Seyedmohammadali Aftabjahani, n/a: No financial relationships to disclose
Background: The role of platelets in coagulation has been well documented. However, their role in fibrinolysis is not as well-defined. We have recently reported that plasminogen binding and activation by tissue-type plasminogen activator (tPA) is enhanced on platelets wben activated by thrombin, and that this process is dependent on fibrin(ogen). This process was also sensitive to the presence of activated thrombin-activatable fibrinolysis inhibitor (TAFIa), suggesting that thrombin-mediated activation of platelets generates a previously unidentified new C-terminal lysine residue., Mass spectrometry identified lysine at position 556 of fibrinogen alpha chain C-domain (αC) was primarily responsible. To further validate this, we used activated platelets obtained from mice lacking fibrinogen (Fg-/-) which had minimal plasminogen binding compared with wild-type (WT). To further define the mechanism of plasminogen binding on platelets, we obtained mice that has fibrinogen with a truncation after amino acid residue 270 in the alpha chain (Fga270), which consequently is lacking the aC domain in fibrinogen; region where this novel C-terminal lysine may be located. We compared plasminogen binding on platelets isolated from Fga270 mice and compared with platelets from Fg-/- and WT mice, with or without TAFIa treatment
METHODS AND RESULTS: Platelets were isolated from WT, Fg-/-, or Fga270 mice blood collected via carotid cannulation. Platelets were then split, and half were activated by thrombin (38 units/mL) for 10 minutes at room temperature. Additionally, either epsilon-aminocaproic acid (εACA) (20mM), a lysine analogue to inhibit plasminogen binding, or TAFIa (18.5 nM) was then added to resting or activated platelets and incubated for 10 minutes at room temperature. Lastly, plasminogen labeled with 5-iodoacetamidofluorescein (5IAF-Pg) was added to all platelets, incubated for 10 minutes at room temperature, fixed, and subjected to analyses by flow cytometry As reported previously, Fg-/- platelets had significantly reduced plasminogen binding even after activation, compared with the WT platelets (Fig. 1). Our new data show that Fga270 platelets behave similarly to Fg-/- platelets for plasminogen binding, further supporting the idea that the αC domain of fibrinogen is responsible. Addition of TAFIa led to a decrease in the activated WT platelets, but it did not affect resting or activated Fg-/- or Fga270 platelets. εACA reduced plasminogen binding in all instances, suggesting that plasminogen binding is mediated through the kringle domains of plasminogen in a lysine-dependent manner
Conclusion: Our data further confirms that the αC domain of fibrinogen alpha chain is primarily responsible for enhanced plasminogen binding observed on platelets when activated with thrombin
Lay Abstract Content: Blood is constantly circulating the body, providing nutrition and oxygen to the cells. Under normal conditions, blood remains in a fluid state and continues to travel through the body through blood vessels. When these blood vessels are damaged, a clot is formed, preventing further blood loss, while restoring vessel integrity. The role of platelets in coagulation is well known but their role in breaking down the clot after vessel repair is not yet well understood. This study investigates the role of platelets in clot breakdown by isolating platelets from mouse models with modified clotting proteins.
