Intravascular Cells and Circulating Microparticles Induce Procoagulant Activity Via Phosphatidylserine Exposure in Venous Thromboembolism and Contribute to Altered Plasma Fibrin Clot Properties
Authors: Kou Yan , Zou Lili , Liang Hui , Hou Li , Li Tao , Zhang Yan , LI Baorong , LIU Yingmiao , Zhang Cong , Liu Ruipeng , Zhao Xinyi , Wang Ying , Jin Jiaqi , Bi Yayan , Shi Jialan , Kou Junjie 
Published: October 2019

Abstract
Introduction:Venous thromboembolism(VTE), encompassing pulmonary embolism (PE) and deep vein thrombosis (DVT), is a major contributor to global morbidity and mortality, especially among cancer patients. However, the factors that contribute to PE, a prevalent and fatal complication of DVT, remain poorly understood.Despite a common pathology relating PE to DVT, this leaves a grey area in the differences among DVT, confirmed PE, or DVT with PE, and the definitive relationship between VTE and this hypercoagulable state.However, relatively little is known about the definitive role of phosphatidylserine (PS), one of the blood constituents in the coagulant pathway, in the hypercoagulability of VTE and their altered plasma fibrin clot properties. Our objectives were to assess the levels of PS exposure on microparticles (MPs), blood cells, endothelial cells(ECs) and cell-derived MPs in each group of VTE and to evaluate their procoagulant activity (PCA), as well as the correlation between initial PS exposure and their altered plasma fibrin clot properties.
Methods:DVT alone (n=27), definitely PE alone (n=22), DVT with PE (n=19) and healthy controls (n=30) were enrolled in the present study. PS exposure on MPs, blood cells and cultured ECs treated with VTE serum in vitro was analyzed with flow cytometry and confocal microscopy. MPs were classified based on their cellular origin: platelets (CD41a+, PMPs), neutrophils (CD66b+, NMPs), endothelial cells(CD31+CD41a-, EMPs), erythrocytes (CD235a+, RMPs), monocytes (CD14+, MMPs), T lymphocytes (CD3+, T LyMPs), and B lymphocytes (CD19+, B LyMPs). PCA was evaluated by clotting time, extrinsic/intrinsic FXa and prothrombinase production assays, as well as fibrin formation assays. Inhibition assays of PCA of PS+MPs, blood cells and ECs were performed by lactadherin. Abnormal fibrin clot properties measured ex vivo in plasma was examined by fibrin clot analysis and scanning electron microscopy (SEM).
Results: There was no significant difference in MP cellular origin between healthy and VTE subjects. However, the total number of PS+MPs was significantly increased (P < 0.001 for total MP and all MP subtypes) in VTE patients compared with healthy controls. In addition, circulating PS+ MPs cooperated with PS+blood cells and cultured ECs to markedly shorten coagulation time (Figure 1A) and dramatically increase FXa/thrombin generation and fibrin formation in each VTE group (all P < 0.05). Confocal microscopy images showed that the FVa/FXa complex and fibrin strands co-stained with PS on ECs. Moreover, blockade of exposed PS on MPs and intravascular cells with lactadherin inhibited PCA by approximately 80% (Figure 1B). Fibrin clot structure was evaluated in 15 VTE patients (DVT=5, PE=5 and DVT with PE=5) and 5 healthy subjects.Compared with those from DVT and PE, clots from plasma of DVT with PE subjects showed lower fiber density and faster clot lysis time, whereas there were no differences in lag time, rate of clot formation and maximum absorbance of turbidity.
Conclusions: Our results suggest that compared with healthy subjects, each group of VTE patients had markedly higher levels of circulating PS+MPs, PS+blood cells. Additional, ECs treated with VTE patient serum had elevated percentages of PS+cells versus those treated with serum from healthy controls. Blockade of PS with lactadherin significantly inhibits PCA of blood cells, MPs and VTE serum-treated ECs. In addition, differences in fibrin clot properties and clot structure among DVT, PE and DVT with PE subjects may provide important insights into the pivotal mechanisms that regulate embolization.

Disclosures
No relevant conflicts of interest to declare.