Andaleb Kholmukhamedov - Academia.edu (original) (raw)

Papers by Andaleb Kholmukhamedov

Research paper thumbnail of Consensus report on markers to distinguish procoagulant platelets from apoptotic platelets: communication from the Scientific and Standardization Committee of the ISTH

Journal of Thrombosis and Haemostasis

Research paper thumbnail of A new fluorescent sensor mitoferrofluor indicates the presence of chelatable iron in polarized and depolarized mitochondria

Journal of Biological Chemistry

Research paper thumbnail of Cytoplasmic Phospholipase A2 Is Essential in GPVI Signaling Initiated Procoagulant Platelet Formation

Blood, Dec 7, 2017

Procoagulant platelets (PP), a subpopulation of highly activated platelets, are essential in hemo... more Procoagulant platelets (PP), a subpopulation of highly activated platelets, are essential in hemostasis by supporting active tenase and prothrombinase complexes on their phosphatidylserine (PSer) externalized surface. When platelets are stimulated with a GPVI agonist, such as convulxin (CVX) or collagen related peptide (CRP), both extracellular calcium entry and mitochondrial permeability transition pore (mPTP) formation are required to mediate the transition to the procoagulant phenotype. Activation of phospholipase A2 (PLA2) is an essential prerequisite for GPVI stimulated thromboxane A2 (TXA2) generation that induces paracrine platelet activation and aggregation. Active PLA2 cleaves the arachidonic acid from the second carbon group of glycerol within the phospholipid membrane. The resultant arachidonic acid, upon downstream enzymatic modification, is modified into TXA2, leukotrienes, and other metabolites. Inhibitors of cyclooxygenases (i.e. aspirin), which prevent arachidonic acid conversion to TXA2 are widely used clinically as antiplatelet agents. Here we investigate the role of PLA2 and its metabolites in inducing GPVI-induced PP formation. Human platelets (106/mL) were activated in the presence of three distinct PLA2 inhibitors: non-specific (PLA2; oleyloxyethyl phosphorylcholine, 1 μM), and specific to inducible (iPLA2; bromoenol lactone, 1 μM) and Ca2+-dependent cytosolic PLA2 (cPLA2; PACOCF3, 1 μM). Examination of platelets in these dilute conditions facilitates both the assessment of autocrine effects of PLA2 and its metabolites and flow cytometry analysis. Platelets were stimulated with different concentrations of GPVI agonists (CRP and CVX), stained (Annexin V for PSer, PAC1 for integrin activation and P-Selectin to assess granule release) and fixed. Additionally pharmacologic inhibitors of the TXA2 receptor (SQ29548, 0.5 μM) and lipoxygenases were utilized. As expected, PP formation in this setting was GPVI signaling and mPTP mediated, as dasatinib (SRC kinase inhibitor, 1 μM) and cyclosporin A (cyclophilin D inhibitor, 5 μM) completely inhibited PP formation. Analysis of platelet activation by flow cytometry demonstrated no effect of PLA2 inhibition on either platelet aggregatory ability or granule release. In contrast, procoagulant platelet formation was significantly decreased in the presence of the non-specific PLA2 inhibitor oleyloxyethyl phosphorylcholine (~59% decrease) and the specific cPLA2 inhibitor PACOCF3 (~51% decrease). Specific iPLA2 inhibition had no effect on any aspect of platelet activation. These results indicate that cPLA2-mediated events are required for autocrine induction of procoagulant platelet formation by GPVI. To begin to interrogate the downstream effectors mediating cPLA29s effects PP formation was examined in the presence of inhibitors of TXA2-mediated platelet activation (SQ29548) and lipoxygenase inhibitors, specifically zileuton, caffeic acid and 8,11,14-eicosatriynoate (all at 1 μM). Inhibition of either of these metabolite-mediated pathways had no effect on GPVI-induced PP formation. In contrast to TXA29s lack of effect on autocrine PP formation, platelet accretion on collagen, presumably mediated by paracrine stimulation of the TXA2 receptor, was decreased by 70% in the presence of SQ29548. These studies identify a novel pathway, mediated through activation of cPLA2, that is essential for GPVI induced PP formation. This cPLA2-mediated pathway is not mediated through the TXA2 receptor, and is distinct from that required for TXA2-mediated paracrine activation of platelet aggregation. Consistent with the findings presented here, previous studies have demonstrated minimal effect of cyclooxygenase inhibition on procoagulant platelet formation. Thus, our studies identify a novel aspirin-insensitive pathway mediating PP formation, and suggest a novel pharmacologic target that could be used for the treatment of procoagulant platelet related pathologic thromboses without affecting proaggregatory phenotype. Disclosures No relevant conflicts of interest to declare.

Research paper thumbnail of Platelet Cyclophilin D-Dependent Events Limit Venous Thrombotic Occlusion and Platelet Accretion

Research paper thumbnail of A New Membrane Potential (ΔΨ)‐Independent Iron Indicator Selectively Detects Mitochondrial Chelatable Iron but Not Calcium in Living Cells

Research paper thumbnail of CD36 Promotes Platelet Phosphatidylserine Exposure through MAP Kinase ERK5

Research paper thumbnail of Platelet reactivity in dyslipidemia: atherothrombotic signaling and therapeutic implications

Reviews in Cardiovascular Medicine, 2021

The risks for adverse thrombotic events, including myocardial infarction, stroke, and deep vein t... more The risks for adverse thrombotic events, including myocardial infarction, stroke, and deep vein thrombosis, are markedly increased in dyslipidemia and other metabolic disorders and are the major cause of death worldwide. Recent evidence points out that increased thrombotic risk in dyslipidemia is mediated by platelets circulating in a pre-activated state. The mechanisms of platelet reactivity in this setting are multifaceted including platelet activation by classic agonist receptor signaling as well as platelet sensitization by pattern recognition receptors. Elevated platelet counts in dyslipidemia due to dysregulation in hematopoiesis also contribute to the overall thrombotic phenotype. Despite recent advancements in antiplatelet and anticoagulation therapies, recurrences of adverse thrombotic events remain to be a large clinical burden. In the light of new knowledge, understanding mechanisms that drive pathologic thrombosis in dyslipidemia, the antithrombotic approach shall be revisited. Here, we discuss potential therapeutic avenues based on the overview of platelet signaling mechanisms that contribute to a prothrombotic phenotype in dyslipidemia.

Research paper thumbnail of A new fluorescent sensor mitoferrofluor indicates the presence of chelatable iron in polarized and depolarized mitochondria

Journal of Biological Chemistry

Research paper thumbnail of Cytoplasmic Phospholipase A2 Is Essential in GPVI Signaling Initiated Procoagulant Platelet Formation

Blood, Dec 7, 2017

Procoagulant platelets (PP), a subpopulation of highly activated platelets, are essential in hemo... more Procoagulant platelets (PP), a subpopulation of highly activated platelets, are essential in hemostasis by supporting active tenase and prothrombinase complexes on their phosphatidylserine (PSer) externalized surface. When platelets are stimulated with a GPVI agonist, such as convulxin (CVX) or collagen related peptide (CRP), both extracellular calcium entry and mitochondrial permeability transition pore (mPTP) formation are required to mediate the transition to the procoagulant phenotype. Activation of phospholipase A2 (PLA2) is an essential prerequisite for GPVI stimulated thromboxane A2 (TXA2) generation that induces paracrine platelet activation and aggregation. Active PLA2 cleaves the arachidonic acid from the second carbon group of glycerol within the phospholipid membrane. The resultant arachidonic acid, upon downstream enzymatic modification, is modified into TXA2, leukotrienes, and other metabolites. Inhibitors of cyclooxygenases (i.e. aspirin), which prevent arachidonic acid conversion to TXA2 are widely used clinically as antiplatelet agents. Here we investigate the role of PLA2 and its metabolites in inducing GPVI-induced PP formation. Human platelets (106/mL) were activated in the presence of three distinct PLA2 inhibitors: non-specific (PLA2; oleyloxyethyl phosphorylcholine, 1 μM), and specific to inducible (iPLA2; bromoenol lactone, 1 μM) and Ca2+-dependent cytosolic PLA2 (cPLA2; PACOCF3, 1 μM). Examination of platelets in these dilute conditions facilitates both the assessment of autocrine effects of PLA2 and its metabolites and flow cytometry analysis. Platelets were stimulated with different concentrations of GPVI agonists (CRP and CVX), stained (Annexin V for PSer, PAC1 for integrin activation and P-Selectin to assess granule release) and fixed. Additionally pharmacologic inhibitors of the TXA2 receptor (SQ29548, 0.5 μM) and lipoxygenases were utilized. As expected, PP formation in this setting was GPVI signaling and mPTP mediated, as dasatinib (SRC kinase inhibitor, 1 μM) and cyclosporin A (cyclophilin D inhibitor, 5 μM) completely inhibited PP formation. Analysis of platelet activation by flow cytometry demonstrated no effect of PLA2 inhibition on either platelet aggregatory ability or granule release. In contrast, procoagulant platelet formation was significantly decreased in the presence of the non-specific PLA2 inhibitor oleyloxyethyl phosphorylcholine (~59% decrease) and the specific cPLA2 inhibitor PACOCF3 (~51% decrease). Specific iPLA2 inhibition had no effect on any aspect of platelet activation. These results indicate that cPLA2-mediated events are required for autocrine induction of procoagulant platelet formation by GPVI. To begin to interrogate the downstream effectors mediating cPLA29s effects PP formation was examined in the presence of inhibitors of TXA2-mediated platelet activation (SQ29548) and lipoxygenase inhibitors, specifically zileuton, caffeic acid and 8,11,14-eicosatriynoate (all at 1 μM). Inhibition of either of these metabolite-mediated pathways had no effect on GPVI-induced PP formation. In contrast to TXA29s lack of effect on autocrine PP formation, platelet accretion on collagen, presumably mediated by paracrine stimulation of the TXA2 receptor, was decreased by 70% in the presence of SQ29548. These studies identify a novel pathway, mediated through activation of cPLA2, that is essential for GPVI induced PP formation. This cPLA2-mediated pathway is not mediated through the TXA2 receptor, and is distinct from that required for TXA2-mediated paracrine activation of platelet aggregation. Consistent with the findings presented here, previous studies have demonstrated minimal effect of cyclooxygenase inhibition on procoagulant platelet formation. Thus, our studies identify a novel aspirin-insensitive pathway mediating PP formation, and suggest a novel pharmacologic target that could be used for the treatment of procoagulant platelet related pathologic thromboses without affecting proaggregatory phenotype. Disclosures No relevant conflicts of interest to declare.

Research paper thumbnail of Procoagulant Platelets

Platelets [Working Title], 2020

There are two well-known subpopulations of activated platelets: proaggregatory and procoagulant. ... more There are two well-known subpopulations of activated platelets: proaggregatory and procoagulant. Procoagulant platelets represent a subpopulation of activated platelets, which are morphologically and functionally distinct from pro-aggregatory ones. Although various names have been used to describe these platelets in the literature (CoaT, CoaTed, highly activated, ballooned, capped, etc.), there is a consensus on their phenotypic features including exposure of high levels of phosphatidylserine (PSer) on the surface; decreased aggregatory and adhesive properties; support of active tenase and prothrombinase complexes; maximal generation by co-stimulation of glycoprotein VI (GPVI) and protease-activated receptors (PAR). In this chapter, morphologic and functional features of procoagulant platelets, as well as the mechanisms of their formation, will be discussed.

Research paper thumbnail of Calcium Independent Events Trigger VWF-Dependent Shear-Induced Procoagulant Platelet Formation

Blood, 2016

Elevated shear stress initiates platelet phosphatidylserine (PSer) externalization. Key intracell... more Elevated shear stress initiates platelet phosphatidylserine (PSer) externalization. Key intracellular mechanisms mediating the initiation of PSer externalization in apoptotic (Bax/Bak) and agonist-stimulated (sustained cytosolic calcium and cyclophilin D-mediated mPTP formation) platelets have been identified. In contrast, little is known about the intracellular processes mediating shear-induced procoagulant platelet (SiPP) formation. To investigate the intracellular mechanisms regulating SiPP formation, either human or murine platelets were sheared on a plate-cone viscometer for 3 min at 10,000 s-1 in various conditions as indicated. Platelets were differentially labeled and evaluated by flow cytometry, and platelets with PSer externalization were defined as SiPPs. In shear conditions engagement of VWF with GPIb-a induces unfolding of the membrane proximal GPIb-a mechanosensing domain. That this triggering mechanism is essential for SiPP formation was supported by the marked inhibi...

Research paper thumbnail of Platelet Cyclophilin D-Dependent Events Limit Venous Thrombotic Occlusion and Platelet Accretion

Research paper thumbnail of A New Membrane Potential (ΔΨ)‐Independent Iron Indicator Selectively Detects Mitochondrial Chelatable Iron but Not Calcium in Living Cells

Research paper thumbnail of CD36 Promotes Platelet Phosphatidylserine Exposure through MAP Kinase ERK5

Research paper thumbnail of Platelet CD36 Promotes ERK5 and Caspase-Dependent Procoagulant Phosphatidylserine Externalization and In Vivo Fibrin Formation in Dyslipidemia

Blood, 2018

Thrombosis during atherosclerotic plaque erosion or rupture is a major cause of death and disabil... more Thrombosis during atherosclerotic plaque erosion or rupture is a major cause of death and disability in individuals with cardiovascular disease. Circulating oxidized lipids are risk factors for this condition and are carried in oxidized low-density lipoprotein particles (oxLDL). CD36 is a pattern recognition receptor that recognizes oxLDL and consequently promotes a prothrombotic platelet phenotype. We previously reported that recognition of oxLDL by CD36 promotes activation of redox sensitive signaling pathways. This phenotype promotes phosphatidylserine (PSer) externalization. However, signaling events downstream of CD36 are unclear. We hypothesize that CD36 signaling through the MAP kinase redox sensor ERK5 promotes a procoagulant phenotype that supports arterial thrombosis in dyslipidemia. Platelets isolated and washed from healthy human donors were stimulated with oxLDL and were stained with fluorophore-conjugated Annexin V to detect exposure of procoagulant PSer. OxLDL-stimula...

Research paper thumbnail of Platelet reactivity in dyslipidemia: atherothrombotic signaling and therapeutic implications

Reviews in Cardiovascular Medicine, 2021

The risks for adverse thrombotic events, including myocardial infarction, stroke, and deep vein t... more The risks for adverse thrombotic events, including myocardial infarction, stroke, and deep vein thrombosis, are markedly increased in dyslipidemia and other metabolic disorders and are the major cause of death worldwide. Recent evidence points out that increased thrombotic risk in dyslipidemia is mediated by platelets circulating in a pre-activated state. The mechanisms of platelet reactivity in this setting are multifaceted including platelet activation by classic agonist receptor signaling as well as platelet sensitization by pattern recognition receptors. Elevated platelet counts in dyslipidemia due to dysregulation in hematopoiesis also contribute to the overall thrombotic phenotype. Despite recent advancements in antiplatelet and anticoagulation therapies, recurrences of adverse thrombotic events remain to be a large clinical burden. In the light of new knowledge, understanding mechanisms that drive pathologic thrombosis in dyslipidemia, the antithrombotic approach shall be revisited. Here, we discuss potential therapeutic avenues based on the overview of platelet signaling mechanisms that contribute to a prothrombotic phenotype in dyslipidemia.

Research paper thumbnail of Procoagulant Platelets

Platelets [Working Title], 2020

There are two well-known subpopulations of activated platelets: proaggregatory and procoagulant. ... more There are two well-known subpopulations of activated platelets: proaggregatory and procoagulant. Procoagulant platelets represent a subpopulation of activated platelets, which are morphologically and functionally distinct from pro-aggregatory ones. Although various names have been used to describe these platelets in the literature (CoaT, CoaTed, highly activated, ballooned, capped, etc.), there is a consensus on their phenotypic features including exposure of high levels of phosphatidylserine (PSer) on the surface; decreased aggregatory and adhesive properties; support of active tenase and prothrombinase complexes; maximal generation by co-stimulation of glycoprotein VI (GPVI) and protease-activated receptors (PAR). In this chapter, morphologic and functional features of procoagulant platelets, as well as the mechanisms of their formation, will be discussed.

Research paper thumbnail of Delayed Wound Healing in Mice with Impaired Procoagulant Platelet Formation

Blood, 2018

Platelets are well recognized as a key player in primary hemostasis and thrombosis, but their rol... more Platelets are well recognized as a key player in primary hemostasis and thrombosis, but their role in the subsequent initiation of tissue repair is less well characterized. Within the hemostatic thrombus, ballooned platelets reminiscent of the procoagulant or necrotic platelet are first observed in the periphery (extravascular and in contact with collagen) and subsequently throughout the platelet plug. The key role of cyclophilin D (CypD) in the regulation of platelet mitochondrial permeability transition pore (mPTP) and procoagulant platelet formation is well established. Furthermore, the cyclophilin inhibitor cyclosporine is known to both block procoagulant platelet formation in vitroand to result in delayed wound healing in patients. While the consequences of CypD's absence have been studied in vivo in models of venous and arterial thrombosis, the effects on wound healing and tissue repair have not been investigated. Here we investigated the role of CypD-dependent events in t...

Research paper thumbnail of Calcium Independent Events Trigger VWF-Dependent Shear-Induced Procoagulant Platelet Formation

Blood, 2016

Elevated shear stress initiates platelet phosphatidylserine (PSer) externalization. Key intracell... more Elevated shear stress initiates platelet phosphatidylserine (PSer) externalization. Key intracellular mechanisms mediating the initiation of PSer externalization in apoptotic (Bax/Bak) and agonist-stimulated (sustained cytosolic calcium and cyclophilin D-mediated mPTP formation) platelets have been identified. In contrast, little is known about the intracellular processes mediating shear-induced procoagulant platelet (SiPP) formation. To investigate the intracellular mechanisms regulating SiPP formation, either human or murine platelets were sheared on a plate-cone viscometer for 3 min at 10,000 s-1 in various conditions as indicated. Platelets were differentially labeled and evaluated by flow cytometry, and platelets with PSer externalization were defined as SiPPs. In shear conditions engagement of VWF with GPIb-a induces unfolding of the membrane proximal GPIb-a mechanosensing domain. That this triggering mechanism is essential for SiPP formation was supported by the marked inhibi...

Research paper thumbnail of Platelet CD36 Promotes ERK5 and Caspase-Dependent Procoagulant Phosphatidylserine Externalization and In Vivo Fibrin Formation in Dyslipidemia

Blood, 2018

Thrombosis during atherosclerotic plaque erosion or rupture is a major cause of death and disabil... more Thrombosis during atherosclerotic plaque erosion or rupture is a major cause of death and disability in individuals with cardiovascular disease. Circulating oxidized lipids are risk factors for this condition and are carried in oxidized low-density lipoprotein particles (oxLDL). CD36 is a pattern recognition receptor that recognizes oxLDL and consequently promotes a prothrombotic platelet phenotype. We previously reported that recognition of oxLDL by CD36 promotes activation of redox sensitive signaling pathways. This phenotype promotes phosphatidylserine (PSer) externalization. However, signaling events downstream of CD36 are unclear. We hypothesize that CD36 signaling through the MAP kinase redox sensor ERK5 promotes a procoagulant phenotype that supports arterial thrombosis in dyslipidemia. Platelets isolated and washed from healthy human donors were stimulated with oxLDL and were stained with fluorophore-conjugated Annexin V to detect exposure of procoagulant PSer. OxLDL-stimula...

Research paper thumbnail of Consensus report on markers to distinguish procoagulant platelets from apoptotic platelets: communication from the Scientific and Standardization Committee of the ISTH

Journal of Thrombosis and Haemostasis

Research paper thumbnail of A new fluorescent sensor mitoferrofluor indicates the presence of chelatable iron in polarized and depolarized mitochondria

Journal of Biological Chemistry

Research paper thumbnail of Cytoplasmic Phospholipase A2 Is Essential in GPVI Signaling Initiated Procoagulant Platelet Formation

Blood, Dec 7, 2017

Procoagulant platelets (PP), a subpopulation of highly activated platelets, are essential in hemo... more Procoagulant platelets (PP), a subpopulation of highly activated platelets, are essential in hemostasis by supporting active tenase and prothrombinase complexes on their phosphatidylserine (PSer) externalized surface. When platelets are stimulated with a GPVI agonist, such as convulxin (CVX) or collagen related peptide (CRP), both extracellular calcium entry and mitochondrial permeability transition pore (mPTP) formation are required to mediate the transition to the procoagulant phenotype. Activation of phospholipase A2 (PLA2) is an essential prerequisite for GPVI stimulated thromboxane A2 (TXA2) generation that induces paracrine platelet activation and aggregation. Active PLA2 cleaves the arachidonic acid from the second carbon group of glycerol within the phospholipid membrane. The resultant arachidonic acid, upon downstream enzymatic modification, is modified into TXA2, leukotrienes, and other metabolites. Inhibitors of cyclooxygenases (i.e. aspirin), which prevent arachidonic acid conversion to TXA2 are widely used clinically as antiplatelet agents. Here we investigate the role of PLA2 and its metabolites in inducing GPVI-induced PP formation. Human platelets (106/mL) were activated in the presence of three distinct PLA2 inhibitors: non-specific (PLA2; oleyloxyethyl phosphorylcholine, 1 μM), and specific to inducible (iPLA2; bromoenol lactone, 1 μM) and Ca2+-dependent cytosolic PLA2 (cPLA2; PACOCF3, 1 μM). Examination of platelets in these dilute conditions facilitates both the assessment of autocrine effects of PLA2 and its metabolites and flow cytometry analysis. Platelets were stimulated with different concentrations of GPVI agonists (CRP and CVX), stained (Annexin V for PSer, PAC1 for integrin activation and P-Selectin to assess granule release) and fixed. Additionally pharmacologic inhibitors of the TXA2 receptor (SQ29548, 0.5 μM) and lipoxygenases were utilized. As expected, PP formation in this setting was GPVI signaling and mPTP mediated, as dasatinib (SRC kinase inhibitor, 1 μM) and cyclosporin A (cyclophilin D inhibitor, 5 μM) completely inhibited PP formation. Analysis of platelet activation by flow cytometry demonstrated no effect of PLA2 inhibition on either platelet aggregatory ability or granule release. In contrast, procoagulant platelet formation was significantly decreased in the presence of the non-specific PLA2 inhibitor oleyloxyethyl phosphorylcholine (~59% decrease) and the specific cPLA2 inhibitor PACOCF3 (~51% decrease). Specific iPLA2 inhibition had no effect on any aspect of platelet activation. These results indicate that cPLA2-mediated events are required for autocrine induction of procoagulant platelet formation by GPVI. To begin to interrogate the downstream effectors mediating cPLA29s effects PP formation was examined in the presence of inhibitors of TXA2-mediated platelet activation (SQ29548) and lipoxygenase inhibitors, specifically zileuton, caffeic acid and 8,11,14-eicosatriynoate (all at 1 μM). Inhibition of either of these metabolite-mediated pathways had no effect on GPVI-induced PP formation. In contrast to TXA29s lack of effect on autocrine PP formation, platelet accretion on collagen, presumably mediated by paracrine stimulation of the TXA2 receptor, was decreased by 70% in the presence of SQ29548. These studies identify a novel pathway, mediated through activation of cPLA2, that is essential for GPVI induced PP formation. This cPLA2-mediated pathway is not mediated through the TXA2 receptor, and is distinct from that required for TXA2-mediated paracrine activation of platelet aggregation. Consistent with the findings presented here, previous studies have demonstrated minimal effect of cyclooxygenase inhibition on procoagulant platelet formation. Thus, our studies identify a novel aspirin-insensitive pathway mediating PP formation, and suggest a novel pharmacologic target that could be used for the treatment of procoagulant platelet related pathologic thromboses without affecting proaggregatory phenotype. Disclosures No relevant conflicts of interest to declare.

Research paper thumbnail of Platelet Cyclophilin D-Dependent Events Limit Venous Thrombotic Occlusion and Platelet Accretion

Research paper thumbnail of A New Membrane Potential (ΔΨ)‐Independent Iron Indicator Selectively Detects Mitochondrial Chelatable Iron but Not Calcium in Living Cells

Research paper thumbnail of CD36 Promotes Platelet Phosphatidylserine Exposure through MAP Kinase ERK5

Research paper thumbnail of Platelet reactivity in dyslipidemia: atherothrombotic signaling and therapeutic implications

Reviews in Cardiovascular Medicine, 2021

The risks for adverse thrombotic events, including myocardial infarction, stroke, and deep vein t... more The risks for adverse thrombotic events, including myocardial infarction, stroke, and deep vein thrombosis, are markedly increased in dyslipidemia and other metabolic disorders and are the major cause of death worldwide. Recent evidence points out that increased thrombotic risk in dyslipidemia is mediated by platelets circulating in a pre-activated state. The mechanisms of platelet reactivity in this setting are multifaceted including platelet activation by classic agonist receptor signaling as well as platelet sensitization by pattern recognition receptors. Elevated platelet counts in dyslipidemia due to dysregulation in hematopoiesis also contribute to the overall thrombotic phenotype. Despite recent advancements in antiplatelet and anticoagulation therapies, recurrences of adverse thrombotic events remain to be a large clinical burden. In the light of new knowledge, understanding mechanisms that drive pathologic thrombosis in dyslipidemia, the antithrombotic approach shall be revisited. Here, we discuss potential therapeutic avenues based on the overview of platelet signaling mechanisms that contribute to a prothrombotic phenotype in dyslipidemia.

Research paper thumbnail of A new fluorescent sensor mitoferrofluor indicates the presence of chelatable iron in polarized and depolarized mitochondria

Journal of Biological Chemistry

Research paper thumbnail of Cytoplasmic Phospholipase A2 Is Essential in GPVI Signaling Initiated Procoagulant Platelet Formation

Blood, Dec 7, 2017

Procoagulant platelets (PP), a subpopulation of highly activated platelets, are essential in hemo... more Procoagulant platelets (PP), a subpopulation of highly activated platelets, are essential in hemostasis by supporting active tenase and prothrombinase complexes on their phosphatidylserine (PSer) externalized surface. When platelets are stimulated with a GPVI agonist, such as convulxin (CVX) or collagen related peptide (CRP), both extracellular calcium entry and mitochondrial permeability transition pore (mPTP) formation are required to mediate the transition to the procoagulant phenotype. Activation of phospholipase A2 (PLA2) is an essential prerequisite for GPVI stimulated thromboxane A2 (TXA2) generation that induces paracrine platelet activation and aggregation. Active PLA2 cleaves the arachidonic acid from the second carbon group of glycerol within the phospholipid membrane. The resultant arachidonic acid, upon downstream enzymatic modification, is modified into TXA2, leukotrienes, and other metabolites. Inhibitors of cyclooxygenases (i.e. aspirin), which prevent arachidonic acid conversion to TXA2 are widely used clinically as antiplatelet agents. Here we investigate the role of PLA2 and its metabolites in inducing GPVI-induced PP formation. Human platelets (106/mL) were activated in the presence of three distinct PLA2 inhibitors: non-specific (PLA2; oleyloxyethyl phosphorylcholine, 1 μM), and specific to inducible (iPLA2; bromoenol lactone, 1 μM) and Ca2+-dependent cytosolic PLA2 (cPLA2; PACOCF3, 1 μM). Examination of platelets in these dilute conditions facilitates both the assessment of autocrine effects of PLA2 and its metabolites and flow cytometry analysis. Platelets were stimulated with different concentrations of GPVI agonists (CRP and CVX), stained (Annexin V for PSer, PAC1 for integrin activation and P-Selectin to assess granule release) and fixed. Additionally pharmacologic inhibitors of the TXA2 receptor (SQ29548, 0.5 μM) and lipoxygenases were utilized. As expected, PP formation in this setting was GPVI signaling and mPTP mediated, as dasatinib (SRC kinase inhibitor, 1 μM) and cyclosporin A (cyclophilin D inhibitor, 5 μM) completely inhibited PP formation. Analysis of platelet activation by flow cytometry demonstrated no effect of PLA2 inhibition on either platelet aggregatory ability or granule release. In contrast, procoagulant platelet formation was significantly decreased in the presence of the non-specific PLA2 inhibitor oleyloxyethyl phosphorylcholine (~59% decrease) and the specific cPLA2 inhibitor PACOCF3 (~51% decrease). Specific iPLA2 inhibition had no effect on any aspect of platelet activation. These results indicate that cPLA2-mediated events are required for autocrine induction of procoagulant platelet formation by GPVI. To begin to interrogate the downstream effectors mediating cPLA29s effects PP formation was examined in the presence of inhibitors of TXA2-mediated platelet activation (SQ29548) and lipoxygenase inhibitors, specifically zileuton, caffeic acid and 8,11,14-eicosatriynoate (all at 1 μM). Inhibition of either of these metabolite-mediated pathways had no effect on GPVI-induced PP formation. In contrast to TXA29s lack of effect on autocrine PP formation, platelet accretion on collagen, presumably mediated by paracrine stimulation of the TXA2 receptor, was decreased by 70% in the presence of SQ29548. These studies identify a novel pathway, mediated through activation of cPLA2, that is essential for GPVI induced PP formation. This cPLA2-mediated pathway is not mediated through the TXA2 receptor, and is distinct from that required for TXA2-mediated paracrine activation of platelet aggregation. Consistent with the findings presented here, previous studies have demonstrated minimal effect of cyclooxygenase inhibition on procoagulant platelet formation. Thus, our studies identify a novel aspirin-insensitive pathway mediating PP formation, and suggest a novel pharmacologic target that could be used for the treatment of procoagulant platelet related pathologic thromboses without affecting proaggregatory phenotype. Disclosures No relevant conflicts of interest to declare.

Research paper thumbnail of Procoagulant Platelets

Platelets [Working Title], 2020

There are two well-known subpopulations of activated platelets: proaggregatory and procoagulant. ... more There are two well-known subpopulations of activated platelets: proaggregatory and procoagulant. Procoagulant platelets represent a subpopulation of activated platelets, which are morphologically and functionally distinct from pro-aggregatory ones. Although various names have been used to describe these platelets in the literature (CoaT, CoaTed, highly activated, ballooned, capped, etc.), there is a consensus on their phenotypic features including exposure of high levels of phosphatidylserine (PSer) on the surface; decreased aggregatory and adhesive properties; support of active tenase and prothrombinase complexes; maximal generation by co-stimulation of glycoprotein VI (GPVI) and protease-activated receptors (PAR). In this chapter, morphologic and functional features of procoagulant platelets, as well as the mechanisms of their formation, will be discussed.

Research paper thumbnail of Calcium Independent Events Trigger VWF-Dependent Shear-Induced Procoagulant Platelet Formation

Blood, 2016

Elevated shear stress initiates platelet phosphatidylserine (PSer) externalization. Key intracell... more Elevated shear stress initiates platelet phosphatidylserine (PSer) externalization. Key intracellular mechanisms mediating the initiation of PSer externalization in apoptotic (Bax/Bak) and agonist-stimulated (sustained cytosolic calcium and cyclophilin D-mediated mPTP formation) platelets have been identified. In contrast, little is known about the intracellular processes mediating shear-induced procoagulant platelet (SiPP) formation. To investigate the intracellular mechanisms regulating SiPP formation, either human or murine platelets were sheared on a plate-cone viscometer for 3 min at 10,000 s-1 in various conditions as indicated. Platelets were differentially labeled and evaluated by flow cytometry, and platelets with PSer externalization were defined as SiPPs. In shear conditions engagement of VWF with GPIb-a induces unfolding of the membrane proximal GPIb-a mechanosensing domain. That this triggering mechanism is essential for SiPP formation was supported by the marked inhibi...

Research paper thumbnail of Platelet Cyclophilin D-Dependent Events Limit Venous Thrombotic Occlusion and Platelet Accretion

Research paper thumbnail of A New Membrane Potential (ΔΨ)‐Independent Iron Indicator Selectively Detects Mitochondrial Chelatable Iron but Not Calcium in Living Cells

Research paper thumbnail of CD36 Promotes Platelet Phosphatidylserine Exposure through MAP Kinase ERK5

Research paper thumbnail of Platelet CD36 Promotes ERK5 and Caspase-Dependent Procoagulant Phosphatidylserine Externalization and In Vivo Fibrin Formation in Dyslipidemia

Blood, 2018

Thrombosis during atherosclerotic plaque erosion or rupture is a major cause of death and disabil... more Thrombosis during atherosclerotic plaque erosion or rupture is a major cause of death and disability in individuals with cardiovascular disease. Circulating oxidized lipids are risk factors for this condition and are carried in oxidized low-density lipoprotein particles (oxLDL). CD36 is a pattern recognition receptor that recognizes oxLDL and consequently promotes a prothrombotic platelet phenotype. We previously reported that recognition of oxLDL by CD36 promotes activation of redox sensitive signaling pathways. This phenotype promotes phosphatidylserine (PSer) externalization. However, signaling events downstream of CD36 are unclear. We hypothesize that CD36 signaling through the MAP kinase redox sensor ERK5 promotes a procoagulant phenotype that supports arterial thrombosis in dyslipidemia. Platelets isolated and washed from healthy human donors were stimulated with oxLDL and were stained with fluorophore-conjugated Annexin V to detect exposure of procoagulant PSer. OxLDL-stimula...

Research paper thumbnail of Platelet reactivity in dyslipidemia: atherothrombotic signaling and therapeutic implications

Reviews in Cardiovascular Medicine, 2021

The risks for adverse thrombotic events, including myocardial infarction, stroke, and deep vein t... more The risks for adverse thrombotic events, including myocardial infarction, stroke, and deep vein thrombosis, are markedly increased in dyslipidemia and other metabolic disorders and are the major cause of death worldwide. Recent evidence points out that increased thrombotic risk in dyslipidemia is mediated by platelets circulating in a pre-activated state. The mechanisms of platelet reactivity in this setting are multifaceted including platelet activation by classic agonist receptor signaling as well as platelet sensitization by pattern recognition receptors. Elevated platelet counts in dyslipidemia due to dysregulation in hematopoiesis also contribute to the overall thrombotic phenotype. Despite recent advancements in antiplatelet and anticoagulation therapies, recurrences of adverse thrombotic events remain to be a large clinical burden. In the light of new knowledge, understanding mechanisms that drive pathologic thrombosis in dyslipidemia, the antithrombotic approach shall be revisited. Here, we discuss potential therapeutic avenues based on the overview of platelet signaling mechanisms that contribute to a prothrombotic phenotype in dyslipidemia.

Research paper thumbnail of Procoagulant Platelets

Platelets [Working Title], 2020

There are two well-known subpopulations of activated platelets: proaggregatory and procoagulant. ... more There are two well-known subpopulations of activated platelets: proaggregatory and procoagulant. Procoagulant platelets represent a subpopulation of activated platelets, which are morphologically and functionally distinct from pro-aggregatory ones. Although various names have been used to describe these platelets in the literature (CoaT, CoaTed, highly activated, ballooned, capped, etc.), there is a consensus on their phenotypic features including exposure of high levels of phosphatidylserine (PSer) on the surface; decreased aggregatory and adhesive properties; support of active tenase and prothrombinase complexes; maximal generation by co-stimulation of glycoprotein VI (GPVI) and protease-activated receptors (PAR). In this chapter, morphologic and functional features of procoagulant platelets, as well as the mechanisms of their formation, will be discussed.

Research paper thumbnail of Delayed Wound Healing in Mice with Impaired Procoagulant Platelet Formation

Blood, 2018

Platelets are well recognized as a key player in primary hemostasis and thrombosis, but their rol... more Platelets are well recognized as a key player in primary hemostasis and thrombosis, but their role in the subsequent initiation of tissue repair is less well characterized. Within the hemostatic thrombus, ballooned platelets reminiscent of the procoagulant or necrotic platelet are first observed in the periphery (extravascular and in contact with collagen) and subsequently throughout the platelet plug. The key role of cyclophilin D (CypD) in the regulation of platelet mitochondrial permeability transition pore (mPTP) and procoagulant platelet formation is well established. Furthermore, the cyclophilin inhibitor cyclosporine is known to both block procoagulant platelet formation in vitroand to result in delayed wound healing in patients. While the consequences of CypD's absence have been studied in vivo in models of venous and arterial thrombosis, the effects on wound healing and tissue repair have not been investigated. Here we investigated the role of CypD-dependent events in t...

Research paper thumbnail of Calcium Independent Events Trigger VWF-Dependent Shear-Induced Procoagulant Platelet Formation

Blood, 2016

Elevated shear stress initiates platelet phosphatidylserine (PSer) externalization. Key intracell... more Elevated shear stress initiates platelet phosphatidylserine (PSer) externalization. Key intracellular mechanisms mediating the initiation of PSer externalization in apoptotic (Bax/Bak) and agonist-stimulated (sustained cytosolic calcium and cyclophilin D-mediated mPTP formation) platelets have been identified. In contrast, little is known about the intracellular processes mediating shear-induced procoagulant platelet (SiPP) formation. To investigate the intracellular mechanisms regulating SiPP formation, either human or murine platelets were sheared on a plate-cone viscometer for 3 min at 10,000 s-1 in various conditions as indicated. Platelets were differentially labeled and evaluated by flow cytometry, and platelets with PSer externalization were defined as SiPPs. In shear conditions engagement of VWF with GPIb-a induces unfolding of the membrane proximal GPIb-a mechanosensing domain. That this triggering mechanism is essential for SiPP formation was supported by the marked inhibi...

Research paper thumbnail of Platelet CD36 Promotes ERK5 and Caspase-Dependent Procoagulant Phosphatidylserine Externalization and In Vivo Fibrin Formation in Dyslipidemia

Blood, 2018

Thrombosis during atherosclerotic plaque erosion or rupture is a major cause of death and disabil... more Thrombosis during atherosclerotic plaque erosion or rupture is a major cause of death and disability in individuals with cardiovascular disease. Circulating oxidized lipids are risk factors for this condition and are carried in oxidized low-density lipoprotein particles (oxLDL). CD36 is a pattern recognition receptor that recognizes oxLDL and consequently promotes a prothrombotic platelet phenotype. We previously reported that recognition of oxLDL by CD36 promotes activation of redox sensitive signaling pathways. This phenotype promotes phosphatidylserine (PSer) externalization. However, signaling events downstream of CD36 are unclear. We hypothesize that CD36 signaling through the MAP kinase redox sensor ERK5 promotes a procoagulant phenotype that supports arterial thrombosis in dyslipidemia. Platelets isolated and washed from healthy human donors were stimulated with oxLDL and were stained with fluorophore-conjugated Annexin V to detect exposure of procoagulant PSer. OxLDL-stimula...