Carl Högberg

Mild hypothermia does not attenuate platelet aggregation and may even increase ADP-stimulated platelet aggregation after clopidogrel treatment

BackgroundMild hypothermia is currently standard of care for cardiac arrest patients in many hospitals and a common belief is that hypothermia attenuates platelet aggregation. We wanted to examine the effects of clopidogrel on platelet aggregation during hypothermia.MethodsPlatelet reactivity at 37°C and 33°C was evaluated by light transmission aggregometry and vasodilator-stimulated phosphoprotein (VASP) in blood from healthy volunteers before, and 24 hours after, a 600 mg loading dose of clopidogrel.ResultsCollagen, 5-HT, epinephrine, U46619 and ADP-induced platelet aggregation was unaltered or even increased by hypothermia. After clopidogrel, there was a significant increase in platelet aggregation for 5 and 20 μM ADP at 33°C compared to 37°C (46 ± 5 vs. 34 ± 5% and 58 ± 4 vs. 47 ± 4%, p < 0.001, n = 8). Hypothermia also increased ADP-induced aggregation after pretreatment with the P2Y1 antagonist MRS2500. The decreased responsiveness to clopidogrel during hypothermia could be overcome by addition of the reversible P2Y12 antagonist AZD6140. ADP-induced inhibition of VASP-phosphorylation was unaffected by hypothermia both in the presence and absence of clopidogrel. A dose-response curve for ADP-induced platelet aggregation revealed increased potency for ADP during hypothermia with no difference in efficacy.ConclusionMild hypothermia did not attenuate platelet aggregation, instead it even increased ADP-stimulated platelet aggregation after clopidogrel treatment. Dual platelet inhibition with aspirin and a P2Y12 receptor antagonist is probably needed for patients with acute coronary syndromes treated with mild hypothermia, and it is possible that future ADP blockers could be of benefit.

The reversible oral P2Y12 antagonist AZD6140 inhibits ADP-induced contractions in murine and human vasculature

OBJECTIVES The platelet ADP P2Y(12) receptor which is a target for the antithrombotic drug clopidogrel is also distributed on vascular smooth muscle cells and stimulate contraction. This study investigates whether AZD6140, in contrast to clopidogrel, can inhibit ADP-mediated arterial contractions. METHODS Mice were treated with clopidogrel, 50 mg/kg, 24 and 2 h before experiment. Thoracic aorta ring segments from both clopidogrel-treated (n=5) and untreated (n=4) mice were mounted in myograph baths. Contractions of human left internal mammary arteries (IMA) and small arteries were studied in an identical manner. RESULTS Clopidogrel treatment per os did not inhibit contractions by the stable ADP analogue 2-MeSADP (10 microM). However, addition of 1 microM AZD6140 in vitro inhibited ADP contraction (% of maximal contraction by 60 mM K(+)) both in the clopidogrel-treated, from 64% to 32% (P=0.002) and in the untreated group, from 59% to 33% (P=0.015). 2-MeSADP contractions in human IMA and small arteries were inhibited by AZD6140. CONCLUSIONS The antiplatelet drug AZD6140 blocks the contractile effects of ADP in both murine and human vasculature. These effects of AZD6140 could be beneficial in the management of conditions in which vasospasm may play a role.

Antibodies targeting human IL1RAP (IL1R3) show therapeutic effects in xenograft models of acute myeloid leukemia

Significance Acute myeloid leukemia (AML) is a hematologic malignancy with poor survival. Current treatment with chemotherapy does not target the leukemic cells specifically and is associated with severe side effects. Here we demonstrate that antibodies directed at the cell surface molecule IL-1 receptor accessory protein (IL1RAP), expressed on immature AML cells, show strong antileukemic effects in mice transplanted with human AML cells and that the mechanism behind the cell killing is through recruitment of effector cells. Using antibodies against IL1RAP also capable of blocking IL-1 signaling, we show that the proliferation of human AML cells can be inhibited, providing a second mode of action of IL1RAP antibodies. These results provide critical evidence in support of a rapid clinical development of an antibody-based anti-IL1RAP therapy in AML. Acute myeloid leukemia (AML) is associated with a poor survival rate, and there is an urgent need for novel and more efficient therapies, ideally targeting AML stem cells that are essential for maintaining the disease. The interleukin 1 receptor accessory protein (IL1RAP; IL1R3) is expressed on candidate leukemic stem cells in the majority of AML patients, but not on normal hematopoietic stem cells. We show here that monoclonal antibodies targeting IL1RAP have strong antileukemic effects in xenograft models of human AML. We demonstrate that effector-cell–mediated killing is essential for the observed therapeutic effects and that natural killer cells constitute a critical human effector cell type. Because IL-1 signaling is important for the growth of AML cells, we generated an IL1RAP-targeting antibody capable of blocking IL-1 signaling and show that this antibody suppresses the proliferation of primary human AML cells. Hence, IL1RAP can be efficiently targeted with an anti-IL1RAP antibody capable of both achieving antibody-dependent cellular cytotoxicity and blocking of IL-1 signaling as modes of action. Collectively, these results provide important evidence in support of IL1RAP as a target for antibody-based treatment of AML.

Succinate independently stimulates full platelet activation via cAMP and phosphoinositide 3-kinase-β signaling

Summary.  Background: The citric cycle intermediate succinate has recently been identified as a ligand for the G‐protein‐coupled receptor (GPCR) SUCNR1. We have previously found that this receptor is one of the most highly expressed GPCRs in human platelets. Objective: The aim of this study was to investigate the role of SUCNR1 in platelet aggregation and to explore the signaling pathways of this receptor in platelets. Methods and Results: Using real‐time‐PCR, we demonstrated that SUCNR1 is expressed in human platelets at a level corresponding to that of the P2Y1 receptor. Light transmission aggregation experiments showed dose‐dependent aggregation induced by succinate, reaching a maximum response at 0.5 mm. The effect of succinate on platelet aggregation was confirmed with flow cytometry, showing increased surface expression of activated glycoprotein IIb–IIIa and P‐selectin. Intracellular SUCNR1 signaling was found to result in decreased cAMP levels, Akt phosphorylation mediated by phosphoinositide 3‐kinase‐β activation, and receptor desensitization. Furthermore, succinate‐induced platelet aggregation was demonstrated to depend on Src, generation of thromboxane A2, and ATP release. Platelet SUCNR1 is subject to desensitization through both homologous and heterologous mechanisms. In addition, the P2Y12 receptor inhibitor ticagrelor completely prevented platelet aggregation induced by succinate. Conclusions: Our experiments show that succinate induces full aggregation of human platelets via SUCNR1. Succinate‐induced platelet aggregation depends on thromboxane A2 generation, ATP release, and P2Y12 activation.

Farnesyl pyrophosphate is an endogenous antagonist to ADP-stimulated P2Y12 receptor-mediated platelet aggregation.

Farnesyl pyrophosphate (FPP) is an intermediate in cholesterol biosynthesis, and it has also been reported to activate platelet LPA (lysophosphatidic acid) receptors. The aim of this study was to investigate the role of extracellular FPP in platelet aggregation. Human platelets were studied with light transmission aggregometry, flow cytometry and [³⁵S]GTPγS binding assays. As shown previously, FPP could potentiate LPA-stimulated shape change. Surprisingly, FPP also acted as a selective insurmountable antagonist to ADP-induced platelet aggregation. FPP inhibited ADP-induced expression of P-selectin and the activated glycoprotein (Gp)IIb/IIIa receptor. FPP blocked ADP-induced inhibition of cAMP accumulation and [³⁵S]GTPγS binding in platelets. In Chinese hamster ovary cells expressing the P2Y₁₂ receptor, FPP caused a rightward shift of the [³⁵S]GTPγS binding curve. In Sf9 insect cells expressing the human P2Y₁₂ receptor, FPP showed a concentration-dependent, although incomplete inhibition of [³H]PSB-0413 binding. Docking of FPP in a P2Y₁₂ receptor model revealed molecular similarities with ADP and a good fit into the binding pocket for ADP. In conclusion, FPP is an insurmountable antagonist of ADP-induced platelet aggregation mediated by the P2Y₁₂ receptor. It could be an endogenous antithrombotic factor modulating the strong platelet aggregatory effects of ADP in a manner similar to the use of clopidogrel, prasugrel or ticagrelor in the treatment of ischaemic heart disease.

Modeling chronic myeloid leukemia in immunodeficient mice reveals expansion of aberrant mast cells and accumulation of pre-B cells

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm that, if not treated, will progress into blast crisis (BC) of either myeloid or B lymphoid phenotype. The BCR-ABL1 fusion gene, encoding a constitutively active tyrosine kinase, is thought to be sufficient to cause chronic phase (CP) CML, whereas additional genetic lesions are needed for progression into CML BC. To generate a humanized CML model, we retrovirally expressed BCR-ABL1 in the cord blood CD34+ cells and transplanted these into NOD-SCID (non-obese diabetic/severe-combined immunodeficient) interleukin-2-receptor γ-deficient mice. In primary mice, BCR-ABL1 expression induced an inflammatory-like state in the bone marrow and spleen, and mast cells were the only myeloid lineage specifically expanded by BCR-ABL1. Upon secondary transplantation, the pronounced inflammatory phenotype was lost and mainly human mast cells and macrophages were found in the bone marrow. Moreover, a striking block at the pre-B-cell stage was observed in primary mice, resulting in an accumulation of pre-B cells. A similar block in B-cell differentiation could be confirmed in primary cells from CML patients. Hence, this humanized mouse model of CML reveals previously unexplored features of CP CML and should be useful for further studies to understand the disease pathogenesis of CML.

Agonistic targeting of TLR1/TLR2 induces p38 MAPK-dependent apoptosis and NFκB-dependent differentiation of AML cells

Acute myeloid leukemia (AML) is associated with poor survival, and there is a strong need to identify disease vulnerabilities that might reveal new treatment opportunities. Here, we found that Toll-like receptor 1 (TLR1) and TLR2 are upregulated on primary AML CD34+CD38- cells relative to corresponding normal bone marrow cells. Activating the TLR1/TLR2 complex by the agonist Pam3CSK4 in MLL-AF9-driven human AML resulted in induction of apoptosis by p38 MAPK-dependent activation of Caspase 3 and myeloid differentiation in a NFκB-dependent manner. By using murine Trp53-/-MLL-AF9 AML cells, we demonstrate that p53 is dispensable for Pam3CSK4-induced apoptosis and differentiation. Moreover, murine AML1-ETO9a-driven AML cells also were forced into apoptosis and differentiation on TLR1/TLR2 activation, demonstrating that the antileukemic effects observed were not confined to MLL-rearranged AML. We further evaluated whether Pam3CSK4 would exhibit selective antileukemic effects. Ex vivo Pam3CSK4 treatment inhibited murine and human leukemia-initiating cells, whereas murine normal hematopoietic stem and progenitor cells (HSPCs) were relatively less affected. Consistent with these findings, primary human AML cells across several genetic subtypes of AML were more vulnerable for TLR1/TLR2 activation relative to normal human HSPCs. In the MLL-AF9 AML mouse model, treatment with Pam3CSK4 provided proof of concept for in vivo therapeutic efficacy. Our results demonstrate that TLR1 and TLR2 are upregulated on primitive AML cells and that agonistic targeting of TLR1/TLR2 forces AML cells into apoptosis by p38 MAPK-dependent activation of Caspase 3, and differentiation by activating NFκB, thus revealing a new putative strategy for therapeutically targeting AML cells.

Interleukin 4 induces apoptosis of acute myeloid leukemia cells in a Stat6 dependent manner

Cytokines provide signals that regulate immature normal and acute myeloid leukemia (AML) cells in the bone marrow microenvironment. We here identify interleukin 4 (IL4) as a selective inhibitor of AML cell growth and survival in a cytokine screen using fluorescently labeled AML cells. RNA-sequencing of the AML cells revealed an IL4-induced upregulation of Stat6 target genes and enrichment of apoptosis-related gene expression signatures. Consistent with these findings, we found that IL4 stimulation of AML cells induced Stat6 phosphorylation and that disruption of Stat6 using CRISPR/Cas9-genetic engineering rendered cells partially resistant to IL4-induced apoptosis. To evaluate whether IL4 inhibits AML cells in vivo, we expressed IL4 ectopically in AML cells transplanted into mice and also injected IL4 into leukemic mice; both strategies resulted in the suppression of the leukemia cell burden and increased survival. Notably, IL4 exposure caused reduced growth and survival of primary AML CD34+CD38− patient cells from several genetic subtypes of AML, whereas normal stem and progenitor cells were less affected. The IL4-induced apoptosis of AML cells was linked to Caspase-3 activation. Our results demonstrate that IL4 selectively induces apoptosis of AML cells in a Stat6-dependent manner—findings that may translate into new therapeutic opportunities in AML.

Transgenic expression of human cytokines in immunodeficient mice does not facilitate myeloid expansion of BCR-ABL1 transduced human cord blood cells

Several attempts have been made to model chronic myeloid leukemia (CML) in a xenograft setting but expansion of human myeloid cells in immunodeficient mice has proven difficult to achieve. Lack of cross-reacting cytokines in the microenvironment of the mice has been proposed as a potential reason. In this study we have used NOD/SCID IL2–receptor gamma deficient mice expressing human SCF, IL-3 and GM-CSF (NSGS mice), that should be superior in supporting human, and particularly, myeloid cell engraftment, to expand BCR-ABL1 expressing human cells in order to model CML. NSGS mice transplanted with BCR-ABL1 expressing cells became anemic and had to be sacrificed due to illness, however, this was not accompanied by an expansion of human myeloid cells but rather we observed a massive expansion of human T-cells and macrophages/histiocytes. Importantly, control human cells without BCR-ABL1 expression elicited a similar reaction, although with a slight delay of disease induction, suggesting that while BCR-ABL1 contributes to the inflammatory reaction, the presence of normal human hematopoietic cells is detrimental for NSGS mice.

Succinate independently stimulates full platelet activation via cAMP and phosphoinositide 3-kinase-β signaling: Succinate independently stimulates full platelet activation

Summary.  Background: The citric cycle intermediate succinate has recently been identified as a ligand for the G‐protein‐coupled receptor (GPCR) SUCNR1. We have previously found that this receptor is one of the most highly expressed GPCRs in human platelets. Objective: The aim of this study was to investigate the role of SUCNR1 in platelet aggregation and to explore the signaling pathways of this receptor in platelets. Methods and Results: Using real‐time‐PCR, we demonstrated that SUCNR1 is expressed in human platelets at a level corresponding to that of the P2Y1 receptor. Light transmission aggregation experiments showed dose‐dependent aggregation induced by succinate, reaching a maximum response at 0.5 mm. The effect of succinate on platelet aggregation was confirmed with flow cytometry, showing increased surface expression of activated glycoprotein IIb–IIIa and P‐selectin. Intracellular SUCNR1 signaling was found to result in decreased cAMP levels, Akt phosphorylation mediated by phosphoinositide 3‐kinase‐β activation, and receptor desensitization. Furthermore, succinate‐induced platelet aggregation was demonstrated to depend on Src, generation of thromboxane A2, and ATP release. Platelet SUCNR1 is subject to desensitization through both homologous and heterologous mechanisms. In addition, the P2Y12 receptor inhibitor ticagrelor completely prevented platelet aggregation induced by succinate. Conclusions: Our experiments show that succinate induces full aggregation of human platelets via SUCNR1. Succinate‐induced platelet aggregation depends on thromboxane A2 generation, ATP release, and P2Y12 activation.