Pro- and non-coagulant forms of non-cell-bound tissue factor in vivo

Background: Concentrations of non-cell-bound (NCB; soluble) tissue factor (TF) are elevated in blood collecting in the pericardial cavity of patients during cardiopulmonary bypass (CPB). Previously, we reported microparticles supporting thrombin generation in such blood samples. In this study we investigated the extent of microparticle association of the NCB form of TF in pericardial and systemic blood, and whether this microparticle-associated form is active in thrombin generation compared with non-microparticle-bound, (fluid-phase) TF. Methods: Systemic and pericardial blood samples were collected before and during CPB from six patients undergoing cardiac surgery. Microparticles were isolated by differential centrifugation and their thrombin-generating capacity measured in a chromogenic assay. Microparticle-associated and fluid-phase forms of NCB TF were measured by ELISA. Microparticle-associated TF was visualized by flow cytometry. Results: In pericardial samples, 45-77% of NCB TF was microparticle-associated, and triggered factor VII (FVII)mediated thrombin generation in vitro. Microparticles from systemic samples triggered thrombin generation independently of FVII, except at the end of bypass (P=0.003). The fluid-phase form of TF did not initiate thrombin generation. Both forms of NCB TF were, at least in part, antigenically cryptic. Conclusions: We demonstrate the occurrence of two forms of NCB TF. One form, which is microparticle-associated, supports thrombin generation via FVII. The other form, which is fluid-phase, does not stimulate thrombin formation. We hypothesize that the microparticle-associated form of NCB TF may be actively involved in postoperative thromboembolic processes when pericardial blood is returned into the patient

Aprotinin administration in the pericardial cavity does not prevent platelet activation

Aprotinin is frequently administered systemically to patients undergoing cardiopulmonary bypass to inhibit activation of platelets and plasma protein systems and thus reduce postoperative blood loss. Two reports on local aprotinin administration, that is, into the pericardial cavity, also indicated improvement in postoperative blood loss, but the underlying mechanism was not investigated. We previously reported the disappearance of glycoprotein Ib from the platelet surface and the appearance of platelet-derived microparticles in the pericardial cavity of patients undergoing cardiopulmonary bypass as signs of platelet activation. Here, we investigated whether such local aprotinin administration reduced platelet activation. In a double-blind study, 6 patients received aprotinin (500,000 KIU) into the pericardial cavity during the operation and 7 patients received a placebo. Platelet surface glycoprotein Ib expression, concentration of microparticles, and concentration of complexes of platelets with leukocytes, erythrocytes, or each other, were measured by flow cytometry. We confirmed the reduced glycoprotein Ib expression and the increased concentration of microparticles in the pericardial cavity, as previously reported, and found no increased concentration of platelet complexes. However, no differences between aprotinin and placebo treatments were observed in these platelet activation parameters in the pericardial cavity or the systemic circulation. We conclude that administration of aprotinin into the pericardial cavity during cardiopulmonary bypass and at concentrations similar to the systemic application does not reduce platelet activation in that compartment or the systemic circulatio

Human cell-derived microparticles promote thrombus formation in vivo in a tissue factor-dependent manner

Background: Circulating microparticles of various cell types are present in healthy individuals and, in varying numbers and antigenic composition, in various disease states. To what extent these microparticles contribute to coagulation ill vivo is unknown. Objectives: To examine the in vivo thrombogenicity of human microparticles. Methods: Microparticles were isolated from pericardial blood of cardiac surgery patients and venous blood of healthy individuals. Their numbers, cellular source, and tissue factor (TF) exposure were determined using flow cytometry. Their in vitro procoagulant properties were studied in a fibrin generation test, and their in vivo thrombogenicity in a rat model. Results: The total number of microparticles did not differ between pericardial samples and samples from healthy individuals (P = 0.786). In both groups. microparticles from platelets, erythrocytes, and granulocytes exposed TF. Microparticle-exposed TF antigen levels were higher in pericardial compared with healthy individual samples (P = 0.036). Pericardial microparticles were strongly procoa.gulant in vitro and highly thrombogenic in a venous stasis thrombosis model in rats, whereas microparticles from healthy individuals were not [thrombus weights 24.8 (12.2-41.3)mg vs. 0 (0-24.3)mg median and range; P 0.05]. The thrombogenicity of the microparticles correlated strongly with their TF exposure (r=0.9524, P=0.001). Conclusions: Human cell-derived microparticles promote thrombus formation in vivo in a TF-dependent manner. They might be the direct cause of an increased thromboembolic tendency in various patient group