Fatal hemorrhage due to a spontaneous factor V inhibitor with lupus anticoagulant properties.

: Factor V inhibitors are rare and have varied clinical presentations. We report on a 76-year-old female admitted to the hospital for pneumonia and treated with multiple antibiotics. Her baseline prothrombin time was 15.6 s and the activated partial thromboplastin time was 35 s. On admission day 10, she developed arm weakness and brain imaging showed a subdural hematoma. The prothrombin time was now 59.1 s with an activated partial thromboplastin time of more than 160 s and a normal thrombin time. A mixing study did not correct the clotting times and coagulation factor assays showed a nonspecific inhibition pattern. Only factor V activity remained low with serial dilutions, however, and a 70 Bethesda Unit inhibitor was identified. Aggressive supportive care was initiated but the patient succumbed to the effects of the intracranial hemorrhage. Factor V inhibitors may display lupus anticoagulant properties and may cause catastrophic bleeding. Our case illustrates that these inhibitors can arise quickly and supports an association with antibiotics.

Molecular basis of purinergic signal metabolism by ectonucleotide pyrophosphatase/phosphodiesterases 4 and 1 and implications in stroke.

NPP4 is a type I extracellular membrane protein on brain vascular endothelium inducing platelet aggregation via the hydrolysis of Ap3A, whereas NPP1 is a type II extracellular membrane protein principally present on the surface of chondrocytes that regulates tissue mineralization. To understand the metabolism of purinergic signals resulting in the physiologic activities of the two enzymes, we report the high resolution crystal structure of human NPP4 and explore the molecular basis of its substrate specificity with NPP1. Both enzymes cleave Ap3A, but only NPP1 can hydrolyze ATP. Comparative structural analysis reveals a tripartite lysine claw in NPP1 that stabilizes the terminal phosphate of ATP, whereas the corresponding region of NPP4 contains features that hinder this binding orientation, thereby inhibiting ATP hydrolysis. Furthermore, we show that NPP1 is unable to induce platelet aggregation at physiologic concentrations reported in human blood, but it could stimulate platelet aggregation if localized at low nanomolar concentrations on vascular endothelium. The combined studies expand our understanding of NPP1 and NPP4 substrate specificity and range and provide a rational mechanism by which polymorphisms in NPP1 confer stroke resistance.

NPP4 is a procoagulant enzyme on the surface of vascular endothelium.

Ap3A is a platelet-dense granule component released into the extracellular space during the second wave of platelet aggregation on activation. Here, we identify an uncharacterized enzyme, nucleotide pyrophosphatase/phosphodiesterase-4 (NPP4), as a potent hydrolase of Ap3A capable of stimulating platelet aggregation and secretion. We demonstrate that NPP4 is present on the surface of vascular endothelium, where it hydrolyzes Ap3A into AMP and ADP, and Ap4A into AMP and ATP. Platelet aggregation assays with citrated platelet-rich plasma reveal that the primary and secondary waves of aggregation and dense granule release are strongly induced by nanomolar NPP4 in a concentration-dependent manner in the presence of Ap3A, while Ap3A alone initiates a primary wave of aggregation followed by rapid disaggregation. NPP2 and an active site NPP4 mutant, neither of which appreciably hydrolyzes Ap3A, have no effect on platelet aggregation and secretion. Finally, by using ADP receptor blockade we confirm that NPP4 mediates platelet aggregation via release of ADP from Ap3A and activation of ADP receptors. Collectively, these studies define the biologic and enzymatic basis for NPP4 and Ap3A activity in platelet aggregation in vitro and suggest that NPP4 promotes hemostasis in vivo by augmenting ADP-mediated platelet aggregation at the site of vascular injury.