The role of antiplatelet therapy in the management of acute coronary syndromes.

The benefit of aspirin use in the emergent care of acute coronary syndromes (ACS) has been well-established. Recent studies have further demonstrated the importance of antiplatelet therapy in the acute setting, primarily with the use of intravenous glycoprotein IIb/IIIa receptor inhibitors. Aspirin and the thienopyridines (ticlopidine and clopidogrel) are oral antiplatelet agents that interfere with platelet activation in complementary, but separate pathways. Combination therapy of aspirin with other antiplatelet agents has demonstrated a benefit for the management of ACS. This article reviews the pathophysiology of platelet activation in ACS, landmark trials regarding antiplatelet agents, and the current recommendations for the use of both intravenous and oral antiplatelet agents in the management of patients with ACS.

Aging of human glyceraldehyde-3-phosphate dehydrogenase is dependent on its subcellular localization.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), long considered a traditional glycolytic protein, displays multiple activities independent of its role in energy generation. This functional diversity is dependent on its membrane, cytoplasmic or nuclear localization. GAPDH is encoded by one active gene and is synthesized as a single 37 kDa protein without alternate splicing. Accordingly, the identical protein would be present in each subcellular fraction. The accumulation of post-translational errors in protein structure as a function of oxidative stress is thought to provide a basic molecular mechanism for the aging process. Thus, during aging, the GAPDH protein should contain the identical degree of oxidative sequence alteration irrespective of its distribution. This would result in equivalent effects on GAPDH activity. However, conformational differences in GAPDH structure due to its subcellular protein, nucleic acid or membrane interactions could affect its degree of modification thereby selectively affecting its function. For that reason, we examined the subcellular expression and intracellular activity of GAPDH as a function of human aging. Subcellular GAPDH expression was quantitated by immunoblot analysis in fetal and senior human cells (postnuclear, nuclear, perinuclear). GAPDH activity was determined by in vitro assay. We now report that the aging of human GAPDH was subcellular dependent. Reductions of nuclear and postnuclear GAPDH activity in senior cells were twofold lower than that observed for the perinuclear protein. In contrast, the subcellular expression of the GAPDH protein was age-independent. These results suggest the possibility that subcellular interactions may mitigate oxidative stress-induced GAPDH modification in human aging. Such selective effects on GAPDH could affect its functional diversity.

Subcellular analysis of aberrant protein structure in age-related neurodegenerative disorders.

Subcellular interactions of neurodegenerative disease proteins may provide a basic molecular mechanism underlying neuronal disorders. Each protein may also exhibit activities related to normal cell structure and function. It may be necessary to develop methodologies to distinguish between normal and abnormal intracellular interactions of such proteins in human cells. The latter would result in distinct perturbations in cell function depending both on the specific protein or nucleic acid interactions as well as its subcellular localization. Individual neurodegenerative disorders may be characterized by distinct alterations in subcellular neuronal protein structure and function. We developed as a basic experimental paradigm a novel human cell system to identify and examine such abnormal neuronal protein structures. The basic rationale is that neurodegenerative protein interactions would result in the formation of intracellular high molecular weight (HMW) complexes in cells from afflicted individuals. Following cell fractionation these unique structures could be detected by gradient sedimentation coupled with immunoblot analysis. They would not be observed in age matched control normal human cells. We now report that this procedure has been successfully used to determine a unique subcellular alteration of beta-amyloid precursor protein (beta-APP) structure in Alzheimer's disease (AD) cells. The latter was not observed in normal cells. Similar structural alterations were observed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein known to bind to beta-APP in vitro. The utility of this model system to interrelate aberrant protein interactions of neurodegenerative disease proteins and their subcellular localization is considered.

Subcellular localization of human glyceraldehyde-3-phosphate dehydrogenase is independent of its glycolytic function.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered a classical glycolytic protein involved exclusively in cytosolic energy production. However, recent evidence suggests that it is a multifunctional protein displaying diverse activities distinct from its conventional metabolic role. These new roles for GAPDH may be dependent on its subcellular localization, oligomeric state or on the proliferative state of the cell. GAPDH is encoded by a single gene without alternate splicing. The regulatory mechanisms are unknown through which an individual GAPDH molecule fulfills its non-glycolytic functions or is targeted to a specific intracellular localization. Accordingly, as a first step to elucidate these subcellular regulatory mechanisms, we examined the interrelationship between the intracellular expression of the GAPDH protein and its glycolytic function in normal human fetal and senior cells. GAPDH localization was determined by immunoblot analysis. Enzyme activity was quantitated by in vitro biochemical assay. We now report that the subcellular expression of GAPDH was independent of its classical glycolytic function. In particular, in both fetal and senior cells, considerable GADPH protein was present in intracellular domains characterized by significantly reduced catalysis. Gradient analysis indicated that this lower activity was not due to the dissociation of tetrameric GAPDH. These results suggest that human cells contain significant intracellular levels of enzymatically inactive GAPDH which is age-independent. The possibility is considered that the functional diversity of GAPDH may be mediated either by posttranslational alteration or by subcellular protein:protein and/or protein:nucleic acid interactions.

Subcellular alteration of glyceraldehyde-3-phosphate dehydrogenase in Alzheimer's disease fibroblasts.

The regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been implicated both in age-related neurodegenerative disease and in apoptosis. Previous in vitro studies suggest an interaction between GAPDH and the beta-amyloid precursor protein (beta-APP), a protein directly involved in Alzheimer's disease (AD). New studies indicate that GAPDH is a multidimensional protein with diverse membrane, cytoplasmic, and nuclear functions; each is distinct from its role in glycolysis. The nuclear functions of GAPDH include a role in apoptosis that requires its translocation to the nucleus. Accordingly, beta-APP-GAPDH interactions, altering GAPDH structure in vivo, may affect energy generation, inducing hypometabolism, a characteristic AD phenotype. Because GAPDH is a multifunctional protein, pleiotropic effects may also occur in a variety of fundamental cellular pathways in AD cells. This may include unique GAPDH-RNA interactions. We report here the identification of a high-molecular-weight (HMW) GAPDH species present exclusively in the postnuclear fraction of AD cells. The latter is characterized by reduced GAPDH activity. The HMW GAPDH species was not detected in postnuclear age-matched control (AMC) fractions nor in AD whole-cell preparations. Each is characterized by normal GAPDH activity. By definition, the preparation of whole-cell extracts entails the destruction of subcellular structure. The latter findings indicate that the dissociation of the GAPDH protein from the HMW species restores its enzymatic activity. Thus, these results reveal a new, unique intracellular phenotype in AD cells. The functional consequences of subcellular alteration in GAPDH structure in AD cells are considered.

Alteration of intracellular structure and function of glyceraldehyde-3-phosphate dehydrogenase: a common phenotype of neurodegenerative disorders?

Recent evidence reveals that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is not simply a classical glycolytic protein of little interest. Instead, it is a multifunctional protein with diverse cytoplasmic, membrane and nuclear activities. Significantly, each activity is separate and distinctfrom its role in energy production. Its nuclear activities include its emerging role in apoptosis especially in neuronal cells. GAPDH translocates into the nucleus during programmed cell death. Introduction of antisense GAPDH sequences reduces apoptosis and prevents its nuclear translocation. Independent analyses demonstrate that GAPDH may be involved in the cellular phenotype of age-related neurodegenerative disorders. GAPDH binds uniquely in vitro to the beta-amyloid precursor protein (betaAPP), to huntingtin as well as to other triplet repeat neuronal disorder proteins. In Parkinson's disease (PD) cells, immunofluorescent data suggests the co-l localization of GAPDH and alpha-synuclein in Lewy bodies. Drugs used to treat PD bind specifically to GAPDH. Our recent findings (Mazzola and Sirover, 2001) demonstrate a subcellular reduction in GAPDH glycolytic activity in Alzheimer's disease (AD) and in Huntington's disease (HD) cells. The latter may be due to intracellular alteration of GAPDH structure (Mazzola and Sirover 2002). We discuss the hypothesis that the intracellularformation of GAPDH: neuronal protein complexes may represent an emerging cellular phenotype of neurodegenerative disorders. The cytoplasmic binding of neuronal proteins to GAPDH could affect energy production. Nuclear interactions could affect its apoptotic activity. Other functions of this multidimensional protein may also be inhibited. Experimental paradigms to test this hypothesis are considered.

Alteration of nuclear glyceraldehyde-3-phosphate dehydrogenase structure in Huntington's disease fibroblasts.

The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) may be involved in neuronal disease and in programmed cell death. Recent investigations indicate an in vitro physical association between GAPDH and huntingtin, the mutated protein in Huntington's disease (HD). Previous studies reveal the functional diversity of GAPDH as a membrane, cytoplasmic and nuclear protein. These activities are independent of its classical glycolytic function. Thus, huntingtin-GAPDH interactions could affect not only energy production but also result in pleiotropic effects involving various biochemical pathways in HD cells. We now report the identification of a nuclear high molecular weight (HMW) GAPDH species in Huntington's disease cells. In contrast, nuclei from age-matched control normal human cells did not contain the HMW GAPDH species. Further, this GAPDH structure was not observed in HD whole cell sonicates which are characterized by normal GAPDH activity. The disruption of intracellular structure is implicit in the preparation of whole cell sonicates. Therefore, these results suggest that the dissociation of the GAPDH protein from its high molecular weight structure results in the recovery of its function. These findings reveal a singular, new subcellular phenotype in HD cells. As such, they indicate an interrelationship between nuclear GAPDH function and huntingtin localization in this CAG expansion neuronal disease.