The present study demonstrates that a thrombin-specific ACPP detects inflammatory demyelination in vivo. We show strong association between thrombin activity and BBB disruption, microglial activation, inflammatory demyelination, and axonal damage. Intriguingly, thrombin activation begins early at disease onset, prior to demyelination, and correlates with disease progression. Together with prior studies showing BBB disruption and deposition of fibrin early in EAE and MS,[1, 3, 6] these results suggest that activation of the coagulation cascade occurs at the earliest signs of inflammatory activity in lesions prior to demyelination.
Association of thrombin activity with sites of increased vascular permeability might indicate that the main source of thrombin in EAE is plasma-derived. It is possible that upon BBB disruption fibrinogen together with prothrombin enter the CNS and local activation of thrombin results in fibrin deposition. However, because prothrombin RNA can be expressed by neurons and microglia, we cannot rule out the possibility that prothrombin might also be produced in situ in neuroinflammation. Pharmacological studies have demonstrated a pathogenic role of thrombin in EAE, as its inhibition by hirudin protects from neurological signs, demyelination, and axonal damage. Thrombin might be pathogenic by catalyzing the formation of fibrin, which is a major activator of microglia and an enabler of inflammatory demyelination.[5, 6] Our data show that thrombin activation can be detected at sites of fibrin deposition both at onset and at the peak of neuroinflammation. Studies using mice genetically deficient for fibrinogen or fibrin inhibitors that do not interfere with thrombin activation show protection from EAE,[5, 6, 8] suggesting that generation of fibrin is a major pathogenic mechanism of thrombin activation. However, it is also possible that thrombin might exert fibrin-independent effects through activation of protease-activated receptors, which are expressed in several CNS cells. Future studies will determine the relative contribution of local thrombin synthesis and the mechanisms of action of thrombin in neuroinflammation.
Our work is a proof-of-principle study using a fluorescently labeled thrombin ACPP to demonstrate that detection of coagulation activity can be exploited for clinical detection of neuroinflammatory lesions. Protease-specific probes appear to be excellent sensors of disease activity, as we previously showed in cancer.[14, 15, 17, 23] In contrast to cancer, molecular probes to detect specific constituents of MS plaques have not been developed. ACPPs are ideal for clinical application, as they can be generated to carry a fluorescent dye, gadolinium (Gd), or both, allowing for multimodal detection of protease activity in vivo. For example, we showed that high levels of Gd were retained in tumors when Gd-labeled MMP-ACPPs were used, resulting in enhanced T1 contrast that lasted for several days. In MS, Gd-enhanced MRI remains the main clinical tool for lesion detection. More sensitive, targeted, and instructive strategies employing advanced molecular probes could significantly enhance the early detection of MS lesions, possibly even in preclinical stages. In that regard, the ACPP technology might offer an advantage over the detection of passively diffusing Gd, as it would allow local signal enhancement by cellular probe uptake, specifically in areas with increased proteolytic activity. Therefore, MRI using a Gd-fused thrombin-ACPP may improve sensitivity, as it would detect a concentrated amount of probe possibly even around a small or slowly leaking vessel, where a new lesion may initiate. Future studies will determine the pharmacokinetic properties of a Gd-labeled thrombin-specific ACPP and exploit its translation in MS. If successful, this technology could be further used for early patient diagnosis and therapeutic intervention, and also for rapid evaluation of patient response to treatments. Because BBB disruption and fibrin deposition are prominent not only in MS, but also in other CNS diseases such as Alzheimer disease, spinal cord injury, and brain trauma,[4, 8, 9, 25] sensitive molecular sensors of coagulation activity could also prove to be invaluable clinical tools for detecting early pathological manifestations in CNS injuries and neurodegenerative diseases.