Dear colleagues,

I investigate a therapeutic peptide bearing a proteolytic cleavage site that is specific to the protease thrombin. I saw significant degradation rates of the peptide, being cleaved at this particular site in a stability assay with Heparin plasma and EDTA plasma, respectively.

Now my question is: Would it make sense that degradation is due to thrombin activity, although the plasma contains different anticoagulants?

EDTA basically prevents the coagulation cascade to proceed by complexing Ca2+. Here I can imagine that normal in vivo levels of thrombin, although very little of course, could still be active in plasma. I found a paper of T.W. Stief that reports a thrombin activity of 5.5 mIU/mL in EDTA plasma. [1]

However, Heparin accelerates the formation of thrombin-antithrombin complexes and therefore participates in direct inhibition of proteolytic activity. Here I imagine all trace thrombin activity should be lost. However, I do see thrombin specific cleavage. Unfortunately, I could not find a paper that makes a statement about thrombin activity in heparin plasma.

I would be very happy about any advice or comment on my reasoning.

Kind regards,

Roland Böttger

[1] T.W. Stief (2006). Specific Determination of Plasmatic Thrombin Activity. Clinical and applied thrombosis/hemostasis, 12(3), 24-329. DOI: 10.1177/1076029606291381