We used an in vivo thrombosis model to evaluate the role of thiol isomerases in thrombus formation in vivo. In a laser injury model, we found that PDI antigen was detected before the onset of platelet and fibrin accumulation. Moreover, infusion of 2 structurally dissimilar PDI inhibitors blocked both platelet and fibrin accumulation in this model.
The source of the PDI that appears during thrombus formation remains to be determined. Because PDI has not been detected in plasma by others (20
) or ourselves (our unpublished observations), the PDI visualized during thrombus formation presumably derives from cells that release PDI upon activation or injury. Activated platelets can release catalytically active PDI (2
); thus, platelet PDI may contribute to the accumulating PDI antigen associated with the thrombus. PDI released from activated or injured endothelial cells or other vessel wall components might also participate.
The ability of PDI inhibitors to abolish fibrin formation strongly suggests that PDI plays a necessary role in local thrombin generation in the laser injury model. Moreover, because fibrin deposition in Par4–/– mice is independent of platelets or requires minimal platelet activation or accumulation, the dramatic effect of PDI inhibitors on fibrin accumulation in Par4–/– mice suggests that these inhibitors may function by a platelet-independent mechanism.
Tissue factor antigen can be detected at the site of vessel wall injury immediately following laser insult in our model (21
). In vitro studies have led to the proposal that PDI may be associated with an encrypted form of tissue factor and may regulate tissue factor activity (9
). Thus, inhibition of extracellular PDI is a potential mechanism for the platelet-independent inhibition of thrombin generation and fibrin deposition that we observed after laser-induced vessel wall injury in the presence of PDI inhibitors. However, the concept that thiol isomerase–catalyzed oxidation of an allosteric disulfide bond in tissue factor leads to the activation of tissue factor has been controversial for 4 reasons: (a) Bach et al. demonstrated that purified full-length tissue factor has no free thiols in the extracellular domain (22
); (b) oxidants such as mercuric chloride that modify tissue factor activity might target a critical chemical moiety other than a free thiol on tissue factor (16
); (c) PDI might be a required cofactor for tissue factor coagulant activity but not participate as an oxidoreductase (10
); and (d) reexamination of this disulfide switching mechanism in tissue factor, albeit with different cells and different experimental conditions, has not confirmed the original report (23
). Thus, while PDI might promote coagulation by switching a disulfide bond in tissue factor, we do not exclude other mechanisms and other PDI targets.
The observation in Par4–/–
mice that PDI inhibitors blocked formation of juxtamural platelet thrombi, whose formation does not require platelet activation by thrombin, suggests that PDI inhibitors may do more than simply reduce thrombin generation. Previous in vitro work has shown that specific platelet receptor activities can be modified by inhibition of PDI. Blocking thiol isomerase activity with inhibitory anti-PDI antibodies, low–molecular weight sulfhydryl reagents, or bacitracin all prevent activation of the fibrinogen receptor αIIb
after platelet stimulation with a variety of agonists in vitro (6
). It has been proposed that activation of αIIb
involves disulfide rearrangement with the number of free sufhydryls, localized to the disulfide bonded knot between residues 400 and 650, increasing in the β3
subunit upon activation (27
). This receptor undergoes a conformational alteration during conversion from the low-affinity to the high-affinity form of the integrin. More recently, Cys663–Cys687 was identified as a potential allosteric disulfide bond (1
), and mutation of Cys663 and Cys687 to alanine resulted in a constitutively active αIIb
). How alteration of these bonds might relate to inside-out and outside-in signaling pathways is unknown. Nonetheless, it is possible that the thrombin-independent effects of PDI inhibitors in the laser model relate to altering the function of the fibrinogen receptor.
The observation that PDI accumulated at a site of laser injury and the dramatic effect of acute administration of PDI inhibitors on inhibiting thrombus formation and prolonging tail bleeding time in mice strongly suggest a key role for PDI in fibrin deposition and platelet accumulation during thrombus formation in vivo. Furthermore, PDI represents a unique antithrombotic target for novel pharmaceuticals because its inhibition blocks both platelet thrombus formation and fibrin formation. However, enthusiasm for this approach must be tempered pursuant to careful evaluation in animal models of the possible risk-benefit ratio of such interventions.