Role of 12-LOX in the Platelet Storage Lesion

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.

Platelet activation and reactivity in a large cohort of patients with Gaucher disease

Patients with Gaucher disease (GD) are at increased risk of bleeding and have varying degrees of thrombocytopenia, making the analysis of platelet function difficult. This study aimed to provide a clinically relevant quantitative assessment of platelet function and determine its relationship with bleeding and GD-related data. Methods: Unstimulated and stimulated platelet function was measured by whole blood flow cytometry of platelet surface activated αIIbβ3 integrin (detected with monoclonal antibody PAC1), P-selectin (CD62P), and lysosomal-associated membrane protein (LAMP3/CD63) in 149 GD patients. Results: GD patients had a higher level of unstimulated CD63 expression than healthy subjects, which was mildly correlated with glucosylsphingosine (lyso-Gb1) levels (r 0.17, p-value 0.042). Splenectomized GD patients had a higher level of unstimulated αIIbβ3 integrin and P-selectin expression. Reduced platelet reactivity (-2 SD of reference range) was found in 79 (53%, 95% CI 44%-61%) patients, of whom 10 (6.7%, 95% CI 3.3%-12%) had more severe platelet dysfunction. In a multivariate model, only lyso-Gb1 levels were associated with the more severe platelet dysfunction. Fifty-four (49%) of 128 adult patients who completed the bleeding tendency questionnaire reported positive bleeding history. In a multivariate logistic model, older age (OR (95% CI), 1.05 (1.01-1.1)) and low P-selectin reactivity (OR (95% CI), 2.03 (1.25-3.35)) were associated with more than one bleeding manifestation. Conclusion: Flow cytometry enables the study of platelet function in thrombocytopenic GD patients. A platelet degranulation defect, but not αIIbβ3 integrin activation defect, is associated with clinical bleeding. In vivo increased CD63 expression may be related to GD-related inflammation.

An alternate covalent form of platelet αIIbβ3 integrin that resides in focal adhesions and has altered function

The αIIbβ3 integrin receptor coordinates platelet adhesion, activation and mechanosensing in thrombosis and haemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in about one in three integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbβ3 is pre-determined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts (BHK) transfected with recombinant integrin. From co-immunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in BHK cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signalling but extended residency time in focal adhesions due to reduced rate of clathrin-mediated integrin internalisation and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein-2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbβ3 integrin receptor is produced in different covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that this alternate integrin form and function is also conserved.

Extract of Seaweed Codium fragile Inhibits Integrin αIIbβ3-Induced Outside-in Signaling and Arterial Thrombosis

Seaweeds are thought to be promising candidates for functional foods and to help prevent thrombotic and related cardiovascular diseases. Codium fragile (Suringer) Hariot has been traditionally used as a culinary ingredient, and it possesses a range of biological activities, including the inhibition of platelet function. However, the mechanism of this inhibition is unclear. The aim of this study was to examine the inhibitory effect of C. fragile in platelet function. The antiplatelet activity of C. fragile on agonist-activated platelet aggregation, granule secretion, calcium mobilization, platelet spreading, and clot retraction was assessed. The phosphorylation of c-Src, Syk, PLCγ2, and several proteins involving in the αIIbβ3 integrin outside-in signaling pathway were also studied in thrombin and CRP-stimulated platelets. The antithrombotic effect was investigated in mice using ferric chloride-induced arterial thrombus formation in vivo. Transection tail bleeding time was used to evaluate whether C. fragile inhibited primary hemostasis. The main components and contents of C. fragile ethanol extract were confirmed by GC-MS analysis. C. fragile significantly impaired agonist-induced platelet aggregation granule secretion, calcium mobilization, platelet spreading, and clot retraction. Biochemical analysis revealed that C. fragile inhibited the agonist-induced activation of c-Src, Syk, and PLCγ2, as well as the phosphorylation of PI3K, AKT, and mitogen-activated protein kinases (MAPKs). The inhibitory effect of C. fragile resulted from an inhibition of platelet αIIbβ3 integrin outside-in signal transduction during cell activation. Oral administration of C. fragile efficiently blocked FeCl3-induced arterial thrombus formation in vivo without prolonging bleeding time. GC-MS analysis revealed that phytol was the main constituent and the total content of isomers was 160.8 mg/kg. Our results demonstrated that C. fragile suppresses not only the inside-out signaling of αIIbβ3 integrin but also outside-in signal transmission. Therefore, C. fragile could be an effective antiplatelet therapeutic candidate.

New Insight into Regulation of αIIbβ3 Integrin Signaling by Filamin A

Filamin (FLN) regulates many cell functions through its scaffolding activity cross-linking cytoskeleton and integrins. FLN was shown to inhibit integrin activity, but the exact mechanism remains unclear. Here, we report on the regulation of platelet integrin αIIbβ3 signaling by the FLNa subtype. Three FLNa deletion mutants were overexpressed in the erythro-megakaryocytic leukemic cell line HEL: Del1 which lacks the N-terminal CH1-CH2 domains mediating the FLNa-actin interaction; Del2 lacking the immunoglobulin-like (Ig) repeat 21 which mediates the FLNa-β3 interaction; and Del3 lacking the C-terminal Ig repeat 24, responsible for FLNa dimerization and interaction with the small Rho GTPase involved in actin cytoskeleton reorganisation. Fibrinogen binding to HEL cells in suspension and talin-β3 interaction in cells adherent to immobilized fibrinogen were assessed using the PKC agonist (phorbol 12-myristate 13-acetate) also to activate αIIbβ3. Our results show that FLNa-actin and FLNa-β3 interactions negatively regulate αIIbβ3 activation. Moreover FLNa-actin interaction represses Rac activation, contributing to the negative regulation of αIIbβ3 activation. In contrast, the FLNa dimerization domain which maintains Rho inactive, was found to negatively regulate αIIbβ3 outside-in signaling. We conclude that FLNa negatively controls αIIbβ3 activation by regulating actin polymerization and restraining activation of Rac, as well as outside-in signaling by repressing Rho.

Genetic predictors of arterial hypertension in young patients with metabolic syndrome

The paper presents the results of studying the polymorphism of the genes rs1799752 of the ACE gene, αIIbβ3 integrin, rs1378942 of the CSK gene in the development of arterial hypertension in young patients with metabolic syndrome. Arterial hypertension as a component of the metabolic syndrome was detected in 15.0% of young patients. The occurrence of mutant alleles of the studied genes among the examined patients is quite high: thus, the carriage of the homozygous DD variant is 21.6%, and the carriage of the mutant D allele of the ACE gene is 47.4%. A high risk of developing hypertension in patients with MS was detected in carriers of the T rs1378942 CSK allele - 54.8%, which was most often observed in a combination of polymorphic loci of the ACE and CSK genes (p = 0.0053).