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

Ehrlich, Paul H.; Sobel, Joan H.; Moustafa, Zeinab A.; Canfield, Robert E.

doi:10.1021/bi00287a004

Cited by 22 publications

(21 citation statements)

References 30 publications

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“…This report details the characteristics of the cross-linked derivative, aXLCNBr, used for immunization and describes the features of one monoclonal antibody, fortuitously discovered, that appears to react with a portion of fibronectin involved in cross-linking to the a chain of fibrin. The following paper (Ehrlich et al, 1983) describes two additional antibodies raised against the same immunogen; these recognize defined segments within CNBr VI11 and CNBr to the solid phase. p: I-RAM IgG displacement was measured (see VI11 (Au 442 and 472) via disulfide interchange occurring under the influence of the reducing agents employed for the stabilization of activated factor XI11 during clot formation.…”

Section: Discussionmentioning

confidence: 99%

“…We also detail the immunochemical characterization of a monoclonal antibody, generated against this immunogen, which reacts with fibronectin and appears to recognize a determinant located within the region of fibronectin that is involved in cross-linking to fibrin. An accompanying report (Ehrlich et al, 1983) describes the charmonitored for 280-nm absorbance and radioactivity. Fractions containing high molecular weight material were pooled and lyophilized (Figure 1 B) , When CNBr-digested fibrin was used as a source of aXLCNBr, high molecular weight fragments were isolated and then purified free of disulfide-containing contaminants according to conditions described by Doolittle et al (1977) in their studies of a-polymer derivatives.…”

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confidence: 99%

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Monoclonal antibody to the region of fibronectin involved in crosslinking to human fibrin

Sobel¹,

Ehrlich²,

Birken³

et al. 1983

Biochemistry

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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)

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Monoclonal antibody to the region of fibronectin involved in crosslinking to human fibrin

Sobel¹,

Ehrlich²,

Birken³

et al. 1983

Biochemistry

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“…These included the anti-fibrinogen mAbs, F-103 and F-102, that bind to epitopes within the COOH-terminal two-thirds of the A␣ chain (A␣ 259 -276 and A␣ 563-578, respectively) (32,33) and the anti-peptide mAb, AP-102 (27). F-103-and F-102-Sepharoses were prepared as previously detailed (34), and these same procedures were used for the construction of AP-102-Sepharose; approximately 3 mg of IgG were coupled per ml of gel (38 -58 nmol antibody combining sites/ml).…”

Section: Methodsmentioning

confidence: 99%

Identification of the α Chain Lysine Donor Sites Involved in Factor XIIIa Fibrin Cross-linking

Sobel

Gawinowicz

1996

Journal of Biological Chemistry

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Biochemical studies of fibrin cross-linking were conducted to identify the specific A␣ chain lysine residues that potentially serve as Factor XIII a amine donor substrates during ␣ polymer formation. A previously characterized Factor XIII a 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 NH 2 -terminal cross-linking domain of ␣ 2 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 ␣ chain CNBr fragments (within A␣ 208 -610) and one ␥ chain CNBr fragment (␥ 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 ␣ 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 ␣ 208 -610 had incorporated the peptide probe, whereas ␥ chain donor activity was due solely to peptide cross-linking at (␥) Lys , and/or Lys 219 responsible for the remaining proportion (2-5%, each). The collective findings extend current models proposed for the mechanism of ␣ polymer formation, raise questions concerning the physiological role of multiple ␣ 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 ␣ chain cross-linking.The structure-function relationships involved in fibrinogen's transition to the cross-linked fibrin gel that forms the hemostatically active portion of a thrombus has been the subject of intense investigation for more than two decades. During this time, the complete primary structure of this large molecule has been elucidated (1-6), its domainal architecture has been characterized (7-9), and the mechanisms involved in the initial events of the transition, i.e. thrombin cleavage and fibrin polymerization, have been defined (as reviewed in Ref. 10). Although these aspects of fibrinogen biochemistry are well understood, less is known about the final stages of fibrin formation in which Factor XIII a cross-links are introduced between neighboring fibrin molecules to stabilize the alignments created during polymerization.Factor XIII a , or plasma transglutaminase, catalyzes the introduction of ⑀-(␥-glutamy...

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“…The development and characterization of mAbs F-102 (anti-A␣-(563-578)) and F-103 (anti-A␣-(259 -276)), raised against a purified CNBr derivative of the ␣ polymer component of cross-linked fibrin, have been previously described (23,24). mAb 5A2 (anti-A␣-529 -539)) was produced following immunization with a preparation of partially purified CNBr fibrin derivatives and selected based on its preferential binding to fibrin, as distinct from fibrinogen, in a direct-binding screening ELISA on antigen-coated wells.…”

Section: Monoclonal Antibodies To Defined A␣ Chain Regionsmentioning

confidence: 99%

Comparative Structural and Functional Features of the Human Fibrinogen αC Domain and the Isolated αC Fragment

Rudchenko

Trakht

Sobel

1996

Journal of Biological Chemistry

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The ␣C domain of fibrinogen (A␣-(220 -610)) plays a central role in maintaining hemostasis by serving as a substrate for factor XIII a and plasmin. Monoclonal antibodies that recognize eight distinct epitopes within the COOH-terminal two-thirds of the A␣ chain were employed as structural probes to: 1) isolate the human ␣C domain, 2) compare the topography of the eight epitopes within the ␣C domain of intact fibrinogen and in purified ␣C fragments, and 3) explore the degree to which the ␣C domain's role as a factor XIII a substrate in intact fibrinogen is preserved within the structure of isolated ␣C fragments. Five antibodies were raised against small, synthetic peptide immunogens (A␣-(220 -230), A␣-(425-442), A␣-(487-498), and A␣-(603-610)), and three were generated against larger cyanogen bromide (A)␣ chain derivatives with each epitope subsequently localized to discrete A␣ chain sequences (A␣-(259 -276), A␣-(529 -539), and A␣-(563-578)). Human ␣C preparations were isolated from mild plasmin digests of fibrinogen by successive chromatography on concanavalin A-Sepharose, anti-A␣-(425-442)-Sepharose, and Superdex-75 fast protein liquid chromatography. Immunochemical characterization indicated that the NH 2 -terminal residue of ␣C fragments was either A␣-220 or A␣-231 and that, although the extreme COOH-terminal region, A␣-(603-610), was absent, all molecules were intact at least through A␣-(563-578). Solution phase competitive assays indicated that the release of the ␣C domain from intact fibrinogen was associated with several conformational changes, e.g. in the vicinity of A␣-(220 -230), A␣-(259 -276), A␣-(487-498), and A␣-(529 -539), but that the relative accessibility of other localized structures remained unchanged, e.g. A␣-(425-442) and A␣-(563-578). Immunoblotting analysis of ␣C cross-linking in vitro revealed that isolated ␣C fragments could serve as a substrate for factor XIII a . Immunoblotting studies of the A␣ chain proteolysis that occurs during thrombolytic therapy indicated that ␣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 ␣C domain and its functional implications and also draw attention to the as yet unexplored role of ␣C fragments in the pathophysiology of thrombosis and hemostasis.The past decade has witnessed a growing interest in the structure of the fibrinogen ␣C domain, i.e. the COOH-terminal two-thirds of the A␣ chain that extends from the coiled-coil portion of each half of the dimeric fibrinogen molecule. Elucidation of this region's structural features is of considerable interest given its multifunctional role in maintaining hemostasis. Factor XIII a cross-linking sites included within the ␣C domain are responsible for the covalent bonds created between fibrin ␣ chains and between ␣ chains and ␣ 2 PI 1 that lead to the formation of a highly cross-linked ␣ polymer network (1-6). The re...

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

Cited by 22 publications

References 30 publications

Monoclonal antibody to the region of fibronectin involved in crosslinking to human fibrin

Monoclonal antibody to the region of fibronectin involved in crosslinking to human fibrin

Identification of the α Chain Lysine Donor Sites Involved in Factor XIIIa Fibrin Cross-linking

Comparative Structural and Functional Features of the Human Fibrinogen αC Domain and the Isolated αC Fragment

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