Biochemical and functional consequences of dissociation of the platelet membrane glycoprotein IIb-IIIa complex

Abstract

The platelet membrane glycoproteins, IIb and IIIa, form a Ca2+- dependent heterodimer complex that functions as the fibrinogen receptor in activated platelets to mediate platelet aggregation. Little is known about factors that affect the IIb-IIIa complex within the platelet membrane. It has been observed that platelets incubated with ethylene glycol tetra-acetic acid (EGTA) at 37 degrees C are unable to aggregate or to bind monoclonal antibodies specific for the IIb-IIIa complex. To determine whether this is due to a dissociation of IIb from IIIa, we developed a method for quantitating the complex on nondenaturing, polyacrylamide gradient gels. Platelets were surface-labeled with 125I and then solubilized and electrophoresed in 0.2% Triton and 10 mmol/L CHAPS. Under these conditions and in the presence of 1 mmol/L Ca2+, glycoproteins IIb and IIIa migrated on the gels as a discrete band at Rf = 0.33. Protein that was eluted from this band bound to an immunoaffinity column specific for the IIb-IIIa complex. In contrast, when the IIb-IIIa complex was solubilized and then dissociated with EGTA, the discrete band at Rf = 0.33 was no longer present, and IIb and IIIa were now found in a broad band at Rf = 0.45 to 0.50. To study IIb and IIIa within the surface membrane, the 125I-labeled platelets were first incubated with 0.5 mmol/L EGTA (1 nmol/L free Ca2+) at 22 degrees C and then solubilized in the absence of EGTA. The IIb and IIIa from these platelets migrated at Rf = 0.33, indicating the presence of the intact IIb-IIIa complex. In contrast, when the platelets were incubated at 37 degrees C for one hour with the EGTA, the discrete band at Rf = 0.33 representing the IIb-IIIa complex gradually disappeared. This phenomenon could not be reversed by adding Ca2+ back to the platelets before solubilization and electrophoresis. This loss of the IIb-IIIa complex from intact platelets was accompanied by (a) a progressive and irreversible decrease in adenosine diphosphate (ADP)-induced platelet aggregation and (b) decreased binding of a complex-dependent monoclonal antibody to the platelets. These studies demonstrate that when platelets are exposed to low Ca2+ at 37 degrees C, the IIb-IIIa heterodimer complexes in their surface membranes are irreversibly disrupted. Because intact IIb-IIIa complexes are required for platelet aggregation, the loss of these complexes may account for the failure of these platelets to aggregate in response to ADP.

Ligand Binding to GPIIb-IIIa: A Status Report

“…Subunit association is noncovalent, and calcium is required to maintain the heterodimeric structure. [12][13][14] In a notable contribution, Calvete et al 15,16 have recently placed the disulfide linkages within GPIIb and GPIIIa. Mature GPIIIa consists of 762 amino acids.…”

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“…The concentration of ionized calcium can also in_uence the formation of GPIIb/IIIa heterodimers [23], and the GPIIb/IIIa and brinogen interaction [5]. Human platelets incubated with the extracellular calcium chelators, EDTA and EGTA, undergo morphological changes [1] and fail to aggregate [2,3,24], or bind to~brinogen in response to ADP [9,20,22]. Winters et al [25] demonstrated that the binding of MoAb 10E5, a complex speci~c anti-GPIIb/IIIa antibody, to the platelets was reduced by 80% under the conditions of complete absence of calcium and a temperature of 37ЊC.…”

“…The ex vivo platelet aggregation assay utilizes trisodium citrate as the conventional anticoagulant for the collection of whole blood to be used for the preparation of platelet-rich plasma (PRP). Unfortunately, the reduction or removal of the ionized calcium concentration from the PRP by trisodium citrate induces a morphological change in the platelets [1] and in_uences the stability of the GPIIb/IIIa complex [2][3][4] and the divalent cation-dependent binding of RGD ligands to the GPIIb/IIIa receptor [5,6]. Under such circumstances, the ex vivo platelet aggreagation studies may falsely reveal a greater antiplatelet potency of a particular antagonist of the glycoprotein receptor.…”

Ligands to the platelet fibrinogen receptor glycoprotein IIb-IIIa do not affect agonist-induced second messengers Ca2+ or cyclic AMP

“…4 shows the results of similar experiments (excitation wavelength 360 nm) conducted using ADP (10,M) as agonist. MnCl2 was added before addition of ADP, since the opening of divalent channels appears to be more transient when ADP is the agonist (Shattil et al, 1985;Rink, 1988). In all cases, ADP-induced Mn2+ influx was not detectably altered by the presence of the monoclonal Platelets were prelabelled with [3H]adenine as indicated in the Materials and methods section and challenged with (a) albolabrin (400 nm final) or (b) elegantin (400 nm final) in the absence or presence of a phosphodiesterase inhibitor (5 mM-IBMX).…”

“…The fibrinogen receptor activity of the complex is only expressed in activated platelets and is essential for platelet aggregation. Based on studies of platelets from thrombasthenic patients, which lack GPIIb-IIIa, and studies with complex-specific monoclonal antibodies, it was proposed that the integrity of the GPIIb-IIIa dimer is important for Ca2+ homeostasis in unstimulated human platelets (Brass, 1984(Brass, , 1985Shattil et al, 1985). Using other monoclonal antibodies directed against GPIIb-IIIa, Powling & Hardisty (1985) investigated the possibility that this complex might serve as a Ca2+ channel in stimulated platelets.…”