Loss of PIP5KIbeta demonstrates that PIP5KI isoform-specific PIP2 synthesis is required for IP3 formation

The three isoforms of PIP5KI (α, β, and γ) synthesize PI4,5P₂ (PIP₂) by phosphorylating PI4P. Therefore, it is not clear why platelets, like all eukaryotic cells, have more than one isoform. To test the hypothesis that PIP5KI isoforms have nonoverlapping functions, we generated a murine line containing a null mutation of PIP5KIβ and analyzed the effect on platelet signaling. PIP5KIβ-null mice had normal platelet counts. In contrast to platelets lacking PIP5KIα, platelets lacking PIP5KIβ exhibited impaired aggregation accompanied by disaggregation. Although platelets lacking PIP5KIβ had only a moderate deficiency of PIP₂ under basal conditions, they had a striking deficiency in PIP₂ synthesis and IP₃ formation after thrombin stimulation. We have also observed that platelets lacking both PIP5KIα and PIP5KIβ have a complete loss of thrombin-induced IP₃ synthesis even though they still contain PIP5KIγ, the predominant PIP5KI isoform in platelets. These results demonstrate that PIP5KIβ, like PIP5KIα, contributes to the rapid synthesis of a pool of PIP₂ that is required for second-messenger formation, whereas the pool of PIP₂ synthesized by PIP5KIγ does not contribute to this process. Additionally, we found that PIP5KIβ-null platelets failed to form arterial thrombi properly in vivo. Together, these data demonstrate that PIP5KIβ is required for rapid PIP₂ synthesis, second-messenger production, and stable platelet adhesion under shear in vivo. These results also demonstrate that after stimulation of a G protein-coupled receptor, IP₃ is completely derived from a rapidly synthesized discrete pool of PIP₂ synthesized by PIP5KIα and PIP5KIβ.     

Continuous signaling via PI3K isoforms beta and gamma is required for platelet ADP receptor function in dynamic thrombus stabilization

Signaling from collagen and G protein-coupled receptors leads to platelet adhesion and subsequent thrombus formation. Paracrine agonists such as ADP, thromboxane, and Gas6 are required for platelet aggregate formation. We hypothesized that thrombi are intrinsically unstable structures and that their stabilization requires persistent paracrine activity and continuous signaling, maintaining integrin alpha(IIb)beta3 activation. Here, we studied the disassembly of human and murine thrombi formed on collagen under high shear conditions. Platelet aggregates rapidly disintegrated (1) in the absence of fibrinogen-containing plasma; (2) by blocking or inhibiting alpha(IIb)beta3; (3) by blocking P2Y12 receptors; (4) by suppression of phosphoinositide 3-kinase (PI3K) beta. In murine blood, absence of PI3Kgamma led to formation of unstable thrombi, leading to dissociation of multiplatelet aggregates. In addition, blocking PI3Kbeta delayed initial thrombus formation and reduced individual platelet-platelet contact. Similarly without flow, agonist-induced aggregation was reversed by late suppression of P2Y12 or PI3K isoforms, resulting in single platelets that had inactivated alpha(IIb)beta3 and no longer bound fibrinogen. Together, the data indicate that continuous outside-in signaling via P2Y12 and both PI3Kbeta and PI3Kgamma isoforms is required for perpetuated alpha(IIb)beta3 activation and maintenance of a platelet aggregate. This novel concept of intrinsic, dynamic thrombus instability gives possibilities for the use of antiplatelet therapy.     

Negative feedback regulation of Rac in leukocytes from mice expressing a constitutively active phosphatidylinositol 3-kinase gamma

Polarization of chemotaxing cells depends on positive feedback loops that amplify shallow gradients of chemoattractants into sharp intracellular responses. In particular, reciprocal activation of phosphatidylinositol 3-kinases (PI3Ks) and small GTPases like Rac leads to accumulation, at the leading edge, of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP3). Mice carrying a "knockin" allele of the G protein-coupled receptor (GPCR)-activated PI3Kgamma, encoding a plasma membrane-targeted protein appeared normal, but their leukocytes showed GPCR-uncoupled PIP3 accumulation. In vivo, the mutation increased proliferation and decreased apoptosis, leading to leukocytosis and delayed resolution of inflammation in wound healing. Mutant leukocytes showed significantly impaired directional cell migration in response to chemoattractants. Stimulated mutant macrophages did not polarize PIP3 and showed a shortened Rac activation because of enhanced PI3K-dependent activation of RacGAPs. Together with the finding that chemoattractants stimulate a PIP3-dependent GAP activation in wild-type macrophages, these results identify a molecular mechanism involving PI3K- and RacGAP-dependent negative control of Rac that limits and fine-tunes feedback loops promoting cell polarization and directional motility.     

The inositol polyphosphate 4-phosphatase forms a complex with phosphatidylinositol 3-kinase in human platelet cytosol

Inositol polyphosphate 4-phosphatase (4-phosphatase) is an enzyme that catalyses the hydrolysis of the 4-position phosphate from phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. In human platelets the formation of this phosphatidylinositol, by the actions of phosphatidylinositol 3-kinase (PI 3-kinase), correlates with irreversible platelet aggregation. We have shown previously that a phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase forms a complex with the p85 subunit of PI 3-kinase. In this study we investigated whether PI 3-kinase also forms a complex with the 4-phosphatase in human platelets. Immunoprecipitates of the p85 subunit of PI 3-kinase from human platelet cytosol contained 4-phosphatase enzyme activity and a 104-kDa polypeptide recognized by specific 4-phosphatase antibodies. Similarly, immunoprecipitates made using 4-phosphatase-specific antibodies contained PI 3-kinase enzyme activity and an 85-kDa polypeptide recognized by antibodies to the p85 adapter subunit of PI 3-kinase. After thrombin activation, the 4-phosphatase translocated to the actin cytoskeleton along with PI 3-kinase in an integrin- and aggregation-dependent manner. The majority of the PI 3-kinase/4-phosphatase complex (75%) remained in the cytosolic fraction. We propose that the complex formed between the two enzymes serves to localize the 4-phosphatase to sites of PtdIns(3,4)P2 production.     

Phosphatidylinositol 3-kinase gamma is a critical mediator of myocardial ischemic and adenosine-mediated preconditioning

Ischemic preconditioning (IPC) is a potent cellular protective mechanism whereby brief periods of sublethal ischemia protect the myocardium from prolonged ischemia-induced injury. We demonstrate the selective role of phosphatidylinositol 3-kinase (PI3K) isoforms in IPC. Hearts from PI3Kgamma knockout mice (PI3Kgamma(-/-)) displayed poorer functional recovery and greater tissue injury following IPC compared to wild-type and PI3Kgamma(+/-) hearts. Examination of the cell-signaling pathways revealed restored phosphorylation levels of Akt and glycogen synthase kinase (GSK)3beta in wild-type hearts, which were abolished in PI3Kgamma(-/-) hearts subjected to IPC. Inhibition of GSK3beta by LiCl reversed the loss in protection in PI3Kgamma(-/-) hearts. In contrast, mice expressing a cardiac-specific kinase-deleted PI3Kalpha (PI3KalphaDN) were resistant to injury induced by 30 minutes of ischemia followed by 40 minutes of reperfusion. Furthermore, the resistance of PI3KalphaDN hearts to ischemia/reperfusion correlated with the persistent expression of p110gamma and was blocked by the PI3K inhibitor wortmannin, suggesting the possible enhanced cell signaling through the PI3Kgamma pathway. These results demonstrate the importance of the PI3Kgamma-Akt-GSK3beta signaling pathway in IPC. Selective activation of myocardial PI3Kgamma may be an attractive target for the treatment of ischemic heart disease.     

GSK3beta is a negative regulator of platelet function and thrombosis

Glycogen synthase kinase (GSK)3beta is a ser-thr kinase that is phosphorylated by the kinase Akt. Although Akt has been shown to regulate platelet function and arterial thrombosis, its effectors in platelets remain unknown. We show here that agonist-dependent phosphorylation of GSK3beta in platelets is Akt dependent. To determine whether GSK3beta regulates platelet function, platelets from mice lacking a single allele of GSK3beta were compared with those of wild-type (WT) controls. GSK3beta+/- platelets demonstrated enhanced agonist-dependent aggregation, dense granule secretion, and fibrinogen binding, compared with WT platelets. Treatment of human platelets with GSK3 inhibitors renders them more sensitive to agonist-induced aggregation, suggesting that GSK3 suppresses platelet function in vitro. Finally, the effect of GSK3beta on platelet function in vivo was evaluated using 2 thrombosis models in mice. In the first, 80% of GSK3beta+/- mice (n=10) formed stable occlusive thrombi after ferric chloride carotid artery injury, whereas the majority of wild-type mice (67%) formed no thrombi (n=15). In a disseminated thrombosis model, deletion of a single allele of GSK3beta in mice conferred enhanced sensitivity to thrombotic insult. Taken together, these results suggest that GSK3beta acts as a negative regulator of platelet function in vitro and in vivo.     

Thrombin-induced platelet aggregation,phosphoinositide metabolism and protein phosphorylation in NIDDM patients treated by diet,sulphonylurea or insulin

Summary We studied thrombin-induced metabolism of phosphoinositide, protein phosphorylation and platelet aggregation in platelets from 32 NIDDM patients and 12 control subjects. To clarify the effect of diet, sulphonylureas, or insulin treatment, the subjects were divided into three groups based on the type of treatment. Thrombin-induced platelet aggregation was measured with an aggregometer. Low-dose thrombin (0.25 U/ml)-stimulated platelet aggregation in diabetic patients was significantly increased compared with the control subjects. Platelet aggregation in the sulphonylurea and insulin groups was significantly lower than in the diet group. On the other hand, in platelets incubated with [³²P]orthophosphate, thrombin-induced incorporation of ³²P radioactivity into phosphatidic acid (PA) was significantly lower in the sulphonylurea and insulin groups than in the diet group. Thrombin-induced incorporation of [³²P] radioactivity into phosphatidylinositol (PIP) for 10 s was significantly higher in the sulphonylurea group than in the diet group. There were no differences in thrombin-induced 47 kDa protein phosphorylation between platelets from the diet, sulphonylurea, or insulin groups. These results suggest that sulphonylureas and insulin induce suppression of thrombin-induced activation of phospholipase C, which mediates hydrolysis of PIP and PIP₂ and production of PA, which leads to inhibition of platelet aggregation.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - IDDM insulin-dependent diabetes mellitus - PA phosphatidic acid - PI phosphatidylinositol - PIP phosphatidylinositol 4-monophosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - TPA 12-O-tetradecanoylphorbol-13-actate - IP₃ inositol 1,4,5-trisphophate - E/I expiration/inspiration - HPLTC high performance thin layer chromatography     

In contrast to fibrinogen or fibrin,peptide and peptide mimetic binding to alpha does not cause outside-in signalling as judged by measurements of phosphatidylinositol 4,5-bisphosphate beta (GPIIb-IIIa) IIb 3

Binding of ligands, including RGD-containing peptides, to the platelet fibrinogen receptor, integrin alpha beta has been reported to cause outside-in signals, which result in clustering of occupied receptors and changes in conformation of the receptor and its cytoplasmic tails. Thus, the peptides that are usually used as inhibitors may function as partial agonists. Binding of ligand, fibrinogen or polymerizing fibrin, to platelets with activated alpha beta causes decreases in phosphatidylinositol 4,5-bisphosphate (PIP ), which may affect actin organization. IIb 3 Whether or not binding to unactivated platelets of the peptide RGDS, the fibrinogen gamma -chain C-terminal dodecapeptide (H12), or a high affinity RGD mimetic SC-54701B affects phosphoinositide metabolism was tested. Although incubation of RGDS (230 mu M), dodecapeptide (400 mu M) or SC-54701B (10 mu M) with platelets for 2 min caused trends towards decreases in PIP , no significant decreases were found. As a positive control, 2 SC-54701B was shown to inhibit the decrease in PIP in ADP-stimulated platelets at concentrations consistent 2 with the reported IC of 0.12 mu M. Thus, binding of peptides or the high affinity RGD mimetic does not 50 generate a sufficient signal to affect the signalling pathway that is involved phosphoinositide metabolism. IIb 3 2     

Polyphosphoinositide Changes in Rabbit Platelets Stimulated with Platelet Activating Factor During the Formation of Platelet-fibrin Clots

Phosphoinositide metabolism in rabbit platelets prelabelled with [(32)P]phosphate and [(3)H]inositol was stimulated by platelet activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) with stirring at 200 rpm for 120 s in the presence of polymerising fibrin produced by the action of batroxobin (B. atrox) (also referred to by the proprietary name Reptilase) on fibrinogen. Under these conditions platelet-fibrin clots formed and retracted around the stirring bar. Phosphoinositides were extracted with chloroform: methanol: HC1. The role of the secretion of platelet granule contents in the phosphoinositide changes was examined by comparison of the effects of 1 nM PAF which did not cause secretion, with 50 nM PAF which caused extensive secretion. Stimulation of platelets with PAF in the presence of polymerising fibrin caused a greater decrease in the amount and labelling of extractable phosphatidylinositol 4,5-bisphosphate (PIP(2)) than was observed with platelets stimulated in the presence of fibrinogen. With 1 nM PAF, the decrease (1.26 ± 0.11 nmol/10(9) platelets) in amount of extractable PIP(2) when platelets were stimulated in the presence of polymerising fibrin compared with in the presence of fibrinogen was accounted for by an increase in the amount of phosphatidylinositol 4-phosphate (PIP). With 50 nM PAF, the decrease in amount of extractable PIP(2) (1.09±0.11 nmol/10(9) platelets) was not accounted for by an increase in the amount of PIP; the decrease in the amount of [(3)H]inositol label in PIP(2) in platelets stimulated in the presence of polymerising fibrin was accounted for by the sum of the increases in PIP labelling and the label associated with interfacial protein from the lipid extractions. When fibrin polymerisation was blocked with glycyl-L-prolyl-L-arginyl-L-proline (GPRP), the large decrease in extractable PIP(2) and the increase in the association of label with the interfacial protein did not occur. Thus, both the formation of a fibrin network, and the changes that accompany the secretion of granule contents, are necessary for the association of the (3)H-labelled material with interfacial protein. Blocking thromboxane A(2) formation had no effect on the changes in response to 50 nM PAF. Although PAF stimulated phospholipase C, resulting in increases in amount and (32)P-labelling of phosphatidic acid and (3)H-labelling of inositol bisphosphate and inositol phosphate, the increases were similar in the presence of polymerising fibrin or fibrinogen. Thus, further stimulation of phospholipase C does not occur in association with clot formation. The specific radioactivities of labelling with [(3)H]inositol of the phosphoinositides in unstimulated platelets differed (PIP(2)> phosphatidylinositol (PI) > PIP). Upon stimulation of the platelets with 1 nM PAF, the specific radioactivity of PIP rose above that of PI and toward that of PIP(2), indicating that the increase in PIP was due to degradation of PIP(2). Thus, the large decrease in extractable PIP(2) and increase in formation of PIP caused by the presence of polymerising fibrin appear to be due to increased degradation of PIP(2) to PIP.     

Phosphatidylinositol-3-kinase-gamma is integral to homing functions of progenitor cells

Endothelial progenitor cells (EPCs) and hematopoietic progenitor cells are recruited to ischemic regions, improving neovascularization. beta1 and beta2 integrins play a crucial role for progenitor cell homing to ischemic tissues. Integrin activity is regulated by chemokines and their respective G protein-coupled receptors. The phosphatidylinositol-3-kinase catalytic subunit gamma (PI3Kgamma) is the PI3K isoform that selectively transduces signals from G protein-coupled receptors. Here, we investigated the role of PI3Kgamma as a signaling intermediate in the chemokine-induced integrin-dependent homing functions of progenitor cells. A pharmacological PI3Kgamma inhibitor significantly reduced chemokine-induced chemotaxis and stromal cell-derived factor (SDF)1alpha-induced transmigration of human EPCs. Moreover, the PI3Kgamma inhibitor significantly reduced SDF1alpha-induced adhesion of EPCs to intercellular adhesion molecule-1 and human umbilical vein endothelial cell monolayers. These findings were corroborated with Lin(-) bone marrow-derived progenitor cells from PI3Kgamma-deficient mice that displayed reduced SDF1alpha-induced migration and intercellular adhesion molecule-1 adhesion as compared with wild-type cells. Pharmacological inhibition or genetic ablation of PI3Kgamma reduced SDF1alpha-induced integrin activation in human EPCs and in murine Lin(-) BM-derived progenitor cells, respectively. In vivo, the homing of PI3Kgamma-deficient Lin(-) progenitor cells to ischemic muscles after intravenous infusion in the model of hindlimb ischemia and their neovascularization-promoting capacity was reduced as compared with wild-type cells. In conclusion, PI3Kgamma is integral to the integrin-dependent homing of progenitor cells.     

In contrast to fibrinogen or fibrin, peptide and peptide mimetic binding to alphaIIbbeta3 (GPIIb-IIIa) does not cause outside-in signalling as judged by measurements of phosphatidylinositol 4,5-bisphosphate

Binding of ligands, including RGD-containing peptides, to the platelet fibrinogen receptor, integrin alphaIIb beta3 has been reported to cause outside-in signals, which result in clustering of occupied receptors and changes in conformation of the receptor and its cytoplasmic tails. Thus, the peptides that are usually used as inhibitors may function as partial agonists. Binding of ligand, fibrinogen or polymerizing fibrin, to platelets with activated alphaIIbbeta3 causes decreases in phosphatidylinositol 4,5-bisphosphate (PIP ), which may affect actin organization. Whether or not binding to unactivated platelets of the peptide RGDS, the fibrinogen gamma -chain C-terminal dodecapeptide (H12), or a high affinity RGD mimetic SC-54701B affects phosphoinositide metabolism was tested. Although incubation of RGDS (230 microM), dodecapeptide (400 microM) or SC-54701B (10 microM) with platelets for 2 min caused trends towards decreases in PIP , no significant decreases were found. As a positive control, 2 SC-54701B was shown to inhibit the decrease in PIP in ADP-stimulated platelets at concentrations consistent 2 with the reported IC50 of 0.12 microM. Thus, binding of peptides or the high affinity RGD mimetic does not 50 generate a sufficient signal to affect the signalling pathway that is involved phosphoinositide metabolism.     

Altered heart rate and sinoatrial node function in mice lacking the cAMP regulator phosphoinositide 3-kinase-gamma

Ablation of the enzyme phosphoinositide 3-kinase (PI3K)gamma (PI3Kgamma(-/-)) in mice increases cardiac contractility by elevating intracellular cAMP and enhancing sarcoplasmic reticulum Ca(2+) handling. Because cAMP is a critical determinant of heart rate, we investigated whether heart rate is altered in mice lacking PI3Kgamma. Heart rate was similar in anesthetized PI3Kgamma(-/-) and wild-type (PI3Kgamma(+/+)) mice. However, IP injection of atropine (1 mg/kg), propranolol (1 mg/kg), or both drugs in combination unmasked elevated heart rates in PI3Kgamma(-/-) mice, suggesting altered sinoatrial node (SAN) function. Indeed, spontaneous action potential frequency was approximately 35% greater in SAN myocytes isolated from PI3Kgamma(-/-) mice compared with PI3Kgamma(+/+) mice. These differences in action potential frequency were abolished by intracellular dialysis with the cAMP/protein kinase A antagonist Rp-cAMP but were unaffected by treatment with ryanodine to inhibit sarcoplasmic reticulum Ca(2+) release. Voltage-clamp experiments demonstrated that elevated action potential frequencies in PI3Kgamma(-/-) SAN myocytes were more strongly associated with cAMP-dependent increases in L-type Ca(2+) current (I(Ca,L)) than elevated hyperpolarization-activated current (I(f)). In contrast, I(Ca,L) was not increased in working atrial myocytes, suggesting distinct subcellular regulation of L-type Ca(2+) channels by PI3Kgamma in the SAN compared with the working myocardium. In summary, PI3Kgamma regulates heart rate by the cAMP-dependent modulation of SAN function. The effects of PI3Kgamma ablation in the SAN are unique from those in the working myocardium.     

Binding of polymerizing fibrin to integrin alpha(IIb)beta(3) on chymotrypsin-treated rabbit platelets decreases phosphatidylinositol 4,5-bisphosphate and increases cytoskeletal actin

Incubation of polymerizing fibrin with washed, chymotrypsin-treated (CT) rabbit platelets resulted in the formation of platelet-fibrin clots, a decrease of 27.4% (P < 0.001, n = 20) in the amount of phosphatidylinositol 4,5-bisphosphate (PIP) and an increase of 37.2% (P < 0.001, n = 20) in phosphatidylinositol 4-phosphate (PIP(2)), apparently because of a shift in the equilibria between PIP(2) and PIP toward PIP. In contrast, incubation of fibrinogen with CT-platelets resulted in agglutination of platelets, but no changes in the phosphoinositides. Preincubation of CT-platelets with staurosporine (1 microM) to inhibit protein phosphorylation, okadaic acid (1 microM) to inhibit protein phosphatases, genistein (100 microM) to inhibit protein tyrosine phosphorylation, PGE (10 microM) to increase cAMP and cause Ca2+ sequestration or with wortmannin (50 nM) to inhibit phosphoinositide 3-kinase, did not inhibit the polymerizing fibrin-induced decrease in PIP2 and increase in PIP. Preincubation with cytochalasin E (CE, 5 microM) inhibited the decrease in PIP(2) by 57% (P < 0.01, n = 8), but not completely. CE did not affect the resting levels of PIP2. Thus, the state of the actin cytoskeleton appears to affect signalling from the integrin receptor alpha(IIb)beta(3) to the enzymes of phosphoinositide interconversion. Platelet cytoskeleton actin content increased by 16.4 4.1% (P < 0.01, n = 5) because of polymerizing fibrin binding to CT-platelets. This is the first demonstration of a pathway involving a decrease in PIP(2) caused by binding of polymerizing fibrin to alpha(IIb)beta(3) and an associated increase in cytoskeletal actin, which may be involved in reorganization of the cytoskeleton for platelet-mediated clot retraction.     

PI3Kgamma (PI3Kgamma) is essential for efficient induction of CXCR3 on activated T cells

The gamma isoform of PI3Kinase (PI3Kgamma) controls leukocyte chemotaxis by participating in GPCR signaling, and by regulating cellular polarization. Here we show that PI3Kgamma is required for efficient induction of CXC chemokine receptor 3 (CXCR3) on T cells upon activation. T cells from PI3Kgamma(-/-) mice up-regulated CXCR3 less efficiently than wild-type controls both upon activation in vitro as well as during Leishmania mexicana infection. Inhibition of PI3Kinases using wortmannin and LY294002 or blockade of PI3Kgamma activity using a selective inhibitor or PI3Kgamma siRNA suppressed induction of CXCR3 on T cells following activation. Levels of CXCR3 and T-bet mRNA were significantly lower in PI3Kgamma inhibitor-treated T cells, indicating that PI3Kgamma may control CXCR3 expression in part through induction of T-bet. These results reveal a novel role for PI3Kgamma in the induction of CXCR3 on T cells and suggest that PI3Kgamma may regulate leukocyte chemotaxis by controlling the expression of chemokine receptors.     

Control of thrombus embolization and fibronectin internalization by integrin alpha IIb beta 3 engagement of the fibrinogen gamma chain

Fibrin(ogen) deficiency (Fg-/-) was shown previously to be compatible with rapid thrombus growth within injured arterioles, but platelet fibronectin content was increased and newly formed thrombi were unstable. To further define the role of fibrin(ogen) in thrombus formation and stabilization, platelet biology was examined in mice expressing a form of fibrinogen that clots normally but lacks the gamma chain C-terminal binding site for alpha IIb beta 3 (Fg gamma Delta 5). Thrombus growth within the arterioles of Fg gamma Delta 5 mice appeared faster than in wild-type mice despite a far greater emboli formation. Unlike Fg-/- mice, the emboli were relatively small and released from the top of thrombi, rather than by fracture at the vessel wall. The fibronectin content in Fg gamma Delta 5 platelets was also dramatically increased through a beta 3 integrin-dependent mechanism. The following has been concluded: (1) Fibrin formation contributes to, but is not sufficient for, the stabilization of arterial thrombi. Platelet receptor engagement of the C-terminal of the Fg gamma chain contributes to the stable incorporation of platelets into thrombi. (2) Alternative ligands to fibrinogen can support efficient thrombus growth. (3) Fibrinogen is internalized through alpha IIb beta 3 engagement of the fibrinogen gamma chain element, and this interaction secondarily controls the fibronectin content of platelets.     

Identification of minor metabolites of phospholipid signal molecules in [(32)P]P(i)-labelled platelets

Incubation of blood platelets with (32)P-labelled inorganic phosphate for 60 min leads to incorporation of radioisotope mainly into phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP) and phosphatidyl-4,5-bisphosphate (PIP(2)) in resting platelets and into phosphatidic acid (PA) in activated platelets. Small amounts of other important phosphoinositide isomers also become labelled following platelet activation, among them the 3-phosphorylated derivatives. In addition, several other faintly labelled spots are visible on TLC separations. Three of these lipids have now been identified as lysophosphatidylinositol (lysoPI), lysophosphatidic acid (lysoPA) and CDP-diacylglcerol (CDP-DAG).[(32)P]LysoPI was present in resting and activated platelets, whereas [(32)P]lysoPA and [(32)P]CDP-DAG were observed only upon platelet activation. The phosphoinositide cycle turns over without accumulation of [(32)P]PA and [(32)P]CDP-DAG in resting platelets. A large increase (as much as 40-fold) in the steady-state level of [(32)P]PA is seen in thrombin-activated platelets. A slight increase in the steady-state levels of [(32)P]CDP-DAG is accompanied by a similar increase in [(32)P]PI and larger increases in [(32)P]PIP and [(32)P]PIP(2) (about 50%), which is indicative of a general increase in flux in the PPI cycle. Elevation of CDP-DAG levels is probably only a reflection of increased flux, whereas lysoPA and lysoPI have been reported to have diverse signalling functions in various cells.     

Identification of minor metabolites of phospholipid signal molecules in [ 32 P]P i -labelled platelets

Incubation of blood platelets with 32 P-labelled inorganic phosphate for 60 min leads to incorporation of radioisotope mainly into phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP) and phosphatidyl-4,5-bisphosphate (PIP 2 ) in resting platelets and into phosphatidic acid (PA) in activated platelets. Small amounts of other important phosphoinositide isomers also become labelled following platelet activation, among them the 3-phosphorylated derivatives. In addition, several other faintly labelled spots are visible on TLC separations. Three of these lipids have now been identified as lysophosphatidylinositol (lysoPI), lysophosphatidic acid (lysoPA) and CDP-diacylglcerol (CDP-DAG).[ 32 P]LysoPI was present in resting and activated platelets, whereas [ 32 P]lysoPA and [ 32 P]CDP-DAG were observed only upon platelet activation. The phosphoinositide cycle turns over without accumulation of [ 32 P]PA and [ 32 P]CDP-DAG in resting platelets. A large increase (as much as 40-fold) in the steady-state level of [ 32 P]PA is seen in thrombin-activated platelets. A slight increase in the steady-state levels of [ 32 P]CDP-DAG is accompanied by a similar increase in [ 32 P]PI and larger increases in [ 32 P]PIP and [ 32 P]PIP 2 (about 50%), which is indicative of a general increase in flux in the PPI cycle. Elevation of CDP-DAG levels is probably only a reflection of increased flux, whereas lysoPA and lysoPI have been reported to have diverse signalling functions in various cells.     

Polyphosphoinositide Changes in Rabbit Platelets Stimulated with Platelet Activating Factor During the Formation of Platelet-fibrin Clots

Phosphoinositide metabolism in rabbit platelets prelabelled with [³²P]phosphate and [³H]inositol was stimulated by platelet activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) with stirring at 200 rpm for 120 s in the presence of polymerising fibrin produced by the action of batroxobin (B. atrox) (also referred to by the proprietary name Reptilase) on fibrinogen. Under these conditions platelet-fibrin clots formed and retracted around the stirring bar. Phosphoinositides were extracted with chloroform: methanol: HC1. The role of the secretion of platelet granule contents in the phosphoinositide changes was examined by comparison of the effects of 1 nM PAF which did not cause secretion, with 50 nM PAF which caused extensive secretion. Stimulation of platelets with PAF in the presence of polymerising fibrin caused a greater decrease in the amount and labelling of extractable phosphatidylinositol 4,5-bisphosphate (PIP₂) than was observed with platelets stimulated in the presence of fibrinogen. With 1 nM PAF, the decrease (1.26 ± 0.11 nmol/10⁹ platelets) in amount of extractable PIP₂ when platelets were stimulated in the presence of polymerising fibrin compared with in the presence of fibrinogen was accounted for by an increase in the amount of phosphatidylinositol 4-phosphate (PIP). With 50 nM PAF, the decrease in amount of extractable PIP₂ (1.09±0.11 nmol/10⁹ platelets) was not accounted for by an increase in the amount of PIP; the decrease in the amount of [³H]inositol label in PIP₂ in platelets stimulated in the presence of polymerising fibrin was accounted for by the sum of the increases in PIP labelling and the label associated with interfacial protein from the lipid extractions. When fibrin polymerisation was blocked with glycyl-L-prolyl-L-arginyl-L-proline (GPRP), the large decrease in extractable PIP₂ and the increase in the association of label with the interfacial protein did not occur. Thus, both the formation of a fibrin network, and the changes that accompany the secretion of granule contents, are necessary for the association of the ³H-labelled material with interfacial protein. Blocking thromboxane A₂ formation had no effect on the changes in response to 50 nM PAF. Although PAF stimulated phospholipase C, resulting in increases in amount and ³²P-labelling of phosphatidic acid and ³H-labelling of inositol bisphosphate and inositol phosphate, the increases were similar in the presence of polymerising fibrin or fibrinogen. Thus, further stimulation of phospholipase C does not occur in association with clot formation. The specific radioactivities of labelling with [³H]inositol of the phosphoinositides in unstimulated platelets differed (PIP₂> phosphatidylinositol (PI) > PIP). Upon stimulation of the platelets with 1 nM PAF, the specific radioactivity of PIP rose above that of PI and toward that of PIP₂, indicating that the increase in PIP was due to degradation of PIP₂. Thus, the large decrease in extractable PIP₂ and increase in formation of PIP caused by the presence of polymerising fibrin appear to be due to increased degradation of PIP₂ to PIP.     

Inositol polyphosphate multikinase is a physiologic PI3-kinase that activates Akt/PKB

The second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), formed by the p110 family of PI3-kinases, promotes cellular growth, proliferation, and survival, in large part by activating the protein kinase Akt/PKB. We show that inositol polyphosphate multikinase (IPMK) physiologically generates PIP(3) as well as water soluble inositol phosphates. IPMK deletion reduces growth factor-elicited Akt signaling and cell proliferation caused uniquely by loss of its PI3-kinase activity. Inhibition of p110 PI3-kinases by wortmannin prevents IPMK phosphorylation and activation. Thus, growth factor stimulation of Akt signaling involves PIP(3) generation through the sequential activations of the p110 PI3-kinases and IPMK. As inositol phosphates inhibit Akt signaling, IPMK appears to act as a molecular switch, inhibiting or stimulating Akt via its inositol phosphate kinase or PI3-kinase activities, respectively. Drugs regulating IPMK may have therapeutic relevance in influencing cell proliferation.