The function of von Willebrand factor (VWF) is regulated by proteolysis, which limits its multimeric size and ability to tether platelets. The importance of ADAMTS13 metalloprotease in VWF regulation is demonstrated by the association between severe deficiency of ADAMTS13 and thrombotic thrombocytopenic purpura (TTP). However, ADAMTS13 activity levels do not always correlate with the clinical course of TTP, suggesting that other proteases could be important in regulating VWF. We identified 4 leukocyte proteases that cleave the synthetic VWF substrate FRETS-VWF73 and multimeric VWF. Elastase and proteinase 3 (PR3) cleave multimeric VWF and FRETS-VWF73 at the V¹⁶⁰⁷-T¹⁶⁰⁸ peptide bond; cathepsin G and matrix metalloprotease 9 cleave VWF substrates at the Y¹⁶⁰⁵-M¹⁶⁰⁶ and M¹⁶⁰⁶-V¹⁶⁰⁷ bonds, respectively. Isolated intact human neutrophils cleave FRETS-VWF73 at the V¹⁶⁰⁷-T¹⁶⁰⁸ peptide bond, suggesting that elastase or PR3 expressed on leukocyte surfaces might cleave VWF. In the presence of normal or ADAMTS13-deficient plasma, cleavage of FRETS-VWF73 by resting neutrophils is abolished. However, activated neutrophils retain proteolytic activity toward FRETS-VWF73 in the presence of plasma. Although the in vivo relevance remains to be established, these studies suggest the existence of a “hot spot” of VWF proteolysis in the VWF A2 domain, and support the possibility that activated leukocytes may participate in the proteolytic regulation of VWF.