Human neutrophil peptides (HNPs) or alpha (α)-defensins are a family of small antimicrobial peptides which play important roles in innate immunity against invading bacteria, fungi, and viruses. HNPs contain 29-33 amino acid residues which share a distinct pattern of disulfide bonding. HNPs can be further subdivided into myeloid (HNPs 1-4) and enteric (HD5-6) forms. HNPs-1, -2, and -3 are structurally identical except for the first amino acid residue and are predominantly expressed in human neutrophils from which they are released at sites of infection or inflammation. Previous studies have demonstrated that HNP1 promotes thrombus formation. However, the mechanisms underlying its prothrombotic effects are not fully understood. In the present study, we demonstrate that HNP-1, -2, and -3 all inhibit plasma-derived and recombinant ADAMTS13 activity in a concentration-dependent manner as determined by the cleavage of FRETS-VWF73 (Fig. 1A & 1B) and multimeric VWF under denaturing conditions. At final concentrations of ~10-15 µM, purified and synthetic HNP-1, -2, and -3 completely abolish ADAMTS13Õs ability to cleave FRETS-VWF73 (Fig. 1A & 1B). The concentrations required for complete inhibition of proteolytic cleavage of pre-denatured VWF by ADAMTS13 using the urea dialysis method is higher, likely resulting from the removal of peptides from the reaction. HNP-1 binds to ultra large VWF released from endothelial cells, soluble multimeric VWF, GST-VWF73 peptide, and ADAMTS13 as determined by cultured endoethelial cells in a microfluidic channels and by surface plasmon resonance. The affinity (the dissociation constant, KD) for HNP-1 to bind VWF, GST-VWF73, and ADAMTS13 is 8.0 micro mol/L, 1.0 micro mol/L, and 3.2 micro mol/L, respectively. Sequence analysis reveals that the amino acid residues of HNP-1, -2, and -3 all contain a RRY motif that is also found in the spacer domain (i.e. 659RRYGEEY665) of ADAMTS13. We hypothesize that competition by HNPs with ADAMTS13 for binding to VWF-A2 domain mediates their inhibition. As shown, a deletion or alanine substitution of RRY within HNP1 nearly abolishes its ability to inhibit ADAMTS13 activity determined by the cleavage of FRETS-VWF73 (in Fig. 1C) and multimeric VWF under denaturing conditions. Similarly, HNP-beta and aliphatic (with no aromatic rings directly on the nitrogen atom) HNP-1 exhibit no inhibitory activity on ADAMTS13 (Fig. 1C). To further demonstrate the inhibitory activity of HNP1 towards ADAMTS13 under more physiological conditions, a BioFlux microfluidic system is employed. Addition of purified (native) HNP-1 (6-15 micro mol/L) to D-phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK) anticoagulated whole blood dramatically augments the rate of platelet adhesion and aggregation to the fibrillar collagen-coated surface under arterial shear stress (approximately 100 dyne per square centimeter). These results indicate that HNP-1 plays a role in the inhibition of VWF proteolysis by ADAMTS13 under flow. We conclude that HNP-1, -2, and -3 released from activated neutrophils at sites of infection or inflammation could significantly augment thrombus formation by inhibiting the local or residual plasma ADAMTS13 activity, when the circulating ADAMTS13 activity is already at critically low levels as in cases of hereditary or acquired autoimmune TTP and HUS, thereby triggering the onset of thrombotic complications. (*authors contribute equally to this work).