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.

Targeted Proteomics Reveals That the Dominant Mechanism of Gpibα Loss during Platelet Storage Depends on Temperature of Storage

Background: Platelet transfusion is a potentially lifesaving procedure, used for both prophylactic and therapeutic indications. Platelets can be stored at room temperature (RT) for up to 7 days in air-permeable bags. Platelet function diminishes during storage, a phenomenon known as the storage lesion. We and others have shown that platelets can be stored for extended periods of time at 4°C and still show acceptable in vitro function while limiting bacterial growth. In the present study, we used proteomics to examine the changes in human platelets stored at RT and 4°C with a focus on the glycoprotein (GP) Ib-IX-V complex, the key receptor for platelet adhesion at sites of vessel injury. Study Design/Method: Platelet units from healthy donors were stored in 100% plasma with or without agitation (at 22°C or 4°C, respectively) at a concentration of 3x1011/L and sampled on days 0, 3, 7, and 14. Microparticles were detected by flow cytometry as described previously. For proteomic analysis, platelets were washed and digested with trypsin. Tryptic peptides were analyzed by nanoflow liquid chromatography electrospray ionization tandem mass spectrometry (nano LC-MS/MS). MS/MS spectra were searched against the human protein database using Proteome Discoverer 2.4 software. A student t-test test was used to determine significant differences in analytes amongst the different storage groups. Results/Finding: Under both storage conditions, GPIbα and GPV decreased significantly over storage time. However, comparison of the decline in these proteins to GPIbβ, GPIX, and other membrane proteins indicated that the mechanisms for this decline differ in the two conditions. At RT, the decrease in GPIbα and GPV appears to be largely proteolytic, given that only a minor concomitant decrease in surface level was seen in the protease-insensitive GPIX and a slight increase in GPIbβ. In addition, a comparable decrease in GPIbα level was not observed when a cytoplasmic GPIbα peptide was assayed, suggesting the extracellular portion had been proteolytically removed. In contrast, at 4°C the decline in GPIbα and GPV was accompanied by a modest decrease in GPIX, and only a small decrease in the ratio of extracellular to cytoplasmic to GPIbα peptide. These results suggested that, at 4°C, in addition to proteolysis, which was attenuated as compared to RT storage, another mechanism was responsible for removal of full-length GPIbα and other polypeptides. One such mechanism that could explain this would be loss of membrane from the platelets during 4°C storage. Indeed, we found that extracellular vesicles accumulated in the platelet supernatant during 4°C to a much higher level than at RT storage. Summary: One of the hallmarks of the platelet storage lesion at RT is shedding of surface membrane proteins including GPIbα. Previous studies in stored mouse and human platelets revealed a cleavage mechanism dependent on the metalloproteinase ADAM17. However, whether GPIbα is lost by the same mechanism in cold-stored human platelets was unknown. Our targeted proteomics analysis confirms that proteolysis is a major cause of GPIbα loss at RT, but is a less prominent mechanism at 4°C. However, another mechanism for membrane protein loss is more prominent at the lower temperature: microvesiculation. Thus, these studies provide new insights into the platelet storage lesion and suggest that measures to prevent them will have to be tailored to the dominant mechanism operating at a particular storage temperature. Disclosures No relevant conflicts of interest to declare.

Fibrinogen-Mediated Platelet Microaggregate Formation in Stored Whole Blood

Introduction: Whole blood is regaining popularity in resuscitation after hemorrhage. The platelet storage lesion that has been observed in apheresis platelets also exists in whole blood, where platelet count and aggregation response decline rapidly after collection and during storage in the blood bank. Platelet microaggregates form in unfiltered whole blood, resulting in relatively large but unstable thrombus formation in a flow model over collagen. We hypothesized that the presence of fibrinogen activates platelets within the whole blood unit and predisposes them toward premature aggregate formation. Methods: Fresh whole blood (WB) was collected in citrate tubes (4.3%, 10.9 mM) from healthy donors (n=4) according to an institutionally approved standard operating procedure. Blood was centrifuged at 200-g to collect the platelet-rich plasma (PRP). The remaining red blood cell (RBC) fraction was centrifuged at 2000-g to pellet RBCs which were washed with HEPES-buffered saline and repelleted; platelet-poor plasma (PPP) was saved. The PRP fraction was centrifuged with 1 µM prostacyclin at 700-g, and the platelet pellet was washed with Tyrode's buffer, repelleted, and resuspended in either fibrinogen-deficient plasma (Fg-DP) or PPP. Platelets were combined with washed RBCs to generate a reassembled whole blood sample containing 150,000 platelets/µl and a hematocrit of 40%. Samples were added to minibags and stored at 4 °C for 14 days. On days of testing (0, 3, 7, 14), 1 ml aliquots were stained with 1 µM calcein-AM for 30 min at 37 °C and run on the BioFlux 1000 microfluidics platform with arterial flow rates generating a shear rate of 980 s-1 over a collagen surface. Fluorescence microscopy images were captured and quantified over 10 min to observe platelet adhesion to the surface. Complete blood count (CBC) was measured on each aliquot. In a follow-up study, unmanipulated, stored whole blood was passed through a standard transfusion filter (200 µm pores) to determine the effects of removing microaggregates prior to microfluidics flow testing at each time point. Results: CBC results are in Figure 1, illustrating the decline of platelet count over storage as previously observed. Figure 2 shows representative images captured at the end of the 10 min flow period. In samples containing normal plasma, thrombus formation is impaired by day 3 of storage with aggregate structures appearing immediately in the microscopy images. These aggregates attach and release periodically over the course of flow, an effect that persisted through day 14. In samples reconstructed with Fg-DP, no microaggregates were observed during storage, although the density and size of thrombi diminished over time. In filtered whole blood samples, thrombus formation on days 3 and 7 more closely resembled the Day 0 fresh samples, but aggregates appeared on day 14 despite filtration. Discussion: WB contains all of the elements required for resuscitation after hemorrhage, however the platelet storage lesion may affect its function. The interaction of platelets and fibrinogen in a relatively static environment results in activation and aggregation of platelets. In apheresis units, studies have shown that using a platelet additive solution to dilute fibrinogen results in better platelet performance for longer periods of storage. Further study is required to determine the clinical significance of aggregate formation and whether mitigation strategies should be explored. Disclosures No relevant conflicts of interest to declare.

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

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.

Down-regulation of platelet adhesion receptors is a controlling mechanism of thrombosis, while also affecting post-transfusion efficacy of stored platelets

Physiologically, upon platelet activation, uncontrolled propagation of thrombosis is prevented by regulating mechanisms which affect the expression and function of either platelet adhesion receptors or integrins. Receptor ectodomain shedding is an elective mechanism which is mainly involved in down-regulation of adhesion receptors GPIbα and GPVI. Platelet integrin αIIbβ3 can also be modulated with a calpain-dependent proteolytic cleavage. In addition, activating signals may induce the internalization of expressed receptors to selectively down-regulate their intensity. Alternatively, further activation of platelets is associated with microvesiculation as a none-selective mechanism which leads to the loss of membrane- bearing receptors. In a non-physiological condition, the storage of therapeutic platelets has also shown to be associated with the unwilling activation of platelets which triggers receptors down-regulation via aforementioned different mechanisms. Notably, herein the changes are time-dependent and not controllable. While the expression and shedding of pro-inflammatory molecules can induce post-transfusion adverse effects, stored-dependent loss of adhesion receptors by ectodomain shedding or microvesiculation may attenuate post-transfusion adhesive functions of platelets causing their premature clearance from circulation. In its first part, the review presented here aims to describe the mechanisms involved in down-regulation of platelet adhesion receptors. It then highlights the crucial role of ectodomain shedding and microvesiculation in the propagation of “platelet storage lesion” which may affect the post-transfusion efficacy of platelet components.

The surface markers and survival rate of platelets during storage at 4°C: The influence of sodium octanoate

Background: Storage of platelet concentrates (PCs) at room temperature (20-24°C) limits its storage time to 5 days due to the destructive effects of platelet storage lesion (PSL) and bacterial contamination. Although prolonged storage of platelets (PLTs) at 4°C reduces the likelihood of bacterial contamination and PSL levels, it is accompanied by an increase in the clearance rate and changes in the surface markers of PLTs. The goal of this study was to evaluate the effects of sodium octanoate (SO) as a stabilizer on PLTs during storage at 4°C. Materials and Methods: In this experimental study, PCs were divided into three portions and stored for 5 days at 3 different conditions, including 20-24°C, 4°C temperature, and 4°C in presence of SO. PLTs enumeration was performed using an automated hematology analyzer. To measure the metabolic activity and survival rate of PLTs, the water-soluble tetrazolium salt (WST-1) assay was performed. The activity of lactate dehydrogenase enzyme (LDH) was measured by a biochemical analyzer. Additionally, the levels of PLT glycoprotein Ibα (GPIbα) and CD62P (P-selectin) were measured on PLTs by flow cytometry technique. Results: PLTs count was higher in SO-treated (4°C) PLTs than two other studied samples. Additionally, the viability was higher in the SO-treated PLTs than that in other groups. LDH amount was lower in the SO-treated PLTs than that in other groups (P>0.05). GPIbα expression was significantly higher in SO-treated PLTs than that other groups (P<0.05). On the other hand, the expression of CD62P was lower at 4°C in PLTs in the presence of SO (P>0.05). Conclusions: SO could modulate the effects of cold temperatures on PLTs. Furthermore, we found that the survival of platelets was better maintained in the presence of SO at 4°C.

Intrinsic apoptosis circumvents the functional decline of circulating platelets but does not cause the storage lesion

Key Points BAK/BAX depletion in murine platelets reveals that intrinsic apoptosis is not required for the development of the platelet storage lesion. Restriction of platelet life span by intrinsic apoptosis is pivotal to maintain a functional, hemostatically reactive platelet population.