An Arg-Gly-Asp sequence within thrombin promotes endothelial cell adhesion

Area of Science:

• Biochemistry
• Cell Biology
• Vascular Biology

Background:

• Thrombin is crucial for hemostasis but also influences wound healing, inflammation, and atherosclerosis.
• Its role in direct endothelial cell (EC) regulation is not fully understood.

Purpose of the Study:

• To investigate the ability of modified thrombin molecules to induce EC adhesion, spreading, and cytoskeletal changes.
• To identify the specific molecular mechanisms and receptors involved in thrombin-mediated EC interactions.

Main Methods:

• Chemical modification of thrombin (e.g., nitration) to create analogues like NO2-alpha-thrombin.
• Assays for EC adhesion, spreading, and cytoskeletal reorganization.
• Inhibition studies using antibodies, peptides (RGD, Loop B, CB67-129), and thrombin inhibitors (ATIII, hirudin).
• Immunofluorescence microscopy to visualize cytoskeletal elements and receptor localization (vitronectin receptor alpha v beta 3, fibronectin receptor alpha 5 beta 1).

Main Results:

• NO2-alpha-thrombin significantly enhanced EC adhesion, spreading, and actin microfilament assembly.
• EC adhesion was mediated by the vitronectin receptor (alpha v beta 3) and blocked by RGD-containing peptides and anti-alpha v beta 3 antibodies.
• Thrombin incubated at 37°C also induced EC attachment, suggesting exposure of a binding site.
• Native thrombin showed minimal EC attachment activity.

Conclusions:

• Modified thrombin, under specific conditions, can expose an RGD domain for interaction with the EC vitronectin receptor (alpha v beta 3).
• This interaction promotes EC adhesion, spreading, and cytoskeletal reorganization, contributing to vascular wall repair.
• The study defines a new bioregulatory role for thrombin and a novel EC recognition mechanism.