Mary T. Walsh

Prothrombotic Consequences of the Oxidation of Fibrinogen and their Inhibition by Aspirin.

Oxidant stress leads to covalent oxidative modification of several plasma proteins, chief among which is fibrinogen. Aspirin can nonenzymatically acetylate fibrinogens lysine residues, the functional groups most susceptible to oxidative modification. Because oxidation of fibrinogen may occur in the atheromatous environment, we studied the effects of oxidative modification on fibrinogen function and the consequences of acetylation by aspirin on fibrinogens susceptibility to oxidation and functional properties. We exposed fibrinogen to Fe3+ ascorbate for 1 hour and showed that the carbonyl/protein molar ratio increased from 0.71 ± 0.18 to 2.86 ± 0.50 mol carbonyl/mol protein (P < 0.02) with an accompanying reduction in the α-helical content of the protein from 34% to 29%. Exposure of fibrinogen to aspirin led to acetylation of lysine residues and inhibition of oxidation. Oxidized fibrinogen was more readily able to form fibrin, and acetylation prevented this enhancement of clot formation. Oxidized fibrinogen also supported platelet aggregation better than did native, unoxidized fibrinogen, and acetylation of fibrinogen prior to oxidation prevented the enhanced platelet aggregation. Oxidized fibrinogen was less effective in stimulating plasminogen activation by tissue-type plasminogen activator (t-PA), with a catalytic efficiency that was reduced by 88% compared with native, unoxidized fibrinogen; acetylated fibrinogen, by contrast, enhanced plasminogen activation by t-PA with a catalytic efficiency that was increased by 18% compared with native, unoxidized fibrinogen (P < 0.05) and was increased by 51% compared with oxidized fibrinogen(P < 0.05). Acetylation prevented the reduction in catalytic efficiency induced by oxidation. These data show that oxidized fibrinogen manifests prothrombotic effects that can be prevented by acetylation and suggest that inhibition of fibrinogen oxidation may be an additional antithrombotic benefit of aspirin therapy.

Cellular Response of Cardiac Fibroblasts to Amyloidogenic Light Chains

Amyloidoses are a group of disorders characterized by abnormal folding of proteins that impair organ function. We investigated the cellular response of primary cardiac fibroblasts to amyloidogenic light chains and determined the corresponding change in proteoglycan expression and localization. The cellular response to 11 urinary immunoglobulin light chains of kappa1, lambda6, and lambda 3 subtypes was evaluated. The localization of the light chains was monitored by conjugating them to Oregon Green 488 and performing live cell confocal microscopy. Sulfation of the proteoglycans was determined after elution over Q1-columns with a single-step salt gradient (1.5 mol/L NaCl) via dimethylmethylene blue. Light chains were detected inside cells within 4 hours and demonstrated perinuclear localization. Over 80% of the cells showed intracellular localization of the amyloid light chains. The light chains induced sulfation of the secreted glycosaminoglycans, but the cell fraction possessed only minimal sulfation. Furthermore, the light chains caused a translocation of heparan sulfate proteoglycan to the nucleus. The conformation and thermal stability of light chains was altered when they were incubated in the presence of heparan sulfate and destabilization of the amyloid light chains was detected. These studies indicate that internalization of the light chains mediates the expression and localization of heparan sulfate proteoglycans.

Biophysical analysis of normal transthyretin: Implications for fibril formation in senile systemic amyloidosis

Transthyretin (TTR) is a plasma protein that transports thyroid hormone andretinot binding protein-vitamin A complex. Eighty-four variants of TTR have been identified and seventy-four are associated with familial amyloidotic polyneuropathy. Normal TTR is the major protein found in the fibrillar deposits in the heart at time of autopsy of individuals with senile systemic amyloidosis. The mechanism by which normally soluble TTR deposits as organ-damaging, insoluble, pathological fibrils late in life is unknown. Understanding the mechanism of fibrillogenesis of normal TTR is critical to the design of clinical treatments aimed at retardation, prevention, or reversal of fibril deposition. We have employed a biophysical approach to explore the hypothesis that an instability in a particular secondary or tertiary structure plays a role in the ability of normal TTR to form fibrils at physiological pH. Using far UV circular dichroic (CD) spectroscopy as a function of temperature we have identified simultaneous, cooperative, reversible structural changes in the β-sheet and α-helical regions. The flexible short, surface-located loops undergo an irreversible conformational change at a lower temperature. Spectra before and after heating are different, particularly in the wavelength region associated with these loops, strongly suggesting that the major portion of TTR returns to its initial conformation while the loops do not. Near UV CD reveals partially reversible and irreversible changes in tertiary structure. Using calorimetry to directly measure the enthalpy associated with these changes, two peaks are observed, with further analysis suggesting conformational intermediates. Precipitates from heated samples reveal pre-fibrillar morphology by negative stain electron microscopy. These biophysical studies suggest that heat-induced conformational rearrangements enable normal TTR to assemble into prefibrils at physiological pH.

Extraction and purification of decorin from corneal stroma retain structure and biological activity

We developed a method to purify decorin core protein from tissue with the goal of preserving its native structure and biological function. Currently, most procedures rely on the use of denaturing reagents potentially altering the biological activity. Decorin was purified from corneal stromas without the use of detergents or chaotropic reagents. Proteoglycans isolated using anion exchange chromatography on Q-Sepharose were treated with chondroitinase ABC. Decorin was isolated by a second Q-Sepharose chromatography with affinity chromatographies on heparin-Sepharose and concanavalin A-Sepharose. SDS-PAGE revealed a 98.4% pure 44kDa protein identified as decorin with a yield of 35mg per 100 bovine corneas. Identification was confirmed by NanoESI and MALDI qTOF. The novel inclusion of 20% propylene glycol in extraction and column buffers resulted in recoveries of proteoglycans comparable with those observed with detergents and urea. Purified decorin did alter the rate of fibrillogenesis of type I collagen and inhibited the lateral fusion of collagen fibrils. It also bound to [125I]TGF-beta1 with an apparent K(d) of 40nM. Circular dichroism spectroscopy of decorin displayed the spectra of alpha-helices and beta-pleated sheets consistent with those obtained from recombinant decorin. Urea-induced unfolding was cooperative and reversible while thermal denaturation caused irreversible unfolding. Native decorin can be purified from tissue in quantity and quality for biophysical, biochemical, and biological assays.

[33] Reassembly of low-density lipoproteins

Abstract The methodologies described here for the selective and sequential reassembly of model LDL particles, although in many instances still in the developmental stages, will undoubtedly provide a basis on which further advances in LDL reassembly will be made. Reassembled LDL complexes of defined lipids and apoB provide well-defined model systems in which to study the molecular interactions and structural organization of LDL, including the lipid-lipid interactions in the particle core, the lipid-lipid and lipid-protein interactions which determine the surface organization and protein conformation, and the interactions between the core and surface components. These reassembled LDL complexes should serve as important models to study the delivery of isotopically labeled lipids with differing physical properties to cells in order to investigate the metabolic complexity of intracellular LDL catabolism and its relationship to positive cholesterol balance and atherogenesis.

A novel amyloidogenic variant of apolipoprotein AI: implications for a conformational change leading to cardiomyopathy.

Amyloidosis is a broad term for a group of diseases which have in common the extracellular deposition of pathological insoluble fibrillar proteins in organs and tissues. 1 Although unrelated in primary sequence and metabolic roles, almost all amyloid proteins exhibit β-pleated sheet secondary structure in their normal environments and green birefringence on polarization microscopy after Congo red staining, and deposit as nonbranching, insoluble, highly ordered fibrils. 2,3 The protein in the fibril and the pathogenesis of the disease is unique for each type of amyloid. 1 The mechanism(s) by which such a diverse array of proteins with different metabolic roles become insoluble and deposit as pathological fibrils, damaging tissues and organs and ultimately causing death, are unknown.

Alterations in the secondary structure of mutant transthyretins associated with familial amyloidotic polyneuropathy after proteolysis by neutrophil serine proteases

Transthyretin (TTR), a tetrameric protein with two extensive β-sheets in each monomer, is the precursor protein of the amyloid fibril deposits in persons with an autosomal dominantly inherited disease termed familial amyloidotic polyneuropathy (FAP orATTR). The TTR isolated from four affected heterozygous individuals, each with a different mutation or at a different stage of disease was examined with respect to alteration in conformation after exposure to human neutrophil elastase (HNE) or cathepsin G by circular dichroism (CD), and their spectra compared to normal.Normal untreated TTR exhibits a negative minimum at 214 nm by CD analysis. After enzymatic digestion, minor changes are observed. The mutant TTRs differ from normal with a more marked alteration in β conformation after digestion. The degree of CD alteration of mutant TTR digested with HNE seemed to correlate with the severity of disease in each kinship. In addition, mutant TTRs that are more hydrophilic have more marked CD alteration.

Secondary Structure Changes in Mutant Transthyretins after Proteolysis by Neutrophilic Serine Proteases

The secondary structure of plasma transthyretin (TTR) from patients with familial amyloidotic polyneuropathy was studied with respect to alteration in the amount of beta conformation after exposure to proteolytic enzymes of the neutrophilic serine protease family. We examined TTR from four affected individuals, each with a different mutation or at a different stage of disease. After exposure to human neutrophilic elastase (HNE) or cathepsin G, circular dichroic (CD) spectra for each was compared to normal.

Secondary and tertiary structure of apolipoproteins.

The advent of these and other high-powered techniques for the detailed study of apoLP organization will allow us to obtain a high resolution picture of apoLP conformation both in solution and on native lipoprotein particles.