Blood-borne tissue factor: another view of thrombosis

Abstract

Arterial thrombosis is considered to arise from the interaction of tissue factor (TF) in the vascular wall with platelets and coagulation factors in circulating blood. According to this paradigm, coagulation is initiated after a vessel is damaged and blood is exposed to vessel-wall TF. We have examined thrombus formation on pig arterial media (which contains no stainable TF) and on collagen-coated glass slides (which are devoid of TF) exposed to flowing native human blood. In both systems the thrombi that formed during a 5-min perfusion stained intensely for TF, much of which was not associated with cells. Antibodies against TF caused approximately 70% reduction in the amount of thrombus formed on the pig arterial media and also reduced thrombi on the collagen-coated glass slides. TF deposited on the slides was active, as there was abundant fibrin in the thrombi. Factor VIIai, a potent inhibitor of TF, essentially abolished fibrin production and markedly reduced the mass of the thrombi. Immunoelectron microscopy revealed TF-positive membrane vesicles that we frequently observed in large clusters near the surface of platelets. TF, measured by factor Xa formation, was extracted from whole blood and plasma of healthy subjects. By using immunostaining, TF-containing neutrophils and monocytes were identified in peripheral blood; our data raise the possibility that leukocytes are the main source of blood TF. We suggest that blood-borne TF is inherently thrombogenic and may be involved in thrombus propagation at the site of vascular injury.

Figure 1

(A) TF staining (brown) of a thrombus formed on pig aortic media. Note the absence of staining of the media and the positive staining of the thrombus and some of the neutrophils; much of the immunostained (pAb-TF) TF appears to be extracellular and associated with fibrin. (B) Microthrombi on collagen-coated glass slide stained by using pAb-TF (brown). Note the 1- to 2-μm granules, which stain intensely. (C) Microthrombi on collagen-coated glass slide stained for CD-18 (brown). Note the staining of the granules. (D) Thrombus (purple) formed on pig media perfused with native human blood. (E) Pig media perfused with native human blood containing pAb-TF; note the markedly diminished thrombi.

Figure 2

(A) Collagen-coated glass slide perfused with native human blood and stained for fibrin (brown). A fibrin network as well as microthrombi are evident. (B) Same as A but the TF inhibitor FVII_ai was included in the flow. Compared with A, the fibrin staining is minimal, and the thrombus mass is considerably smaller. (C) Glass slides coated with pAb-TF and perfused with anticoagulated whole blood shows deposition of leukocytes. Both neutrophils and macrophages are positive (pAb-TF stain).

Figure 3

Electron micrograph of thrombi that were deposited on collagen-coated glass slide. ImmunoGold staining for TF shows that vesicular structures adjacent to the platelet membrane are positive. (Inset) A magnification (×4) of a stained vesicle.