Protein S and factor V in regulation of coagulation on platelet microparticles by activated protein C

Somajo, Sofia; Koshiar, Ruzica Livaja; Norström, Eva; Dahlbäck, Björn

doi:10.1016/j.thromres.2014.04.031

Cited by 30 publications

(32 citation statements)

References 55 publications

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“…However, we could not detect any APC-independent effect of protein S on the surface of eryMPs in the TGA. Inclusion of an antibody against protein S in absence of APC did not affect the thrombin generation curve as shown before using both phospholipids [44] and platelet-derived MPs [20]. Platelets contain TFPIα, which is released upon activation [49], [50].…”

Section: Discussionmentioning

confidence: 84%

Erythrocyte-Derived Microparticles Supporting Activated Protein C-Mediated Regulation of Blood Coagulation

et al. 2014

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Elevated levels of erythrocyte-derived microparticles are present in the circulation in medical conditions affecting the red blood cells. Erythrocyte-derived microparticles expose phosphatidylserine thus providing a suitable surface for procoagulant reactions leading to thrombin formation via the tenase and prothrombinase complexes. Patients with elevated levels of circulating erythrocyte-derived microparticles have increased thrombin generation in vivo. The aim of the present study was to investigate whether erythrocyte-derived microparticles are able to support the anticoagulant reactions of the protein C system. Erythrocyte-derived microparticles were isolated using ultracentrifugation after incubation of freshly prepared erythrocytes with the ionophore A23187 or from outdated erythrocyte concentrates, the different microparticles preparations yielding similar results. According to flow cytometry analysis, the microparticles exposed phoshatidylserine and bound lactadherin, annexin V, and protein S, which is a cofactor to activated protein C. The microparticles were able to assemble the tenase and prothrombinase complexes and to stimulate the formation of thrombin in plasma-based thrombin generation assay both in presence and absence of added tissue factor. The addition of activated protein C in the thrombin generation assay inhibited thrombin generation in a dose-dependent fashion. The anticoagulant effect of activated protein C in the thrombin generation assay was inhibited by a monoclonal antibody that prevents binding of protein S to microparticles and also attenuated by anti-TFPI antibodies. In the presence of erythrocyte-derived microparticles, activated protein C inhibited tenase and prothrombinase by degrading the cofactors FVIIIa and FVa, respectively. Protein S stimulated the Arg306-cleavage in FVa, whereas efficient inhibition of FVIIIa depended on the synergistic cofactor activity of protein S and FV. In summary, the erythrocyte-derived microparticle surface is suitable for the anticoagulant reactions of the protein C system, which may be important to balance the initiation and propagation of coagulation in vivo.

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Section: Discussionmentioning

confidence: 84%

Erythrocyte-Derived Microparticles Supporting Activated Protein C-Mediated Regulation of Blood Coagulation

et al. 2014

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“…It is therefore unlikely that platelet‐associated EPCR plays a major role in APC‐mediated anticoagulation on the platelet surface. In contrast, platelet microparticles can support FVa inactivation by APC , and platelet‐derived EPCR may contribute to counterbalancing the procoagulant effects of microparticles.…”

Section: Discussionmentioning

confidence: 99%

Human platelets express endothelial protein C receptor, which can be utilized to enhance localization of factor VIIa activity

Fager

Machlus

Ezban

et al. 2018

Journal of Thrombosis and Haemostasis

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Background High-dose factor VIIa (FVIIa) is routinely used as an effective bypassing agent to treat hemophilia patients with inhibitory antibodies that compromise factor replacement. However, the mechanism by which FVIIa binds activated platelets to promote hemostasis is not fully understood. FVIIa-DVQ is an analog of FVIIa with enhanced tissue factor (TF)-independent activity and hemostatic efficacy relative to FVIIa. Our previous studies have shown that FVIIa-DVQ exhibits greater platelet binding, thereby suggesting that features in addition to lipid composition contribute to platelet-FVIIa interactions. Objectives Endothelial cell protein C receptor (EPCR) also functions as a receptor for FVIIa on endothelial cells. We therefore hypothesized that an interaction with EPCR might play a role in platelet-FVIIa binding. Methods/results In the present study, we used flow cytometric analyses to show that platelet binding of both FVIIa and FVIIa-DVQ is partially inhibited in the presence of excess protein C or an anti-EPCR antibody. This decreased binding results in a corresponding decrease in the activity of both molecules in FXa and thrombin generation assays. Enhanced binding to EPCR was sufficient to account for the increased platelet binding of FVIIa-DVQ compared with wild-type FVIIa. As EPCR protein expression has not previously been shown in platelets, we confirmed the presence of EPCR in platelets using immunofluorescence, flow cytometry, immunoprecipitation, and mass spectrometry. Conclusions This work represents the first demonstration that human platelets express EPCR and suggests that modulation of EPCR binding could be utilized to enhance the hemostatic efficacy of rationally designed FVIIa analogs.

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“…Platelet Microparticle Preparation and Labeling-Platelets were purified from pooled fresh human citrated blood collected from healthy volunteers as described (2) with the permission of the local ethical board at Lund University. MP formation was induced by stimulating platelets at a concentration of 100 ϫ 10 6 /ml with 5.9 M calcium ionophore (A23187, Life Technologies, Inc.) or 0.5 units/ml thrombin and 25 g/ml collagen (Chrono-Log Corp.) in activation buffer (140 mM NaCl, 2.5 mM KCl, 0.1 mM MgCl 2 , 3 mM CaCl 2 , 60 mM Hepes, 0.5 mM NaH 2 PO 4 , 5.5 mM glucose, and 10 mM HCO 3 , pH 7.4), for 25 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Upon activation, platelets shed small, 100-nm to 1-m vesicles, often referred to as microparticles (PMPs), 2 microvesicles or extracellular vesicles. These PMPs expose a hundredfold more phosphatidylserine (PS) on their surface than activated platelets themselves, and by supporting the activation of factor X and prothrombin, they are highly pro-coagulant (1).…”

mentioning

confidence: 99%

“…These PMPs expose a hundredfold more phosphatidylserine (PS) on their surface than activated platelets themselves, and by supporting the activation of factor X and prothrombin, they are highly pro-coagulant (1). However, the activated protein C system together with protein S has the ability to down-regulate their pro-coagulant phenotype, thereby creating a balance between coagulation and anti-coagulation (2). In addition to plasma membrane-derived PMPs, platelets also shed exosomes upon activation.…”

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confidence: 99%

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The Gas6-Axl Protein Interaction Mediates Endothelial Uptake of Platelet Microparticles

Happonen

Tran

Mörgelin

et al. 2016

Journal of Biological Chemistry

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Upon activation, platelets release plasma membrane-derived microparticles (PMPs) exposing phosphatidylserine on their surface. The functions and clearance mechanism of these microparticles are incompletely understood. As they are pro-coagulant and potentially pro-inflammatory, rapid clearance from the circulation is essential for prevention of thrombotic diseases. The tyrosine kinase receptors Tyro3, Axl, and Mer (TAMs) and their ligands protein S and Gas6 are involved in the uptake of phosphatidylserine-exposing apoptotic cells in macrophages and dendritic cells. Both TAMs and their ligands are expressed in the vasculature, the functional significance of which is poorly understood. In this study, we investigated how vascular TAMs and their ligands may mediate endothelial uptake of PMPs. PMPs, generated from purified human platelets, were isolated by ultracentrifugation and labeled with biotin or PKH67. The uptake of labeled microparticles in the presence of protein S and Gas6 in human aortic endothelial cells and human umbilical vein endothelial cells was monitored by flow cytometry, Western blotting, and confocal/electron microscopy. We found that both endothelial cell types can phagocytose PMPs, and by using TAM-blocking antibodies or siRNA knockdown of individual TAMs, we show that the uptake is mediated by endothelial Axl and Gas6. As circulating PMP levels were not altered in Gas6 ؊/؊ mice compared with Gas6 ؉/؉ mice, we hypothesize that the Gas6-mediated uptake is not a means to clear the bulk of circulating PMPs but may serve to locally phagocytose PMPs generated at sites of platelet activation and as a way to effect endothelial responses.

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Protein S and factor V in regulation of coagulation on platelet microparticles by activated protein C

Cited by 30 publications

References 55 publications

Erythrocyte-Derived Microparticles Supporting Activated Protein C-Mediated Regulation of Blood Coagulation

Erythrocyte-Derived Microparticles Supporting Activated Protein C-Mediated Regulation of Blood Coagulation

Human platelets express endothelial protein C receptor, which can be utilized to enhance localization of factor VIIa activity

The Gas6-Axl Protein Interaction Mediates Endothelial Uptake of Platelet Microparticles

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