ABNORMAL TYROSINE PHOSPHORYLATION LINKED TO A DEFECTIVE INTERACTION BETWEEN ADP AND ITS RECEPTOR ON PLATELETS

ADP, a primary stimulus of platelets. binds to one or more populations of receptors on the platelet surface. These receptors re linked to di screte activation pathways. Both G proteins and tyrosine kinases have been implicated in the cellular responses to this agonist. We have stu died a patient with a congenital abnormality of ADP-induced platelet a ggregation in an effort to gain information on the signalling pathways used by ADP. Immunoblotting with a broadly reactive rabbit antibody r ecognizing the GTP-binding domain of G protein alpha-subunits, and wit h rabbit antibodies specific for G(i)alpha 1-3, and G alpha 12 all sho wed normal reactivity when tested against he patient's platelets. The phosphorylation of proteins was studied using an anti-phosphotyrosine MoAb (4G10) and platelets stimulated in a platelet aggregometer with A DP, a thromboxane A(2) mimetic (IBOP), TRAP-14-mer peptide and alpha-t hrombin. With normal platelets, a time-dependent phosphorylation of se veral bands in the 60 to 130 kDa mel. wt. range was observed with all agonists. For the patient, minimal aggregation and little or no phosph orylation of proteins of 80-85 kDa (cortactin), 100-105 kDa and 125-13 0 kDa were seen in response to ADP. The aggregation and phosphorylatio n responses were slightly modified in the presence of low doses of thr ombin but were normal with high doses. Aggregation and tyrosine phosph orylation were virtually absent with IBOP, a finding reproduced when n ormal platelets were incubated with IBOP and the CP/CPK ADP scavenging system, thereby underlining the role of ADP in the response to IBOP. Our results show that the ADP receptor pathway deficient in the patien t is linked to a selective tyrosine phosphorylation response.