Eli Lilly and Company Insulins - A Century of Innovation.

Eli Lilly and Company has played a pivotal role in the development of insulin products since its discovery in 1921. Through their dedication to pharmaceutical innovation, Josiah K. Lilly Sr. and George HA Clowes, in close collaborations with the University of Toronto, made insulin commercially available in 1923. Other innovations include the development and commercialization of the first biosynthetic human insulin, a rapid-acting insulin analog and analog mixtures. Lilly has advanced the field of knowledge with significant efforts toward developing a hepatic preferential basal insulin. Other important insulin projects include the first concentrated rapid-acting insulin analog, clinical studies supporting the use of highly concentrated human insulin, and an advanced clinical development program for an ultra-rapid insulin analog. Lilly's commitment to people affected with diabetes remains strong and will continue into the future through collaborative research, innovative product development and investing in advanced technologies.

Basal insulin peglispro: Overview of a novel long-acting insulin with reduced peripheral effect resulting in a hepato-preferential action.

Basal insulin peglispro (BIL) is a novel basal insulin with a flat, prolonged activity profile. BIL has been demonstrated in a dog model, in healthy men and in patients with type 1 diabetes (T1D) to have significant hepato-preferential action resulting from reduced peripheral activity. In the IMAGINE-Phase 3 clinical trial program, more than 6000 patients were included, of whom ~3900 received BIL. Of the 7 pivotal IMAGINE trials, 3 studies were double-blinded and 3 were in T1D patients. BIL consistently demonstrated a greater HbA1c reduction, less glycaemic variability and a clinically relevant reduction in the rates of nocturnal hypoglycaemia across comparator [glargine and isophane insulin (NPH)] studies. Trials using basal/bolus regimens had higher rates of total hypoglycaemia with BIL due to higher rates of daytime hypoglycaemia. Severe hypoglycaemia rates were similar to comparator among both patients with T1D or type 2 diabetes (T2D). T1D patients lost weight compared with glargine (GL). Patients with T2D tended to gain less weight with BIL than with glargine. Compared to glargine, BIL was associated with higher liver fat, triglycerides and alanine aminotransferase (ALT) levels, including a higher frequency of elevation of ALT ≥3 times the upper limit of normal, but without severe, acute drug-induced liver injury. Injection site reactions, primarily lipohypertrophy, were more frequent with BIL. In conclusion, BIL demonstrated better glycaemic control with reduced glucose variability and nocturnal hypoglycaemia but higher triglycerides, ALT and liver fat relative to conventional comparator insulin. The hepato-preferential action of BIL with reduced peripheral activity may account for these findings.

Contrasting weight changes with LY2605541, a novel long-acting insulin, and insulin glargine despite similar improved glycaemic control in T1DM and T2DM.

AIMS: The basal insulin analogue LY2605541, a PEGylated insulin lispro with prolonged duration of action, was previously shown to be associated with modest weight loss in Phase 2, randomized, open-label trials in type 2 (N=288) and type 1 (N=137) diabetes mellitus (T2DM and T1DM), compared with modest weight gain with insulin glargine. Exploratory analyses were conducted to further characterize these findings. METHODS: Pearson correlations between change in body weight and other variables were calculated. Continuous variables were analysed using a mixed linear model with repeated measurements. Proportions of subjects with weight loss were analysed using Fisher's exact test for T2DM and Nagelkerke's method for T1DM. RESULTS: Weight loss was more common in LY2605541-treated patients than in patients treated with insulin glargine (T2DM: 56.9 vs. 40.2%, p=0.011; T1DM: 66.1 vs. 40.3%, p<0.001). More LY2605541-treated patients experienced ≥5% weight loss compared to patients treated with glargine (T2DM: 4.8 vs. 0%, p=0.033; T1DM: 11.9 vs. 0.8%, p<0.001). In both the T1DM and T2DM studies, weight change did not correlate with baseline body mass index (BMI), or change in HDL-cholesterol in either treatment group. No consistent correlations were found across both studies between weight change and any of the variables assessed; however, weight change was significantly correlated with hypoglycaemia rate in glargine-treated T2DM patients. CONCLUSION: In two Phase 2 trials, improved glycaemic control with long-acting basal insulin analogue LY2605541 is associated with weight loss in previously insulin-treated patients. This weight change is independent of baseline BMI or hypoglycaemia.

Crystal structure of the obese protein leptin-E100.

Mutations in the obese gene (OB) or in the gene encoding the OB receptor(OB-R) result in obesity, infertility and diabetes in a variety of mouse phenotypes. The demonstration that OB protein (also known as leptin) can normalize body weight in ob/ob mice has generated enormous interest. Most human obesity does not appear to result from a mutant form of leptin: rather, serum leptin concentrations are increased and there is an apparent inability to transport it to the central nervous system (CNS). Injection of leptin into the CNS of overfed rodents resistant to peripheral administration was found to induce biological activity. Consequently, for the leptin to act as a weight-lowering hormone in human obesity, it appears that appropriate concentrations must be present in the CNS. This places a premium on understanding the structure of the hormone in order to design more potent and selective agonists. Here we report the crystal structure at 2.4A resolution of a human mutant OB protein (leptin-E100) that has comparable biological activity to wild type but which crystallizes more readily. The structure reveals a four-helix bundle similar to that of the long-chain helical cytokine family.

Assembly and dissociation of human insulin and LysB28ProB29-insulin hexamers: a comparison study.

PURPOSE: Investigations into the kinetic assembly and dissociation of hexameric LysB28ProB29-human insulin (LysPro), a rapid-acting insulin analog produced by the sequence inversion of amino acids at positions B28 and B29, were designed to explain the impact that the sequence inversion has on the formulation and pharmacokinetics of the insulin analog. METHODS: The kinetics of phenolic ligand binding to human insulin and LysPro were studied by stopped-flow spectroscopy. The kinetics of R6 hexamer disruption were studied by extraction of Co(II) with EDTA. RESULTS: Phenolic ligand binding to human insulin yielded rate constants for a fast and slow phase that increased with increasing ligand concentration and are attributed to the T6 --> T3R3 and T3R3 --> R6 transitions, respectively. However, the kinetics of phenolic ligand binding with LysPro was dominated by rates of hexamer assembly. The kinetic differences between the insulin species are attributed to alterations at the monomer-monomer interface in the dimer subunit of the LysPro analog. The extraction of Co(II) from both hexameric complexes by EDTA chelation is slow at pH 8.0 and highly dependent on ligand concentration. Cobalt extraction from LysPro was pH dependent. Of the various phenolic ligands tested, the relative affinities for binding to the human and LysPro hexamer are resorcinol > phenol > m-cresol. CONCLUSIONS: The extraction data support the formation of an R6-type LysPro hexamer under formulation conditions, i.e., in the presence of divalent metal and phenolic ligand, that is similar in nature to that observed in insulin. However, the formation kinetics of LysPro identify a radically different monomeric assembly process that may help explain the more rapid pharmacokinetics observed with the hexameric formulation of LysPro insulin relative to human insulin.

Physicochemical basis for the rapid time-action of LysB28ProB29-insulin: dissociation of a protein-ligand complex.

The rate-limiting step for the absorption of insulin solutions after subcutaneous injection is considered to be the dissociation of self-associated hexamers to monomers. To accelerate this absorption process, insulin analogues have been designed that possess full biological activity and yet have greatly diminished tendencies to self-associate. Sedimentation velocity and static light scattering results show that the presence of zinc and phenolic ligands (m-cresol and/or phenol) cause one such insulin analogue, LysB28ProB29-human insulin (LysPro), to associate into a hexameric complex. Most importantly, this ligand-bound hexamer retains its rapid-acting pharmacokinetics and pharmacodynamics. The dissociation of the stabilized hexameric analogue has been studied in vitro using static light scattering as well as in vivo using a female pig pharmacodynamic model. Retention of rapid time-action is hypothesized to be due to altered subunit packing within the hexamer. Evidence for modified monomer-monomer interactions has been observed in the X-ray crystal structure of a zinc LysPro hexamer (Ciszak E et al., 1995, Structure 3:615-622). The solution state behavior of LysPro, reported here, has been interpreted with respect to the crystal structure results. In addition, the phenolic ligand binding differences between LysPro and insulin have been compared using isothermal titrating calorimetry and visible absorption spectroscopy of cobalt-containing hexamers. These studies establish that rapid-acting insulin analogues of this type can be stabilized in solution via the formation of hexamer complexes with altered dissociation properties.

Hierarchical modeling of phenolic ligand binding to 2Zn--insulin hexamers.

Phenolic ligands, e.g., phenol and m-cresol, bind to 2Zn(II)-insulin hexamers and induce a conformational change at the N-terminus of the B-chain for each monomer. The binding of these phenolic ligands to 2Zn(II)-insulin hexamers has been studied by isothermal titrating calorimetry (ITC). The binding isotherms were modeled and thermodynamic parameters were quantified using a novel, flexible algorithm that permitted the development of a hierarchical series of physical models. With the insulin hexamer represented as a dimer of trimers, the modeling demonstrated that ligand binding is highly cooperative in nature, both intra- and inter-trimer. The isotropic inter-trimer cooperativity was dominant and negative in every system studied, with initial binding constants typically an order of magnitude greater for the binding of ligands to the first trimer relative to the second. The inter-trimer cooperatively estimated from the modeling of solution calorimetry data is consistent with a T6 <--> T3R3 <--> R6 equilibrium first proposed from crystallographic investigations. Intra-trimer cooperatively was present only in the enthalpy coefficient space, not in the equilibrium coefficient space, and therefore, less of a factor. The order of binding affinity for the ligands studied in resorcinol >> phenol > or = m-cresol as determined from their overall free energies of binding to the 2Zn(II)-insulin hexamer (-26.6, -23.4, and -23.4 kcal/mol, respectively) and their intrinsic binding constants (8780, 5040, and 3370 L/mol, respectively) at 14 degrees C. The temperature dependence of phenol binding to 2Zn(II)-insulin hexamer was modeled. Increasing temperature decreased the magnitude of both the intrinsic binding constant and the inter-trimer was cooperatively. The second phase of the ITC binding profile was also found to be highly temperature dependent. At lower temperatures the second phase is endothermic but gradually decreases with increasing temperature and subsequently becomes exothermic. This effect is attributed to loss of water from the hydration shell of the insulin hexamer with increasing temperature and consequently reduces the entropic contributions to the T <--> R transition in the phenol/2Zn(II)-insulin hexamer system.

Role of C-terminal B-chain residues in insulin assembly: the structure of hexameric LysB28ProB29-human insulin.

BACKGROUND: LysB28ProB29-human insulin (Humalog), a fully potent insulin analog in which the prolyl, lysyl sequence at the C-terminal end of the B-chain is inverted, exhibits a decreased association of monomers to dimers leading to rapid in vivo absorption. This provides important benefits for the insulin-requiring diabetic. In spite of its monomeric nature, LysB28ProB29-human insulin can exist as a discrete hexameric structure in the presence of both zinc and phenol. Studies of the crystal structure of LysB28ProB29-human insulin in a hexameric complex were initiated to gain a molecular understanding of the effect of the sequence inversion on the analog's self-association properties and, consequently, its in vivo efficacy. RESULTS: Under the conditions reported, LysB28ProB29-human insulin crystallized as a T3Rf3 hexamer that is isomorphous with the uncomplexed T3Rf3 native human insulin hexamer previously known as '4Zn insulin'. The three-dimensional structure of the T3Rf3 hexamer was determined by X-ray crystallographic methods to a resolution of 2.3 A. The prolyl, lysyl sequence inversion leads to local conformational changes at the C termini of the B-chains which eliminate two critical hydrophobic interactions and weaken two terminal beta-sheet hydrogen bonds that stabilize the dimer. CONCLUSIONS: The loss of these native dimer interactions weakens the hexameric LysB28ProB29-human insulin complex formed in the presence of phenolic ligands. Thus, it is hypothesized that the diffusion of the phenolic ligands from the site of injection results in the dissociation of hexamers directly to monomers, thereby maintaining the rapid time-action of the monomeric analog in spite of the hexameric conformation in therapeutic formulations.

Reversible adsorption of soluble hexameric insulin onto the surface of insulin crystals cocrystallized with protamine: an electrostatic interaction.

Mixing pharmaceutical preparations of soluble neutral regular insulin solution (NRI) and neutral protamine Hagedorn (NPH) crystalline insulin suspension leads to a reduction in the measurable amount of soluble insulin in the formulation supernatant. However in spite of the loss in soluble insulin, the time-actions of these components have been shown, in clinical trials, to be unaffected. The interaction between these different physical forms of insulin has been studied using reversed-phase HPLC, isothermal titrating calorimetry, and Doppler electrophoretic light scattering analysis. Sorbent surface and solution perturbation studies revealed that the NRI adsorbs to the surface of the NPH crystal with an equilibrium constant ranging from 10(4) M-1 to 10(7) M-1, depending on the protamine concentration, pH, ionic strength, and temperature. This adsorption behavior suggests that the binding is mediated by electrostatic interactions arising between the positively-charged NPH crystal and the negatively-charged NRI hexamer. Doppler electrophoretic light scattering results, used to probe the pH-dependent surface charge of NPH and soluble insulin hexamer, support the conclusion that electrostatic interactions mediate the adsorption process. Adsorption studies under physiological conditions indicate that the elevated temperature and ionic strength, in a subcutaneous depot, are sufficient to lead to the dissociation of the NRI/NPH complex that exists in these NPH mixture formulations.

Amide hydrogen exchange of the central B-chain helix within the T- and R-states of insulin hexamers.

Comparative analysis of the 1H-NMR spectra of human insulin shows that in the presence of the allosteric ligand, phenol, the tertiary structure of the protein is altered as evidenced by the decreased rate of amide hydrogen-deuterium exchange. In particular, exchange of amide protons in residues of the B-chain helix (B9-B20) are significantly affected suggesting either a stabilization of this helix or a reduction in the solvent accessibility of the helix in the R-state. This paper exemplifies the exchange rates of two amides (ValB18 and TyrB16) from this helix which decrease by approximately 400-fold as a result of this ligand induced conformational transition.

Conformational studies of a peptide corresponding to a region of the C-terminus of ribonuclease A: implications as a potential chain-folding initiation site.

Conformational properties of the OT-16 peptide, the C-terminal 20 amino acids of RNase A, were examined by nonradiative energy transfer. A modified OT-16 peptide was prepared by solid-phase synthesis with the inclusion of diaminobutyric acid (DABA) at the C-terminus. The OT-16-DABA peptide was labeled with a fluorescent 1,5-dimethylaminonaphthalene sulfonyl (dansyl, DNS) acceptor at the N-terminal amine and a fluorescent naphthoxyacetic acid (NAA) donor at the gamma-amine of the DABA located at the C-terminus of the peptide by using an orthogonal protection scheme. Energy transfer was monitored in DNS-OT-16-DABA-NAA by using both fluorescence intensity (sensitized emission) and lifetime (donor quenching) experiments. The lifetime data indicate that the peptide system is a dynamic, flexible one. A detailed analysis, based on a dynamic model that includes a skewed Gaussian function to model the equilibrium distribution of interprobe distances and a mutual diffusion coefficient between the two probes to model conformational dynamics in the peptide [Beechem & Haas (1989) Biophys. J. 55, 1225.], identified the existence of a partially ordered structure (relatively narrow distribution of interprobe distances) at temperatures greater than or equal to 20 degrees C in the absence of denaturant. The width and the position of the average of the distributions decrease with increasing temperature, in this range; this suggests that the structure is stabilized by hydrophobic interactions. In addition, the peptide undergoes cold denaturation at around 1.5 degrees C as indicated by broadening of the distance distribution. The addition of 6 M guanidine hydrochloride (Gdn-HCl) also broadens the distance distribution significantly, presumably by eliminating the hydrophobic interactions and unfolding the peptide. The results of the analysis of the distance distribution demonstrate that (1) nonradiative energy transfer can be used to study the conformational dynamics of peptides on the nanosecond time scale, (2) a partially ordered structure of OT-16-DABA exists in solution under typical refolding conditions, and (3) structural constraints (presumably hydrophobic interactions) necessary for the formation of a chain-folding initiation site in RNase A are also present in the OT-16-DABA peptide in the absence of denaturant and are disrupted by Gdn-HCl.

The kinetic assembly of the intrinsic bovine factor X activation system.

The activation of bovine factor X by bovine factors IXa alpha and IXa beta has been examined under conditions of progressive assembly of the complete intrinsic activation system, i.e., factor X/factor IXa/Ca2+/phospholipid (PL)/factor VIIIa. In the presence of Ca2+, and the absence of PL and factor VIIIa, factor IXa alpha is a more efficient enzyme than factor IXa beta toward factor X activation, primarily due to the much higher kcat for the factor IXa alpha-catalyzed reaction. Analysis of the steady-state kinetic properties, after addition of PL (mixtures of phosphatidylcholine/phosphatidylserine) to the factor X/factor IXa/Ca2+ activation system, shows that the mechanism most closely follows a nonessential activation scheme, where the true substrate is the factor X/Ca2+/PL complex. The presence of PL results in a large (1-2 orders of magnitude) increase of the kcat for factor IXa beta, but does not substantially affect the steady-state kinetic constants of the factor IXa alpha-catalyzed reaction. Examination of the steady-state activation kinetics of factor X, after addition of factor VIIIa to factor X/factor IXa/Ca2+/PL, demonstrates that the mechanism is most consistent with a nonessential activation scheme of fluid phase substrate (factor X) being activated by a PL-bound enzyme system (factor IXa/Ca2+/factor VIIIa/PL). The presence of factor VIIIa stimulated the rates of factor X activation by factor IXa beta/Ca2+/PL by 1-2 orders of magnitude. Qualitatively similar behavior was noted for the factor IXa alpha-catalyzed activation. The results of this manuscript show that, in the presence of Ca2+ and absence of other cofactors, factor IXa alpha is a much more efficient enzyme for factor X activation, as compared to factor IXa beta. This is likely due to effects on the system resulting from covalent retention of the negatively charged activation peptide, by factor IXa alpha. However, the enzymatic activity of factor IXa beta shows a far better response to cofactors, particularly PL, than factor IXa alpha, thereby rendering factor IXa beta the more efficient enzyme in the complete intrinsic activation system.

Circular dichroism analysis of the secondary structures of bovine blood coagulation factor IX, factor X, and prothrombin.

Analysis of the far-ultraviolet circular dichroism spectrum of bovine blood coagulation factor IX reveals the presence of approximately 14% helical structures 26% beta-sheets, 20% beta-turns, and 40% coils. These values are essentially the same for the activation products of this zymogen, factor IX alpha alpha and factor IX alpha beta. Similar analysis for bovine factor X permits calculation of these secondary structural as approximately 11% helices, 31% beta-structures, 22% beta-turns, and 36% random structures. Bovine prothrombin contains approximately 12% helical structures, 35% beta-structures, 24% beta-turns, and 29% coils. None of these values is substantially altered as a result of increase of the pH from 7.4 to 10.5, or upon addition of Ca2+ to a concentration of at least 20 mM. Analysis of the near-ultraviolet spectra of factor IX and prothrombin suggests that several aromatic amino acid residues and the disulfide bond present in their gamma-carboxyglutamic acid-containing regions are exposed to solvent and are perturbed by the above pH adjustment and Ca2+ addition. Similar effects are observed in the case of factor X; in addition, the Trp residue at the amino terminus of the heavy chain appears to be influenced by the above pH alteration. The results reported in this paper show that these vitamin K-dependent blood coagulation proteins are similar in their ordered secondary structures, which are dominated by beta-sheets and beta-turns. Their overall secondary structures are not influenced by Ca2+ binding and are stable to alkaline pH changes. However, these same environmental alterations appear to be effective probes of aromatic residues in the gamma-carboxyglutamic acid regions.

Prediction of the secondary structures of bovine blood coagulation factor IX, factor X, and prothrombin.

The secondary structures of bovine blood coagulation factors IX and X, as well as that of bovine prothrombin, were predicted on the basis of a computerized combination of the Chou-Fasman and Burgess algorithms. Refinements in the predictions were made after consideration of the content of various secondary structures, as determined by circular dichroism studies of these same proteins. The final turn assignments were in good agreement with those assigned with use of an algorithm involving pattern matching of beta-turns in proteins of known structure.

The genetic relationships between the kringle domains of human plasminogen, prothrombin, tissue plasminogen activator, urokinase, and coagulation factor XII.

A computer-based statistical evaluation of the optimal alignments of the kringle domains of human plasminogen, human prothrombin, human tissue plasminogen activator, human urokinase, and human coagulation Factor XIIa, as well as the putative kringle of human haptoglobin, has been performed. A variety of different alignments has been examined and scores calculated in terms of the number of standard deviations (SD) of a given match from randomness. With the exception of human haptoglobin, it was found that very high alignment scores (8.9-23.0 SD from randomness) were obtained between each of the kringles, with the kringle 1 and kringle 5 regions of human plasminogen displaying the highest similarity, and the S kringle of human prothrombin and the human Factor XII kringle showing the least similarity. The relationships obtained were employed to construct an evolutionary tree for the kringles. The predicted alignments have also allowed nucleotide mutations in these regions to be evaluated more accurately. For those regions for which nucleotide sequences are known, we have employed the maximal alignments from the protein sequences to assess nucleotide sequence similarities. It was found that a range of approximately 40-55% of the nucleotide bases were placed at identical positions in the kringles, with the highest number found in the alignment of the two kringles of human tissue plasminogen activator and the lowest number in the alignment of the S kringle of prothrombin with the second kringle of tissue plasminogen activator. From both protein and nucleotide alignments, we conclude that haptoglobin is not statistically homologous to any other kringle. Secondary structural comparisons of the kringle regions have been predicted by a combination of the Burgess and Chou-Fasman methods. In general, the kringles display a very high number of beta-turns, and very low alpha-helical contents. From analysis of the predicted structures in relationship to the functional properties of these domains, it appears as though many of their functional differences can be related to possible conformational alterations resulting from amino acid substitutions in the kringles.

Secondary structure predictions of human plasminogen and the bovine prothrombin kringle loops.

Secondary structural predictions, based upon the statistical methodology of Chou and Fasman, for the kringle loops of human plasminogen and bovine prothrombin suggest a "winding staircase" pattern of beta-turns, spaced by short regions of ordered and coil structures. Analysis of the predicted structures of the regions containing the two His (113 and 387) and Asp (136 and 410) residues in plasminogen kringles 1 and 4, which have been found to be important in binding the ligand, epsilon-aminocaproic acid, shows that all are localized at the same positions on beta-turns. In addition, both of the two Asp residues occur at the end of homologous nonapeptide regions common to all of the five human plasminogen and two bovine prothrombin kringles, indicating evolutionary conservation to preserve biologically critical conformations. Examination of the protein conformation in the region of Asn288, the residue which is glycosylated in one of the two circulating variants of human plasminogen, shows that it most likely exists in a position which may present topographical hindrance to post-translational attachment of carbohydrate, thus, possibly, explaining the incomplete glycosylation of human plasminogen with complex-type carbohydrate.

Examination of the secondary structure of the kringle 4 domain of human plasminogen.

The structure of a small region of human plasminogen (F4), consisting of amino acid residues Val354-Ala439 and containing its kringle 4 (K4) domain (residues Cys357-Cys434), has been predicted from Chou-Fasman calculations and hydropathy profiles, and compared to circular dichroism (CD) measurements on the isolated fragment. Calculations, by the Chou-Fasman method, of the probabilities of various types of secondary structures that exist in this region reveal that no helical structures are present. Of the total of 86 amino acid residues present in this K4-containing peptide region, 37% can adopt conformations of beta-pleated sheets, 48% of the amino acids can exist in beta-turns, and 15% of the residues can be present as coils. The structure of F4 in dilute aqueous solution has been experimentally evaluated by CD measurements. At pH = 7.4, in dilute salt solutions, a total of 64% beta-structures, 30% beta-turns, and 6% coiled structures is estimated to be present in this peptide region. Consideration of the marginal stability of many of the conformational regions of F4, as predicted by Chou-Fasman calculations, suggests that secondary structural flexibility is present in this fragment, which could result in ready adoption of new conformations. The hydropathy profile of F4 has been determined and suggests that this polypeptide is highly hydrophilic, especially in the regions of residues His387-Tyr396 and Cys406-Lys413. Thus, it appears as though a large portion of the surface of F4 can be exposed to solvent in its native conformation.

The interaction of bovine factor IX, its activation intermediate, factor IX alpha, and its activation products, factor IXa alpha and factor IXa beta, with acidic phospholipid vesicles of various compositions.

The interactions of bovine factor IX, its activation intermediate, Factor IX alpha, and its activation products, Factor IXa alpha and Factor IXa beta, with phospholipid vesicles, of mean radius of approx. 30 nm, containing various amounts of phosphatidylserine (PS) and phosphatidylcholine (PC), have been examined. For Factor IX, Factor IX alpha, Factor IXa alpha and Factor IXa beta, the dissociation constants, at saturating levels of Ca2+, are independent of the PS concentration in the vesicle after levels of 20-30% (w/w) have been reached, and attain minimum values of approx. 1.7, 1.7, 0.7 and 1.0 microM, respectively, with vesicles containing 50% PS. The amount of protein bound per vesicle particle is independent of the PS content, above 20% PS, for Factor IX and Factor IXa beta, with values of approx. 995-1197 and 1128-1566 molecules/vesicle, respectively. With Factor IX alpha, a dependence on the amount of protein bound with the content of PS is seen, which ranges from 338 to 619 molecules/vesicle with membranes containing 30-50% PS. For Factor IXa alpha, no regularity is noted and a range of 583-1083 molecules of protein/vesicle is observed with the systems employed. Examination of the radii of the proteins on the vesicle demonstrates that Factors IX alpha and IXa alpha occupy considerably more of the surface than do Factors IX and IXa beta, suggesting that a reason for the decreased number of binding sites for the former two proteins on the vesicle may be related to their greater surface spatial requirements.

Interspecies cross-reactivity of monoclonal antibodies to various epitopes of human plasminogen.

The immunological cross-reactivities of three conformationally specific monoclonal antibodies to distinct epitopes on human plasminogen toward plasminogens purified from 14 additional species have been examined. Antibody 10-F-1, which is produced against an epitope on the kringle 4 region of human plasminogen, shows a high degree (greater than 80%) of cross-reactivity against baboon, goat, monkey, ovine, and rabbit plasminogens; more limited (20-50%) cross-reactivity against bovine, equine, goose, guinea pig, mouse, rat, and porcine plasminogens; and little comparable cross-reactivity against canine and chicken plasminogens. Antibody 10-H-2, generated to an epitope of the kringles 1-3 region of human plasminogen, shows extensive cross-reactivity (72%) only toward monkey plasminogen, more limited (22-35%) cross-reactivity toward equine and rabbit plasminogens, and much less cross-reactivity toward any other of the above plasminogens. Antibody 10-V-1, also produced against an epitope on the kringle 1-3 region of human plasminogen, which is distinct from the 10-H-2 epitope, shows extensive cross-reactivity (72-100%) with baboon, monkey, and rabbit plasminogens; more limited cross-reactivity with equine (48%) and mouse (28%) plasminogens; and a low level of such reactivity with the remaining plasminogens. These studies show that the extent of interspecies cross-reactivity of various plasminogens greatly depends upon the epitope in question. The K4 region of these molecules appears more extensively conserved than the K1-3 region, at least in regard to the particular epitopes examined in this study.