Critical roles for the phosphatidylinositide 3-kinase isoforms p110β and p110γ in thrombopoietin-mediated priming of platelet function

Abstract Thrombopoietin (TPO) enhances platelet activation through activation of the tyrosine kinase; JAK2 and the lipid kinase phosphatidylinositide 3-kinase (PI3K). The aim of our study was to identify the PI3K isoforms involved in mediating the effect of TPO on platelet function and elucidate the underlying mechanism. We found that p110β plays an essential role in TPO-mediated (i) priming of protease-activated receptor (PAR)-mediated integrin αIIbβ3 activation and α-granule secretion, (ii) synergistic enhancement of PAR-mediated activation of the small GTPase RAP1, a regulator of integrin activation and (iii) phosphorylation of the PI3K effector Akt. More importantly, the synergistic effect of TPO on phosphorylation of extracellular-regulated kinase (ERK1/2) and thromboxane (TxA2) synthesis was dependent on both p110β and p110γ. p110β inhibition/deletion, or inhibition of p110γ, resulted in a partial reduction, whereas inhibiting both p110β and p110γ completely prevented the synergistic effect of TPO on ERK1/2 phosphorylation and TxA2 synthesis. The latter was ablated by inhibition of MEK, but not p38, confirming a role for ERK1/2 in regulating TPO-mediated increases in TxA2 synthesis. In conclusion, the synergistic effect of TPO on RAP1 and integrin activation is largely mediated by p110β, whereas p110β and p110γ contribute to the effect of TPO on ERK1/2 phosphorylation and TxA2 formation

The Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ) Binder Rasa3 Regulates Phosphoinositide 3-kinase (PI3K)-dependent Integrin α IIb β 3 Outside-in Signaling

The class I PI3K family of lipid kinases plays an important role in integrin αIIbβ3 function, thereby supporting thrombus growth and consolidation. Here, we identify Ras/Rap1GAP Rasa3 (GAP1IP4BP) as a major phosphatidylinositol 3,4,5-trisphosphate-binding protein in human platelets and a key regulator of integrin αIIbβ3 outside-in signaling. We demonstrate that cytosolic Rasa3 translocates to the plasma membrane in a PI3K-dependent manner upon activation of human platelets. Expression of wild-type Rasa3 in integrin αIIbβ3-expressing CHO cells blocked Rap1 activity and integrin αIIbβ3-mediated spreading on fibrinogen. In contrast, Rap1GAP-deficient (P489V) and Ras/Rap1GAP-deficient (R371Q) Rasa3 had no effect. We furthermore show that two Rasa3 mutants (H794L and G125V), which are expressed in different mouse models of thrombocytopenia, lack both Ras and Rap1GAP activity and do not affect integrin αIIbβ3-mediated spreading of CHO cells on fibrinogen. Platelets from thrombocytopenic mice expressing GAP-deficient Rasa3 (H794L) show increased spreading on fibrinogen, which in contrast to wild-type platelets is insensitive to PI3K inhibitors. Together, these results support an important role for Rasa3 in PI3K-dependent integrin αIIbβ3-mediated outside-in signaling and cell spreading

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Lysophosphatidylinositol-acyltransferase-1 (LPIAT1) is required to maintain physiological levels of PtdIns and PtdInsP(2) in the mouse.

We disrupted the gene encoding lysophosphatidylinositol-acyltransferase-1 (LPIAT1) in the mouse with the aim of understanding its role in determining cellular phosphoinositide content. LPIAT1(-/-) mice were born at lower than Mendelian ratios and exhibited a severe developmental brain defect. We compared the phospholipid content of livers and brains from LPIAT1(-/-) and LPIAT1(+/+) littermates by LC-ESI/MS. In accord with previous studies, the most abundant molecular species of each phosphoinositide class (PtdIns, PtdInsP, PtdInsP2 and PtdInsP3) possessed a C38∶4 complement of fatty-acyl esters (C18∶0 and C20∶4 are usually assigned to the sn-1 and sn-2 positions, respectively). LPIAT1(-/-) liver and brain contained relatively less of the C38∶4 species of PtdIns, PtdInsP and PtdInsP2 (dropping from 95-97% to 75-85% of the total species measured for each lipid class) and relatively more of the less abundant species (PtdInsP3 less abundant species were below our quantification levels). The increases in the less abundant PtdIns and PtdInsP2 species did not compensate for the loss in C38∶4 species, resulting in a 26-44% reduction in total PtdIns and PtdInsP2 levels in both brain and liver. LPIAT1(-/-) brain and liver also contained increased levels of C18∶0 lyso-PtdIns (300% and 525% respectively) indicating a defect in the reacylation of this molecule. LPIAT1(-/-) brain additionally contained significantly reduced C38∶4 PC and PE levels (by 47% and 55% respectively), possibly contributing to the phenotype in this organ. The levels of all other molecular species of PC, PE, PS and PA measured in the brain and liver were very similar between LPIAT1(-/-) and LPIAT1(+/+) samples. These results suggest LPIAT1 activity plays a non-redundant role in maintaining physiological levels of PtdIns within an active deacylation/reacylation cycle in mouse tissues. They also suggest that this pathway must act in concert with other, as yet unidentified, mechanisms to achieve the enrichment observed in C38∶4 molecular species of phosphoinositides

Effect of LPIAT1 knockout on liver phospholipid molecular species.

<p>Livers from 13 day old littermates expressing (LPIAT1^+/+ (WT)) or lacking (LPIAT1^−/− (KO)) LPIAT1 were homogenized and lipids extracted from 0.5 mg wet weight as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Targeted molecular species of PC (<b>A</b>), PE (<b>B</b>), PS (<b>C</b>) and PA (<b>D</b>) were detected by MRM mass spectrometric analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Data are expressed as moles/mg protein, normalized to relevant internal standards. Shown are mean ± SD, n = 4 for both WT and KO. Data were analyzed by T-test.</p

Effect of LPIAT1 knockout on brain and liver lyso-phospholipid molecular species.

<p>Brains (<b>A</b>) or livers (<b>B</b>) from 13 day old littermates expressing (LPIAT1^+/+ (WT)) or lacking (LPIAT1^−/− (KO)) LPIAT1 were ground, homogenized and lipids extracted from 0.5 mg wet weight as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Lyso-phospholipids were targeted and were detected by MRM mass spectrometric anaylsis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Data are expressed as moles/mg protein, normalized to relevant internal standards. Shown are mean ± SD, n = 4 for both WT and KO. Data were analyzed by T-test. *p≤0.05, **p≤0.005.</p

Effect of LPIAT1 knockout on brain phospholipid molecular species.

<p>Brains from 13 day old littermates expressing (LPIAT1^+/+ (WT)) or lacking (LPIAT1^−/− (KO)) LPIAT1 were homogenized and lipids extracted from 0.5 mg wet weight as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Targeted molecular species of PC (<b>A</b>), PE (<b>B</b>), PS (<b>C</b>) and PA (<b>D</b>) were detected by MRM mass spectrometric analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Data are expressed as moles/mg protein, normalized to relevant internal standards. Shown are mean ± SD, n = 4 for both WT and KO. Data were analyzed by T-test. *p≤0.05, **p≤0.005.</p

Effect of LPIAT1 knockout on brain phosphoinositide lipid molecular species.

<p>Brains from 13 day old littermates expressing (LPIAT1^+/+ (WT)) or lacking (LPIAT1^−/− (KO)) LPIAT1 were homogenised and lipid extracted from 0.5 mg wet weight as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Targeted molecular species of PtdIns (<b>A</b>), PtdInsP (<b>B</b>), PtdInsP₂ (<b>C</b>) and PtdInsP₃ (<b>D</b>) were detected by MRM mass spectrometric analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058425#s2" target="_blank">Materials and Methods</a>. Data are expressed as moles/mg protein, normalized to relevant internal standards. Shown are mean ± SD, n = 4 for both WT and KO. Data were analyzed by T-test. *p≤0.05, **p≤0.005.</p

Numbers of genotypes in litters from LPIAT1^+/− crosses.

<p>Numbers of genotypes in litters from LPIAT1^+/− crosses.</p