Quasielastic light-scattering studies on human fibrinogen and fibrin. I. Fibrinogen

A quasielastic light-scattering system has been constructed to study human fibrinogen. The first phase of the investigation was an attempt to clarify the shape of the firbinogen molecule using diffusion methods. The translational diffusion coefficient was measured as 2.04 ± 0.09 × 10^?7 cm² sec^?1. Aggregation is suggested as the reason for a lower value previously obtained using this technique. Diffusion indicated the molecule was rigid and did not dissociate at low concentration, low ionic strength, or 37°C. Thermal denaturation was observed at 40°C. At 3°C, a second thermal instability was discovered.     

Polymerization of the tubulin-colchicine complex: relation to microtubule assembly

The polymerization of purified tubulin-colchicine complex, which results in polymers different from microtubules under microtubule-promoting conditions, has been characterized. It proceeds as a nucleated condensation polymerization, requires Mg2+, and is inhibited by small concentrations of Ca2+. Polymerization requires GTP binding, but GDP is inhibitory. The GTPase activity proceeds, but it is unlinked to polymerization. The thermodynamic characteristics of the growth reaction, namely, the apparent changes of free energy, enthalpy, entropy, heat capacity, and preferential interaction with H+ and Mg2+, are very similar to those of microtubule assembly. It is proposed that the interactions responsible for the two types of polymerization are very similar and that the molecular mechanism of microtubule inhibition by colchicine may consist in a drug-induced distortion of the normal protomer bonding geometry.     

Polymerization of rod-like macromolecular monomers studied by stopped-flow, multiangle light scattering: set-up, data processing, and application to fibrin formation

Many biological supramolecular structures are formed by polymerization of macromolecular monomers. Light scattering techniques can provide structural information from such systems, if suitable procedures are used to collect the data and then to extract the relevant parameters. We present an experimental set-up in which a commercial multiangle laser light scattering photometer is linked to a stopped-flow mixer, allowing, in principle, the time-resolved extrapolation of the weight-average molecular weight M(w) and of the z-average square radius of gyration (z) of the polymers from Zimm-like plots. However, if elongated structures are formed as the polymerization proceeds, curved plots rapidly arise, from which M(w) and (z) cannot be recovered by linear fitting. To verify the correctness of a polynomial fitting procedure, polydisperse collections of rod-like or worm-like particles of different lengths, generated at various stages during bifunctional polycondensations of rod-like macromolecular monomers, were considered. Then, the angular dependence of their time-averaged scattered intensity was calculated in the Rayleigh-Gans-Debye approximation, with random and systematic noise also added to the data. For relatively narrow size distributions, a third-degree polynomial fitting gave satisfactory results across a broad range of conversion degrees, yielding M(w) and (z) values within 2% and no greater than 10-20%, respectively, of the calculated values. When more broad size distributions were analyzed, the procedure still performed well for semiflexible polymers, but started to seriously underestimate both M(w) and (z) when rigid rod-like particles were analyzed, even at relatively low conversion degrees. The data were also analyzed in the framework of the Casassa approximation, from which the mass per unit length of the polymers can be derived. These procedures were applied to a set of data taken on the early stages of the thrombin-catalyzed polymerization of fibrinogen, a rod-like macromolecule approximately 50 nm long. The polymers, grown in the absence of Ca(2+) by rate-limiting amounts of thrombin, appeared to be characterized by a much broader size distribution than the one expected for a classical Flory bifunctional polycondensation, and they seem to behave as relatively flexible worm-like double-stranded chains. Evidence for the formation of fibrinogen-fibrin monomer complexes is also inferred from the time dependence of the mass/length ratio. However, our data are also compatible with the presence of limited amounts of single-stranded structures in the very early stages, either as a secondary, less populated pathway, or as transient intermediates to the classical double-stranded fibrils.     

Quasielastic light-scattering studies on human fibrinogen and fibrin. II. Fibrin polymerization

The shape of fibrin intermediate polymers as well as the rate of fibrinogen polymerization was studied using diffusion measured by quasielastic light scattering. After their length distribution was narrowed by gel filtration, the polymers yielded translational and rotational diffusion coefficients of 0.37 ± 0.05 × 10^?7 cm² sec^?1 and 142 ± 32 sec^?1, respectively. Theoretical considerations indicated the polymers to be rigid rods. The rate of polymerization of fibrinogen monitored by diffusion paralleled that provided by simulataneous intensity measurements. Both monitors indicated polymerization occurs most rapidly at 30°C.     

Fibrin polymerization studied by static and dynamic light-scattering as a function of fibrinopeptide A release

The polymerization of fibrin induced by the enzyme thrombin was investigated in the pregelation phase by combining measurements of the release of the fibrinopeptides A with static and dynamic light-scattering at scattering angles ranging from 5°–150°. Without making any assumptions about the polymer distribution, one is led to the following conclusions: The aggregates are rodlike; the Flory-Stockmayer model is to be preferred over the percolation model, i.e., cyclic bonds are infrequent; in the early stages the experiments indicate a functionality f = 2 (number of reactive binding sites per monomer) that increases with increasing release of fibrinopeptides A, eventually approaching the value f = 4; the number of binding sites involved in a bond is about twice the number of the released fibrinopeptides A; and in the polymers the monomer units aggregate end-to-end in the very early stage and then in a staggered overlap.     

A mechanism of inhibition of fibrin self-assembly by a peptide inhibitor

The amino acid composition of fibrin self-assembly inhibitors isolated from the blood plasma of man, ox, albino rat and from frog muscle tissue was established. Using a human blood plasma inhibitor, it was found that the inhibition of fibrin self-assembly is due to the inhibitor interaction with monomeric fibrin in the sites of domain D which become accessible after fibrinogen activation by thrombin.     

Integrated light-scattering spectroscopy, a sensitive probe for peptide-vesicle binding: application to the membrane-bound colicin E1 channel peptide.

Integrated light-scattering (ILS) spectroscopy was used to monitor the binding of the colicin E1 channel peptide to POPC:POPG large unilamellar vesicles (LUV; 60:40, mol:mol) at acidic pH (3.5). Binding conditions were chosen such that nearly all of the channel peptide was bound to the vesicles with little free peptide remaining in solution. The increase in vesicle size upon the insertion of the channel peptide was measured by performing a discrete inversion technique on data obtained from an ILS spectrometer. Vesicle size number distributions were determined for five different systems having peptide/vesicle ratios of approximately 0, 77, 154, 206, and 257. The experiment was repeated four times (twice at two different vesicle concentrations) to determine reproducibility. The relative changes in vesicle radius upon peptide binding to the membrane vesicles was remarkably reproducible even though these changes represented only a few nanometers. A comparison of vesicle size number distributions in the absence of bound peptide was made between ILS and dynamic light scattering (DLS) data and showed similar results. However, DLS was incapable of detecting the small changes due to peptide-induced vesicle swelling. The membrane-bound volume of the colicin E1 channel peptide was approximately 177 +/- 22 nm3. These data indicate that in the absence of a membrane potential (closed channel state) the colicin E1 channel peptide inserts into the membrane resulting in a significant displacement of the lipid bilayer as evidenced from the dose-dependent increase in the vesicle radius.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylcholine release during burst-patterned stimulation of a sympathetic ganglion: its relation to transmission efficiency

The time-course of increase in acetylcholine (ACh) output during burst-patterned preganglionic stimulation of the cat superior cervical ganglion has been determined. The increase in output corresponded closely with the increase in transmission efficiency found earlier in the stellate ganglion subjected to similar preganglionic stimulation. ACh output however continued to increase following the time of attainment of full neuronal recruitment. The results indicate that the increase in ACh output may largely account for the increase in transmission; but they leave unexplained the synaptic function of the continuing increase in transmitter release.     

Ghrelin: a new peptide regulating growth hormone release and food intake

Ghrelin, identified as an endogenous ligand for the growth hormone secretagogue receptor, is a 28 amino acid peptide hormone possessing an unusual octanoyl group on the serine in position 3, crucial for its biological activity. Ghrelin is predominantly produced by the stomach but also by many other tissues such as pituitary, hypothalamus, duodenum, jejunum, ileum, colon, lung, heart, pancreas, kidney, and testis. In addition to stimulation of GH release, ghrelin stimulates appetite and food intake, enhancing fat mass deposition and weight gain. Besides these main actions, ghrelin regulates gastric motility and acid secretion, exerts cardiovascular and anti-inflammatory effects, modulates cell proliferation and influences endocrine and exocrine pancreatic secretion, as well as glucose and lipid metabolism. Therefore, ghrelin agonists and antagonists might be valuable for some clinical aspects.     

A freeze-dried fibrin disc as a biodegradable drug release matrix.

A fibrin clot loaded with soluble tetracycline (TET) was prepared and lyophilized to make discs of a size and shape to use as a drug delivery matrix. On subcutaneous implantation of these discs in mice, they were found to have degraded in 15 days as evidenced by gross and histological examination. The in vitro discharge kinetics of tetracycline from the disc into phosphate buffered saline (PBS) and human serum were compared. It was observed that the release rate of tetracycline from the matrix into serum remained steady from day 1 to day 12, maintaining sufficient concentration that may be required to control microbial growth in the medium. Two different concentrations of fibrinogen were used to fabricate discs denoted as FG200 and FG100, and in both cases the retention rate was comparable when the study medium was serum. In contrast, when suspended in PBS instead of serum, the delivery of the drug into the medium was found to be high for up to the 3rd day when a sharp decline in discharge was observed. The fibrinogen used is a factor that determines not only the longevity of discharge but also fibrinolysis. The degradation of the disc in vitro was visible when the discs were suspended in the buffer, and correspondingly fibrin degradation product (FDP) measured in the medium using an antibody-based assay system was high. Fibrin disc is haemostatic and biodegradable in vivo, and in vitro release of a small molecule at a controlled rate is demonstrated here. Hence, it may be a suitable candidate as a drug delivery implant for short-term use.     

Frequency and release flux of calcium sparks in rat cardiac myocytes: a relation to RYR gating

Cytosolic calcium concentration in resting cardiac myocytes locally fluctuates as a result of spontaneous microscopic Ca²⁺ releases or abruptly rises as a result of an external trigger. These processes, observed as calcium sparks, are fundamental for proper function of cardiac muscle. In this study, we analyze how the characteristics of spontaneous and triggered calcium sparks are related to cardiac ryanodine receptor (RYR) gating. We show that the frequency of spontaneous sparks and the probability distribution of calcium release flux quanta of triggered sparks correspond quantitatively to predictions of an allosteric homotetrameric model of RYR gating. This model includes competitive binding of Ca²⁺ and Mg²⁺ ions to the RYR activation sites and allosteric interaction between divalent ion binding and channel opening. It turns out that at rest, RYRs are almost fully occupied by Mg²⁺. Therefore, spontaneous sparks are most frequently evoked by random openings of the highly populated but rarely opening Mg₄RYR and CaMg₃RYR forms, whereas triggered sparks are most frequently evoked by random openings of the less populated but much more readily opening Ca₂Mg₂RYR and Ca₃MgRYR forms. In both the spontaneous and the triggered sparks, only a small fraction of RYRs in the calcium release unit manages to open during the spark because of the limited rate of Mg²⁺ unbinding. This mechanism clarifies the unexpectedly low calcium release flux during elementary release events and unifies the theory of calcium signaling in resting and contracting cardiac myocytes.