Early Endosomal GTPase Rab5 (Ras-Related Protein in Brain 5) Regulates GPIbß (Glycoprotein Ib Subunit ß) Trafficking and Platelet Production In Vitro.

OBJECTIVE: Maturation of megakaryocytes culminates with extensive membrane rearrangements necessary for proplatelet formation. Mechanisms required for proplatelet extension and origin of membranes are still poorly understood. GTPase Rab5 (Ras-related protein in brain 5) regulates endocytic uptake and homotypic fusion of early endosomes and regulates phosphatidylinositol 3-monophosphate production important for binding of effector proteins during early-to-late endosomal/lysosomal maturation. Approach and Results: To investigate the role of Rab5 in megakaryocytes, we expressed GFP (green fluorescent protein)-coupled Rab5 wild type and its point mutants Q79L (active) and N133L (inactive) in primary murine fetal liver-derived megakaryocytes. Active Rab5 Q79L induced the formation of enlarged early endosomes, while inactive Rab5 N133L caused endosomal fragmentation. Consistently, an increased amount of transferrin internalization in Rab5 Q79L was impaired in Rab5 N133L expressing megakaryocytes, when compared with GFP or Rab5 wild type. Moreover, trafficking of GPIbß (glycoprotein Ib subunit beta), a subunit of major megakaryocytes receptor and membrane marker, was found to be mediated by Rab5 activity. While GPIbß was mostly present along the plasma membrane, and within cytoplasmic vesicles in Rab5 wild type megakaryocytes, it accumulated in the majority of Rab5 Q79L enlarged endosomes. Conversely, Rab5 N133L caused mostly GPIbß plasma membrane retention. Furthermore, Rab5 Q79L expression increased incorporation of the membrane dye (PKH26), indicating higher membrane content. Finally, while Rab5 Q79L increased proplatelet production, inactive Rab5 N133L strongly inhibited it and was coupled with a decrease in late endosomes/lysosomes. Localization of GPIbß in enlarged endosomes was phosphatidylinositol 3-monophosphate dependent. CONCLUSIONS: Taken together, our results demonstrate that Rab5-dependent endocytosis plays an important role in megakaryocytes receptor trafficking, membrane formation, and thrombopoiesis.

The Toll-Like Receptor 2 Ligand Pam2CSK4 Activates Platelet Nuclear Factor-κB and Bruton's Tyrosine Kinase Signaling to Promote Platelet-Endothelial Cell Interactions.

Circulating platelets establish a variety of immunological programs and orchestrate inflammatory responses at the endothelium. Platelets express the innate immunity family of Toll-like receptors (TLRs). While TLR2/TLR1 ligands are known to activate platelets, the effects of TLR2/TLR6 ligands on platelet function remain unclear. Here, we aim to determine whether the TLR2/TLR6 agonists Pam2CSK4 and FSL-1 activate human platelets. In addition, human umbilical vein endothelial cells (HUVECs) and platelets were co-cultured to analyze the role of platelet TLR2/TLR6 on inflammation and adhesion to endothelial cells. Pam2CSK4, but not FSL-1, induced platelet granule secretion and integrin αIIbß3 activation in a concentration-dependent manner. Moreover, Pam2CSK4 promoted platelet aggregation and increased platelet adhesion to collagen-coated surfaces. Mechanistic studies with blocking antibodies and pharmacologic inhibitors demonstrated that the TLR2/Nuclear factor-κB axis, Bruton's-tyrosine kinase, and a secondary ADP feedback loop are involved in Pam2CSK4-induced platelet functional responses. Interestingly, Pam2CSK4 showed cooperation with immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling to enhance platelet activation. Finally, the presence of platelets increased inflammatory responses in HUVECs treated with Pam2CSK4, and platelets challenged with Pam2CSK4 showed increased adhesion to HUVECs under static and physiologically relevant flow conditions. Herein, we define a functional role for platelet TLR2-mediated signaling, which may represent a druggable target to dampen excessive platelet activation in thrombo-inflammatory diseases.

Inhibition of Phosphodiesterase 3A by Cilostazol Dampens Proinflammatory Platelet Functions.

OBJECTIVE: platelets possess not only haemostatic but also inflammatory properties, which combined are thought to play a detrimental role in thromboinflammatory diseases such as acute coronary syndromes and stroke. Phosphodiesterase (PDE) 3 and -5 inhibitors have demonstrated efficacy in secondary prevention of arterial thrombosis, partially mediated by their antiplatelet action. Yet it is unclear whether such inhibitors also affect platelets' inflammatory functions. Here, we aimed to examine the effect of the PDE3A inhibitor cilostazol and the PDE5 inhibitor tadalafil on platelet function in various aspects of thromboinflammation. Approach and results: cilostazol, but not tadalafil, delayed ex vivo platelet-dependent fibrin formation under whole blood flow over type I collagen at 1000 s-1. Similar results were obtained with blood from Pde3a deficient mice, indicating that cilostazol effects are mediated via PDE3A. Interestingly, cilostazol specifically reduced the release of phosphatidylserine-positive extracellular vesicles (EVs) from human platelets while not affecting total EV release. Both cilostazol and tadalafil reduced the interaction of human platelets with inflamed endothelium under arterial flow and the release of the chemokines CCL5 and CXCL4 from platelets. Moreover, cilostazol, but not tadalafil, reduced monocyte recruitment and platelet-monocyte interaction in vitro. CONCLUSIONS: this study demonstrated yet unrecognised roles for platelet PDE3A and platelet PDE5 in platelet procoagulant and proinflammatory responses.

Monoamine Oxidase as a Potential Biomarker of the Efficacy of Treatment of Mental Disorders.

The review summarizes the results of our own studies and published data on the biological markers of psychiatric disorders, with special emphasis on the activity of platelet monoamine oxidase. Pharmacotherapy studies in patients with the mixed anxiety-depressive disorder and first episode of schizophrenia have shown that the activity of platelet monoamine oxidase could serve as a potential biomarker of the efficacy of therapeutic interventions in these diseases.

Endothelial microparticle-associated protein disulfide isomerase increases platelet activation in diabetic coronary heart disease.

BACKGROUND: Endothelial microparticles (EMPs) carrying the protein disulfide isomerase (PDI) might play a key role in promoting platelet activation in diabetes. This study aimed to examine the activation of platelets, the amounts of MPs, PMPs, and EMPs, and the concentration and activity of PDI in patients with diabetic coronary heart disease (CHD) and non-diabetic CHD. METHODS: Patients with CHD (n=223) were divided as non-diabetic CHD (n=121) and diabetic CHD (n=102). Platelet activation biomarkers, circulating microparticles (MPs), the concentration of protein disulfide isomerase (PDI), and MP-PDI activity were determined. The effect of EMPs on platelet activation was investigated in vitro. Allosteric GIIb/IIIa receptors that bind to PDI were detected by a proximity ligation assay (PLA). RESULTS: Platelet activation, platelet-leukocyte aggregates, circulating MPs, EMPs, PDI, and MP-PDI activity in the diabetic CHD group were significantly higher than in the non-diabetic CHD group (P<0.05). Diabetes (P=0.006) and heart rate <60 bpm (P=0.047) were associated with elevated EMPs. EMPs from diabetes increased CD62p on the surface of the platelets compared with the controls (P<0.01), which could be inhibited by the PDI inhibitor RL90 (P<0.05). PLA detected the allosteric GIIb/IIIa receptors caused by EMP-PDI, which was also inhibited by RL90. CONCLUSIONS: In diabetic patients with CHD, platelet activation was significantly high. Diabetes and heart rate <60 bpm were associated with elevated EMPs and simultaneously increased PDI activity on EMP, activating platelets through the allosteric GPIIb/IIIa receptors.

Antiplatelet Activity of Coumarins: In Vitro Assays on COX-1.

Atherosclerotic cardiovascular disease is the leading cause of death in developed countries. Therefore, there is an increasing interest in developing new potent and safe antiplatelet agents. Coumarins are a family of polyphenolic compounds with several pharmacological activities, including platelet aggregation inhibition. However, their antiplatelet mechanism of action needs to be further elucidated. The aim of this study is to provide insight into the biochemical mechanisms involved in this activity, as well as to establish a structure-activity relationship for these compounds. With this purpose, the antiplatelet aggregation activities of coumarin, esculetin and esculin were determined in vitro in human whole blood and platelet-rich plasma, to set the potential interference with the arachidonic acid cascade. Here, the platelet COX activity was evaluated from 0.75 mM to 6.5 mM concentration by measuring the levels of metabolites derived from its activity (MDA and TXB2), together with colorimetric assays performed with the pure recombinant enzyme. Our results evidenced that the coumarin aglycones present the greatest antiplatelet activity at 5 mM and 6.5 mM on aggregometry experiments and inhibiting MDA levels.

PCSK9 Biology and Its Role in Atherothrombosis.

It is now about 20 years since the first case of a gain-of-function mutation involving the as-yet-unknown actor in cholesterol homeostasis, proprotein convertase subtilisin/kexin type 9 (PCSK9), was described. It was soon clear that this protein would have been of huge scientific and clinical value as a therapeutic strategy for dyslipidemia and atherosclerosis-associated cardiovascular disease (CVD) management. Indeed, PCSK9 is a serine protease belonging to the proprotein convertase family, mainly produced by the liver, and essential for metabolism of LDL particles by inhibiting LDL receptor (LDLR) recirculation to the cell surface with the consequent upregulation of LDLR-dependent LDL-C levels. Beyond its effects on LDL metabolism, several studies revealed the existence of additional roles of PCSK9 in different stages of atherosclerosis, also for its ability to target other members of the LDLR family. PCSK9 from plasma and vascular cells can contribute to the development of atherosclerotic plaque and thrombosis by promoting platelet activation, leukocyte recruitment and clot formation, also through mechanisms not related to systemic lipid changes. These results further supported the value for the potential cardiovascular benefits of therapies based on PCSK9 inhibition. Actually, the passive immunization with anti-PCSK9 antibodies, evolocumab and alirocumab, is shown to be effective in dramatically reducing the LDL-C levels and attenuating CVD. While monoclonal antibodies sequester circulating PCSK9, inclisiran, a small interfering RNA, is a new drug that inhibits PCSK9 synthesis with the important advantage, compared with PCSK9 mAbs, to preserve its pharmacodynamic effects when administrated every 6 months. Here, we will focus on the major understandings related to PCSK9, from its discovery to its role in lipoprotein metabolism, involvement in atherothrombosis and a brief excursus on approved current therapies used to inhibit its action.

Bioenergetic and inflammatory systemic phenotypes in Alzheimer's disease APOE ε4-carriers.

We examined the impact of an APOE ε4 genotype on Alzheimer's disease (AD) subject platelet and lymphocyte metabolism. Mean platelet mitochondrial cytochrome oxidase Vmax activity was lower in APOE ε4 carriers and lymphocyte Annexin V, a marker of apoptosis, was significantly higher. Proteins that mediate mitophagy and energy sensing were higher in APOE ε4 lymphocytes which could represent compensatory changes and recapitulate phenomena observed in post-mortem AD brains. Analysis of the lipid synthesis pathway found higher AceCSI, ATP CL, and phosphorylated ACC levels in APOE ε4 lymphocytes. Lymphocyte ACC changes were also observed in post-mortem brain tissue. Lymphocyte RNAseq showed lower APOE ε4 carrier sphingolipid Transporter 3 (SPNS3) and integrin Subunit Alpha 1 (ITGA1) expression. RNAseq pathway analysis revealed APOE ε4 alleles activated inflammatory pathways and modulated bioenergetic signaling. These findings support a relationship between APOE genotype and bioenergetic pathways and indicate platelets and lymphocytes from APOE ε4 carriers exist in a state of bioenergetic stress. Neither medication use nor brain-localized AD histopathology can account for these findings, which define an APOE ε4-determined molecular and systemic phenotype that informs AD etiology.

Protein kinase A activity and NO are involved in the regulation of crucian carp (Carassius carassius) red blood cell osmotic fragility.

Activation of the cAMP pathway by ß-adrenergic stimulation and cGMP pathway by activation of guanylate cyclase substantially affects red blood cell (RBC) membrane properties in mammals. However, whether similar mechanisms are involved in RBC regulation of lower vertebrates, especially teleosts, is not elucidated yet. In this study, we evaluated the effects of adenylate cyclase activation by epinephrine and forskolin, guanylate cyclase activation by sodium nitroprusside, and the role of Na+/H+-exchanger in the changes of osmotic fragility and regulatory volume decrease (RVD) response in crucian carp RBCs. Western blot analysis of protein kinase A and protein kinase G substrate phosphorylation revealed that changes in osmotic fragility were regulated via the protein kinase A, but not protein kinase G signaling pathway. At the same time, the RVD response in crucian carp RBCs was not affected either by activation of adenylate or guanylate cyclase. Adenylate cyclase/protein kinase A activation significantly decreased RBC osmotic fragility, i.e., increased cell rigidity. Inhibition of Na+/H+-exchanger by amiloride had no effect on the epinephrine-mediated decrease of RBC osmotic fragility. NO donor SNP did not activate guanylate cyclase, however affected RBCs osmotic fragility by protein kinase G-independent mechanisms. Taken together, our data demonstrated that the cAMP/PKA signaling pathway and NO are involved in the regulation of crucian carp RBC osmotic fragility, but not in RVD response. The authors confirm that the study has no clinical trial.

Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT): Targeting Pathomechanisms with Bruton Tyrosine Kinase Inhibitors.

A series of cases with rare thromboembolic incidents including cerebral sinus vein thrombosis (some of them fatal) and concomitant thrombocytopenia occurring shortly after vaccination with the coronavirus disease 2019 (COVID-19) vaccine AZD1222 (Vaxzevria) have caused significant concern and led to its temporary suspension in many countries. Immediate laboratory efforts in four of these patients have identified a tentative pathomechanism underlying this syndrome termed initially vaccine-induced prothrombotic immune thrombocytopenia (VIPIT) and renamed recently vaccine-induced immune thrombotic thrombocytopenia (VITT). It encompasses the presence of platelet-activating antibodies to platelet factor-4/heparin complexes, possibly emulated by polyanionic constituents of AZD1222, and thus resembles heparin-induced thrombocytopenia (HIT). Because these immune complexes bind and activate platelets via Fcγ receptor IIA (FcγRIIA), high-dose intravenous immunoglobulin G has been suggested for treatment of VITT in addition to non-heparin anticoagulants. Here we propose inhibitors of Bruton tyrosine kinase (Btk) approved for B cell malignancies (e.g., ibrutinib) as another therapeutic option in VITT, as they are expected to pleiotropically target multiple pathways downstream of FcγRIIA-mediated Btk activation, for example, as demonstrated for the effective inhibition of platelet aggregation, dense granule secretion, P-selectin expression and platelet-neutrophil aggregate formation stimulated by FcγRIIA cross-linking. Moreover, C-type lectin-like receptor CLEC-2- and GPIb-mediated platelet activation, the interactions and activation of monocytes and the release of neutrophil extracellular traps, as encountered in HIT, could be attenuated by Btk inhibitors. As a paradigm for emergency repurposing of approved drugs in COVID-19, off-label use of Btk inhibitors in a low-dose range not affecting haemostatic functions could thus be considered a sufficiently safe option to treat VITT.

PAR-4/Ca2+-calpain pathway activation stimulates platelet-derived microparticles in hyperglycemic type 2 diabetes.

BACKGROUND: Patients with type 2 diabetes (T2DM) have a prothrombotic state that needs to be fully clarified; microparticles (MPs) have emerged as mediators and markers of this condition. Thus, we investigate, in vivo, in T2DM either with good (HbA1c ≤ 7.0%; GGC) or poor (HbA1c > 7.0%; PGC) glycemic control, the circulating levels of MPs, and in vitro, the molecular pathways involved in the release of MPs from platelets (PMP) and tested their pro-inflammatory effects on THP-1 transformed macrophages. METHODS: In 59 T2DM, and 23 control subjects with normal glucose tolerance (NGT), circulating levels of CD62E+, CD62P+, CD142+, CD45+ MPs were determined by flow cytometry, while plasma levels of ICAM-1, VCAM-1, IL-6 by ELISA. In vitro, PMP release and activation of isolated platelets from GGC and PGC were investigated, along with their effect on IL-6 secretion in THP-1 transformed macrophages. RESULTS: We found that MPs CD62P+ (PMP) and CD142+ (tissue factor-bearing MP) were significantly higher in PGC T2DM than GGC T2DM and NGT. Among MPs, PMP were also correlated with HbA1c and IL-6. In vitro, we showed that acute thrombin exposure stimulated a significantly higher PMP release in PGC T2DM than GGC T2DM through a more robust activation of PAR-4 receptor than PAR-1 receptor. Treatment with PAR-4 agonist induced an increased release of PMP in PGC with a Ca2+-calpain dependent mechanism since this effect was blunted by calpain inhibitor. Finally, the uptake of PMP derived from PAR-4 treated PGC platelets into THP-1 transformed macrophages promoted a marked increase of IL-6 release compared to PMP derived from GGC through the activation of the NF-kB pathway. CONCLUSIONS: These results identify PAR-4 as a mediator of platelet activation, microparticle release, and inflammation, in poorly controlled T2DM.

Hydroxytyrosol as anti-parkinsonian molecule: Assessment using in-silico and MPTP-induced Parkinson's disease model.

3-Hydroxytyrosol (HXT) is a natural polyphenol present in extra virgin olive oil. It is a key component of Mediterranean diet and is known for its strong antioxidant activity. The present study evaluated the potential of HXT as an anti-parkinsonian molecule in terms of its ability to inhibit MAO-B and thereby maintaining dopamine (DA) levels in Parkinson's disease (PD). In-silico molecular docking study followed by MMGBSA binding free energy calculation revealed that HXT has a strong binding affinity for MAO-B in comparison to MAO-A. Moreover, rasagiline and HXT interacted with the similar binding sites and modes of interactions. Additionally, molecular dynamics simulation studies revealed stable nature of HXT-MAO-B interaction and also provided information about the amino acid residues involved in binding. Moreover, in vitro studies revealed that HXT inhibited MAO-B in human platelets with IC50 value of 7.78 µM. In vivo studies using MPTP-induced mouse model of PD revealed increase in DA levels with concomitant decrease in DA metabolites (DOPAC and HVA) on HXT treatment. Furthermore, MAO-B activity was also inhibited on HXT administration to PD mice. In addition, HXT treatment prevented MPTP-induced loss of DA neurons in substantia nigra and their nerve terminals in the striatum. HXT also attenuated motor impairments in PD mice assessed by catalepsy bar, narrow beam walk and open field tests. Thus, the present findings reveal HXT as a potential inhibitor of MAO-B, which may be used as a lead molecule for the development of therapeutics for PD.

Shear and Integrin Outside-In Signaling Activate NADPH-Oxidase 2 to Promote Platelet Activation.

[Figure: see text].

Assessment of the effects of Syk and BTK inhibitors on GPVI-mediated platelet signaling and function.

Spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (BTK) play critical roles in platelet physiology, facilitating intracellular immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling downstream of platelet glycoprotein VI (GPVI) and GPIIb/IIIa receptors. Small molecule tyrosine kinase inhibitors (TKIs) targeting Syk and BTK have been developed as antineoplastic and anti-inflammatory therapeutics and have also gained interest as antiplatelet agents. Here, we investigate the effects of 12 different Syk and BTK inhibitors on GPVI-mediated platelet signaling and function. These inhibitors include four Syk inhibitors, Bay 61-3606, R406 (fostamatinib), entospletinib, TAK-659; four irreversible BTK inhibitors, ibrutinib, acalabrutinib, ONO-4059 (tirabrutinib), AVL-292 (spebrutinib); and four reversible BTK inhibitors, CG-806, BMS-935177, BMS-986195, and fenebrutinib. In vitro, TKIs targeting Syk or BTK reduced platelet adhesion to collagen, dense granule secretion, and alpha granule secretion in response to the GPVI agonist cross-linked collagen-related peptide (CRP-XL). Similarly, these TKIs reduced the percentage of activated integrin αIIbß3 on the platelet surface in response to CRP-XL, as determined by PAC-1 binding. Although all TKIs tested inhibited phospholipase C γ2 (PLCγ2) phosphorylation following GPVI-mediated activation, other downstream signaling events proximal to phosphoinositide 3-kinase (PI3K) and PKC were differentially affected. In addition, reversible BTK inhibitors had less pronounced effects on GPIIb/IIIa-mediated platelet spreading on fibrinogen and differentially altered the organization of PI3K around microtubules during platelets spreading on fibrinogen. Select TKIs also inhibited platelet aggregate formation on collagen under physiological flow conditions. Together, our results suggest that TKIs targeting Syk or BTK inhibit central platelet functional responses but may differentially affect protein activities and organization in critical systems downstream of Syk and BTK in platelets.

Role of Rho-GTPases in megakaryopoiesis.

Megakaryocytes (MKs) are the bone marrow (BM) cells that generate blood platelets by a process that requires: i) polyploidization responsible for the increased MK size and ii) cytoplasmic organization leading to extension of long pseudopods, called proplatelets, through the endothelial barrier to allow platelet release into blood. Low level of localized RHOA activation prevents actomyosin accumulation at the cleavage furrow and participates in MK polyploidization. In the platelet production, RHOA and CDC42 play opposite, but complementary roles. RHOA inhibits both proplatelet formation and MK exit from BM, whereas CDC42 drives the development of the demarcation membranes and MK migration in BM. Moreover, the RhoA or Cdc42 MK specific knock-out in mice and the genetic alterations in their down-stream effectors in human induce a thrombocytopenia demonstrating their key roles in platelet production. A better knowledge of Rho-GTPase signalling is thus necessary to develop therapies for diseases associated with platelet production defects.Abbreviations: AKT: Protein Kinase BARHGEF2: Rho/Rac Guanine Nucleotide Exchange Factor 2ARP2/3: Actin related protein 2/3BM: Bone marrowCDC42: Cell division control protein 42 homologCFU-MK: Colony-forming-unit megakaryocyteCIP4: Cdc42-interacting protein 4mDIA: DiaphanousDIAPH1; Protein diaphanous homolog 1ECT2: Epithelial Cell Transforming Sequence 2FLNA: Filamin AGAP: GTPase-activating proteins or GTPase-accelerating proteinsGDI: GDP Dissociation InhibitorGEF: Guanine nucleotide exchange factorHDAC: Histone deacetylaseLIMK: LIM KinaseMAL: Megakaryoblastic leukaemiaMARCKS: Myristoylated alanine-rich C-kinase substrateMKL: Megakaryoblastic leukaemiaMLC: Myosin light chainMRTF: Myocardin Related Transcription FactorOTT: One-Twenty Two ProteinPACSIN2: Protein Kinase C And Casein Kinase Substrate In Neurons 2PAK: P21-Activated KinasePDK: Pyruvate Dehydrogenase kinasePI3K: Phosphoinositide 3-kinasePKC: Protein kinase CPTPRJ: Protein tyrosine phosphatase receptor type JRAC: Ras-related C3 botulinum toxin substrate 1RBM15: RNA Binding Motif Protein 15RHO: Ras homologousROCK: Rho-associated protein kinaseSCAR: Suppressor of cAMP receptorSRF: Serum response factorSRC: SarcTAZ: Transcriptional coactivator with PDZ motifTUBB1: Tubulin ß1VEGF: Vascular endothelial growth factorWAS: Wiskott Aldrich syndromeWASP: Wiskott Aldrich syndrome proteinWAVE: WASP-family verprolin-homologous proteinWIP: WASP-interacting proteinYAP: Yes-associated protein.

Heparanase expression and activity are increased in platelets during clinical sepsis.

BACKGROUND: Heparanase (HPSE) is the only known mammalian enzyme that can degrade heparan sulfate. Heparan sulfate proteoglycans are essential components of the glycocalyx, and maintain physiological barriers between the blood and endothelial cells. HPSE increases during sepsis, which contributes to injurious glyocalyx degradation, loss of endothelial barrier function, and mortality. OBJECTIVES: As platelets are one of the most abundant cellular sources of HPSE, we sought to determine whether HPSE expression and activity increases in human platelets during clinical sepsis. We also examined associations between platelet HPSE expression and clinical outcomes. PATIENTS/METHODS: Expression and activity of HPSE was determined in platelets isolated from septic patients (n = 59) and, for comparison, sex-matched healthy donors (n = 46) using complementary transcriptomic, proteomic, and functional enzymatic assays. Septic patients were followed for the primary outcome of mortality, and clinical data were captured prospectively for septic patients. RESULTS: The mRNA expression of HPSE was significantly increased in platelets isolated from septic patients. Ribosomal footprint profiling, followed by [S35] methionine labeling assays, demonstrated that HPSE mRNA translation and HPSE protein synthesis were significantly upregulated in platelets during sepsis. While both the pro- and active forms of HPSE protein increased in platelets during sepsis, only the active form of HPSE protein significantly correlated with sepsis-associated mortality. Consistent with transcriptomic and proteomic upregulation, HPSE enzymatic activity was also increased in platelets during sepsis. CONCLUSIONS: During clinical sepsis HPSE, translation, and enzymatic activity are increased in platelets. Increased expression of the active form of HPSE protein is associated with sepsis-associated mortality.

Rho GTPase regulation of reactive oxygen species generation and signalling in platelet function and disease.

Platelets are master regulators and effectors of haemostasis with increasingly recognized functions as mediators of inflammation and immune responses. The Rho family of GTPase members Rac1, Cdc42 and RhoA are known to be major components of the intracellular signalling network critical to platelet shape change and morphological dynamics, thus playing a major role in platelet spreading, secretion and thrombus formation. Initially linked to the regulation of actomyosin contraction and lamellipodia formation, recent reports have uncovered non-canonical functions of platelet RhoGTPases in the regulation of reactive oxygen species (ROS), where intrinsically generated ROS modulate platelet function and contribute to thrombus formation. Platelet RhoGTPases orchestrate oxidative processes and cytoskeletal rearrangement in an interconnected manner to regulate intracellular signalling networks underlying platelet activity and thrombus formation. Herein we review our current knowledge of the regulation of platelet ROS generation by RhoGTPases and their relationship with platelet cytoskeletal reorganization, activation and function.

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.

Targeting Thymidine Phosphorylase With Tipiracil Hydrochloride Attenuates Thrombosis Without Increasing Risk of Bleeding in Mice.

OBJECTIVE: Current antiplatelet medications increase the risk of bleeding, which leads to a clear clinical need in developing novel mechanism-based antiplatelet drugs. TYMP (Thymidine phosphorylase), a cytoplasm protein that is highly expressed in platelets, facilitates multiple agonist-induced platelet activation, and enhances thrombosis. Tipiracil hydrochloride (TPI), a selective TYMP inhibitor, has been approved by the Food and Drug Administration for clinical use. We tested the hypothesis that TPI is a safe antithrombotic medication. Approach and Results: By coexpression of TYMP and Lyn, GST (glutathione S-transferase) tagged Lyn-SH3 domain or Lyn-SH2 domain, we showed the direct evidence that TYMP binds to Lyn through both SH3 and SH2 domains, and TPI diminished the binding. TYMP deficiency significantly inhibits thrombosis in vivo in both sexes. Pretreatment of platelets with TPI rapidly inhibited collagen- and ADP-induced platelet aggregation. Under either normal or hyperlipidemic conditions, treating wild-type mice with TPI via intraperitoneal injection, intravenous injection, or gavage feeding dramatically inhibited thrombosis without inducing significant bleeding. Even at high doses, TPI has a lower bleeding side effect compared with aspirin and clopidogrel. Intravenous delivery of TPI alone or combined with tissue plasminogen activator dramatically inhibited thrombosis. Dual administration of a very low dose of aspirin and TPI, which had no antithrombotic effects when used alone, significantly inhibited thrombosis without disturbing hemostasis. CONCLUSIONS: This study demonstrated that inhibition of TYMP, a cytoplasmic protein, attenuated multiple signaling pathways that mediate platelet activation, aggregation, and thrombosis. TPI can be used as a novel antithrombotic medication without the increase in risk of bleeding.

NADPH Oxidases Are Required for Full Platelet Activation In Vitro and Thrombosis In Vivo but Dispensable for Plasma Coagulation and Hemostasis.

OBJECTIVE: Using 3KO (triple NOX [NADPH oxidase] knockout) mice (ie, NOX1-/-/NOX2-/-/NOX4-/-), we aimed to clarify the role of this family of enzymes in the regulation of platelets in vitro and hemostasis in vivo. Approach and Results: 3KO mice displayed significantly reduced platelet superoxide radical generation, which was associated with impaired platelet aggregation, adhesion, and thrombus formation in response to the key agonists collagen and thrombin. A comparison with single-gene knockouts suggested that the phenotype of 3KO platelets is the combination of the effects of the genetic deletion of NOX1 and NOX2, while NOX4 does not show any significant function in platelet regulation. 3KO platelets displayed significantly higher levels of cGMP-a negative platelet regulator that activates PKG (protein kinase G). The inhibition of PKG substantially but only partially rescued the defective phenotype of 3KO platelets, which are responsive to both collagen and thrombin in the presence of the PKG inhibitors KT5823 or Rp-8-pCPT-cGMPs, but not in the presence of the NOS (NO synthase) inhibitor L-NG-monomethyl arginine. In vivo, triple NOX deficiency protected against ferric chloride-driven carotid artery thrombosis and experimental pulmonary embolism, while hemostasis tested in a tail-tip transection assay was not affected. Procoagulatory activity of platelets (ie, phosphatidylserine surface exposure) and the coagulation cascade in platelet-free plasma were normal. CONCLUSIONS: This study indicates that inhibiting NOXs has strong antithrombotic effects partially caused by increased intracellular cGMP but spares hemostasis. NOXs are, therefore, pharmacotherapeutic targets to develop new antithrombotic drugs without bleeding side effects.