The role of DL and DH degradation products in the reactions of the plasmin hydrolysis of fibrinogen and fibrin]

The mechanism of effect of plasmin hydrolysis degradation products, fragments DL and DH, on fibrinolysis and fibrinogenolysis processes was investigated on the basis of their various structural and functional characteristics. Using electrophoresis of unreduced samples and degradation products concentrations changing kinetics, DH was shown to be the only fragment which possessed an antifibrinolytic effect. Rauleigh's light scattering analysis indicated the ability of fragments DL and DH to co-form with plasminogen reversible equimolar complexes.     

Terminal plasmin degradation products D and E of duck fibrinogen and their effect on polymerization of fibrin

Duck fibrinogen (Mr 320 000) treated with streptokinase-activated human plasminogen in the presence of calcium ions was hydrolysed to terminal core fragments D and E. They were isolated from the digest by: (1) ion-exchange chromatography on DEAE-cellulose, (2) gel filtration on Sephadex G-100, and (3) affinity chromatography with the use of fibrin monomers coupled to CNBr-activated Sepharose. When the native D fragment, D1 was additionally digested by plasmin in the presence of EDTA, more degraded forms D2 and D3 appeared. Molecular weight of D1, D2, D3 and E estimated on SDS-polyacrylamide gel electrophoresis is 100 000, 89 000, 80 000 and 50 000, respectively. It was found that after reduction with 2-mercaptoethanol the fragments D1 and D3 consisted each of three polypeptide chains: alpha, beta, gamma: the gamma-chain of D3 remnant was more degraded (Mr 24 000) as compared with the gamma-chain of D1 remnant (Mr 42 000). Polymerization of both duck and pig fibrin monomers was inhibited by fragments D1 but not by D3.     

A model of proteolysis of the fragment DH (95 kD) of the fibrinogen molecule

A detailed analysis of further proteolytic degradation of fibrinogen fragment DH (Mr 95 kDa) was performed. Two new proteolytic fragments DLA and DL derived from DH-fragment have been purified and analyzed. The results obtained allow to propose a general scheme of DH-fragment proteolysis by stepwise splitting of their individual domains. The borders and molecular masses of these domains were evaluated on the base of proteolysis data.     

An amphiphilic structure of the ninth component of human complement. Evidence from analysis of fragments produced by alpha-thrombin

Purified human C9 was treated separately with three proteolytic enzymes: trypsin, plasmin, and alpha-thrombin, and the digestion products were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Trypsin initially cleaved the Mr = 71,000 C9 to produce a Mr = 47,000 fragment plus numerous smaller fragments and prolonged digestion reduced the molecule to small polypeptides. Plasmin produced a Mr = 37,000 fragment which was stable to further digestion, plus fragments smaller than Mr = 10,000. Human alpha-thrombin cleaved C9 (7.8% carbohydrate) at a single internal site to produce a Mr = 37,000 fragment (11.3% carbohydrate) and a Mr = 34,000 fragment (3.9% carbohydrate). Statistical analysis of the amino acid compositions of the fragments and alkaline polyacrylamide gel electrophoresis showed that C9 is highly amphiphilic; the Mr = 34,000 fragment contains a majority of the acidic amino acids and migrates rapidly on alkaline gels; the Mr = 37,000 fragment is hydrophobic with a slow electrophoretic mobility. The two fragments remain noncovalently associated, but were separated by sodium dodecyl sulfate-hydroxylapatite chromatography. The NH2-terminal sequence analysis of native C9, of alpha-thrombin-cleaved C9, and for the isolated fragments showed that the acidic Mr = 34,000 fragment is the NH2-terminal C9a domain and the more hydrophobic Mr = 37,000 fragment is the carboxyl-terminal C9b domain. Hemolytic activity of C9 was unaffected by alpha-thrombin cleavage.     

Binding phenomena of isolated unique plasmic degradation products of human cross-linked fibrin.

Proteolysis of human cross-linked fibrin by plasmin results in the formation of a DD . E complex, and Fragments DD and E as the major degradation products. Three species of Fragment E, which differ both in molecular weights (E1, Mr = 60,000; E2, Mr = 55,000; E3, Mr = 50,000) and in charge, have been isolated from a digest of cross-linked fibrin. Each Fragment E species reacts with monospecific anti-E antiserum. Fragments E1 and E2 bind with Fragment DD to form a DD . E complex but Fragment E3 is inactive. This binding is specific since these Fragments E do not bind to fibrinogen or to degradation products of fibrinogen or of noncross-linked fibrin. Fragments E1 and E2 incubated with plasmin are degraded to Fragment E3, suggesting that the three species represent sequential degradation products. Plasmin-treated Fragments E1 and E2 no longer bind with Fragment DD; therefore, it appears that the peptides cleaved from Fragment E2 by plasmin contain or modify the sites responsible for complex formation. On the other hand, Fragment DD binds not only to Fragments E1 and E2, but also to fibrinogen, Fragments X (Stage 1), X (Stage 2), Y, and NH2-terminal disulfide knot, but only after thrombin treatment, suggesting that Fragment DD binds to complementary sites on the NH2-terminal region of fibrinogen which are exposed after thrombin treatment.     

Evidence for preferential proteolytic cleavage of one of the two fibronectin subunits and for immunological localization of a site distinguishing them

Purified plasma fibronectin was digested sequentially by thrombin and cathepsin G or by cathepsin G alone and the degradation products and their gelatin-binding and heparin-binding fractions were analyzed in NaDodSO4-polyacrylamide gel electrophoresis followed by immunoblotting with a defined monoclonal anti-fibronectin antibody. In early cathepsin G digests, several gelatin-binding fragments were detected: a few large (Mr greater than or equal to 150 000) polypeptides and fragments of Mr = 85 000, 72 000, 64 000 and 40 000. The 85 000-Mr and 64 000-Mr fragments appeared as closely spaced doublets and reacted with the antibody while the 72 000-Mr and 40 000-Mr fragments did not. Therefore the 64 000-Mr fragments are likely to be derived from the 85 000-Mr fragments. Three large fragments that bound to heparin, but not to gelatin were detected: Mr = 145 000, 135 000 and 120 000. Of these only the 135 000-Mr peptide reacted with the antibody. When fibronectin was digested with thrombin, polypeptides of Mr = 180 000-200 000 and a 30 000-Mr NH2-terminal fragment were produced. Cathepsin G added to this mixture further cleaved the fragments to a digestion pattern resembling that obtained from intact fibronectin except that the 85 000-Mr and 64 000-Mr fragments appeared as single bands and the amount of the 72 000-Mr fragment was reduced. The results suggest that thrombin cleaves the 30 000-Mr fragment preferentially from the NH2-terminal end of one of the two subunits of fibronectin and that the 85 000-Mr, 72 000-Mr and 64 000-Mr fragments obtained by the additional cathepsin G digestion were derived from the other chain. The results are consistent with the model that the antigenic determinant resides 72 000-85 000 Da from the NH2-terminus and is cleaved by cathepsin G alternatively at one of its sides. Thus, the components of the 85 000-Mr and 64 000-Mr doublets are derived from different subunits and the region located by the antibody may be responsible for the difference in their migration in the polyacrylamide gel.     

Subunit structure of the dihydrolipoyl transacylase component of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Mapping of the lipoyl-bearing domain by limited proteolysis

To characterize the lipoyl-bearing domain of the dihydrolipoyl transacylase (E2) component, purified branched-chain alpha-keto acid dehydrogenase complex from bovine liver was reductively acylated with [U-14C] alpha-ketoisovalerate in the presence of thiamin pyrophosphate and N-ethylmaleimide. Digestion of the modified complex with increasing concentrations of trypsin sequentially cleaved the E2 polypeptide chain (Mr = 52,000) into five radiolabeled lipoyl-containing fragments in the order of L1 (Mr = 28,000), L2 (Mr = 24,500), L3 (Mr = 21,000), L4 (Mr = 15,000) to L5 (Mr = 14,000) as determined by the autoradiography of sodium dodecyl sulfate-polyacrylamide gel. In addition, a lipoate-free inner E2 core consisting of fragment A (Mr = 26,000) and fragment B (Mr = 22,000) was produced. Fragment A contains the active site for transacylation reaction and fragment B is the subunit-binding domain. Fragment L5 and fragment B were stable and resistant to further tryptic digestion. Mouse antiserum against E2 reacted only with fragments L1, L2, and L3, and did not bind fragments L4, L5, A, and B as judged by immunoblotting analysis. The anti-E2 serum strongly inhibited the overall reaction catalyzed by the complex, but was without effect on the transacylation activity of E2. Measurement of incorporation of [1-14C]isobutyryl groups into the E2 subunit indicated the presence of 1 lipoyl residue/E2 chain. Based on the above data, a model is proposed in which the lipoyl-bearing domain is connected to the inner E2 core via a trypsin-sensitive hinge. The lipoyl-bearing domain contains five consecutive tryptic sites (L1 to L5), with the L1 site in the hinge region, and the L5 site next to the terminal lipoyl-binding sequence. An exposed and antigenic region is located between L1 and L4 tryptic sites of the lipoyl-bearing domain. The region accounts for about 24% of the E2 chain length. Binding of antibodies to this region probably impairs the mobility of the lipoyl-containing polypeptide, resulting in an interruption of the active-site interactions that are necessary for the overall reaction. The lack of antigenicity and resistance to tryptic digestion indicate a highly folded conformation for fragment L5, the limit polypeptide carrying the single lipoyl residue.     

Characterization of a fragment of bovine von Willebrand factor that binds to platelets

Bovine von Willebrand factor was digested with human plasmin in order to isolate and characterize a fragment that can bind to human platelets. A terminal plasmin digest of bovine von Willebrand factor is composed of five fragments, ranging in relative molecular weight (Mr) from 250,000 to 35,000. The major fragment has a Mr of 250,000 and consists of four disulfide-linked polypeptide chains with Mr from 69,000 to 35,000. The Mr 69,000 and 49,000 polypeptides possess carbohydrate moieties, as indicated by their reaction with periodate-Schiff reagent. Gel filtration studies suggest that, at physiological ionic strength, four of the Mr 250,000 fragments associate into a limited noncovalent oligomer. Monoclonal antibodies were prepared against native von Willebrand factor and used to characterize the distribution of epitopes on native vWF and the Mr 250,000 major fragment. Two of the monoclonal antibodies that recognize the major fragment (2 and H-9) inhibit platelet agglutination. The Mr 250,000 fragment binds to human platelets, and the binding is inhibited by monoclonal antibodies 2 and H-9. The Mr 250,000 fragment does not agglutinate platelets, consistent with a requirement for high molecular weight oligomers of von Willebrand factor for platelet agglutination. The Mr 250,000 fragment can compete with intact, bovine von Willebrand factor for binding to human platelets. However, its affinity is one-tenth that of intact von Willebrand factor.     

Proteolysis of factor Va by factor Xa and activated protein C

Bovine Factor Va, produced by selective proteolytic cleavage of Factor V by thrombin, consists of a heavy chain (D chain) of Mr = 94,000 and a light chain (E chain) of Mr = 74,000. These peptides are noncovalently associated in the presence of divalent metal ion(s). Each chain is susceptible to proteolysis by activated protein C and by Factor Xa. Sodium dodecyl sulfate electrophoretic analysis indicates that cleavage of the E chain by either activated protein C or Factor Xa yields two major fragments: Mr = 30,000 and Mr = 48,000. Amino acid sequence analysis indicates that the Mr = 30,000 fragments have identical NH2-terminal sequences and that this sequence corresponds to that of intact E chain. The Mr = 48,000 fragments also have identical NH2-terminal sequences, indicating that activated protein C and Factor Xa cleave the E chain at the same position. Sodium dodecyl sulfate electrophoretic analysis indicates that activated protein C cleavage of the D chain yields two products: Mr = 70,000 and Mr = 24,000. Amino acid sequence analysis indicates that the Mr = 70,000 fragment has the same NH2-terminal sequence as intact D chain, whereas the Mr = 24,000 fragment does not. Factor Xa cleavage of the D chain also yields two products: Mr = 56,000 and Mr = 45,000. The Mr = 56,000 fragment corresponds to the NH2-terminal end of the D chain and Factor V. Functional studies have shown that both chains of Factor Va may be entirely cleaved to products by Factor Xa without loss of activity, whereas activated protein C cleavage results in loss of activity. Since activated protein C and Factor Xa cleave the E chain at the same position, the cleavage of the D chain by activated protein C is responsible for the inactivation of Factor Va.     

Isolation and characterization of a heparin-binding domain from the amino terminus of platelet thrombospondin

Calcium-replete thrombospondin has been purified from outdated platelets using heparin-Sepharose affinity chromatography, gelatin-Sepharose to remove fibronectin, and gel filtration to eliminate low-molecular-weight heparin-binding proteins. Edman degradation of six different preparations revealed the amino-terminal sequence of thrombospondin (TSP) to be Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-. This sequence was obtained in initial yields as high as 85%, indicating that no blocked chains are present. Cleavage of calcium-replete TSP with thermolysin or plasmin results in the production of relatively stable fragments. Chromatography of these digests on heparin-Sepharose followed by elution with 0.6 M NaCl affords purification of an Mr 25,000 fragment from the thermolysin digest and an Mr 35,000 fragment from the plasmin digest. The binding of these fragments to heparin-Sepharose does not require divalent metal ions. Neither fragment is disulfide-bonded to other fragments present in the digests. The heparin-binding domains from both digests have similar amino acid compositions and their tryptic peptide maps on high performance liquid chromatography are identical with the exception of one peptide unique to each fragment. Automated Edman degradation in a vapor-phase sequenator of the thermolytic heparin-binding domain electroeluted from sodium dodecyl sulfate-gels indicates that the heparin-binding domain resides at the amino terminus of the Mr 180,000 TSP peptide chain.     

Domain structure of human plasma and cellular fibronectin. Use of a monoclonal antibody and heparin affinity to identify three different subunit chains

The domain structure of human plasma fibronectin was investigated by using heparin-binding and antibody reactivity of fibronectin and its proteolytically derived fragments. Digestion of human plasma fibronectin with a combination of trypsin and cathepsin D produced six major fragments. Affinity chromatography showed that one fragment (Mr 45 000) binds to gelatin and three fragments (Mr 31 000, 36 000, and 61 000) bind to heparin. The 31K fragment corresponds to NH2-terminal fragments isolated from other species. The 36K and 61K fragments are derived from a region near the C-terminus of the molecule and appear to be structurally related as demonstrated by two-dimensional peptide maps. A protease-sensitive fragment (Mr 137 000), which binds neither gelatin nor heparin but which has been shown previously to be chemotactic for cells [Postlethwaite, A. E., Keski-Oja, J., Balian, G., & Kang, A. H. (1981) J. Exp. Med. 153, 494-499], separates the NH2-terminal heparin- and gelatin-binding fragments from the C-terminal 36K and 61K heparin-binding fragments. A monoclonal antibody to fibronectin that recognized the 61K heparin-binding fragment was used to isolate a sixth fragment (Mr 34 000) that did not bind to heparin or gelatin and that represents a difference between the 61K and 36K heparin-binding fragments. Cathepsin D digestion produced an 83K heparin-binding, monoclonal antibody reactive fragment that contains the interchain disulfide bond(s) linking the two fibronectin chains at their C-termini. The data indicate that plasma fibronectin is a heterodimeric molecule consisting of two very similar but not identical chains (A and B). In contrast, enzymatic digestion of cellular fibronectin produced a 50K heparin-binding fragment lacking monoclonal antibody reactivity which suggests that the cellular fibronectin subunit is similar to the plasma A chain in enzyme susceptibility but contains a larger heparin-binding domain. A model relating the differences in the three fibronectin polypeptides to differences in published cDNA sequences is presented.     

Alpha 2-antiplasmin Enschede is not an inhibitor, but a substrate, of plasmin.

alpha 2-Antiplasmin Enschede is a variant of alpha 2-antiplasmin which has lost its ability to inhibit plasmin irreversibly and which is associated with a haemorrhagic disorder [Kluft et al. (1987) J. Clin. Invest. 80, 1391-1400]. The abnormal protein was purified from the plasma of a homozygous patient and subjected to one-dimensional peptide mapping using papain for digestion. A slightly abnormally migrating polypeptide (Mr 17,000) was found which represented the C-terminal part of the molecule (the N-terminus of the polypeptide corresponded to Gly-338 in normal alpha 2-antiplasmin) and which contained the reactive centre. The interaction of plasmin with alpha 2-antiplasmin Enschede was studied by adding plasmin to plasma of the homozygous patient. SDS/polyacrylamide-gel electrophoresis and immunoblotting showed that no complex persisted, but that the abnormal alpha 2-antiplasmin was cleaved into two fragments of Mr 56,000 and 14,000 respectively. The latter fragment co-migrated with the post-complex peptide, which is cleaved from normal alpha 2-antiplasmin during complex-formation with plasmin. In a purified system, catalytic amounts of plasmin rapidly cleaved alpha 2-antiplasmin Enschede into the aforementioned fragments. In kinetic studies alpha 2-antiplasmin Enschede reversibly and temporarily inhibited the plasmin-catalysed hydrolysis of D-valyl-L-leucyl-L-lysine p-nitroanilide ('S-2251') as a competitive inhibitor (Ki,app. 35 nM). It was concluded that alpha 2-antiplasmin Enschede apparently forms a normal complex with plasmin. The complex is, however, not stable, but disintegrates rapidly to a cleaved form of alpha 2-antiplasmin Enschede and active plasmin. The abnormal protein thus behaves like a substrate, instead of an inhibitor, of plasmin.     

Binding of Glu-plasminogen by fibrinogen and byproducts of its proteolysis

The ability of the native form of plasminogen (Glu-plasminogen) to form complexes with fibrinogen and its fragments immobilized on CNBr-agarose was studied. It was found that unlike Lys-plasminogen, the native form of the proenzyme does not bind to fibrinogen agarose. Limited proteolysis of fibrinogen by plasmin involving alpha C-domains results in the appearance of Glu-plasminogen binding sites at fibrinogen surface. The X2 fragment of fibrinogen binds to about 0.5 moles of Glu-plasminogen at an equimolar ratio of the interacting proteins. Under these conditions, the amount of bound Glu-plasminogen does not increase as a result of subsequent hydrolysis of fibrinogen down to end products, fragments E and D. It was found that Glu-plasminogen interacts with both E- and D-fragments of fibrinogen. Similar to Lys-plasminogen, Glu-plasminogen exhibits a high affinity for the E-fragment. The maximal quantity of the bound protein under the given experimental conditions is 2 moles per mole of the immobilized E-fragment. The interaction of Glu-plasminogen with the E-fragment is mediated by the lysine-binding sites of the proenzyme with a high and low affinity [Kd = 1.8.10(-6) and 7.5.10(-5) M, respectively]. Glu-plasminogen, unlike Lys-plasminogen, shows a low affinity for the D-fragment (Kd = 2.10(-5) M). Glu-plasminogen cannot be adsorbed by arginine-binding sites at the DH fragment-agarose.     

A cleavage-associated neoantigenic marker for a gamma chain site in the NH2-terminal aspect of the fibrinogen molecule.

The E fragment, derived from the NH2-terminal aspect of fibrinogen by plasmin cleavage (fg-E), possesses two generically distinct sets of antigenic expressions. The major set of antigens is expressed by the parent molecule as indicated by the capacity of a major subpopulation of antibodies present in antiserum to fg-E and reactive with fg-E to: (a) react with fibrinogen, and (b) be specifically absorbed by fibrinogen but appears following proteolysis with plasmin. These cleavage associated neoantigens (fg-E-neo) specifically react with a minor subpopulation of antibodies present in antiserum to fg-E.E fragments isolated after varying exposures to plasmin all expressed fg-E-neo, but early E fragments exhibited quantitatively less neoantigenic expression than more extensively degraded E fragments. The entire fg-E-neo expression is recovered on a single isolated constituent chain of the E fragment, and immunochemical analysis with antiserum to the isolated constituent chain-bearing fg-E-neo identifies it as a derivative of the gamma chain constituent, exhibits marked stability to physicochemical denaturation and enzymatic degradation. These properties suggest that the neoantigen may be associated with a specific amino acid sequence which is exposed by the cleavage process. The identification and localization of fg-E-neo provides a specific molecular marker site for the characterization of structural and conformational changes associated with catabolism and function of fibrinogen.     

Enzymatic modifications of human plasma fibronectin in relation to opsonizing activity

Plasma fibronectin is one of the largest plasma proteins (Mr approximately 440 000), comprising two approximately equal polypeptide chains which are held together by a disulfide linkage near the C-terminal end of the molecule. The binding of gelatinized latex beads to liver slices as well as the internalization of these particles by macrophages, in the presence of heparin, is greatly enhanced by fibronectin. The question as to whether the entire covalent structure of fibronectin was necessary for opsonizing activity was approached by limited proteolytic degradations of the molecule. Patterns of controlled digestion with trypsin, cathepsin D, Staphylococcus aureus protease, and plasmin all indicate that the minimal unit necessary for retention of opsonic activity is some large (Mr 200 000 and 190 000) single-chain entity. Treatment with plasmin proved to be the most reliable procedure for generating the active split product which could be readily separated from the inactive, disulfide-containing C-terminal fragment. Incorporation of dansylcadaverine into plasma fibronectin (3.5 mol/mol of protein) by fibronoligase (coagulation factor XIIIa) did not affect the opsonic activity of the protein.     

Conformational and structural modulation of the NH2-terminal regions of fibrinogen and fibrin associated with plasmin cleavage.

Conformational and structural modulations of the NH2-terminal region of fibrinogen and fibrin associated with plasmin cleavage have been examined utilizing specific antibody probes. The E region derived from the NH2-terminal aspects of fibrinogen undergoes complex structural and conformational changes throughout the cleavage process as indicated by differences in the quantitative and qualitative expression of antigenic determinants by the E region of each isolated cleavage fragment. When the range of antigenic determinants recognized by the antibody probe is limited to a specific molecular marker on the gamma chain within the E region, fg-E-neo, evidence for a systematic and progressive modulation of this site during plasmin cleavage is observed. Fg-E-neo undergoes progressive exposure as the cleavage of fibrinogen proceeds from X to Y to D:E complex. Separation of the D:E complex into its constituent, D and E fragments, is associated with further exposure of fg-E-neo determinants. The sequential cleavage of fibrin by plasmin also leads to progressive exposure of the fg-E-neo site; however, comparison of corresponding fragments derived from fibrinogen and fibrin reveals significant differences in the character of fg-E-neo expression. Immunochemical differences between fibrin and fibrinogen E fragments are not abolished by further exposure of the fragments to plasmin, are apparently not due to the presence or absence of fibrinopeptides, and are maintained following denaturation and renaturation of the fragments. These results suggest that the differential expression of fg-E-neo by the E fragments may be primarily dependent upon differences in amino acid compositions of the fragments.     

Characterization of an apparently lower molecular weight gamma-chain variant in fibrinogen Kyoto I. The replacement of gamma-asparagine 308 by lysine which causes accelerated cleavage of fragment D1 by plasmin and the generation of a new plasmin cleavage site

Congenitally abnormal fibrinogen Kyoto I with impaired fibrin monomer polymerization contains a normal gamma-chain and a gamma-chain variant (gamma Kyoto I) that has an apparently lower Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the Laemmli system (Laemmli, U. K. (1970) Nature 227, 680-685) but migrates with apparently normal Mr in the Weber and Osborn system (Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412). Reverse-phase high performance liquid chromatographic analyses of the cyanogen bromide or lysyl endopeptidase cleavage fragments of the purified gamma-chains of fibrinogen Kyoto I showed the presence of peptides not seen from normal fibrinogen. Amino acid sequence analysis of these peptides indicated that gamma Asn308 of the gamma-chain variant is replaced by lysine. Purified fragment D1 of fibrinogen Kyoto I also contains two types of D1 gamma-remnants: normal and apparently lower Mr types. Abnormal fragment D1 is cleaved faster to fragments D2 and D3 by plasmin in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) than normal fragment D1, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting using anti-gamma-chain monoclonal antibody. Analysis of peptides released from fragment D1 by plasmin in the presence of EGTA demonstrated the cleavage of the gamma Lys308-Gly309 bond. Fragment D1 of fibrinogen Kyoto I has normal calcium binding properties. The data suggest that a region or conformation containing gamma Asn308 affects the polymerization of fibrin monomers and that the gamma Asn308----Lys replacement causes a conformational change in the gamma-chain which results in the accelerated cleavage of gamma Lys356-Ala357 and gamma Lys302-Phe303 bonds by plasmin and also results in the generation of a new plasmin cleavage site between Lys308 and Gly309 in the presence of EGTA. During these studies, we found that part of the gamma Lys212-Glu213 bond in fragment D1 is cleaved by plasmin in the presence of EGTA.     

Reactivity of chemically cross-linked fibrinogen and its fragments D toward the staphylococcal clumping receptor

It has been established that the binding domain for the staphylococcal clumping receptor exists in fragment D of human fibrinogen [Hawiger J., Timmons, S., Strong, D. D., Cottrell, B. A., Riley, M., & Doolittle, R. F. (1982) Biochemistry 21, 1407; Strong, D. D., Laudano, A., Hawiger, J., & Doolittle, R. F. (1982) Biochemistry 21, 1414]. To examine the role of valency in the adhesive function of fibrinogen, its fragments were prepared by digestion with plasmin in the presence of calcium and purified by a two-step chromatographic procedure. Fragments D1 and E did not induce the staphylococcal clumping reaction. After they were prepared in oligomeric form by chemical cross-linking with glutaraldehyde, fragment D1 (Mr 94,000) became functionally reactive toward the staphylococcal clumping receptor, and fragment D3 (Mr 75,000) and fragment E (Mr 50,000) remained inactive. Fragment D dimer derived from enzymatic cross-linking was not reactive. Human fibrinogen cross-linked with glutaraldehyde usually reached a 250 times higher reactivity toward the staphylococcal clumping receptor, depending on the condition of the cross-linking reaction. It is concluded that the valency of fibrinogen in regard to its receptor binding domain and the availability of this domain are essential for the staphylococcal clumping reaction.     

Complex formation of platelet membrane glycoproteins IIb and IIIa with the fibrinogen D domain

Glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) form a macromolecular complex on the activated platelet surface which contains the fibrinogen-binding site necessary for normal platelet aggregation. To identify the specific region of the fibrinogen molecule responsible for its interaction with the GPIIb-GPIIIa complex, purified fragment D1 (Mr = 100,000) and fragment E (Mr = 50,000) were prepared from plasmin digests of purified human fibrinogen. In addition, the polypeptide chain subunits A alpha, B beta, and gamma of fibrinogen were prepared. Using an enzyme-linked immunosorbent assay we have demonstrated that isolated fragment D1 in a solid phase system forms a complex with a mixture of GPIIb and GPIIIa. The binding of the GPIIb-GPIIIa mixture to fragment D1-coated plates reached saturation at 8 nM and to fibrinogen-coated plates at 24 nM. Isolated A alpha, B beta, and gamma chains were not reactive with added glycoproteins. Fragment E coated directly on plastic plates or immobilized on antibody-coated plastic plates did not form a complex with GPIIb-GPIIIa. Only fluid phase fibrinogen and fragment D1 but not fragment E were inhibitory toward formation of a complex between solid phase fibrinogen and GPIIb-GPIIIa. Isolated A alpha, B beta, and gamma chains at concentrations equivalent to fluid phase fibrinogen were inactive. Binding of fragment D1 but not fragment E to the GPIIb-GPIIIa complex was also demonstrated by rocket immunoelectrophoresis of the membrane glycoprotein mixture through a gel containing the individual fragments and subsequent autoradiography of the complex following exposure to 125I-anti-fibrinogen. These observations with isolated platelet membrane glycoproteins provide strong evidence that each of the D domains of the fibrinogen molecule interacts directly with the GPIIb-GPIIIa complex on the activated platelet surface, thus allowing formation of a tertiary molecular "bridge" across the surface of two adjacent activated platelets.     

Subunit structure of bovine milk xanthine oxidase. Effect of limited cleavage by proteolytic enzymes on activity and structure.

Bovine milk xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) has been purified by a modified method without the use of proteases, and its structure has been analyzed by polyacrylamide gel electrophoresis. Native xanthine oxidase is found to consist of only two polypeptide chains A with molecular weights of 150 000 each. These chains have NH2-terminal methionine. Limited proteolysis with trypsin, chymotrypsin, or subtilisin at pH 8 did not affect molecular weight and activities of the enzyme while each of the A chains was cleaved under these conditions to three fragments C, E, and F with molecular weights of 92 00, 42 000 and 20 000, respectively. These fragments remained bound to each other and were relatively resistant to subsequent proteolysis. The isolation of xanthine oxidase in the presence of pancreatin as described by Hart et al. (1970, Biochem. J. 116, 851) gives partially digested enzyme composed mainly of chains C, E (Mr 35 000) and a small component (Mr approx. 15 0-0). The action of subtilisin on xanthine oxidase at pH 11 resulted in complete digestion of E chains, FAD separation, and total loss of xanthine:oxygen oxidoreductase activity while xanthine:indophenol oxidoreductase activity was relatively little affected. The residual enzyme has a molecular weight of about 200 000, is composed mainly of two C chains (and may probably contain F and/or proteolytic fragments of low molecular weight), contains molybdenum, and does not contain FAD.