A plasmin-activatable thrombin inhibitor reduces experimental thrombosis and assists experimental thrombolysis in murine models.

The leech protein hirudin is a potent natural thrombin inhibitor. Its potential as an antithrombotic agent is limited by its promotion of bleeding. We attempted to modify this profile by positioning albumin and a plasmin cleavage site on its N-terminus, in recombinant protein HSACHV3 [comprising hirudin variant 3 (HV3) fused to the C-terminus of human serum albumin (HSA) via a plasmin cleavage site (C)], Previously we showed that HSACHV3 inhibited thrombin in a plasmin-dependent manner, and that, unlike HV3, it did not increase bleeding in vivo when administered to mice.

Prothrombin complex concentrates reduce blood loss in murine coagulopathy induced by warfarin, but not in that induced by dabigatran etexilate.

Background: Both established oral anticoagulants such as warfarin and newer agents such as dabigatran etexilate (DE) effectively prevent thromboembolic disease, but may provoke bleeding. Limited clinical data exist linking oral anticoagulant reversal and bleeding tendency, as opposed to surrogate laboratory markers.

Objective:  To quantify bleeding in warfarin-anticoagulated and DE-anticoagulated mice by tail transection with or without pretreatment with potential reversal agents: prothrombin complex concentrate (PCC); activated PCC (APCC); recombinant factor VIIa (rFVIIa); or murine fresh-frozen plasma (FFP).

In vivo clearance of alpha-1 acid glycoprotein is influenced by the extent of its N-linked glycosylation and by its interaction with the vessel wall.

Alpha-1 acid glycoprotein (AGP) is a highly glycosylated plasma protein that exerts vasoprotective effects. We hypothesized that AGP's N-linked glycans govern its rate of clearance from the circulation, and followed the disappearance of different forms of radiolabeled human AGP from the plasma of rabbits and mice. Enzymatic deglycosylation of human plasma-derived AGP (pdAGP) by Peptide: N-Glycosidase F yielded a mixture of differentially deglycosylated forms (PNGase-AGP), while the introduction of five Asn to Gln mutations in recombinant Pichia pastoris-derived AGP (rAGP-N(5)Q) eliminated N-linked glycosylation.

Reduction of thrombus size in murine models of thrombosis following administration of recombinant α1-proteinase inhibitor mutant proteins.

The variant serpin α1-PI M358R inhibits thrombin and other proteases such as activated protein C (APC) and factor XIa. We previously described recombinant proteins HAPI M358R (α1-PI M358R containing an N-terminal extension corresponding to residues 1-75 of heparin cofactor II) and HAPI RCL5 (HAPI M358R with F352-I356 and I360 substituted for the corresponding residues of antithrombin), with enhanced selectivity for thrombin over APC inhibition. We tested the hypotheses that these recombinant proteins would limit thrombosis in three mouse models, and that the HAPI chimeric proteins would be more effective than α1-PI M358R.

Comparison of methods for the purification of alpha-1 acid glycoprotein from human plasma.

Alpha-1 acid glycoprotein (AGP) is a highly glycosylated, negatively charged plasma protein suggested to have anti-inflammatory and/or immunomodulatory activities. Purification of AGP could be simplified if methods that exploit its high solubility under chemically harsh conditions could be demonstrated to leave the protein in its native conformation. Procedures involving exposure of AGP to hot phenol or sulphosalicylic acid (SSA) were compared to solely chromatographic methods.