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 polyethylene glycol (PEG) chain length (0, 2, 5, and 10-30 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 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. Biotechnol. Bioeng. 2011; 108: 2424-2433. (C) 2011 Wiley Periodicals, Inc.