RESUMEN
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).
RESUMEN
We report a case of Glanzmann thrombasthenia in a Pakistani child whose platelets express less than 10% of the normal amount of alphaIIb beta3 on their surface. Single-stranded conformation polymorphism analysis of the exons of the patient's alphaIIb and beta3 genes showed an abnormality in exon 4 of the beta3 gene. Direct sequence analysis showed that the patient was homozygous for a T --> G nucleotide substitution in this exon, resulting in the replacement of a highly conserved Leu at position 117 with Trp. Heterologous expression of alphaIIb beta3 containing the beta3 mutation in COS-1 cells confirmed the pathogenicity of the Leu117 --> Trp substitution and showed that it resulted in the intracellular retention of malfolded alphaIIb beta3 heterodimers. Additional site-directed mutagenesis at position 117 indicated that, although the smaller hydrophobic amino acid Val could be substituted for the wild-type Leu, the larger hydrophobic amino acids Trp and Phe or the charged amino acids Asp and Lys were not tolerated. These studies indicate that Leu117 in beta3 plays a critical role in attaining the correct folded conformation of alphaIIb beta3. These studies also suggest that the hydrophobic side chain of Leu117 is likely folded into the interior of beta3, where it serves to stabilize internal packing of the protein and determines its overall shape.
Asunto(s)
Plaquetas/metabolismo , Leucina/genética , Mutación , Oligopéptidos/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Trombastenia/genética , Triptófano/genética , Alelos , Secuencia de Aminoácidos , Animales , Células COS , Citometría de Flujo , Humanos , Lactante , Datos de Secuencia Molecular , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Conformación Proteica , Propiedades de SuperficieRESUMEN
The integrin alpha IIb beta 3, a calcium-dependent heterodimer, plays a critical role in platelet aggregation. The alpha IIb subunit of the heterodimer contains four highly conserved putative calcium-binding domains in its extracellular portion. During studies of the molecular basis of Glanzmann thrombasthenia in a child of mixed Caucasian background whose platelets expressed little alpha IIb beta 3 on their surface, we found the patient heterozygous for a two amino acid deletion in the fourth alpha IIb calcium-binding domain. When this alpha IIb mutant was expressed in COS-1 cells, we found that the deletion did not interfere with the assembly of alpha IIb beta 3 heterodimers, but altered their conformation such that they were neither recognized by the heterodimer-specific antibody A2A9 nor able to undergo further intracellular processing or transport to the cell surface. These results suggest that the calcium-binding domains in alpha IIb play an important role maintaining the overall conformation of alpha IIb beta 3. To confirm this suggestion, we deleted each of the four 12 amino acid calcium-binding domains in alpha IIb by in vitro mutagenesis and expressed the mutants along with beta 3 in COS-1 cells. Each construct formed a heterodimer with beta 3, but none of the heterodimers interacted with A2A9 or underwent further intracellular processing. These data indicate that the calcium-binding domains in alpha IIb are not involved in alpha IIb beta 3 heterodimer formation, but their presence is required for the intracellular transport of alpha IIb beta 3 to the cell surface.
Asunto(s)
Calcio/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Conformación Proteica , Eliminación de Secuencia , Trombastenia/genética , Adulto , Alelos , Secuencia de Aminoácidos , Animales , Antígenos de Plaqueta Humana/química , Antígenos de Plaqueta Humana/genética , Antígenos de Plaqueta Humana/inmunología , Secuencia de Bases , Sitios de Unión , Transporte Biológico , Plaquetas/metabolismo , Línea Celular Transformada , Chlorocebus aethiops , Clonación Molecular , Epítopos/química , Epítopos/inmunología , Femenino , Citometría de Flujo , Humanos , Recién Nacido , Masculino , Datos de Secuencia Molecular , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
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.
Asunto(s)
Plaquetas/metabolismo , Fibrinógeno/química , Fibrinógeno/metabolismo , Oligopéptidos/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Adenosina Difosfato/metabolismo , Sitio Alostérico , Animales , Linfocitos B/metabolismo , Células CHO , Carcinógenos , Adhesión Celular , Cricetinae , ADN Complementario/metabolismo , Ditiotreitol/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Ligandos , Agregación Plaquetaria , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/química , Acetato de Tetradecanoilforbol , TransfecciónRESUMEN
Decreased expression of functional alphaIIbbeta3 complexes on the platelet surface produces Glanzmann thrombasthenia. We have identified mutations of alphaIIb(P145) in 3 ethnically distinct families affected by Glanzmann thrombasthenia. Affected Mennonite and Dutch patients were homozygous and doubly heterozygous, respectively, for a P(145)A substitution, whereas a Chinese patient was doubly heterozygous for a P(145)L substitution. The mutations affect expression levels of surface alphaIIbbeta3 receptors on their platelets, which was confirmed by co-transfection of alphaIIb(P145A) and beta3 cDNA constructs in COS-1 cells. Each mutation also impaired the ability of alphaIIbbeta3 on affected platelets to interact with ligands. Moreover, when alphaIIb(P145A) and beta3 were stably coexpressed in Chinese hamster ovary cells, alphaIIbbeta3 was readily detected on the cell surface, but the cells were unable to adhere to immobilized fibrinogen or to bind soluble fluorescein isothiocyanate-fibrinogen after alphaIIbbeta3 activation by the activating monoclonal antibody PT25-2. Nonetheless, incubating affected platelets with the peptide LSARLAF, which binds to alphaIIb, induced PF4 secretion, indicating that the mutant alphaIIbbeta3 retained the ability to mediate outside-in signaling. These studies indicate that mutations involving alphaIIb(P145 )impair surface expression of alphaIIbbeta3 and that the alphaIIb(P145A) mutation abrogates ligand binding to the activated integrin. A comparative analysis of other alphaIIb mutations with a similar phenotype suggests that these mutations may cluster into a single region on the surface of the alphaIIb and may define a domain influencing ligand binding. (Blood. 2000;95:180188)