Affimer reagents assist in uncovering the architecture of blood clots

The top of your scab is a highly specialised structure with novel functions uncovered in a new paper (link is external) published in the Journal of Clinical Investigation. Scientists led by Professor Ariëns from the University of Leeds, UK explored the architecture of blood clots, in particular the clot surface that forms an interface between the host and environment, using Affimer reagents to help understand this essential protective mechanism.

Haemostasis is important in preventing blood loss and infection at sites of injury in the body and involves a close interplay between coagulation and platelets. Activated platelets and red blood cells are contained within clots, with the highly elastic 3D fibrin network holding the clot structure together. Dense networks of thin fibres and increased rigidity increase the risk of thrombosis, while loose clot networks with thick fibres and reduced rigidity are associated with bleeding, so ensuring the correct assembly of clots is key to their function.

Following the observation of a biofilm across the clot surface in scanning electron microscopy, Ariëns and his team set out to decipher its structure, composure and function. SDS-PAGE and confocal microscopy analysis of the clot biofilm constituents revealed fibrin as the key component. The fibrin in this film appeared not to be arranged into fibres typical of the rest of the clot architecture, rather it forms a continuous sheet covering the clot surface. 

Combining fibrinogen and thrombin to assess whether fibrin alone can form clots yielded identical structures to that of clots from whole blood plasma. Further investigation varying the concentrations of thrombin and fibrinogen showed alterations to the thickness and amount of fibrin within the surface film. Optimal film formation occurred at lower concentrations of both thrombin and fibrinogen, where the longer clotting time allowed increases in fibrin building up at the surface before the underlying clot network was formed. 

As part of these investigations into the formation of the clot surface, blood samples from patients with dysfibrinogenemia and afibrinogenemia were used. To prevent changes in the structure of the clot film with fluorescently labelled fibrinogen, an Alexa488-fibrinogen specific Affimer reagent was incubated with patient plasma samples to bind fibrinogen before clotting was initiated. The resulting clots were imaged via laser scanning confocal microscopy to show patients with dysfibrinogenemia formed films that were thicker and contained more fibrin than normal plasma, while afibrinogenemia patient samples failed to form clots. This demonstrated that fibrin is sufficient to form a film covering the blood clot at the air interface with a different structure to the remainder of the clot, but this film structure can be modulated by various conditions.

The scientists further examined the fibrin film of blood clots to show that the film is initiated by the exposure of blood to the air interface, and that the film is able to dramatically slow bacterial growth preventing their movement into the wound site and allowing the full clot to develop underneath this film. They also showed that the biofilm protects against cell loss from the clot. This unique and novel mechanism in haemostasis throws new light on the structure and function of the fibrin biofilm on the clot surface. Exploiting this new understanding could offer improved healing from injuries, and suggests an underlying cause for the potential increased risk of persistent infections from minor dermal abrasions in afibrinogenemia patients who fail to form the clot surface correctly.