Thromboxane-insensitive dog platelets have impaired activation of phospholipase C due to receptor-linked G protein dysfunction.

Abstract

Human platelet thromboxane A2/prostaglandin H2 (TXA2/ PGH2) receptors are linked to phosphoinositide-specific phospholipase C (PI-PLC) via a G protein tentatively identified as a member of the Gq class. In contrast, platelet thrombin receptors appear to activate PI-PLC via other unidentified G pro-, teins. Platelets from most dogs are TXA2 insensitive (TXA2-); i.e., they do not aggregate irreversibly or secrete although they bind TXA2, but they respond normally to thrombin. In contrast, a minority of dogs have TXA2-sensitive (TXA2+) platelets that are responsive to TXA2. To determine the mechanism responsible for TXA2-platelets, we evaluated receptor activation of PI-PLC. Equilibrium binding of TXA2/PGH2 receptor agonists, 1251IBOP and 13H1U46619, and antagonist, 13H1SQ29,548, revealed comparable high-affinity binding to TXA2-, TXA2+, and human platelets. U46619-induced PI-PLC activation was impaired in TXA2-platelets as evidenced by reduced (a) phosphorylation of the 47-kD substrate of protein kinase C, (b) phosphatidic acid (PA) formation, (c) rise in cytosolic calcium concentration, and (d) inositol 1,4,5 trisphosphate (IP3) formation, while thrombin-induced PI-PLC activation was not impaired. GTPase activity stimulated by U46619, but not by thrombin, was markedly reduced in TXA2-platelets. Antisera to Gq class a subunits abolished U46619-induced GTPase activity in TXA2-, TXA2+, and human platelets. Direct G protein stimulation by GTPyS yielded significantly less PA and IP3 in TXA2-platelets. Immunotransfer blotting revealed comparable quantities of Gq class a-subunits in all three platelet types. Thus, TXA2-dog platelets have impaired PI-PLC activation in response to TXA2/PGH2 receptor agonists secondary to G protein dysfunction, presumably involving a

Effect of adenosine A2 receptor stimulation on platelet activation–aggregation: Differences between canine and human models

“…We found in pilot experiments that U46619 failed to trigger platelet activation-aggregation in dog blood. This observation is in agreement with some previous reports [34,35], and has been attributed to a genetic defect in G protein function that purportedly renders most dogs refractory to thromboxane A 2 receptor stimulation [36,37]. In contrast, others have successfully used U46619 in canine models [38], while in our hands IBOP effectively stimulated platelet activation-aggregation.…”

Platelet signal transduction defect with Gα subunit dysfunction and diminished Gα q in a patient with abnormal platelet responses

“…In contrast, the observed abnormality in response to several agonists in our patient suggests an aberration in signal transduction pathways common to different agonists. Moreover, in the canine model (17), although a defect in the G q family was postulated, immunoblotting studies showed normal levels of G q in the platelet membranes. In our patient the diminished agoniststimulated GTPase activity is associated with decreased binding of GTP and with decreased amount of immunoreactive G␣ q in platelet membranes.…”

“…Moreover, there is evidence that PLC-␤ isozymes can be activated by ␤␥ subunits independent of action of ␣ q subunit (4,12,13). Abnormalities in G-protein-coupled signal transduction pathways have been described in several human disease states (14)(15)(16), and in dog platelets with impaired responses to thromboxane A 2 (17).…”

Platelet Activation Via Trimeric GTP-Binding Proteins

“…The presence of G )3 in platelets is still uncertain (26). G] 1 has been found in dog but not in human platelets (27). The a-subunit of G 11 shows subtle species differences making identification with antibodies unreliable.…”

“…In platelets from patients with non-insulin dependent diabetis a down regulation of Gj-2 and Gj-3 is found resulting in abnormal regulation of adenylyl cyclase and disturbed platelet functions (34). In addition, there is indirect evidence for abnormal G-protein function in pla telets that fail to respond to thromboxane A 2 (27).…”