Phosphatidylserine-exposing blood and endothelial cells contribute to the hypercoagulable state in essential thrombocythemia patients

Abstract

The mechanisms of thrombogenicity in essential thrombocythemia (ET) are complex and not well defined. Our objective was to explore whether phosphatidylserine (PS) exposure on blood cells and endothelial cells (ECs) can account for the increased thrombosis and distinct thrombotic risks among mutational subtypes in ET. Using flow cytometry and confocal microscopy, we found that the levels of PS-exposing erythrocytes, platelets, leukocytes, and serum-cultured ECs were significantly higher in each ET group [JAK2, CALR, and triple-negative (TN) (all P < 0.001)] than those in controls. Among ET patients, those with JAK2 mutations showed higher levels of PS-positive erythrocytes, platelets, neutrophils, and serum-cultured ECs than TN patients or those with CALR mutations, which show similar levels. Coagulation function assays showed that higher levels of PS-positive blood cells and serum-cultured ECs led to markedly shortened coagulation time and dramatically increased levels of FXa, thrombin, and fibrin production. This procoagulant activity could be largely blocked by addition of lactadherin (approx. 70% inhibition). Confocal microscopy showed that the FVa/FXa complex and fibrin fibrils colocalized with PS on ET serum-cultured ECs. Additionally, we found a relationship between D-dimer, prothrombin fragment F1 + 2, and PS exposure. Our study reveals a previously unrecognized link between hypercoagulability and exposed PS on cells, which might also be associated with distinct thrombotic risks among mutational subtypes in ET. Thus, blocking PS-binding sites may represent a new therapeutic target for preventing thrombosis in ET.

Thrombin Generation and Cancer: Contributors and Consequences

“…As the PS exposure on the external membrane of platelets is a prerequisite for the assembly of coagulation factors on platelets and their associated EVs, flow cytometry-based assays to detect PS expression are often used as a surrogate for measurement of procoagulant platelets. Confocal microscopy can be used to visualise PS exposure as well as the binding of FVa and FXa on the membranes of platelets and EVs [75]. Procoagulant activity contributed by platelets and platelet EVs can be assessed by commercially available functional coagulation tests such as the CAT assay (see Section 3) and STA Procoag-PPL assay (Diagnostica Stago, Asnieres sur Seine, France) (using platelet-free but EV-containing plasma to assess procoagulant activity contributed by EVs).…”

“…Procoagulant activity contributed by platelets and platelet EVs can be assessed by commercially available functional coagulation tests such as the CAT assay (see Section 3) and STA Procoag-PPL assay (Diagnostica Stago, Asnieres sur Seine, France) (using platelet-free but EV-containing plasma to assess procoagulant activity contributed by EVs). In addition, procoagulant activity of isolated platelets or platelet EVs can also be measured with in-house assays using either a single stage clotting assay or a 2-stage amidolytic assay to quantify the generation of thrombin and FXa [75,76,77]. Recently, a novel assay utilising GSAO [(4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid] and P-selectin permits the direct identification of procoagulant platelets in whole blood by flow cytometry [78] and this may be useful in future studies of cancer thrombosis.…”

La thrombose au cours des néoplasies myéloprolifératives

“…L'augmentation de marqueurs plasmatiques d'activation plaquettaire (P-sélectine soluble, ligand de CD40, bêta-thromboglobuline, facteur plaquettaire 4, thromboxane A2) constitue un autre argument en faveur de l'activation plaquettaire au cours des néoplasies myéloprolifératives. Une augmentation d'expression de phosphatidylsérine (PS) à la surface des plaquettes des patients [21] ainsi qu'un taux plus élevé de plaquettes immatures ou réticulées [22], qui ont un pouvoir hémostatique accru, ont également été montrés. Cependant, l'hétérogénéité des cohortes de patients (traitements cytoréducteurs, types de mutations initiatrices, variations de charges alléliques, traitements anti-agrégant plaquettaire ou…”

“…Le profil d'activation est plus marqué en cas de mutation JAK2V617F [19,20]. Chez les patients présentant une thrombocytémie essentielle ou une polyglobulie de Vaquez, l'expression du facteur tissulaire (FT) à la surface des neutrophiles et des monocytes [19] est également plus importante, de même que celle de la phosphatidylsérine, à la surface des neutrophiles [21]. Le contenu de ces cellules en élastase neutrophile, et les concentrations plasmatiques en élastase neutrophile et en myéloperoxydase sont aussi augmentés chez les patients [27].…”

“…La génération de thrombine, reflet plus global de l'activation de la coagulation, a aussi été évaluée chez les patients présentant une thrombocytémie essentielle, avec une augmentation, particulièrement chez les patients présentant la mutation JAK2V617F [56]. Enfin, l'étude de l'expression de la phosphatidylsérine à la surface des cellules sanguines des patients atteints de néoplasies myéloprolifératives a montré qu'elle était surexprimée au cours de la thrombocytémie essentielle, avec, en conséquence, un raccourcissement des temps de coagulation et une augmentation de l'activation du facteur X et de la génération de thrombine et de fibrine [21]. Les microparticules (MP) sont des vésicules extracellulaires constituées de fragments de membrane cytoplasmique libérés par les cellules sanguines et les cellules endothéliales dans le milieu extracellulaire.…”