Abstract-ADP plays a central role in regulating platelet function. It induces platelet aggregation via the activation of 2 major ADP receptors, P2Y 1 and P2Y 12 . We have investigated the role of P2Y 12 in platelet adhesion and thrombus formation under physiological flow by using blood from a patient with a defect in the gene encoding P2Y 12 . Anticoagulated blood from the patient and from healthy volunteers was perfused over collagen-coated coverslips. The patient's thrombi were smaller and consisted of spread platelets overlying platelets that were not spread, whereas control thrombi were large and densely packed. Identical platelet surface coverage, aggregate size, and morphology were found when a P2Y 12 antagonist, N 6 -(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-␤,␥-dichloromethylene ATP (also known as AR-C69931 MX), was added to control blood. The addition of a P2Y 1 antagonist (adenosine-3Ј,5Ј-diphospate) to control blood resulted in small, but normally structured, thrombi. Thus, the ADP-P2Y 12 interaction is essential for normal thrombus buildup on collagen. The patient's blood also showed reduced platelet adhesion on fibrinogen, which was not due to changes in morphology. Comparable results were found by using control blood with AR-C69931 MX and also with adenosine-3Ј,5Ј-diphospate. This suggested that P2Y 12 and P2Y 1 were both involved in platelet adhesion on immobilized fibrinogen, thereby revealing it as ADP dependent. This was confirmed by complete inhibition on the addition of creatine phosphate/creatine phosphokinase. Key Words: ADP receptors Ⅲ platelet adhesion Ⅲ collagen Ⅲ fibrinogen Ⅲ thrombus formation under flow P latelets play an important role in primary hemostasis via adhesion, aggregation, and subsequent thrombus formation on collagen exposed at the site of vascular damage. Perturbations of this system may lead to pathological thrombus formation and vascular occlusion, resulting in stroke, myocardial infarction, or cerebral infarction. Under conditions of high shear, platelet thrombus formation is dependent on the interaction between von Willebrand factor and platelet glycoprotein Ib. This interaction leads to platelet activation and conformational changes in the platelet integrin ␣ IIb ␤ 3 , 1 which is followed by the binding of fibrinogen to the integrin and the formation of stable bridges between aggregating platelets. 2 The ␣ IIb ␤ 3 receptor has been the recent target for antithrombotic agents such as abciximab, eptifibatide, and tirofiban as adjunctive therapy to decrease the ischemic complications of percutaneous coronary interventions and/or unstable angina. 3 During the past decade, ADP receptors on the platelet membrane have also become a target for antithrombotic strategies with compounds such as ticlopidine, 4 clopidogrel, 4,5 and the AR-C compounds. 6,7 ADP is an important agonist, released from damaged vessels and red blood cells, that induces platelet aggregation through the activation of ␣ IIb ␤ 3 . Another major source of ADP, as illustrated by the platelet functio...

Background The anti-phospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with persistent presence of anti-phospholipid antibodies (aPL). Laboratory criteria include aPL detection by coagulation tests for lupus anticoagulant (LAC) or solid phase assays measuring anti-β2 glycoprotein I (aβ2GPI) or anti-cardiolipin (aCL) immunoglobulin (Ig) G/IgM antibodies. External quality control programs illustrate that commercially available aPL assays produce variable results. Objective We aimed to investigate the agreement and diagnostic accuracy of solid phase assays. Materials and Methods In this multi-centre study, 1,168 patient samples were tested on one site for aCL and aβ2GPI IgG/IgM antibodies by four solid phase test systems. Samples included APS patients, controls and monoclonal antibodies (MoAB) against different epitopes of β2GPI. LAC was determined by the local centre. Results aCL IgM assays resulted in the most discrepancies (60%), while aCL IgG and aβ2GPI IgM assays resulted in lower discrepancies (36%), suggesting better agreement. Discrepant samples displayed lower median aPL titers. Dependent on the solid phase test system, odds ratios (ORs) for thrombosis and pregnancy morbidity ranged from 1.98 to 2.56 and 3.42 to 4.78, respectively. Three platforms showed lower sensitivity for MoAB directed against the glycine (Gly) 40-arginine (Arg) 43 epitope of domain I of β2GPI. Conclusion Poor agreement was observed between different commercially available aCL and aβ2GPI IgG/IgM assays, hampering uniformity in the identification of aPL-positive patients. Clinical association was globally concordant between solid phase test systems considering results of the four aPL together. An assay sensitive in detecting the MoAB against Gly40-Arg43 of domain I of β2GPI reached the highest OR for thrombosis.

We conclude that perfusion studies with patient blood are of added value in the diagnostic process, which resulted in identification of a novel molecular defect in the P2Y(12) gene of a patient with haemorrhagic diathesis.