Platelet Surface Research Articles

Platelet Toll-Like-Receptor-2 and -4 Mediate Different Immune-Related Responses to Bacterial Ligands.

Background Like immune cells, platelets express toll-like receptors (TLRs) on their surface membrane. TLR2 and TLR4 are able to recognize bacterial antigens and have the potential to influence hemostatic functions and classical intracellular signaling pathways. This study investigated the role of TLR2 and TLR4 for immune-related functions in human platelets. Materials and Methods Washed platelets and neutrophils were prepared from fresh human peripheral blood. Basal-, Pam3CSK4- (as TLR2 agonist) and Lipopolysaccharides (LPS; as TLR4 agonist) -induced CD62P expression, fibrinogen binding and TLR2 or TLR4 expression, intracellular reactive oxygen species (ROS) production in H 2 DCFDA-loaded platelets and uptake of fluorescence-labeled TLR ligands, and fluorophore-conjugated fibrinogen were evaluated by flow cytometry. Analysis of platelet–neutrophil complexes was performed after coincubation of washed platelets and neutrophils in the presence and absence of TLR2 or TLR4 agonists on poly-L-lysine coated surfaces, followed by immunostaining and immunofluorescence imaging. Results Pam3CSK4 rapidly and transiently increased TLR2 and TLR4 expression. Over the course of 30 minutes after activation with Pam3CSK4 and LPS, the expression of both receptors decreased. Pam3CSK4-stimulated intracellular ROS production and the uptake of TLR ligands or fibrinogen much stronger than LPS. Besides, TLR4 activation led to a significant increase of platelet–neutrophil contacts. Conclusion Stimulation leads to rapid mobilization of TLR2 or TLR4 to the platelet surface, presumably followed by receptor internalization along with bound TLR ligands. After activation, platelet TLR2 and TLR4 mediate different immune-related reactions. In particular, TLR2 induces intracellular responses in platelets, whereas TLR4 initiates interactions with other immune cells such as neutrophils.

Anchoring IgG-degrading enzymes to the surface of platelets selectively neutralizes antiplatelet antibodies

AbstractImmune thrombocytopenia (ITP) is an acquired bleeding disorder characterized by immunoglobulin G (IgG)–mediated platelet destruction. Current therapies primarily focus on reducing antiplatelet antibodies using immunosuppression or increasing platelet production with thrombopoietin mimetics. However, there are no universally safe and effective treatments for patients presenting with severe life-threatening bleeding. The IgG-degrading enzyme of Streptococcus pyogenes (IdeS), a protease with strict specificity for IgG, prevents IgG-driven immune disorders in murine models, including ITP. In clinical trials, IdeS prevented IgG-mediated kidney transplant rejection; however, the concentration of IdeS used to remove pathogenic antibodies causes profound hypogammaglobulinemia, and IdeS is immunogenic, which limits its use. Therefore, this study sought to determine whether targeting IdeS to FcγRIIA, a low-affinity IgG receptor on the surface of platelets, neutrophils, and monocytes, would be a viable strategy to decrease the pathogenesis of antiplatelet IgG and reduce treatment-related complications of nontargeted IdeS. We generated a recombinant protein conjugate by site-specifically linking the C-terminus of a single-chain variable fragment from an FcγRIIA antibody, clone IV.3, to the N-terminus of IdeS (scIV.3-IdeS). Platelets treated with scIV.3-IdeS had reduced binding of antiplatelet IgG from patients with ITP and decreased platelet phagocytosis in vitro, with no decrease in normal IgG. Treatment of mice expressing human FcγRIIA with scIV.3-IdeS reduced thrombocytopenia in a model of ITP and significantly improved the half-life of transfused platelets expressing human FcγRIIA. Together, these data suggest that scIV.3-IdeS can selectively remove pathogenic antiplatelet IgG and may be a potential treatment for patients with ITP and severe bleeding.

Exogenous human α-Synuclein acts in vitro as a mild platelet antiaggregant inhibiting α-thrombin-induced platelet activation

α-Synuclein (αSyn) is a small disordered protein, highly conserved in vertebrates and involved in the pathogenesis of Parkinson’s disease (PD). Indeed, αSyn amyloid aggregates are present in the brain of patients with PD. Although the pathogenic role of αSyn is widely accepted, the physiological function of this protein remains elusive. Beyond the central nervous system, αSyn is expressed in hematopoietic tissue and blood, where platelets are a major cellular host of αSyn. Platelets play a key role in hemostasis and are potently activated by thrombin (αT) through the cleavage of protease-activated receptors. Furthermore, both αT and αSyn could be found in the same spatial environment, i.e. the platelet membrane, as αT binds to and activates platelets that can release αSyn from α-granules and microvesicles. Here, we investigated the possibility that exogenous αSyn could interfere with platelet activation induced by different agonists in vitro. Data obtained from distinct experimental techniques (i.e. multiple electrode aggregometry, rotational thromboelastometry, immunofluorescence microscopy, surface plasmon resonance, and steady-state fluorescence spectroscopy) on whole blood and platelet-rich plasma indicate that exogenous αSyn has mild platelet antiaggregating properties in vitro, acting as a negative regulator of αT-mediated platelet activation by preferentially inhibiting P-selectin expression on platelet surface. We have also shown that both exogenous and endogenous (i.e. cytoplasmic) αSyn preferentially bind to the outer surface of activated platelets. Starting from these findings, a coherent model of the antiplatelet function of αSyn is proposed.

A RARE CASE OF GLANZMANN THOMBASTHENIA

Background: Glanzmanns Thrombasthenia (GT) is a GPIIb/IIIa platelet surface receptor condition that is defined by a lack of platelet aggregation, either qualitatively or quantitatively. This receptors physiological function is to bind various sticky plasma proteins that aid platelet adhesion and aggregation, ensuring the development of thrombus at sites of vascular damage. Patient of GT typically presents with menorrhagia, easy bruising, epistaxis, and gum bleeding.Prolonged untreated or unsuccessfully treated hemorrhagia associated with GlanzmannsThrombasthenia may be life threatening. Case Description: A 12 years old girl presented with bleeding since 12 days with complaints of generalized weakness. The patient was then investigated to find the cause of puberty menorrhagia. Several blood investigations were performed, and a final diagnosis of Glanzmanns Thrombasthenia was made. Objective:To highlight a rare case of Glanzmanns Thrombasthenia with β-Thalassemia trait in a 12 years old girl with puberty menorrhagia.

Role of Plasminogen Activation System in Platelet Pathophysiology: Emerging Concepts for Translational Applications.

Traditionally, platelets have been exclusively considered for their procoagulant and antifibrinolytic effects during normal activation of hemostasis. Effectively, activated platelets secrete coagulation factors, expose phosphatidylserine, and promote thrombin and fibrin production. In addition to procoagulant activities, platelets confer resistance of thrombi to fibrinolysis by inducing clot retraction of the fibrin network and release of huge amounts of plasminogen activator inhibitor-1, which is the major physiologic inhibitor of the fibrinolytic cascade. However, the discovery of multiple relations with the fibrinolytic system, also termed Plasminogen Activation System (PAS), has introduced new perspectives on the platelet role in fibrinolysis. Indeed, the activated membrane surface of platelets provides binding sites on which fibrinolytic enzymes can be activated. This review discusses the evidence of the profibrinolytic properties of platelets through the description of PAS components and related proteins that are contained in or bind to platelets. Our analyses of literature data lead to the conclusion that in the initial phase of the hemostatic process, antifibrinolytic effects prevail over profibrinolytic activity, but at later stages, platelets might enhance fibrinolysis through the engagement of PAS components. A better understanding of spatial and temporal characteristics of platelet-mediated fibrinolysis during normal hemostasis could improve therapeutic options for bleeding and thrombotic disorders.

Neutrophil extracellular traps regulate ischemic stroke brain injury.

Ischemic stroke prompts a strong inflammatory response, which is associated with exacerbated outcomes. In this study, we investigated mechanistic regulators of neutrophil extracellular trap (NET) formation in stroke and whether they contribute to stroke outcomes. NET-forming neutrophils were found throughout brain tissue of ischemic stroke patients, and elevated plasma NET biomarkers correlated with worse stroke outcomes. Additionally, we observed increased plasma and platelet surface–expressed high-mobility group box 1 (HMGB1) in stroke patients. Mechanistically, platelets were identified as the critical source of HMGB1 that caused NETs in the acute phase of stroke. Depletion of platelets or platelet-specific knockout of HMGB1 significantly reduced plasma HMGB1 and NET levels after stroke, and greatly improved stroke outcomes. We subsequently investigated the therapeutic potential of neonatal NET-inhibitory factor (nNIF) in stroke. Mice treated with nNIF had smaller brain infarcts, improved long-term neurological and motor function, and enhanced survival after stroke. nNIF specifically blocked NET formation without affecting neutrophil recruitment after stroke. Importantly, nNIF also improved stroke outcomes in diabetic and aged mice and was still effective when given 1 hour after stroke onset. These results support a pathological role for NETs in ischemic stroke and warrant further investigation of nNIF for stroke therapy.

Platelet TAU is Associated with Changes in Depression and Alzheimer's Disease.

Platelets (thrombocytes) are small anuclear cells that play an important role in blood clotting. They are activated and dysfunctional in brain disorders, such as Alzheimer's disease (AD) and depression. Platelets express the amyloid-precursor protein (APP) and release beta-amyloid40 into the blood. Recent evidence reports that platelets also express the microtubule-associated protein tau. In this study, we further characterized the molecular appearance of tau and examined its alterations in patients with neurocognitive impairment. Platelets were isolated from patients with AD, mild cognitive impairment (MCI) or depression and compared to healthy controls. Subsequently, FACS analysis was employed to characterize platelets for platelet surface P-selectin (CD62P). In order to enhance the detection levels, samples were pooled (15 samples per group) and analyzed by Lumipulse Assay, Western blots, and mass spectrometry. Tau is expressed in human platelets and tau levels were decreased in platelets isolated from patients with AD and depression. Additionally, phospho-tau-181 was slightly increased in patients with depression. We show that tau is highly fragmented (20-40 kDa) in the platelet extracts using Western blot analysis. The mass spectrometry data did not show a clear identification of tau in the pooled platelet samples. Our data reveal that tau is found in platelets, possibly in a highly fragmented form. Tau levels may be used as a potential diagnostic approach to differentiate AD and depression from healthy controls.

Differential Inhibition of Platelet Reactivity by Dual Therapy With Aspirin and Low-Dose Rivaroxaban in Peripheral Arterial Disease: A Pilot Study.

Patients with peripheral arterial disease (PAD) benefit from combination therapy with acetylsalicylic acid (ASA, 100 mg, one time per day) plus low-dose rivaroxaban (2.5 mg, two times per day) compared to ASA monotherapy. In particular, major adverse cardiac and limb events were significantly reduced after peripheral endovascular revascularization (EVR). In this pilot study, the platelet activation status in vivo and platelet reactivity in vitro were longitudinally analyzed by flow cytometric assays and calibrated automated thrombography in platelet-rich plasma (PRP) from 10 patients with PAD receiving ASA (100 mg, one time per day) before EVR, ASA plus clopidogrel (75 mg, one time per day) after EVR, and ASA plus rivaroxaban (2.5 mg, two times per day) during a long-term follow-up. Platelet responsiveness to clopidogrel was compared to additional 10 patients with stable PAD and clopidogrel (75 mg, one time per day) monotherapy. ASA plus rivaroxaban treatment resulted in a significantly decreased thrombin peak in PRP for two triggers, namely, low concentration of tissue factor (TF) and thrombin, compared to ASA monotherapy. TF-controlled thrombin generation was additionally characterized by a significantly prolonged lag time in PRP and platelet-free plasma during ASA plus rivaroxaban combination therapy. In comparison, ASA plus clopidogrel treatment presented a significant reduction of the thrombin peak in PRP, which was less pronounced than during subsequent ASA plus rivaroxaban therapy. Platelet responsiveness to clopidogrel was observed for 60% of patients receiving ASA plus clopidogrel and clopidogrel monotherapy, respectively. Blocking of CD36 on the platelet surface further reduced the thrombin peak in PRP induced by TF for all three therapy regimes. Platelet activation in vivo and in response to the GPVI-agonist convulxin or thrombin in vitro was similar, whereas integrin αIIbβ3 activation and α-granule release induced by the PAR-1 activating peptide TRAP-6 were significantly diminished during ASA plus rivaroxaban treatment compared to ASA monotherapy. In conclusion, the data of this pilot study indicate an inhibitory effect of rivaroxaban on the thrombin propagation phase of CD36-sensitive platelet thrombin formation in patients with PAD treated with ASA plus rivaroxaban combination therapy, which is associated with decreased PAR-1 but not thrombin-mediated platelet activation.

Platelet Pharmacytes for the Hierarchical Amplification of Antitumor Immunity in Response to Self-Generated Immune Signals.

Systemic immunosuppression mediated by tumor-derived exosomes is an important cause for the resistance of immune checkpoint blockade (ICB) therapy. Herein, self-adaptive platelet (PLT) pharmacytes are engineered to mediate cascaded delivery of exosome-inhibiting siRNA and anti-PD-L1 (aPDL1) toward synergized antitumor immunity. In the pharmacytes, polycationic nanocomplexes (NCs) assembled from Rab27 siRNA (siRab) and a membrane-penetrating polypeptide are encapsulated inside the open canalicular system of PLTs, and cytotoxic T lymphocytes (CTLs)-responsive aPDL1 nanogels (NGs) are covalently backpacked on the PLT surface. Upon systemic administration, the pharmacytes enable prolonged blood circulation and active accumulation to tumors, wherein PLTs are activated to liberate siRab NCs, which efficiently transfect tumor cells, silence Rab27a, and inhibit exosome secretion. The immunosuppression is thus relieved, leading to the activation, proliferation, and tumoral infiltration of cytotoxic T cells, which trigger latent aPDL1 release. As such, the competitive aPDL1 exhaustion by PD-L1-expressing exosomes is minimized to sensitize ICB. Synergistically, siRab and aPDL1 induce strong antitumor immunological response and memory against syngeneic murine melanoma. This study reports a bioinspired mechanism to resolve the blood circulation/cell internalization contradiction of polycationic siRNA delivery systems, and renders an enlightened approach for the spatiotemporal enhancement of antitumor immunity.

Activation mechanism dependent surface exposure of cellular factor XIII on activated platelets and platelet microparticles

BackgroundPlatelets contain a high amount of potentially active A subunit dimer of coagulation factor XIII (cellular FXIII; cFXIII). It is of cytoplasmic localization, not secreted, but becomes translocated to the surface of platelets activated by convulxin and thrombin (CVX+Thr). ObjectiveTo explore the difference in cFXIII translocation between receptor mediated and non‐receptor mediated platelet activation and if translocation can also be detected on platelet‐derived microparticles. Our aim was also to shed some light on the mechanism of cFXIII translocation. MethodsGel‐filtered platelets were activated by CVX+Thr or Ca2+‐ionophore (calcimycin). The translocation of cFXIII and phosphatidylserine (PS) to the surface of activated platelets and platelet‐derived microparticles was investigated by flow cytometry, immunofluorescence, and immune electron microscopy. Fluo‐4‐AM fluorescence was used for the measurement of intracellular Ca2+ concentration. ResultsReceptor mediated activation by CVX+Thr exposed cFXIII to the surface of more than 60% of platelets. Electron microscopy revealed microparticles with preserved membrane structure and microparticles devoid of labeling for membrane glycoprotein CD41a. cFXIII was observed on both types of microparticles but was more abundant in the absence of CD41a. Rhosin, a RhoA inhibitor, significantly decreased cFXIII translocation. Non‐receptor mediated activation of platelets by calcimycin elevated intracellular Ca2+ concentration, induced the translocation of PS to the surface of platelets and microparticles, but failed to expose cFXIII. ConclusionsThe elevation of intracellular Ca2+ concentration is sufficient for the translocation of PS from the internal layer of the membrane, while the translocation of cFXIII from the platelet cytoplasm requires additional receptor mediated mechanism(s).

Abstract 316: Hydroxytyrosol, An Olive Oil Phenol, Reduces Ex Vivo Thrombin-induced Platelet Activation

Background and Aims: Olive oil consumption is associated with lower cardiovascular disease (CVD) risk. We recently demonstrated that frequent olive oil consumption was inversely associated with thrombin-induced platelet activation ex vivo . While various olive oil phenolic compounds, notably hydroxytyrosol, have been shown to reduce human platelet aggregation ex vivo , little is known about how olive oil affects platelet activation, a key mediator of atherothrombosis. Therefore, we sought to investigate whether hydroxytyrosol exposure affects platelet activation. Methods and Results: Ten healthy, non-smoking men and women (25.0±1.6 years) with normal body mass index (BMI 22.2±3.3 kg/m 2 ) were recruited for ex vivo platelet studies. Individuals on antiplatelet or anticoagulation medications or medications known to influence lipid levels were excluded. Subjects attended a single visit at NYU Langone where platelet-rich plasma (PRP) was isolated and incubated with three different concentrations of hydroxytyrosol or ethanol vehicle for 60 minutes at room temperature. PRP was then incubated with bovine thrombin for 5 minutes followed by anti-CD62P for 15 minutes in the dark. Platelet activation was determined by platelet surface expression of P-selectin, assessed via flow cytometry. E x vivo exposure to hydroxytyrosol inhibited thrombin-induced platelet P-selectin expression in a dose-dependent manner (Figure). Conclusions: We demonstrate that hydroxytyrosol exposure reduces thrombin-induced platelet P-selectin expression in a dose-response fashion. The dose response seen in this ex vivo platelet activation study is consistent with our clinical observation that more frequent olive oil intake was associated with reduced ex vivo thrombin-induced P-selectin expression. This laboratory study suggests a putative mechanism underlying our clinical observation as well as the established inverse association of olive oil consumption with CVD.

Changes in glycans on platelet microparticles released during storage of apheresis platelets are associated with phosphatidylserine externalization and phagocytosis.

Platelets shed platelet microparticles (PMP) when activated or stored. As the removal of sialic acid (desialylation) promotes platelet uptake and clearance from the circulation, similar mechanisms for PMP uptake were hypothesized. The aim of the study was to investigate the role of surface glycans in the in vitro uptake of PMP from stored platelet components. Apheresis platelet components were stored in 40% plasma/60% SSP+ and sampled on day 1, 5, and 7 post-collection. PMP were characterized by staining with annexin-V (AnV) for phosphatidylserine (PS)-exposure, CD41 antibody, and fluorescently labeled glycan-binding lectins using flow cytometry. The procoagulant function of PMP following desialylation by neuraminidase treatment was assessed by AnV binding and a procoagulant phospholipid assay. PMP were isolated and stained with Deep Red, and phagocytosis by HepG2 cells was measured. Isolated PMP were deglycosylated with neuraminidase and galactosidase to assess the involvement of glycans in mediating phagocytosis. While the overall platelet surface glycan profile was unchanged during storage, PS+ platelets were sialylated, indicating different glycoproteins were changed. In contrast, sialic acid was removed from PS+ and CD41+ PMP, which specifically lost α-2,3-linked sialic acid during platelet storage. PMP were phagocytized by HepG2 cells, and PMP from platelets stored for 7 days were phagocytized to a lesser extent than on day 1. Desialylation by neuraminidase induced PS-exposure on PMP, decreased PPL clotting time, and increased PMP phagocytosis. PMP glycans change during platelet storage. Desialylation influences the procoagulant function of PMP and phagocytosis by HepG2 cells.

Dysregulation in the Expression of Platelet Surface Receptors in Acute Coronary Syndrome Patients-Emphasis on P2Y12.

Simple SummaryAcute Coronary Syndrome is a disease of the circulatory system characterized by the partial or complete blockage of coronary arteries. In thrombosis, a major role is played by platelets—the smallest, anucleate, morphotic elements in the bloodstream. Platelets are involved in the process of hemostasis, which ensures the continuity of the blood vessel by forming a clot that prevents blood loss. Unique structures of blood platelets ensure their high reactivity in the vascular microenvironment due to the interaction with many biologically active molecules, which is recognized by the surface receptors. The research carried out in the manuscript is aimed at detecting potential changes at the molecular level in platelet surface receptors that could constitute the potential importance of the occurrence of Acute Coronary Syndrome. The obtained results indicate that the P2Y12 receptor, which is the main target of antiplatelet therapy, is expressed more frequently among patients. In addition, we have shown that at the genetic level, quantitative changes also occur in the case of other receptors that are important in the activation of platelets. The following manuscript suggests the potential mechanisms responsible for the differences between patients and healthy donors due to a better understanding of the molecular causes of Acute Coronary Syndrome pathogenesis.The pathological conditions caused by blood platelet activation constitute a fundamental core in the pathogenesis of Acute Coronary Syndrome (ACS). The hyperactivity of platelets in ACS is well-documented, but there is still little research into the molecular basis of phenotypic changes in platelet functionality. To expand the knowledge of this phenomenon, we analyzed the disturbances in the expression of several key platelet receptors and the aspect of regulating potential abnormalities. Platelet surface receptors are responsible for maintaining the hemostatic balance, platelet interaction with immune cells, and support of the coagulation cascade leading to occlusion of the vessel lumen. Due to their prominent role, platelet receptors constitute a major target in pharmacological treatment. Our work aimed to identify the molecular alteration of platelet surface receptors, which showed augmented mRNA expression of P2Y12, GP1BB, ITGA2B, and ITGB3 and increased protein concentrations of P2Y12 and GP IIb/IIIa in ACS. The upregulation of the P2Y12 level was also confirmed by confocal and cytometric visualization. Furthermore, we evaluated the expression of two microRNAs: miR-223-3p and miR-126-3p, which were suggested to regulate platelet P2Y12 expression. Results of our study present new insight into the molecular background of ACS.

Novel salivary antihemostatic activities of long-form D7 proteins from the malaria vector Anopheles gambiae facilitate hematophagy

To successfully feed on blood, hematophagous arthropods must combat the host's natural hemostatic and inflammatory responses. Salivary proteins of blood-feeding insects such as mosquitoes contain compounds that inhibit these common host defenses against blood loss, including vasoconstriction, platelet aggregation, blood clotting, pain, and itching. The D7 proteins are some of the most abundantly expressed proteins in female mosquito salivary glands and have been implicated in inhibiting host hemostatic and inflammatory responses. Anopheles gambiae, the primary vector of malaria, expresses three D7 long-form and five D7 short-form proteins. Previous studies have characterized the AngaD7 short-forms, but the D7 long-form proteins have not yet been characterized in detail. Here, we characterized the A. gambiae D7 long-forms by first determining their binding kinetics to hemostatic agonists such as leukotrienes and serotonin, which are potent activators of vasoconstriction, edema formation, and postcapillary venule leakage, followed by ex vivo functional assays. We found that AngaD7L1 binds leukotriene C4 and thromboxane A2 analog U-46619; AngaD7L2 weakly binds leukotrienes B4 and D4; and AngaD7L3 binds serotonin. Subsequent functional assays confirmed AngaD7L1 inhibits U-46619-induced platelet aggregation and vasoconstriction, and AngaD7L3 inhibits serotonin-induced platelet aggregation and vasoconstriction. It is therefore possible that AngaD7L proteins counteract host hemostasis by scavenging these mediators. Finally, we demonstrate that AngaD7L2 had a dose-dependent anticoagulant effect via the intrinsic coagulation pathway by interacting with factors XII, XIIa, and XI. The uncovering of these interactions in the present study will be essential for comprehensive understanding of the vector-host biochemical interface.

Growth Differentiation Factor-15 Correlates Inversely with Protease-Activated Receptor-1-Mediated Platelet Reactivity in Patients with Left Ventricular Assist Devices.

Growth differentiation factor (GDF)-15 inhibits platelet activation, prevents thrombus formation, and has been linked to bleeding events. This was a prospective study including 51 left-ventricular assist device (LVAD) patients on aspirin and phenprocoumon. Platelet surface expression of activated glycoprotein (GP) IIb/IIIa was assessed by flow cytometry, and platelet aggregation was measured by multiple electrode aggregometry (MEA) in response to arachidonic acid (AA), adenosine diphosphate (ADP), and thrombin receptor-activating peptide (TRAP), a protease-activated-receptor-1 (PAR-1) agonist. GDF-15 was determined with a commercially-available assay. There was a trend towards an inverse correlation of GDF-15 with activated GPIIb/IIIa in response to TRAP (r = −0.275, p = 0.0532) but not in response to AA and ADP. Moreover, GDF-15 correlated with MEA TRAP (r = −0.326, p = 0.0194), whereas it did not correlate with MEA ADP and MEA AA. In a second step, GDF-15 levels in the fourth quartile were defined as high GDF-15. Patients with high GDF-15 showed significantly lower TRAP-inducible platelet aggregation by MEA compared to patients in the first quartile (63 AU vs. 113 AU, p = 0.0065). In conclusion, in LVAD patients receiving state-of-the-art antithrombotic therapy, GDF-15 correlates inversely with residual platelet reactivity via PAR-1.

IgG fractions from patients with antiphospholipid syndrome and systemic lupus erythematosus bind to platelets, but do not affect collagen-induced platelet activation

ABSTRACT Anti-beta-2 glycoprotein 1 (anti-β2GP1) is an antiphospholipid antibody found in patients with antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). Its presence commonly is associated with thrombosis; however, the mechanisms of interaction of anti-β2GP1 antibodies and platelets remain unclear. We investigated the effects of APS and SLE patient-derived IgG fractions on collagen-mediated platelet aggregation and examined the binding of patient-derived IgG to platelets before and after activation by collagen. IgG fractions, 150, 200, 300 or 350 µg/ml, isolated from 11 patients with APS and SLE were incubated with two sets of platelet-rich plasma (PRP) in the incubation wells of an aggregometer. The first set was activated by collagen and the other set was incubated for an additional 10 min. All platelets were collected by centrifugation and fixed in cell blocks. We assessed binding of IgG to platelets using immunocytochemistry (ICC). Patient-derived IgG fractions did not affect collagen-induced platelet aggregation. ICC staining using anti-human IgG antibodies demonstrated that patient-derived IgG fractions had greater affinity for non-activated platelets than those activated by 0.75 µg/ml collagen. Patient-derived IgG fractions bound to the surface of platelets and potentially could be internalized by platelets. IgG fractions from APS and SLE patients may sensitize non-activated platelets, which could increase platelet reactivity and thrombotic risk in patients. We did not detect secondary effects of patient-derived IgG fractions.

Triple apheresis platelet concentrate quality after pneumatic tube system, conveyor box, and courier transport: An observational study.

Background and AimsPlatelets are prone to activation from handling; they are therefore transported as gently as possible, most commonly by courier. Speedier methods like pneumatic tube system (PTS) transport could improve patient care but may subject platelets to mechanical stress. To test the impact of mechanical stress caused by transport, we compared a PTS with a conveyor box and courier transport on apheresis platelet function.MethodsFourteen apheresis platelet concentrate triple donations were analyzed by light transmission aggregometry (LTA), rotational thrombelastometry (ROTEM), and flow cytometry before and after indoor transport over 800 m by PTS, conveyor, and courier, respectively, while recording shocks and vibrations with a high‐frequency acceleration data logger. Shock index scores were calculated as shock intensity (g‐force) times frequency.ResultsThe shock index was 81 for courier, 6279 for conveyor, and 9075 for PTS. Flow cytometry revealed no significant difference in platelet surface expression of CD62p before (16%) and after transport via courier (15%), conveyor (14%), or PTS (16%). LTA with adenosine phosphate and thrombin receptor‐activating peptide‐6 resulted in comparable platelet aggregation for courier, conveyor, and PTS. ROTEM assays showed no relevant differences in coagulation time, clot formation time, and maximum clot firmness between transport modes.ConclusionThough the mechanical challenge was smallest with courier transport, there were no significant differences in platelet activation or aggregation between the three transport modes. These data contradict restrictions on the use of PTSs for platelet concentrate transport.

Effects of the ITGA2B Nonsense Mutation (c.2659C > T, p.Q887X) on Platelet Function in a Mouse Model of Glanzmann's Thrombasthenia Generated with CRISPR/Cas9 Technology

To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane. The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbβ3 on the surface of platelet. The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbβ3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05). A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.

IDF21-0650 Platelet responses and activation to agonist stimulation in type 2 diabetes mellitus

Background: Diabetes is a leading cause of mortality worldwide, with type 2 diabetes mellitus (T2DM) the most common. T2DM is a multifactorial disease with genetic and environmental causes leading to an increase in mortality linked to cardiovascular disease and coagulation. Platelet hyperactivation, increased coagulability and decreased thrombolysis are central to cardiovascular complications. Platelet hyperreactivity is associated with increased levels of thromboxane A2 and an upregulation of P2Y12 receptors on the platelet surface, binding adenosine diphosphate (ADP).

Triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1) reveals platelet activation in preeclampsia

Triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1) is a membrane protein receptor found in α-granules of megakaryocytes and platelets. Upon platelet activation TLT-1 is rapidly relocated to the surface of platelets. In plasma, a soluble form of TLT-1 (sTLT-1) is present. Plasma levels of sTLT-1 are significantly elevated in thrombotic diseases. In the present study, we investigated to whether TLT-1 reflects platelet activation in pregnant women with preeclampsia. We studied 30 preeclamptic patients who were matched with 30 normotensive pregnant women and 30 non-pregnant controls. Basal TLT-1, P-selectin, and CD63 expressions on platelets were analyzed with the use of flow-cytometry (FCM). Platelet reactivity was induced by thrombin receptor activation peptide and determined by FCM. Plasma concentrations of sTLT-1 and soluble P-selectin (sP-selectin) were measured by an enzyme-linked immunosorbent assay. Results show that basal platelet expression of TLT-1, P-selectin and CD63 were increased in women with preeclampsia (PE) compared with normotensive pregnant women (NP). Platelets from PE women and NP women were more responsive compared to from nonpregnant women controls (NC), and which was demonstrated by increased expression of TLT-1, P-selectin, and CD63 upon stimulation in vitro. Plasma concentration of sTLT-1 was greater in PE women compared to NP women and NC women. Plasma sP-selectin level was higher in pregnant women than in nonpregnant women, but there were no significant differences between PE and NP women. In summary, our results revealed that platelet activation is prominent in preeclampsia, TLT-1 reflects platelet activation and may be a useful indicator for preeclampsia.