Platelet Activation Status Research Articles

Platelet-Dependent Inflammatory Dysregulation in Patients with Stages 4 or 5 Chronic Kidney Disease: A Mechanistic Clinical Study.

Chronic kidney disease (CKD) is characterized by dysregulated inflammation that worsens with CKD severity. The role of platelets in modulating inflammation in stage 4 or 5 CKD remains unexplored. We investigated whether there are changes in platelet-derived thromboinflammatory markers in CKD with dual antiplatelet therapy (DAPT; aspirin 81 mg/d plus P2Y12 inhibitor). In a mechanistic clinical trial, we compared platelet activation markers (aggregation and surface receptor expression), circulating platelet-leukocyte aggregates, leukocyte composition (monocyte subtypes and CD11b surface expression), and plasma cytokine profile (45 analytes) of non-CKD controls (n=26) and CKD outpatients (n=48) with a glomerular filtration rate (GFR) <30 ml/min per 1.73 m2 on 2 weeks of DAPT. Patients with CKD demonstrated a reduced mean platelet count, elevated mean platelet volume, reduced platelet-leukocyte aggregates, reduced platelet-bound monocytes, higher total non-classic monocytes in the circulation, and higher levels of IL-1RA, VEGF, and fractalkine (all P<0.05). There were no differences in platelet activation markers between CKD and controls. Although DAPT reduced platelet aggregation in both groups, it had multifaceted effects on thromboinflammatory markers in CKD, including a reduction in PDGF levels in all CKD individuals, reductions in IL-1β and TNF-α levels in select CKD individuals, and no change in a number of other cytokines. Significant positive correlations existed for baseline IL-1β, PDGF, and TNF-α levels with older age, and for baseline TNF-α levels with presence of diabetes mellitus and worse albuminuria. Mean change in IL-1β and PDGF levels on DAPT positively correlated with younger age, mean change in TNF-α levels with higher GFR, and mean changes in PDGF, and TRAIL levels correlated with worse albuminuria. Minimum spanning trees plot of cytokines showed platelet-derived CD40L had a large reduction in weight factor after DAPT in CKD. Additionally, platelet-derived IL-1β and PDGF were tightly correlated with other cytokines, with IL-1β as the hub cytokine. Attenuated interactions between platelets and leukocytes in the CKD state coincided with no change in platelet activation status, an altered differentiation state of monocytes, and heightened inflammatory markers. Platelet-derived cytokines were one of the central cytokines in patients with CKD that were tightly correlated with others. DAPT had multifaceted effects on thromboinflammation, suggesting that there is platelet-dependent and -independent inflammation in stage 4 or 5 CKD.

Theme and Variation: Structuring Thrombus Formation from Jugular/Arterial Puncture Wounds to Occlusive Clots in a Mouse Model

Hemostasis in veins and arteries has been assumed to be different processes, e.g., white vs. red clots. We took the unbiased experimental approaches of 3D EM across full clot volumes and high-resolution heat mapping of platelet activation state to probe for commonalities and variations using controlled mouse models. Remarkably, the thrombi formed, 5 min post-puncture, in the jugular vein and femoral artery showed considerable consistency in organization. There were vaulted cavities within the puncture wound and apparent extravascular capping of the puncture hole to produce bleeding cessation. Notable differences in the venous thrombi were a greater activation of platelets lining the surfaces of the vaulted columns, an outcome that we attribute to lowr flow rates resulting in the longer retention of activating signals, and in the "hanging drop” nature of the arterial thrombi anchored to the extrema with an outward, extravascular bulge of the thrombus. We attribute these variations to arterial vessel wall properties and higher flow rates. At 20 min post-puncture, the intravascular crown of the venous wound thrombi was enriched in loosely adherent platelets in a low activation state. Similarly, the outer portion of the intravascular arterial thrombus was rich in low activation platelets including obviously discoid platelets. Strikingly, the arterial thrombus was subject to rebleeding and ate 20 min displayed fractured extravascular features indicative of an unstable clot. The puncture wound thrombi form in the relatively small volume of the immediate wound site. In contrast, occlusive clots fill the cylindrical volume of the vessel. To us, the overall structure of the wound thrombi suggested a predictive hypothesis for induced occlusive clotting. We propose that 1) the exposed intima lining the arterial vessel wall provides a cylindrical substratum for the generation of a tightly adherent platelet layer anchoring the forming clot and 2) the central volume of the clot resembles the intravascular crown of the puncture wound thrombus. Hence, the prediction of a central volume of the occlusive clot consisting of loosely adherent to discoid shaped platelets. That was result found for FeCl3 induced femoral clots by wide area-TEM (WA-TEM) i.e., a central volume rich in discoid shaped platelet and few red blood cells were trapped in the tightly adherent rim of the clot.. Using sequential block face SEM, we have begun to image and volume render carotid clots by sequential block face SEM. Analysis of 5,000 sequential images from the downstream half of the carotid clot, showed that the structure gradually tapered in a conical manner and contains few red blood cells. We propose that there are indeed organizational themes shared by the puncture wound thrombus in venous or arterial settings and the occlusive clot in which vascular damage is restricted to the endothelial cell layer. Work was supported by NIH grants (BS) R01 HL155519 and P20 HL146373 (Joel Bennett, principal investigator), R35 HL150118 (SWW) and by intramural NIBIB research program (RDL). An occlusive FeCl3 clot in the carotid artery of a C57BL/6 mouse imaged by sequential block face SEM over a length of 1.2 millimeters.A. cross section of the clot 200 microns in from the mid-occlusion starting point for sectioning. B. Zoom of A) to illustrate the low level of trapped red blood cells within the clot. C. cross section of the clot 900 microns in from the starting point showing how the clot has now tapered down and fills a minor portion of the artery. D. Rendering of 1.2 millimeters of the clot (blue), vessel wall (green) and in gray a single plane image. Flow was from left to right. Note that the clot progressively tapers to a point in the downstream direction.Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Patent foramen ovale (PFO) and migraine: role of platelet Tissue Factor expression and oxidative stress. The LEARNER study

Abstract Background Migraine is a chronic neurovascular disorder with a multifactorial aetiology. A strong relationship links migraine with aura (MHA) and patent foramen ovale (PFO), with migraine regression after PFO closure. Increased platelet aggregation and oxidative stress were documented in migraineurs. Current guidelines suggest the use of aspirin or P2Y12-antagonists. To date, however, no clear mechanisms connecting MHA to PFO has been demonstrated. Purpose To perform a comprehensive analysis of platelet activation, inflammation and oxidative stress status in 78 aspirin-treated MHA-patients before (T0) and 6-months after (T1) PFO closure (LEARNER Study-NCT03521193-clinicaltrials.gov). Primary endpoint was migraine regression rate in relation to these parameters. Methods P-selectinpos-, activated-glycoproteinIIbIIIa (aGPIIbIIIa)pos-, Tissue Factor (TF)pos-, reactive oxygen species (ROS)pos-platelets, platelet-leukocyte aggregates (PLA) and microvesicles (MVs) were evaluated by flow cytometry; thrombin generation (TG) by Calibrated Automated Thrombogram (CAT) assay; oxidative stress status by mass spectrometry. Twelve aspirin-treated-healthy subjects (HS) were enrolled for comparison. To test the effect of PFO patients' plasma on platelet activation, blood from HS was plasma-depleted and replaced with a pool of plasma from PFO patients. Results Migraine resolution occurred in 69.7%, significant reduction in 27%, while no effect was observed in 2 patients (3.2%). Only ROSpos-platelets, and TFpos-platelets and -MVs were significantly higher at T0, sustaining a TG capacity that was associated with an altered blood GSSG/GSH (Oxidized/Reduced Glutathione) ratio. This phenotype reverted to HS levels at T1 (Fig. 1). MHA-PFO plasma, added to HS blood, mirrored the in vivo platelet activation and N-acetylcysteine blunted it. In vitro GSSG treatment of HS platelets reproduced the in vivo condition. Aspirin had little effect on the platelet prothrombotic phenotype which was effectively inhibited by a P2Y12-antagonist (Fig.2). Conclusions MHA-PFO patients show a platelet-associated prothrombotic phenotype, characterized by a marked thrombin generation capacity sustained by an elevated number of platelets and MVs expressing a functionally active Tissue Factor and sustained by altered oxidative stress status. This phenotype, not fully controlled by aspirin but by P2Y12-antagonism, reverted after PFO closure together with a complete migraine remission. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Health

SGLT-2 inhibition by empagliflozin exerts neutral effects on experimental arterial thrombosis in a murine model of low-grade inflammation

Abstract Introduction Low-grade inflammation couples dysmetabolic states to insulin resistance and atherosclerotic cardiovascular disease (ASCVD). Selective sodium-glucose co-transporter 2 (SGLT-2) inhibition by empagliflozin improves clinical outcomes in patients with ASCVD independently of glucose-lowering. Yet, its mechanism of action remains largely undetermined. Purpose We aimed to test whether empagliflozin affects arterial thrombus formation in baseline conditions or low-grade inflammation, a systemic milieu shared among patients with ASCVD. Methods Sixteen-week-old C57BL/6 mice were randomly assigned to acute administration of empagliflozin (25 mg/kg BW) or vehicle, of which a subgroup was pre-treated biweekly over 4 weeks with super-low-dose lipopolysaccharide (LPS; 5 ng/kg BW), before carotid thrombosis was induced by photochemical injury. The translational value of these findings was investigated in primary human aortic endothelial cells (HAECs) and plasma samples of patients randomized to empagliflozin therapy. Results The between-group difference in doppler-flow probe detected time-to-occlusion (TTO) remained within the predefined equivalence margin (Δ=|10.50|), irrespective of low-grade inflammation (95% confidence interval [CI], −9.82 to 8.85 and −9.20 to 9.69), while glucose dropped by 1.64 and 4.84 mmol/l, respectively (Fig. 1). Ex vivo platelet aggregometry suggests similar platelet activation status, corroborated by unchanged circulating platelet-factor 4 (PF4) plasma levels. In concert, carotid PAI-1 expression and TF activity remained unaltered upon SGLT-2 inhibition, and no difference in plasma D-dimer levels was detected, suggesting comparable coagulation cascade activation and fibrinolytic activity (Fig. 1). In HAECs pre-treated with LPS, empagliflozin neither changed TF activity nor PAI-1 expression (Fig. 2). Accordingly, among patients with established ASCVD or at high cardiovascular (CV) risk randomized to 10 mg empagliflozin daily signatures of thrombotic (i.e., TF) and fibrinolytic activity (i.e., PAI-1) remained unchanged, while plasma glucose declined significantly during 3 months of follow-up (Fig. 2). Conclusion SGLT-2 inhibition by empagliflozin does not impact experimental arterial thrombus formation, neither under baseline conditions nor during sustained low-grade inflammation, and has no impact on proxies of thrombotic/fibrinolytic activity in patients with ASCVD. The beneficial pleiotropic effects of empagliflozin are likely independent of pathways mediating thrombosis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science FoundationSwiss Heart FoundationFoundation for Cardiovascular Research–Zurich Heart House

Long COVID-19 syndrome: association of cardiopulmonary impairment with a persistent platelet activation

Abstract Background A considerable proportion of patients do not fully recover from COVID-19 infection and report symptoms that persist beyond the initial phase of infection: this condition is defined long-COVID-19 syndrome (LCS). LCS can involve lungs as well as several extrapulmonary organs, including the cardiovascular system. The risk and 1-year burden of cardiovascular diseases (CVD) is increased in COVID-19 survivors, even in subjects at low risk of CVD. Recently, we documented that acute COVID-19 infection induces altered platelet activation state characterized by a prothrombotic phenotype and by the formation of platelet-leukocyte aggregates (PLA), that may be involved in the pulmonary microthrombi found in autoptic specimens. No data are yet available on the contribution of platelet activation to residual pulmonary impairment and procoagulant potential in LCS patients. Purpose To study platelet activation status, microvesicle (MV) profile, platelet thrombin generation capacity (pTGC) in LCS patients enrolled at 6 months after resolution of the acute phase (6mo-FU), compared to acute COVID-19 infection patients. Methods 6mo-FU COVID-19 patients (n=24) with established LCS were enrolled at Centro Cardiologico Monzino. Residual pulmonary impairment was assessed by Cardiopulmonary Exercise Testing (CPET) and 64-rows-CT scan evaluation. Platelet activation (P-selectin, Tissue Factor [TF] and PLA) and MV profile were assessed by flow cytometry; pTGC by calibrated automated thrombogram. 46 patients enrolled during acute COVID-19 infection and 46 healthy subjects (HS) were used for comparison. Results Dispnea in LCS patients was confirmed by CPET showing compromised alveolus-capillary membrane diffusion and residual pulmonary impairment. TF+-platelet and -MV levels were 3-fold (1.5% [1.2–2.9] vs 2.4% [1.6–5.7]) and 2-fold (217/μl [137–275] vs 435/μl [275–633]) lower at 6mo-FU compared to acute phase, being comparable to HS. pTGC behaved similarly. At 6mo-FU, the MV profile, in terms of total number and cell origin, returned to physiological levels. Conversely, although lower than that measured in acute phase, a 2.5-fold higher platelet P-selectin expression (6.9% [3–13.5] vs 11.7% [5.2–18.9]) and PLA formation (35.5% [27.4–46.8] vs 67.7% [45.7–85.3]) was observed at 6mo-FU compared to HS. Interestingly, a significant correlation between PLA formation and residual pulmonary impairment was observed (r=−0.423; p=0.02). Conclusion These data strengthen the hypothesis that the presence of PLA in the bloodstream, and thus also in the pulmonary microcirculation, may contribute to support pulmonary dysfunction still observed in LCS patients. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Health (Ricerca Corrente 2020 MPP COVID4)

Platelets and Sera from Donors of Convalescent Plasma after Mild COVID-19 Show No Procoagulant Phenotype.

Coronavirus disease-2019 (COVID-19) is associated with increased thromboembolic complications. Long-term alteration in the coagulation system after acute COVID-19 infection is still a subject of research. Furthermore, the effect of sera from convalescent subjects on platelets is not known. In this study, we investigated platelet phenotype, coagulation, and fibrinolysis in COVID-19 convalescent plasma (CCP) donors and analyzed convalescent sera-induced effects on platelets. We investigated CCP donors who had a history of mild COVID-19 infection and donors who did not have COVID-19 were used as controls. We analyzed phosphatidylserine (PS) externalization, CD62p expression, and glycoprotein VI (GPVI) shedding both in platelet-rich plasma (PRP) and after incubation of washed healthy platelets with donors' sera using flow cytometry. Coagulation and fibrinolysis systems were assessed with thromboelastometry. Forty-seven CCP donors (22 males, 25 females; mean age (±SD): 41.4 ± 13.7 years) with a history of mild COVID-19 infection were included. Median duration after acute COVID-19 infection was 97 days (range, 34-401). We did not find an increased PS externalization, CD62p expression, or GPVI shedding in platelets from CCP donors. Sera from CCP donors did not induce PS externalization or GPVI shedding in healthy platelets. Sera-induced CD62p expression was slightly, albeit statistically significantly, lower in CCP donors than in plasma donors without a history of COVID-19. One patient showed increased maximum clot firmness and prolonged lysis time in thromboelastometry. Our findings suggest that procoagulant platelet phenotype is not present after mild COVID-19. Furthermore, CCP sera do not affect the activation status of platelets.

Investigation of shear-induced platelet activation in ventricular assist device

Due to the scarcity of organ donations, ventricular assist devices are the most accessible treatment for patients with advanced heart failure. Since their development, these devices have helped thousands of patients and could have helped even more had it not been for some of the complications they still experience. Among the most common complications are thrombosis and hemolysis. The purpose of this paper is to numerically investigate the effect of the blade angle and the blade count on the hydraulic properties of a newly designed ventricular assist device, as well as the potential for shear-induced platelet activation. The study was conducted on several models with different blade angles and blade counts using a variety of rotational speeds. Analysis of the obtained results showed a significant improvement in the pressure rise and the hydraulic efficiency in models with higher blade angle and lower blade count. In contrast, the other models showed slight improvement or deterioration of the hydraulic performance. In terms of shear-induced platelet activation, although the performance of all models was within an acceptable range, models with a higher blade angle and lower blade count had the lowest average platelet activation state.

β-Amyloid promotes platelet activation and activated platelets act as bridge between risk factors and Alzheimer’s disease

Alzheimer’s disease (AD) is an evolving challenge that places an enormous burden on families and society. The presence of obvious brain β-amyloid (Aβ) deposition is a premise to diagnose AD, which induces the subsequent tau hyperphosphorylation and neurodegeneration. Platelets are the primary source of circulating amyloid precursor protein (APP). Upon activation, they can secrete significant amounts of Aβ into the blood, which can be actively transported to the brain across the blood-brain barrier and promote amyloid deposition. In this review, we summarized the changes in the platelet APP metabolic pathway in patients with AD and further comprehensively explored the targets and downstream events of Aβ-activated platelets. In addition, we attempted to clarify whether patients with AD are in a state of general platelet activation, with inconsistent results. Considering the increasingly evident bidirectional relationship between AD and vascular events, we speculate that the AD pathology alone seems to be insufficient to induce the general activation of platelets; however, the intervention of third-party factors, such as atherosclerosis, exposes the extracellular matrix and leads to platelet activation, further promoting AD progression. Therefore, we proposed a framework in which the relationship between platelets and AD is indirect and mediated by vascular factors. Therapies targeting platelets and interventions for vascular risk factors are likely to contribute to the prevention and treatment of AD.

Assessment of on-treatment platelet reactivity at high and low shear stress and platelet activation status after the addition of dipyridamole to aspirin in the early and late phases after TIA and ischaemic stroke

BackgroundData are limited on the ability of dipyridamole to additionally inhibit platelet function/reactivity in ischaemic cerebrovascular disease (CVD) patients on aspirin. AimsTo assess inhibition of platelet function/reactivity and platelet activation with dipyridamole in CVD. MethodsThis prospective, observational study assessed TIA/ischaemic stroke patients before (baseline; N = 60), at 14 ±7 days (14d, N = 39) and ≥ 90 days (90d, N = 31) after adding dipyridamole to aspirin. Platelet function/reactivity at high shear stress (PFA-100® C-ADP) and low shear stress (VerifyNow® P2Y12 and Multiplate® ADP assays), and platelet activation status (% expression of CD62P, CD63 and leucocyte-platelet complexes on whole blood flow cytometry) were quantified. ‘Dipyridamole-high on-treatment platelet reactivity (HTPR)’ was defined as failure to inhibit ADP-induced platelet aggregation +/- adhesion compared with the patient's baseline on aspirin monotherapy by more than twice the coefficient-of-variation of the assay after adding dipyridamole to aspirin. ResultsDipyridamole-HTPR was identified in 71.4–75% of patients on PFA-100 C-ADP, 83.9–86.8% of patients on VerifyNow P2Y12, and 81.5–83.3% of patients on Multiplate ADP assays. There were no changes in CD62P/CD63 expression (P ≥ 0.18), or consistent changes in leucocyte-platelet complexes in CVD patients overall at 14d or 90d vs. baseline after commencing dipyridamole. Monocyte-platelet complexes increased in the patient subgroup with dipyridamole-HTPR at 14d and 90d on PFA-100, and at 14d on VerifyNow (P ≤ 0.04), but not in those without dipyridamole-HTPR. DiscussionAdditional antiplatelet effects of dipyridamole are detectable under high and low shear stress conditions with user-friendly platelet function/reactivity tests ex vivo. Increasing circulating monocyte-platelet complexes over time are associated with dipyridamole-HTPR.

P979: EXOSOMES IN POLYCYTHEMIA VERA: “MINI PLATELETS“ WITH THROMBOGENIC POTENTIAL

Background: Polycythemia vera (PV) is characterized by clonal proliferation of myeloid progenitor cells that is caused by activating mutation in the Janus Kinase (JAK)2 gene. Patients with PV tend to develop thrombotic complications across the vasculature tree. Yet, how PV-clonal cells induce pro-thrombotic environment is still under investigation. Secreted by all types of cells, exosomes are nano-scaled particles which carry a molecular cargo that reflects their cell of origin. Exosomes travel in blood, may reach distant sites and affect various physiological and pathological processes. These features led us to think that PV-derived exosomes (hereafter PV-exosomes) carry the mutated JAK2 oncogene and contribute to the thrombotic manifestations seen in PV. Aims: Determine whether PV-exosomes carry mutated JAK2 transcripts. Determine the prothrombotic potential of PV-exosomes and their effect on endothelium. Methods: Exosomes were isolated from eythroleukemia (HEL 92.1.7) cell line (positive for mutated JAK2) and from patients with PV by serial ultracentrifugation. To confirm the presence of exosomes, electron microscopy images were obtained and to quantify their concentration nanoscale tracking analysis was applied (Malvern). The exosomes were stained with the FM-4-11 lipophilic dye and their uptake by HUVECs and HaCaT cells was followed by flow cytometry. To determine the presence of JAK2 transcript, a 465 bp long DNA segment within exon14 of JAK2 was amplified by qRT-PCR and by Sanger sequencing JAK2 mutational status was determined. Thrombin generation assay (TGA) (Ceveron®, Alpha, Technoclone) was used to assess the thrombotic potential of PV-exosomes. Platelet activation status was assessed by the expression of PAC-1, CD-62 and CD-63 in platelets and Trans Epithelial Electric Resistance (TEER) (Electric cell substrate impedance sensing device, Applied Biophysics) was used to assay endothelial dysfunction in HUVECs. Results: We isolated exosomes from the sera of 24 healthy donors and 31 patients with PV. We found the presence of the mutated JAK2 mRNA in all samples confirming that the mutated JAK2 transcript is packaged into sera-exosomes. Yet, while healthy donors’ derived exosomes carry only the wild type JAK2 mRNA, we detected both the wild type and the mutated JAK2 isoforms in patients with PV. We showed that PV-exosomes induced thrombin generation. Then we compared PV-exosomes and healthy-individuals’ derived exosomes and found that thrombin generation potential of PV-exosomes is higher in all measured parameters (p< 0.05 in all parameters). Furthermore, using various platelet markers (CD62, CD63, PAC1) we confirmed that PV-exosomes induced platelet activation. Together, these findings suggest that PV-exosomes may promote thrombosis directly. Under physiological conditions the integrity of the vascular wall prevents uncontrolled thrombi. Therefore, we wondered whether PV-exosomes induce endothelial dysfunction, and in this way contribute indirectly to the pro-thrombotic phenotype. PV-exosomes were taken up by HUVEC endothelial cells in a dose- and time- dependent manner and TEER assay revealed that exosomal uptake by HUVECs induced endothelial dysfunction by breaking the tight junction between adjacent endothelial cells. Summary/Conclusion: Exosomes may act as “mini platelets” that promote thrombosis in patients with PV. PV-exosomes may promote thrombosis either directly by activating platelets and inducing thrombin generation or indirectly by inducing endothelial dysfunction.

The Platelet Electrome: Evidence for a Role in Regulation of Function and Surface Interaction

Background: Platelets protect the body from injury through formation of blood clots, changing from a normal, quiescent state to becoming “activated” in response to external stimuli such as chemical cues, shear stress, and temperature. This causes changes in shape, increased adhesion, and alteration of electrical properties such as membrane potential Vm and zeta potential ζ. These electrical phenomena have been regarded as largely unconnected; for example, changes in ζ have been attributed solely to alteration of surface lipid concentration. However, recent reports suggest that cells can alter ζ electrostatically by alteration of Vm in red blood cells. We hypothesized that if platelets also modulate ζ through Vm, this may provide an alternative mechanism to alter cell-cell interaction. Materials and Methods: We investigated platelets stored at different temperatures (4°C, 22°C, and 37°C) for 24 h, which is known to alter platelet behavior and electrical properties, and compared these with analyses of freshly harvested platelets. These four conditions exhibited unique sets of electrical properties (Vm, ζ, membrane conductance Geff, and cytoplasm conductivity σcyto), as well as surface exposure of the adhesion molecule P-selectin. These were analyzed to identify correlations between electrical parameters and platelet activation state. Results: Many parameters exhibit pairwise correlation across all four conditions, in particular between ζ and Geff, and between Vm and σcyto. Furthermore, when the electrical behavior of platelets stored at 4°C (known to activate the cells) was removed from the analysis, additional relationships were observed among the remaining conditions, including those connecting ζ and Vm to the amount of P-selectin binding. Conclusion: Results suggest that Vm may mechanistically alter the availability of cationic molecules at the cell surface, a process never reported before, with implications for our wider understanding of cell-molecule and cell-cell interaction.

Flow cytometry based platelet activation markers and state of inflammation among subjects with type 2 diabetes with and without depression

Type 2 Diabetes Mellitus (T2DM) and Major Depressive Disorder (MDD) are highly disabling disorders associated with a multitude of vascular complications. Platelets are known to play a role in the pathogenesis of vascular complications in both T2DM and MDD. These complications could increase in patients with comorbid diabetes and depression. To quantify and compare flow cytometry based platelet activation markers and the inflammatory state between individuals of diabetes with depression, individuals of diabetes without depression and healthy controls. Out of 114 participants, each study group contained 38 participants in diabetic group, diabetics with depression group and matched control group. Diabetes was diagnosed with the American Diabetes Association (ADA) criteria. Screening of MDD was done with Patient Health Questionnaire 2 (PHQ2) and severity of depression assessed with Hamilton Depression Rating (HAM-D) scale. Platelet markers CD41, CD42b, CD62P and CD63 were assayed using flow cytometer. Platelet count, surface expression of platelet activation markers CD62P and CD63, hs-CRP, insulin and HOMA-IR score differed significantly between the groups. Post hoc analysis showed significantly high CD63 expression in patients with comorbid diabetes and depression compared to those having diabetes without depression. Patients with comorbid diabetes and depression have enhanced platelet hyperactivation and a pro inflammatory state which increases susceptibility to vascular complications.

Variation in Platelet Activation State in Pre-Donation Whole Blood: Effect of Time of Day and ABO Blood Group

BackgroundWhilst there has been investigation into the effect of time of the day on platelet activation and function in healthy individuals, there is a lack of studies in the literature to examine this relationship among platelet donors.MethodsWe assessed the extent of platelet activation by percentage of platelets with surface-expressed P-selectin and flow cytometry in samples of whole blood from a group of qualified platelet donors (n = 84).ResultsThe mean (SD) percentage of activated platelets in the pre-donation blood samples was 1.85 ± 1.57% (range 0.2–7.5%). In univariate analyses, the percentage of activated platelets was significantly and inversely correlated with the collection time (ie, the time of day blood samples were collected) (r = –0.35, p = 0.001) and positively correlated to mean platelet volume (MPV) (r = 0.29, p = 0.008). A weaker positive correlation was also observed with ABO blood group (r = 0.228, p = 0.036). Analysis of the collection time as a categorical variable showed a greater degree of activated platelets in samples collected between 8:00 h and 10:00 h than in samples collected during the hours of >10:00 h ≤14:00 h (2.5 ± 1.8 versus 1.1 ± 0.74, p < 0.001). In the adjusted linear regression model, collection time was a significant independent predictor of platelet activation state in whole blood (β = –0.26; p < 0.001), as did ABO blood group (β = 0.55; p = 0.019).ConclusionOur results show that collection time is the most important predictor of platelet activation state in pre-donation whole blood among platelet donors. This work may have implications for optimizing the timing of platelet donation.

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 Function Is Associated With Dementia Risk in the Framingham Heart Study.

BackgroundVascular function is compromised in Alzheimer disease (AD) years before amyloid and tau pathology are detected and a substantial body of work shows abnormal platelet activation states in patients with AD. The aim of our study was to investigate whether platelet function in middle age is independently associated with future risk of AD.Methods and ResultsWe examined associations of baseline platelet function with incident dementia risk in the community‐based FHS (Framingham Heart Study) longitudinal cohorts. The association between platelet function and risk of dementia was evaluated using the cumulative incidence function and inverse probability weighted Cox proportional cause‐specific hazards regression models, with adjustment for demographic and clinical covariates. Platelet aggregation response was measured by light transmission aggregometry. The final study sample included 1847 FHS participants (average age, 53.0 years; 57.5% women). During follow‐up (median, 20.5 years), we observed 154 cases of incident dementia, of which 121 were AD cases. Results from weighted models indicated that platelet aggregation response to adenosine diphosphate 1.0 µmol/L was independently and positively associated with dementia risk, and it was preceded in importance only by age and hypertension. Sensitivity analyses showed associations with the same directionality for participants defined as adenosine diphosphate hyper‐responders, as well as the platelet response to 0.1 µmol/L epinephrine.ConclusionsOur study shows individuals free of antiplatelet therapy with a higher platelet response are at higher risk of dementia in late life during a 20‐year follow‐up, reinforcing the role of platelet function in AD risk. This suggests that platelet phenotypes may be associated with the rate of dementia and potentially have prognostic value.

Investigation of the influence of blade configuration on the hemodynamic performance and blood damage of the centrifugal blood pump.

The design and optimization of centrifugal blood pumps are crucial for improved extracorporeal membrane oxygenation system performance. Secondary flow passages are common in centrifugal blood pumps, allowing for a high volume of unstable flow. Traditional design theory offers minimal guidance on the design and optimization of centrifugal blood pumps, so it's critical to understand how design parameter variables affect hydraulic performance and hemocompatibility. Computational fluid dynamics (CFD) was employed to investigate the effects of blade number, blade wrap angle, blade thickness, and splitters on pressure head, hemolysis, and platelet activation state. Eulerian and Lagrangian features were used to analyze the flow fields and hemocompatibility metrics such as scalar shear stress, velocity distribution, and their correlation. The equalization of frictional and flow losses allow impellers with more blades and smaller wrap angles to have higher pressure heads, whereas the trade-off between the volume of high scalar shear stress and exposure time allows impellers with fewer blades and larger blade wrap angles to have a lower HI; there are configurations that increase the possibility of platelet activation for both number of blades and wrap angles. The hydraulic performance and hemocompatibility of centrifugal blood pumps are not affected by blade thickness. Compared to the main blades, splitters can improve the blood compatibility of a centrifugal blood pump with a small reduction in pressure head, but there is a trade-off between the length and location of the splitter that suppresses flow losses while reducing the velocity gradient. According to correlation analysis, pressure head, HI, and the volume of high shear stress were all substantially connected, and exposure time had a significant impact on HI. The platelet activation state was influenced by the average scalar shear stress and the volume of low velocity. The findings reveal the impact of design variables on the performance of centrifugal blood pumps with secondary flow passages, as well as the relationship between hemocompatibility, hydraulic performance, and flow characteristics, and are useful for the design and optimization of this type of blood pump, as well as the prediction of clinical complications.

Migraine in Patients Undergoing PFO Closure: Characterization of a Platelet-Associated Pathophysiological Mechanism: The LEARNER Study

The association between migraine and patent foramen ovale (PFO) has been documented. We aimed to investigate platelet activation, prothrombotic phenotype, and oxidative stress status of migraineurs with PFO on 100mg/day aspirin, before and 6months after PFO closure. Data show that, before PFO closure, expression of the classical platelet activation markers is comparable in patients and aspirin-treated healthy subjects. Conversely, MHA-PFO patients display an increased prothrombotic phenotype (higher tissue factorpos platelets and microvesicles and thrombin-generation potential), sustained by an altered oxidative stress status. This phenotype, which is more controlled by P2Y12-blockade than by aspirin, reverted after PFO closure together with a complete migraine remission. (pLatelEts And MigRaine iN patEnt foRamen Ovale [LEARNER]; NCT03521193).

Platelet activation state in early stages of COVID-19.

Platelet activation at the early stage of COVID-19 is poorly described. The need for antiplatelet therapy in patients with COVID-19 remains controversial. We characterized the platelet activation profile in hospitalized patients at the early stage of COVID-19 using the modified prothrombinase Platelet Activation State (PAS) Assay. Sixteen patients admitted to the emergency department of the IRCCS San Raffaele Hospital (Milan, Italy) between February 8 and April 2021 were enrolled. All patients presented with respiratory symptoms and tested positive for severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2). Platelet activation was measured via the PAS Assay within 24 hours from patients' hospital admission. Data were compared with those measured in N.=24 healthy subjects (controls). Platelet activation was significantly higher in COVID-19 patients with respect to controls (PAS=0.63 [0.58-0.98%] vs. 0.46 [0.40-0.65%], respectively; P=0.03). Of note, highest PAS values were measured in the two patients with the worst clinical outcome, i.e., death because of respiratory failure (PAS=2.09% and 1.20%, respectively). No differences in standard coagulation parameters were noted between these two patients and those who were later discharged home. This study provides evidence of significant platelet activation state at the early stage of COVID-19 and suggests that the patient-specific platelet activation profile is a reliable clinical marker to stratify COVID-19 patients at high risk of poor clinical outcome who might potentially benefit from antiplatelet therapy.

Ramifications of Vorticity on Aggregation and Activation of Platelets in Bi-Leaflet Mechanical Heart Valve: Fluid-Structure-Interaction Study.

Bileaflet mechanical heart valves (BMHV) are widely implanted to replace diseased heart valves. Despite many improvements in design, these valves still suffer from various complications, such as valve dysfunction, tissue overgrowth, hemolysis, and thromboembolism. Thrombosis and thromboembolism are believed to be initiated by platelet activation due to contact with foreign surfaces and nonphysiological flow patterns. The implantation of the valve causes nonphysiological patterns of vortex shedding behind the leaflets. This study signifies the importance of vorticity in platelet activation and aggregation in BMHV implants. A two-phase model with the first Eulerian phase for blood and the second discrete phase for platelets is used here. The generalized cross model of viscosity has been used to simulate the non-Newtonian viscosity of blood. A fluid-structure-interaction model has been used to simulate the motion of leaflets. This study has also estimated the platelet activation state (PAS), which is the mathematical estimation of the degree of activation of platelets due to flow-induced shear stresses that cause thrombus formation. The regions in the fluid domain with a higher vorticity field have been found to contain platelets with relatively higher PAS than regions with relatively lower vorticity fields. Also, this study has quantitatively reported the effect of vorticity on platelet aggregation. Platelet densities in fluid areas with higher vorticity are higher than densities in fluid areas with lower vorticity, indicating that highly activated platelets aggregate in areas with stronger vorticity.

Agitation-dependent biomechanical forces modulate GPVI receptor expression and platelet adhesion capacity during storage

BackgroundContinuous agitation during storage slows down the platelet storage lesions. However, in special circumstances, manual-mixing can be alternatively used to store products for short time periods without compromising platelet quality. Based on this finding, and given the role of shear stress in modulating receptor expression, we were interested in comparing the levels of platelet adhesion receptor, GPVI and platelet adhesion capacity under each storage condition.MethodsPlatelet concentrates (PCs) were divided into three groups: continuously-agitated PCs (CAG-PCs) with or without PP2 (Src kinase inhibitor) and manually-mixed PCs (MM-PCs). Platelet count/MPV, swirling, GPVI and P-selectin expression, GPVI shedding, platelet adhesion/spreading to collagen were examined during 5 days of storage.ResultsWhile MM- and CAG-PCs showed similar levels of P-selectin expression, GPVI expression was significantly elevated in MM-PCs with lower GPVI shedding/expression ratios, enhanced platelet adhesion/spreading and swirling in manually-mixed PCs. Of note, CAG-PCs treated with PP2 also demonstrated lower P-selectin expression and GPVI shedding, higher GPVI expression and attenuated swirling and spreading capability.ConclusionGiven the comparable platelet activation state in MM and CAG-PCs as indicated by P-selectin expression, enhanced platelet adhesion/spreading in MM-PCs, along with relatively higher GPVI expression here, supports previous studies demonstrating a role for biomechanical forces in modulating GPVI-dependent function. Thus, lower GPVI expression in CAG-PCs may be due to shear forces induced by agitation, which keeps this receptor down-regulated while also attenuating platelet adhesion/spreading capacities during storage. Low platelet function in PP2-CAG-PCs also highlights the importance of Src-kinases threshold activity in maintaining platelets quality.