Platelet Agonists Research Articles

Fermentation Enhances the Anti-Inflammatory and Anti-Platelet Properties of Both Bovine Dairy and Plant-Derived Dairy Alternatives

Within the present study, the effects of fermentation on the anti-inflammatory and anti-platelet properties of both homemade and commercially purchased bovine dairy and almond, coconut, and rice-based dairy alternatives were evaluated. The extracted total lipids (TL) from homemade and commercially purchased fermented and unfermented bovine, almond, coconut, and rice-based products were further separated into their neutral lipids (NL) and polar lipids (PL) fractions by counter current distribution. The TL, PL, and NL of each sample were assessed in human platelets against the inflammatory and thrombotic mediator, platelet-activating factor (PAF), and the well-established platelet agonist, adenosine 5′ diphosphate (ADP). In all samples, the PL fractions showed significantly stronger inhibitory effects against human platelet aggregation induced by PAF or ADP, in comparison to the TL and NL, with higher specificity against PAF. PL of all fermented products (bovine yogurt and fermented dairy alternatives from almond, rice, and coconut), exhibited the strongest anti-inflammatory and anti-platelet potency, in comparison to PL from their initial pasteurized materials (bovine milk and rice, almond, and coconut-based dairy alternative drinks). PL of the pasteurized rice-based drink and, especially PL from the novel homemade rice-based fermented product (HMFRD), showed the strongest anti-PAF and anti-ADP potency compared to all samples, with anti-PAF activity being most potent overall. The unfermented pasteurized coconut-based drink showed the lowest anti-inflammatory and anti-platelet potency, and the bovine and almond-based fermented products showed an intermediate effect. Further lipidomics with LC-MS analysis of all these PL fractions revealed that fermentation altered their fatty acid content in a way that decreased their degree of saturation and increased the content of unsaturated fatty acids, thus providing a rationale for the stronger anti-inflammatory and anti-platelet potency of the more unsaturated PL fractions of the fermented products. This study has shown that fermentation alters the fatty acid content and the bio-functionality of the PL bioactives in both fermented bovine dairy and plant-based dairy alternatives, and subsequently improved their anti-inflammatory and anti-platelet functional properties.

Assay validity of point-of-care platelet function tests in thrombocytopenic blood samples.

IntroductionPoint-of-care (POC) platelet function tests are faster and easier to perform than in-depth assessment by flow cytometry. At low platelet counts, however, POC tests are prone to assess platelet function incorrectly. Lower limits of platelet count required to obtain valid test results were defined and a testing method to facilitate comparability between different tests was established.Materials and methodsWe assessed platelet function in whole blood samples of healthy volunteers at decreasing platelet counts (> 100, 80-100, 50-80, 30-50 and < 30 x109/L) using two POC tests: impedance aggregometry and in-vitro bleeding time. Flow cytometry served as the gold standard. The number of platelets needed to reach 50% of the maximum function (ED50) and the lower reference limit (EDref) were calculated to define limits of test validity.ResultsThe minimal platelet count required for reliable test results was 100 x109/L for impedance aggregometry and in-vitro bleeding time but only 30 x109/L for flow cytometry. Comparison of ED50 and EDref showed significantly lower values for flow cytometry than either POC test (P value < 0.05) but no difference between POC tests nor between the used platelet agonists within a test method.ConclusionCalculating the ED50 and EDref provides an effective way to compare values from different platelet function assays. Flow cytometry enables correct platelet function testing as long as platelet count is > 30 x109/L whereas impedance aggregometry and in-vitro bleeding time are inconsistent unless platelet count is > 100 x109/L.

Effects of ex vivo blood anticoagulation and preanalytical processing time on the proteome content of platelets

BackgroundEx vivo assays of platelet function critically inform mechanistic and clinical hematology studies, where effects of divergent blood processing methods on platelet composition are apparent, but unspecified. ObjectiveHere, we evaluate how different blood anticoagulation options and processing times affect platelet function and protein content ex vivo. MethodsParallel blood samples were collected from healthy human donors into sodium citrate, acid citrate dextrose, EDTA or heparin, and processed over an extended time course for functional and biochemical experiments, including platelet proteome quantification with multiplexed tandem mass tag (TMT) labeling and triple quadrupole mass spectrometry (MS). ResultsEach anticoagulant had time‐dependent effects on platelet function in whole blood. For instance, heparin enhanced platelet agonist reactivity, platelet‐monocyte aggregate formation and platelet extracellular vesicle release, while EDTA increased platelet α‐granule secretion. Following platelet isolation, TMT‐MS quantified 3357 proteins amongst all prepared platelet samples. Altogether, >400 proteins were differentially abundant in platelets isolated from blood processed at 24 h versus 1 h post‐phlebotomy, including proteins pertinent to membrane trafficking and exocytosis. Anticoagulant‐specific effects on platelet proteomes included increased complement system and decreased α‐granule proteins in platelets from EDTA‐anticoagulated blood. Platelets prepared from heparinized blood had higher levels of histone and neutrophil‐associated proteins in a manner related to neutrophil extracellular trap (NET) formation and platelet:NET interactions in whole blood ex vivo. ConclusionOur results demonstrate that different anticoagulants routinely used for blood collection have varying effects on platelets ex vivo, where methodology‐associated alterations in platelet proteome may influence mechanistic, translational and biomarker studies.

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.

Measurement of procoagulant platelets provides mechanistic insight and diagnostic potential in heparin‐induced thrombocytopenia

BackgroundHeparin‐induced thrombocytopenia (HIT) is a prothrombotic, immune‐mediated adverse drug reaction associated with high rates of thrombosis‐related morbidity and mortality caused by FcγRIIa‐activating pathogenic antibodies to PF4‐heparin. Procoagulant platelets are a platelet subset that promote thrombin generation, are clinically relevant in prothrombotic diseases, and are formed when platelet G‐protein‐coupled receptor (GPCR) and ITAM‐linked receptors are co‐stimulated. ObjectivesWe examined the procoagulant platelet response of healthy donors to platelet agonists in the presence of HIT plasma and determined the contribution of FcγRIIa. Patients/MethodsOur previously established flow cytometry‐based procoagulant platelet assay was modified to incorporate plasma samples, performed using FcγRIIa‐responsive donor platelets. Plasma samples were serotonin‐release assay–confirmed HIT (HIT+), or negative on HIT screening. ResultsIn response to GPCR stimulation, only HIT+ plasma produced a heparin‐dependent sensitization that required active FcγRIIa. As a potential diagnostic tool, the procoagulant platelet assay achieved 98% accuracy in identifying clinically verified HIT when performed blinded to the diagnoses of a validation cohort. Samples inducing a higher procoagulant platelet response were more likely from patients with thrombotic complications. Thrombin stimulation markedly increased the procoagulant platelet response with HIT+ plasma that was heparin independent and only partially reversed by FcγRIIa blockade, possibly reflecting ongoing thrombotic risk after heparin cessation. ConclusionsWe demonstrate that HIT plasma together with platelet agonists increased the procoagulant platelet proportions, which may contribute to thrombotic risk in HIT. Targeting procoagulant platelet activation may represent a novel treatment strategy. This assay may be a rapid, clinically relevant functional assay for accurately detecting pathological HIT antibodies.

Galectin‐9 activates platelet ITAM receptors glycoprotein VI and C‐type lectin‐like receptor‐2

BackgroundPlatelets are multifunctional cellular mediators in many physiological and pathophysiological processes such as thrombosis, angiogenesis, and inflammation. Several members of galectins, a family of carbohydrate‐binding proteins with a broad range of immunomodulatory actions, have been reported to activate platelets. ObjectiveIn this study, we investigated the role of galectin‐9 (Gal‐9) as a novel ligand for platelet glycoprotein VI (GPVI) and C‐type lectin‐like receptor 2 (CLEC‐2). MethodsPlatelet spreading, aggregation, and P‐selectin expression in response to Gal‐9 were measured in washed platelet suspensions via static adhesion assay, light transmission aggregometry, and flow cytometry, respectively. Solid‐phase binding assay and protein phosphorylation studies were utilized to validate the interaction between Gal‐9 and GPVI, and immunoprecipitation for detecting CLEC‐2 phosphorylation. Wild‐type (WT), GPVI‐knockout (Gp6−/−), and GPVI and CLEC‐2‐double knockout (Gp6−/−/Gp1ba‐Cre‐Clec1bfl/fl) mice were used. ResultsWe have shown that recombinant Gal‐9 stimulates aggregation in human and mouse washed platelets dose‐dependently. Platelets from both species adhere and spread on immobilized Gal‐9 and express P‐selectin. Gal‐9 competitively inhibited the binding of human recombinant D1 and D2 domains of GPVI to collagen. Gal‐9 stimulated tyrosine phosphorylation of CLEC‐2 and proteins known to lie downstream of GPVI and CLEC‐2 including spleen tyrosine kinase and linker of activated T cells in human platelets. GPVI‐deficient murine platelets exhibited significantly impaired aggregation in response to Gal‐9, which was further abrogated in GPVI and CLEC‐2‐double‐deficient platelets. ConclusionsWe have identified Gal‐9 as a novel platelet agonist that induces activation through interaction with GPVI and CLEC‐2.

Anti‐platelet factor 4 immunoglobulin G levels in vaccine‐induced immune thrombocytopenia and thrombosis: Persistent positivity through 7 months

BackgroundAnti‐platelet factor 4 (PF4) antibodies that activate platelets via FcγRIIA drive the pathophysiology of vaccine‐induced immune thrombocytopenia and thrombosis (VITT). Evolution of these antibodies and their ability to activate platelets after initial treatment remains unknown. ObjectivesTo assess how clinical and platelet parameters, anti‐PF4 antibody levels, and patient serum reactivity changes during follow‐up after VITT presentation. MethodsWe describe cases of seven discharged VITT patients that were followed from diagnosis up to 280 days (range 199–280) after vaccination. We measured anti‐PF4 antibodies and PF4 levels in patient serum during follow‐up and tested the ability of patient serum to activate healthy donor platelets and patient platelets over time. ResultsAnti‐PF4 immunoglobulin G antibody levels are very high at diagnosis (0.9–2.6 OD) and remain relatively high (>1.0 OD) in all patients, except one treated with rituximab, at 7 months post vaccination. All patients were on direct oral anticoagulants throughout follow‐up and no patients had recurrent thrombosis. Patients’ platelets during follow‐up have normal FcγRIIA levels and responsiveness to platelet agonists. Patient diagnostic serum strongly activated control platelets, either alone or with PF4. Most follow‐up serum alone was weaker at stimulating donor and patient platelets. However, follow‐up serum beyond 150 days still strongly activated platelets with PF4 addition in three patients. Patient serum PF4 levels were lower than controls at diagnosis but returned within normal range by day 50. ConclusionsExplanations for reduced platelet activation during follow‐up, despite similar total anti‐PF4 antibody levels, remains unclear. Clinical implications of persistent anti‐PF4 antibodies in VITT require further study.

Identification of novel antiplatelet agents by targeting Glycoprotein VI: A combined virtual screening study

Cardiovascular diseases are the primary reason for deaths in the world. However, antiplatelet drugs in the market have limitations in use because of the risk of increased bleeding and other side effects that impair primary homeostasis. Therefore, safe and effective antithrombotic drugs are needed for the treatment of plaque formation in blood vessels. Glycoprotein VI (GPVI) is a platelet major collagen receptor and a target for potent and safe antithrombotic therapy. We designed this study based on the molecular interaction pattern of previously published GPVI receptor antagonists within the reported binding site. We selected sixteen hit compounds from a large chemical database that contains>6 million in-stock compounds by following a combined virtual screening. Then, we evaluated their inhibitory effects on platelet aggregation induced by GPVI receptor agonists (collagen, collagen related peptide (CRP), convulxin) and the most potent platelet agonist, thrombin, in vitro by using washed human platelets. IC50 values of compounds 1 and 2 are, respectively, 0.35 μM and 1.01 μM for collagen, 0.80 μM and 1.92 μM for CRP, 195.2 and 7.24 μM for convulxin and 81.38 and 51.74 μM for thrombin. We identified compounds 1 and 2 as the most promising antiplatelet agents out of sixteen compounds. Additionally, compounds 1 and 2 may serve as promising starting points and shed light on the design of new, potent and selective GPVI receptor antagonists.

Chronic edible dosing of Δ9-tetrahydrocannabinol (THC) in nonhuman primates reduces systemic platelet activity and function.

Cannabis usage has steadily increased as acceptance is growing for both medical and recreational reasons. Medical cannabis is administered for treatment of chronic pain based on the premise that the endocannabinoid system signals desensitize pain sensor neurons and produce anti-inflammatory effects. The major psychoactive ingredient of cannabis is Δ9-tetrahydrocannabinol (THC) that signals mainly through cannabinoid receptor-1 (CBr), which is also present on nonneuron cells including blood platelets of the circulatory system. In vitro, CBr-mediated signaling has been shown to acutely inhibit platelet activation downstream of the platelet collagen receptor glycoprotein (GP)VI. The systemic effects of chronic THC administration on platelet activity and function remain unclear. This study investigates the effects of chronic THC administration on platelet function using a nonhuman primate (NHP) model. Our results show that female and male NHPs consuming a daily THC edible had reduced platelet adhesion, aggregation, and granule secretion in response to select platelet agonists. Furthermore, a change in bioactive lipids (oxylipins) was observed in the female cohort after THC administration. These results indicate that chronic THC edible administration desensitized platelet activity and function in response to GPVI- and G-protein coupled receptor-based activation by interfering with primary and secondary feedback signaling pathways. These observations may have important clinical implications for patients who use medical marijuana and for providers caring for these patients.

Neutrophil Cathepsin G Enhances Thrombogenicity of Mildly Injured Arteries via ADP-Mediated Platelet Sensitization.

Inhalation of particulate matter in polluted air causes direct, size-restricted passage in the circulation and pronounced lung inflammation, provoking platelet activation and (non)-fatal cardiovascular complications. To determine potency and mechanism of platelet sensitization via neutrophil enzymes, we performed in vitro aggregation studies in washed human platelets and in murine and human blood, in the presence of elastase, cathepsin G and regular platelet agonists, present in damaged arteries. The impact of both enzymes on in vivo thrombogenicity was studied in the same thrombosis mouse model, previously having demonstrated that neutrophil activation enhances peripheral thrombogenicity. At 0.05 U/mL, cathepsin G activated washed human platelets via PAR1, whereas at 0.35 U/mL, aggregation occurred via PAR4. In Swiss mouse platelet-rich plasma no aggregation occurred by cathepsin G at 0.4 U/mL. In human and murine blood, aggregations by 0.05–0.1 U/mL cathepsin G were similar and not PAR-mediated, but platelet aggregation was inhibited by ADP antagonists, advocating cathepsin G-released ADP in blood as the true agonist of sustained platelet activation. In the mouse thrombosis model, cathepsin G and elastase amplified mild thrombogenicity at blood concentrations that activated platelets in vitro. This study shows that cathepsin G and elastase secreted in the circulation during mild air pollution-induced lung inflammation lyse red blood cell membrane proteins, leading to ADP-leakage into plasma, sensitizing platelets and amplifying their contribution to cardiovascular complications of ambient particle inhalation.

Platelet SHARPIN regulates platelet adhesion and inflammatory responses through associations with αIIbβ3 and LUBAC

AbstractPlatelets form hemostatic plugs to prevent blood loss, and they modulate immunity and inflammation in several ways. A key event during hemostasis is activation of integrin αIIbβ3 through direct interactions of the β3 cytoplasmic tail with talin and kindlin-3. Recently, we showed that human platelets express the adapter molecule Shank-associated RH domain interacting protein (SHARPIN), which can associate directly with the αIIb cytoplasmic tail and separately promote NF-κB pathway activation as a member of the Met-1 linear ubiquitination activation complex (LUBAC). Here we investigated the role of SHARPIN in platelets after crossing Sharpin flox/flox (fl/fl) mice with PF4-Cre or GPIbα-Cre mice to selectively delete SHARPIN in platelets. SHARPIN-null platelets adhered to immobilized fibrinogen through αIIbβ3, and they spread more extensively than littermate control platelets in a manner dependent on feedback stimulation by platelet adenosine diphosphate (ADP) (P < .01). SHARPIN-null platelets showed increased colocalization of αIIbβ3 with talin as assessed by super-resolution microscopy and increased binding of soluble fibrinogen in response to submaximal concentrations of ADP (P < .05). However, mice with SHARPIN-null platelets showed compromised thrombus growth on collagen and slightly prolonged tail bleeding times. Platelets lacking SHARPIN also showed reduced NF-κB activation and linear ubiquitination of protein substrates upon challenge with classic platelet agonists. Furthermore, the loss of platelet SHARPIN resulted in significant reduction in inflammation in murine models of colitis and peritonitis (P < .01). Thus, SHARPIN plays differential and context-dependent roles in platelets to regulate important inflammatory and integrin adhesive functions of these anucleate cells.

Conditional CRISPR‐mediated deletion of Lyn kinase enhances differentiation and function of iPSC‐derived megakaryocytes

BackgroundThrombocytopenia leading to life‐threatening excessive bleeding can be treated by platelet transfusion. Currently, such treatments are totally dependent on donor‐derived platelets. To support future applications in the use of in vitro‐derived platelets, we sought to identify genes whose manipulation might improve the efficiency of megakaryocyte production and resulting hemostatic effectiveness. Disruption of Lyn kinase has previously been shown to improve cell survival, megakaryocyte ploidy and TPO‐mediated activation in mice, but its role in human megakaryocytes and platelets has not been examined. MethodsTo analyze the role of Lyn at defined differentiation stages during human megakaryocyte differentiation, conditional Lyn‐deficient cells were generated using CRISPR/Cas9 technology in iPS cells. The efficiency of Lyn‐deficient megakaryocytes to differentiate and become activated in response to a range of platelet agonists was analyzed in iPSC‐derived megakaryocytes. ResultsTemporally controlled deletion of Lyn improved the in vitro differentiation of hematopoietic progenitor cells into mature megakaryocytes, as measured by the rate and extent of appearance of CD41+CD42+ cells. Lyn‐deficient megakaryocytes also demonstrated improved hemostatic effectiveness, as reported by their ability to mediate clot formation in rotational thromboelastometry. Finally, Lyn‐deficient megakaryocytes produced increased numbers of platelet‐like particles (PLP) in vitro. ConclusionsConditional deletion of Lyn kinase increases the hemostatic effectiveness of megakaryocytes and their progeny as well as improving their yield. Adoption of this system during generation of in vitro‐derived platelets may contribute to both their efficiency of production and their ability to support hemostasis.

Isotopically nonstationary 13C metabolic flux analysis in resting and activated human platelets

Platelet metabolism is linked to platelet hyper- and hypoactivity in numerous human diseases. Developing a detailed understanding of the link between metabolic shifts and platelet activation state is integral to improving human health. Here, we show the first application of isotopically nonstationary 13C metabolic flux analysis to quantitatively measure carbon fluxes in both resting and thrombin activated platelets. Metabolic flux analysis results show that resting platelets primarily metabolize glucose to lactate via glycolysis, while acetate is oxidized to fuel the tricarboxylic acid cycle. Upon activation with thrombin, a potent platelet agonist, platelets increase their uptake of glucose 3-fold. This results in an absolute increase in flux throughout central metabolism, but when compared to resting platelets they redistribute carbon dramatically. Activated platelets decrease relative flux to the oxidative pentose phosphate pathway and TCA cycle from glucose and increase relative flux to lactate. These results provide the first report of reaction-level carbon fluxes in platelets and allow us to distinguish metabolic fluxes with much higher resolution than previous studies.

A Multi-Institution Retrospective Study to Assess Bleeding Phenotype Among Patients with Platelet Function Disorders

▪Introduction: Platelet function disorders (PFD) clinically manifest with wide variability in mucocutaneous bleeding and significant hemorrhage post-surgery or trauma. The overall prevalence of PFD is not known, as there have not been large population-based studies. Treatment of these patients vary based on their bleeding phenotype. Additionally, the exact bleeding phenotype of many qualitative platelet defects are not well described in literature. This study aims to describe the bleeding phenotype among patients with different (PFD).Methods: This is a retrospective study among patients with PFD conducted at 3 Hemophilia Treatment centers - HOG Center for Bleeding and Clotting Disorders of CHOA, Children's Mercy hospital HTC and Rainbow babies & Children's hospital HTC. Institutional IRB approval was obtained at all 3 institutions. We collected data on demographics, bleeding symptoms at presentation, bleeding episodes, management of these bleeds over a 6-year time period (2015-2020).Results: We identified 131 patients with PFDs at 3 institutions. This included 67 males (51.2%) and 64 females (48.8%). Among 131 patients, 72 patients (55%) had a defect in platelet agonist interaction/receptor defect (ADP/Epinephrine/Collagen/TXA2/Arachidonic acid), 37 patients (28.2%) had delta storage pool defect, 8 patients (6.1%) had Glanzmann thrombasthenia, 7 patients (5.3%) had a platelet release defect, 3 patients (2.3%) with an alpha granule defect, 2 patients (1.5%) with Bernard Soulier syndrome and 1 patient (0.76%) with Wiskott Aldrich syndrome. The most common bleeding symptoms at presentation were epistaxis (40.4%), followed by easy bruising (31.3%), heavy menstrual bleeding (15.2%), gum bleeding (6.87%) and gastrointestinal bleeding (4.58%). From 2015-2020, a total of 162 bleeds were documented, and 68 patients (51.9%) with at least 1 documented episode of bleeding. 67.2% of these bleeds were spontaneous, 12.3% were secondary to trauma, 4.9% after a dental procedure, 2.5% after surgery and 0.6% after child birth. The most common type of bleeding episode in diagnosed patients included epistaxis (50%), heavy menstrual bleeding (17.9%), skin/soft tissue bleed (5.5%), gastrointestinal (5.5%) and dental/tooth related (4.9%). 93 bleeding episodes (57.4%) required some form of treatment in various settings - home (73%), clinic (15%), emergency room (7.5%), hospitalization (14%) and ICU stay (2%). Treatments included antifibrinolytics (68.8%), recombinant factor VIIa (11.8%), desmopressin (9.6%), hormonal therapy (9.6%) and platelet transfusions (5.3%).Conclusions: Our study helps characterize the bleeding phenotype and management in patients with various PFD. This data is crucial in understanding the burden of illness among different types of PFD, and to understand health care utilization to better serve the needs of these poorly characterized patients. DisclosuresSidonio: Sanofi, Takeda, Octapharma, Bayer, Biomain, Grifols, Kedrion, Genentech. Catalyst, Guardian Therapeutics, Novo Nordisk, Hema Biologics, Uniqure.: Consultancy, Honoraria. Silvey: Genentech: Speakers Bureau; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees. Ahuja: XaTek, Inc: Patents & Royalties; Sanofi: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: DSMB member ; Genentech: Membership on an entity's Board of Directors or advisory committees.

Electron Microscope Characterization of Platelet Activation State Reveals That Wound Closure and P2Y 12 Receptors Are Major Early Determinants of Thrombus Structure in a Venous Puncture Model

Vascular damage presents in many forms and varying geometries. Nevertheless, the platelet response to endothelial damage to the blood vessel wall, be it through a prick or a full puncture wound, is thought to be staged by a qualitatively similar temporal variance in signaling agonists. For example, endothelial damage in the microvasculature is thought to be initially dominated by thrombin and later by platelet released ADP and thromboxane. The same temporal sequence in signaling has been proposed to exist in a profusely bleeding puncture wound 1. If so, platelet morphology, a gold standard of platelet activation state, could provide a strong readout of temporally distinct signaling effects. Platelet morphology has long been considered to be a reliable indicator of a strong agonist such as thrombin acting through PAR receptors that produces a rounded, pseudopod extending, degranulated, highly adhesive platelet versus weaker agonists such as ADP or thromboxane acting through P2Y 12 receptors to produce a less adhesive, somewhat rounded platelet. A testable prediction of existing hemostasis models is that temporal staging of signaling leads to temporal differences in platelet morphology within the forming/remodeling thrombus. Such hypothesized temporal differences in signaling are clinically significant as they form the basis for hypothesizing phenotypically distinct outcomes for direct acting anti-coagulants (DOACs) affecting thrombin versus anti-platelet drugs affecting P2Y 12, ADP receptors.Advances in imaging, e.g., wide area transmission electron microscopy (WA-TEM), make possible the local determination of platelet activation state with high precision 2. Taking a mouse jugular vein puncture wound model 1,2, we found that all morphologically recognized platelet activation states were present early, 1 min post puncture, with loosely bound discoid shaped platelets being the most peripherally located. For bleeding, early-stage puncture wound, these loosely adherent, low activation state platelets were located on both intravascular and extravascular thrombus aggregates. Once the puncture wound is closed, loosely adherent platelets were only found on the intravascular surfaces of the thrombus. We propose that this result is most consistent with a platelet conversion model in which new loosely adherent platelets rapidly convert to tightly packed platelets. As the thrombus remodels, 5 and 20 min post-puncture, the thrombus continued to accumulate platelets both intravascularly and extravascularly. Peripheral, discoid shaped platelets provided a source for intravascular thrombus growth. However, any subsequent extravascular thrombus growth must be due to platelet migration. Significantly, we found that cangrelor, a direct acting P2Y 12 inhibitor, stalled thrombus formation/remodeling at an early stage (Figure 1A,C,E see also ref 1,2). By WA-TEM, the accumulation of discoid-shaped, loosely adherent platelets appeared to be enhanced in a cangrelor treated 5 min thrombus (Figure 1E,F). We suggest that P2Y 12 receptors must act early in thrombus formation with the conversion of discoid to more activated platelets being most affected. In contrast, a 5-min post puncture dabigatran (DOAC) treated showed deformed architecture with inhibition of the accumulation of discoid shaped platelets/rounded loosely adherent platelets being most affected (Figure 1D,F, see also ref 2). Accumulation of degranulated platelets appeared to be lessened in both cangrelor and dabigatran treated thrombi. We propose that the simplest explanation of these results is that multiple signaling pathways act in parallel with select activation states being more dependent on one pathway than another. Clinically, our results suggest that P2Y 12 inhibitors can affect thrombus formation at early time points in addition to the late time points projected by current models.1. Tomaiuolo M., Matzko C.N., Posentud-Fuentes I., Weisel J.W., Brass L.F. & Stalker T.J. Interrelationships between structure and function during the hemostatic response to injury. Proc Natl Acad Sci USA. 116. 2243-2252 (2019).2. Rhee, Pokrovskaya I.D.,BallK., LingK., VedanapartiY., CohenJ., CruzD., ZhaoO.S., AronovaM.A., ZhangG., Kamykowski J.A., LeapmanR.D., & StorrieB. Venous puncture wound hemostasis results in a vaulted thrombus structured by locally nucleated platelet aggregates. Commun. Biol., accepted. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Iron Repletion Increases Platelet Aggregation and Reduces Platelet Count in Iron Deficient Premenopausal Women

Background: Iron deficiency, heavy menstrual bleeding, and anemia are interrelated conditions with high prevalence in women's health. Together, they impact an estimated 310 million premenopausal women worldwide. Iron deficiency has been associated with development of anemia as well as thrombocytosis. However, the effects of iron deficiency on platelet function remain unknown.Objective: We set out to investigate the impact of IV iron repletion on platelet function, platelet count, and other blood cell indices in iron deficient premenopausal women.Methods: We conducted a prospective single-center study of iron deficient premenopausal women who underwent iron repletion with a single dose of 1000 mg of low molecular weight iron. Pre-infusion and post-infusion blood samples were obtained for laboratory analysis. Standard of care monitoring of iron indices and complete blood counts were also included in the analysis. Pre-infusion rates of anemia and thrombocytosis were calculated using minimum hemoglobin and peak platelet count in the 6 months preceding iron infusion. Platelet function was quantified by Fluorescence-Activated Cell Sorting (FACS) quantification of platelet activation marker antibodies for GPIIb/IIIa (PAC1) and P-selectin (CD62P) after exposure to multiple platelet agonists. Platelet aggregation was assessed by flow of anticoagulated whole blood at a shear rate of 300 s -1 for 5 minutes through chambers coated with type I collagen, imaged with differential interference contrast optics, and quantitatively represented through computation of percent total surface coverage. Pre-infusion and post-infusion cell indices and aggregation data were compared by paired samples t-test. All statistical analyses were performed in GraphPad Prism (Version 8.0.0).Results: Thirteen patients were included in the analysis. Heavy menstrual bleeding was the suspected cause of iron deficiency in 83% of patients. Thrombocytosis was present in 15% of patients at pre-infusion and no patients at post-infusion. Average ferritin was 14 µg/L at pre-infusion and 126 µg/L at post-infusion. The peak platelet count within 6 months pre-infusion was 309 K/mm 3 (±89) vs. 274 (±64) K/mm 3 at post-infusion (p<0.05). The average change to 6-month pre-infusion peak and post-infusion platelet count was -35.2 K/mm 3 (95%CI: -66.2, -5.23). The mean 6-month minimum hemoglobin was 11.9 (±1.9) g/dL vs. 13.3 (±1.1) g/dL at post-infusion (p<0.005). Acquisition of FACS platelet reactivity data are ongoing, with preliminary results for the first 7 consecutively enrolled study patients displayed in Figure 1. Platelet aggregation measured at pre-infusion showed 14% surface coverage, with a significant increase to 29% at post-infusion (p < 0.05). None of the 13 women experienced a thrombotic event during the study period.Conclusion: Correction of iron deficiency results in decreased platelet count and improves platelet aggregation over collagen. Iron repletion may also improve platelet reactivity in response to physiologic agonists. These findings may suggest that iron deficiency impairs hemostasis and that correction of low iron may be even more critical for women with heavy menstrual bleeding. [Display omitted] DisclosuresShatzel: Aronora Inc,: Consultancy.

GRK2 Regulates ADP Signaling in Platelets Via P2Y 1 and P2Y 12

AbstractVenous thromboembolism (VTE), heart attack, and stroke are all diseases in which platelets play a role, through inappropriate platelet activation and subsequent thrombus formation. Most platelet agonists activate platelets via G protein-coupled receptors (GPCRs), which are targeted by many antiplatelet drugs. Along with thrombin and TxA 2, ADP has long been recognized for its important role in hemostasis and thrombosis. It activates platelets via GPCRs, P2Y 1 and P2Y 12. However, little is known about the negative feedback mechanisms governing P2Y receptor-mediated platelet activation and thrombus formation.Here, we provide the first evidence that GPCR kinase 2 (GRK2) serves this regulatory role during platelet activation and thrombus formation by using a platelet-specific GRK2 deletion mouse model and a GRK2-specific inhibitor in human platelets. Deletion of GRK2 in mouse platelets causes increased platelet accumulation following laser-induced injury in cremaster muscle arterioles, particularly in the shell region of thrombi. In addition, this deletion increases ADP-induced pulmonary thromboembolism. GRK2 -/- platelets also have increased platelet aggregation in response to ADP, but not to PAR4 receptor agonist, TxA 2, or convulxin. Underlying these changes in GRK2 -/- platelets is an increase in Ca 2+ mobilization, Akt phosphorylation, and Rap1 activation in response to ADP, and an attenuated rise of cAMP levels in response to ADP in the presence of prostaglandin I 2. Furthermore, platelet aggregation can be restored in GRK2 -/- platelets in response to ADP re-stimulation, indicating that GRK2 contributes to ADP receptor desensitization. To further assess the role of GRK2 in the P2Y 12 signaling pathway in vivo, we examine laser-induced thrombus formation in WT and GRK2 -/- mice treated with the P2Y 12 antagonist, cangrelor. Cangrelor treatment eliminates the phenotypic difference in platelet accumulation between WT and GRK2 -/- mice in response to injury. Using a specific GRK2 inhibitor, pharmacologic inhibition of GRK2 activity in human platelets results in an increase in platelet activation in response to ADP. Finally, our biochemical studies show that GRK2 binds to endogenous Gβγ subunits during platelet activation.Taken together, we have demonstrated for the first time that 1) GRK2 plays a negative regulatory role in platelet activation by attenuating ADP-dependent signaling, 2) it does this by limiting P2Y 1 and P2Y 12-mediated signaling, 3) GRK2 interacts with Gβγ and functions as a signaling hub in platelets for fine-tuning GPCR signaling, and 4) although the potential inhibition of GRK2 can be beneficial for treatment of heart diseases, maintaining GRK2 activity in platelets could be beneficial for prevention of thrombotic diseases. DisclosuresNo relevant conflicts of interest to declare.

Effects of Platelet Agonists and Priming on the Formation of Platelet Populations.

Platelets from healthy donors display heterogeneity in responsiveness to agonists. The response thresholds of platelets are controlled by multiple bioactive molecules, acting as negatively or positively priming substances. Higher circulating levels of priming substances adenosine and succinate, as well as the occurrence of hypercoagulability, have been described for patients with ischaemic heart disease. Here, we present an improved methodology of flow cytometric analyses of platelet activation and the characterisation of platelet populations following activation and priming by automated clustering analysis. Platelets were treated with adenosine, succinate, or coagulated plasma before stimulation with CRP-XL, 2-MeSADP, or TRAP6 and labelled for activated integrin α IIb β 3 (PAC1), CD62P, TLT1, CD63, and GPIX. The Super-Enhanced Dmax subtraction algorithm and 2% marker (quadrant) setting were applied to identify populations, which were further defined by state-of-the-art clustering techniques (tSNE, FlowSOM). Following activation, five platelet populations were identified: resting, aggregating (PAC1 + ), secreting (α- and dense-granules; CD62P + , TLT1 + , CD63 + ), aggregating plus α-granule secreting (PAC1 + , CD62P + , TLT1 + ), and fully active platelet populations. The type of agonist determined the distribution of platelet populations. Adenosine in a dose-dependent way suppressed the fraction of fully activated platelets (TRAP6 > 2-MeSADP > CRP-XL), whereas succinate and coagulated plasma increased this fraction (CRP-XL > TRAP6 > 2-MeSADP). Interestingly, a subset of platelets showed a constant response (aggregating, secreting, or aggregating plus α-granule secreting), which was hardly affected by the stimulus strength or priming substances.

Development of a DHA-Losartan hybrid as a potent inhibitor of multiple pathway-induced platelet aggregation

Despite the scientific progression in the prevention and treatment of cardiovascular diseases (CVDs) they remain the leading cause of mortality and disability worldwide. The classic treatment involves the simultaneous dosing of two antiplatelet drugs, aspirin and clopidogrel/prasugrel. However, besides drug resistance, severe side effects have been also manifested including acute bleeding and toxicity. Thus, new therapeutic agents with enhanced efficacy and diminished side effects are of importance. Towards this end, omega-3 (ω-3) fatty acids have demonstrated potent efficacy against CVDs through inhibiting platelet aggregation that bears a pivotal role in atherothrombosis. Another factor that displays a critical role in the pathogenesis of cardiovascular diseases is the renin-angiotensin system (RAS), and especially the AT1R blocker losartan that has been reported to exert antiplatelet activity mediated by this receptor. Along these lines, we envisaged developing a molecular hybrid consisted of docosahexaenoic acid (ω-3 fatty acid) and losartan, that could exert a notable antiplatelet effect against CVDs. The design and synthesis of the new DHA-losartan hybrid, designated DHA-L, bestowed with the additive properties of the parent compounds, is reported. In silico studies were first exploited to validate the potential of DHA-L to retain losartan’s ability to bind AT1R. The antiplatelet activity of DHA-L was evaluated against in vitro platelet aggregation induced by several platelet agonists. Notably, the hybrid illustrated a pleiotropic antiplatelet profile inhibiting platelet aggregation through multiple platelet activation pathways including P2Y12, PAR-1 (Protease-Activated Receptor-1), PAF (Platelet Activating Factor), COX-1 (cyclooxygenase-1) and collagen receptors. The stability of DHA-L in human plasma and in a wide range of pH values was also evaluated over time using an HPLC protocol. The hybridization approach described herein could pave the way for the development of novel potent multitargeted therapeutics with enhanced antiplatelet profile. Communicated by Ramaswamy H. Sarma

Analysis of microvascular thrombus mechanobiology with a novel particle-based model

Platelet accumulation at the site of a vascular injury is regulated by soluble platelet agonists, which induce various types of platelet responses, including integrin activation and granule secretion. The interplay between local biochemical cues, mechanical interactions between platelets and macroscopic thrombus dynamics is poorly understood.Here we describe a novel computational model of microvascular clot formation for the detailed analysis of thrombus mechanics. We adopt a previously developed two-dimensional particle-based model focused on the thrombus shell formation and revise it to introduce the platelet agonists. Blood flow is simulated via a computational fluid dynamics approach. In order to model soluble platelet activators, we apply Langevin dynamics to a large number of non-dimensional virtual particles. Taking advantage of the available data on platelet dense granule secretion kinetics, we model platelet degranulation as a stochastic agonist-dependent process.The new model qualitatively reproduces the enhanced thrombus formation due to dense granule secretion, in line with in vivo findings, and provides a mechanism for the thrombin confinement at the early stages of clot formation. Our calculations also predict that the release of platelet dense granules results in the additional mechanical stabilization of the inner layers of thrombus. Distribution of the inter-platelet forces throughout the aggregate reveals multiple weak spots in the outer regions of a thrombus, which are expected to result in the mechanical disruptions at the later stages of clot formation.