Our goal is to characterize unstudied pathways of disease biochemically, biophysically, and physiologically. We are especially interested in biochemical pathways directly resulting in disease phenotypes that are amenable to biophysical study, and to translational intervention.
To this end, we are currently studying a family of extracellular enzymes (the nucleotide pyrophosphatase/phosphodiesterase – or ENPP – family) that create or destroy extracellular signals governing (among other things) hemostasis and stroke, bone mineralization, angiogenesis, cancer cell proliferation, and cancer cell survival. The family consists of seven members in humans, and we use x-ray crystallography and biochemistry to define the substrate range and enzymatic mechanism, and to understand the molecular basis of substrate specificity. Insights gained biochemically and biophysically are used to understand the enzyme’s physiologic role and scope of action, and to characterize diseases of enzyme deficiency or excess. We study the physiologic pathways governed by ENPPs using in vitro and in vivo methods, include cell biology, transgenic animals, platelet aggregation studies, and cancer cell proliferation and metastasis assays.
We recently discovered that NPP4 is present on brain vascular endothelium and is capable of inducing irreversible platelet aggregation through the hydrolysis of a small molecule released by platelets upon degranulation (Ap3A), and proposed a role for NPP4 in central hemostasis and stroke. We next demonstrated that NPP1 was also capable Ap3A hydrolysis and promoting irreversible platelet aggregation, a finding of significant interest when viewed in light an early (1988) publication reporting NPP1 on the capillaries in brain vasculature, but not on capillaries elsewhere. Taken together, the findings support a role of NPP1 in central hemostasis, and account for the mechanism by which recent reported polymorphisms in NPP1 confer stroke protection to children with sickle cell anemia, who are at significant increased risk for stroke (see press release). We have recently put forward the idea that loss of function mutations in NPP1 confer stroke protection, in addition to NPP1s established role in ATP hydrolysis and bone mineralization. These findings, if validated, have significant implications for the development of novel anti-thrombotic agents.
We are now validating the role of NPP1 and NPP4 in central hemostasis using transgenic animals and mouse models of hemostasis and stroke, and developing targeted agents against the enzymes using structure aided drug design to validate their proposed physiologic roles. Finally, we are using protein-engineering methods to shift substrate specificity and alter substrate range in order to better understand substrate discrimination and selectivity in the NPP family.