Salmon-derived thrombin inhibits development of chronic pain through an endothelial barrier protective mechanism dependent on APC

Abstract

Many neurological disorders are initiated by blood-brain barrier breakdown, which potentiates spinal neuroinflammation and neurodegeneration. Peripheral neuropathic injuries are known to disrupt the blood-spinal cord barrier (BSCB) and to potentiate inflammation. But, it is not known whether BSCB breakdown facilitates pain development. In this study, a neural compression model in the rat was used to evaluate relationships among BSCB permeability, inflammation and pain-related behaviors. BSCB permeability increases transiently only after injury that induces mechanical hyperalgesia, which correlates with serum concentrations of pro-inflammatory cytokines, IL-7, IL-12, IL-1α and TNF-α. Mammalian thrombin dually regulates vascular permeability through PAR1 and activated protein C (APC). Since thrombin protects vascular integrity through APC, directing its affinity towards protein C, while still promoting coagulation, might be an ideal treatment for BSCB-disrupting disorders. Salmon thrombin, which prevents the development of mechanical allodynia, also prevents BSCB breakdown after neural injury and actively inhibits TNF-α-induced endothelial permeability in vitro, which is not evident the case for human thrombin. Salmon thrombin's production of APC faster than human thrombin is confirmed using a fluorogenic assay and APC is shown to inhibit BSCB breakdown and pain-related behaviors similar to salmon thrombin. Together, these studies highlight the impact of BSCB on pain and establish salmon thrombin as an effective blocker of BSCB, and resulting nociception, through its preferential affinity for protein C.

Keywords: Activated protein C; Blood–brain barrier; Nerve root injury; Pain; Thrombin.

Fig. 1. Peripheral neural injury causes early BSCB breakdown and inflammation corresponding to the onset of behavioral hypersensitivity

(A) A 15-minute nerve root compression significantly decreases (^p<0.001) the withdrawal threshold in the ipsilateral forepaw on each day compared to corresponding baseline thresholds. That 15-minute compression also significantly decreases the withdrawal threshold compared to sham overall (p=0.018) and on individual days 1 and 7 (*p<0.042). Thresholds in the contralateral forepaw are not different between groups.Data are shown as mean±SD. (B) Representative images depict intense IgG labeling only in the ipsilateral dorsal horn on day 1 after 15min. Minimal bilateral IgG labeling is evident on day 1 for 3min and sham. (C) The percent IgG labeling in the ipsilateral spinal cord significantly increases at day 1 after 15min compared to sham (*p<0.001) and 3min (**p<0.001). By day 7, ipsilateral IgG significantly decreases (^p<0.001) compared to day 1. Data are mean±SD. (D) On day 1, withdrawal threshold positively correlates to the serum concentration of four pro-inflammatory cytokines: IL-7, IL-12, IL-1α and TNF-α (see Table 1). Ipsilateral spinal TNF-α immunoreactivity also increases in areas of BSCB breakdown on day 1 only for 15min.

Fig. 2. Salmon thrombin prevents vascular disruption dependent on protein C

(A) Mechanical allodynia (quantified by the number of paw withdrawals) is significantly reduced (**p=0.0148) on day 1 after compression treated with salmon thrombin (15min+STh) compared to human thrombin (15min+HTh). Allodynia for 15min+HTh is significantly greater (*p=0.016) than sham and not different from vehicle-treatment with compression (15min+veh). (B) There is minimal IgG labeling after 15min+STh in the ipsilateral dorsal horn on day 1, and is significantly less than 15min+HTh (*p<0.0001) and 15min+veh (#p=0.0009). Both 15min+HTh and 15min+veh exhibit robust ipsilateral spinal IgG labeling that is significantly greater (**p<0.0065) than the labeling in the respective contralateral sides. (C) HUVEC-lined microchannels exhibit increased FITC-dextran flux into the surrounding collagen when treated with TNF-α with or without serum. Salmon thrombin (TNF-α+STh) significantly reduces (*p=0.0073) TNF-α-induced flux compared to human thrombin (TNF-α+HTh) only in the presence of serum. TNF-α+STh and TNF-α+HTh are not different in serum-free conditions. (D) STh produces APC significantly faster (*p=0.008) than HTh. All data are mean±SD.

Fig. 3. Blocking BSCB breakdown with intravenous APC inhibits nociception after neural injury

(A) Spinal IgG is reduced after APC treated 15-minute compression (15min+APC) compared to compression alone (15min). (B) A 15min compression significantly increases (**p=0.028) spinal IgG in the ipsilateral dorsal horn compared to the contralateral dorsal horn on day 1. Ipsilateral IgG for 15min+APC is significantly lower (*p<0.001) than 15min on day 1 and is not different from respective contralateral values. (C) Forepaw withdrawal threshold at day 1 is significantly reduced (*p=0.024) by a 15min compression compared to sham, whereas, 15min+APC exhibits a significantly higher (**p=0.012) withdrawal threshold than 15min, which is not different sham. All data represented as mean±SD.

Fig. 4. Protein structure for thrombin derived from fish and from human differs in the autolysis loop, which partially controls thrombin’s specificity for protein C

Aligned amino acid sequences for the B chains of human thrombin (HTh) and fish thrombin (Salmo gairdneri, FTh) exhibit a key divergence in the autolysis loop (blue). A crystal structure for HTh (magenta) bound to a small peptide of protein C (PDB:4DT7) and the created homology model for FTh (orange) have strong alignment (RMSD=0.51Å over 265 residues) shown in ribbon representation. The autolysis loop (in blue) does not retain the same structure between species. The residues within both autolysis loops are represented below as sticks to visualize predicted close interactions with protein C (green cage structure).