Binding of GTP[35S], a nonhydrolyzable analog of GTP, to platelet membranes was measured by the method of Wieland and Jacobs (37)

Binding of GTP[35S], a nonhydrolyzable analog of GTP, to platelet membranes was measured by the method of Wieland and Jacobs (37). platelet membranes showed a decrease in Gq (<50%) but not Gi, Gz, G12, and G13. These studies provide evidence for a hitherto undescribed defect in human platelet G-protein -subunit function leading to impaired platelet responses, and they provide further evidence for a major role of Gq in thrombin-induced responses. G proteins play a major role in signal transduction from the surface heptahelical receptors to effector systems upon platelet activation, and they regulate Terfenadine downstream responses such as aggregation and secretion (1C4). G proteins are a family of heterotrimeric proteins (made up of , , and subunits) which mediate the interactions between agonist receptors and intracellular enzymes, such as adenylyl cyclase, phospholipase C (PLC), and phospholipase A2 Terfenadine (PLA2). Signaling mechanisms involve a cycle in which CGDP complex dissociates after replacement of GDP by GTP to produce CGTP, which then activates the effector molecule. Because of its intrinsic GTPase activity, the subunit hydrolyzes GTP and reassociates with subunit with termination of the activation process. Multiple forms of G have been identified in platelets (1) and are grouped in families; these include Gs, Gi (Gi1, Gi2, Gi3, Gz), Gq (Gq, G11, G14, G15, G16), and G12 families (G12, G13). Gs and Gi mediate the interaction with adenylyl cyclase. There is evidence for both remarkable specificity and potential redundancy in G proteins that mediate interaction between receptors and effectors (1C4). Thromboxane A2-induced activation of PLC- in platelets is mediated by pertussis toxin-insensitive Gq (5, 6). Thrombin activates PLC FGF-18 by both pertussis toxin-sensitive (possibly Gi2) (7, 8) and -insensitive mechanisms (Gq) (9, 10). Recently, G12 and G13 (members of the G12 family) have been shown to play a role during platelet activation with thrombin and thromboxane A2 (11). 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 (14C16), and in dog platelets with impaired responses to thromboxane A2 (17). Congenital abnormalities in platelet aggregation and secretion in response to activation with surface-receptor-mediated agonists may arise by diverse mechanisms, including abnormalities in the surface receptors, membrane glycoproteins, and deficiency of dense Terfenadine and granule contents (18C22). However, these well recognized abnormalities are observed in a small proportion of patients with abnormal platelet dysfunction; in the Terfenadine majority of such patients, the underlying mechanisms leading to the dysfunction are unknown (18). Evidence is becoming available that at least in some of these patients the primary abnormalities may lie in the signaling mechanisms that follow receptor activation and precede the ultimate responses of aggregation or secretion (18, 19). We have previously reported (23) a patient with diminished Terfenadine platelet aggregation and secretion in response to multiple agonists despite presence of normal dense granule stores. Further studies showed that receptor-mediated release of arachidonic acid from phospholipids (23) and calcium mobilization (24) were impaired upon platelet activation. We postulated that these abnormal responses may arise due to a defect in signal transduction mechanisms. To delineate the platelet defect in this patient, we investigated receptor-stimulated G-protein function and report an abnormality in G subunit function associated with a decrease in immunoreactive Gq in platelets.? To our knowledge a human platelet G-protein defect has hitherto not been described. MATERIALS AND METHODS Patient Information and Previous Studies. The patient is a 46-year-old white female with mild life-long mucocutaneous bleeding diathesis associated with prolonged bleeding times and normal platelet counts (23). The patients daughter and father may also have a history of easy bruising. Previous studies in the patient showed the following: (for 15 min at room temperature and was washed with Tyrodes buffer (pH.