Factor VIII accelerates proteolytic cleavage of von Willebrand factor by ADAMTS13

Abstract

Proteolytic processing of von Willebrand factor (VWF) by ADAMTS13 metalloproteinase is crucial for normal hemostasis. In vitro, cleavage of VWF by ADAMTS13 is slow even at high shear stress and is typically studied in the presence of denaturants. We now show that, under shear stress and at physiological pH and ionic strength, coagulation factor VIII (FVIII) accelerates, by a factor of Ϸ10, the rate of specific cleavage at the Tyr 1605 -Met 1606 bond in VWF. Multimer analysis reveals that FVIII preferentially accelerates the cleavage of high-molecular-weight multimers. This rate enhancement is not observed with VWF predenatured with 1.5 M guanidine. The ability of FVIII to enhance VWF cleavage by AD-AMTS13 is rapidly lost after pretreatment of FVIII with thrombin. A FVIII derivative lacking most of the B domain behaves equivalently to full-length FVIII. In contrast, a derivative lacking both the B domain and the acidic region a3 that contributes to the highaffinity interaction of FVIII with VWF exhibits a greatly reduced ability to enhance VWF cleavage. Our data suggest that FVIII plays a role in regulating proteolytic processing of VWF by ADAMTS13 under shear stress, which depends on the high-affinity interaction between FVIII and its carrier protein, VWF.thrombotic thrombocytopenic purpura ͉ von Willebrand factor cleaving metalloprotease N ormal hemostasis requires the proteolytic processing of newly synthesized and secreted von Willebrand factor (VWF) in plasma by the metalloproteinase ADAMTS13. This process reduces VWF multimer size by cleavage at the Tyr 1605 -Met 1606 bond in the A2 subunit (1). Defective proteolytic processing of VWF multimers due to hereditary and/or acquired deficiency of ADAMTS13 activity results in the accumulation of ''unusually large'' multimers of VWF in plasma and disseminated microthrombi (2). These are characteristic pathological features of thrombotic thrombocytopenic purpura.ADAMTS13 cleaves VWF in a relatively inefficient way, and its preference for cleavage of the larger multimers is considered to lie in their greater susceptibility to deformation under shear stress or under flow while tethered to platelets (3). Previous studies have used a variety of manipulations to enhance cleavage including the use of ''low concentrations'' of urea (4) or guanidine hydrochloride (5) and prolonged incubation of denatured VWF with ADAMTS13 at low ionic strength, alkaline pH, and high concentrations of various divalent cations such as Ba 2ϩ , Ca 2ϩ , and Zn 2ϩ (1,4,6). These strategies are reminiscent of the extreme conditions used for observable cleavage of the coagulation zymogens in the absence of membranes and the appropriate cofactors that are now known to be essential for their efficient activation (7). Only very small enhancing effects of heparin and glycoprotein Ib on cleavage of denatured VWF have been reported previously (8). Thus, efficient processing of VWF by ADAMTS13 under more physiological conditions may be further regulated by a cofactor or by accessory components that are y...

Factor VIII and Platelets Synergistically Accelerate Cleavage of von Willebrand Factor by ADAMTS13 under Fluid Shear Stress

“…Shim et al (24) and we (22) have demonstrated that lyophilized platelets or coagulation factor VIII (FVIII) alone at physiological concentrations can increase the proteolytic cleavage of VWF by ADAMTS13 by ϳ2-3-fold under mechanically induced fluid shear stresses (22,24). In this study, we show that the combination of the two (FVIII and platelets) at physiological concentrations synergistically accelerates the proteolytic cleavage of VWF by ADAMTS13 under similar conditions.…”

“…Patients with aortic stenosis, which generates high flow shear stress in circulation, do exhibit increased VWF proteolysis and reduced VWF multimer size compared with healthy individuals (20,21), suggesting that increased VWF proteolysis is correlated with increased fluid shear stress in vivo. However, under in vitro assay conditions, fluid shear stress alone (22, 23) has only a modest effect on proteolytic cleavage of soluble multimeric VWF by ADAMTS13, suggesting that other proteins or nonprotein cofactors or cellular components may be required for efficient proteolytic processing of soluble multimeric VWF by ADAMTS13.Shim et al (24) and we (22) have demonstrated that lyophilized platelets or coagulation factor VIII (FVIII) alone at physiological concentrations can increase the proteolytic cleavage of VWF by ADAMTS13 by ϳ2-3-fold under mechanically induced fluid shear stresses (22,24). In this study, we show that the combination of the two (FVIII and platelets) at physiological concentrations synergistically accelerates the proteolytic cleavage of VWF by ADAMTS13 under similar conditions.…”

“…The purity of VWF was determined by 8% SDS-PAGE and Coomassie Blue staining. The multimer distribution was determined by 1% mini-agarose gel electrophoresis and Western blotting as described previously (22,25). The amount of purified VWF was determined by A 280 nm corrected by A 320 nm (1 corrected A 280 nm ϭ approximately 1 mg/ml) (26).…”

“…Patients with aortic stenosis, which generates high flow shear stress in circulation, do exhibit increased VWF proteolysis and reduced VWF multimer size compared with healthy individuals (20,21), suggesting that increased VWF proteolysis is correlated with increased fluid shear stress in vivo. However, under in vitro assay conditions, fluid shear stress alone (22,23) has only a modest effect on proteolytic cleavage of soluble multimeric VWF by ADAMTS13, suggesting that other proteins or nonprotein cofactors or cellular components may be required for efficient proteolytic processing of soluble multimeric VWF by ADAMTS13.…”

ADAMTS13 content in plasma‐derived factor VIII/von Willebrand factor concentrates

“…This may be especially true on the basis of in vitro studies indicating that FVIII contained in this and other concentrates may physiologically facilitate the cleavage of VWF by ADAMTS13 [19]. Koate V R -DVI has a higher content of functional ADAMTS13 than other tested VWF-FVIII concentrates.…”