Platelets were stimulated with collagen or convulxin (gift of Ken Clemetson) in the absence of fibrinogen under stirring conditions in a Model 700 Lumi-aggregometer for up to 5 minutes

Platelets were stimulated with collagen or convulxin (gift of Ken Clemetson) in the absence of fibrinogen under stirring conditions in a Model 700 Lumi-aggregometer for up to 5 minutes. activation in vitro and in vivo. Human platelets treated with the AADACL1 inhibitor JW480 or the AADACL1 substrate 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG) exhibited decreased platelet aggregation, granule secretion, Ca2+ flux, and PKC phosphorylation. Decreased aggregation and secretion were rescued by exogenous adenosine 5-diphosphate, indicating that AADACL1 likely functions to induce dense granule secretion. Experiments with P2Y12?/? and CalDAG GEFI?/? mice revealed that the P2Y12 PLX-4720 pathway is the predominate target of HAG-mediated inhibition of platelet aggregation. HAG itself displayed weak agonist properties and likely mediates its inhibitory effects via conversion to a phosphorylated metabolite, HAGP, which directly interacted with the C1a domains of 2 distinct PKC isoforms and blocked PKC kinase activity in vitroFinally, AADACL1 inhibition in rats reduced platelet aggregation, protected against FeCl3-induced arterial thrombosis, and delayed tail bleeding time. In summary, our data support a model whereby AADACL1 inhibition shifts the platelet ether lipidome to an inhibitory axis of HAGP accumulation that impairs PKC activation, granule secretion, and recruitment of platelets to sites of vascular damage. Visual Abstract Open in a separate window Introduction Platelets respond rapidly to many physiological and pathological stressors, including arterial injury, inflammation, atherosclerotic plaque rupture, and tumor growth. Activated platelets form homotypic (platelet-platelet) and heterotypic (platelet-leukocyte) aggregates that adhere to sites of vascular damage to prevent blood loss in response to physiological cues (hemostasis) or in response to pathological stimuli (thrombosis). For both of these processes, the platelets most abundant surface receptor, the IIb3 integrin, is converted to an active conformation that facilitates intracellular signaling, fibrinogen binding, and secretion of bioactive molecules (eg, adenosine 5-diphosphate [ADP], growth factors, and cytokines) from intracellular granules that amplify initial signals and recruit additional platelets to the site of injury. Platelet granule secretion amplifies activation through intracellular molecules, including Rap GTPases, and protein kinases, such as protein kinase C (PKC) isoforms, which are triggered downstream of phospholipase C, via the phospholipase C products diacylglycerol (DAG) and inositol 1,4,5 triphosphate. DAG binds directly to several PKCs, whereas inositol 1,4,5 triphosphate induces intracellular Ca2+ launch1 to help activate calcium-sensitive PKCs and additional molecules. Human being platelets communicate 3 PKC subfamilies that play nonredundant and antagonistic tasks in secretion: standard isoforms (PKC, PKC, and PKCII), novel isoforms (PKC, PKC, and PKC), and atypical isoforms (PKC and PKC).2 Mouse platelets lacking PKC fail to secrete or dense granule material.3 Moreover, small molecule PKC inhibitors suppress platelet secretion, which is consistent with genetic data showing a positive part for PKC in regulating secretion from FAE both and dense granules, which contain proteins or small molecules (eg, ADP), respectively.4 Interestingly, PKC has been implicated as both a positive and a negative regulator of platelet secretion, depending on which agonist receptor is activated,5,6 but how its precise function is integrated with other PKCs is unresolved. Rules of PKC isoforms is definitely a multistep process including lipid and/or calcium signaling. Conventional PKC activation requires DAG binding to tandem C1a and C1b domains in the N-terminus and Ca2+ binding to the C2 website to relieve autoinhibition.7,8 PKCs will also be regulated by ether lipids, such as 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG), which was originally discovered like a precursor to the vasoactive agonist, platelet activating element. HAG is more stable than DAG,9 can reportedly inhibit or activate PKC kinase activity in vitro,9-14 and may block PKC translocation to intracellular membranes, most likely via competition with DAG.15,16 Direct HAG binding to PKC C1 domains has been inferred, but unlike DAG or other PKC activators, HAG alone does not increase PKC activity, which suggests a distinct regulatory mechanism.17,18 To identify unique molecular events that regulate human platelet activation, we previously found out a HAG hydrolase, arylacetamide.Bleeding time was recorded as the time at which blood flow stopped and did not continue for 30 mere seconds. activation in vitro and in vivo. Human being platelets treated with the AADACL1 inhibitor JW480 or the AADACL1 substrate 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG) exhibited decreased platelet aggregation, granule secretion, Ca2+ flux, and PKC phosphorylation. Decreased aggregation and secretion were rescued by exogenous adenosine 5-diphosphate, indicating that AADACL1 likely functions to induce dense granule secretion. Experiments with P2Y12?/? and CalDAG GEFI?/? mice exposed the P2Y12 pathway is the predominate target of HAG-mediated inhibition of platelet aggregation. HAG itself displayed fragile agonist properties and likely mediates its inhibitory effects via conversion to a phosphorylated metabolite, HAGP, which directly interacted with the C1a domains of 2 unique PKC isoforms and clogged PKC kinase activity in vitroFinally, AADACL1 inhibition in rats reduced platelet aggregation, safeguarded against FeCl3-induced arterial thrombosis, and delayed tail bleeding time. In summary, our data support a model whereby AADACL1 inhibition shifts the platelet ether lipidome to an inhibitory axis of HAGP build up that impairs PKC activation, granule secretion, and recruitment of platelets to sites of vascular damage. Visual Abstract Open in a separate window Intro Platelets respond rapidly to many physiological and pathological stressors, including arterial injury, swelling, atherosclerotic plaque rupture, and tumor growth. Activated platelets form homotypic (platelet-platelet) and heterotypic (platelet-leukocyte) aggregates that abide by sites of vascular damage to prevent blood loss in response to physiological cues (hemostasis) or in response to pathological stimuli (thrombosis). For both of these processes, the platelets most abundant surface receptor, the IIb3 integrin, is definitely converted to an active conformation that facilitates intracellular signaling, fibrinogen binding, and secretion of bioactive molecules (eg, adenosine 5-diphosphate [ADP], growth factors, and cytokines) from intracellular granules that amplify initial signals and recruit additional platelets to the site of injury. Platelet granule secretion amplifies activation through intracellular molecules, including Rap GTPases, and protein kinases, such as protein kinase C (PKC) isoforms, which are triggered downstream of phospholipase C, via the phospholipase C products diacylglycerol (DAG) and inositol 1,4,5 triphosphate. DAG binds directly to several PKCs, whereas inositol 1,4,5 triphosphate induces intracellular Ca2+ launch1 to help activate calcium-sensitive PKCs and additional molecules. Human being platelets communicate 3 PKC subfamilies that play nonredundant and antagonistic tasks in secretion: standard isoforms (PKC, PKC, and PKCII), novel isoforms (PKC, PKC, and PKC), and atypical isoforms (PKC and PKC).2 Mouse platelets lacking PKC fail to secrete or dense granule material.3 Moreover, small molecule PKC inhibitors suppress platelet secretion, which is consistent with genetic data showing a positive part for PKC in regulating secretion from both and dense granules, which contain proteins or small molecules (eg, ADP), respectively.4 Interestingly, PKC has been implicated as both a positive and a negative regulator of platelet secretion, depending on which agonist receptor is activated,5,6 but how its precise function is integrated with other PKCs is unresolved. Rules of PKC isoforms is definitely a multistep process including lipid and/or calcium signaling. Conventional PKC activation requires DAG binding to tandem C1a and C1b domains in the N-terminus and Ca2+ binding to the C2 website to relieve autoinhibition.7,8 PKCs will also be regulated by ether lipids, such as 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG), which was originally discovered as a precursor to the vasoactive agonist, platelet activating factor. HAG is more stable than DAG,9 can reportedly inhibit or activate PKC kinase activity in vitro,9-14 and can block PKC translocation to intracellular membranes, most likely via competition with DAG.15,16 Direct HAG binding to PKC C1 domains has been inferred, but unlike DAG or other PKC activators, HAG alone does not increase PKC activity, which suggests a distinct regulatory mechanism.17,18 To identify unique molecular events that regulate human platelet activation, we previously discovered a HAG hydrolase, arylacetamide deacetylase-like 1 (AADACL1/NCEH1), via competitive activityCbased protein profiling.19-21 We implicated AADACL1 via its lipid substrate, HAG, as an important regulator of human platelet aggregation and thrombus formation ex vivo, but how.Integrated peak areas were utilized for relative quantitation, and HAGP deacetylation (% lipid hydrolysis) was calculated as product/(product + substrate) 100 (**< .001 for AADACL1 vs control for HAG; n = 3). accumulation of ether lipids that impact PKC signaling networks crucial for platelet activation in vitro and in vivo. Human platelets treated with the AADACL1 inhibitor JW480 or the AADACL1 substrate 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG) exhibited decreased platelet aggregation, granule secretion, Ca2+ flux, and PKC phosphorylation. Decreased aggregation and secretion were rescued by exogenous adenosine 5-diphosphate, indicating that AADACL1 likely functions to induce dense granule secretion. Experiments with P2Y12?/? and CalDAG GEFI?/? mice revealed that this P2Y12 pathway is the predominate target of HAG-mediated inhibition of platelet aggregation. HAG itself displayed poor agonist properties and likely mediates its inhibitory effects via conversion to a phosphorylated metabolite, HAGP, which directly interacted with the C1a domains of 2 unique PKC isoforms and blocked PKC kinase activity in vitroFinally, AADACL1 inhibition in rats reduced platelet aggregation, guarded against FeCl3-induced arterial thrombosis, and delayed tail bleeding time. In summary, our data support a model whereby AADACL1 inhibition shifts the platelet ether lipidome to an inhibitory axis of HAGP accumulation that impairs PKC activation, granule secretion, and recruitment of platelets to sites of vascular damage. Visual Abstract Open in a separate window Introduction Platelets respond rapidly to many physiological and pathological stressors, including arterial injury, inflammation, atherosclerotic plaque rupture, and tumor growth. Activated platelets form homotypic (platelet-platelet) and heterotypic (platelet-leukocyte) aggregates that adhere to sites of vascular damage to prevent blood loss in response to physiological cues (hemostasis) or in response to pathological stimuli (thrombosis). For both of these processes, the platelets most abundant surface receptor, the IIb3 integrin, is usually converted to an active conformation that facilitates intracellular signaling, fibrinogen binding, and secretion of bioactive molecules (eg, adenosine 5-diphosphate [ADP], growth factors, and cytokines) from intracellular granules that amplify initial signals and recruit additional platelets to the site of injury. Platelet granule secretion amplifies activation through intracellular molecules, including Rap GTPases, and protein kinases, such as protein kinase C (PKC) isoforms, which are activated downstream of phospholipase C, via the phospholipase C products diacylglycerol (DAG) and inositol 1,4,5 triphosphate. DAG binds directly to several PKCs, whereas inositol 1,4,5 triphosphate induces intracellular Ca2+ release1 to help activate calcium-sensitive PKCs and other molecules. Human platelets express 3 PKC subfamilies that play nonredundant and antagonistic functions in secretion: standard isoforms (PKC, PKC, and PKCII), novel isoforms (PKC, PKC, and PKC), and atypical isoforms (PKC and PKC).2 Mouse platelets lacking PKC fail to secrete or dense granule contents.3 Moreover, small molecule PKC inhibitors suppress platelet secretion, which is consistent with genetic data showing a positive role for PKC in regulating secretion from both and dense granules, which contain proteins or small molecules (eg, ADP), respectively.4 Interestingly, PKC has been implicated as both a positive and a negative regulator of platelet secretion, depending on which agonist receptor is activated,5,6 but how its precise function is integrated with other PKCs is unresolved. Regulation of PKC isoforms is usually a multistep process including lipid and/or calcium signaling. Conventional PKC activation requires DAG binding to tandem C1a and C1b domains in the N-terminus and Ca2+ binding to the C2 domain name to relieve autoinhibition.7,8 PKCs are also regulated by ether lipids, such as 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG), which was originally discovered as a precursor to the vasoactive agonist, platelet activating factor. HAG is more stable than DAG,9 can reportedly inhibit or activate PKC kinase activity in vitro,9-14 and can block PKC translocation to intracellular membranes, most likely via competition with DAG.15,16 Direct HAG binding to PKC C1 domains has been inferred, but unlike DAG or other PKC activators, HAG alone does not increase PKC activity, which suggests a distinct regulatory mechanism.17,18 To identify unique molecular events that regulate human platelet activation, we previously discovered a HAG hydrolase, arylacetamide deacetylase-like 1 (AADACL1/NCEH1), via competitive activityCbased protein profiling.19-21 We implicated AADACL1 via its lipid substrate, HAG, as an important regulator of human platelet aggregation and thrombus formation ex vivo, but how AADACL1 regulates these crucial platelet functions or how AADACL1 contributes to in vivo physiology was unknown. Here, PLX-4720 we provide compelling evidence that this AADACL1 substrate HAG is usually converted to a phosphorylated species 1-O-hexadecyl-2-acetyl-sn-glycerol-3-phosphate (HAGP) over time and that HAGP negatively regulates platelet granule secretion via direct inhibition of PKC isoforms. We also show that HAG itself has agonist properties. Moreover, irreversible AADACL1 inhibition protects against rat arterial thrombosis and delays hemostasis in rats, both of which require platelet activation. Collectively, these data reveal a novel platelet lipid signaling node that regulates PKC signaling networks critical for platelet aggregation, secretion, and clot formation in vivo. Methods PLX-4720 Human and rodent platelet isolation Resting human platelets were isolated by gel filtration or centrifugation as explained previously20 and in the supplemental Data. Mouse and rat platelets were isolated as explained in Stefanini et al22 and in the supplemental.(G) Platelets were treated with the indicated concentrations of HAG, and aggregation was observed for 4 short minutes postaddition of HAG without collagen (n = 3). GEFI?/? mice exposed how the P2Y12 pathway may be the predominate focus on of HAG-mediated inhibition of platelet aggregation. HAG itself shown weakened agonist properties and most likely mediates its inhibitory results via transformation to a phosphorylated metabolite, HAGP, which straight interacted using the C1a domains of 2 specific PKC isoforms and clogged PKC kinase activity in vitroFinally, AADACL1 inhibition in rats decreased platelet aggregation, shielded against FeCl3-induced arterial thrombosis, and postponed tail bleeding period. In conclusion, our data support a model whereby AADACL1 inhibition shifts the platelet ether lipidome for an inhibitory axis of HAGP build up that impairs PKC activation, granule secretion, and recruitment of platelets to sites of vascular harm. Visual Abstract Open up in another window Intro Platelets respond quickly to numerous physiological and pathological stressors, including arterial damage, swelling, atherosclerotic plaque rupture, and tumor development. Activated platelets type homotypic (platelet-platelet) and heterotypic (platelet-leukocyte) aggregates that abide by sites of vascular harm to prevent loss of blood in response to physiological cues (hemostasis) or in response to pathological stimuli (thrombosis). For both these procedures, the platelets most abundant surface area receptor, the IIb3 integrin, can be converted to a dynamic conformation that facilitates intracellular signaling, fibrinogen binding, and secretion of bioactive substances (eg, adenosine 5-diphosphate [ADP], development elements, and cytokines) from intracellular granules that amplify preliminary indicators and recruit extra platelets to the website of damage. Platelet granule secretion amplifies activation through intracellular substances, including Rap GTPases, and proteins kinases, such as for example proteins kinase C (PKC) isoforms, that are triggered downstream of phospholipase C, via the phospholipase C items diacylglycerol (DAG) and inositol 1,4,5 triphosphate. DAG binds right to many PKCs, whereas inositol 1,4,5 triphosphate induces intracellular Ca2+ launch1 to greatly help activate calcium-sensitive PKCs and additional molecules. Human being platelets communicate 3 PKC subfamilies that play non-redundant and antagonistic jobs in secretion: regular isoforms (PKC, PKC, and PKCII), book isoforms (PKC, PKC, and PKC), and atypical isoforms (PKC and PKC).2 Mouse platelets lacking PKC neglect to secrete or thick granule material.3 Moreover, little molecule PKC inhibitors suppress platelet secretion, which is in keeping with hereditary data showing an optimistic part for PKC in regulating secretion from both and thick granules, that have proteins or little substances (eg, ADP), respectively.4 Interestingly, PKC continues to be implicated as both an optimistic and a poor regulator of platelet secretion, based on which agonist receptor is activated,5,6 but how its precise function is integrated with other PKCs is unresolved. Rules of PKC isoforms can be a multistep procedure concerning lipid and/or calcium mineral signaling. Conventional PKC activation needs DAG binding to tandem C1a and C1b domains in the N-terminus and Ca2+ binding towards the C2 site to alleviate autoinhibition.7,8 PKCs will also be regulated by ether lipids, such as for example 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG), that was originally discovered like a precursor towards the vasoactive agonist, platelet activating element. HAG is even more steady than DAG,9 can apparently inhibit or activate PKC kinase activity in vitro,9-14 and may stop PKC translocation to intracellular membranes, probably via competition with DAG.15,16 Direct HAG binding to PKC C1 domains continues to be inferred, but unlike DAG or other PKC activators, HAG alone will not increase PKC activity, which implies a definite regulatory mechanism.17,18 To recognize unique molecular events that control human platelet activation, we previously found out a HAG hydrolase, arylacetamide deacetylase-like 1 (AADACL1/NCEH1), via competitive activityCbased protein profiling.19-21 We implicated AADACL1 via its lipid substrate, HAG,.In keeping with our earlier outcomes with isolated human being platelets,20 JW480 treatment dose-dependently inhibited aggregation of purified rat platelets (supplemental Shape 5B-C). by exogenous adenosine 5-diphosphate, indicating that AADACL1 most likely features to induce thick granule secretion. Tests with P2Y12?/? and CalDAG GEFI?/? mice exposed how the P2Y12 pathway may be the predominate focus on of HAG-mediated inhibition of platelet aggregation. HAG itself shown weakened agonist properties and most likely mediates its inhibitory results via transformation to a phosphorylated metabolite, HAGP, which straight interacted using the C1a domains of 2 specific PKC isoforms and clogged PKC kinase activity in vitroFinally, AADACL1 inhibition in rats decreased platelet aggregation, shielded against FeCl3-induced arterial thrombosis, and postponed tail bleeding period. In conclusion, our data support a model whereby AADACL1 inhibition shifts the platelet ether lipidome for an inhibitory axis of HAGP build up that impairs PKC activation, granule secretion, and recruitment of platelets to sites of vascular harm. Visual Abstract Open up in another window Intro Platelets respond quickly to numerous physiological and pathological stressors, including arterial damage, swelling, atherosclerotic plaque rupture, and tumor development. Activated platelets type homotypic (platelet-platelet) and heterotypic (platelet-leukocyte) aggregates that abide by sites of vascular harm to prevent loss of blood in response to physiological cues (hemostasis) or in response to pathological stimuli (thrombosis). For both these procedures, the platelets most abundant surface PLX-4720 area receptor, the IIb3 integrin, can be converted to a dynamic conformation that facilitates intracellular signaling, fibrinogen binding, and secretion of bioactive substances (eg, adenosine 5-diphosphate [ADP], development elements, and cytokines) from intracellular granules that amplify preliminary indicators and recruit extra platelets to the website of damage. Platelet granule secretion amplifies activation through intracellular molecules, including Rap GTPases, and protein kinases, such as protein kinase C (PKC) isoforms, which are activated downstream of phospholipase C, via the phospholipase C products diacylglycerol (DAG) and inositol 1,4,5 triphosphate. DAG binds directly to several PKCs, whereas inositol 1,4,5 triphosphate induces intracellular Ca2+ release1 to help activate calcium-sensitive PKCs and other molecules. Human platelets express 3 PKC subfamilies that play nonredundant and antagonistic roles in secretion: conventional isoforms (PKC, PKC, and PKCII), novel isoforms (PKC, PKC, and PKC), and atypical isoforms (PKC and PKC).2 Mouse platelets lacking PKC fail to secrete or dense granule contents.3 Moreover, small molecule PKC inhibitors suppress platelet secretion, which is consistent with genetic data showing a positive role for PKC in regulating secretion from both and dense granules, which contain proteins or small molecules (eg, ADP), respectively.4 Interestingly, PKC has been implicated as both a positive and a negative regulator of platelet secretion, depending on which agonist receptor is activated,5,6 but how its precise function is integrated with other PKCs is unresolved. Regulation of PKC isoforms is a multistep process involving lipid and/or calcium signaling. Conventional PKC activation requires DAG binding to tandem C1a and C1b domains in the N-terminus and Ca2+ binding to the C2 domain to relieve autoinhibition.7,8 PKCs are also regulated by ether lipids, such as 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG), which was originally discovered as a precursor to the vasoactive agonist, platelet activating factor. HAG is more stable than DAG,9 can reportedly inhibit or activate PKC kinase activity in vitro,9-14 and can block PKC translocation to intracellular membranes, most likely via competition with DAG.15,16 Direct HAG binding to PKC C1 domains has been inferred, but unlike DAG or other PKC activators, HAG alone does not increase PKC activity, which suggests a distinct regulatory mechanism.17,18 To identify unique molecular events that regulate human platelet activation, we previously discovered a HAG hydrolase, arylacetamide deacetylase-like 1 (AADACL1/NCEH1), via competitive activityCbased protein profiling.19-21 We implicated AADACL1 via its lipid substrate, HAG, as an important regulator of human platelet aggregation and thrombus formation ex vivo, but how AADACL1 regulates these crucial platelet functions or how AADACL1 contributes to in vivo physiology was unknown. Here, we provide compelling evidence that the AADACL1 substrate HAG is converted to a phosphorylated species 1-O-hexadecyl-2-acetyl-sn-glycerol-3-phosphate (HAGP) over time and that HAGP negatively regulates platelet granule secretion via direct inhibition of PKC isoforms. We also show that HAG itself has agonist properties. Moreover, irreversible AADACL1 inhibition protects against rat arterial thrombosis and delays hemostasis in rats, both of which require platelet.