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Biomedical subjects

G R Matsueda

Publications and source records attributed to G R Matsueda.

At least 37 records · Page 2Linked to original sources
PubMed1992

Platelet factor XIII increases the fibrinolytic resistance of platelet-rich clots by accelerating the crosslinking of alpha 2-antiplasmin to fibrin.

Platelet clots resist fibrinolysis by plasminogen activators. We hypothesized that platelet factor XIII may enhance the fibrinolytic resistance of platelet-rich clots by catalyzing the crosslinking of alpha 2-antiplasmin (alpha 2AP) to fibrin. Analysis of plasma clot structure by polyacrylamide gel electrophoresis and immunoblotting revealed accelerated alpha 2AP-fibrin crosslinking in platelet-rich compared with platelet-depleted plasma clots. A similar study of clots formed with purified fibrinogen (depleted of factor XIII activity), isolated platelets, and specific factor XIII inhibitors indicated that this accelerated crosslinking was due to the catalytic activity of platelet factor XIII. Moreover, when washed platelets were aggregated by thrombin, there was evidence of platelet factor XIII-mediated crosslinking between platelet alpha 2AP and platelet fibrin(ogen). Specific inhibition (by a monoclonal antibody) of the alpha 2AP associated with washed platelet aggregates accelerated the fibrinolysis of the platelet aggregate. Thus in platelet-rich plasma clots, and in thrombin-induced platelet aggregates, platelet factor XIII actively formed alpha 2AP-fibrin crosslinks, which appeared to enhance the resistance of platelet-rich clots to fibrinolysis.

Blood Coagulation Factors↗
PubMed1992

Soluble, cross-linked fibrin(ogen) hybrid oligomers do not stimulate t-PA conversion of plasminogen.

Cross-linked hybrid oligomers of fibrinogen and fibrin are found in plasma from fibrinaemic patients and normal individuals as well as in preparations of purified human fibrinogen. The present study was undertaken to see if such hybrid oligomers have the same stimulatory effect on tissue plasminogen activator (t-PA) conversion of plasminogen as do polymeric and monomeric fibrin. Hybrid oligomeric fibrin(ogen) material was provided by subjecting purified human fibrinogen to gel filtration in urea-containing buffer at pH 5.6. Well separated fractions of hybrid oligomeric material and monomeric fibrinogen were thus obtained. Some of this material was converted to soluble polymeric or monomeric fibrin using insolubilized thrombin. Hybrid polymeric fibrin, polymeric fibrin or monomeric fibrin were then added to citrated, normal plasma to 2.5 or 5 per cent of the plasma fibrinogen concentration. The added material was kept in solution by plasma fibrinogen. The "COA-SET Fibrin Monomer Test" (Kabi,Stocholm,Sweden), based on the ability of fibrin monomers to enhance t-PA mediated plasminogen-plasmin conversion, was used to compare the potential stimulatory effect of the preparations above. The results led to the following conclusions: 1) Cross-linked, soluble fibrin(ogen) hybrid polymers in a concentration of 5 per cent of plasma fibrinogen concentration (w/w) do not stimulate t-PA. 2) Thrombin conversion of the fibrin-fibrinogen hybrid material resulted in an increase in the rate of t-PA mediated plasminogen conversion, corresponding to the one observed with equivalent (w/w) amounts of fibrin monomers. Compared on a mole to mole basis, fibrin oligomers are more powerful than fibrin monomers as stimulators of t-PA activity.

Chromatography, Gel↗
PubMed1992

Availability of the B beta(15-21) epitope on cross-linked human fibrin and its plasmic degradation products.

The binding of radiolabeled monoclonal antifibrin antibody 59D8 (specific for fibrin but not fibrinogen) to a series of degraded fibrin clots showed that the availability of the B beta(15-21) epitope (against which 59D8 had been raised) was inversely proportional to the extent of clot lysis. Examination of digest supernatants revealed that the B beta(15-21) epitope was released from clots as a high molecular weight degradation product in the presence of calcium ions but that the generation of low molecular weight peptides occurred in the absence of calcium ions. To address the question of epitope accessibility, we compared levels of fibrin clot binding among four radioactively labeled antibodies: antifibrin monoclonal antibody 59D8, two antifibrinogen monoclonal antibodies that cross-reacted with fibrin, and an affinity-purified polyclonal antifibrinogen antibody. We expected that the antifibrinogen antibodies would show enhanced binding to clots in comparison with the antifibrin antibody. However, the epitope accessibility experiments showed that all four antibody preparations bound fibrin clots at comparable levels. Taken together, these studies demonstrated that one fibrin-specific epitope, B beta(15-21), remains available on clots as they undergo degradation by plasmin and, importantly, that the epitope is not solubilized at a rate faster than the rate at which the clot is itself solubilized. The availability of the B beta(15-21) epitope during the course of plasminolysis assures the potential utility of antifibrin antibodies such as 59D8 for detecting thrombi and targeting plasminogen activators.

Antibodies, Monoclonal↗
PubMed1992

Early cross-linked fibrin in human plasma contains alpha-polymers with intact fibrinopeptide A.

alpha-polymer formation, as opposed to gamma-chain dimerization has been considered a relatively late event in factor XIII-induced fibrin stabilization. Recently it has been shown, however, that plasma from healthy individuals and from patients with fibrinaemia contains small amounts of soluble fibrin/fibrinogen oligomers interlinked through dimerized gamma-chains as well as cross-linked alpha-chains. The present work was carried out to see if these early alpha-chain polymers also arise during coagulation of plasma in vitro. Plasma samples from healthy individuals, prepared by immediate centrifugation of blood collected without anticoagulant, were allowed to clot spontaneously for varying periods. The plasma clots were solubilized in SDS-urea-mercaptoethanol and samples were subjected to SDS-PAGE and Western blotting using polyclonal antibodies to human fibrinogen, or monoclonal antibodies specific either for A alpha/alpha-chains, for fibrinopeptide A-containing chains, for the N-terminus of the fibrin beta-chain or for the gamma-chains. Fibrin/fibrinogen oligomers were seen to form long before visible gelation of plasma. These oligomers were cross-linked through gamma-chain dimerization, but also through A alpha- or alpha-chain polymerization. The number and amount of alpha-polymers containing A alpha-chains increased immediately after clot formation, but these disappeared about 20 min later, due to complete removal of fibrinopeptide A (FPA) by thrombin. It is concluded that alpha-polymer formation is a very early event during plasma coagulation in vitro, and that both A alpha- and alpha-chains are involved.

Blood Coagulation↗
PubMed1992

Immunoelectrophoretic and immunohistochemical characterizations of fibrinogen derivatives in atherosclerotic aortic intimas and vascular prosthesis pseudo-intimas.

Cadaveric aortic intimas with uncomplicated atherosclerosis were examined to determine the distribution and polypeptide chain composition of fibrinogen-related protein. Immunohistochemical staining showed deposits rich in fibrinopeptides A and B. The deposits were usually disseminated throughout intimas of moderate thickness < 0.7 mm, but were distributed focally in elongate patches bounded both lumenally and medially by deposit-free tissue in thick atheromas. Saline extracts generally showed undegraded monomers and dimers by electrophoresis. The residual protein contained A alpha and gamma-chains that were cross-linked predominantly (>80%) into unresolved high M(r) (>200 kd) derivatives, whereas B beta-chains were left non-cross-linked, as occurs in late stages of cross-linking by transglutaminases. The resolved components had electrophoretic mobilities corresponding to characteristic products of both factor XIIIa and tissue-transglutaminase. A greater incorporation of alpha- rather than gamma-chains into cross-linked products implicated tissue-transglutaminase as contributing heavily. By contrast, vascular graft pseudo-intimas and a cadaveric clot were rich in degraded fibrin devoid of fibrinopeptide A, and cross-linked in patterns typical of XIIIa with gamma 2 dimers constituting the principal product. The findings indicate that the fibrinogen in the aortic intima is comparatively well protected from thrombin and plasmin, and that much of it is deposited through direct cross-linking by tissue-transglutaminase without being converted to fibrin.

Antibodies, Monoclonal↗
PubMed1991

Inhibition of factor XIII activation by an anti-peptide monoclonal antibody.

As the final enzyme in the coagulation cascade, activated fibrin stabilizing factor or factor XIII catalyzes the intermolecular cross-linking of fibrin chains. To study this enzyme in plasma, we derived a monoclonal antibody (MAb 309) against a peptide sequence (NH2-G-V-N-L-Q-E-F-C-COOH) in the thrombin activation site of factor XIII. Radioimmunoassays indicate that MAb 309 binds specifically to both platelet and plasma factor XIII. Peptide inhibition studies demonstrate that the MAb binds equally well to the factor XIII (FXIII) zymogen and the active form of FXIII (FXIIIa). In immunoblots of whole platelet lysates, MAb 309 binds only to FXIII and does not cross-react with other proteins. In saturation binding studies, the antibody shows a binding avidity of (1.75 +/- 0.35) x 10(9) M-1. MAb 309 also inhibited 99% of apparent FXIIIa activity in a standard transglutaminase assay. SDS-PAGE analysis of fibrin clots showed that MAb 309 inhibited fibrin gamma-gamma cross-linking. Moreover, MAb 309 accelerated the lysis of plasma clots, consistent with inhibition of fibrin-fibrin and fibrin-alpha 2-antiplasmin cross-linking. Immunoblotting experiments revealed that MAb 309 affected apparent FXIIIa activity by inhibiting the thrombin activation of the FXIII zymogen. In addition to its utility as a specific probe for the FXIII a-subunit, the strategy used to obtain MAb 309 may be used to generate MAbs that inhibit the activation of other coagulation factor zymogens.

Amino Acid Sequence↗
PubMed1991

A discrete sequence in a platelet integrin is involved in ligand recognition.

Platelet membrane glycoprotein IIb-IIIa (gpIIb-IIIa; alpha IIb-beta 3), the most prominent member of the integrin family of adhesion receptors on these cells, mediates platelet aggregation by binding fibrinogen and is critical in thrombosis and haemostasis. A short amino-acid sequence at the carboxy terminus of the gamma chain of fibrinogen is recognized by gpIIb-IIIa and peptides containing this sequence are selectively crosslinked to residues 294-314 of gpIIb. Here we show that an 11-residue peptide from this region of gpIIb inhibits platelet aggregation and binding of fibrinogen to platelets and to purified gpIIb-IIIa, and that it interacts directly with fibrinogen. These results implicate this segment of gpIIb-IIIa in the ligand-binding function of the receptor. Moreover, as this region is highly conserved among integrins, it may have a general function in ligand recognition by this broadly distributed family of adhesion receptors.

Amino Acid Sequence↗
PubMed1991

Antibody-detectable changes in fibrinogen adsorption affecting platelet activation on polymer surfaces.

Reactivity of platelets with an artificial surface exposed to whole blood is correlated with the concentration of adsorbed fibrinogen detectable by antifibrinogen antibodies. To examine the effect on platelets of the organization (distribution, orientation, conformation) of fibrinogen adsorbed on a hydrophobic surface, we studied the binding of polyclonal and monoclonal antifibrinogen antibodies to polyalkyl methacrylate polymers previously exposed to purified fibrinogen solution or diluted plasma and compared the results with platelet retention in methacrylate bead columns. There was an increase in platelet retention following diluted plasma pretreatment, which was eliminated by a polyclonal antibody against fibrinogen or against a gamma-(395-411) peptide from fibrinogen and was reduced by monoclonal antibodies (4A5, 4-2) against other COOH-terminal gamma-chain epitopes. Monoclonal antibody 10E5 against the fibrinogen receptor GpIIb/IIIa totally inhibited platelet retention in the bead columns. Our data suggest that different methacrylate polymers induce different changes in adsorbed fibrinogen, which may interfere with its interaction with platelets, and that platelet retention in a methacrylate bead column involves interaction of the COOH-terminal end of the gamma-chain of adsorbed fibrinogen with platelet GpIIb/IIIa receptors.

Adsorption↗
PubMed1991

Localization and visualization of pulmonary emboli with radiolabeled fibrin-specific monoclonal antibody.

Indium-111-labeled monoclonal antibody 64C5 specific for the beta-chain of fibrin monomer was used to image canine (n = 6) experimental pulmonary emboli (at least one barium-thrombin and one copper-coil induced clot per dog). Uptake of 111In-64C5 and 125I-control-DIG26-11 were compared in 10 clots (7 barium-thrombin and 3 copper-coil) identified in the lungs. There was no difference in the blood clearance of 111In-64C5 and 125I-DIG26-11. Uptake of 111In-64C5 (0.183 +/- 0.105, mean %ID/g) was greater than 125I-DIG26-11 (0.024 +/- 0.025) in pulmonary clots (p less than 0.001). Mean thrombus to blood ratios at 24 hr were 6.78:1 for 64C5 and 0.57:1 for DIG26-11. The clots visualized in vivo were larger (0.315 +/- 0.381 g) than clots not visualized (0.089 +/- 0.098). Negative images were recorded in three dogs with pulmonary emboli, injected with 111In-labeled control monoclonal antibody 3H3. These data suggest that 111In-labeled antifibrin can detect large pulmonary emboli in vivo.

Animals↗
PubMed1990

Bispecific monoclonal antibodies produced by somatic cell fusion increase the potency of tissue plasminogen activator.

Bispecific monoclonal antibodies that bind simultaneously to human fibrin and tissue plasminogen activator (tPA) enhance the fibrinolytic potency of tPA. Two bispecific antibodies (F36.23 and F32.1) were generated by somatic cell fusion. Antibody F36.23 derives its tPA binding from monoclonal anti-tPA antibody TCL8 and its fibrin binding from monoclonal antifibrin antibody 59D8. After purification from cell supernatants and ascites by two steps of affinity chromatography, hybrid-hybridoma bispecific antibody F36.23 simultaneously bound tPA and fibrin in solution and in solid-phase assays. In an assay for the lysis of human fibrin monomer, F36.23 increased the fibrinolytic potency of tPA by 5 to 10 fold, regardless of whether the bispecific antibody had been combined with the tPA before or during the assay. Bispecific F36.23 F(ab')2 also bound tPA and fibrin simultaneously, and the enhancement in fibrinolysis in the presence of F36.23 F(ab')2 was identical to that in the presence of intact F36.23. The second bispecific antibody, F32.1, was produced by an alternative strategy that has a wider potential for application in other systems. Hybridoma bispecific antibody F32.1 was derived from the fusion of immune splenocytes (in mice immunized with a synthetic oligopeptide representing the amino terminus of the alpha-chain of human fibrin) with the anti-tPA cell line TCL8. The properties of hybridoma bispecific antibody F32.1 and its F(ab')2 were indistinguishable from those of hybrid-hybridoma bispecific antibody F36.23 in solid-phase binding assays and in assays of fibrinolysis. Bispecific antibodies produced by somatic cell fusion, particularly in the form of F(ab')2, may have potential for use in clinical thrombolysis.

Animals↗
PubMed1990

A bispecific antibody enhances the fibrinolytic potency of single-chain urokinase.

A monoclonal antibody specific for an epitope at the amino terminus of the beta chain of fibrin and a monoclonal antibody that binds both one- and two-chain high molecular weight urokinase were chemically cross-linked [using N-succinimidyl 3-(2-pyridyldithio)propionate and 2-iminothiolane]. The chemically modified material was heterogeneous, ranging in molecular size from tetramers to monomers and containing the two antibodies in various ratios. Nevertheless, fractions of a molecular size larger than a monomer were capable of binding fibrin and urokinase simultaneously in a radioimmunoassay. These fractions also enhanced fibrinolysis by high molecular weight single-chain urokinase (scuPA) by 50-fold and plasma clot lysis by 5-fold. Whereas scuPA significantly decreased the concentration of fibrinogen in plasma clot assay supernatants, scuPA in association with the bispecific antibody did not.

Antibodies, Monoclonal↗
PubMed1990

Synergistic fibrinolysis: combined effects of plasminogen activators and an antibody that inhibits alpha 2-antiplasmin.

To improve the efficacy of plasminogen activators, we produced a monoclonal antibody (RWR) that inhibits human alpha 2-antiplasmin (alpha 2AP). In addition to inhibiting alpha 2AP in plasma, RWR binds to and inhibits fibrin cross-linked alpha 2AP and reproduces the "spontaneous" clot lysis that is the hallmark of human alpha 2AP deficiency. By inhibiting the inactivation of plasmin by alpha 2AP, RWR interacts synergistically with plasminogen activators to increase the potency (for 50% clot lysis) of urokinase by 80-fold, tissue plasminogen activator by 27-fold, and streptokinase by 20-fold. Yet, for a given amount of fibrinolysis, the combination of RWR and lower doses of plasminogen activator leads to less fibrinogen consumption than is obtained with higher, equipotent doses of plasminogen activator alone. These results suggest a strategy for increasing the efficacy of plasminogen activators. More generally, this approach to amplifying enzymatic activity by immunoneutralizing an inhibitor may be useful in other biologic processes that are rigidly governed by inhibitors.

Antibodies, Monoclonal↗
PubMed1990

Inhibition of clot-bound alpha 2-antiplasmin enhances in vivo thrombolysis.

Recent experiments in vitro have shown that inhibition of human alpha 2-antiplasmin by a monoclonal antibody (MAb RWR) markedly enhances clot lysis by plasminogen activators. To extend these studies in vivo, we tested whether inhibition of clot or fibrin-bound alpha 2-antiplasmin by MAb RWR could enhance the lysis of a human clot by tissue-type plasminogen activator (t-PA) in a rabbit jugular vein thrombosis model. Compared with a saline placebo or a control antibody, MAb RWR significantly increased thrombolysis by endogenous plasminogen activator in rabbits to which no t-PA was administered (p less than 0.05). In rabbits that received t-PA, the combination of MAb RWR and t-PA caused significantly greater thrombolysis than equivalent doses of t-PA alone (p less than 0.05). However, compared with equipotent doses of t-PA alone, the combination of MAb RWR and t-PA did not increase the nonspecific consumption of fibrinogen. These experiments suggest that the combination of an alpha 2-antiplasmin inhibitor and a plasminogen activator could be a more potent thrombolytic strategy.

Animals↗
PubMed1990

Antibody-targeted thrombolytic agents.

Plasminogen activator therapy for acute myocardial infarction has become standard medical practice. Bleeding complications, however, limit the utility of the currently available agents. This article reviews how the tools of immunology, molecular biology and protein engineering are being used to develop safer and more effective plasminogen activators.

Fibrinolysis↗
PubMed1989

Antibody-directed fibrinolysis. An antibody specific for both fibrin and tissue plasminogen activator.

An investigation was made to determine whether it is possible to attract tissue plasminogen activator (tPA) to the site of a thrombus by means of an antibody with affinites for both tPA and fibrin. A bispecific antibody conjugate was constructed by cross-linking two monoclonal antibodies: one specific for tPA, the other specific for fibrin. The bispecific antibody enhanced fibrinolysis by capturing tPA at the site of a fibrin deposit. In an in vitro quantitative fibrinolysis assay, the relative fibrinolytic potency of tPA bound to the bispecific antibody was 13 times greater than that of tPA and 200 times greater than that of urokinase. When fibrin was treated with the bispecific antibody before being mixed with tPA, the relative fibrinolytic potency of tPA was enhanced 14-fold. This capture also occurred when the concentration of tPA present in the assay was less than the concentration of tPA present in normal human plasma. In a human plasma clot assay, samples containing both the bispecific antibody and tPA exhibited significantly more lysis than did samples containing tPA alone. In spite of the increased clot lysis effected by the presence of bispecific antibody, there was no significant increase in fibrinogen or alpha 2-antiplasmin degradation at equal tPA concentrations. The ability of the bispecific antibody to concentrate exogenous tPA in vivo was then examined in the rabbit jugular vein model. Systemic infusion of a small amount of tPA (10,000 units) produced no significant increment in thrombolysis over the level of spontaneous lysis (14 +/- 8%). However, the simultaneous infusion of 10,000 units of tPA and 2 mg of bispecific antibody resulted in 42 +/- 14% (p less than 0.01) lysis. These results suggest that a molecule capable of binding both fibrin and tPA with high affinity could enhance thrombolysis in the circulation by capturing endogenous tPA.

Animals↗
PubMed1989

Innovative approaches to plasminogen activator therapy.

Plasminogen activator therapy for acute myocardial infarction has become standard medical practice. Bleeding complications, however, limit the utility of the currently available agents. This article reviews how the tools of molecular biology and protein engineering are being used to develop safer and more effective plasminogen activators.

Humans↗
PubMed1989

Enhanced immunogenicity of a T cell immunogenic peptide by modifications of its N and C termini.

The modification of the terminal ionizable charges of an immunogenic peptide, HEL (46-61), was found to greatly increase the immunogenicity of the peptide. The modified peptide had 100- to 1000-fold enhanced activity in both in vitro and in vivo T cell assays. The mechanism of the enhancement was investigated by determining the binding affinities to I-Ak as well as circular dichroism (CD) studies. The native and enhanced peptides had indistinguishable binding affinities, as well as similar kinetics. The CD studies revealed that in aqueous solution, neither peptide had any detectable helicity; however, the addition of trifluoroethanol did result in significant helicity; with the two peptides being indistinguishable. These same modifications were also shown to enhance other immunogenic peptides if they contained a basic carboxy-terminal amino acid residue. Thus, by modifying the termini of T cell epitopes, their immunogenicity can be dramatically increased, but the molecular basis for this enhancement is still unclear.

Amino Acid Sequence↗