Figure 1.

Pathophysiology of ADP-induced platelet function in normal subjects and in the studied patients. (A) Effects of antagonists of P2Y₁ (MRS2216, 25 μM), P2Y₁₂ (AR-C69931MX, 1 μM) and integrin αIIbβ3 (MoAb 10E5, 10 μg/mL) on ADP (5 μM)-induced platelet aggregation in platelet-rich plasma ADP, added alone or in combination to normal platelet-rich plasma (PRP); maximal amplitude of platelet aggregation in control PRP (left tracing) was 78%. (B) Regulation of ADP-induced platelet shape change and aggregation. ADP binding to Gq-coupled P2Y1 activates the PLCβ isoform, to form IP3, which releases Ca²⁺ from stores. Ca²⁺ induces: 1) platelet shape change through activation of MLCK and phosphorylation of myosin light chain (MLC); 2) platelet aggregation through rapid CalDAG-GEFI-dependent activation of the small GTPase Rap1 to Rap1-GTP, which, through the cooperation of talin and kindlin3 promotes the binding of adhesive proteins to αIIbβ3 and platelet aggregation. This process is regulated by Ras GTPase-activating protein 3 (RASA3), which hydrolyses Rap1-GTP to inactive Rap1-GDP; RASA3 is inactivated by the Gi-coupled platelet ADP receptor P2Y₁₂, allowing sustained Rap1 signalling and full platelet aggregation. PLCβ: phosphlipase Cβ; IP3: inositol trsiphosphate; MLCK: myosin light chain kinase; pMLC: phosphorylated myosin light chain; CalDAG-GEFI: calcium- and DAG-regulated guanine exchange factor-1; PI3K=phosphoinositide 3-kinase; DTS=dense tubular system; AC: adenylyl cyclase; cAMP: cyclic adenosine monophosphate. (C) Platelet aggregation in citrate PRP from patient II-1 (see Figure 2A-B) and a healthy control, induced by ADP at the indicated concentrations; maximal amplitude of platelet aggregation induced by ADP 10 μM was 80% in healthy control and 15% in patient II-1. (D) Effects of ADP and epinephrine, at the indicated concentrations, on PGE1 (1 μM)-induced increase in platelet cAMP of II-1 and healthy controls (means± standard deviation [SD], n=21). (E) Platelet aggregation in citrate PRP from patients II-3, II-4 and II-5 (see Figure 2A-B), induced by ADP at the indicated concentrations; maximal amplitudes of platelet aggregation were 87% and 89% in patient II-3 and 25% and 80% in patient II-4.