Abnormal inside-out signal transduction-dependent activation of glycoprotein IIb-IIIa in a patient with impaired pleckstrin phosphorylation

Abstract

Platelet-agonist interaction results in activation of glycoprotein (GP) IIb-IIIa complex and fibrinogen binding, a prerequisite for platelet aggregation. Fibrinogen binding exposes new antibody binding sites on GPIIb-IIIa (ligand-induced binding sites: LIBS). Signal transduction events, including pleckstrin phosphorylation by protein kinase C (PKC), are considered to regulate GPIIb-IIIa activation. We studied a 16-year- old white male with lifelong mucocutaneous bleeding manifestations and abnormal platelet aggregation and secretion in response to multiple agonists. Pleckstrin phosphorylation was diminished in response to platelet-activating factor (PAF; 4 and 400 nmol/L) and thrombin (0.05 U/mL). Binding of monoclonal antibodies (MoAbs) 10E5 and A2A9, which bind to both resting and activated GPIIb-IIIa, was normal. Binding of MoAb PAC1, which binds to only activated GPIIb-IIIa, was diminished upon activation with PAF, adenosine diphosphate (ADP), thrombin receptor agonist peptide (SFLLRN), A23187, and 1,2-dioctonylglycerol (DiC8). Signal transduction-dependent LIBS expression (studied using MoAb 62) induced by ADP, SFLLRN, and DiC8 and signal transduction- independent LIBS expression induced by RGDS peptide or disintegrin albolabrin were normal or minimally decreased, indicating the presence of intact ligand binding sites. We conclude that the patient's platelets have a defect in inside-out signal transduction-dependent GPIIb-IIIa activation due to an upstream defect in the signal transduction mechanisms rather than in the GPIIb-IIIa complex itself. Our findings extend the spectrum of congenital mechanisms leading to impaired aggregation from defects in GPIIb-IIIa per se to aberrations in signaling mechanisms.

Defective acid hydrolase secretion in RUNX1 haplodeficiency: Evidence for a global platelet secretory defect

“…The right panel shows the increase in the radioactivity in free arachidonic acid, reflecting the release of free arachidonic acid upon platelet activation aggregation responses were markedly diminished in response to multiple agonists. 6 This provides a mechanism for the markedly decreased aggregation responses. Impaired thromboxane production compromises responses to platelet agonists.…”

“…[2][3][4][5] In addition, abnormalities in mechanisms that regulate secretion have been reported in some patients, 2,3,5 including in myosin light chain and pleckstrin phosphorylation, and decreased platelet PKCθ. 6,7 Platelet mRNA expression profiling of a patient with RUNX1 mutation 8 and other studies 7 have shown downregulation of several genes, including MYL9, PRKQ ALOX12 and PF4, and some of these are direct transcriptional targets of RUNX1, providing evidence for aberrations in multiple platelets mechanisms, including in those regulating platelet responses to activation. 2,[9][10][11][12] Platelets possess three distinct types of granules-dense and α-granules, and acid hydrolase (AH)-bearing vesicles (lysosomes), whose contents are secreted on platelet activation.…”

Combined defect in membrane expression and activation of platelet GPIIb–IIIa complex without primary sequence abnormalities in myeloproliferative disease

“…These include abnormalities in the integrin per se , resulting in impaired ligand binding or its activation and in the upstream signalling events that regulate GPIIb–IIIa function, unrelated to any abnormality in the integrin. Evidence for the latter type of signalling‐related abnormality in GPIIb–IIIa activation has been shown in some patients (Gabbeta et al , 1996; 1997).…”

“…LIBS expression on receptor activation is a signal transduction‐dependent process; however, under certain conditions (e.g. exposure to small RGD‐containing compounds), it can be induced in a signal transduction‐independent manner (Du et al , 1991; Gabbeta et al , 1996).…”

“…Flow cytometric analysis of platelet surface glycoproteins These analyses were performed using platelet‐rich plasma as described previously (Gabbeta et al , 1996). For GPIIb–IIIa, platelets (50 μl at a concentration of 1 × 10 8 /ml) were incubated with FITC‐conjugated monoclonal antibody 10E5 (50 μg/ml), A2A9 (50 μg/ml), PAC‐1 (50 μg/ml) or Ab33 (anti‐LIBS; 50 μg/ml) in the absence or presence of a specific agonist or disintegrin (Echistatin) at room temperature for 15 min without stirring.…”