P-selectin functions as an adhesion molecule (27
) but was subsequently shown to have a role in fibrin formation (52
). In a baboon arteriovenous shunt model of thrombosis, blocking antibodies against P-selectin not only inhibited leukocyte accumulation in the developing thrombus but also decreased fibrin formation. The molecular and cellular basis for this experimental observation was not clear at the time, but it was thought that leukocytes might generate tissue factor upon stimulation more rapidly in vivo than in the in vitro systems used to explore de novo tissue factor biosynthesis in stimulated cells (53
). Although a relationship of P-selectin to fibrin formation was secure, the basis for the inhibition of fibrin formation by anti–P-selectin antibodies remained unknown.
Using genetically altered mice and digital intravital microscopy imaging, this question was revisited (55
). Tissue factor antigen and fibrin were observed throughout the thrombus generated in WT mice (Figure ), a result that confirmed earlier in vitro experiments (56
). However, minimal tissue factor antigen or fibrin was observed in thrombi generated in either P-selectin–null mice or PSGL-1–null mice (55
). These results were similar to those obtained in the baboon thrombosis model using anti–P-selectin antibodies to block P-selectin action. We hypothesized that tissue factor and PSGL-1 must be physically coupled. Although this is true of activated monocytes, where tissue factor and PSGL-1 reside on the plasma membrane (57
), there is no evidence that such monocytes circulate constitutively in blood (58
). Furthermore, leukocytes do not interact with developing thrombi as rapidly as fibrin deposition begins (38
). Rather, leukocyte microparticles might provide the basis for this observation. Leukocyte microparticles, first identified in 1994 (59
), could express both tissue factor and PSGL-1 if derived from monocytes. Indeed, a population of microparticles exists in the circulation that is positive for both tissue factor antigen and PSGL-1 antigen. Using a monocyte-like cell line, fluorescently labeled microparticles were generated and infused into mice. During thrombus formation, microparticles accumulated in the thrombi of WT mice. In contrast, no accumulation was observed in P-selectin–null mice.
These results are consistent with a model in which circulating microparticles expressing tissue factor and PSGL-1 accumulate in the developing thrombus via the interaction of P-selectin with PSGL-1. This delivers and concentrates tissue factor in the thrombus, leading to a critical concentration that can initiate blood coagulation (Figure ). Numerous groups have reported tissue factor antigen in platelet-poor plasma, with levels varying from 100 to 150 pg/ml. However, Butenas et al. have recently reopened this issue (58
). They report no detectable tissue factor activity in whole blood, no tissue factor antigen associated with unstimulated mononuclear cells in whole blood, and a level of tissue factor activity that cannot exceed 20 fM, equivalent to about 1 pg/ml, and is more likely lower. Since these authors demonstrate that 1 pg/ml of active tissue factor rapidly clots whole blood, it would seem that blood tissue factor concentration is much lower than 1 pg/ml and that a manyfold concentration of tissue factor within the thrombus is a critical component for the initiation of blood coagulation. Alternatively, an inactive form of tissue factor may undergo some form of activation to its biologically functional form.
Figure 2 Model of P-selectin/PSGL-1–mediated tissue factor accumulation during thrombus formation. (A) Leukocyte microparticles (red) circulate constitutively in the blood under resting conditions. These microparticles express tissue factor (TF) and PSGL-1 (more ...)
Tissue factor resides in 3 distinct compartments: (a) the surface of extravascular cells, (b) the vessel wall, and (c) blood microparticles. Upon stimulation, both endothelial cells and monocytes have the capacity to express tissue factor. To determine whether tissue factor associated with blood microparticles contributes to fibrin formation during thrombosis in vivo, 1 strain of chimeric mice in which tissue factor was associated with the vessel wall but not the blood microparticles and another strain of chimeric mice in which tissue factor was associated with the blood microparticles but not the vessel wall were prepared (60
). Such mice were generated by bone marrow transplantation of WT mice, with normal levels of tissue factor in both the vessel wall and blood microparticles, and low–tissue factor mice, with about 1% of the normal level of tissue factor (61
). Chimeras generated by transplantation of low–tissue factor bone marrow into WT mice showed platelet thrombi containing markedly reduced tissue factor and fibrin (60
). Conversely, chimeras generated by transplantation of WT bone marrow into low–tissue factor mice rescued tissue factor accumulation and fibrin generation in the platelet thrombus. These results emphasize that within the context of this in vivo model, fibrin propagation is dependent on tissue factor derived from blood microparticles. Both vessel wall tissue factor and microparticle tissue factor appear to contribute to thrombus formation. In thrombosis models where there is no vessel wall tissue factor (56
), where vessel wall injury causes vessel wall tissue factor to predominate (62
), or where there is no blood flow and thus the deposition of microparticles is eliminated (62
), the balance between the contribution of vessel wall tissue factor and that of microparticle tissue factor can be altered, giving varying results. Likely, different pathologies associated with thrombosis may also differentially impact on the contributions of tissue factor from the vessel wall and from blood microparticles.