HSP47 inhibitor Col003 attenuates thromboinflammation after cerebral ischemia-reperfusion by suppressing GPVI-mediated CD84 shedding in platelets

Background: Thromboinflammation is a major factor exacerbating neurological damage after reperfusion in ischemic stroke, with platelet Collagen receptor Glycoprotein VI (GPVI) activation-induced CD84 shedding and its mediated CD4 + T cell migration playing a key role. Our previous work demonstrated that the HSP47 inhibitor Col003 suppresses platelet activation by inhibiting GPVI signalling, thereby downregulating platelet-derived Reactive Oxygen Species (ROS) and inflammatory mediators such as P-selectin. However, its potential to modulate thromboinflammatory responses after cerebral ischemia-reperfusion by regulating CD84 shedding downstream of GPVI remains unclear. This study aims to investigate how Col003 mitigates thromboinflammatory responses after cerebral ischemia-reperfusion, potentially offering a novel therapeutic strategy for ischemic stroke.

Method: The middle cerebral artery occlusion (MCAO) model was employed to simulate ischemic stroke in rats. Reperfusion was performed 2 h after ischemia onset. Col003 was administered via tail vein immediately upon reperfusion. A solvent control group received an equivalent volume of DMSO (0.4% final concentration), and a sham-operated group was also established. Cerebral infarction volume was assessed via TTC staining, and neurological deficit severity was evaluated using the Bederson neurological function score. Histopathological changes, cerebral microthrombosis, and neuronal injury were observed through HE staining and Nissl staining. Immunofluorescence staining detected platelet (CD42) and leukocyte (CD45) adhesion in microvessels (CD31) and CD4 + T cell infiltration on the infarct side. Western blot assessed inflammatory factor expression (TNF-α, IL-1β) in the infarct zone and peripheral blood platelet CD84 levels. Enzyme-linked immunosorbent assay (ELISA) measured serum sCD84 levels. Concurrently, in vitro platelet activation was simulated using Collagen (a GPVI agonist) to mimic in vivo conditions, with a GPVI inhibitor serving as a parallel control. The study observed that Col003 mitigated platelet CD84 shedding by inhibiting GPVI effects.

Results: Col003 treatment significantly improved neurological deficits in MCAO rats, reduced infarct volume, decreased cerebral microthrombus formation and CD4 + T cell infiltration, and downregulated TNF-α and IL-1β expression. Col003 markedly decreased serum soluble CD84 (sCD84) levels while increasing platelet CD84 expression, suggesting inhibition of CD84 shedding. In vitro experiments have demonstrated that Col003 produces functional outcomes similar to those of GPVI inhibitors ((S)-S007-1558, HY-144390), significantly reducing collagen-induced CD84 shedding from platelets.

Conclusion: The HSP47 inhibitor Col003 suppresses GPVI-mediated shedding of CD84 from platelets, alleviates platelet-mediated inflammatory responses and cerebral microthrombus formation, and reduces the infiltration of CD4 + T cells and Other immune cells in the cerebral infarct region. These results indicate that targeting HSP47 produces functional effects similar to those achieved by inhibiting the GPVI pathway, thereby providing a novel potential target and therapeutic strategy for ischemic stroke.