Elevated thrombospondin 1 (TSP1) is a prevalent factor, via cognate receptor CD47, in the pathogenesis of cardiovascular conditions, including ischemia-reperfusion injury (IRI) and pulmonary arterial hypertension (PAH). Moreover, TSP1/CD47 interaction has been found to be associated with platelet hyperaggregability and impaired nitric oxide response, exacerbating progression in IRI and PAH. Pathological TSP1 in circulation arises as a target of our novel therapeutic approach. Our “proof-of-concept” pharmacological strategy relies on recombinant human CD47 peptide (rh-CD47p) as a decoy receptor protein (DRP) to specifically bind TSP1 and neutralize TSP1-impaired vasorelaxation, strongly implicated in IRI and PAH. The binding of rh-CD47p and TSP1 was first verified as the primary mechanism via Western blotting and further quantified with modified ELISA, which also revealed a linear molar dose-dependent interaction. Ex vivo, pretreatment protocol with rh-CD47p (rh-CD47p added prior to TSP1 incubation) demonstrated a prophylactic effect against TSP1-impairment of endothelium-dependent vasodilation. Post-treatment set-up (TSP1 incubation prior to rh-CD47p addition), mimicking pre-existing excessive TSP1 in PAH, reversed TSP1-inhibited vasodilation back to control level. Dose titration identified an effective molar dose range (approx. ≥1:3 of tTSP1:rh-CD47p) for prevention of/recovery from TSP1-induced vascular dysfunction. Our results indicate the great potential for proposed novel decoy rh-CD47p-therapy to abrogate TSP1-associated cardiovascular complications, such as PAH.

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Hemostatic abnormalities mainly decreased blood flow and increased stasis, and platelet hyper-aggregation increase thrombotic risk subsequent atrial fibrillation (AF) episodes. Excess elevation of extracellular glycoprotein thrombospondin 1 (TSP1) in circulation has emerged to predict prognosis of ischemic stroke. Moreover, TSP1 is found strongly associated with platelet hyper-aggregability and impaired nitric oxide response, through its cognate receptor CD47, thus exacerbating ischemic progression in AF patients. TSP1 arises as a prospective target of our candidate medicine. Our work provides “proof-of-concept” of a novel pharmacological strategy, relying on soluble recombinant human CD47 peptide (rh-CD47p) to serve as a decoy receptor to specifically bind TSP1, and consequently neutralize TSP1-impaired vasorelaxation. Our pilot studies were initially conducted with isolated mouse thoracic aorta. A pre-treatment with rh-CD47p (added 15 min prior to TSP1 incubation) showed a significant amelioration of TSP1-impaired endothelium-dependent vasodilation ( p <0.0001, rh-CD47p+TSP1 vs. TSP1, n=6), indicating a prophylactic effect by rh-CD47p against vasoconstriction enhanced by excess TSP1. A post-treatment set-up, where TSP1 incubation was started 15 min prior to rh-CD47p addition to mimic pre-existing high level of TSP1 in ischemic stroke, exhibited a marked reversal of TSP1-inhibited vasodilation back to the same level as controls ( p <0.0001, rh-CD47p+TSP1 vs. TSP1, n=4). Dose titration of rh-CD47p in molar ratios to TSP1 in both set-ups identified the dose range of rh-CD47p for prevention/recovery from TSP1-induced vascular dysfunction. Binding of rh-CD47p and TSP1 was first verified as the primary mechanism via Western Blot, and further quantified with a modified ELISA assay. A linear correlation was found between % of rh-CD47p bound to TSP1 and dose of rh-CD47p added (r 2 =0.8). Current results established the ground of rh-CD47p as a unique pharmacologic moiety for drug development to limit vasoconstriction and improve vasodilation against TSP1 in excess, thus showing great potential for proposed novel decoy CD47-coated formulation to abrogate/mitigate TSP1-associated cardiovascular complications in AF patients.