Platelet Aggregation Formation Research Articles

Sinomenium acutum Modulates Platelet Aggregation and Thrombus Formation by Regulating the Glycoprotein VI-Mediated Signalosome in Mice.

Sinomenium acutum (SA) has long been used as a traditional medicine in China, Japan, and Korea to treat a wide range of diseases. It has been traditionally used to ameliorate inflammation and improve blood circulation. However, its role in platelet activation has not been thoroughly investigated. Hence, we conducted this study to assess the potential inhibitory effect of SA on platelet aggregation and thrombus formation. The antiplatelet activities of SA were evaluated by assessing platelet aggregation, granular secretion, intracellular Ca2+ mobilization, and the Glycoprotein (GP) VI-mediated signalosome. The thrombosis and bleeding time assays were used to investigate the effect of SA (orally administered at 50 and 100 mg/kg for seven days) in mice. SA treatment at concentrations of 50, 100, and 200 μg/mL significantly reduced GPVI-mediated platelet aggregation, granular secretion, and intracellular Ca2+ mobilization. Further biochemical studies revealed that SA inhibited spleen tyrosine kinase, phospholipase Cγ2, phosphatidylinositol 3-kinase, and AKT phosphorylation. Interestingly, oral administration of SA efficiently ameliorated FeCl3-induced arterial thrombus formation without prolonging the tail bleeding time. These findings suggest that SA has beneficial effects in thrombosis and hemostasis. Therefore, SA holds promise as an effective therapeutic agent for the treatment of thrombotic diseases.

NLRP3 Inflammasome-Mediated Platelet Activation and Thrombus Formation in Sickle Cell Mice Can be Targeted By the BTK Inhibitor Ibrutinib

Introduction: Platelets are abnormally activated in sickle cell disease (SCD) and are involved in promoting vascular occlusions and thrombosis. The NOD like receptor protein 3 (NLRP3) inflammasome is a critical inflammatory mediator that has recently been found to be upregulated in platelets in SCD. Our recent discovery of Bruton Tyrosine Kinase (BTK) as a regulator of the NLRP3 inflammasome has provided an opportunity to suppress NLRP3 inflammasome activation in platelets via the BTK inhibitor ibrutinib. The role of the NLRP3 inflammasome in regulating platelet activation in SCD and its potential targetability through BTK inhibition is poorly understood. Methods: Townes SCD mice, which express human sickle hemoglobin (HbSS), and control mice (HbAA) at age 16 to 28 weeks were used for this study. Where indicated, mice were injected with the NLRP3 inhibitor MCC950, the BTK inhibitor ibrutinib or vehicle control 4 hours prior to blood collection. NLRP3 inflammasome activation was assessed in isolated platelets with fluorescent labeled inhibitor of caspase-1 (FLICA) assay and immunofluorescence staining of intraplatelet NLRP3 and the adaptor apoptosis-associated speck-like protein containing a CARD (ASC) with confocal microscopy. We measured platelet surface expression of P-selectin (CD62P), platelet ATP secretion, platelet spreading, platelet aggregation, and in vitro thrombus formation using flow cytometry, luminescence, scanning electron microscopy, whole blood impedance aggregometry, and a flow chamber with blood perfused at shear rates of 1,700 s -1, respectively. Isolated platelets or blood samples were treated with the NLRP3 activator nigericin where indicated. Collagen was used as platelet agonist. Data are shown as mean ± SD. Results: We found increased platelet P-selectin expression (CD62P%: 74.57 ± 3.03 vs 35.18 ± 0.73, p<0.01), platelet ATP secretion (AUC: 16.24 ± 2.17 vs 3.16 ± 0.94, p<0.01), platelet spreading (spreading area, µm 2: 5.47 ± 1.97 vs 2.35 ± 0.23, p<0.01), platelet aggregation (AUC: 68.18 ± 6.84 vs 36.28 ± 3.29, p<0.01), and thrombus formation (thrombus area %: 64.24 ± 14.64 vs 27.44 ± 5.99, p<0.05) in blood from SCD mice as compared to control mice. NLRP3:ASC speck formation (Pearson coefficient: 0.62 ± 0.03 vs 0.38 ± 0.02, p<0.001) and caspase-1 activation levels (RFUs: 476.6 ± 54.84 vs 237.8 ± 50.09, p<0.001) were elevated in platelets from SCD mice as compared to control mice, indicating platelet NLRP3 inflammasome activation in SCD mice. Next, we investigated the role of the NLRP3 inflammasome in regulating platelet function in SCD mice. Treatment of mice with the NLRP3 inhibitor MCC950 resulted in a reduction of upregulated platelet P-selectin expression (CD62P%: 33.55 ± 17.21 vs 74.23 ± 9.95, p<0.001), platelet ATP secretion (AUC: 5.98 ± 1.2 vs 13 ± 2.91, p<0.001), platelet spreading (spreading area, µm 2: 2.42 ± 0.75 vs 5.47 ± 1.97, p<0.001), platelet aggregation (AUC: 39.85 ± 2.5 vs 64.65 ± 6.5, p<0.001), and thrombus formation (thrombus area %: 41.63 ± 7.78 vs 70.22 ± 5.5, p<0.001) (MCC950 vs vehicle) in SCD mice but had no effect in control mice. We next investigated the effect of the BTK inhibitor ibrutinib. Injection of ibrutinib interfered with elevated platelet NLRP3 inflammasome activation in SCD mice, as shown by quantification of intraplatelet NLRP3:ASC specks (Pearson coefficient: 0.56 ± 0.04 vs 0.72 ± 0.03, p<0.001) and platelet caspase-1 activity (RFUs: 193 ± 55.24 vs 386.8 ± 88.08, p<0.001) (ibrutinib vs vehicle) but had no effect in control mice. Ibrutinib treatment decreased upregulated platelet P-selectin (CD62P%: 37.81 ± 10.65 vs 64.74 ± 17.73, p<0.01), platelet ATP secretion (AUC: 6.78 ± 0.24 vs 13.9 ± 1.12, p<0.001), platelet spreading (spreading area, µm 2: 2.32 ± 0.79 vs 6.45 ± 0.99, p<0.001), platelet aggregation (AUC: 40.08 ± 3.56 vs 62.13 ± 3.99, p<0.001), and thrombus formation (thrombus area, %: 44.42 ± 8.24 vs 66.97 ± 11.83, p<0.001) (ibrutinib vs vehicle) in SCD mice. The NLRP3 activator nigericin reversed these inhibitory effects of ibrutinib in SCD mice, indicating that ibrutinib interferes with platelet activation and thrombus formation in SCD mice through the NLRP3 inflammasome. Conclusion: The NLRP3 inflammasome is a critical mediator of platelet activation and thrombus formation in SCD mice, which is targetable by the BTK inhibitor ibrutinib, making it a potentially useful therapeutic approach for managing the prothrombotic state in SCD.

The inhibitory effects of glabridin on human platelet aggregation and thrombus formation

The inhibitory effects of glabridin on human platelet aggregation and thrombus formation

Evaluating the impact of transient shear stress on platelet activation, adhesion, and aggregation with microfluidic chip technique.

When nonphysiological stenosis occurs, the transient high shear stress formed in vessels increases the risk of thrombosis and is a potential factor for cardiovascular diseases. But the platelet adhesion and aggregation behavior at nonphysiological post-stenosis and its affecting factors are not fully understood yet. In this experiment, platelet aggregation on collagen and fibrinogen at different shear stresses and different hematocrits were observed by microfluidic technology. Platelet activation (P-selectin, glycoprotein IIb/IIIa) and monocyte-platelet aggregate (MPA) levels under different shear stresses were analyzed by flow cytometry. On fibrinogen, platelets aggregate more at higher shear stress conditions. While on collagen, it becomes more difficult for platelets to form stable aggregation at higher shear stress conditions. If platelets adhere initially at low shear stress, stable platelet aggregation can be formed at subsequent high shear stress. Moreover, when the shear stress increases, platelet activity markers (P-selectin, glycoprotein IIb/IIIa and MPAs) increase significantly. Hematocrit affects the degree of platelet aggregation, and the influence of hematocrit is obvious at high shear stress. Transient high shear stress (46 ms) can effectively activate platelets. Platelet aggregation behavior was different for coated fibrinogen and collagen protein. Stable platelet adhesion at post-stenosis is more dependent on fibrinogen and platelet aggregation is stable on both fibrinogen and collagen. Hematocrit can significantly affect the formation of platelet aggregation.

Flow Chamber Analyses in Cardiovascular Research: Impact of Platelets and the Intercellular Crosstalk with Endothelial Cells, Leukocytes, and Red Blood Cells.

Platelets are main drivers of thrombus formation. Besides platelet aggregate formation, platelets interact with different blood cells such as red blood and white blood cells (RBCs, WBCs) and endothelial cells (ECs), to promote thrombus formation and inflammation. In the past, the role of different proteins in platelet adhesion, activation, and aggregate formation has been analyzed using platelets/mice with a genetic loss of a certain protein. These knock-out mouse models have been investigated for changes in experimental arterial thrombosis or hemostasis. In this review, we focused on the Maastricht flow chamber, which is a very elegant tool to analyze thrombus formation under flow using whole blood or different blood cell components of genetically modified mice. Besides, the interaction of platelets with RBCs, WBCs, and ECs under flow conditions has been evaluated with regard to thrombus formation and platelet-mediated inflammation. Importantly, alterations in thrombus formation as emerged in the flow chamber frequently reflect arterial thrombosis in different mouse models. Thus, the results of flow chamber experiments in vitro are excellent indicators for differences in arterial thrombosis in vivo. Taken together, the Maastricht flow chamber can be used to (1) determine the severity of platelet alterations in different knock-out mice; (2) analyze differences in platelet adhesion, aggregation, and activation; (3) investigate collagen and non-collagen-dependent alterations of thrombus formation; and (4) highlight differences in the interaction of platelets with different blood/ECs. Thus, this experimental approach is a useful tool to increase our understanding of signaling mechanisms that drive arterial thrombosis and hemostasis.

OC 58.1 Circulating Endoglin Decreases Platelet Aggregation and Thrombus Formation through Interaction with the αIIbβ3 Integrin

OC 58.1 Circulating Endoglin Decreases Platelet Aggregation and Thrombus Formation through Interaction with the αIIbβ3 Integrin

A-154 Cost-effectiveness of On-site Vs Send-out Measurement of ADAMTS13 Activity in the Management of Acquired Thrombotic Thrombocytopenic Purpura

Abstract Background Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy (TMA) caused by congenital or acquired severe deficiency of disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13), an enzyme cleaving von Willebrand factor (VWF). ADAMTS-13 deficiency leads to the accumulation of ultra-large VWF multimers, and subsequent platelet aggregation and microthrombi formation. Aside clinical symptoms, the diagnosis of TTP is based thrombocytopenia, hemolytic anemia with the presence of schistocytes, and severely reduced activity of ADAMTS-13 (<10%). As TTP is a life-threatening condition, it is of great importance to shorten the time to the treatment, and according to the French recommendations, the triplet therapy including plasma exchange (PEX), rituximab, caplacizumab, plus corticosteroids, must be administered to patients with suspected TTP until the results of ADAMTS-13 was obtained, and be pursued only in the case of deficiency. Therefore, a rapid ADAMTS-13 testing is critical for early diagnosis and optimal management of acute TTP. Methods We compared the cost-effectiveness of a strategy based on on-site measurement of ADAMTS-13 activity using a rapid fully automated chemiluminescent assay (HemosIL AcuStar ADAMTS-13) vs its centralized measurement in a reference laboratory, as it is currently the case at our institution. For that purpose, we created two scenarios based on either its local measurement with a maximum time-to-result of 16 h (assay performed during daytime, 7/7) or its centralized measurement with a median time-to-result of 4 days (range: 2–8, as demonstrated from the 2018–2021 estimates). The triplet therapy was hypothesized to be started on admission and stopped if ADAMTS-13 activity was >10%. In case of ADAMTS-13 activity <10%, an inhibitor screening would be performed at the reference laboratory, as this assay was not expected to be locally implemented. For the calculation, we considered the costs of reagent, shipping/handling of samples to the reference laboratory, laboratory staff workload, reimbursement rates, and treatments administered in patients with suspected TTP until availability of the ADAMTS-13 result. Results Based on 2018 to 2021 estimates, there was a mean of 60 prescriptions of ADAMTS-13 activity per year. Most were for the follow-up of TTP patients and for patients with non-TTP TMA. Only seven were for the diagnosis of acute TTP, as suggested by the French score, which was confirmed in four cases (mean values). The local measurement of ADAMTS-13 activity was found to be cost-effective with a 16%-reduction of the costs compared to its centralized measurement in those seven patients with suspected TTP during the first 4 days. Conclusion Together with its short turnaround time (33 min) and its full automation, our results suggest that the HemosIL AcuStar ADAMTS-13 activity assay could be an assay of choice to rapidly measure ADAMTS-13 activity in the plasma and thus to establish the diagnosis of acute TTP in emergency settings. Considering the local costs, using the strategy based on the local evaluation of ADAMTS-13 activity led to a 16%-reduction of the overall costs during the first 4 days after admission of the seven patients with suspected acute TTP.

Aging Is Associated With Organ-Specific Alterations in the Level and Expression Pattern of von Willebrand Factor.

VWF (von Willebrand factor) is an endothelial-specific procoagulant protein with a major role in thrombosis. Aging is associated with increased circulating levels of VWF, which presents a risk factor for thrombus formation. Circulating plasma, cellular protein, and mRNA levels of VWF were determined and compared in young and aged mice. Major organs were subjected to immunofluorescence analyses to determine the vascular pattern of VWF expression and the presence of platelet aggregates. An in vitro model of aging, using extended culture time of endothelial cells, was used to explore the mechanism of age-associated increased VWF levels. Increased circulating plasma levels of VWF with elevated levels of larger multimers, indicative of VWF functional activity, were observed in aged mice. VWF mRNA and cellular protein levels were significantly increased in the brains, lungs, and livers but not in the kidneys and hearts of aged mice. Higher proportion of small vessels in brains, lungs, and livers of aged mice exhibited VWF expression compared with young, and this was concomitant with increased platelet aggregate formation. Prolonged culture of endothelial cells resulted in increased cell senescence that correlated with increased VWF expression; VWF expression was specifically detected in senescent cultured endothelial cells and abolished in response to p53 knockdown. A significantly higher proportion of VWF expressing endothelial cells in vivo exhibited senescence markers SA-β-Gal (senescence-associated β-galactosidase) and p53 in aged mouse brains compared with that of the young. Aging elicits a heterogenic response in endothelial cells with regard to VWF expression, leading to organ-specific increase in VWF levels and alterations in vascular tree pattern of expression. This is concomitant with increased platelet aggregate formation. The age-associated increase in VWF expression may be modulated through the process of cell senescence, and p53 transcription factor contributes to its regulation.

Covid- 19 Vaccine-induced Myocarditis.

Myocarditis and pericarditis are rare adverse reactions, more commonly seen in young males after receiving the second dose of an mRNA vaccine. However, the benefits of vaccination heavily outweigh the risk of these side effects. In addition, vaccination boosters are effective against the newest, more infective variants. Therefore we expect more vaccines to be administered in the following years. The objective of this study is to review the current understanding of the mechanism, diagnosis, and treatment of myocarditis and pericarditis. Proposed mechanisms include molecular mimicry against the S protein and hypersensitivity reactions with mRNA vaccines and platelet aggregation and thrombus formation in cardiac blood vessels with adenoviral vaccines. Diagnosis of myocarditis is based on clinical findings, cardiac enzymes, ECG, MRI, and echocardiographic findings. Management includes NSAIDs and cardiovascular support in selected cases with ventricular dysfunction. Most patients have a mild presentation with preservation of cardiac function and recover entirely within seven days; the average hospital stay is three days. Long-term complications are infrequent.

Research and Clinical Approaches to Assess Platelet Function in Flowing Blood.

Platelet adhesion and activation is fundamental to the formation of a hemostatic response to limit loss of blood and instigate wound repair to seal a site of vascular injury. The process of platelet aggregate formation is supported by the coagulation system driving injury-proximal formation of thrombin, which converts fibrinogen to insoluble fibrin. This highly coordinated series of molecular and membranous events must be routinely achieved in flowing blood, at vascular fluid shear rates that place significant strain on molecular and cellular interactions. Platelets have long been recognized to be able to slow down and adhere to sites of vascular injury and then activate and recruit more platelets that forge and strengthen adhesive ties with the vascular wall under these conditions. It has been a major challenge for the Platelet Research Community to construct experimental conditions that allow precise definition of the molecular steps occurring under flow. This brief review will discuss work to date from our group, as well as others that has furthered our understanding of platelet function in flowing blood.

Hydrophobic Polystyrene-Modified Gelatin Enhances Fast Hemostasis and Tissue Regeneration in Traumatic Brain Injury.

Hemostatic sealant is required to deal with blood loss, especially in the scenario of traumatic brain injury (TBI), which presents high rates of morbidity and disability. Hemostasis in surgery with traditional gelatin-based sealants often leads to blood loss and other issues in brain because of the hydrophilic gelatin swelling. Herein, hydrophobic effects on the hemostasis in TBI surgery are studied by tuning the chain length of polystyrene (PS) onto methylacrylated gelatin (Gel-MA). The hydrophobicity and hemostatic efficiency can be tuned by controlling the length of PS groups. The platelet activation of modified sealants Gel-MA-2P, Gel-MA-P, and Gel-MA-0.5P is as much as 17.5, 9.1, and 2.1 times higher than Gel-MA in vitro. The hemostatic time of Gel-MA-2P, Gel-MA-P, and Gel-MA-0.5P groups is 2.0-, 1.6-, and 1.1-folds faster than that in Gel-MA group in TBI mice. Increased formation of fibrins and platelet aggregation can also be observed in vitro by scanning electron microscopy. Animal's mortality is lowered by 46%, neurologic deficiency is reduced by 1.5 times, and brain edema is attenuated by 10%. Protein expression is further investigated to exhibit toxic iron-related processes caused by delayed hemostasis and activation of platelets via PI3K/PKC-α signaling. The hydrophobic Gel-MA has the potential in hemostatic TBI and promotes nervous system recovery in brain with the potentials in clinics.

Effect of pathological high shear exposure time on platelet activation and aggregation.

Circulating platelets are sometimes exposed to high shear rate environments due to vascular stenosis, and the effect of transiently elevated pathological high shear rates on platelet activation and aggregation function has not been clarified. The aim of this study was to investigate the effect of pathological high shear rate (8302s-1) exposure time (3.16-25.3 ms) on platelet activation and aggregation function. In addition, by adding active ingredients of antiplatelet drugs such as ASA (an active ingredient of aspirin), Ticagrelor, Tirofiban and GP1BA (platelet membrane protein GPIb inhibitor) in vitro, we studied TXA2, P2Y12-ADP, GPIIb/IIIa-fibrinogen and GPIb /IX/V-vWF receptor pathways to determine platelet activation function mediated by pathological high shear rate. In this study, we designed a set of microfluidic chips with stenosis lengths of 0.5 mm, 1 mm, 2 mm, 3 mm, and 4 mm, all with 80% stenosis, to generate pathological high shear forces that can act at different times. The whole blood flowing through the microchannels was collected by perfusion of sodium citrate anticoagulated whole blood at a physiological arterial shear rate (1500 s-1), and the expression levels of platelet surface activation markers (P-selectin and GP IIb/IIIa) and the degree of platelet aggregation were analyzed by flow cytometry; platelet aggregation patterns were observed by microscopic examination of blood smears. The results showed that shearing significantly increased platelet activation and aggregation levels compared to un-sheared whole blood, and the activation and aggregation levels increased with increasing duration of pathological high shear rate. In vitro inhibition studies showed that ASA barely inhibited the expression of P-selectin and PAC-1 on the platelet surface; Ticagrelor effectively inhibited the expression of both P-selectin and PAC-1; Tirofiban significantly inhibited the expression of PAC-1 on the platelet surface and slightly inhibited the expression of P-selectin; GP1BA significantly inhibited the expression of both. Our results suggest that transient pathological high shear rate (8302s-1) exposure can induce platelet activation in a time-dependent manner; however, the mechanism is more complex and may be due to the following reasons: transient elevated pathological high shear rate activates platelets through the GPIb/IX/V-vWF receptor pathway, and after platelet activation, its surface membrane protein GPIIb/IIIa receptors activate platelets through fibrinogen to form platelet-platelet aggregates, and further activation of active substances such as ADP and TXA2 released by platelet alpha particles, which contribute to the formation of irreversible platelet aggregation.

Evaluating von Willebrand factor and ADAMTS13 levels in thalassemia major patients and assessing a possible association with Thrombospondin-1.

Alterations in the endothelium and endothelial adhesion proteins such as von Willebrand factor (vWF) play major roles in hypercoagulability in thalassemia. vWF protein release leads to platelet aggregation and thrombi formation at the site of vascular injury. It is then degraded by the proteolytic enzyme ADAMTS13. Thrombospondin-1 is a multifactorial glycoprotein, which was reported to compete with ADAMTS13 for sites of vWF proteolysis. In this study, levels of vWF, ADAMTS13, and TSP-1 proteins were determined in β-thalassemia major patients. A possible association between TSP-1 and vWF and ADAMTS-13 was also evaluated. The study was conducted on 80 β-thalassemia major patients and 80 age and sex matched healthy controls. The 80 patients were sub-divided into two groups; splenectomised and non-splenectomised. vWF, ADAMTS13 and TSP-1 plasma level were measured using ELISA technique. There was no significant difference in vWF and TSP-1 levels between patients and controls (p > 0.05). However, ADAMTS13 levels and ADAMTS13 activity/vWF antigen ratio were significantly higher in patients compared to controls (p < 0.05). VWF antigen and TSP-1 level were significantly higher in splenectomised patients (p = 0.025 and p < 0.001, respectively). We also observed a significant decrease in ADAMTS13 activity/vWF antigen ratio among splenectomised compared to non- splenectomised patients (p = 0.019). Correlation analysis showed a significant negative correlation between TSP-1 and vWF Collagen Binding Activity (r = -0.394, p = 0.021) and a positive correlation with ADAMTS13 activity/vWF antigen ratio (r = 0.356, p = 0.039) in splenectomised compared to non- splenectomised patients. Our findings highlight the adequacy of patient management protocols for β-TM in Kuwait as patients presented with comparable levels of platelets, vWF and TSP-1 compared to normal controls. The reported increase in ADAMTS13 in patients may be required to maintain normal levels of vWF. Although no active thrombotic episodes were reported at the time of the study, the significant rise in platelets, vWF:Ag and TSP-1 levels in splenectomised patients may indicate a tendency towards hypercoagulability. Monitoring of splenectomised patients is recommended.

MAGT1 Deficiency Dysregulates Platelet Cation Homeostasis and Accelerates Arterial Thrombosis and Ischemic Stroke in Mice.

MAGT1 (magnesium transporter 1) is a subunit of the oligosaccharide protein complex with thiol-disulfide oxidoreductase activity, supporting the process of N-glycosylation. MAGT1 deficiency was detected in human patients with X-linked immunodeficiency with magnesium defect syndrome and congenital disorders of glycosylation, resulting in decreased cation responses in lymphocytes, thereby inhibiting the immune response against viral infections. Curative hematopoietic stem cell transplantation of patients with X-linked immunodeficiency with magnesium defect causes fatal bleeding and thrombotic complications. We studied the role of MAGT1 deficiency in platelet function in relation to arterial thrombosis and hemostasis using several in vitro experimental settings and in vivo models of arterial thrombosis and transient middle cerebral artery occlusion model of ischemic stroke. MAGT1-deficient mice (Magt1-/y) displayed accelerated occlusive arterial thrombus formation in vivo, a shortened bleeding time, and profound brain damage upon focal cerebral ischemia. These defects resulted in increased calcium influx and enhanced second wave mediator release, which further reinforced platelet reactivity and aggregation responses. Supplementation of MgCl2 or pharmacological blockade of TRPC6 (transient receptor potential cation channel, subfamily C, member 6) channels, but not inhibition of store-operated calcium entry, normalized the aggregation responses of Magt1-/y platelets to the control level. GP (glycoprotein) VI activation of Magt1-/y platelets resulted in hyperphosphorylation of Syk (spleen tyrosine kinase), LAT (linker for activation of T cells), and PLC (phospholipase C) γ2, whereas the inhibitory loop regulated by PKC (protein kinase C) was impaired. A hyperaggregation response to the GP VI agonist was confirmed in human platelets isolated from a MAGT1-deficient (X-linked immunodeficiency with magnesium defect) patient. Haploinsufficiency of TRPC6 in Magt1-/y mice could normalize GP VI signaling, platelet aggregation, and thrombus formation in vivo. These results suggest that MAGT1 and TRPC6 are functionally linked. Therefore, deficiency or impaired functionality of MAGT1 could be a potential risk factor for arterial thrombosis and stroke.

P67: An Evaluation of Aspirin and Platelet Activation in the Presence of Shear Stress

Background: Mechanical circulatory support (MCS) causes high levels of shear stress, which could lead to complications such as platelet activation and thrombosis. Aspirin is an anti-platelet drug that functions through COX-1 inhibition, however, this may not be sufficient to reduce activation via mechanical stimuli. As such, this study evaluated the efficiency of Aspirin to reduce the downstream effects of platelet functioning and the impact this may have on shear stress-induced thrombosis. Methods: Human blood was treated with and without Aspirin (4.5 μg/mL) and subjected to VAD-like shear stress for 4 hours. Platelets were analyzed by flow cytometry, using CD42b and CD62P, to assess multifactorial parameters. Positive events were normalized and assessed for platelet activation, granularity (size), as well as the number of platelet-platelet aggregates and platelet-derived extracellular vesicles (EVs). Results: Aspirin moderately reduced platelet activation and platelet granularity compared to the untreated control, suggesting little to no protective effects against mechanical stimulation. However, the presence of Aspirin had a greater effect on reducing the number of platelet aggregates and platelet-derived EVs in the presence of VAD-like shear stress. Conclusion: The use of Aspirin (and COX-1 inhibition) may prove ineffective for reducing platelet activation by mechanical stimuli in high-risk patients. However, Aspirin may impair the downstream formation of pro-thrombotic platelet aggregates and EVs, thereby reducing the risk of thrombosis in MCS patients. As Aspirin is observed to provide some protection, further research into combination therapy with additional anti-platelet drugs is needed to overcome the negative impact of mechanical stimulation.

Platelet zinc status regulates prostaglandin-induced signaling, altering thrombus formation

BackgroundApproximately 17.3% of the global population exhibits an element of zinc (Zn2+) deficiency. One symptom of Zn2+ deficiency is increased bleeding through impaired hemostasis. Platelets are crucial to hemostasis and are inhibited by endothelial-derived prostacyclin (prostaglandin I2 [PGI2]), which signals via adenylyl cyclase (AC) and cyclic adenosine monophosphate signaling. In other cell types, Zn2+ modulates cyclic adenosine monophosphate concentrations by changing AC and/or phosphodiesterase activity. ObjectivesTo investigate if Zn2+ can modulate platelet PGI2 signaling. MethodsPlatelet aggregation, spreading, and western blotting assays with Zn2+ chelators and cyclic nucleotide elevating agents were performed in washed platelets and platelet-rich plasma conditions. In vitro thrombus formation with various Zn2+ chelators and PGI2 was assessed in whole blood. ResultsIncubation of whole blood or washed platelets with Zn2+ chelators caused either embolization of preformed thrombi or reversal of platelet spreading, respectively. To understand this effect, we analyzed resting platelets and identified that incubation with Zn2+ chelators elevated pVASPser157, a marker of PGI2 signaling. In agreement that Zn2+ affects PGI2 signaling, addition of the AC inhibitor SQ22536 blocked Zn2+ chelation–induced platelet spreading reversal, while addition of Zn2+ blocked PGI2-mediated platelet reversal. Moreover, Zn2+ specifically blocked forskolin-mediated AC reversal of platelet spreading. Finally, PGI2 inhibition of platelet aggregation and in vitro thrombus formation was potentiated in the presence of low doses of Zn2+ chelators, increasing its effectiveness in inducing platelet inhibition. ConclusionZn2+ chelation potentiates platelet PGI2 signaling, elevating PGI2’s ability to prevent effective platelet activation, aggregation, and thrombus formation.

Evaluation of total leukocytes count by flow cytometry with Beckman Coulter® DXH900 in the presence of platelet aggregates

The Beckman Coulter® DXH900 uses the impedance method to measure the total leukocytes count. In presence of platelet aggregates, the device identifies the structural changes and associates an alarm with the leukocytes result. The aim of this study was to evaluate the influence of platelet aggregates using the principle of flow cytometry as a second assessment of the white blood cell count. Total leukocytes count was evaluated in 49 specimens with presence of platelet aggregates and 32 without anomaly. The differences between total leukocyte count by the two automatic methods (impedance and flow cytometry) and the microscopic method were compared. Without platelet aggregates, the median values were 5.6 (microscopic cell count), 5.4 (impedance) and 5.4 (flow cytometry) and no discordance was observed. In presence of platelet aggregates, the median values were 5.6, 6.4 and 5.1 respectively. The graphical analysis with the allowable total error range of ± 25.7% showed substantial analytical discrepancies (15/49) using impedance method while the flow cytometry method revealed minor disagreements (3/49). Analytical discordances versus WBC reference ranges showed 88% agreement and a substantial Kappa coefficient of 0.70 by impedance, while the flow cytometry method had 94% agreement and a perfect Kappa coefficient of 0.83. The formation of platelet aggregates increased the total leukocyte count performed DXH900 impedance method. Our study has shown that DXH 900 flow cytometry method may be an alternative to exclude the presence of pseudoleukocytosis. In case flags are generated, the microscopic method may be needed for the confirmation of WBC count.

Low-dose rivaroxaban: can cardiovascular events be reduced?

Despite available effective guideline-based preventive therapies, patients with vascular diseases remain at high-risk of recurrent ischaemic events. Novel therapeutic strategies are therefore needed in order to further reduce the residual risk that is present in these high-risk patients. The Cardiovascular Outcomes for People using Anticoagulation Strategies trial demonstrated that, in patients with chronic coronary artery disease (CAD) and peripheral artery disease (PAD), a combination of rivaroxaban 2.5 mg/bid (vascular dose) and acetylsalicylic acid (ASA) 100 mg once daily, the so-called dual pathway inhibition (DPI), reduced cardiovascular death, stroke, or myocardial infarction by 24% and mortality by 18%, as compared with ASA-alone. The rationale that can explain the improvement of cardiovascular outcome is that platelet aggregation and fibrin formation are involved in arterial thrombosis and rivaroxaban is able to target both ways and has a synergic effect with ASA. The aim of this review is to discuss the potential mechanisms and added benefits of DPI, in patients with PAD and CAD.

The Subtilisin-Like Protease Furin Regulates Hemin-Dependent Ectodomain Shedding of Glycoprotein VI.

Hemolysis results in release of free hemoglobin and hemin liberation from erythrocytes. Hemin has been described to induce platelet activation and to trigger thrombosis. We evaluated the effect of hemin on platelet function and surface expression of the platelet collagen receptor glycoprotein VI (GPVI). Isolated platelets were stimulated with increasing concentrations of hemin. We found that hemin strongly enhanced platelet activation, aggregation, and aggregate formation on immobilized collagen under flow. In contrast, we found that surface expression of GPVI was significantly reduced upon hemin stimulation with high hemin concentrations indicating that hemin-induced loss of surface GPVI does not hinder platelet aggregation. Loss of hemin-induced surface expression of GPVI was caused by shedding of the ectodomain of GPVI as verified by immunoblotting and is independent of the GPVI or CLEC-2 mediated ITAM (immunoreceptor-tyrosine-based-activation-motif) signaling pathway as inhibitor studies revealed. Hemin-induced GPVI shedding was independent of metalloproteinases such as ADAM10 or ADAM17, which were previously described to regulate GPVI degradation. Similarly, concentration-dependent shedding of CD62P was also induced by hemin. Unexpectedly, we found that the subtilisin-like proprotein convertase furin controls hemin-dependent GPVI shedding as shown by inhibitor studies using the specific furin inhibitors SSM3 and Hexa-D-arginine. In the presence of SSM3 and Hexa-D-arginine, hemin-associated GPVI degradation was substantially reduced. Further, SSM3 inhibited hemin-induced but not CRP-XL-induced platelet aggregation and thrombus formation, indicating that furin controls specifically hemin-associated platelet functions. In summary, we describe a novel mechanism of hemin-dependent GPVI shedding and platelet function mediated by furin.

Tyrame [N-(3-hydroxy-1:3:5(10)-estratrien-17β-yl)-4-hydroxy-phenethylamine], antithrombotic aminoestrogen that decreases microvesicle formation.

Estrogens that are used as contraceptives or in replacement therapy are associated with an increase in the risk for developing thrombosis, mainly during the first year of treatment and in women with associated risk factors. To synthesize, characterize and identify the anticoagulant, antiplatelet aggregation and microvesicle-reducing effect of the new aminoestrogen Tyrame. CD1 strain mice were used, which had Tyrame (0, 1 and 2 mg/100 g) subcutaneously administered. At 24 h, a blood sample was obtained to determine whole-blood clotting time, microvesicles concentration and inhibitory effect on platelet aggregation. Blood clotting time increased up to 1.5 times in comparison with the control. Platelet aggregation inhibition had different magnitude depending on the agonist agent employed, and was complete with collagen. Both effects had a dose-dependent relationship. The microvesicles decreased up to six times with respect to the control. Tyrame reduces platelet aggregation and microvesicle formation, which emphasizes its potential therapeutic utility as an estrogen free of thrombotic effects.