Role In Platelet Function Research Articles

Role of platelet autophagy in cardiovascular diseases

Platelet hyperactivity leads to thrombosis, the primary cause of cardiovascular and cerebrovascular adverse events. Emerging evidence suggests that autophagy plays a fundamental role in platelet function, both at baseline and in response to stress. Autophagy is an intracellular mechanism devoted to the removal and recycling of damaged cytoplasmic cargoes. Basal level of autophagy ensures physiological platelet aggregation and hemostasis. Autophagy is impaired in platelets isolated from subjects at high cardiovascular risk whereas autophagy stimulation reduces platelet hyperactivity, along with an overall amelioration of oxidative stress status. In this mini-review, we explore the current literature on the role of autophagy in platelet biology and its relevance as a therapeutic target for counteracting cardiovascular diseases.

Role and recent progress of P2Y12 receptor in cancer development.

P2Y12 receptor (P2Y12R) is an adenosine-activated G protein-coupled receptor (GPCR) that plays a central role in platelet function, hemostasis, and thrombosis. P2Y12R activation can promote platelet aggregation and adhesion to cancer cells, promote tumor angiogenesis, and affect the tumor immune microenvironment (TIME) and tumor drug resistance, which is conducive to the progression of cancers. Meanwhile, P2Y12R inhibitors can inhibit this effect, suggesting that P2Y12R may be a potential therapeutic target for cancer. P2Y12R is involved in cancer development and metastasis, while P2Y12R inhibitors are effective in inhibiting cancer. However, a new study suggests that long-term use of P2Y12R inhibitors may increase the risk of cancer and the mechanism remains to be explored. In this paper, we reviewed the structural and functional characteristics of P2Y12R and its role in cancer. We explored the role of P2Y12R inhibitors in different tumors and the latest advances by summarizing the basic and clinical studies on the effects of P2Y12R inhibitors on tumors.

Abstract 140: Exploring Protein Kinase C Theta (PKCθ) Signaling In The Generation Of Procoagulant Platelet: Insights From Phosphoproteomics And Systems Analysis

Background: Platelet activation plays a pivotal role in physiological hemostasis and thrombosis. Protein Kinase C theta (PKCθ) isoforms exert significant regulatory influence on platelet activation. While specific PKC family members have well-defined roles in platelet function, the roles of PKCθ remain less explored. Objectives: To determine mechanisms of PKCθ signaling that orchestrate procoagulant platelet generation. Methods: Platelets were prepared from healthy human donors for ex vivo experiments. Platelet signaling and function were studied following stimulation with GPVI agonist crosslinked collagen-related peptide (CRP-XL). To investigate PKCθ inhibition mechanisms in platelets, small molecule inhibitors targeting the PKCθ (CC-90005 and C20) were employed. Results: From phosphoproteomics, we noted prominent phosphorylation on the C-terminus PKCθ at Ser685. Pretreatment of platelets with inhibitors specific PKC isoforms revealed that PKCα/β, PKCθ, and PKCΔ activities determine PKCθ Ser685 phosphorylation. From the PKC Inhibitors Activity Curves, we noted a robust and selective inhibitory impact of CC-90005 on PKCθ, with specificity surpassing that of C20. Parallel platelet function analyses revealed that inhibiting PKCθ had minimal impact on platelet spreading and aggregation. However, PKCθ inhibition in the presence of Ca 2+ upregulated platelet phosphatidylserine (PS) exposure, as measured by flow cytometry. This was accompanied by an increase in platelet procoagulant activity, where PKCθ inhibition reduced the lag time for fibrin formation. Furthermore, fluorescence microscopy also found that PKCθ inhibition significantly augmented the proportion of procoagulant platelets and microparticles. Additionally, compared to the control group, the use of cyclosporin A (CsA) reduced the production of procoagulant platelets, but the addition of CC-90005 or C20 rescued this phenomenon, with signal intensity surpassing that of the control group. Conclusions: Our study proposes a model wherein PKCΔ and PKCα/β activities induce the phosphorylation of PKCθ at S685, influencing mitochondrial calcium signaling and supporting procoagulant platelet generation.

Early onset neonatal thrombocytopenia is associated with severe intraventricular hemorrhage in very preterm infants: A retrospective cohort study.

The role of platelet function in the development of intraventricular hemorrhage is still a subject of debate. In this study, we aimed to determine whether there is an association between platelet indices in the first week of life and severity of intraventricular hemorrhage in very preterm infants. Preterm infants born < 30 weeks of gestation in our hospital were retrospectively evaluated. Platelet parameters, including platelet counts, mean platelet volume, platelet distribution width, and platelet mass were retrieved at two different time points: the initial value on the first day of life and the value closest to the end of the first week of life. The infants were categorized according to the findings of cranial ultrasonography as; no intraventricular hemorrhage, mild or severe intraventricular hemorrhage. Totally, 1051 infants were evaluated. The mean gestational age and birth weight for the entire cohort were 27.9±1.6 weeks and 1058±247 g, respectively. Infants in the severe intraventricular hemorrhage group had significantly lower gestational age (p < 0.001) and birthweight (p < 0.001) compared to other two groups. Furthermore, there were significant differences in platelet count and platelet mass between the groups at two time intervals. However, logistic regression analysis revealed that only platelet count of < 100×109/L on the first postnatal day was independently associated with the severity of intraventricular hemorrhage. There is an association between platelet count of < 100×109/L on the first postnatal day and severe intraventricular hemorrhage in very preterm infants.

Celastrol inhibits platelet function and thrombus formation

IntroductionCelastrol is an active pentacyclic triterpenoid extracted from Tripterygium wilfordii and has anti-inflammatory and anti-tumor properties. Whether Celastrol modulates platelet function remains unknown. Our study investigated its role in platelet function and thrombosis. MethodsHuman platelets were isolated and incubated with Celastrol (0, 1, 3 and 5 μM) at 37 °C for 1 h to measure platelet aggregation, granules release, spreading, thrombin-induced clot retraction and intracellular calcium mobilization. Additionally, Celastrol (2 mg/kg) was intraperitoneally administrated into mice to evaluate hemostasis and thrombosis in vivo. ResultsCelastrol treatment significantly decreased platelet aggregation and secretion of dense or alpha granules induced by collagen-related peptide (CRP) or thrombin in a dose-dependent manner. Additionally, Celastrol-treated platelets showed a dramatically reduced spreading activity and decreased clot retraction. Moreover, Celastrol administration prolonged tail bleeding time and inhibited formation of arterial/venous thrombosis. Furthermore, Celastrol significantly reduced calcium mobilization. ConclusionCelastrol inhibits platelet function and venous/arterial thrombosis, implying that it might be utilized for treating thrombotic diseases.

The Actin Bundling Protein L-Plastin Mediates Platelet Force Generation and Thrombosis

Within minutes of activation, platelets dramatically change their shape, forming filopodia and lamellipodia, aggregate with other platelets or different cells, and can increase their surface area up to 4 times. This relies on a dynamic actin cytoskeleton, regulated by many actin binding proteins in a time- and spatial-specific manner. Deficiency or inhibition of several actin binding proteins in platelets have been shown with impaired platelet spreading and reduced thrombosis. Recently, we found that an actin bundling protein, L-plastin, is expressed in platelets. Its function in platelets has not been studied. In megakaryocytes, we showed that L-plastin is responsible for maintaining cytoskeletal stiffness; in this study we sought to determine L-plastin's role in platelet functions, including thrombosis. We examined the role of L-plastin in platelets in vivo and ex vivo using L-plastin deficient ( Lcp1 -/-) male and female mice. First, we used the collagen/epinephrine induced pulmonary embolism (PE) model and found that survival times were significantly shorter in Lcp1 -/- mice than in their littermate wildtype (WT) controls (Mean±SEM WT 202±12 sec, Lcp1 -/- 139.5±14 sec, P=0.004), suggesting accelerated thrombosis upon L-plastin deficiency in vivo. Second, we examined L-plastin's role in arterial thrombosis in vivo using the laser-induced cremaster arteriole thrombosis mode. Platelets were labeled with anti-GPIb IgG derivative X488 and the thrombosis formation after laser injury was recorded. Preliminary data indicate that thrombi in Lcp1 -/- mice are larger than those in WT mice. Third, we examined L-plastin's role in platelet adhesion using a microfluidics assay with DiOC6-labelled whole blood. At a shear rate of 200/s, the Lcp1 -/- mice had significantly increased platelet adhesion to both collagen and fibrinogen matrices. Fourth, to evaluate the effect of L-plastin on platelet aggregation in vitro, we stimulated washed platelets with 3 different concentrations of thrombin and measured the platelet aggregation (i.e., 0.1, 0.5 and 1 U/mL). When platelets are stimulated with 0.5U/mL or 1U/mL thrombin, platelets from WT and Lcp1 -/- mice showed comparable aggregation kinetics. At 0.1U/mL thrombin, however, preliminary data showed increased aggregation in the Lcp1 -/- group. The difference between the low and high concentration of thrombin indicates potential regulation of the GPIba mediated actin cytoskeleton rearrangement during platelet aggregation. We next examined under low thrombin concentration, the platelet actin cytoskeleton reorganization and platelet spreading. We seeded washed platelets on a fibrinogen matrix with the presence of thrombin (0.1U/mL). Platelets were then fixed and stained for F-actin and imaged using immunofluorescent confocal microscopy. The spreading area per platelet was calculated using FIJI software. Platelets from Lcp1 -/- mice showed significantly increased average membrane area at 30 min (Median WT 689 pixels, Lcp1 -/- 866 pixels, P&amp;lt;0.0001). This suggests that the actin rearrangement in platelets upon fibrinogen activation and thrombin stimulation is inhibited by L-plastin. Lastly, we examined whether L-plastin is involved in platelet force generation during the actin reorganization. We used a recently developed “black dot” assay. Briefly, a fluorescent pattern (i.e., black dots) was microcontact printed onto a flexible polydimethylsiloxane substrate and coated with fibrinogen. Platelets were seeded onto the matrix and fixed after 30 min. The contractile forces of individual platelets were measured based on the deformation of the fluorescent micropattern as illustrated in Fig 1A. The contractile force in platelets from Lcp1 -/- mice is decreased with altered actin distribution pattern compared to WT mice (Fig 1. Contractile force per platelet, Mean±SEM WT 14.78±0.86nN, Lcp1 -/- 12.03±0.67nN, P=0.01). In summary, we found accelerated thrombosis in Lcp1 -/- mice, associated with accelerated platelet adhesion and spreading ex vivo. Mechanistically, our preliminary data suggest this is due to impaired actin bundle formation and decreased platelet contractile force. L-plastin may be unique among actin binding proteins.

Identification of the Role of SEL1L in Platelet Function with Implications for Atypical Equine Thrombasthenia in Thoroughbred Horses

Atypical Equine Thrombasthenia (AET) is a recessive heritable platelet disorder in horses due to aberrant platelet signaling after thrombin stimulation, preventing platelets from fully activating or efficiently binding to fibrinogen, leading to prolonged bleeding. To date, AET has only been identified in the Thoroughbred horse breed. A prevalence study performed at one breeding farm found that one in every 150 Thoroughbreds was affected. Despite the negative effects on horse health and racing performance, the underlying etiology of this unique platelet disorder is unknown. Here we show that a variant in SEL1L, a gene which encodes a protein known to play a pivotal role in endoplasmic reticulum-associated protein degradation (ERAD) and not previously known to be involved in platelet function, leads to AET. In order to identify the underlying genetic mechanism, we performed whole genome sequencing on five affected, one equivocal, and eleven control Thoroughbred horses. A whole-genome association study was conducted, and associated variants were filtered by determining if they were in or near genes known to be expressed in platelets. This evaluation identified three putative loci: a missense variant in SEL1L (p.Ile604Val), a 1.6kb deletion in a long non-coding RNA ( AL355838.1) and a 73bp intronic deletion in VIPAS39. The long non-coding RNA AL355838.1 did not show expression in horse platelets, and there was no differential expression of SEL1L or VIPAS39 mRNA identified between affected and unaffected individuals. However, a significant difference in SEL1L protein expression was observed in AET-affected horses' platelets. Flow cytometry and immunofluorescence studies demonstrated that SEL1L is located intracellularly in equine platelets, but not in the α-granule, and moves to the surface of the platelet upon activation with thrombin. Although the template bleeding time was normal, platelets from horses that were homozygous for the SEL1L variant showed defective spreading on collagen. To support SEL1L as the causative gene and dissect the underlying mechanisms, we undertook studies in three additional models. Differentiation of human megakaryocytes revealed the presence of two SEL1L protein isoforms, p100 and p38, and both showed increased expression during megakaryopoiesis, although only p100 was delivered to mature platelets. We perfused whole blood from Mx1-Cre +; Sel1l fl/fl conditional knockouts and controls through a collagen-coated microfluidic chamber. We found that significantly fewer platelets from knockout mice bound to the collagen as compared to control siblings. Finally, a knockout zebrafish line was developed by deleting exons 3-20 using CRISPR/Cas9 mediated genome editing. Similar to the mouse, laser-mediated arterial endothelial injury of 5-day old mutant larvae resulted in significantly fewer thrombocytes (the fish platelet equivalent) adhering to the injured vessel wall as compared to wild-type siblings. Overall, these data support the conclusion that the SEL1L missense variant leads to AET, and that SEL1L has a previously undescribed and conserved role in platelet function, particularly the ability of platelets/thrombocytes to adhere to exposed collagen at sites of endothelial injury. These results will allow the Thoroughbred industry to easily diagnose AET-affected horses and make appropriate decisions for their racing and breeding careers. These data also provide an excellent introduction to further investigation into the understudied and potential role of ERAD proteins in human platelet disorders.

α-Synuclein is the major platelet isoform but is dispensable for activation, secretion, and thrombosis

Platelets play many roles in the vasculature ensuring proper hemostasis and maintaining integrity. These roles are facilitated, in part, by cargo molecules released from platelet granules via Soluble NSF Attachment Protein Receptor (SNARE) mediated membrane fusion, which is controlled by several protein-protein interactions. Chaperones have been characterized for t-SNAREs (i.e. Munc18b for Syntaxin-11), but none have been clearly identified for v-SNAREs. α-Synuclein has been proposed as a v-SNARE chaperone which may affect SNARE-complex assembly, fusion pore opening, and thus secretion. Despite its abundance and that it is the only isoform present, α-synuclein’s role in platelet secretion is uncharacterized. In this study, immunofluorescence showed that α-synuclein was present on punctate structures that co-stained with markers for α-granules and lysosomes and in a cytoplasmic pool. We analyzed the phenotype of α-synuclein−/− mice and their platelets. Platelets from knockout mice had a mild, agonist-dependent secretion defect but aggregation and spreading in vitro were unaffected. Consistently, thrombosis/hemostasis were unaffected in the tail-bleeding, FeCl3 carotid injury and jugular vein puncture models. None of the platelet secretory machinery examined, e.g. the v-SNAREs, were affected by α-synuclein’s loss. The results indicate that, despite its abundance, α-synuclein has only a limited role in platelet function and thrombosis.

Role of microRNAs and their downstream target transcription factors in zebrafish thrombopoiesis

Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, a comprehensive study on the microRNAs and their targets has not been undertaken. Zebrafish thrombocytes could be used as a model to study their role in megakaryocyte maturation and platelet function because thrombocytes have both megakaryocyte features and platelet properties. In our laboratory, we identified 15 microRNAs in thrombocytes using single-cell RNA sequencing. We knocked down each of these 15 microRNAs by the piggyback method and found knockdown of three microRNAs, mir-7148, let-7b, and mir-223 in adult zebrafish led to an increase in the percentage of thrombocytes. Functional thrombocyte analysis using plate tilt assay showed no modulatory effect of the three microRNAs on thrombocyte aggregation/agglutination. We also found enhanced thrombosis using arterial laser thrombosis assay in a group of zebrafish larvae after mir-7148, let-7b, and mir-223 knockdowns. These results suggested mir-7148, let-7b, and mir-223 are repressors for thrombocyte production. We then explored miRWalk database for let-7b downstream targets and then selected those that are expressed in thrombocytes, and from this list based on their role in differentiation selected 14 genes, rorca, tgif1, rfx1a, deaf1, zbtb18, mafba, cebpa, spi1a, spi1b, fhl3b, ikzf1, irf5, irf8, and lbx1b that encode transcriptional regulators. The qRT-PCR analysis of expression levels of the above genes following let-7b knockdown showed changes in the expression of 13 targets. We then studied the effect of the 13 targets on thrombocyte production and identified 5 genes, irf5, tgif1, irf8, cebpa, and rorca that showed thrombocytosis and one gene, ikzf1 that showed thrombocytopenia. Furthermore, we tested whether mir-223 regulates any of the above 13 transcription factors after mir-223 knockdown using qRT-PCR. Six of the 13 genes showed similar gene expression as observed with let-7b knockdown and 7 genes showed opposing results. Thus, our results suggested a possible regulatory network in common with both let-7b and mir-223. We also identified that tgif1, cebpa, ikzf1, irf5, irf8, and ikzf1 play a role in thrombopoiesis. Since the ikzf1 gene showed a differential expression profile in let-7b and mir-223 knockdowns but resulted in thrombocytopenia in ikzf1 knockdown in both adults and larvae we also studied an ikzf1 mutant and showed the mutant had thrombocytopenia. Taken together, these studies showed that thrombopoiesis is controlled by a network of transcription regulators that are regulated by multiple microRNAs in both positive and negative manner resulting in overall inhibition of thrombopoiesis.

PAR4 Inhibition Reduces Coronary Artery Atherosclerosis and Myocardial Fibrosis in SR-B1/LDLR Double Knockout Mice.

SR-B1 (scavenger receptor class B type 1)/LDLR (low-density lipoprotein receptor) double knockout mice fed a high-fat, high-cholesterol diet containing cholate exhibit coronary artery disease characterized by occlusive coronary artery atherosclerosis, platelet accumulation in coronary arteries, and myocardial fibrosis. Platelets are involved in atherosclerosis development, and PAR (protease-activated receptor) 4 has a prominent role in platelet function in mice. However, the role of PAR4 on coronary artery disease in mice has not been tested. We tested the effects of a PAR4 inhibitory pepducin (RAG8) on diet-induced aortic sinus and coronary artery atherosclerosis, platelet accumulation in atherosclerotic coronary arteries, and myocardial fibrosis in SR-B1/LDLR double knockout mice. SR-B1/LDLR double knockout mice were fed a high-fat, high-cholesterol diet containing cholate and injected daily with 20 mg/kg of either the RAG8 pepducin or a control reverse-sequence pepducin (SRQ8) for 20 days. Platelets from the RAG8-treated mice exhibited reduced thrombin and PAR4 agonist peptide-mediated activation compared with those from control SRQ8-treated mice when tested ex vivo. Although aortic sinus atherosclerosis levels did not differ, RAG8-treated mice exhibited reduced coronary artery atherosclerosis, reduced platelet accumulation in atherosclerotic coronary arteries, and reduced myocardial fibrosis. These protective effects were not accompanied by changes in circulating lipids, inflammatory cytokines, or immune cells. However, RAG8-treated mice exhibited reduced VCAM-1 (vascular cell adhesion molecule 1) protein levels in nonatherosclerotic coronary artery cross sections and reduced leukocyte accumulation in atherosclerotic coronary artery cross sections compared with those from SRQ8-treated mice. The PAR4 inhibitory RAG8 pepducin reduced coronary artery atherosclerosis and myocardial fibrosis in SR-B1/LDLR double knockout mice fed a high-fat, high-cholesterol diet containing cholate. Furthermore, RAG8 reduced VCAM-1 in nonatherosclerotic coronary arteries and reduced leukocyte and platelet accumulation in atherosclerotic coronary arteries. These findings identify PAR4 as an attractive target in reducing coronary artery disease development, and the use of RAG8 may potentially be beneficial in cardiovascular disease.

Antiplatelet Effect of Daphnetin Is Regulated by cPLA2-Mediated Thromboxane A2 Generation in Mice.

Coumarin derivatives have been recognized for their antithrombotic, anti-inflammatory, and antioxidant properties, and daphnetin is one of the natural coumarin derivatives isolated from Daphne Koreana Nakai. Although the pharmacological value of daphnetin is well documented in diverse biological activities, its antithrombotic effect has not been studied to date. Here, we characterized the role and underlying mechanism of daphnetin in the regulation of platelet activation using murine platelets. In order to check the effect of daphnetin on platelet function, we first measured the effect of daphnetin on platelet aggregation and secretion. Collagen-induced platelet aggregation and dense granule secretion were partially inhibited by daphnetin. Interestingly, 2-MeSADP-induced secondary waves of aggregation and secretion were completely inhibited by daphnetin. It is known that 2-MeSADP-induced secretion and the resultant secondary wave of aggregation are mediated by the positive feedback effect of thromboxane A2 (TxA2) generation, suggesting the important role of daphnetin on TxA2 generation in platelets. Consistently, daphnetin did not affect the 2-MeSADP-induced platelet aggregation in aspirinated platelets where the contribution of TxA2 generation was blocked. Additionally, platelet aggregation and secretion induced by a low concentration of thrombin, which is affected by the positive feedback effect of TxA2 generation, were partially inhibited in the presence of daphnetin. Importantly, 2-MeSADP- and thrombin-induced TxA2 generation was significantly inhibited in the presence of daphnetin, confirming the role of daphnetin on TxA2 generation. Finally, daphnetin significantly inhibited 2-MeSADP-induced cytosolic phospholipase A2 (cPLA2) and ERK phosphorylation in non-aspirinated platelets. Only cPLA2 phosphorylation, not ERK phosphorylation, was significantly inhibited by daphnetin in aspirinated platelets. In conclusion, daphnetin plays a critical role in platelet function by inhibiting TxA2 generation through the regulation of cPLA2 phosphorylation.

Characterization of molecular basis of anti‐platelet effect of daphnetin

Coumarin derivates have been known for their antithrombotic, anti‐inflammatory, and antioxidant activities and daphnetin is one of the natural coumarin derivatives isolated from Daphne Korean Nakai. Although the pharmacological value of daphnetin is well documented in diverse biological activities, its antithrombotic effect is not studied till date.We investigated the functional role of daphnetin and its molecular basis on the regulation of platelet activation using murine platelets.In order to check the effect of daphnetin on GPVI‐mediated platelet response, we first measured the effect of daphnetin on collagen‐induced platelet aggregation and secretion. Collagen‐induced platelet aggregation and dense granule secretion were partially inhibited in the presence of daphnetin. Interestingly, 2‐MeSADP‐induced secondary wave of aggregation and secretion were completely inhibited in the presence of daphnetin. It has been known that 2‐MeSADP‐induced secretion and the resultant secondary wave of aggregation are mediated by positive‐feedback effect of thromboxane A2 (TxA2) generation, suggesting the important role of daphnetin on TxA2 generation in platelets. Consistently, daphnetin did not affect the 2‐MeSADP‐induced platelet aggregation in aspirin‐treated platelets where the contribution of TxA2 generation is blocked. Additionally, AYPGKF‐induced platelet aggregation and secretion were inhibited in the presence of daphnetin by blocking the positive‐feedback effect of TxA2 generation. Moreover, 2‐MeSADP‐induced TxA2 generation was completely inhibited in the presence of daphnetin confirming the role of daphnetin on TxA2 generation. Finally, 2‐MeSADP‐induced ERK phosphorylation was inhibited in the presence of daphnetin.We conclude that daphnetin plays a role in platelet function by inhibiting the TxA2 generation through the regulation of ERK phosphorylation.

Antiplatelet Activity of Tetramethylpyrazine via Regulation of the P2Y12 Receptor Downstream Signaling Pathway

Background Tetramethylpyrazine (TMP) is an alkaloid in Chinese herbal medicine, which possesses antiplatelet activity. TMP inhibits platelet activation in many ways. The platelet P2Y12 receptor for adenosine 5′ diphosphate (ADP) plays a central role in platelet function, hemostasis, and thrombosis. Here, we investigated the inhibitory effect of TMP on P2Y12 receptor-related platelet function. Methods The inhibitory potential of TMP was assessed using agonist-induced platelet aggregation, flow cytometric analysis of CD62p expression, PAC-1 activation, and fibrin clot retraction. After the P2Y12 receptor-related signaling pathway was inhibited using the blocker, platelet activation was studied by platelet aggregation, CD62p expression, and PAC-1 activation. The secretion of cyclic adenosine monophosphate (cAMP) was measured using enzyme-linked immunosorbent assay (ELISA), and the expression of signaling pathway protein, phosphorylation of vasodilator-stimulated phosphoprotein, and phosphorylation of Akt were investigated using western blotting. The release of platelet inflammatory mediators was measured using ELISA. Results TMP had an antiplatelet effect by inhibiting ADP-induced aggregation, P-selectin secretion, and glycoprotein (GP) IIb/IIIa expression and reducing the release of atherosclerotic-related inflammatory mediators (sCD40L and IL-1β). TMP decreased the area of clot retraction, reflecting inhibition of GPIIb/IIIa activation. TMP inhibited adenosine diphosphate-induced platelet activation via increased cAMP production, VASPser157 phosphorylation, and Akt dephosphorylation. Conclusion TMP selectively inhibits ADP-induced platelet activation via P2Y12 receptor-related signaling pathways.

Deletion of Grin1 in mouse megakaryocytes reveals NMDA receptor role in platelet function and proplatelet formation

AbstractThe process of proplatelet formation (PPF) requires coordinated interaction between megakaryocytes (MKs) and the extracellular matrix (ECM), followed by a dynamic reorganization of the actin and microtubule cytoskeleton. Localized fluxes of intracellular calcium ions (Ca2+) facilitate MK-ECM interaction and PPF. Glutamate-gated N-methyl-D-aspartate receptor (NMDAR) is highly permeable to Ca2+. NMDAR antagonists inhibit MK maturation ex vivo; however, there are no in vivo data. Using the Cre-loxP system, we generated a platelet lineage–specific knockout mouse model of reduced NMDAR function in MKs and platelets (Pf4-Grin1−/− mice). Effects of NMDAR deletion were examined using well-established assays of platelet function and production in vivo and ex vivo. We found that Pf4-Grin1−/− mice had defects in megakaryopoiesis, thrombopoiesis, and platelet function, which manifested as reduced platelet counts, lower rates of platelet production in the immune model of thrombocytopenia, and prolonged tail bleeding time. Platelet activation was impaired to a range of agonists associated with reduced Ca2+ responses, including metabotropic like, and defective platelet spreading. MKs showed reduced colony and proplatelet formation. Impaired reorganization of intracellular F-actin and α-tubulin was identified as the main cause of reduced platelet function and production. Pf4-Grin1−/− MKs also had lower levels of transcripts encoding crucial ECM elements and enzymes, suggesting NMDAR signaling is involved in ECM remodeling. In summary, we provide the first genetic evidence that NMDAR plays an active role in platelet function and production. NMDAR regulates PPF through a mechanism that involves MK-ECM interaction and cytoskeletal reorganization. Our results suggest that NMDAR helps guide PPF in vivo.

EP017: GNE gene variants associated with thrombocytopenia with or without GNE myopathy

GNE Myopathy is a rare, degenerative skeletal muscle disease, first presenting with anterior tibialis weakness then progressing proximally, relatively sparing the quadriceps. Symptoms usually develop in early adulthood (between 20-40 years) and include bilateral foot drop and rimmed vacuoles on muscle biopsy. Most patients become wheelchair dependent within a decade after onset. Upper limbs are usually involved around a decade later than the lower limbs. GNE myopathy is caused by biallelic variants in GNE, which encodes the rate limiting bifunctional enzyme in sialic acid synthesis, UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), leading to decreased sialic acid production. Sialic acid is a negatively charged sugar, that serves as the terminal sugar of many N- and O-linked glycoproteins; it serves many functions in cell interactions and signaling. Recently, GNE variants have been associated with thrombocytopenia, with or without manifestations of myopathy. To investigate this phenomenon, we performed a literature review and analyzed data of patients in our GNE myopathy prospective natural history (NH) study. Data were collected through a prospective NH study of individuals with GNE myopathy (NCT1417533). Patients are admitted to the NIH Clinical Center for investigations, including physical function tests, questionnaires, blood analysis (including a complete blood count at each visit), and cardiac and pulmonary function tests. Patient platelet data were compared to normal values. For the literature review, two authors performed a search of all reports including GNE and thrombocytopenia. Clinical manifestations as well as variants identified were compared to those of our cohort. We analyzed a total of 126 platelet counts obtained from 51 GNE myopathy patients (61% female), between 21 and 65 years of age at their baseline to our NH study, with at least one platelet count obtained during subsequent visits. The average platelet count for females was 251 X 109 cells/L (range 144–390) and 205 X 109 cells/L for males (range 103-300), which are within the normal range for our laboratory (173–369 and 161-347, respectively). However, these values are lower than the expected means of 271 for females (p < 0.001) or 254 for males (p < 0.0001), based upon our lab’s normal ranges. A total of 8 individuals were below the normal range at some point, but above the pathologic thrombocytopenia cutoff (100). None presented signs of thrombocytopenia. The literature review yielded 25 patients with thrombocytopenia (17 without myopathy and 8 with myopathy) and biallelic GNE variants. The GNE variants in the 25 patients included 7 novel variants not reported in individuals with GNE myopathy and 11 variants previously associated with GNE myopathy. The novel variants were not localized to specific functional GNE domains but were scattered across the entire GNE gene and found in Chinese, European, Pakistani, Thai, and Palestinian Arab populations. None of our 51 NH study patients had any of the 18 GNE variants associated with thrombocytopenia. Although there was no evident thrombocytopenia in our NH study cohort, the average platelet count was slightly lower than expected, suggesting a potential role of GNE in platelet survival. Previous studies indicate that sialic acid has a significant role in platelet function. Mice deficient in a sialyltransferase had decreased sialic acid and platelet survival times, leading to thrombocytopenia. Patients with GNE myopathy and thrombocytopenia had increased reticulated platelets, suggesting that pathogenic GNE variants may contribute to platelet surface hyposialylation, resulting in increased platelet clearance by the Ashwell-Morell receptor.

Increased Let-7e expression is associated with long-term all-cause mortality and antiplatelet treatment in patients with type 2 diabetes mellitus

Abstract Background In the light of growing prevalence of type 2 diabetes mellitus (T2DM), efforts are made to discover novel biomarkers. MicroRNAs (miRNAs-miR) are non-coding RNAs used in various processes involved in regulating gene expression which play a role in platelet function. Purpose To analyze the ability of platelet-derived miRNAs in prediction of mortality and response to antiplatelet treatment among T2DM-patients. Methods 252 diabetic subjects were enrolled and were receiving either acetylsalicylic acid (ASA) 75mg (65%) or 150 mg (15%) or clopidogrel (19%). Plasma miR-126, miR-223, miR-125a-3p and Let-7e expressions were assessed by qRT-PCR and compared between the patients who survived and those who died. Median observation time was 5.9 years. Adjusted Cox-regression analysis was used for prediction of mortality. Differential miRNAs expression due to different antiplatelet treatment was analyzed. Results ROC curve analysis revealed increasing concentrations of miR-126, Let-7e and miR-125a-3p levels had a diagnostic ability for prediction of long-term all-cause mortality (c-index=0.75, p&amp;lt;0.001; 0.72, p&amp;lt;0.001; 0.72, p=0.001, respectively). Multivariate Cox regression model revealed high miR-126 and Let-7e expressions which were strong and independent predictors of all-cause long-term mortality (HR=5.08, 95% CI: 1.92–13.43; p=0.001; HR=5.94, 95% CI: 1.98–17.79; p=0.001,respectively). After including all miRNAs into one multivariate Cox regression model, only Let-7e was predictive of future occurrence of long-term all-cause death (HR=7.83, 95% CI: 1.2–51.1; p=0.032). MiR-126, Let-7e and miR-223 expressions in the clopidogrel group were significantly higher than in the ASA group (p=0.014; p=0.013; p=0.028, respectively). Conclusions Let-7e expression is a strong and independent predictor of long-term all-cause mortality among patients with T2DM. MiR-223, miR-126 and Let-7e present significant interactions with antiplatelet treatment and clinical outcomes. Funding Acknowledgement Type of funding sources: None. Figure 1Table 1

The secreted tyrosine kinase VLK is essential for normal platelet activation and thrombus formation

AbstractTyrosine phosphorylation of extracellular proteins is observed in cell cultures and in vivo, but little is known about the functional roles of tyrosine phosphorylation of extracellular proteins. Vertebrate lonesome kinase (VLK) is a broadly expressed secretory pathway tyrosine kinase present in platelet α-granules. It is released from platelets upon activation and phosphorylates substrates extracellularly. Its role in platelet function, however, has not been previously studied. In human platelets, we identified phosphorylated tyrosines mapped to luminal or extracellular domains of transmembrane and secreted proteins implicated in the regulation of platelet activation. To determine the role of VLK in extracellular tyrosine phosphorylation and platelet function, we generated mice with a megakaryocyte/platelet-specific deficiency of VLK. Platelets from these mice are normal in abundance and morphology but have significant changes in function both in vitro and in vivo. Resting and thrombin-stimulated VLK-deficient platelets exhibit a significant decrease in several tyrosine phosphobands. Results of functional testing of VLK-deficient platelets show decreased protease-activated receptor 4–mediated and collagen-mediated platelet aggregation but normal responses to adenosine 5′-diphosphate. Dense granule and α-granule release are reduced in these platelets. Furthermore, VLK-deficient platelets exhibit decreased protease-activated receptor 4–mediated Akt (S473) and Erk1/2 (T202/Y204) phosphorylation, indicating altered proximal signaling. In vivo, mice lacking VLK in megakaryocytes/platelets display strongly reduced platelet accumulation and fibrin formation after laser-induced injury of cremaster arterioles compared with control mice but with normal bleeding times. These studies show that the secretory pathway tyrosine kinase VLK is critical for stimulus-dependent platelet activation and thrombus formation, providing the first evidence that a secreted protein kinase is required for normal platelet function.

Lipids and Antiplatelet Therapy: Important Considerations and Future Perspectives.

Lipids play an essential role in platelet functions. It is known that polyunsaturated fatty acids play a role in increasing platelet reactivity and that the prothrombotic phenotype plays a crucial role in the occurrence of major adverse cardiovascular events. The ongoing increase in cardiovascular diseases’ incidence emphasizes the importance of research linking lipids and platelet function. In particular, the rebound phenomenon that accompanies discontinuation of clopidogrel in patients receiving dual antiplatelet therapy has been associated with changes in the lipid profile. Our many years of research underline the importance of reduced HDL values for the risk of such a rebound effect and the occurrence of thromboembolic events. Lipids are otherwise a heterogeneous group of molecules, and their signaling molecules are not deposited but formed “on-demand” in the cell. On the other hand, exosomes transmit lipid signals between cells, and the profile of such changes can be monitored by lipidomics. Changes in the lipid profile are organ-specific and may indicate new drug action targets.

Influence of Antiplatelet Agents on the Lipid Composition of Platelet Plasma Membrane: A Lipidomics Approach with Ticagrelor and Its Active Metabolite.

Lipids contained in the plasma membrane of platelets play an important role in platelet function. Modifications in the lipid composition can fluidify or rigidify the environment around embedded receptors, in order to facilitate the access of the receptor by the drug. However, data concerning the lipid composition of platelet plasma membrane need to be updated. In addition, data on the impact of drugs on plasma membrane composition, in particular antiplatelet agents, remain sparse. After isolation of platelet plasma membrane, we assessed, using lipidomics, the effect of ticagrelor, a P2Y12 antagonist, and its active metabolite on the lipid composition of these plasma membranes. We describe the exact lipid composition of plasma membrane, including all sub-species. Ticagrelor and its active metabolite significantly increased cholesterol and phosphatidylcholine ether with short saturated acyl chains 16:0/16:0, and decreased phosphatidylcholine, suggesting overall rigidification of the membrane. Furthermore, ticagrelor and its active metabolite decreased some arachidonylated plasmalogens, suggesting a decrease in availability of arachidonic acid from the membrane phospholipids for synthesis of biologically active mediators. To conclude, ticagrelor and its active metabolite seem to influence the lipid environment of receptors embedded in the lipid bilayer and modify the behavior of the plasma membrane.

Influence of long-term proteasome inhibition on platelet responsiveness mediated by bortezomib

Introduction: Although platelets contain a full proteasome system, its role in platelet function is not completely understood yet. Since the proteasome system may be involved in time-delayed processes, platelet responsiveness was investigated after long-term, bortezomib-mediated proteasome inhibition.Materials and Methods: Citrate-anticoagulated whole blood was stored with 5 nM and 1 μM bortezomib for 24 h. Consecutively, aggregation was measured by light transmission in platelet-rich-plasma (PRP). Flow cytometry was performed to determine phosphorylation levels of the vasodilator-stimulated phosphoprotein (VASP), fibrinogen binding, PAC1-antibody binding and purinergic receptor expression in PRP, P2Y12 activity or glycoprotein (GP) Ib and IIb expression in whole blood. P2Y1 and P2X1 activities were assessed by calcium flux-induced fluorescence in washed platelets. Using PRP, adherent platelets on fibrinogen-, collagen- and ristocetin-coated surfaces were visualized and quantified by immunostaining.Results: Under bortezomib, VASP phosphorylation was less inducible and nitric oxide-induced inhibition of fibrinogen binding was slightly reduced. Proteasome inhibition did not tamper adenosine diphosphate-mediated aggregation or purinergic receptor expression and activity. Induced expression of activated fibrinogen receptors and fibrinogen binding were not significantly influenced by incubation with bortezomib for 24 h. Aggregation values with threshold agonist concentrations were increased under bortezomib. Despite unchanged GPIb expression, bortezomib-treated platelets showed enhanced adhesion on coated surfaces.Conclusions: In platelets incubated for 24 h, bortezomib mediates a slight attenuation of inhibitory signaling, associated with facilitated platelet aggregation using threshold agonist concentrations and enhanced adhesion on agonist-coated surfaces.