Human neutrophil peptide‐1 inhibits thrombus formation under arterial flow via its terminal free cysteine thiols

Abstract

Biological activity of human neutrophil peptide (HNP)‐1 in hemostasis under physiological conditions is not fully understood. HNP‐1 inhibits the adhesion/aggregation of murine platelets on a fibrillar collagen surface or an activated endothelial cell surface under flow. The anti‐adhesion activity appears to depend on the terminal free thiols of HNP‐1, which may inhibit VWF‐VWF lateral associations. Our results suggest a protective role and potential novel therapeutic use of HNP‐1 for arterial thrombosis. Summary BackgroundHuman neutrophil peptides (HNPs), also known as α‐defensins, are released from degranulated neutrophils and play an important role in innate immunity. However, their biological roles in hemostasis under flow are not fully explored. ObjectiveThis study aims to determine the role of HNP‐1 on platelet adhesion and aggregation on a collagen surface or ultra large von Willebrand factor (ULVWF) on endothelium under flow and elucidate the structural elements required for its activity. MethodsAnticoagulated whole blood from wild‐type or Adamts13−/− mice was incubated with a fluorescein‐conjugated anti‐human CD41 in the presence of increasing concentrations of a synthetic HNP‐1 and perfused over a collagen surface or a tumor necrosis factor (TNF)‐α activated murine endothelial cell surface under arterial flow. The rate of accumulation and the final surface coverage of fluoresceinated murine platelets or the rate of forming platelet‐decorated ULVWF strings were determined using the BioFlux microfluidic system. ResultsHNP‐1 inhibited the rate and final coverage of fluorescein‐labeled murine platelets on a fibrillar collagen surface under flow (100 dyne/cm2) in a concentration‐dependent manner and the anti‐adhesive activity of HNP‐1 depended on its terminal free cysteine thiols. HNP‐1 (20 μM) also dramatically inhibited the formation of platelets‐decorated ULVWF strings on TNF‐α activated murine endothelial surface under arterial flow. ConclusionsOur results demonstrate for the first time an antiplatelet adhesion or antithrombotic activity of HNP‐1; this activity depends on its terminal free thiols, likely affecting VWF‐VWF lateral associations. These findings may suggest a potential novel therapeutic strategy for arterial thrombosis.

Histone-induced thrombotic thrombocytopenic purpura in adamts13−/− zebrafish depends on von Willebrand factor

“…Plasma HNP1-3, (30,31) histone-DNA complexes, (29) and other inflammatory mediators are significantly increased in patients with acute iTTP. HNP1-3 may be prothrombotic (31,32) or anti-thrombotic, (33) depending on the context and their redox status. Increased plasma levels of histone-DNA complexes correlate with low platelet counts and the disease severity.…”

Insights Into Immunothrombosis: The Interplay Among Neutrophil Extracellular Trap, von Willebrand Factor, and ADAMTS13

“…Surprisingly, HNPs also exhibit antithrombotic effects. HNP1 inhibits the adhesion and aggregation of murine platelets on the collagen-coated surface or on TNF-α activated endothelial cells under arterial shear stress in the absence of ADAMTS13 ( 138 ). Under arterial flow, VWF multimers are activated by high shear stress and expose the free cysteine thiols that facilitate VWF-VWF lateral association by forming new covalent disulfide bonds ( 139 ), leading to the formation of larger and thicker VWF multimer networks and the recruitment of more blood cells.…”

“…Under arterial flow, VWF multimers are activated by high shear stress and expose the free cysteine thiols that facilitate VWF-VWF lateral association by forming new covalent disulfide bonds ( 139 ), leading to the formation of larger and thicker VWF multimer networks and the recruitment of more blood cells. HNP1 inhibits the formation of UL-VWF strings via the direct binding of its free cysteine thiols to free cysteine thiols in VWF ( 138 ).…”

State-of-the-art review - A review on snake venom-derived antithrombotics: Potential therapeutics for COVID-19-associated thrombosis?

“…Several of the low molecular mass polypeptides from snake venoms (see Table 3 ) have demonstrated appreciable antithrombotic activity. With the advancement of cost-effective automated protein synthesis technology, a synthetic biology approach for the production of antithrombotic proteins and enzymes will overcome the cumbersome task of isolating and purifying protein/polypeptide from natural resources [152] , [153] , [154] . A similar approach may also be applied for the production of antithrombotic snake venom proteins and polypeptides.…”