The role of inositol polyphosphate 4-phosphatase 1 in platelet function using a weeble mouse model.

Platelet activation plays an important role in the development and course of cardiovascular disease. It is triggered by the interaction of subendothelial matrix-bound and/or soluble agonists with platelet surface receptors causing a series of morphological and biochemical changes leading to the recruitment of additional platelets and formation of stable platelet aggregates. In addition to events causing initial activation and recruitment of platelets, signaling continues post-aggregation that promotes stability of the thrombus (Brass et al., 2004). Platelet levels of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) increase dramatically in response to agonist stimulation in an aggregation-dependent manner (Kucera et al., 1990; Nolan et al., 1990; Sultan et al., 1990). Furthermore the increase in platelet PtdIns(3,4)P2 occurs late in platelet aggregation and correlates with the irreversible phase of platelet aggregation (Sorisky et al., 1992; Sultan et al., 1991; Trumel et al., 1999), suggesting that PtdIns(3,4)P2 mediates the stabilization of platelet aggregates. However little is known about the regulation of PtdIns(3,4)P2 in platelets. PtdIns(3,4)P2 can be formed by three different routes 1) by direct phosphorylation of PtdIns(4)P by phosphatidylinositol 3-kinase (EC2.7.1.153) (PI 3-K) 2) by PI 3-K using PtdIns(4,5)P2 as a substrate followed by dephosphorylation by a 5-phosphatase (EC3.1.3.56) and, 3) by the action of type I PtdIns(4)P phosphate kinase (EC2.7.1.68) using PI 3-K as substrate (Zhang et al., 1998), shown diagramatically in figure 1. It is well documented that different PI 3-K isoforms play important roles in both early and later stages of platelet aggregation (Jackson et al., 2006). It has been shown that that pharmacologic inhibition of PI 3-K prevents agonist-induced formation of PtdIns(3,4)P2 (Kovacsovics et al., 1995; Schoenwaelder et al., 2007). Furthermore addition of PI 3-K inhibitors after the onset of platelet aggregation induces a decline in PtdIns(3,4)P2 and disaggregation of platelets, supporting a role for PtdIns(3,4)P2 in the stabilization of aggregates. However, platelet agonist-dependent activation of PI 3-K mediates an increase in both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 making approaches using pharmacologic inhibition or genetic disruption of PI 3-K unsuitable to distinguish the contributions of the individual 3-phosphorylated phosphoinositides to platelet signaling. The levels of inositol lipids in cells and their distribution in discrete cellular compartments are regulated by the balance of the enzymatic activities of kinases and phosphatases. Figure 1 Pathways for the formation of PtdIns (3,4)P2. The major route of PtdIns(3,4)P2 hydrolysis is the removal of the D4 phosphate by the enzymes cloned and characterized in our lab (Norris et al., 1997; Norris et al., 1995), inositol polyphosphate 4-phosphatase type I (EC3.1.3.40) and type II (EC3.1.3.66) that are magnesium-independent phosphatases. Over their entire sequence type I and type II 4-phosphatases are 37% identical (Norris et al., 1997). The active site region is more highly conserved, and contains a consensus sequence found in other magnesium-independent phosphatases (Zhang et al., 1994). The consensus sequence CX5 RT/S, is conserved throughout 4-ptases from C. elegans to humans as shown in figure 2. These enzymes do not catalyze the hydrolysis of lipids other than PtdIns(3,4)P2 and therefore provide unique means for the study of this lipid in platelet activation. We have shown that 4-ptase I forms a complex with PI 3-K in platelets which localizes the complex to sites of PtdIns(3,4)P2 production (Munday et al., 1999). Figure 2 Alignment of active sites of 4ptases. We postulate that PtdIns(3,4)P2 is important for platelet function and will study this using a mouse model. We previously showed that an antibody that reacts with 4-phosphatases immunoprecipitates all of the PtdIns(3,4)P2 hydrolyzing activity from human platelets (Munday et al., 1999). An early indication that PtdIns(3,4)P2 was important for platelet function was the work of Norris (Norris et al., 1997). It was shown that calpain caused degradation of recombinant 4-phosphatase I in vitro thereby inactivating it. It was also shown that activation of human platelets with either calcium ionophore or thrombin led to proteolysis of endogenous platelet 4-phosphatase I. If calpeptin, a cell-permeable inhibitor of calpain, was included in these experiments no proteolysis was seen. The levels of PtdIns(3,4)P2 in platelets were lower when calpeptin was included, indicating that 4-phosphatase I was important for controlling the levels of PtdIns(3,4)P2 during platelet activation. A naturally occurring mutation in type I 4-phosphatase is a single nucleotide deletion which is found in the weeble mouse. These animals suffer from severe neurodegeneration and die within the first weeks of life. Therefore such mutant mice cannot be used to study platelet function. We circumvented this problem by creating chimeric mice by bone marrow transplantation of weeble fetal liver cells into lethally irradiated wild type mice. These mice lack 4-phosphatase in bone marrow derived cells including platelets. The mice are viable, but lack platelet 4-phosphatase I.