Exposure to Perfluoroalkyl Chemicals and Cardiovascular Disease: Experimental and Epidemiological Evidence

Polyfluoro- and perfluoro–alkyl substances (PFAS) are organic chemicals extensively used worldwide for industry and consumer products. Due to their chemical stability, PFAS represent a major cause of environmental pollution. PFAS accumulate in animal and human blood and tissues exerting their toxicity. We performed a review of the epidemiological studies exploring the relationship between exposure to PFAS and thromboembolic cardiovascular disease. An increase in cardiovascular disease or death related to PFAS exposure has been reported from cross-sectional and longitudinal observational studies with evidence concerning the relation with early vascular lesions and atherosclerosis. Several studies indicate an alteration in lipid and glucose metabolism disorders and increased blood pressure as a possible link with cardiovascular thromboembolic events. We also examined the recent evidence indicating that legacy and new PFAS can be incorporated in platelet cell membranes giving a solid rationale to the observed increase risk of cardiovascular events in the populations exposed to PFAS by directly promoting thrombus formation. Exposure to PFAS has been related to altered plasma membrane fluidity and associated with altered calcium signal and increased platelet response to agonists, both in vitro and ex vivo in subjects exposed to PFAS. All the functional responses are increased in platelets by incorporation of PFAS: adhesion, aggregation, microvesicles release and experimental thrombus formation. These findings offer mechanistic support the hypothesis that platelet-centred mechanisms may be implicated in the increase in cardiovascular events observed in populations chronically exposed to PFAS.

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Characterization of platelet functionality in pediatric patients with kaposiform hemangioendothelioma / Kasabach-Merritt phenomenon

10.22541/au.163949732.29741152/v1 ◽

2021 ◽

Author(s):

Alexey Martyanov ◽

Ivan Tesakov ◽

Olga An ◽

Julia-Jessica Korobkin ◽

Anastasia Ignatova ◽

...

Keyword(s):

Parallel Plate ◽

Ex Vivo ◽

Thrombus Formation ◽

Lower Boundary ◽

Calcium Mobilization ◽

Control Group ◽

Functional Responses ◽

Kaposiform Hemangioendothelioma ◽

Normal Ranges ◽

Platelet Receptor

Background. Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy commonly associated with Kasabach-Merritt phenomenon (KMP) that includes thrombocytopenia and coagulation dysfunction. Platelet receptor CLEC-2 -tumor cell podoplanin interaction is considered the key mechanism of thrombocytopenia in KMP, however, the effect of long-term exposure to podoplanin on platelet function is unknown. Procedure. Here we examined blood samples from six patients with KHE and one KMP. Platelet calcium signaling and functional responses to conventional activation and CLEC-2 stimulation were analyzed by continuous and endpoint live cell flow cytometry. Platelet aggregation in response to ADP or rhodocytin was analyzed by low-angle light scattering approach (LaSca). Additionally, ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Results. We demonstrate that in KHE/KMP platelet functional responses to strong stimulation were on the lower boundary of age-matched normal ranges, while calcium mobilization and fibrinogen binding upon stimulation with ADP alone were significantly lower than control values. Platelet di-aggregate formation in response to ADP was also diminished in most of the patients. Formation of platelet aggregates in collagen-coated parallel plate flow chambers was also noticeably lower than in the age-matched control group. Calcium mobilization in response to CLEC-2 stimulation was unaltered in the patients and could be blocked by low-molecular-weight inhibitors, 2CP and HB125. Conclusions. While platelet responsiveness in KHE/KMP is moderately altered, their CLEC-2 receptors remain functional and respond to inhibition. Therefore, our findings suggest that CLEC-2-targeting molecules are new potential agents in therapeutic management of this life-threatening condition.

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Loss of zinc transporters ZIP1 and ZIP3 augments platelet reactivity in response to G protein-coupled receptor agonists and accelerates thrombus formation in vivo

10.1101/2021.11.19.469234 ◽

2021 ◽

Author(s):

Amro Elgheznawy ◽

Patricia Oeftering ◽

Maximilian Englert ◽

Friederike Kaiser ◽

Charly Kusch ◽

...

Keyword(s):

G Protein ◽

Ex Vivo ◽

Platelet Reactivity ◽

Thrombus Formation ◽

Platelet Response ◽

G Protein Coupled Receptor ◽

Icp Ms ◽

And Function ◽

G Protein Coupled

Zinc (Zn2+) is considered as an important mediator for thrombosis and haemostasis. However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these well-known Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly delayed and less efficient Zn2+ release upon thrombin-stimulated platelet activation. This resulted in a hyperactive platelet response not only in response to thrombin, but also towards other G protein-coupled receptor (GPCR) agonists. Immunoreceptor tyrosine-based activation (ITAM)-coupled receptor agonist signalling, however, was unaffected. Augmented GPCR responses were accompanied by enhanced Ca2+ signalling and PKC activation. Further functional analysis of ZIP1/3 double deficient mice revealed enhanced platelet aggregation, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function.

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Observation of granulocyte function during ex vivo thrombus formation for patients with ANKRD26-associated thrombocytopenia

Pediatric Hematology/Oncology and Immunopathology ◽

10.24287/1726-1708-2020-19-1-27-34 ◽

2020 ◽

Vol 19 (1) ◽

pp. 27-34

Author(s):

D. S. Morozova ◽

A. A. Martyanov ◽

M. A. Panteleev ◽

P. A. Zharkov ◽

D. V. Fedorova ◽

...

Keyword(s):

Intracellular Signaling ◽

Ex Vivo ◽

Thrombus Formation ◽

Published Data ◽

Platelet Activity ◽

Functional Responses ◽

Movement Velocity ◽

Plane Flow ◽

Healthy Donors

ANKRD26-associated thrombocytopenia is a non-syndromic hereditary thrombocytopenia for which there are currently no formal diagnostic criteria. It is known that the probability of myeloid leukemia in patients with pathogenetic variants in the ANKRD26 gene significantly increases, however, studies of the functioning of granulocytes in this pathology have not been conducted. Aims: Analysis of the functioning of granulocytes and platelets during ex vivo thrombosis in patients with ANKRD26-associated thrombocytopenia. The study was approved by the Independent Ethics Committee of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. Two patients and 10 healthy volunteers were included in the study. Intracellular signaling and platelet functional responses were observed by continuous flow cytometry. Ex vivo thrombus formation and granulocyte functioning were observed on a fluorescence microscope in parallel-plane flow chambers containing fibrillar collagen. Upon physiological activation (ADP, collagen) of patients’ platelets in vitro, there were no significant differences between the platelets of patients and healthy donors. However, the observed ex vivo size of platelet aggregates was significantly reduced in comparison with healthy donors and published data on patients with other thrombocytopenias. The observed number and activity (movement velocity) of granulocytes of patients was within normal values. However, significant morphological differences were observed for granulocytes of patients compared with granulocytes of healthy donors: there was an increased spreading of granulocytes, in particular, expressed in a large number of thin pseudopodia, as well as an increased curvature of the motion trajectories of granulocytes. Ex vivo observation of thrombus formation in patients with ANKRD26- associated thrombocytopenia, a significantly reduced thrombus size is observed with normal platelet activity and increased variability in the shape of granulocytes.

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Impaired Glycoprotein VI-Mediated Signaling and Platelet Functional Responses in CD45 Knockout Mice

Thrombosis and Haemostasis ◽

10.1055/s-0039-1692422 ◽

2019 ◽

Vol 119 (08) ◽

pp. 1321-1331

Author(s):

Vaishali V. Inamdar ◽

John C. Kostyak ◽

Rachit Badolia ◽

Carol A. Dangelmaier ◽

Bhanu Kanth Manne ◽

...

Keyword(s):

Catalytic Domain ◽

Ex Vivo ◽

Thrombus Formation ◽

Tyrosine Phosphatase ◽

Receptor Protein ◽

Functional Responses ◽

Glycoprotein Vi ◽

Extracellular Region ◽

C Terminus

Background and Objective CD45 is a receptor protein tyrosine phosphatase present on the surface of all hematopoietic cells except for erythrocytes and platelets. Proteomics studies, however, have demonstrated the presence of a CD45 c-terminal catalytic peptide in platelets. Therefore, we investigated the functional role of this truncated isoform of CD45 in platelets, which contains the c-terminal catalytic domain but lacks the extracellular region. Methods and Results We used an antibody specific to the c-terminus of CD45 to confirm the presence of a truncated CD45 isoform in platelets. We also examined ex vivo and in vivo platelet function using CD45 knockout (KO) mice. Aggregation and secretion mediated by the glycoprotein VI (GPVI) receptor was impaired in CD45 KO platelets. Consequently, CD45 KO mice had impaired hemostasis indicated by increased tail bleeding times. Also, using a model of pulmonary embolism we showed that CD45 KO mice had defective in vivo thrombus formation. Next, we investigated whether or not the truncated isoform of CD45 had a role in GPVI signaling. The full-length isoform of CD45 is known to regulate Src family kinase (SFK) activation in lymphocytes. We find a similar role for the truncated isoform of CD45 in platelets. SFK activation was impaired downstream of the GPVI receptor in the CD45 KO murine platelets. Consequently, Syk, PLCγ2, and pleckstrin phosphorylations were also impaired in CD45 KO murine platelets. Conclusion We conclude that the truncated CD45 isoform regulates GPVI-mediated signaling and platelet functional responses by regulating SFK activation.

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Characterization of platelet functionality in pediatric patients with kaposiform hemangioendothelioma / Kasabach-Merritt phenomenon

10.22541/au.163949732.29741152/v2 ◽

2021 ◽

Author(s):

Alexey Martyanov ◽

Ivan Tesakov ◽

Olga An ◽

Julia-Jessica Korobkin ◽

Anastasia Ignatova ◽

...

Keyword(s):

Parallel Plate ◽

Ex Vivo ◽

Thrombus Formation ◽

Lower Boundary ◽

Calcium Mobilization ◽

Control Group ◽

Functional Responses ◽

Kaposiform Hemangioendothelioma ◽

Normal Ranges ◽

Platelet Receptor

Background. Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy commonly associated with Kasabach-Merritt phenomenon (KMP) that includes thrombocytopenia and coagulation dysfunction. Platelet receptor CLEC-2 -tumor cell podoplanin interaction is considered the key mechanism of thrombocytopenia in KMP, however, the effect of long-term exposure to podoplanin on platelet function is unknown. Procedure. Here we examined blood samples from 7 patients with KHE/KMP. Platelet calcium signaling and functional responses to conventional activation and CLEC-2 stimulation were analyzed by continuous and endpoint live cell flow cytometry. Platelet aggregation in response to ADP or rhodocytin was analyzed by low-angle light scattering approach (LaSca). Additionally, ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Results. We demonstrate that in KHE/KMP platelet functional responses to strong stimulation were on the lower boundary of age-matched normal ranges, while calcium mobilization and fibrinogen binding upon stimulation with ADP alone were significantly lower than control values. Platelet di-aggregate formation in response to ADP was also diminished in most of the patients. Formation of platelet aggregates in collagen-coated parallel plate flow chambers was also noticeably lower than in the age-matched control group. Calcium mobilization in response to CLEC-2 stimulation was unaltered in the patients and could be blocked by low-molecular-weight inhibitors, 2CP and HB125. Conclusions. While platelet responsiveness in KHE/KMP is moderately altered, platelet CLEC-2 receptors remain functional and respond to inhibition. Therefore, our findings suggest that CLEC-2-targeting molecules are new potential agents in therapeutic management of this life-threatening condition.

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Antithrombotic Effects and Bleeding Time Prolongation with Synthetic Platelet GPIIb/llla Inhibitors in Animal Models of Platelet-Mediated Thrombosis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642390 ◽

1994 ◽

Vol 71 (01) ◽

pp. 095-102 ◽

Cited By ~ 43

Author(s):

Désiré Collen ◽

Hua Rong Lu ◽

Jean-Marie Stassen ◽

Ingrid Vreys ◽

Tsunehiro Yasuda ◽

...

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Bolus Injection ◽

Cell Injury ◽

Ex Vivo ◽

Thrombus Formation ◽

Femoral Vein ◽

Thrombotic Occlusion ◽

Antithrombotic Effects

SummaryCyclic Arg-Gly-Asp (RGD) containing synthetic peptides such as L-cysteine, N-(mercaptoacetyl)-D-tyrosyl-L-arginylglycyl-L-a-aspartyl-cyclic (1→5)-sulfide, 5-oxide (G4120) and acetyl-L-cysteinyl-L-asparaginyl-L-prolyl-L-arginyl-glycyl-L-α-aspartyl-[0-methyltyrosyl]-L-arginyl-L-cysteinamide, cyclic 1→9-sulfide (TP9201) bind with high affinity to the platelet GPIIb/IIIa receptor.The relationship between antithrombotic effect, ex vivo platelet aggregation and bleeding time prolongation with both agents was studied in hamsters with a standardized femoral vein endothelial cell injury predisposing to platelet-rich mural thrombosis, and in dogs with a carotid arterial eversion graft inserted in the femoral artery. Intravenous administration of G4120 in hamsters inhibited in vivo thrombus formation with a 50% inhibitory bolus dose (ID50) of approximately 20 μg/kg, ex vivo ADP-induccd platelet aggregation with ID50 of 10 μg/kg, and bolus injection of 1 mg/kg prolonged the bleeding time from 38 ± 9 to 1,100 ± 330 s. Administration of TP9201 in hamsters inhibited in vivo thrombus formation with ID50 of 30 μg/kg, ex vivo platelet aggregation with an ID50 of 50 μg/kg and bolus injection of 1 mg/kg did not prolong the template bleeding time. In the dog eversion graft model, infusion of 100 μg/kg of G4120 over 60 min did not fully inhibit platelet-mediated thrombotic occlusion but was associated with inhibition of ADP-induccd ex vivo platelet aggregation and with prolongation of the template bleeding time from 1.3 ± 0.4 to 12 ± 2 min. Infusion of 300 μg/kg of TP9201 over 60 min completely prevented thrombotic occlusion, inhibited ex vivo platelet aggregation, but was not associated with prolongation of the template bleeding time.TP9201, unlike G4120, inhibits in vivo platelet-mediated thrombus formation without associated prolongation of the template bleeding time.

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Further Studies on the Mechanisms for the Antithrombotic Effects of Sulfated Polysaccharides in Rabbits

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647027 ◽

1988 ◽

Vol 60 (02) ◽

pp. 188-192 ◽

Cited By ~ 15

Author(s):

F A Ofosu ◽

F Fernandez ◽

N Anvari ◽

C Caranobe ◽

F Dol ◽

...

Keyword(s):

Antithrombin Iii ◽

Ex Vivo ◽

Thrombus Formation ◽

Minimum Weight ◽

Sulfated Polysaccharides ◽

Pentosan Polysulfate ◽

Heparin Cofactor Ii ◽

Thrombin Inhibition ◽

The Relationship ◽

Prothrombin Activation

SummaryA recent study (Fernandez et al., Thromb. Haemostas. 1987; 57: 286-93) demonstrated that when rabbits were injected with the minimum weight of a variety of glycosaminoglycans required to inhibit tissue factor-induced thrombus formation by —80%, exogenous thrombin was inactivated —twice as fast in the post-treatment plasmas as the pre-treatment plasmas. In this study, we investigated the relationship between inhibition of thrombus formation and the extent of thrombin inhibition ex vivo. We also investigated the relationship between inhibition of thrombus formation and inhibition of prothrombin activation ex vivo. Four sulfated polysaccharides (SPS) which influence coagulation in a variety of ways were used in this study. Unfractionated heparin and the fraction of heparin with high affinity to antithrombin III potentiate the antiproteinase activity of antithrombin III. Pentosan polysulfate potentiates the activity of heparin cofactor II. At less than 10 pg/ml of plasma, all three SPS also inhibit intrinsic prothrombin activation. The fourth agent, dermatan sulfate, potentiates the activity of heparin cofactor II but fails to inhibit intrinsic prothrombin activation even at concentrations which exceed 60 pg/ml of plasma. Inhibition of thrombus formation by each sulfated polysaccharides was linearly related to the extent of thrombin inhibition achieved ex vivo. These observations confirm the utility of catalysis of thrombin inhibition as an index for assessing antithrombotic potential of glycosaminoglycans and other sulfated polysaccharides in rabbits. With the exception of pentosan polysulfate, there was no clear relationship between inhibition of thrombus formation and inhibition of prothrombin activation ex vivo.

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