ABSTRACT
To determine the molecular basis for the insensitivity of rat alpha(IIb)beta(3) to inhibition by RGD-containing peptides, hybrids of human and rat alpha(IIb)beta(3) and chimeras of alpha(IIb)beta(3) in which alpha(IIb) was composed of portions of human and rat alpha(IIb) were expressed in Chinese hamster ovary cells and B lymphocytes, and the ability of the tetrapeptide RGDS to inhibit fibrinogen binding to the various forms of alpha(IIb)beta(3) was measured. These measurements indicated that sequences regulating the sensitivity of alpha(IIb)beta(3) to RGDS are located in the seven amino-terminal repeats of alpha(IIb). Moreover, replacing the first three or four (but not the first two) repeats of rat alpha(IIb) with the corresponding human sequences enhanced sensitivity to RGDS, whereas replacing the first two or three repeats of human alpha(IIb) with the corresponding rat sequences had little or no effect. Nevertheless, RGDS bound to Chinese hamster ovary cells expressing alpha(IIb)beta(3) regardless whether the alpha(IIb) in the heterodimers was human, rat, or a rat-human chimera. These results indicate that the sequences determining the sensitivity of alpha(IIb)beta(3) to RGD-containing peptides are located in the third and fourth amino-terminal repeats of alpha(IIb). Because RGDS binds to both human and rat alpha(IIb)beta(3), the results suggest that differences in RGDS sensitivity result from differences in the allosteric changes induced in these repeats following RGDS binding.
Subject(s)
Blood Platelets/metabolism , Fibrinogen/chemistry , Fibrinogen/metabolism , Oligopeptides/chemistry , Platelet Glycoprotein GPIIb-IIIa Complex/chemistry , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Adenosine Diphosphate/metabolism , Allosteric Site , Animals , B-Lymphocytes/metabolism , CHO Cells , Carcinogens , Cell Adhesion , Cricetinae , DNA, Complementary/metabolism , Dithiothreitol/pharmacology , Dose-Response Relationship, Drug , Humans , Inhibitory Concentration 50 , Ligands , Platelet Aggregation , Protein Binding , Protein Structure, Tertiary , Rats , Recombinant Fusion Proteins/chemistry , Tetradecanoylphorbol Acetate , TransfectionABSTRACT
Platelets are derived from megakaryocytic and have a critical role in thrombus formation. Megakaryocytes are terminally differentiated marrow cells that are derived from the pluripotent hematopoietic stem cell (1). These extremely large, polyploid cells demarcate their cytoplasm, giving rise to circulating platelets. Following vascular injury, platelets adhere to the site of injury through von Willebrand factor (vWF) and the platelet membrane glycoprotein (GP) Ib/IX complex. The platelets become activated, and aggregate with other activated platelets through fibrinogen and the platelet membrane αIIb/ß3 (GPIIb/IIIa) integrin complex. In addition, platelets contain unique granules called α-granules that contain important factors involved in normal coagulation. These factors include factor VIII, vWF, factor V, Multimerin, fibrinogen, factor XIII, factor XI, thrombospondin, fibronectin, ß-thromboglobulin (ßTG) and platelet factor 4 (PF4). Some of these factors are actively synthesized in megakaryocytes, some are actively transported through clatherin pits, and some are endocytosed (2,3,9).
ABSTRACT
The most recent major advances in the study of programmed cell death (PCD) in plants include the observation that peptide inhibitors of caspases inhibit the hypersensitive response. Nitric oxide has been shown to be required for the induction of disease related PCD. Mutant analysis has led to the cloning of the first genes involved in PCD related disease resistance, LSD1 and MLO.
Subject(s)
Apoptosis/genetics , Arabidopsis Proteins , Apoptosis/drug effects , Arabidopsis/cytology , Arabidopsis/genetics , DNA-Binding Proteins/genetics , Mutation , Plant Proteins/genetics , Salicylic Acid/pharmacology , Signal Transduction , Transcription Factors/geneticsABSTRACT
Arabidopsis Isd1 mutants are hyperresponsive to cell death initiators and fail to limit the extent of cell death. Superoxide is a necessary and sufficient signal for cell death propagation. Thus, LSD1 monitors a superoxide-dependent signal and negatively regulates a plant cell death pathway. We isolated LSD1 via its map position. The predicted LSD1 protein contains three zinc finger domains, defined by CxxCxRxxLMYxxGASxVxCxxC. These domains are present in three additional Arabidopsis genes, suggesting that LSD1 defines a zinc finger protein subclass. LSD1 is constitutively expressed, consistent with the mutant phenotype. Alternate splicing gives rise to a low abundance mRNA encoding an extra five amino-terminal amino acids. We propose that LSD1 regulates transcription, via either repression of a prodeath pathway or activation of an antideath pathway, in response to signals emanating from cells undergoing pathogen-induced hypersensitive cell death.