Fibrinogen Fragments Research Articles

Production of analytical quantities of recombinant proteins in Chinese hamster ovary cells using sodium butyrate to elevate gene expression

Sodium butyrate was used to enhance expression levels and thereby facilitate the generation of analytical quantities of nine different tissue plasminogen activator (tPA) analogues expressed under the control of the cytomegalovirus immediate early (CMV IE) promoter by the Chinese hamster ovary (CHO) mammalian expression system. Production involved growth in roller bottles, using serum free or low serum media formulations, together with repetitive, sodium butyrate inductions. Average inductions in the presence of sodium butyrate ranged from 2 to 9-fold relative to uninduced controls, using cell lines with no previous butyrate exposure. Retardation of growth rate by butyrate minimized the need to split cells during the production runs, extending longevity of roller bottles containing cells secreting at induced levels. SDS-PAGE analyses indicate a consistently high percentage of single-chain material. Measurements of specific activity and fibrinogen fragment enhancement for one of the analogues demonstrate that neither of these two critical parameters are affected by production in the presence of butyrate. Induction kinetic data and growth curves for the expression of sCD4 under control of the SV40 early promoter demonstrate that the benefits of butyrate can be realized with different promoters and heterologous genes, and are additive when used in conjunction with an amplified cell line constitutively expressing at elevated elevels. This work demonstrates the practical application of sodium butyrate in the production of analytical quantities of protein from the CHO expression system, and suggests a role for sodium butyrate in commercial scale processes as well.

Selective recognition of adhesive sites in surface-bound fibrinogen by glycoprotein IIb-IIIa on nonactivated platelets

We demonstrate that unstimulated platelets attach to immobilized fibrinogen in a selective process mediated by the membrane glycoprotein (GP) complex IIb-IIIa (alpha IIb beta 3). The initial attachment, independent of platelet activation, is followed by spreading and irreversible adhesion even in the presence of activation inhibitors. Using fibrinogen fragments derived from plasmin digestion, we found that unstimulated platelets do not attach to immobilized fragment E, which contains an Arg-Gly-Asp sequence at A alpha 95-97, and adhere to fragments X and D, both containing the gamma 400-411 dodecapeptide adhesion sequence, less efficiently than to intact fibrinogen. Thus, the carboxyl terminus of the A alpha chain, missing in the "early" fragment X used in these studies, appears to be involved in the interaction of fibrinogen with unstimulated platelets. In contrast, activated platelets adhere to immobilized fibrinogen and fragments X, D, and E in a time-dependent and equivalent manner. Although activated platelets adhere to immobilized vitronectin, fibronectin, and von Willebrand factor through GP IIb-IIIa, unstimulated platelets fail to adhere to vitronectin and have only a limited capacity to adhere to fibronectin and von Willebrand factor. These results demonstrate that GP IIb-IIIa on unstimulated platelets displays a recognition specificity for attachment to immobilized adhesive proteins that is distinct from that seen following platelet activation. Thus, unstimulated platelets selectively interact with fibrinogen, and the initial attachment is followed by spreading and irreversible adhesion in the absence of exogenous agonists. This process may be regulated by plasmin cleavage of the fibrinogen A alpha chain and may play an important role during normal hemostasis and during the pathological development of thrombotic vascular occlusions.

Development and Application of Assays for Elastase‐Specific Fibrinogen Fragmentsa

Annals of the New York Academy of SciencesVolume 624, Issue 1 p. 154-166 Development and Application of Assays for Elastase-Specific Fibrinogen Fragmentsa JEFFREY I. WEITZ, Corresponding Author JEFFREY I. WEITZ Department of Medicine McMaster University and Hamilton Civic Hospitals Research Centre Hamilton, OntarioDr. Jeffrey Weitz, Henderson General Hospital, 711 Concession Street, Hamilton, Ontario, L8V 1C3, Canada.Search for more papers by this author JEFFREY I. WEITZ, Corresponding Author JEFFREY I. WEITZ Department of Medicine McMaster University and Hamilton Civic Hospitals Research Centre Hamilton, OntarioDr. Jeffrey Weitz, Henderson General Hospital, 711 Concession Street, Hamilton, Ontario, L8V 1C3, Canada.Search for more papers by this author First published: May 1991 https://doi.org/10.1111/j.1749-6632.1991.tb17015.xCitations: 3 a This work was supported by grants from the Medical Research Council of Canada and the Ontario Heart and Stroke Foundation. Dr. Weitz is a Scholar of the Heart and Stroke Foundation of Ontario. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume624, Issue1Pulmonary Emphysema: The Rationale for Therapeutic InterventionMay 1991Pages 154-166 RelatedInformation

Soluble fibrin preparations inhibit the reaction of plasmin with α2-macroglobulin. Comparison with α2-antiplasmin and leupeptin

The kinetics of plasmin inhibition by alpha 2-antiplasmin (alpha 2AP), alpha 2-macroglobulin (alpha 2M) and leupeptin were studied in the presence of fibrin monomer (Fn) and CNBr fragments of fibrinogen (Fg-CNBr). Active plasmin was detected in continuous and discontinuous assays using the chromogenic substrate D-Val-L-Leu-L-Lys p-nitroanilide hydrochloride (S-2251). The two 'fibrin-like' preparations functioned as hyperbolic mixed-type inhibitors of S-2251 hydrolysis. The dissociation constants (KF) for the binding of plasmin to Fn and Fg-CNBr were 22 nM and 17 nM respectively. Fn and Fg-CNBr inhibited the reaction of plasmin with alpha 2AP: the extent of inhibition depended on the fibrin concentration. In the presence of 800 nM-Fn or 800 nM-Fg-CNBr, the experimental second-order rate constant (K"app.) was decreased from 2.4 x 10(7) M-1.s-1 to 1.2 x 10(6) and 5.3 x 10(5) M-1.s-1 respectively. The effect of Fn and Fg-CNBr on the rate of plasmin inhibition by alpha 2M was even greater. The k"app. value was decreased from 4.0 x 10(5) M-1.s-1 to 8.0 x 10(2) and 1.3 x 10(3) M-1.s-1 in the presence of 800 nM-Fn and -Fg-CNBr respectively. By contrast, the fibrin preparations caused only a small change in the rate of plasmin inhibition by leupeptin. The maximum change in k"app. was 3-fold. All plasmin inhibition curves were linear, suggesting that free and fibrin-bound forms of plasmin remained in equilibrium during the course of reaction with proteinase inhibitors. Fn and Fg-CNBr had no effect on the reaction of miniplasmin with S-2251, alpha 2AP or alpha 2M. When 125I-plasmin was incubated with Fg-CNBr and then allowed to react with a premixed solution of alpha 2AP and alpha 2M, the Fg-CNBr did not significantly change the percentage of plasmin bound to alpha 2AP. These experiments demonstrate that the reaction of plasmin with alpha 2M is inhibited by the non-covalent binding of plasmin to fibrin. We propose that plasmin bound to the surface of a clot is protected from inhibition by alpha 2M as well as by alpha 2AP.

CD11c/CD18 on neutrophils recognizes a domain at the N terminus of the A alpha chain of fibrinogen.

Fibrinogen and fibrin serve as adhesive substrates for a variety of cells including platelets, endothelial cells, and leukocytes. Previously, we identified the C terminus of the gamma chain of fibrinogen as the region of the fibrinogen molecule that contains a ligand for CD11b/CD18 (complement receptor 3) on phorbol ester-stimulated polymorphonuclear leukocytes. In contrast, we report here that neutrophils stimulated with tumor necrosis factor adhere to fibrinogen-coated surfaces, but not to human serum albumin-coated surfaces, via the integrin CD11c/CD18 (p150/95). Monoclonal antibodies LeuM5 and 3.9, which are directed against the alpha subunit of CD11c/CD18, but not monoclonal antibodies OKM10 and OKM1, which are directed against the alpha subunit of CD11b/CD18, inhibit the adhesion of tumor necrosis factor-stimulated neutrophils to fibrinogen-coated surfaces. To identify the site on fibrinogen recognized by CD11c/CD18, we have examined the adhesion of tumor necrosis factor-stimulated neutrophils to surfaces coated with various fibrinogen fragments. Stimulated neutrophils adhere to surfaces coated with the N-terminal disulfide knot fragment of fibrinogen or fibrinogen fragment E. Moreover, peptides containing the sequence Gly-Pro-Arg (which corresponds to amino acids 17-19 of the N-terminal region of the A alpha chain of fibrinogen), and monoclonal antibody LeuM5, block tumor necrosis factor-stimulated neutrophil adhesion to fibrinogen and to the N-terminal disulfide knot fragment of fibrinogen. Thus, CD11c/CD18 on tumor necrosis factor-stimulated neutrophils functions as a fibrinogen receptor that recognizes the sequence Gly-Pro-Arg in the N-terminal domain of the A alpha chain of fibrinogen.

Fibronectin decreases the stimulatory effect of fibrin and fibrinogen fragment FCB-2 on plasmin formation by tissue plasminogen activator

Fibronectin is a dimeric glycoprotein (Mr 440,000) involved in many adhesive processes. During blood coagulation it is bound and cross-linked to fibrin. Fibrin binding is achieved by structures (type I repeats) which are homologous to the "finger" domain of tissue plasminogen activator. Tissue plasminogen activator also binds to fibrin via the finger domain and additionally via the "kringle 2" domain. Fibrin binding of tissue plasminogen activator results in stimulation of its activity and plays a crucial role in fibrinolysis. Since fibronectin might interfere with this binding, we studied the effect of fibronectin on plasmin formation by tissue plasminogen activator. In the absence of fibrin, fibronectin had no effect on plasminogen activation. In the presence of stimulating fibrinogen fragment FCB-2, fibronectin increased the duration of the initial lag phase (= time period until maximally stimulated plasmin formation occurs) and decreased the rate of maximal plasmin formation which occurs after that lag phase mainly by increasing the Michaelis constant (Km). These effects of fibronectin were dose-dependent and were similar with single- and two-chain tissue plasminogen activator. They were also observed with plasmin-pretreated FCB-2. An apparent Ki of 43 micrograms/ml was calculated for the inhibitory effect of fibronectin when plasminogen activation by recombinant single-chain tissue plasminogen activator was studied in the presence of 91 micrograms/ml FCB-2. When a recombinant tissue plasminogen activator mutant lacking the finger domain was used in a system containing FCB-2, no effect of fibronectin was seen, indicating that the inhibitory effect of fibronectin might in fact be due to competition of fibronectin and tissue plasminogen activator for binding to fibrin(ogen) via the finger domain.

Interaction of hementin with fibrinogen and fibrin

The giant Amazon leech Haementeria ghilianii manufactures blood anticoagulant which is present in the posterior and anterior salivary glands. The mechanism of blood anticoagulation by Haementeria ghilianii is completely different from that used by Hirudo medicinalis. The anticoagulant is mostly associated with a fibrinogen-degrading proteinase, hementin. However, other inhibitors of blood coagulation are also present in the salivary glands. The salivary gland extract inhibits platelet aggregation that is mostly attributable to the degradation of fibrinogen. Hementin purified by various methods has a molecular weight in the range of 80,000-120,000 and appears to be a metalloproteinase that is regulated by calcium ions. The enzyme degrades both fibrinogen and fibrin. The Michaelis constant for human fibrinogen is 1 microM. The cleavage of the isolated chains of fibrinogen is inefficient implying that the native conformation of the substrate may play a role in the recognition mechanism. The pattern of fibrinogen degradation by hementin resembles that caused by plasmin since products analogous to fragments Y, D and E are generated. However, the unique action of hementin on fibrinogen is in the initial proteolytic attack in the coiled-coil connector region while proteolysis of the alpha-chain is very slow. In consequence, unique fibrinogen fragments are formed that contain the entire COOH-terminus of the alpha-chain. The mechanism of blood anticoagulation by hementin is very efficient since the cleavage of only three peptide bonds in the fibrinogen molecule disassembles its bivalent structure and renders it non-functional.

The Structure and Function of Candida albicans Cell Wall

The wall and outer surface of pathogenic fungi is involved in adhesion and cell shape maintenance, and acts as a barrier to metabolites and drugs. In addition the cell wall contains immunogenic determinants, immunomodulators and secreted enzymes. The cell wall of C. albicans comprises some 30% of the dry weight of the cell and is composed predominantly of mannoproteins and β-glucans with small quantities of chitin, protein and lipid. Transmission electronmicroscopy of the wall reveals a number of different layers; the thickness and number of these layers varies during germ-tube formation and the cell cycle. The outer fuzzy layer of the wall contains receptors that bind plastic, laminin, fibrinogen, and C3 fragments of complement. The β1, 3 and β1, 6 glucans and chitin are the polymers which endow rigidity on the cell wall. These polymers form a microfibrillar scaffolding upon which other macromolecules, particularly mannoproteins are bound.The wall mannoproteins are highly branched polysaccharides primarily composed of mannose units attached to protein through a G 1 cNac dimer bridge and asparagine. There are also smaller numbers of 0-linked mannoproteins where the mannose units are attached to protein through serine and threonine residues. The mannoproteins have a backbone of α-1, 6 linked mannose units to which side chains of mannoses attach via α-1, 2-and occasionally α-1, 3 bonds. The side chains are the major antigenic determinants and the A and B serotypes are distinguished by differences between these side chains. A model for cell wall growth and development will is described.

Tissue plasminogen activator mutants lacking the growth factor domain and the first kringle domain: I: DNA Constructions, Expression in Mammalian Cells, Protein Structure, Fibrin Affinity and Enzymatic Properties

Six variants of tissue-type plasminogen activator (t-PA) were produced in mouse C127 cells using a bovine papilloma virus expression vector. All variants lacked the growth factor (G) domain and the first kringle domain (K1) and three of the variants also lacked the finger domain (F). Furthermore, the specific changes, Lys 277 → Val and Asn 448 → Gln were introduced into some of the molecules. The variants were denoted K2P, K2P(Val277), K2P(Gln448), FK2P, FK2P(Va1277), and FK2P(Gln448). Amino acid sequence analyses revealed that the variants were proteolytically processed in the amino terminus and at Arg 275-Ile 276 in the same way as the full sized molecule. A proteolytically sensitive site was identified in the F domain at Arg 27-Ser 28. The two variants that lacked glycosylation at Asn 448, K2P(GIn448) and FK2P(GIn448), were cleaved at Arg 449-Thr 450, indicating that the oligosaccharide normally present at Asn 448 protects this site against proteolysis. The fibrin affinity for all variants was markedly reduced compared with normal t-PA. The plasminogen activator activity of all variants was stimulated by cyanogen bromide fragments of fibrinogen. In an indirect chromogenic assay K2P and K2P(Val277) showed specific activities that were 23% and 36%, respectively, of that of wild type t-PA, while the corresponding non-glycosylated variant K2P(Gln448) was as active as t-PA. The activity of the three F domain-containing variants were between 88 and 98% of the value determined for t-PA. When the specific activity was determined with the fibrin plate assay all variants were found to have higher specific activities than t-PA (1.8–4.7 fold). The lack of correlation between the activity of t-PA and the variants in these two assays indicate that the reaction mechanism may differ between the variants and wild type t-PA. The kinetic constants K m and k cat were determined for two-chain forms of t-PA and the variants with the chromogenic peptide substrate DIleProArgpNA. The results show that the t-PA heavy chain is not affecting the reaction with small peptide substrates as the K m and k cat values were essentially identical for t-PA, K2P, and FK2P (K m, 0.18–0.21 mM and k cat, 8.8–11.1s −1). The values for the two non-glycosylated variants K2P(Gln448) and FK2P(Gln448) were 0.28mM, 9.4s −1 and 0.24mM, 5.3s −1, respectively. Interestingly, the Val277 variants showed significantly reduced K m values, suggesting that Lys 277 is important for the substrate interaction. For the variant K2P(Val277) K m and k cat were 0.06mM and 6.5s −1, respectively, and for FK2P(Val277) the corresponding values were 0.06mM and 6.0s −1.

Establishment of Solid Phase GPIIb/IIIa Binding Assay Using Biotinylated Fibrinogen as a Ligand

We have established a solid phase binding assay using biotinylated fibrinogen as a ligand. Biotinylation did not alter its binding capacity and affinity to solid phase GPIIb/IIIa. Biotinylated fibrinogen binding, as measured by color development of enzymatic reaction, was specific, time-dependent, reversible, saturable and divalent cation-dependent. Unlabeled fibrinogen and three peptide fragments of fibrinogen, RGDV, RGDS and HHLGGAKQAGDV (gamma-chain 400-411) competitively inhibited the binding with IC50 values of 3.7, 49, 155 and 715nM, respectively. The apparent dissociation constant (Kd) of biotinylated fibrinogen was determined to be 0.33nM.This simple and high-flux solid phase binding assay can be useful to directly detect the binding between fibrinogen and GPIIb/IIIa.

Specific cell components of Bacteroides gingivalis mediate binding and degradation of human fibrinogen

Bacteroides (Porphyromonas) gingivalis, which has been implicated as an etiologic agent in human periodontal diseases, has been shown to bind and degrade human fibrinogen. B. gingivalis strains bind fibrinogen reversibly and with high affinity and bind to a specific region of the fibrinogen molecule that appears to be located between the D and E domains (M. S. Lantz, R. D. Allen, P. Bounelis, L. M. Switalski, and M. Hook, J. Bacteriol. 172:716-726, 1990). We now report that human fibrinogen is bound and then degraded by specific B. gingivalis components that appear to be localized at the cell surface. Fibrinogen binding to bacterial cells occurred at 4, 22, and 37 degrees C. A functional fibrinogen-binding component (Mr, 150,000) was identified when sodium dodecyl sulfate-solubilized bacteria were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with 125I-fibrinogen. Fibrinogen degradation did not occur at 4 degrees C but did occur at 22 and 37 degrees C. When bacteria and iodinated fibrinogen were incubated at 37 degrees C, two major fibrinogen fragments (Mr, 97,000 and 50,000) accumulated in incubation mixture supernatant fractions. Two major fibrinogen-degrading components (Mr, 120,000 and 150,000) have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in substrate-containing gels. Fibrinogen degradation by the Mr-120,000 and -150,000 proteases was enhanced by reducing agents, completely inhibited by N-alpha-p-tosyl-L-lysyl chloromethyl ketone, and partially inhibited by n-ethyl maleimide, suggesting that these enzymes are thiol-dependent proteases with trypsinlike substrate specificity. The fibrinogen-binding component could be separated from the fibrinogen-degrading components by selective solubilization of bacteria in sodium deoxycholate.

Effect of Fibrinogen Fragments D and E on the Adhesive Properties of Human Granulocytes to Venous Endothelial Cells

In hyperfibrinolytic conditions, e.g. in disseminated intravascular coagulation or the adult respiratory distress syndrome, high levels of fibrinogen degradation products (FDPs) D and E are found in human plasma. This study investigates the influence of these fragments on cell attachment of human granulocytes in vitro. While leaving unaffected the adhesion of human umbilical vein endothelial cells (HUVEC) on gelatine-coated glass, both FDP fragments at 50 micrograms/ml inhibited granulocyte attachment to glass as well as to HUVEC monolayers. At the same concentration, the fragments diminished the superoxide release of stimulated granulocytes. These results suggest a modulatory role of pathologically elevated FDPs on the granulocyte function cascade.

Validity of enzyme-linked immunosorbent assays of cross-linked fibrin degradation products as a measure of clot lysis.

Concentrations of cross-linked fibrin degradation products (XL-FDPs) in plasma, measured by enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies (MAbs) raised against fragment D-dimer of cross-linked fibrin, increase when patients are given fibrinolytic agents. Whether XL-FDPs derive from circulating cross-linked fibrin polymers in plasma, compared with clot-associated fibrin, has been questioned because increases in XL-FDP are measured by some assays after fibrinolysis in vitro in the absence of clot. We characterized the source of XL-FDP immunoreactivity in plasma of patients with acute myocardial infarction and ischemic heart disease and the response to plasminogen activation in vitro induced by pharmacological concentrations of tissue-type plasminogen activator (t-PA) and streptokinase. XL-FDPs were measured with two different ELISA. One, "pan-specific tag ELISA," was based on a capture MAb specific for XL-FDP and a tag MAb that recognizes an epitope exposed in the fragment D region of both fibrin and fibrinogen, whereas the other, "fibrin-specific tag ELISA," was based on a capture and tag MAbs both specific for fibrin. After plasminogen activation was induced in vitro in plasma from patients with myocardial infarction, increased concentrations of XL-FDP were measured by the pan-specific tag ELISA; however, concentrations measured with the fibrin-specific tag ELISA were not increased. To determine the mechanism for this discrepancy, plasma was subjected to immunoadsorption with a MAb specific for fragment D-dimer before and after in vitro activation of the fibrinolytic system and immunoblotting with a fragment D-dimer-specific MAb and with the pan-specific MAb. Increased concentrations of fragment D-dimer, as well as fibrinogen fragment D at high concentrations, were recognized by the specific MAb. Non-cross-linked fragments were also shown by immunoblotting with the pan-specific MAb to coprecipitate with cross-linked fibrin fragments. This suggested the increased concentrations of XL-FDP measured by the pan-specific tag ELISA after in vitro activation of the fibrinolytic system were due to detection of non-cross-linked fibrinogen fragments. However, fibrin fragment D-dimer concentrations were found to increase in plasma of 15 patients given t-PA for acute myocardial infarction. We conclude fragment D-dimer in plasma of patients during thrombolysis does not originate from circulating soluble cross-linked fibrin but rather is a marker of solid-phase fibrin dissolution, which may be quantitated with assays based on capture and tag antibodies that do not detect fibrinogen or its degradation products.

Structures involved in the binding of human fibrinogen toCandida albicansgerm tubes

Recent evidence for the interaction between human fibrinogen and Candida albicans germ tubes have led us to attempt to characterize the structures involved. Using 125I-radiolabeled proteins, fibrinogen purified by affinity chromatography and its plasmin degradation products, the binding sites on the fibrinogen molecule appeared to be located specifically in the D-domain. Conversely to the fibrinogen and the fragment D, radiolabeled fragment E, however, did not interact with cells. The binding was time-dependent, saturable and reversible. Scatchard analysis of the data obtained revealed an average of 6000 binding sites per germ tube with dissociation constant (Kd) of 5.2 X 10(-8) M. No potent competition was observed for a range of different proteins and carbohydrates. Fibrinogen fragment D binding proteins were identified using a dithiothreitol-iodoacetamide extract of the fungus. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, one component of 68 kDa was detected. Thus, the presence of fibrinogen binding proteins specifically localized on the cell wall surface of C. albicans germ tubes may constitute one of the factors involved in the development of candidosis.

Structures involved in the binding of human fibrinogen to Candida albicans germ tubes

Recent evidence for the interaction between human fibrinogen and Candida albicans germ tubes have led us to attempt to characterize the structures involved. Using 125I-radiolabeled proteins, fibrinogen purified by affinity chromatography and its plasmin degradation products, the binding sites on the fibrinogen molecule appeared to be located specifically in the D-domain. Conversely to the fibrinogen and the fragment D, radiolabeled fragment E, however, did not interact with cell. The binding was time-dependent, saturable and reversible. Scatchard analysis of the data obtained revealed an average of 6000 binding sites per germ tube with dissociation constant ( K d) of 5.2 × 10 −8 M. No potent competition was observed for a range of different proteins and carbohydrates. Fibrinogen fragment D binding proteins were identified using a dithiothreitol-iodoacetamide extract of the fungus. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, one compotent of 68 kDa was detected. Thus, the presence of fibrinogen binding proteins specifically localized on the cell wall surface of C. albicans germ tubes may constitute one of the factors involved in the development of candidosis.

Evolutionary stability of fibrinogen epitopes implicated in fibrin polymerization and in fibrinolysis

In this work, fibrinogen evolution was analysed by testing the reactivity of fibrinogen from different species with monoclonal antibodies against human fibrinogen fragment D. One epitope concerning the fibrin polymerization site 'a' and two epitopes responsible for tPA binding to fibrin were conserved in all mammalian fibrogens tested but not in crab coagulogen or pleurodella fibrinogen. In these two species, some epitopes which are not implicated in fibrinogen function were conserved. Therefore, we can conclude that polymerizing site 'a' and tPA binding sites have not been modified for at least 80 million years.

A unique proteolytic fragment of human fibrinogen containing the A alpha COOH-terminal domain of the native molecule.

The COOH-terminal portion of the A alpha chain of human fibrinogen is highly susceptible to proteolytic degradation. This property has prevented isolation of the COOH-terminal domain of fibrinogen for the direct investigation of its functional characteristics. Human fibrinogen was degraded with hementin, a fibrinogen-olytic protease from the posterior salivary glands of the leech, Haementeria ghilianii. Two initial fragments, Yhem1 and Dhem1, produced by cleavage through the three polypeptide chains in the connector region, were characterized and shown to retain the entire A alpha COOH-terminal domain. Late cleavages by hementin occurred in the A alpha chain COOH-terminal region to produce fragments Yhem and Dhem with shorter A alpha chain remnants. Fragments Dhem were isolated from an intermediate hementin digest of fibrinogen using anion-exchange chromatography. Fragment Dhem1 was separated further from Dhem fragments with shorter alpha chain remnants by affinity chromatography on immobilized plasma fibronectin. Fragment Dhem1 represents a unique proteolytic fragment of fibrinogen containing an intact A alpha chain COOH-terminal region. NH2-terminal sequence analysis of isolated chains from fragment Dhem1 located hementin cleavage sites in the connector region to A alpha Asn102-Asn103, B beta Lys130-Gln131, and gamma Pro76-Asn77. The specific interaction of fragment Dhem1 with immobilized fibronectin indicated that the binding site probably was located within the COOH-terminal 111 amino acids of the A alpha chain. The overall pattern of fibrinogen cleavage by hementin is similar to that of plasmin, yet hementin cleaves preferably in the coiled-coil connector, sparing the A alpha COOH-terminal domain.

Interaction of integrins alpha v beta 3 and glycoprotein IIb-IIIa with fibrinogen. Differential peptide recognition accounts for distinct binding sites.

Glycoprotein (GP) IIb-IIIa is the major fibrinogen receptor on platelets and participates in platelet aggregation at the site of a wound. Integrin alpha v beta 3, which contains an identical beta-subunit, is expressed on endothelial cells and also serves as a fibrinogen receptor. Here, we demonstrate by several criteria that purified GPIIb-IIIa and integrin alpha v beta 3 bind to distinct sites on fibrinogen. First, a plasmin-generated fragment of fibrinogen lacking the RGD sequence at residues 572-574 retained the ability to bind GPIIb-IIIa, but failed to bind integrin alpha v beta 3. Second, a monoclonal antibody which exclusively recognizes the RGD sequence at fibrinogen A alpha chain residues 572-574 abolished interaction between integrin alpha v beta 3 and fibrinogen, but had only a minimal effect on fibrinogen binding to GPIIb-IIIa. Finally, we show that the difference in recognition of sites on fibrinogen by these two integrins is probably a consequence of their remarkably different ligand binding properties. Peptides corresponding to fibrinogen gamma chain residues 400-411 effectively blocked RGD sequence and fibrinogen binding by GPIIb-IIIa, but had no effect on the ability of integrin alpha v beta 3 to bind these ligands. We also show that integrin alpha v beta 3 has a higher affinity than GPIIb-IIIa for a synthetic hexapeptide containing the RGD sequence. In fact, this RGD-containing peptide was 150-fold more effective at blocking fibrinogen binding to integrin alpha v beta 3 than to GPIIb-IIIa. Collectively, our results demonstrate that integrins alpha v beta 3 and GPIIb-IIIa display qualitative and quantitative differences in their ligand binding properties, as is evident by their ability to interact with synthetic peptides. The ultimate result of these differences is the recognition of distinct sites on fibrinogen by the two integrins. These observations may have relevance in the processes of hemostasis and wound healing.

Increased concentrations of D-dimers in newborn infants.

The concentrations of D-dimers (the D fragments of fibrinogen) were measured in blood from 15 preterm infants, and 45 born at full term, to establish normal ranges. The adult normal range is less than 0.25 mg/l; 31 of the 60 infants (52%) had values less than 0.25 mg/l, in 16 (27%) they were 0.25-0.5, in eight (13%) 0.5-1, in three (5%) 1-2, and in two (3%) 2-4. D-dimer concentrations measured during the neonatal period should be interpreted with caution.

Characterization of a modified human tissue plasminogen activator comprising a kringle-2 and a protease domain.

To study structure/function relationships of tissue plasminogen activator (t-PA) activity, one of the simplest modified t-PA structures to activate plasminogen in a fibrin-dependent manner was obtained by constructing an expression vector that deleted amino acid residues 4-175 from the full-length sequence of t-PA. The expression plasmid was introduced into a Syrian hamster cell line, and stable recombinant transformants, producing high levels of the modified plasminogen activator, were isolated. The resulting molecule, mt-PA-6, comprising the second kringle and serine protease domains of t-PA, produced a doublet of plasminogen activator activity having molecular masses of 40 and 42 kDa. The one-chain mt-PA-6 produced by cultured Syrian hamster cells was purified in high yield by affinity and size exclusion chromatography. The purified mt-PA-6 displayed the same two types of microheterogeneity observed for t-PA. NH2-terminal amino acid sequencing demonstrated that one-chain mt-PA-6 existed in both a GAR and a des-GAR form. Purified mt-PA-6 also existed in two glycosylation forms that accounted for the 40- and 42-kDa doublet of activity produced by the cultured Syrian hamster cells. Separation of these two forms by hydrophobic interaction chromatography and subsequent tryptic peptide mapping demonstrated that both forms contained N-linked glycosylation at Asn448; in addition, some mt-PA-6 molecules were also glycosylated at Asn184. Plasmin treatment of one-chain mt-PA-6 converted it to a two-chain molecule by cleavage of the Arg275-Ile276 bond. This two-chain mt-PA-6, like t-PA, had increased amidolytic activity. The fibrinolytic specific activities of the one- and two-chain forms of mt-PA-6 were similar and twice that of t-PA. The plasminogen activator activity of one-chain mt-PA-6 was enhanced greater than 80-fold by CNBr fragments of fibrinogen, and the one-chain enzyme lysed human clots in vitro in a dose-dependent manner. The ability to produce and purify a structurally simple plasminogen activator with desirable fibrinolytic properties may aid in the development of a superior thrombolytic agent for the treatment of acute myocardial infarction.