Granulocytes do not express but acquire monocyte-derived tissue factor in whole blood: evidence for a direct transfer

Abstract

Unlike unanimous opinion on tissue factor (TF) expression in monocytes, the quest for TF presence in granulocytes has been going on for decades. To study the cell origin and track the blood-borne TF, we assessed TF activity and protein levels, knocked-down endogenous TF expression with small interfering RNA (siRNA), and overexpressed TF-yellow fluorescent protein (TF-YFP) fusion in immunologically isolated human monocytes and granulocytes. Monocytes and, to a much lesser extent, granulocytes isolated from lipopolysaccharide (LPS)/ phorbol 12-myristate-13-acetate (PMA)-stimulated whole blood contained active TF antigen. However, only monocytes possessed significant TF activity and protein levels when stimulated with LPS/PMA in suspension. Reintroduction of TF-silenced monocytes to whole blood led to a profound reduction of LPS/PMA-stimulated TF activity in both mononuclear cell (MNC) and granulocyte fractions. No reduction in TF activity in MNC and granulocyte fractions was observed when TFsilenced granulocytes were reintroduced to whole blood. As shown by immunoblotting, flow cytometry, and confocal microscopy, granulocytes became positive for TF-YFP when isolated from whole blood reconstituted with TF-YFP-expressing monocytes. Together, we pinpoint monocytes as a major source of TF and provide solid experimental evidence for a direct transfer of TF protein from the monocytes to granulocytes in the blood. IntroductionThe source of tissue factor (TF) among circulating blood leukocytes still remains a mystery that is bolstered by several conflicting reports. 1-7 TF is a 45-kDa membrane glycoprotein that binds to factor F VII (FVII)/FVIIa with high affinity. 8 The formation of TF-FVIIa complexes triggers the coagulation cascade by activating both factors IX and X, which leads to thrombin generation and stimulates platelet activation and fibrin deposition. [9][10][11] Under physiologic conditions, TF is absent in the cells and tissues located on the interface with circulating blood. It is constitutively expressed in vascular cell types that have no direct contact with blood, such as adventitial fibroblasts, pericytes, and smooth muscle cells. 12 This mode of expression provides a haemostatic protection should vascular injury occur. In 1989, Drake et al created a concept of TF as being a "haemostatic envelope ready to activate coagulation when vascular integrity is disrupted." 13 Although studies demonstrating the presence of TF on circulating blood cells began in the early 1970s, the question of which blood cell type expresses TF is still a matter of considerable discussion. 14,15 To date, neutrophils, eosinophils, platelets, and even lymphocytes were shown to be positive for TF protein under diverse conditions. [2][3][4][5][6][7] In our present study, we studied TF procoagulant activity and antigen in highly pure resting and stimulated monocytes and granulocytes under conditions of whole blood and plating. Having used small interfering RNA (siRNA)-mediated knockdown of endogenous TF expression in freshly isolated ...

Plasma and IgGs from patients with lupus anticoagulant induce tissue factor in monocytes: a possible risk factor for thrombosis

“…This is in agreement with our previous studies, in which we clearly showed that neutrophils or eosinophils do not synthesize or express TF activity when blood has been exposed to various forms of activation [6,7]. This, however, is a controversial subject, as there are some reports of TF expression by granulocytes [10].…”

Tissue factor–positive neutrophils bind to injured endothelial wall and initiate thrombus formation

“…2,3 Clinical studies have shown that traditional vascular risk factors are associated with leukocyte activation and may predispose to thrombogenesis. 4,5 PMNs may contribute to fibrin generation directly by exposing active tissue factor (TF) 6 and/or indirectly by inactivating the major TF inhibitor, TF pathway inhibitor (TFPI). 2 PMNs may also locally assemble proteins of the factor XII-driven contact pathway.…”

“…41 It is possible, as previously suggested, that TF was transferred from monocytes or monocyte-derived microparticles to PMNs. 6 Massberg et al have recently shown that blood -activated neutrophils can inactivate TFPI via the release of proteases at the site of injury. 2 In this model, the subendothelial matrix is exposed to blood leading to the formation of a platelet thrombus.…”

Leukocyte-Derived Microparticles in Vascular Homeostasis

“…48 TFϩ MonoMPs constitute the second largest pool of thrombogenic MPs after plateletderived MPs (PMPs). 49 -51 TFϩ MonoMPs can interact with neutrophils, 52 transferring to them a procoagulant activity. Indeed, neutrophils are capable of fusion with MPs derived from other cell types.…”