A new direction for anticoagulants: Inhibiting fibrin assembly with PEGylated fibrin knob mimics

Abstract

Current anticoagulants target coagulation Factors upstream from fibrin assembly and polymerization (i.e. formation of fibrin clot). While effective, this approach requires constant patient monitoring since pharmacokinetics and pharmacodynamics vary from patient to patient. To address these limitations, we developed an alternative anticoagulant that effectively inhibits fibrin polymerization. Specifically, we investigated PEGylated fibrin knob `A' peptides, evaluating the effect of both PEG chain length (0, 2, 5, 10, 20, and 30 kDa) and knob peptide sequence (GPRPAAC, GPRPFPAC, and GPRPPERC) on inhibiting fibrin polymerization (i.e. clot formation). Thrombin-initiated clotting assays with purified fibrinogen were performed to compare clot formation with each peptide-PEG conjugate. Results indicated a biphasic effect of PEG chain length whereby active-PEG conjugates demonstrated increasingly enhanced inhibition of fibrin polymerization from 0 to 5 kDa PEG. However, the anticoagulant activity diminished to control levels for PEG chains above 5 kDa. Ultimately, we observed a 10-fold enhancement of anticoagulant activity with active peptides PEGylated with 5 kDa PEG compared to non-PEGylated knob peptides. The sequence of the active peptide significantly influenced the anticoagulant properties only at the highest 1:100 molar ratio where GPRPFPAC-5 kDa PEG and GPRPPERC-5 kDa PEG demonstrated significantly lower percent clottable protein than GPRPAAC-5 kDa PEG. Moreover, human plasma treated with the active 5 kDa PEG conjugate exhibited delayed the prothrombin time to within the therapeutic range specified for oral anticoagulants. Collectively, this study demonstrated the utility of PEGylated fibrin knob peptides as potential anticoagulant therapeutics.

Procoagulant activity induced by vascular injury determines contribution of elevated factor VIII to thrombosis and thrombus stability in mice

“…Regardless, the differences between these findings demonstrate fundamental differences in the hypercoagulable mechanisms of elevated fibrinogen and elevated FVIII, and they suggest antithrombotic strategies could be tailored for a specific hypercoagulability (blocking thrombin generation/activity vs blocking fibrin formation). 46 Our findings on the relationship between vascular injury and effect of FVIII on thrombosis inform epidemiologic studies showing either moderate or no relationship between FVIII and arterial thrombosis. 9,10,12,13 Using the presence of carotid plaque as a marker of mild or moderate disease, Pan et al showed a significant association between heart disease and FVIII level (OR ϭ 2.65).…”

“…Regardless, the differences between these findings demonstrate fundamental differences in the hypercoagulable mechanisms of elevated fibrinogen and elevated FVIII, and they suggest antithrombotic strategies could be tailored for a specific hypercoagulability (blocking thrombin generation/activity vs blocking fibrin formation). 46 …”

A novel platelet lysate hydrogel for endothelial cell and mesenchymal stem cell-directed neovascularization

“…PL and fibrin hydrogels were formed as described above at a volume of 0.5 mL and polymerized in 24-well 1.5 glass bottom tissue culture plates[20,21]. Laser scanning confocal microscopy (LSM 700, Carl Zeiss, Inc.) with a 63X oil immersion objective was performed for visualization of the fibrin network using 5% FITC-labeled fibrinogen (Sigma).…”

Fibrin-based biomaterials: Modulation of macroscopic properties through rational design at the molecular level

“…These mimics can then be used alone, or in conjunction with carrier materials, to modify fibrin structure. We subsequently created a variety of knob mimic constructs including knob A -protein constructs, PEGylated knobs A and B and knob A modified elastin-like peptide (ELP) micelles and characterized their effect on fibrin properties such as polymerization, degradation and mechanical properties and network structure [31, 43, 151–153]. Knob A -protein constructs designed to display the tetrapeptide sequences GPRP, GPRV, GHRP or GSPE (non-binding control) at the N-termini of a protein fragment, specifically the cell binding 9th and 10th type III repeats of fibronectin, were found to stably bind fibrinogen via specific knob:hole interactions and were retained within fibrin matrices for 24 hours, therefore showing promise as a potential method for drug release from fibrin matrices [152].…”

“…These perturbations were not observed in matrices formed in the presence of 7.5 kDa GPRP2 -PEG, 10 kDa and 20 kDa GPRP4 -PEG (Figure 5L, P, R). In additional studies, our group has created PEG- knob constructs with higher affinity for fibrinogen through conjugation of a linear 5 kDa PEG to the GPRPFPAC sequence; addition of this construct to fibrin matrices was found to increase network porosity compared to the peptide sequence alone [151]. Likewise, studies utilizing a PEGylated knob B mimic, AHRPYAAC, found that this construct also enhanced the porosity of the fibrin network (Figure 6) and increased susceptibility to degradation, though interestingly, these constructs resulted in an increase in the complex modulus [43].…”