Abstract
Abstract 363
Phosphoinositides are the phosphorylated forms of phosphatidylinositol. Although they constitute only 1% of platelet membrane phospholipids, phosphoinositides are important contributors to platelet signaling. As an example the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 or PIP2) has been shown to regulate integrin activation, actin assembly, and secretion in platelets. PtdIns(4,5)P2 contributes to these events by directly binding to signaling proteins, and also by serving as a substrate for PLC and PI3K to generate the second messengers Inositol(3,4,5)P3 (Ins(3,4,5)P3) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). The phosphoinositide concentration in different cellular compartments is tightly regulated both in time and space, and their relative amounts of various phosphoinositides change within seconds of agonist stimulation. Given their importance in platelet biology, we sought to better understand the mechanism of phosphoinositide synthesis and function. Class I PhosphatidylInositol Transfer Proteins (PITPs) are a small family of proteins that bind to and transfer phosphoinositide monomers from one cellular compartment to another in an energy independent manner. Although there are no studies on the function of PITPs in hematopoietic cells, the proteins are essential in yeast cells for the biosynthesis and metabolism of phosphoinositides. This suggests that PITPs may also be essential for phosphoinositide metabolism in platelets. There are two dominant Class I PITP family members, PITPα and PITPβ in murine platelets. We introduced conditional loss-of-function mutations into the murine PITPα and PITPβ genes These mice were crossed with transgenic mice expressing Cre recombinase under the control of the platelet specific promoter PF4, thereby generating mice lacking individual PITP isoforms only in platelets and megakaryocytes. Mice lacking either platelet PITP isoform have approximately one-third lower platelet counts than littermate controls, but otherwise have normal blood counts and appear phenotypically normal. We have also generated a few viable double knockout (PITPαfl/fl PITPβfl/fl PF4Cre+) mice and found that these mice produce some platelets even in the absence of both PITP isoforms. Next, we analyzed the concentration of individual phosphoinositides in platelets of the knockout mice by thin layer chromatography. Although loss of either PITP isoform did not affect PtdIns(4)P synthesis in resting or thrombin stimulated platelets, PtdIns(4,5)P2 synthesis was significantly inhibited in platelets lacking either PITPα (∼40% of normal) or PITPβ (∼50% of normal). This suggests that both PITPα and PITPβ are required for normal PtdIns(4,5)P2 synthesis in platelets. To test whether PITP isoforms are critical for second messenger formation, we analyzed the formation of Ins(3,4,5)P3 in response to thrombin stimulation. We found that the PITPα-null mutation causes an almost absence of Ins(3,4,5)P3 formation. The PITPβ-null mutation also caused a profound defect in Ins(3,4,5)P3 formation, albeit less than that seen following PITPα deletion. Nonetheless, both PITPα and PITPβ knockout platelets were able to increase their intracellular calcium concentration following thrombin stimulation to approximately 50% of normal. These data imply that a significant portion of the calcium response in platelets results from the influx of extracellular calcium and not by the Ins(3,4,5)P3–dependent release of calcium from intracellular stores. Further, platelets lacking either PITP isoform exhibited only a moderate (∼50%) defect in Akt phosphorylation – a PtdIns(3,4,5)P3 synthesis-dependent event. Although both Ins(3,4,5)P3 and PtdIns(3,4,5)P3 are derived from PtdIns(4,5)P2, it is remarkable that PITP enzymes are more critical for Ins(3,4,5)P3-synthesis than for PtdIns(3,4,5)P3-synthesis (as suggested by Akt phosphorylation). Finally, we performed pilot experiments of the ex vivo function of platelets lacking both PITP isoforms and found that double knockout platelets had a profound loss of agonist-induced platelet aggregation. Together, our data suggest that PITPα and PITPβ are both critical for the synthesis of essential platelet phosphoinositides and second messengers. Thus, these enzymes may represent novel therapeutic targets for the prevention and treatment of arterial occlusion.
Disclosures:
No relevant conflicts of interest to declare.
Emerging perspectives on multidomain phosphatidylinositol transfer proteins
