state early during therapy, information that could help determine the need for further clinical intervention. Thus, these studies illustrate a rational, targeted, approach towards the development of a novel antifibrinogen MoAb whose application as a structural probe for the region Aalpha 487-498 in vitro and in vivo can provide new insights into the various molecular forms of fibrinogen that circulate under physiologic conditions and in disease.

Amino Acid Sequence↗
PubMed1996

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 Aalpha chain lysine residues that potentially serve as Factor XIIIa amine donor substrates during alpha 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 alpha2 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 alpha chain CNBr fragments (within Aalpha 208-610) and one gamma chain CNBr fragment (gamma 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 alpha 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 alpha 208-610 had incorporated the peptide probe, whereas gamma chain donor activity was due solely to peptide cross-linking at (gamma) Lys406; the alpha chain lysines, Lys556 and Lys580, accounted for 50% of the total alpha 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 alpha polymer formation, raise questions concerning the physiological role of multiple alpha 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 alpha chain cross-linking.

Amino Acid Sequence↗
PubMed1996

Comparative structural and functional features of the human fibrinogen alpha C domain and the isolated alpha C fragment. Characterization using monoclonal antibodies to defined COOH-terminal A alpha chain regions.

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

Amino Acid Sequence↗
PubMed1996

Monoclonal antibody directed to a fibrinogen A alpha #529-539 epitope inhibits alpha-chain crosslinking by transglutaminases.

A monoclonal antibody (5A2) recognizing a segment near the C-terminus of the fibrin(ogen) A alpha-chain (A alpha #529-539) was found to inhibit alpha-chain crosslinking catalyzed by coagulation factor XIIIa and by tissue-transglutaminase. The rapid gamma-chain cross-linking by factor XIIIa was not affected by the antibody. Results obtained from direct binding and competitive immunoassay established that the antigenic determinant recognized by 5A2 was included within the CNBr fragment referred to as CNBr X (A alpha #518-584), and that it survived trypsin digestion but was destroyed by treatment with Staph V-8 protease or chymotrypsin. Reverse-phase (C-18) high performance liquid chromatography (HPLC) was employed to obtain a CNBr X tryptic fingerprint, which was subsequently characterized by compositional and NH2-terminal analysis. Assay of the HPLC column effluent revealed a single peak of 5A2 immunoreactivity that coincided with elution of the eleven-residue tryptic peptide, A alpha #529-539. When this isolated peptide and its parent CNBr fragment were employed as solution phase competitors in the 5A2 immunoassay, the relative cross-reactivities (18.3%, peptide: fragment) indicated that a significant proportion of the 5A2 epitope was preserved within the small peptide. This is a region that is released from fibrinogen early in its degradation by plasmin. Thus, the antibody can be used as a probe for intact fibrin(ogen) and C-terminal (A) alpha-chain fragments, in addition to assessing roles of the A alpha-chain C-terminus in cross-linking.

Animals↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1994

The development of assays for the detection of fibrin(ogen)olysis based on COOH-terminal A alpha chain epitopes.

The COOH-terminal two-thirds of the fibrinogen A alpha chain is a substrate for both factor XIIIa and plasmin and is, therefore, a source of structural markers for the clinical detection of fibrin(ogen)olysis. Monoclonal antibodies (MoAbs) that bind to epitopes within this region (F-102, A alpha 563-578; F-103, A alpha 259-276) have been applied towards the development of two sensitive and specific enzyme-linked immunosorbent assays (ELISAs). The first assay, a capture (F-102)-tag (F-103) ELISA, measures plasma fibrinogen molecules whose A alpha chains are intact. The second assay, a solution phase competitive ELISA based on MoAb F-102, quantifies circulating COOH-terminal A alpha chain degradation products (A alpha FDPs), among the earliest peptides released from fibrinogen during plasmin-mediated fragment X formation. This assay features a novel preliminary plasma absorption step on concanavalin A to recover A alpha FDPs (if present in the sample) in a milieu free of immunologically cross-reactive fibrinogen. Both ELISAs use highly purified fibrinogen as the assay standard for quantitation. In control plasmas, circulating A alpha FDPs accounted for less than 2% of their respective intact fibrinogen A alpha chain concentration, suggesting a physiologic low level of proteolysis occurring at the extreme COOH-terminal portion of the molecule. Plasma A alpha FDPs were elevated (2.3% to 7.8% of their respective intact fibrinogen A alpha chain concentration) in a group of plasma from patients with documented, high serum FDPs (21 to 41 micrograms/mL). Application of the two ELISAs to characterize the course of A alpha chain proteolysis during thrombolytic therapy (TIMI phase 1) indicated that A alpha FDPs were a very early marker of the lytic state (detectable 15 minutes after treatment had been initiated), and that streptokinase and recombinant tissue plasminogen activator appeared to produce significantly different A alpha chain degradation profiles.

Chromatography, Affinity↗
PubMed1994

Further studies on the interaction of migrating keratinocytes with fibrinogen.

If glass implants placed under one edge of a skin wound in the adult newt are coated with fibrinogen (FGN), keratinocytes from the wound periphery migrate onto the implant. To learn more about the site(s) in FGN that permits this migration, we exposed keratinocytes to implants coated with forms of FGN containing modifications or deletions in the 3 most commonly studied cell binding sites; the RGDF sequence at A alpha 95-98, RGDS at A alpha 572-575 and the carboxy terminal 12 amino acids in the gamma A chain. Recombinant FGN with either RGD sequence altered to RGE supported migration as well as unmodified FGN did. Replacement of the carboxy terminal 4 amino acids in the gamma A chain by a 20 amino acid sequence that disrupts the ability of the gamma terminus to mediate platelet aggregation (the gamma' variant) likewise had no effect. Nor did simultaneous antibody blockade of the RGDS, RGDF, and gamma A sites have any effect. At its best, Dhem1, a fragment containing the RGDS and gamma A sites, produced only about half as much migration as the maximum obtained on intact FGN. Dhem2, a fragment differing from Dhem1 only by having a gamma' variant in place of gamma A, was even less active. Two other D fragments, both of which were missing a large part of the A alpha chain, and one of which contained none of the three major binding sites, supported considerable migration, suggesting that loss of the A alpha chain COOH terminus reveals a site that was not exposed in Dhem1 and 2. A alpha chain fragments containing the RGDF or RGDS sequence were active, but a much larger fragment without RGD was inactive. A soluble peptide consisting of the sequence, RGDS, was a potent inhibitor of migration on FGN but RGDF and the gamma A pentapeptide, KQAGD, were minimally effective. Longer versions of these peptides decreased the effectiveness in all cases. These results suggest that under certain circumstances, newt keratinocytes may interact with each of the 3 major binding sites in FGN as well as a site outside these sequences.

Amino Acid Sequence↗
PubMed1992

Low molecular weight fibrinogen degradation products stimulate the release of growth factors from endothelial cells.

Cultured porcine aortic endothelial cells (PAEC) constitutively produce and secrete in their culture medium mitogens collectively called endothelial cell-derived growth factors (EDGFs). Incubation of PAEC with fibrinogen-degradation products (FDPs) obtained by plasmin digestion of highly purified fibrinogen caused an increased release of EDGFs, as assessed by [3H]-thymidine incorporation in 3T3 mouse fibroblasts. The effect was time-dependent and correlated with the degree of fibrinogenolysis. It was accompanied by elongation of the cells. Neither increase in EDGFs release nor cell damage was observed when non-degraded fibrinogen was incubated with endothelial cells. Low molecular weight fibrinogen degradation products (LMWFDPs) (M(r) less than or equal to 10,000), and the higher molecular weight fibrinogen fragments D and E were tested under the same conditions. Only the LMWFDPs caused elongation and damage to PAEC and a marked stimulation (up to 12 fold) of EDGFs release. A low density growth assay revealed that the released EDGFs were mitogenically active on the same PAEC. The activity of the released EDGFs was time and dose dependent on both 3T3 fibroblasts and PAEC, indicating that LMWFDPs caused enhanced release of EDGFs that can act in paracrine and autocrine fashion. This study suggests an additional role for fibrinogenolysis contributing to wound healing, and possibly to atherosclerosis.

Animals↗
PubMed1990

Isolation and partial structural characterization of an equine fibrinogen CNBr fragment that exhibits immunologic cross-reactivity with an A alpha-chain cross-linking region of human fibrinogen.

Immunochemical studies of equine fibrinogen were conducted to characterize the structural basis for the immunologic cross-reactivity observed between human and equine A alpha chains when employing an antiserum to the 26K, human cyanogen bromide (CNBr) fragment, A alpha 241-476 (CNBr VIII). A 38K, equine CNBr fragment that reacts with this antiserum was isolated from CNBr-digested equine fibrinogen by Sephadex G-100 gel filtration. It was further purified by sequential hydrophobic chromatography on phenyl-Sepharose CL-4B, followed by reversed-phased (C-8) high-performance liquid chromatography (HPLC). NH2-Terminal analysis of the purified fragment, designated EqA alpha CNBr, identified one major sequence whose first three residues, E-L-E, were identical with those of human CNBr VIII. Tryptic and staphylococcal protease digests of the equine fragment were resolved by reversed-phase HPLC (C-4, C-18), and the separated components were characterized by amino acid analysis and automated Edman degradation. A total of 34 tryptic and 20 staph protease peptides yielded sequence information that permitted the alignment of 271 equine residues with residues A alpha 241-517 from the COOH-terminal two-thirds of the human A alpha chain so that 63% of the possible matches were identical. Other features of interest included (1) an amino acid substitution in which the methionine residue at A alpha 476 in the human A alpha chain was replaced by a valine residue, thus accounting, in part, for the larger EqA alpha CNBr fragment obtained from the equine molecule, and (2) a region of striking homology in which 36 successive residues, corresponding to A alpha 428-464 in the human A alpha chain, were identical in both species. These findings, together with available structural data for the COOH-terminal portion of the rat and bovine A alpha chains, indicate that the region corresponding to (human) A alpha 240-517 represents a conserved portion of the fibrinogen molecule. This may, in turn, explain the difficulties encountered when trying to raise monoclonal antibodies to cross-linking regions that are contained within the COOH-terminal two-thirds of the human A alpha chain.

Amino Acid Sequence↗
PubMed1988

Early alpha chain crosslinking in human fibrin preparations.

Purified fibrinogen preparations were clotted under crosslinking conditions and the evolution of alpha polymers was examined by Western blotting (SDS-PAGE). Monoclonal antibodies that bind to two localized regions within the COOH-terminal portion of the (A) alpha chains of fibrinogen (F-103, A alpha #259-276; F-102, A alpha #540-554) were used for immunodetection. Three crosslinked components (100K, 168K, 210K) that each exhibited coincident F-102 and F-103 immunoreactivities were evident as early as gamma dimer formation under the in vitro conditions employed. Initial events in the alpha chain crosslinking process appeared to include interactions between relatively intact chains and a variety of degraded ones that shared the structure A alpha #1-276, but differed in the extent to which regions between residues approximately #276 and approximately #539 were preserved. Degraded fibrinogen molecules whose A alpha chains all terminated before A alpha #540-554 were isolated from purified fibrinogen preparations by immunoaffinity chromatography on F-102 Sepharose. Immunoblotting data obtained for the crosslinking capacity of these degraded molecules indicated that early crosslinked components (95 K, 205 K) could form even in the absence of intact alpha chain partners. This crosslinking, moreover, could progress to the polymer stage. These findings demonstrate that some early crosslinking activity is localized exclusively within regions NH2-terminal to A alpha approximately #540-554 and suggest that fibrinogen molecules with partially degraded A alpha chains, which are likely to be circulating under many pathophysiologic conditions, can undergo fibrin stabilization through crosslinking despite a loss of at least 70 COOH-terminal A alpha chain residues.

Blotting, Western↗
PubMed1988

Immunochemical characterization of crosslinked derivatives isolated from alpha chain oligomers formed during early stages of fibrin crosslinking.

Early crosslinked alpha chain oligomers (E alpha XL) that formed within 1/2 h of in vitro clot formation were isolated from preparations of reduced, carboxymethylated fibrin by gel filtration on Sepharose CL-4B. Cyanogen bromide (CNBr) digestion of E alpha XL released a mixture of fragments which could be partially separated by Sephadex G-150 chromatography. Crosslinked and non-crosslinked forms of the A alpha chain peptides, CNBr VIII (A alpha #241-476) and CNBr X (A alpha #518-584) were identified in the column effluent both by radioimmunoassay (RIA) and by immunoblotting using polyclonal antisera (anti-CNBr VIII, anti-CNBr X) and monoclonal antibodies (F-103, binds to A alpha #259-276; F-102, binds to A alpha #540-554), respectively. Immunoaffinity chromatography of the crosslinked material, on F-103, followed by F-102 Sepharose, resulted in the separation of two types of fragments, each of which contained an early alpha chain crosslink. One of these (35-37K) exhibited coincident CNBr VIII and CNBr X immunoreactivities, while the other (54-59K) exhibited only CNBr VIII immunoreactivity, based on immunoblotting and RIA findings. NH2-terminal sequencing and cyanoethylation data provided biochemical evidence for the occurrence of covalent CNBr VIII-X as well as covalent CNBr VIII-VIII interactions during early alpha chain crosslinking. These findings indicate, for the first time, that both glutamine acceptor and lysine donor activities may be localized within CNBr VIII. This information should facilitate the chemical identification of alpha chain residues that partner during factor XIIIa-catalyzed cross-linking and thus enable development of reagents for the generation of fibrin-specific antibodies.

Amino Acids↗
PubMed1986

Immunologic identification of the cleavage products from the A alpha- and B beta-chains in the early stages of plasmin digestion of fibrinogen.

Fragment X components (Mr 225,000 to 333,000) were distinguished on sodium dodecyl sulfate polyacrylamide gels. Western blotting with monoclonal antibodies to A alpha-chain segments demonstrated that the A alpha-chains of fibrinogen and the largest fragment X components (Mr 285,000-340,000) contained both A alpha 259-276 and A alpha 540-554. Fragment X components of Mr 270,000-285,000 contained A alpha 259-276 but lacked A alpha 540-554, whereas the smallest fragment X components (Mr 225,000-270,000) contained neither A alpha 540-554 nor A alpha 259-276. Studies of the small peptides generated during fragment X formation complemented the studies of the large molecules, by demonstrating peptides containing both A alpha 259-276 and A alpha 540-554 (Mr 41,600-41,800 and Mr 38,700-38,900), peptides containing A alpha 540-554 but not A alpha 259-276 (Mr 20,500-21,000 and Mr 17,300-17,500) and peptides containing only A alpha 259-276 (Mr 23,600-24,000 and Mr 20,500-21,000). Cleavage of B beta 1-42 from the amino terminal ends of the B beta-chains, measured with a specific radioimmunoassay, was linear until 1.6 moles per mole of fibrinogen had been released, and coincided with loss of the central and carboxy terminal A alpha-chain regions, i. e. A alpha 259-276 and A alpha 540-554. Based on present and previously reported data, a model is proposed for the evolution of the heterogeneous group of fragment X derivatives from fibrinogen with the simultaneous release of small peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1983

Monoclonal antibody to the region of fibronectin involved in cross-linking to human fibrin.

A cross-link-containing fragment (alpha XLCNBr), derived from the alpha-polymer component of human fibrin following CNBr digestion, has been isolated and characterized. NH2-terminal sequence studies of three alpha XLCNBr derivatives, each prepared by a different method, indicate that the A alpha-chain regions comprised of residues 241-476 (CNBr VIII) and 518-584 (CNBr X) are the major constituents of the cross-linked fragments examined. Evidence for at least two additional sequences suggests that A alpha 208-235 (CNBr V) and A alpha 585-610 (CNBr XI) may have auxiliary roles in alpha-polymer formation. When alpha XLCNBr was used as an immunogen for the production of murine hybridoma cell lines, two groups of antibodies were obtained. The majority of supernatants from primary hybridoma cultures did not discriminate between fibrinogen and alpha XLCNBr and appeared to contain antibodies directed against determinants within either CNBr VIII or CNBr X [see Ehrlich, P. H., Sobel, J. H., Moustafa, Z. A., & Canfield, R. E. (1983) Biochemistry (following paper in this issue)]. Several supernatants from primary hybridoma cultures, however, did exhibit significant binding toward the alpha-polymer derivative in the absence of demonstrable immunoreactivity toward either highly purified fibrinogen or its A alpha-chain peptides, CNBr VIII and CNBr X.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1983

Monoclonal antibodies to alpha-chain regions of human fibrinogen that participate in polymer formation.

Monoclonal antibodies have been generated against a cross-link-containing derivative of alpha polymer (alpha XLCNBr), isolated following CNBr digestion of fibrin [Sobel, J. H., Ehrlich, P. H., Birken, S., Saffran, A. J., & Canfield, R. E. (1983) Biochemistry (preceding paper in this issue)]. One cloned cell line (F-102) was chosen for characterization based on its apparent specificity for the A alpha-chain region A alpha 518-584 (CNBr X). A second line (F-103) was selected because of its anti-A alpha 241-476 (CNBr VIII) properties. These two regions of the A alpha chain have previously been implicated as major contributors to the cross-linking process that leads to alpha-polymer formation. Radioimmunoassays have been developed, employing the immunoglobulins produced by clones F-102 and F-103. These assays have been applied, in conjunction with high-performance liquid chromatography purified tryptic and chymotryptic derivatives of CNBr VIII and CNBr X, to localize the respective determinants involved in antibody binding. In each case, virtually full immunoreactivity was exhibited by both the CNBr fragment and a single tryptic or chymotryptic peptide originating from it. These findings indicate that sequence-specific, rather than conformational, determinants were operative in the generation of antibodies F-102 and F-103. The epitope recognized by F-102 was localized to the region of A alpha 540-554, while the F-103 binding site resided within A alpha 259-276. When these radioimmunoassays were applied to study the relative immunoreactivity exhibited by a variety of fibrinogen derivatives, the results obtained support earlier suggestions that the COOH-terminal portion of the A alpha chain contains regions of random conformation.

Amino Acids↗
PubMed1982

Alpha chain crosslinks of human fibrin: purification and radioimmunoassay development for two A alpha chain regions involved in crosslinking.

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 A alpha chain of human fibrinogen. Amino acid analysis, NH2-terminal sequence determination, and SDS-PAGE indicated that greater than 95% purity of each peptide was achieved. Sensitive and specific radioimmunoassays capable 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 A alpha 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 a alpha polymer, the data provided immunologic confirmation for the involvement of CNBr VIII and CNBr X in alpha chain crosslinking.

Amino Acid Sequence↗