scholarly journals Role of 12-LOX in the Platelet Storage Lesion

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3241-3241
Author(s):  
Hannah Johnson ◽  
Xiaoyun Fu ◽  
Shawn Lawrence Bailey ◽  
Daire Byrne ◽  
Michael Holinstat ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Function ◽  
Circulation Time ◽  
Integrin Activation ◽  
Targeted Metabolomics ◽  
Storage Lesion ◽  
Platelet Storage Lesion ◽  
Platelet Storage ◽  
Αiibβ3 Integrin

Abstract Background: 12-lipoxygenase (12-LOX) is an enzyme abundant in platelets which can contribute to the platelet storage lesion by oxidizing polyunsaturated fatty acids (PUFAs) released from phospholipid membranes. We and others have shown that the PUFA arachidonic acid (AA) and its lipid oxidation products, such as 12-hydroxyeicosatetraenoic acid (12-HETE), accumulate during storage and have inhibitory effects on platelet recovery, survival, and function. However, several PUFAs are substrates for 12-LOX, and their resulting oxylipins may have different effects. We used targeted metabolomics to quantify PUFAs and oxylipins and platelet function assays to characterize function of fresh and stored wild-type (WT) and 12-LOX -/- platelets. Methods: Blood from WT and 12-LOX -/- mice was collected by retro-orbital bleeding. Platelet-rich plasma (PRP) was generated from whole blood. After fresh samples were aliquoted, the remaining PRP was separated in two groups. One group was stored at room temperature with agitation (RT) for 24 hours, and the other for 48 hours. Metabolites were extracted from samples and quantified by targeted metabolomics as described previously. We assessed platelet function by αIIbβ3 integrin activation by flow cytometry. In vivo recovery of function was measured by transfusing stored platelets into UBiC-GFP mice and stimulating platelets with agonists, followed by gating for transfused (GFP-negative) platelets by flow cytometry. For recovery and survival, we traced biotinylated fresh, 24h, or 48h-stored platelets after transfusion in vivo. Results: We quantified metabolites present in platelets by targeted metabolomics to monitor their changes in concentration over storage time. Among the 10 PUFAs and 28 related oxylipins we analyzed, 15 of 38 analytes showed a significant difference in PRP from WT and 12-LOX-/- mouse samples. The major metabolites of 12-LOX include 12-HETE, 12-hydroxyeicosapentaenoic acid (12-HEPE) and 14-hydroxydocosahexaenoic acid (14-HDHA), from AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). 12-HETE, 12-HEPE, and 14-HDHA were only detected at <8 nmol/L levels in fresh PRP from 12-LOX -/- mice compared to 668 ± 409nM, 149 ± 85nM, and 295 ± 154nM from WT mice, respectively. After 24 hours of storage at RT, 12-HETE, 12-HEPE, and 14-HDHA dramatically increased to 29.0±4.2µM, 3.7±1.1µM, and 6.3±0.8µM in PRP from WT mice, respectively. As expected, these same metabolites remained at low nmol/L levels in 12-LOX-/- samples during storage accompanied by a significant increase of their precursors AA, EPA, and DHA due to lack of 12-LOX activity. Interestingly, there was also a significant reduction in 15-HETE, 17-HDHA, and 13-hydroxyoctadecadienoic acid (13-HODE) in the 12-LOX -/- mice compared to the WT mice, which are primarily produced by the 15-LOX enzyme. Additionally, we observed a significant decrease of metabolites mediated via the cyclooxygenase (COX) pathway in PRP from 12-LOX-/- mice, including prostaglandin E2 (PGE2), PGD2, thromboxane B2, and 12-hydroxyheptadecatrienoic acid (12-HHTrE). Function-wise, fresh 12-LOX -/- platelets were less responsive to agonists compared to WT platelets. Surprisingly, after transfusion of fresh 12-LOX -/- platelets, we found comparable αIIbβ3-integrin activation results after 1, 4, and 24h of circulation time. In contrast, 24h and 48h of storage of 12-LOX -/- platelets led to significantly lower pre-activation at baseline and a significantly lower activation response than WT platelets after 1h and 4h of circulation time. No significant differences were observed after 24h of circulation time. We observed a clear trend for longer survival after 24 and 48h of storage. Conclusions: We found many metabolic changes between 12-LOX -/- and WT mice during storage. While the 12-LOX -/- mouse model highlights the primary metabolic differences that occur without 12-LOX activity, other changes, such as differences in COX or additional LOX isoform activity, may attenuate oxylipin production. Functionally, we observed less pre-activation and better survival in functional studies, but this may be due to a combined effect of each of these individual metabolites. Future studies will have to determine the roles of individual oxylipins. Disclosures Stolla: Cerus: Research Funding.

scholarly journals Platelet Storage Temperature Determines Recovery of GPVI-Function In Vivo

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 39-39
Author(s):  
Jeffrey Miles ◽  
Chomkan Usaneerungrueng ◽  
Ava M. Obenaus ◽  
Molly Y Mollica ◽  
Jake R Flynn ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Storage Temperature ◽  
Recovery Of Function ◽  
Platelet Rich Plasma ◽  
Integrin Activation ◽  
Platelet Storage ◽  
Platelet Aggregates ◽  
Contractile Forces

Background: Platelets (PLTs) are currently stored at 22°C (RT, room temperature) for clinical purposes. This approach ensures long circulation time but has numerous downsides, including limited storage time due to the risk of bacterial growth and increased costs due to bacterial testing or pathogen reduction processing. PLTs stored at 4°C were the standard of care in the 1960s and 1970s. In our previous study with healthy volunteers, we showed that humans who received cold-stored PLTs have a significantly weaker response to collagen (an agonist that acts predominantly via GPVI) compared to RT-stored PLTs. If and how cold-stored PLTs recover their function in vivo is poorly understood. Methods: We obtained human PLTs by an apheresis collection and sampled either at baseline (fresh) or after five days at RT or 4°C. To test the response to GPVI-dependent agonists, we stimulated platelet-rich plasma or washed PLTs with collagen and the GPVI-specific agonist convulxin (CVX) and tested for activated integrin and α-degranulation by flow cytometry. Platelet aggregation, in response to GPVI-dependent agonists, was tested by aggregometry. We checked for GPVI expression levels by flow cytometry and for signaling events downstream of GPVI by immunoblotting. To allow for recovery of function in vitro, we incubated either 4°C-stored, or RT-stored PLTs with fresh, platelet-depleted blood for 15min, and perfused the reconstituted whole blood through a microfluidic block and post device to quantify the contractile forces of platelet aggregates. Additionally, we performed platelet force measurements at the single cell level using a traction force microscopy approach. To validate a murine model of platelet storage and transfusion, we replicated functional studies in vitro by testing mouse PLTs for integrin activation and α-degranulation by flow cytometry. Platelet aggregation in response to collagen, CVX, and the GPVI-specific antibody JAQ-1 with crosslinking anti-IgG was also tested. To evaluate the platelet function after transfusion, we obtained whole blood from UbiC-GFP mice and isolated platelet-rich plasma followed by storage for 24 hours at either 4°C or RT. To allow tracking of stored PLTs in vivo, we transfused the UbiC-GFP PLTs into wild-type C57BL/6J mice and tested for integrin activation of endogenous and transfused PLTs. Results: In human PLTs, we found a significantly increased integrin response in 4°C-stored PLTs stimulated with collagen in flow cytometry studies in vitro. Similarly, the aggregation response of 4°C-stored PLTs to collagen was significantly increased compared to RT-stored PLTs in vitro. In line with these findings, we observed more PLCγ2 phosphorylation and Syk phosphorylation at baseline in 4°C-stored PLTs compared to RT-stored PLTs, suggesting more pre-activation downstream of GPVI. However, no differences in PLCγ2 phosphorylation or Syk-phosphorylation were found between RT and 4°C-stored PLTs after stimulation with CVX, and no significant differences in surface expression levels of GPVI were detected between RT and 4°C. Stored platelets in plasma showed superior function after 4°C-storage in aggregation and flow cytometry assays. In contrast, we found similar contractile forces of platelet aggregates when RT-stored or 4°C-stored PLTs were added to platelet-depleted fresh blood. Additionally, at the single cell level, we found a similar magnitude of platelet forces in RT-stored and 4°C-stored PLTs. Similar to human PLTs, mouse PLTs showed significantly more integrin activation, P-selectin exposure, and aggregation in 4°C-stored PLTs compared to RT. To test the recovery of function of stored mouse platelets in vivo, we transfused GFP-positive PLTs into GFP-negative wild-type mice. Contrary to our pre-transfusion results, we found a significantly lower integrin activation response to CVX in 4°C-stored platelets after transfusion, consistent with our previous results in healthy human volunteers. Summary: The in vivo recovery of function of stored PLTs is an underappreciated phenomenon in platelet storage biology, and most studies are solely based on functional in vitro data. Based on our post-transfusion results, storage temperature affects the ability to recover function in vivo significantly in human and mouse platelets. Whether these differences lead to differences in clinical outcomes needs to be investigated in clinical trials. Disclosures Sniadecki: Stasys Medical Corporation: Current equity holder in private company, Other: Co-founder; Curi Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

scholarly journals RhoA Targeting Suppresses Cold-Induced Platelet Lesion and Allows Refrigerated Storage of Functional Human Platelets to Fourteen Days

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 99-99
Author(s):  
Shailaja N Hegde ◽  
Huzoor Akbar ◽  
Shawnagay Nestheide ◽  
Ashley M Wellendorf ◽  
Breanna Bohan ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Research Funding ◽  
Rho Gtpase ◽  
Shape Change ◽  
Human Platelets ◽  
Refrigerated Storage ◽  
Storage Lesion ◽  
Platelet Storage Lesion ◽  
Platelet Storage

Platelet transfusion is required for the support therapy of patients with hematological disorders and cancer. Current practice of donor-derived platelet storage at room temperature (RT) associates with an inherent risk of microbial contamination and a limit of 5-7 day shelf life. Refrigerated platelets are hemostatically superior than RT platelets but their survival in circulation is severely reduced. Storage in cold temperature induces loss of galactosylation/sialylation of platelet GPIb with exposure of b-N-acetyl glucosamine of N-linked glycans, which clusters on the platelet plasma membrane. Upon transfusion, these clusters are recognized by macrophages and hepatocytes resulting in lectin-mediated platelet clearance. We hypothesized that the long-term refrigerated storage lesion depends on the mislocalization of membrane-bound glycosyl- and syalyl-transferases in lipid rafts and endocytotic intermediates of cold-stored platelets. By using a combination of genetic and pharmacological approaches, we investigated whether the three major members of the Rho GTPase family, RHOA, RAC1 and/or CDC42, which are molecular switches regulating actomyosin dynamics and signaling in platelets. We found that cooling of platelets induces a sustained activation of RhoA and Rac1, but not Cdc42 in mouse and human platelets. Inducible genetic deletion of platelet RhoA in mice prevents clearance of cold-stored platelets upon transfusion in wild-type congenic animals (WT: 2±0.5 hrs x fraction of infusate; RhoAΔ/Δ: 8± 0.7 hrs x fraction of infusate; p≤0.05). When human platelets were stored in either plasma or PAS-IIIM (PAS-E) for up to 14 days, the inclusion in the storage medium of a rationally designed RHOA inhibitor G04 can prevent the phagocytosis of 14-day refrigerated platelets by activated macrophages in vitro (RT-stored, vehicle-treated platelets: 20%±4; cold stored, vehicle-treated 70±5%; cold-treated, G04(10μM)- treated: 25±3%; p≤0.001) or in vivo clearance as assessed by the area under the curve of surviving human platelets (hrs x fraction of infusate) transfused into clodronate-treated, sub-lethally irradiated non-obese diabetic, γc-/- (NSG) mice (RT-stored, vehicle-treated platelets: 16±1.0; cold-stored, vehicle-treated: 6±0.5; cold-stored, G04(10μM)-treated: 15±0.6; p≤0.001). This inhibition is reversible by either a wash or a 3-fold dilution that resulted in a reversion of the inhibitory effect on aggregation and in vivo correction of the bleeding time of mice pre-treated with aspirin (RT-stored, vehicle-treated: 45±4 sec.; cold-stored, vehicle-treated: 150±10 sec.; cold-stored, G04(10μM)-treated: 76±3.8 sec.; p≤0.001). Mechanistically, RHOA inhibition prevents the cold-induced cytoskeleton/shape change and spreading on fibrinogen through the prevention of the formation of lipid rafts enriched in glycosyl- and syalyl-transferase activities and the endocytosis of vacuoles enriched in GPIbα. Addition of the lipid raft disruptor b-cyclodextrin, but not of the RHOA downstream effector ROCK inhibitor Fasudil, phenocopies the effect of G04 on the prevention of cold-induced platelet damage. Thus, RHOA is the key mediator of the platelet storage lesion through a ROCK independent mechanism and its reversible inhibition allows the functional maintenance of cold-stored platelets in both survival and hemostatic properties. Our pre-clinical data support the concept that a platelet additive solution containing a low-affinity RHOA inhibitor is useful in preventing platelet storage lesion while fully maintaining their hemostatic function after 14 days of storage. A rationally designed cold storage regimen is highly feasible, which could resolve the platelet clearance problem and meet an urgent need in transfusion medicine for the support therapy of patients with thrombocytopenia or thrombocytopathy. Disclosures Cancelas: Cellphire: Research Funding; Velico: Consultancy, Research Funding; Hemanext: Consultancy, Research Funding; Fresenius-Kabi: Research Funding; Cerus Co.: Research Funding; TerumoBCT: Consultancy, Research Funding; Macopharma Inc: Research Funding; Cytosorbents: Research Funding.

p38 MAPK Inhibition Prevents TACE-Mediated Receptor Shedding and Improves the Hemostatic Function of Stored Platelets.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 990-990
Author(s):  
Matthias Canault ◽  
Daniel Duerschmied ◽  
Alexander Brill ◽  
Ian Sean Patten ◽  
Wolfgang Bergmeier ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Human Platelets ◽  
P38 Map Kinase ◽  
Storage Lesion ◽  
Mitochondrial Injury ◽  
Platelet Storage Lesion ◽  
Platelet Storage ◽  
Metalloproteinase Inhibitors ◽  
Receptor Shedding

Abstract Platelet transfusion is a lifesaving procedure widely used during surgery and chemotherapy. During storage platelets undergo several modifications that reduce their postransfusion survival and functionality. One important feature of this platelet storage lesion (PSL) is the shedding of surface glycoproteins such as GPIbα and GPV. We have recently demonstaretd that metalloproteinase inhibitors prevent storage-induced shedding of adhesion receptors, resulting in markedly improved postransfusion recovery and hemostatic function of platelets in mice. We now demonstrate that TNF-alpha converting enzyme (TACE/ADAM17) mediates receptor shedding from platelet stored for 18 hours at 37ºC or 22ºC. Using pharmacological inhibitors, we show that TACE -dependent shedding of GPIbα and GPV from both mouse and human platelets during storage required p38 MAP kinase signaling. In contrast, protein kinase C, Erk MAPK, and caspases were not involved. Inhibition of p38 MAPK during the storage of mouse platelets also markedly improved their posttransfusion recovery and hemostatic function in vivo. Moreover, p38 MAPK inhibition during storage of human platelets improved their adhesion to collagen under flow. Inhibition of p38 MAPK also prevented TACE-dependent shedding of GPIbα from platelets undergoing mitochondrial injury, a model for PSL. Phosphorylation of p38 MAPK was observed after platelet storage and mitochondrial injury. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

The Platelet Storage Lesion

2010 ◽  
Vol 30 (2) ◽  
pp. 475-487 ◽  
Author(s):  
Dana V. Devine ◽  
Katherine Serrano
Keyword(s):  
Storage Lesion ◽  
Platelet Storage Lesion ◽  
Platelet Storage

Effect of recombinant human megakaryocyte growth and development factor coupled with polyethylene glycol on the platelet storage lesion

Transfusion ◽  
1999 ◽  
Vol 39 (3) ◽  
pp. 258-264 ◽  
Author(s):  
Edward L. Snyder ◽  
Peter Perrotta ◽  
Harvey Rinder ◽  
Laurene Baril ◽  
Janet Nichol ◽  
...  
Keyword(s):  
Polyethylene Glycol ◽  
Growth And Development ◽  
Storage Lesion ◽  
Platelet Storage Lesion ◽  
Platelet Storage ◽  
Development Factor

scholarly journals Influence of γ-Secretase Inhibitor 24-Diamino-5-Phenylthiazole DAPT on Platelet Activation

2016 ◽  
Vol 38 (2) ◽  
pp. 726-736 ◽  
Author(s):  
Guoxing Liu ◽  
Guilai Liu ◽  
Madhumita Chatterjee ◽  
Anja T. Umbach ◽  
Hong Chen ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Blood Platelets ◽  
Annexin V ◽  
Negative Regulator ◽  
Ros Generation ◽  
Integrin Activation ◽  
Forward Scatter ◽  
Secretase Inhibitor ◽  
Αiibβ3 Integrin

Background/Aims: DAPT (24-diamino-5-phenylthiazole) inhibits γ-secretase, which cleaves the signaling molecule CD44, a negative regulator of platelet activation and apoptosis. CD44 is a co-receptor for macrophage migration inhibitory factor (MIF) an anti-apoptotic pro-inflammatory cytokine expressed and released from blood platelets. Whether DAPT influences platelet function, remained, however, elusive. Activators of platelets include collagen related peptide (CRP). The present study thus explored whether DAPT modifies the stimulating effect of CRP on platelet function. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to DAPT (10 µM). Flow cytometry was employed to estimate Orai1 abundance with specific antibodies, cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, generation of reactive oxygen species (ROS) from DCFDA fluorescence, mitochondrial transmembrane potential from TMRE fluorescence, phospholipid scrambling of the cell membrane from annexin-V-binding, relative platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: Exposure of platelets to 2-5 µg/ml CRP was followed by significant increase of Orai1 abundance, [Ca2+]i, and P-selectin abundance, as well as by αIIbβ3 integrin activation, ROS generation, mitochondrial depolarization, enhanced annexin-V-binding, decreased cell volume, and aggregation. All CRP induced effects were significantly blunted in the presence of DAPT. Conclusions: The γ-secretase inhibitor DAPT counteracts agonist induced platelet activation, apoptosis and aggregation.

Platelets: Testing, Dosing and the Storage Lesion—Recent Advances

Hematology ◽  
2006 ◽  
Vol 2006 (1) ◽  
pp. 492-496 ◽  
Author(s):  
Richard M. Kaufman
Keyword(s):  
Ex Vivo ◽  
Storage Lesion ◽  
New Approaches ◽  
Platelet Storage Lesion ◽  
Platelet Storage ◽  
Recent Advances ◽  
The Future ◽  
Limited Supply ◽  
And Function ◽  
Platelet Transfusions

Abstract The demand for platelet transfusions continues to grow. Several complementary approaches that may help meet this demand in the future are reviewed. First, platelet bacterial testing is beginning to allow the extension of platelet storage beyond 5 days. Studies are also underway aimed at better preserving viability and function during ex vivo platelet storage: additive solutions and other approaches are being developed to try to negate the “platelet storage lesion.” Finally, new approaches to dosing platelets may help extend the limited supply.

Sign in / Sign up

Export Citation Format

Share Document