von Willebrand factor (VWF) and the metalloprotease a disintegrin and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13) are present both within endothelial cells (ECs) and in peripheral blood. Calcium concentrations are lower in intracellular compartments (80-400 μM) compared with the extracellular milieu (～1.25 mM). Because low calcium favors VWF A2-domain proteolysis by ADAMTS13， the dependence of proteolysis rates on calcium was assayed both within ECs and in blood. Confocal microscopy studies demonstrate partial perinuclear colocalization of VWF with ADAMTS13 in human umbilical vein ECs (HUVECs). Consequently， low levels (5%-10%) of VWF cleavage products were detected in HUVEC lysates and also culture-supernatant following EC stimulation. This proteolysis occurred before disulfide bond formation. Compared with wild-type VWF A2-domain， calcium-binding mutants including the common von Willebrand disease (VWD) type 2A R1597W mutant were expressed in an open conformation in ECs and were highly susceptible to intracellular proteolysis. Fluorescence resonance energy transfer measurements demonstrate strong calcium-dependent VWF-A2 conformation changes at concentrations <500 μM， with unfolding rates being fourfold higher for monomeric VWF A2-domain compared with multimeric， full-length VWF. Under shear， physiological levels of ADAMTS13 did not cleave VWF strings on HUVECs， unless platelets were attached to stretch these strings under flow. Further， VWF-platelet string cleavage under shear proceeded with equal efficiency in the absence and presence of calcium at shear stress ≥1 dyn/cm. Overall， low calcium levels may promote intracellular VWF proteolysis particularly during VWD type 2A disease. Calcium has a negligible effect on VWF-platelet string proteolysis under physiologically relevant fluid shear.