Activated platelets form protected zones of adhesion on fibrinogen and fibronectin-coated surfaces.

Leukocytes form zones of close apposition when they adhere to ligand-coated surfaces. Because plasma proteins are excluded from these contact zones, we have termed them protected zones of adhesion. To determine whether platelets form similar protected zones of adhesion, gel-filtered platelets stimulated with thrombin or ADP were allowed to adhere to fibrinogen- or fibronectin-coated surfaces. The protein-coated surfaces with platelets attached were stained with either fluorochrome-conjugated goat anti-human fibrinogen or anti-human fibronectin antibodies, or with rhodamine-conjugated polyethylene glycol polymers. Fluorescence microscopy revealed that F(ab')2 anti-fibrinogen (100 kD) did not penetrate into the contact zones between stimulated platelets and the underlying fibrinogen-coated surface, while Fab antifibrinogen (50 kD) and 10 kD polyethylene glycol readily penetrated and stained the substrate beneath the platelets. Thrombin- or ADP-stimulated platelets also formed protected zones of adhesion on fibronectin-coated surfaces. F(ab')2 anti-fibronectin and 10 kD polyethylene glycol were excluded from these adhesion zones, indicating that they are much less permeable than those formed by platelets on fibrinogen-coated surfaces. The permeability properties of protected zones of adhesion formed by stimulated platelets on surfaces coated with both fibrinogen and fibronectin were similar to the zones of adhesion formed on fibronectin alone. mAb 7E3, directed against the alpha IIb beta 3 integrin blocked the formation of protected adhesion zones between thrombin-stimulated platelets and fibrinogen or fibronectin coated surfaces. mAb C13 is directed against the alpha 5 beta 1 integrin on platelets. Stimulated platelets treated with this mAb formed protected zones of adhesion on surfaces coated with fibronectin. These protected zones were impermeable to F(ab')2 antifibronectin but were permeable to 10 kD polyethylene glycol. These results show that activated platelets form protected zones of adhesion and that the size of molecules excluded from these zones depends upon the composition of the matrix proteins to which the platelets adhere. They also show that formation of protected zones of adhesion by platelets requires alpha IIb beta 3 integrins while the permeability properties of these zones of adhesion are regulated by both alpha IIb beta 3 and alpha 5 beta 1 integrins.

Monoclonal antibodies to a synthetic fibrin-like peptide bind to human fibrin but not fibrinogen.

A synthetic heptapeptide from the amino terminus of the beta chain in human fibrin was used as an antigen to produce monoclonal antibodies that bind to fibrin even in the presence of human fibrinogen at the concentration found in plasma. As expected, the antifibrin activity was inhibited by the peptide antigen but not by a control heptapeptide. In a chicken ex vivo circulatory model for fibrin detection, intravenously administered monoclonal antibodies bound to human fibrin-coated disks placed in an extracorporeal chamber. These findings may lead to better methods for identifying deep vein and coronary artery thrombi.

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.

Antibody-directed urokinase: a specific fibrinolytic agent.

A specific fibrinolytic agent was synthesized by covalently coupling urokinase to a monoclonal antibody that was fibrin-specific and did not cross-react with fibrinogen. The antibody was raised against a synthetic peptide representing the seven amino-terminal residues of the beta chain of human fibrin. The urokinase-antifibrin conjugate retained the original binding specificity of the antibody and showed 100-fold increased fibrinolysis in vitro when compared to unmodified urokinase. The presence of human fibrinogen at plasma concentration did not influence these properties.

Biosynthesis and secretion of proatrial natriuretic factor by cultured rat cardiocytes.

Rat atrial natriuretic factor (ANF) is translated as a 152-amino acid precursor preproANF. PreproANF is converted to the 126-amino acid proANF, the storage form of ANF in the atria. ANF isolated from the blood is approximately 25 amino acids long. It is demonstrated here that rat cardiocytes in culture store and secrete proANF. Incubation of proANF with serum produced a smaller ANF peptide. PreproANF seems to be processed to proANF in the atria, and proANF appears to be released into the blood, where it is converted by a protease to a smaller peptide.

Induction of fibrin-specific antibodies by immunization with synthetic peptides that correspond to amino termini of thrombin cleavage sites.

Fibrin-specific antibodies have been produced in rabbits which were immunized with synthetic peptides. Specificity against human fibrin monomer was achieved because the synthetic peptide haptens were derived from sites unique to fibrin as compared with fibrinogen. Two undecapeptides were chemically synthesized according to the amino acid sequence of the amino termini of human fibrin alpha- and beta-chains which are revealed by thrombin cleavage. Rabbits immunized with either an alpha- or beta-chain peptide conjugate produced anti-peptide sera which reacted with fibrin monomer. Following immunoadsorption of the rabbit sera with human fibrinogen-Sepharose, fibrin-specific antibodies were detectable by solid-phase radioimmunoassay that did not react with fibrinogen. Antisera elicited by clotted human fibrin contained antibodies that reacted with fibrin and fibrinogen when treated in a similar manner.

Increased specificity in human cardiac-myosin radioimmunoassay utilizing two monoclonal antibodies in a double sandwich assay.

In some instances, even the increased resolution that may be afforded in immunoassays by the use of monoclonal antibodies fails to effect resolution among molecules that share many epitopes. An immunoradiometric assay that simultaneously measured two different epitopes on the same molecule was devised to overcome this difficulty in the differentiation between cardiac- and skeletal-myosin light chains. Three monoclonal antibodies were examined that were 100% (1C5), 25% (2B9) and 17% (4F10) cross reactive, respectively, between the two antigens. One antibody of the pair to be studied was immobilized to cyanogen bromide-activated Sepharose 4B while the other was iodinated with 125I using the lactoperoxidase method. The antigen was mixed with the immobilized antibody, the labeled antibody was added and the precipitate then washed and counted in a gamma counter. When both antibodies of the pair to be studied (immobilized and labeled) were the same (2B9), no radioactivity above background was bound to the precipitate, indicating that the second antibody could not bind to an already occupied epitope. When two different antibodies were employed, the specificity of the assay increased over that of a single antibody. The cross reactivity of a pair approximated the product of the cross reactivities of the individual antibodies. Thus, 1C5 and 2B9 were 25% cross reactive together, 1C5 and 4F10 17% cross reactive, and 2B9 and 4F10 4.3% cross reactive.

Characterization of a new four-chain coiled-coil: influence of chain length on stability.

Limited information is available on inherent stabilities of four-chain-coils. We have developed a model system to study this folding motif using synthetic peptides derived from sequences contained in the tetramerization domain of Lac repressor. These peptides are tetrameric as judged by both gel filtration and sedimentation equilibrium and the tetramers are fully helical as determined by CD. The four-chain coiled-coils are well folded as judged by the cooperativity of thermal unfolding and by the extent of dispersion in aliphatic chemical shifts seen in NMR spectra. In addition, we measured the chain length dependence of this four-chain coiled-coil. To this end, we developed a general procedure for nonlinear curve fitting of denaturation data in oligomeric systems. The dissociation constants for bundles that contain alpha-helical chains 21, 28, and 35 amino acids in length are 3.1 x 10(-12), 6.7 x 10(-23), and 1.0 x 10(-38) M3, respectively. This corresponds to tetramer stabilities (in terms of the peptide monomer concentration) of 180 microM, 51 nM, and 280 fM, respectively. Finally, we discuss the rules governing coiled-coil formation in light of the work presented here.

Cellular processing of pro-atrial natriuretic factor (pro-ANF): studies using an antiserum that selectively binds ANF-(99-126) after its cleavage from pro-ANF.

Atrial natriuretic factor (ANF) is stored in atrial myocytes as a 15-17K prohormone, but circulates in plasma as a 3K, carboxy (C)-terminal fragment of the prohormone. The tissue location at which the cleavage of pro-ANF to its hormonal form occurs is unknown. In the present study, an immunological approach was taken to address this question. A polyclonal antiserum was generated which recognizes the hormonal form of ANF [ANF-(99-126)] only after its cleavage from the prohormone. This was accomplished by immunizing rabbits with a synthetic peptide corresponding to the seven amino (N)-terminal residues of ANF-(99-126) coupled to carrier protein via a C-terminal cysteine. This antiserum, anti-ANF-(99-105), demonstrated high affinity for ANF-(99-126) (IC50 = 170 pM), but displayed 100-fold less affinity for recombinant pro-ANF [ANF-(2-126)]. The N-terminal specificity of anti-ANF-(99-105) was evident by its failure to bind ANF-(103-126) at concentrations up to 100 nM. The specificity of anti-ANF-(99-105) for the hormonal form of ANF was examined by using thrombin to cleave pro-ANF and testing for the generation of anti-ANF-(99-105) immunoreactivity. Cleavage of atrial pro-ANF or 35S biosynthetically-labeled pro-ANF resulted in the production of immunoreactive material from the prohormone, whereas pro-ANF itself demonstrated no cross-reactivity with anti-ANF-(99-105). Anti-ANF-(99-105) could also recognize ANF released from the isolated perfused rat heart. When anti-ANF-(99-105) was used in immunohistochemical studies of rat atrial myocardium, no staining was observed in unfixed frozen sections. This suggests that proteolytic processing of pro-ANF is not an intracardiocytic event.

Study of the antigenic determinants of human renin.

The primary structure of human renin, recently established from the complementary DNA sequence of its messenger RNA, shows a strong homology to other aspartyl proteases. This homology has permitted the construction of a model of the three-dimensional structure of renin based on the crystallographically determined structures of three aspartyl proteases: penicillopepsin, endothiapepsin, and rhizopuspepsin. Using an algorithm in which a spherical probe approximating the size of the antibody-binding domain (1-nm radius) was allowed to contact the surface of the renin model, we predicted 12 to 15 peptides to be immunogenic epitopes. We synthesized peptides corresponding to three different regions of the model: Cys-Gly-Ser-Asp-Pro-Gln-His-Tyr-Glu-Gly-amide (C-180-188), Tyr-Leu-Leu-Cys-Glu-Asp-Gly-Cys-Leu-Ala-Leu-amide (Y-215-224; disulfide bond between cysteines) and Tyr-Gly-Ser-Ser-Thr-Leu-Leu-Cys-Glu-Asp-Gly-Cys-Leu-Ala-Leu-amide (Y-211-224; disulfide bond between cysteines), and Cys-Tyr-Ser-Ser-Lys-Lys-Leu-Cys-Gly (C-290-296-G; disulfide bond between cysteines). All four peptides were tested for their binding to 11 polyclonal and 7 monoclonal antibodies raised against pure human renin, in both a solution assay and an enzyme-linked immunosorbent assay. Peptides Y-215-224 and Y-211-224 bound to all 11 polyclonal antibodies in the solution assay, and peptide Y211-224 bound to eight of them in the enzyme-linked immunosorbent assay. Therefore, region 211-224 can be identified as a major epitope of the human renin molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Selectivity of angiotensin II antisera.

A significant problem in the immunoassay of angiotensin II is the cross-reactivity of most available antisera with the peptide's metabolic products, (des-Asp1)-angiotensin II and (des-Asp1.Arg2)-angiotensin II. In order to attempt to generate antisera of greater selectivity, a variety of conjugates between angiotensin II or derivative peptides and carrier proteins were examined as immunogens with the aim of generating antisera that would selectively identify the amino terminal region of the peptide. Selectivity for the amino terminus was achieved by either (1) immunization with N-acetylated angiotensin II-amide which had been coupled to rabbit serum albumin by its carboxy terminus, or (2) immunization with angiotensin-(1-7)-heptapeptide which was randomly coupled to thyroglobulin. The antisera produced with the N-acetylated immunogen cross-reacted with the unacetylated ligand (Asn1-Val5)-angiotensin, but did not recognize the human hormone (Asp1,Ile5)-angiotensin. Carboxy-terminal coupling of angiotensin without N-acetylation did not induce selectivity for the amino terminus, nor did a conjugate which was linked to the carrier protein via a diazo bond to His6 of the peptide. These findings may be explained by the fact that N-acetylated angiotensin II resists degradation by amino peptidases and thus retains its structure in the immunogen and by the fact that the (1-7)-heptapeptide has lost the immunodominant carboxy-terminal epitope, thus emphasizing the desired amino terminal determinant.

Synthetic peptide immunogens for the development of a cardiac myosin light chain-1 specific radioimmunoassay.

To determine if the presence of cardiac light chains in blood could be used to detect acute myocardial infarction, we developed a specific light chain immunoassay. A synthetic peptide sequence specific for human cardiac ventricular myosin light chain 1 (VLC1) was synthesized and designated P348. This peptide coupled to keyhole limpet hemocyanin was used as an immunogen to obtain murine monoclonal antibodies specific for VLC1. Five monoclonal antibodies were obtained. One of these designated Mab-8E3 reacted equally well with both the synthetic peptide and VLC1. Although the 8E3 antibody is specific for VLC1, the use of HPLC purification of skeletal muscle myosin light chain 1 demonstrated that VLC1 is present in human skeletal muscle. The clinical utility of the assay was tested in 18 patients with creatine kinase (CK) and ECG documented acute myocardial infarction. VLC1 was below the limit of detection (< 1 ng/ml) in sera obtained from healthy volunteers and patients without myocardial infarction or chest pain. In contrast VLC1 was elevated in the serum of all 18 patients with acute myocardial infarction. Combining the two test results at the time of admission resulted in 83% of patients having detectable serum levels of one or both markers.

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.

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.

A chimeric streptokinase with unexpected fibrinolytic selectivity.

Chimeric 59D8-SK was designed to confer fibrin-selectivity to streptokinase by fusion of the Fab fragment of anti-fibrin antibody 59D8 to the N-terminus of streptokinase (SK: Ile1-Lys414). It was expressed in a mouse hybridoma cell line and purified by affinity chromatography on a 59D8-antigen column. Chimeric 59D8-SK is a disulfide-linked heterodimer composed of an antibody light chain (Mr 27,000) and a N-glycosylated chimeric heavy chain (M(r) 90,000). The fibrin targeting by 59D8 increased plasma clot lysis by 2-fold, but connecting 59D8 to SK has provided 59D8-SK several unique properties: (i) 59D8-SK activated human Glu-plasminogen with a significant lag period that coincided with limited proteolysis of 59D8-SK similar to that observed for wild-type SK. In a kinetic study, both gave very similar kinetic parameters for the activation of Glu-plasminogen even though 59D8-SK was N-glycosylated in its SK portion; (ii) 59D8-SK was relatively inactive in human plasma, compared to SK, but it became activated in the presence of clots; (iii) 59D8-SK lysed clots slowly but completely whereas SK lysed clots rapidly but incompletely. Even though the mechanism behind these new properties is not fully understood, they are characteristics of a second-generation plasminogen activator.

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.

Immunodetection of human fibrin using monoclonal antibody-64C5 in an extracorporeal chicken model.

The potential use of a fibrin-specific monoclonal antibody-64C5 as a thrombus detecting agent was examined in a quantifiable extracorporeal circulatory model using the chicken. This species was selected because antibody-64C5 did not cross-react with chicken fibrin. Human fibrinogen was clotted with human thrombin and factor XIII on filter paper disks, which were then inserted into an ex-vivo chamber that interrupted an extracorporeal loop between a carotid artery and a jugular vein. Antibody-64C5 was injected intravenously and after an equilibration period, disks bearing human fibrin were placed in the extracorporeal chamber and exposed to circulating antibody. Uptake which was quantified by treating the disks with 125I-goat antimouse-(Fab')2 was rapid, with 70 percent of maximal uptake occurring within 10 min. Fibrinolysis, although measurable, had no adverse effect upon uptake of the monoclonal antifibrin-64C5.

Evaluation of monoclonal antifibrin antibodies by their binding to human blood clots.

The primary goal of this study was to develop a method for evaluating fibrin-specific antibodies as thrombus detecting agents. The apparatus and assay conditions were chosen by testing antibody 64C5, which binds to the amino terminus of the fibrin beta chain, for its ability to bind to human blood clots. Using 125I-labeled antibody 64C5, the effects of antibody concentration, clot shape, clot mass, temperature, and flow rate were tested. Increased antibody binding was observed when antibody concentration, clot mass and temperature were increased. Under one set of conditions, six 125I-labeled monoclonal antifibrin antibodies (four specific for the beta chain, two specific for the alpha chain) were tested for their binding to retracted clots of human blood. Two radioiodinated antidigoxin antibodies were used as a control. Beta chain-specific antibody 59D8, which provided the highest level of binding to clot, bound 14-fold better than the control antidigoxin antibody. Neither alpha chain-specific antibody bound to clotted blood. To examine the in vivo relevance of the in vitro binding to clots, the uptake of antibody 64C5 was assessed for its binding to human fibrin clotted within the jugular vein of a rabbit. The correlation coefficient between in vitro and in vivo uptake as a function of clot weight was calculated to be 0.91. Thus, in vitro binding of monoclonal antifibrin antibodies to human blood clots was judged to be a realistic method for the comparison and selection of an ideal antifibrin antibody for detailed in vivo testing.

Characterization of a monoclonal antibody directed against the carboxyl-terminus of human factor XIII. An epitope exposed upon denaturation and conserved across species lines.

By deriving an anti-peptide monoclonal antibody, mAb 7A4, we characterized the relatively unstudied carboxyl-terminal end of the a-chain of human factor XIII, the plasma transglutaminase. MAb 7A4 was directed against the last eight amino acids (Gln-Ile-Gln-Arg-Arg-Pro-Ser-Met) and bound with a dissociation constant of 3.4 x 10(-8)M. In a solid assay format, mAb 7A4 bound equally well to factor XIII obtained from human plasma, platelets or placenta. However, in a solution-phase assay format, the epitope was largely unavailable but could be readily exposed by heat denaturation. Immunoblotting showed that this epitope is conserved among all species of plasma factor XIII tested except rabbit suggesting that the carboxyl-terminus might be an important structural element. Other competitive binding experiments with synthetic peptides as inhibitors pointed toward the final carboxyl-terminal amino acid, Met-731, as an immunochemically important determinant. This was used advantageously to confirm the finding that the carboxyl-terminal Met-731 is largely absent from placental factor XIII (1) as compared to platelet or plasma factor XIII.

A monoclonal antibody against a peptide sequence of fibrinogen gamma chain acts as an inhibitor of factor XIII-mediated crosslinking of human fibrin.

Recurrent hemorrhage has been reported in humans as a result of acquired antibody inhibitors which interfere with the crosslinking of fibrin by factor XIII. One type of these inhibitors (Type III) prevents activated factor XIII from acting on fibrin. We have generated an antifibrin monoclonal antibody, called mAb 4A5, which binds to a peptide sequence at the carboxyl-terminus of human fibrinogen gamma-chains. MAb 4A5 acts like a Type III inhibitor and prevents proper factor XIII-mediated crosslinking. Pre-incubation of fibrinogen or pooled human plasma with mAb 4A5, but not mAb D2 (specific for the carboxyl terminus of fibrin alpha-chains), resulted in clots which are soluble in either 5 M urea or 1% monochloroacetic acid. SDS-PAGE and immunoblotting analysis of these clots confirmed that mAb 4A5 inhibited gamma-chain crosslinking in plasma clots and fibrin clots. Results from a factor XIII activity assay demonstrated that biotinylcadaverine crosslinking into fibrin by factor XIII could be inhibited by mAb 4A5 but not mAb D2, arguing that mAb 4A5 acted by binding the crosslinking site of factor XIII. Studies of the immunoreactivity of these mAbs with 12 different animal species showed that the gamma-chain epitope recognized by mAb 4A5 was more conserved than the alpha-chain epitope recognized by mAb D2. The species fibrinogens, recognized by mAb 4A5 in binding assays, also showed impaired crosslinking when mAb 4A5 was present during the clotting reaction