Rapamycin restrains platelet procoagulant responses via FKBP-mediated protection of mitochondrial integrity.

BACKGROUND AND PURPOSE: Rapamycin is a potent immunosuppressant and anti-proliferative agent used clinically to prevent organ transplant rejection and for coating coronary stents to counteract restenosis. Rapamycin complexes with the immunophilin FKBP12, which subsequently binds and inhibits mTORC1. Despite several reports demonstrating that rapamycin affects platelet-mediated responses, the underlying mechanism of how it alters platelet function is poorly characterised. This study aimed to elucidate the effect of rapamycin on platelet procoagulant responses. EXPERIMENTAL APPROACH: The effect of rapamycin on platelet activation and signalling was investigated alongside the catalytic mTOR inhibitors KU0063794 and WYE-687, and the FKBP12-binding macrolide FK506. KEY RESULTS: Rapamycin affects platelet procoagulant responses by reducing externalisation of the procoagulant phospholipid phosphatidylserine, formation of balloon-like structures and local generation of thrombin. Catalytic mTOR kinase inhibitors did not alter platelet procoagulant processes, despite having a similar effect as rapamycin on Ca2+ signalling, demonstrating that the effect of rapamycin on procoagulant responses is independent of mTORC1 inhibition and not linked to a reduction in Ca2+ signalling. FK506, which also forms a complex with FKBP12 but does not target mTOR, reduced platelet procoagulant responses to a similar extent as rapamycin. Both rapamycin and FK506 prevented the loss of mitochondria integrity induced by platelet activation, one of the central regulatory events leading to PS externalisation. CONCLUSIONS AND IMPLICATIONS: Rapamycin suppresses platelet procoagulant responses by protecting mitochondrial integrity in a manner independent of mTORC1 inhibition. Rapamycin and other drugs targeting FKBP immunophilins could aid the development of novel complementary anti-platelet therapies.

Platelet surface marker analysis by mass cytometry.

Mass cytometry is a next generation flow cytometry technology which analyzes cells one at a time (up to 1000/sec) using mass spectrometry to detect probes labeled with rare-earth metals. Rare-earth metals detected by mass spectrometry have extremely low backgrounds and can be identified with high resolution enabling the routine simultaneous detection of more than 45 probes on each cell without the need for complex compensation matrices. Here we describe a panel of 14 platelet-specific metal-conjugated antibodies (targeting cluster of differentiation [CD] 9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD107a, CD154, glycoprotein [GP] VI and activated integrin αIIbß3) and methods for staining and analysis of platelets by mass cytometry. High dimensional clustering algorithms, which take into account the levels of all 14 markers detected by mass cytometry on each cell, allow identification of platelet subpopulations not previously appreciated. We previously reported that platelet heterogeneity identified by mass cytometry appears similar across healthy donors and consistent over time. High dimensional analysis revealed the presence of a platelet subpopulation with significantly higher levels of surface expression of activated GPIIb-IIIa and P-selectin suggesting this subpopulation may play a greater role in thrombus formation than other platelet subpopulations. Thus, analysis by mass cytometry of platelet heterogeneity and subpopulations may suggest distinct biological roles for different platelet subpopulations and may be useful in evaluating inherited or acquired platelet disorders and platelet function in health and disease.

Critical roles for the phosphatidylinositide 3-kinase isoforms p110ß and p110γ in thrombopoietin-mediated priming of platelet function.

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 p110ß plays an essential role in TPO-mediated (i) priming of protease-activated receptor (PAR)-mediated integrin αIIbß3 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 p110ß and p110γ. p110ß inhibition/deletion, or inhibition of p110γ, resulted in a partial reduction, whereas inhibiting both p110ß 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 p110ß, whereas p110ß and p110γ contribute to the effect of TPO on ERK1/2 phosphorylation and TxA2 formation.

Mass Cytometry Reveals Distinct Platelet Subtypes in Healthy Subjects and Novel Alterations in Surface Glycoproteins in Glanzmann Thrombasthenia.

Mass cytometry (MC) uses mass spectrometry to simultaneously detect multiple metal-conjugated antibodies on single cells, thereby enabling the detailed study of cellular function. Here, for the first time, we applied MC to the analysis of platelets. We developed a panel of 14 platelet-specific metal-tagged antibodies (targeting cluster of differentiation [CD] 9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD107a, CD154, glycoprotein [GP] VI and activated integrin αIIbß3) and compared this panel with two fluorescence flow cytometry (FFC) panels (CD41, CD42b, and CD61; or CD42b, CD62P, and activated integrin αIIbß3) in the evaluation of activation-dependent changes in glycoprotein expression on healthy subject and Glanzmann thrombasthenia (GT) platelets. High-dimensional analysis of surface markers detected by MC identified previously unappreciated subpopulations of platelets in healthy donors. As expected, MC and FFC revealed that GT platelets had significantly reduced CD41, CD61, and activated integrin αIIbß3 surface expression. MC also revealed that surface expression of CD9, CD42a and CD63 were elevated, CD31, CD154 and GPVI were reduced and CD29, CD36, CD42b, CD62P and CD107a were similar on GT platelets compared to healthy donor platelets. In summary, MC revealed distinct platelet subtypes in healthy subjects and novel alterations in surface glycoproteins on GT platelets.

Loss of the insulin receptor in murine megakaryocytes/platelets causes thrombocytosis and alterations in IGF signalling.

AIMS: Patients with conditions that are associated with insulin resistance such as obesity, type 2 diabetes mellitus, and polycystic ovary syndrome have an increased risk of thrombosis and a concurrent hyperactive platelet phenotype. Our aim was to determine whether insulin resistance of megakaryocytes/platelets promotes platelet hyperactivation. METHODS AND RESULTS: We generated a conditional mouse model where the insulin receptor (IR) was specifically knocked out in megakaryocytes/platelets and performed ex vivo platelet activation studies in wild-type (WT) and IR-deficient platelets by measuring aggregation, integrin αIIbß3 activation, and dense and α-granule secretion. Deletion of IR resulted in an increase in platelet count and volume, and blocked the action of insulin on platelet signalling and function. Platelet aggregation, granule secretion, and integrin αIIbß3 activation in response to the glycoprotein VI (GPVI) agonist collagen-related peptide (CRP) were significantly reduced in platelets lacking IR. This was accompanied by a reduction in the phosphorylation of effectors downstream of GPVI. Interestingly, loss of IR also resulted in a reduction in insulin-like growth factor-1 (IGF-1)- and insulin-like growth factor-2 (IGF-2)-mediated phosphorylation of IRS-1, Akt, and GSK3ß and priming of CRP-mediated platelet activation. Pharmacological inhibition of IR and the IGF-1 receptor in WT platelets recapitulated the platelet phenotype of IR-deficient platelets. CONCLUSIONS: Deletion of IR (i) increases platelet count and volume, (ii) does not cause platelet hyperactivity, and (iii) reduces GPVI-mediated platelet function and platelet priming by IGF-1 and IGF-2.

Phosphoinositide 3-kinases p110α and p110ß have differential roles in insulin-like growth factor-1-mediated Akt phosphorylation and platelet priming.

OBJECTIVE: Platelet hyperactivity is a contributing factor in the pathogenesis of cardiovascular disease and can be induced by elevated levels of circulating growth factors, such as insulin-like growth factor-1 (IGF-1). IGF-1 is a primer that cannot stimulate platelet activation by itself, but in combination with physiological stimuli can potentiate platelet functional responses via a phosphoinositide 3-kinase-dependent mechanism. In this study, we explored the role of the phosphoinositide 3-kinase p110α isoform in IGF-1-mediated enhancement of platelet function. APPROACH AND RESULTS: Using a platelet-specific p110α knockout murine model, we demonstrate that genetic deletion, similar to pharmacological inactivation of p110α, did not affect proteinase-activated receptor 4 signaling to Akt/protein kinase B but significantly reduced IGF-1-mediated Akt phosphorylation. The p110ß inhibitor TGX-221 abolished IGF-1-induced Akt phosphorylation in p110α-deficient platelets, demonstrating that both p110α and p110ß contribute to IGF-1-mediated Akt phosphorylation. Genetic deletion of p110α had no effect on IGF-1-mediated increases in thrombus formation on collagen and enhancement of proteinase-activated receptor 4-mediated integrin activation and α-granule secretion. In contrast, pharmacological inhibition of p110α blocked IGF-1-mediated potentiation of integrin activation and α-granule secretion. Functional enhancement by IGF-1 in p110α knockout samples was lost after TGX-221 treatment, suggesting that p110ß drives priming in the absence of the p110α isoform. CONCLUSIONS: Together, these results demonstrate that both p110α and p110ß are involved in Akt signaling by IGF-1, but that it is the p110α isoform that is responsible for IGF-1-mediated potentiation of platelet function.