Platelet Life Span Research Articles

Non-activating ITGB3 mutation in β3 cytoplasmic region causes macrothrombocytopenia with impaired αIIbβ3/RhoA pathway

Almost all mutations of ITGA2B or ITGB3 identified in congenital macrothrombocytopenia induce constitutive activation of αIIbβ3. However, whether concomitant αIIbβ3 activation is essential for macrothrombocytopenia development remains unknown. Recently, we identified the β3(R760C) mutation that does not induce αIIbβ3 activation in a subject with macrothrombocytopenia. The family study showed that macrothrombocytopenia with reduced expression of αIIbβ3 and GPVI appeared to be associated with subjects heterozygous for β3(R760C). We generated β3(R760C) knock-in (KI) mice and investigated the effects of the mutation on platelet/Mgk biology. Macrothrombocytopenia was decreased to 76% and 40% of platelet counts in heterozygous (Hetero) and homozygous (Homo) KI mice, respectively, compared to the wild-type mice. Platelet αIIbβ3 and GPVI expression were decreased in KI mice, and αIIbβ3 activation was not detected on non-stimulated KI platelets. Thus, the Hetero KI mice reproduced the phenotype of the human subject, indicating that the β3(R760C) mutation is responsible for the macrothrombocytopenia. Platelet aggregation, agonist-induced JON/A binding and P-selectin expression were impaired in KI mice. Platelet spreading on fibrinogen was also impaired in Homo mice with ADP or thrombin stimulation. Filopodia and lamellipodia formation was impaired in fibrinogen-adhered megakaryocytes of Homo mice with significantly impaired RhoA activation. Proplatelet formation in Homo mice was impaired with abnormal morphology. In addition, platelet lifespan was shortened in Homo mice. These data indicate that β3(R760C) mutation impairs the inside-out and outside-in signaling of αIIbβ3, and abnormal actin rearrangement and impaired RhoA activation may play major roles in leading to macrothrombocytopenia.

What It Takes To Be a Platelet: Evolving Concepts in Platelet Production.

Platelets are among the most abundant cells within the circulation. Given that the platelet lifespan is 7 to 10 days in humans, a constant production of around 100 billion platelets per day is required. Platelet production from precursor cells called megakaryocytes is one of the most enigmatic processes in human biology. Although it has been studied for over a century, there is still controversy about the exact mechanisms leading to platelet release into circulation. The formation of proplatelet extensions from megakaryocytes into bone marrow sinusoids is the best-described mechanism explaining the origin of blood platelets. However, using powerful imaging techniques, several emerging studies have recently raised challenging questions in the field, suggesting that small platelet-sized structures called buds might also contribute to the circulating platelet pool. How and whether these structures differ from microvesicles or membrane blebs, which have previously been described to be released from megakaryocytes, is still a matter of discussion. In this review, we will summarize what the past and present have revealed about platelet production and whether mature blood platelets might emerge via different mechanisms.

Judicious Transfusion of Platelets Among Neonates: A Systematic Review.

In newborns, especially premature babies, there is a high association between thrombocytopenia and bleeding, particularly intraventricular hemorrhage, which may be due to immaturity. It was usual clinical practice that neonates should be transfused with higher platelet counts than older children or adults to reduce their risk of bleeding. However, after keen observations, we noticed that bleeding and mortality were more common in newborns who received more platelet transfusions. The mechanisms underlying the adverse effects of platelet transfusions in neonates may be due to higher antigenicity and immunological factors. We know that neonatal platelets are hyporeactive; this hyporeactivity is balanced by factors in the neonatal blood that promote coagulation, such as increased hematocrit, von Willebrand factor, and fibrinogen, which, on balance, leads to normal primary neonatal hemostasis. Platelets are very similar to adults in number, but functional capabilities were less, and for the reasons mentioned above, particularly bleeding time was short. Theologically, neonatal platelet lifespan was high to compensate for less production. We started this review because we observed that many babies were not having bleeding symptoms in some instances of severe thrombocytopenia. Many well-active babies are receiving unnecessary transfusions, as human blood is precious, and many young neonatologists are going on protocol-based excessive transfusions. This stimulated us to write a review.

Delayed cold-stored vs. room temperature stored platelet transfusions in bleeding adult cardiac surgery patients—a randomized multicentre pilot study (PLTS-1)

BackgroundPlatelets stored at 1–6 °C are hypothesized to be more hemostatically active than standard room temperature platelets (RTP) stored at 20–24 °C. Recent studies suggest converting RTP to cold-stored platelets (Delayed Cold-Stored Platelets, DCSP) may be an important way of extending platelet lifespan and increasing platelet supply while also activating and priming platelets for the treatment of acute bleeding. However, there is little clinical trial data supporting the efficacy and safety of DCSP compared to standard RTP.MethodsThis protocol details the design of a multicentre, two-arm, parallel-group, randomized, active-control, blinded, internal pilot trial to be conducted at two cardiac surgery centers in Canada. The study will randomize 50 adult (≥ 18 years old) patients undergoing at least moderately complex cardiac surgery with cardiopulmonary bypass and requiring platelet transfusion to receive either RTP as per standard of care (control group) or DCSP (intervention group). Patients randomized to the intervention group will receive ABO-identical, buffy-coat, pathogen-reduced, platelets in platelet additive solution maintained at 22 °C for up to 4 days then placed at 4 °C for a minimum of 24 h, with expiration at 14 days after collection. The duration of the intervention is from the termination of cardiopulmonary bypass to 24 h after, with a maximum of two doses of DCSP. Thereafter, all patients will receive RTP. The aim of this pilot is to assess the feasibility of a future RCT comparing the hemostatic effectiveness of DCSP to RTP (defined as the total number of allogeneic blood products transfused within 24 h after CPB) as well as safety. Specifically, the feasibility objectives of this pilot study are to determine (1) recruitment of ≥ 15% eligible patients per center per month); (2) appropriate platelet product available for ≥ 90% of patients randomized to the cold-stored platelet group; (3) Adherence to randomization assignment (> 90% of patients administered assigned product).DiscussionDCSP represents a promising logistical solution to address platelet supply shortages and a potentially more efficacious option for the management of active bleeding. No prospective clinical studies on this topic have been conducted. This proposed internal pilot study will assess the feasibility of a larger definitive study.Trial registrationNCT 06147531 (clinicaltrials.gov).

EVALUATING THE SAFETY AND THERAPEUTIC EFFICACY OF INTRAVENOUS HYDROGEN NANOBUBBLE INFUSIONS IN A HYPERCHOLESTEROLEMIC RAT MODEL

Hydrogen has emerged as a promising candidate due to its antioxidant properties and potential to modulate electron flow, enhancing mitochondrial energy metabolism and reducing oxidative stress. In this study, hypercholesterolemia male Wistar rats were utilized to investigate the safety and efficacy of intravenous hydrogen nanobubble solution (HNB). The study assessed serum lipid levels, complete blood count, and organ health following HNB administration compared to positive (simvastatin) and negative controls. Bayesian statistics were used to validate the findings. The administration of HNB did not adversely affect the immediate survival of the rats on a high fat diet (HFD), whereas simvastatin significantly reduced survival rates. It showed a dose dependent immunomodulatory effect, reducing white blood cell, lymphocyte, and monocyte counts. It also influenced erythropoiesis, as indicated by increased red blood cell counts and hemoglobin levels. A significant elevation in platelet counts was observed, suggesting potential effects on platelet production or lifespan. The study also noted changes in liver enzyme levels, with HNB indicating potential hepatoprotective effects. Furthermore, HNB treatment decrease in mean fat droplet count, suggesting a mitigation of diet induced hepatic steatosis. The findings suggest that HNB may serve as a protective agent against the deleterious effects of a high-fat diet, offering immunomodulatory and hepatoprotective benefits. This study provides a foundation for considering HNB as a potential therapeutic intervention for hypercholesterolemia and warrants further investigation into the long-term impacts of nanobubble treatment on diet induced pathologies. The consistency of results across traditional statistical significance and Bayesian evidence strengthens the validity of the observed treatment-specific effects.

Mitofusin-2 Regulates Platelet Mitochondria and Function.

Single-nucleotide polymorphisms linked with the rs1474868 T allele (MFN2 [mitofusin-2] T/T) in the human mitochondrial fusion protein MFN2 gene are associated with reduced platelet MFN2 RNA expression and platelet counts. This study investigates the impact of MFN2 on megakaryocyte and platelet biology. Mice with megakaryocyte/platelet deletion of Mfn2 (Mfn2-/- [Mfn2 conditional knockout]) were generated using Pf4-Cre crossed with floxed Mfn2 mice. Human megakaryocytes were generated from cord blood and platelets isolated from healthy subjects genotyped for rs1474868. Ex vivo approaches assessed mitochondrial morphology, function, and platelet activation responses. In vivo measurements included endogenous/transfused platelet life span, tail bleed time, transient middle cerebral artery occlusion, and pulmonary vascular permeability/hemorrhage following lipopolysaccharide-induced acute lung injury. Mitochondria was more fragmented in megakaryocytes derived from Mfn2-/- mice and from human cord blood with MFN2 T/T genotype compared with control megakaryocytes. Human resting platelets of MFN2 T/T genotype had reduced MFN2 protein, diminished mitochondrial membrane potential, and an increased rate of phosphatidylserine exposure during ex vivo culture. Platelet counts and platelet life span were reduced in Mfn2-/- mice accompanied by an increased rate of phosphatidylserine exposure in resting platelets, especially aged platelets, during ex vivo culture. Mfn2-/- also decreased platelet mitochondrial membrane potential (basal) and activated mitochondrial oxygen consumption rate, reactive oxygen species generation, calcium flux, platelet-neutrophil aggregate formation, and phosphatidylserine exposure following dual agonist activation. Ultimately, Mfn2-/- mice showed prolonged tail bleed times, decreased ischemic stroke infarct size after cerebral ischemia-reperfusion, and exacerbated pulmonary inflammatory hemorrhage following lipopolysaccharide-induced acute lung injury. Analysis of MFN2 SNPs in the iSPAAR study (Identification of SNPs Predisposing to Altered ALI Risk) identified a significant association between MFN2 and 28-day mortality in patients with acute respiratory distress syndrome. Mfn2 preserves mitochondrial phenotypes in megakaryocytes and platelets and influences platelet life span, function, and outcomes of stroke and lung injury.

Proposal of New Diagnostic Criteria for Primary Immune Thrombocytopenia

Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized by isolated thrombocytopenia due to accelerated platelet destruction and impaired platelet production. Diagnosis of ITP is still challenging because ITP has been diagnosed by exclusion. Exclusion of thrombocytopenia due to bone marrow failure is especially important in Japan because of high prevalence of aplastic anemia compared to Western countries. Hence, we propose new diagnostic criteria involving the measurement of plasma thrombopoietin (TPO) levels and percentage of immature platelet fraction (RP% or IPF%) as shown in Fig. 1. These biomarkers reflect the pathology of ITP, in which shortened platelet lifespan associated with increased platelet destruction and relatively maintained platelet production. Diagnostic criteria is as follows: 1. ALL of the following *1 ・Thrombocytopenia (＜100,000/μL) ・Absence of anemia (excluding anemia due to bleeding and/or iron deficiency) ・Normal leukocyte count (but may present with mild abnormal leukocyte count) ・No morphological evidence of dysplasia in any blood cell type in a blood smear 2. Normal or slightly increased plasma TPO level (<cutoff) 3. Elevated percentage of immature platelet fraction (RP% or IPF%; > upper limit of normal) 4. Absence of other conditions that potentially cause thrombocytopenia including secondary ITP ⋆2 ● A diagnosis of ITP is made if conditions 1 - 4 above are all met (ITP) ● Cases in which criterion 2 or 3 is not met or unavailable are defined as “possible ITP, pITP”. A diagnosis of ITP can be made if the patient is a glycoprotein (GP)-specific platelet-antibody positive, increased GP-specific platelet antibody-producing B cells, or typical clinical course. (Footnotes) *1 Bone marrow examination is strongly recommended to rule out other diseases if peripheral blood demonstrates any of the following findings: leukocyte count <3,000 or ≥10,000/μL, MCV ≥110, neutrophils <30% or lymphocytes ≥50%, presence of immature leukocytes *2 Includes drug-related thrombocytopenia, radiation injury, aplastic anemia, myelodysplastic syndrome (MDS), paroxysmal nocturnal hemoglobinuria (PNH), systemic lupus erythematosus (SLE), leukemia, malignant lymphoma, bone marrow metastasis of cancer cells, disseminated intravascular coagulation (DIC) , thrombotic thrombocytopenic purpura (TTP), hypersplenism, megaloblastic anemia, sepsis, infections (tuberculosis, etc.), sarcoidosis, and huge hemangioma such as Kasabach-Merritt syndrome. Congenital diseases involving thrombocytopenia include Bernard-Soulier syndrome, Wiskott-Aldrich syndrome, MYH9-related disease and Upshaw-Schulman syndrome. Sensitivity and specificity analysis of this criteria was performed by using data of previously performed prospective study (Kuwana et al. J Thromb Haemost 2006). In this study plasma TPO level were measured by R&D Systems ELISA Kit and RP% were measured by flow cytometry. 112 thrombocytopenic patients who were initially suspected ITP (final diagnosis: ITP 88, aplastic anemia 11, MDS 10, others 3) were included in the study. The sensitivity and specificity of “ITP” who met all 4 conditions of the new criteria were 53.4% (47/88) and 95.8% (23/24), respectively. If cases of diagnosed as “ITP” or “pITP” by the new criteria were considered as ITP, sensitivity and specificity were 94.3% (83/88) and 75.0% (18/24), respectively. More importantly, these criteria exclude aplastic anemia completely from true ITP. For clinical diagnostic use, new CLEIA-based TPO measurement kit (MBL, Japan) has recently been developed in Japan. However, it is still crucial to carefully differentiate ITP from other thrombocytopenic diseases before applying these biomarkers. These new criteria enable us to clearly differentiate ITP from aplastic anemia and other forms of hypoplastic thrombocytopenia, and can be highly useful in clinical practice for avoiding unnecessary bone marrow examination as well as for appropriate selection of treatments.

Abstract P1172: Shear-mediated Remodeling Of Platelet Surface Glycosylation: How Can Platelets Trim Their Sugar Coats?

Mechanical circulatory support (MCS) is associated with bleeding complications often requiring multiple transfusions or reoperation. Hypershear stress within device-supported circulation promotes platelet dysfunction associated with pro-apoptosis, impaired aggregation, and microvesiculation - all contributors to bleeding. Recent studies showed that glycosylation of surface receptors, i.e. “ sugar coat ,” plays a major role in the regulation of platelet function and lifespan. We tested the hypothesis that shear stress promotes platelet surface deglycosylation by increasing platelet glycosidase activity. Methods: Human platelets were obtained from ACD-anticoagulated blood via gel filtration on Sepharose 2B. Platelets were exposed to neuraminidase and continuous shear stress in a hemodynamic shearing device. Platelets were stained with fluorophore-conjugated lectins binding terminal sialic acids (SNA I, MAL II) and galactose (ECA), anti-CD41 and anti-CD42a a/b. Multi-colored flow cytometry was used to quantify platelet surface glycosylation. Neuraminidase and galactosidase activities of intact and lysed platelets were measured using enzyme-specific fluorogenic substrates. Results: Exposure to shear stress and neuraminidase induced a significant decrease in platelet 2,6-sialylation and galactosylation, while 2,3-sialylation remained unaltered. Neuraminidase reinforced shear-mediated platelet desialylation, causing a prominent decrease in SNA binding even at low shear, and increased platelet galactose levels by unmasking the galactose residues capped by sialic acids. Shear stress resulted in a 9.5-fold increase in platelet neuraminidase and a 1.4-fold increase in galactosidase activities vs their basal levels. Neutral neuraminidase and galactosidase were upregulated by shear, while acidic isoforms did not increase, suggesting that other than lysosomal glycosidase pools are involved. Shear stress promotes platelet deglycosylation via upregulation of platelet neuraminidase and galactosidase activities. Developing therapeutic strategies for the preservation of platelet glycosylation offers significant translational potential for the pharmacologic management of MCS platelet dysfunction and bleeding.

Characterization of the human platelet N- and O-glycome upon storage using tandem mass spectrometry

AbstractChanges in surface glycan determinants, specifically sialic acid loss, determine platelet life span. The gradual loss of stored platelet quality is a complex process that fundamentally involves carbohydrate structures. Here, we applied lipophilic extraction and glycan release protocols to sequentially profile N- and O-linked glycans in freshly isolated and 7-day room temperature–stored platelet concentrates. Analytical methods including matrix assisted laser desorption/ionization time-of-flight mass spectrometry, tandem mass spectrometry, and liquid chromatography were used to obtain structural details of selected glycans and terminal epitopes. The fresh platelet repertoire of surface structures revealed diverse N-glycans, including high mannose structures, complex glycans with polylactosamine repeats, and glycans presenting blood group epitopes. The O-glycan repertoire largely comprised sialylated and fucosylated core-1 and core-2 structures. For both N- and O-linked glycans, we observed a loss in sialylated epitopes with a reciprocal increase in neutral structures as well as increased neuraminidase activity after platelet storage at room temperature. The data indicate that loss of sialylated glycans is associated with diminished platelet quality and untimely removal of platelets after storage.

Platelet ageing: A review

Platelet ageing is an area of research which has gained much interest in recent years. Newly formed platelets, often referred to as reticulated platelets, young platelets or immature platelets, are defined as RNA-enriched and have long been thought to be hyper-reactive. This latter view is largely rooted in associations and observations in patient groups with shortened platelet half-lives who often present with increased proportions of newly formed platelets. Evidence from such groups suggests that an increased proportion of newly formed platelets is associated with an increased risk of thrombotic events and a reduced effectiveness of standard anti-platelet therapies. Whilst research has highlighted the existence of platelet subpopulations based on function, size and age within patient groups, the common intrinsic changes which occur as platelets age within the circulation are only just being explored. By understanding the changes that occur during the natural ageing processes of platelets, we may be able to identify the triggers for alterations in platelet life span and platelet reactivity. Here we review research on platelet ageing in the context of health and disease, paying particular attention to the experimental approaches taken and the robustness of conclusions that can be drawn.

Renal Klotho safeguards platelet lifespan in advanced chronic kidney disease through restraining Bcl‐xL ubiquitination and degradation

BackgroundThrombosis and hemorrhage as two opposite pathologies are prevalent within the chronic kidney disease (CKD) population. Platelet homeostasis, which positions centrally in their pathogenesis, varies among the CKD population, while the underlying mechanism is poorly understood. ObjectiveTo investigate the change character and mechanism of platelet homeostasis in CKD and its association with renal Klotho deficiency. MethodsThe change character of platelet homeostasis and its association with renal Klotho deficiency were determined based on a cohort study as well as CKD mice and Klotho‐deficient mice with CKD. The effects on thrombopoiesis and platelet lifespan were examined by flow cytometry and platelet transfer. The underlying mechanism was explored by proteomics, flow cytometry, western blot, and immunoprecipitation. ResultsWe show that platelet count declines both in patient and mouse models with advanced CKD (Adv‐CKD) and is positively associated with circulating Klotho levels. Mechanistically, we identify that ubiquitin ligase UBE2O governs Bcl‐xL ubiquitination and degradation in platelets, whereas Adv‐CKD–induced oxidative stress in platelets stimulates p38MAPK to promote Bcl‐xL phosphorylation, which facilitates UBE2O binding to Bcl‐xL and subsequent Bcl‐xL degradation. Consequently, platelet lifespan is shortened in Adv‐CKD, culminating in platelet count decline. However, kidney‐secreted soluble Klotho protein restricts oxidative stress in platelets, thereby preserving Bcl‐xL expression and platelet lifespan. ConclusionsOur findings uncover the mechanism of platelet count decline in Adv‐CKD and identify renal Klotho as a long‐range regulator of platelet lifespan, which not only provide a molecular mechanism underlying CKD‐associated thrombocytopenia and hemorrhage but also offer a promising therapy choice.

Accelerated Platelet Clearance in Mice with Constitutive Store-Operated Calcium Entry Is Independent of Caldag-GEFI/Rap1 Activation

Calcium (Ca2+) is critical for numerous functions of platelets including integrin activation, granule secretion and phosphatidylserine exposure. Circulating platelets maintain low cytosolic Ca2+ concentrations which rapidly rise upon cellular activation. Ca2+ release from intracellular stores triggers influx of extracellular Ca2+, a process known as store-operated Ca2+ entry (SOCE). Critical for SOCE is Stromal interaction molecule 1 (Stim1), a protein expressed in the dense tubular system membrane. Loss of Stim1 in platelets partially impairs platelet activation and procoagulant function. On the other hand, gain-of-function mutations in Stim1 are associated with thrombocytopenia and bleeding. In platelets, Ca2+ also activates CalDAG-GEFI and in turn Rap1 GTPase, the principal pathway mediating integrin activation. We recently demonstrated that unrestricted activation of Rap1 due to loss of the Rap1 GAP Rasa3 causes severe thrombocytopenia due to CalDAG-GEFI/integrin-dependent platelet clearance. To investigate whether CalDAG-GEFI activation mediates thrombocytopenia seen with constitutive Stim1 activation, we crossed Stim1 gain-of-function mice (Stim1Sax/+) with CalDAG-GEFI-/- mice to obtain Stim1Sax/+ x CalDAG-GEFI+/- or -/-mice. We observed no improvement in platelet count or platelet lifespan in Stim1Sax/+ mice with 1 or 0 copies of CalDAG-GEFI. While Rasa3 mutant platelets are predominantly cleared in the spleen, Stim1Sax/+ platelets accumulated to a greater extent in the lung. These results suggest that reduced platelet survival due to constitutive SOCE is not the result of increased CalDAG-GEFI/Rap1 signaling in circulating platelets.

Shear Stress Promotes Platelet Surface Desialylation, Platelet Count Drop, and Microvesiculation: Do Platelets Need to be Sugar-Coated?

Introduction. Mechanical circulatory support (MCS) devices restore hemodynamics and are lifesaving for patients with advanced heart failure and, recently, for patients with severe COVID-19. Long-term MCS is associated with bleeding complications, often requiring multiple transfusions of blood products or reoperations. To date, due to undefined etiology, device-related bleeding lacks efficient therapeutic management. We have shown that hypershear stress, existing within device-supported circulation promotes platelet dysfunction associated with pro-apoptosis, downregulation of key adhesion receptors, impaired platelet aggregation, and microvesiculation - all contributors to bleeding. Recent studies have also shown that glycosylation of platelet surface receptors, i.e. the platelet "sugar coat," plays a major role in the regulation of platelet function and lifespan. Here, we tested the hypothesis that hypershear stress promotes platelet surface desialylation, thus facilitating platelet count drop and increased microvesiculation. Methods. Human platelets were obtained from ACD-anticoagulated blood of healthy volunteers via gel filtration on Sepharose 2B. Gel-filtered platelets were exposed to neuraminidase and continuous shear stress (30 or 70 dyne/cm2,10 min) in a hemodynamic shearing device. Optionally, platelets were pretreated with oseltamivir, a common neuraminidase inhibitor. Platelets were co-stained with fluorophore-conjugated SNA lectin, binding terminal 2,6-linked sialic acid moieties, and anti-CD41 antibody. Multi-colored flow cytometry was performed to quantify platelet surface sialylation, platelets, and microparticles; fluorescent nanobeads SPHEROTM were used as a size standard. The surface density of platelet sialylation was calculated as median fluorescence intensity normalized to forward scatter indicating platelet size. The platelet neuraminidase activity was quantified using fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid (MUNANA) and was presented as µM of substrate per min. Results. Platelet exposure to both hypershear stress and neuraminidase-1 induced platelet surface desialylation as indicated by the significant decrease of SNA lectins' binding density on platelets (Fig.1A). Platelet desialylation induced by shear stress and neuraminidase was associated with a notable decrease in platelet count and an increase in platelet-derived microparticles (Fig.1B). Interestingly, neuraminidase reinforced shear-mediated desialylation, platelet count drop, and microvesiculation. Oseltamivir slightly inhibited shear-mediated desialylation, preserved platelet count (30% increase vs. shear), and decreased microparticle generation (29% decrease vs. shear). Exposure to 70 dyne/cm2 shear stress resulted in a 9.5-fold increase in platelet neuraminidase activity as compared to basal level: 0.85 ± 0.61 µM/min vs 0.05 ± 0.03 µM/min, respectively (Mean ± SD, p < 0.01, one-way ANOVA). Conclusions. Shear stress promotes platelet surface desialylation with the associated reduction in platelet count and increased microvesiculation. Neuraminidase inhibition restores platelet count and decreases microparticle generation caused by hypershear. Shear-mediated increase of platelet neuraminidase activity is considered a plausible mechanism for shear-mediated platelet deglycosylation. Developing therapeutic strategies for preservation of platelet sialylation offers significant translational potential for pharmacologic management of MCS-related platelet dysfunction and bleeding coagulopathy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The effect of storage period on the platelet levels on whole blood in the blood bank refrigerator

The quality of blood treatment before and after transfusion must be maintained to prevent the potential of transfusion reactions. A blood storage process must meet the requirements that have been set. Platelets play a role in repairing the chain reaction of blood vessel damage and initiating bleeding cessation that results in blood clotting. Therefore, this study aimed to analyze the effect of storage period on platelet levels in whole blood in a blood bank refrigerator using a quasi-experimental method (one-group pre-post test design). The population was 30 donors selected by simple random sampling. Data analysis used was Friedman test. The results showed that there was a significant effect of storage period on platelet levels at 0, 10, 20 and 30 days with a p value &lt;001. The life span of platelets in vivo is about 7-10 days, whereas in vitro (without shaking) is only 3 days. The decrease in platelet levels is possible due to short lifespan effect and changes in cell structure. This can cause the platelet cells morphology to change shape to become irreversible and can cause the loss of platelet viability. The blood storage period with the right duration is needed to maintain blood quality.

CPT2 K79 acetylation regulates platelet life span

AbstractThe short life span of platelets is a major challenge to platelet transfusion services because of the lack of effective intervention. Here, we found that the accumulation of long-chain acylcarnitines (LCACs) is responsible for mitochondrial damage and platelet storage lesion. Further studies showed that the blockade of fatty acid oxidation and the activation of AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase/carnitine palmitoyltransferase 1 (CPT1) pathways that promote fatty acid metabolism are important reasons for the accumulation of LCACs. The excessive accumulation of LCACs can cause mitochondrial damage and a short life span of stored platelets. The mechanism study elucidated that NAD+ exhaustion and the subsequent decrease in sirtuin 3 (Sirt3) activity caused an increase in the level of CPT2 K79 acetylation, which is the primary cause of the blockade of fatty acid oxidation and the accumulation of LCACs. Blocking LCAC generation with the inhibitors of AMPK or CPT1, the agonists of Sirt3, and antioxidants tremendously retarded platelet storage lesion in vitro and prolonged the survival of stored platelets in vivo posttransfusion with single or combined use. In summary, we discovered that CPT2 acetylation attenuates fatty acid oxidation and exacerbates platelet storage lesion and may serve as a new target for improving platelet storage quality.

Fast clearance of platelets in a commonly used mouse model for GPIbα is impeded by an anti‐GPIbβ antibody derivative

BackgroundGlycoprotein (GP)Ibα plays a critical role in regulating platelet clearance. Recently, we identified the mechanosensory domain (MSD) in GPIbα and reported evidence to suggest that unfolding of the GPIbα MSD induces exposure of the Trigger sequence therein and subsequent GPIb‐IX signaling that accelerates platelet clearance. In a commonly used transgenic mouse model, IL4R‐IbαTg, where the Trigger sequence is constitutively exposed, constitutive GPIb‐IX‐mediated cellular signals are present. Clearance of their platelets is also significantly faster than that of wild‐type mice. Previously, an anti‐GPIbβ antibody RAM.1 was developed. RAM.1 inhibits GPIbα‐dependent platelet signaling and activation. Further, RAM.1 also inhibits anti‐GPIbα antibody‐mediated filopodia formation. ObjectiveTo investigate whether RAM.1 can ameliorate trigger sequence exposure‐mediated platelet clearance. MethodsSpontaneous filopodia were measured by confocal microscopy. Other platelet signaling events were measured by flow cytometry. Endogenous platelet life span was tracked by Alexa 488‐labeled anti‐mouse GPIX antibody. ResultTransfected Chinese hamster ovary cells stably expressing the same chimeric IL4R‐Ibα protein complex as in IL4R‐IbαTg mice also constitutively exhibit filopodia, and that such filopodia could be abolished by treatment of RAM.1. Further, transfusion of a recombinant RAM.1 derivative that is devoid of its Fc portion significantly extends the endogenous life span of IL4R‐IbαTg platelets. ConclusionThese results provide the key evidence supporting the causative link of Trigger sequence exposure to accelerated platelet clearance, and suggest that a RAM.1 derivative may be therapeutically developed to treat GPIb‐IX‐mediated thrombocytopenia.

Rasa3 deficiency minimally affects thrombopoiesis but promotes severe thrombocytopenia due to integrin-dependent platelet clearance.

Platelet homeostasis is dependent on a tight regulation of both platelet production and clearance. The small GTPase Rap1 mediates platelet adhesion and hemostatic plug formation. However, Rap1 signaling is also critical for platelet homeostasis as both Rap1 deficiency and uninhibited Rap1 signaling lead to marked thrombocytopenia in mice. Here, we investigated the mechanism by which deficiency in Rasa3, a critical negative regulator of Rap1, causes macrothrombocytopenia in mice. Despite marked morphological and ultrastructural abnormalities, megakaryocytes in hypomorphic Rasa3hlb/hlb (R3hlb/hlb) or Rasa3–/– mice demonstrated robust proplatelet formation in vivo, suggesting that defective thrombopoiesis is not the main cause of thrombocytopenia. Rather, we observed that R3hlb/hlb platelets became trapped in the spleen marginal zone/red pulp interface, with evidence of platelet phagocytosis by macrophages. Clearance of mutant platelets was also observed in the liver, especially in splenectomized mice. Platelet count and platelet life span in Rasa3-mutant mice were restored by genetic or pharmacological approaches to inhibit the Rap1/talin1/αIIbβ3 integrin axis. A similar pattern of splenic clearance was observed in mice injected with anti-αIIbβ3 but not anti–glycoprotein Ibα platelet-depleting antibodies. In summary, we describe a potentially novel, integrin-based mechanism of platelet clearance that could be critical for our understanding of select inherited and acquired thrombocytopenias.

Severe Congenital Thrombocytopenia Characterized by Decreased Platelet Sialylation and Moderate Complement Activation Caused by Novel Compound Heterozygous Variants in GNE.

BackgroundHereditary thrombocytopenias constitute a genetically heterogeneous cause of increased bleeding. We report a case of a 17-year-old boy suffering from severe macrothrombocytopenia throughout his life. Whole genome sequencing revealed the presence of two compound heterozygous variants in GNE encoding the enzyme UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase, crucial for sialic acid biosynthesis. Sialic acid is required for normal platelet life span, and biallelic variants in GNE have previously been associated with isolated macrothrombocytopenia. Furthermore, sialic acid constitutes a key ligand for complement factor H (FH), an important inhibitor of the complement system, protecting host cells from indiscriminate attack.MethodsSialic acid expression and FH binding to platelets and leukocytes was evaluated by flow cytometry. The binding of FH to erythrocytes was assessed indirectly by measuring the rate of complement mediated hemolysis. Complement activation was determined by measuring levels of C3bBbP (alternative pathway), C4d (classical/lectin pathway) and soluble terminal complement complex assays.ResultsThe proband exhibited markedly decreased expression of sialic acid on platelets and leukocytes. Consequently, the binding of FH was strongly reduced and moderate activation of the alternative and classical/lectin complement pathways was observed, together with an increased rate of erythrocyte lysis.ConclusionWe report two previously undescribed variants in GNE causing severe congenital macrothrombocytopenia in a compound heterozygous state, as a consequence of decreased platelet sialylation. The decreased sialylation of platelets, leukocytes and erythrocytes affects the binding of FH, leading to moderate complement activation and increased hemolysis.

Impediment of Gpibα-Mediated Platelet Clearance By an Anti-Gpibβ Antibody Derivative

Background:Glycoprotein (GP)Ib-IX complex plays a critical role in mediating platelet activation and platelet clearance. Recently, we identified the mechanosensory domain (MSD) in the GPIbα subunit, and demonstrated that unfolding of the MSD and subsequent exposure of the Trigger sequence (residues 473-483) therein activates GPIb-IX and induces rapid platelet clearance. This mechanism could explain acute thrombocytopenia induced by activated VWF, anti-GPIbα antibodies, neuraminidase, and ectodomain shedding of GPIbα. Consistently, platelets in IL4R-IbαTg mice, a transgenic strain in which the entire extracellular domain of human GPIbα except the Trigger sequence was replaced with that of the α-subunit of interleukin-4 receptor, exhibit constitutively more filopodia and are cleared much faster than the wild type.Previously, an anti-GPIbβ antibody RAM.1 was developed. RAM.1 significantly inhibits GPIb-IX-mediated filopodia formation and Ca 2+ signaling in platelets. In addition, it could inhibit GPIb-dependent thrombin generation. These results suggest that targeting GPIbβ could inhibit activation of GPIb-IX induced by MSD unfolding.Objectives:To investigate whether targeting GPIbβ with RAM.1 can impede rapid platelet clearance induced by exposed Trigger sequence and ameliorate related thrombocytopenia.Methods:Spontaneous filopodia in platelets and transfected Chinese hamster ovary (CHO) cells were visualized by fluorescence staining of actin and confocal microscopy. Images were quantified by ImageJ. Platelet signaling events, like P-selectin exposure, β-galactose exposure, and Ca 2+ influx, were measured by flow cytometry. Endogenous platelet life span was tracked by Alexa 488-labeled anti-mouse GPIX antibody.Results:CHO cells stably expressing the same mutant GPIb-IX complex in IL4R-IbαTg mouse platelets have been successfully obtained. Like IL4R-IbαTg platelets, these IL4R-IbαTg CHO cells exhibited spontaneous filopodia in the absence of any GPIbα ligands. RAM.1 could inhibit spontaneous filopodia formation in these CHO cells and IL4R-IbαTg platelets (Fig. 1, 2). Compared to wild-type mouse platelets, IL4R-IbαTg platelets constitutively exhibited increased P-selectin exposure, increased β-galactose exposure, and elevated intracellular Ca 2+, all of which could be inhibited by treatment of RAM.1 (Fig. 3). Recombinant RAM.1-GCN4 protein (rRAM.1-GCN4), in which the Fc region of RAM.1 heavy chain was replaced with the GCN4 coiled coil dimerizing sequence, has been generated and used as an alternative of the divalent RAM.1-Fab2. It retained the ability of RAM.1 antibody to inhibit GPIb-IX signaling. Injecting rRAM.1-GCN4 into IL4R-IbαTg mice dramatically improved the life span of endogenous IL4R-IbαTg platelets (Fig. 4).Conclusion:These results demonstrate that the exposed Trigger sequence is sufficient to activate GPIb-IX in transfected CHO cells, and that RAM.1 derivatives can impede GPIbα-mediated rapid platelet clearance. Targeting GPIbβ may be a novel approach to treat GPIb-related thrombocytopenia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mitofusin2 (MFN2) Preserves Mitochondrial Integrity and Function in Megakaryocytes and Platelets

Genome wide association studies (GWAS) have associated mitochondria related loci with platelet numbers, function, and CVD. However, causality has not been established for many of these variants, and their mechanism and functional consequences are unknown. One such variant, MFN2 eQTL rs1474868 (T/T), has been associated with reduced platelet counts and reduced expression (5 fold) of MFN2 RNA in platelets. We show here that the MFN2 T/T variant corresponds with significantly reduced MFN2 protein in platelets. This difference contributes to a significant correlation between MFN2 RNA levels and mitochondrial load and potential in platelets. MFN2 RNA is also reduced by T/T in human cord blood derived megakaryocytes resulting in unfused mitochondria and impaired megakaryopoiesis. Using platelet/megakaryocyte specific Mfn2-/- (Mfn2 KO) mice, we show that Mfn2 impacts platelet numbers, activation and function by regulating mitochondrial energetics. Platelets without Mfn2 had reduced mitochondrial membrane potential and significantly reduced platelet lifespan (P<0.01) that was attributed to an increased rate of phosphatidylserine (PS) flipping (P=0.01). Increased RNA expression with conversely reduced protein expression of Ndufb8 (P<0.01), an index nuclear encoded complex I subunit that is stable only in a fully assembled complex I, suggested a defect in complex I assembly in Mfn2 KO platelets. Furthermore, complex I activity was reduced in Mfn2 KO platelets compared to WT platelets (P<0.01). Both basal and thrombin triggered mitochondrial oxygen consumption rate as assessed by Seahorse analyzer was significantly reduced in Mfn2 KO platelets (1.28 pmol/min/µg protein) compared to WT control platelets (3.06 pmol/min/µg protein). Platelet activation was subtly, yet significantly, decreased in Mfn2 KO platelets compared to WT platelets as assessed by surface expression of activated integrin alpha2b/beta3 and P-selectin. In addition, Mfn2 KO platelets had impaired Ca 2+ signaling, ROS generation, and procoagulant platelet formation (PS +ve platelets), and formed fewer platelet-neutrophil aggregates (PNAs) compared to WT platelets (P=0.01). Consistent with this, we observed significantly prolonged bleeding times in Mfn2 KO mice compared to their WT control littermates (P=0.001). Finally, mice with loss of platelet Mfn2 exhibited a modest reduction in ischemic stroke infarct size after cerebral ischemia-reperfusion that was statistically significant (P<0.01). Taken together these results suggest that MFN2 preserves mitochondrial functions necessary for platelet survival and activity, and that loss of MFN2 leads to accelerated platelet death, dysfunction, and altered hemostasis and thrombosis. DisclosuresRondina: Novartis: Research Funding; Platelet Biogenesis: Membership on an entity's Board of Directors or advisory committees; Acticor Biotech: Membership on an entity's Board of Directors or advisory committees; Platelet Transcriptomics: Patents & Royalties.