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...

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 IntroductionThe hemostatic activity of von Willebrand factor (VWF) is regulated in blood by the metalloprotease ADAMTS13, which cleaves VWF in the A2 domain. 1 The importance of VWF regulation by ADAMTS13 is demonstrated by the close association between severe deficiency of ADAMTS13 activity and thrombotic thrombocytopenic purpura (TTP). 2,3 However, the association between ADAMTS13 activity and TTP is imperfect. Patients with inhibitor-mediated ADAMTS13 deficiency can achieve clinical remission despite persistent severe deficiency of ADAMTS13 activity, 2,4 and not all patients with congenital deficiency of ADAMTS13 develop TTP. 5 Moreover, VWF proteolysis can be paradoxically increased in TTP patients during acute episodes. 6 These observations suggest the existence of other important disease-modifying factors in TTP, in addition to ADAMTS13.Disease-modifying factors in TTP may include other VWFcleaving proteases. Before the discovery of ADAMTS13, the proteases calpain, neutrophil elastase, and cathepsin G were known to cleave VWF. [7][8][9][10][11][12] However, the exact cleavage sites were not determined, and the physiologic relevance of these proteases was unknown. It was known that normal plasma contained proteolytic fragments of VWF having masses of approximately 176 kDa and 140 kDa. The primary cleavage site that gave rise to these fragments was identified as the Y1605-M1606 peptide bond. 13 In studies seeking to identify the responsible protease, Tsai et al 8,9,14 observed that neutrophil proteases cleaved high-molecular-weight VWF into a series of multimers indistinguishable from those found in normal plasma. By contrast, Berkowitz et al 10 concluded that VWF cleavage fragments generated by neutrophil elastase were different from the 176-kDa and 140-kDa fragments found in plasma. The role of leukocyte proteases in VWF regulation remained unresolved, and was later overshadowed by the discovery of ADAMTS13.In this study, we demonstrate that under denaturing and fluid shear stress conditions multiple leukocyte proteases cleave VWF predominantly in the central A2 domain. We also show that activated neutrophils, but not normal neutrophils, retain VWF cleaving activity in the presence of plasma inhibitors, suggesting that leukocyte proteases may regulate VWF function under physiologic conditions. Methods Patients and plasmaBlood samples were obtained from volunteer subjects after informed consent, in accordance with the Declaration of Helsinki, sanctioned by the institutional human research subject committees o...

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Immune-mediated thrombotic thrombocytopenic purpura is characterized by severe thrombocytopenia and microangiopathic hemolytic anemia. It is primarily caused by immunoglobin G type autoantibodies against ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor. However, reliable markers predictive of patient outcomes are yet to be identified. Seventy-three unique patients with a confirmed diagnosis of immune-mediated thrombotic thrombocytopenic purpura between April 2006 and December 2017 were enrolled from the Univeristy of Alabama at Birmingham Medical Center. Clinical information, laboratory values, and a panel of special biomarkers were collected and/or determined. The results demonstrated that the biomarkers associated with endothelial injury (e.g. von Willebrand factor antigen and collagen-binding activity), acute inflammation (e.g. human neutrophil peptides 1-3 and histone/deoxyribonucleic acid complexes), and activation of the complement alternative pathway (e.g. factors Bb and iC3b) were all significantly increased in patients with acute immune-mediated thrombotic thrombocytopenic purpura compared to those in the healthy controls who did not have a hematological disease; moreover, failure to normalize platelet counts within 7 days or failure to markedly reduce serum lactate dehydrogenase by day 5, low total serum protein or albumin, and high serum troponin levels were also predictive of mortality; so were the prolonged activated partial thromboplastin time, high fibrinogen, and elevated serum lactate dehydrogenase, Bb, and sC5b-9 on admission. These results may help to stratify patients for more intensive management; the findings may also provide a framework for future multicenter studies to identify valuable prognostic markers for immune-mediated thrombotic thrombocytopenic purpura.

Previous studies have demonstrated that factor VIII (FVIII) or platelets alone increase cleavage of von Willebrand factor (VWF) by ADAMTS13 under mechanically induced shear stresses. We show in this study that the combination of FVIII and platelets at the physiological concentrations is more effective than either one alone. In the absence of FVIII, lyophilized platelets increase the formation of cleavage product by 2-3-fold. However, in the presence of physiological concentration of FVIII (1 nM), the formation of VWF cleavage product increases dramatically as a function of increasing platelets with the maximal rate enhancement of ϳ8-fold. Conversely, in the presence of a physiological concentration of lyophilized platelets (150 ؋ 10 3 /l), the half-maximal concentration of FVIII required to accelerate VWF proteolysis by ADAMTS13 reduces by ϳ10-fold (to ϳ0.3 nM) compared with that in the absence of platelets (ϳ3.0 nM). Further studies using the FVIII derivative that lacks an acidic region (a3), an antiplatelet glycoprotein 1b␣ IgG, and a purified recombinant VWF-A1 domain or glycoprotein 1b␣-stripped platelets demonstrate that the synergistic rate-enhancing effect of FVIII and platelets depends on their specific binding interactions with VWF. Our findings suggest that FVIII and platelets are cofactors that regulate proteolysis of multimeric VWF by ADAMTS13 under physiological conditions. ADAMTS13, a member of the A Disintegrin And Metalloprotease with ThromboSpondin type repeats (ADAMTS)2 family (1, 2), controls the sizes of von Willebrand factor (VWF) by cleaving VWF at the Tyr 1605 -Met 1606 bond in the central A2 domain (3,4). This proteolytic cleavage appears to be critical for regulating VWF adhesive function and maintaining normal hemostasis (5). The inability to cleave the ultralarge (UL) VWF into the smaller forms due to a hereditary (1, 6 -8) or acquired deficiency (9 -11) of plasma ADAMTS13 results in thrombotic thrombocytopenic purpura, a potentially fatal thrombotic microangiopathy (5, 12, 13). Conversely, excessive proteolytic cleavage of plasma multimeric VWF by ADAMTS13 leads to a certain subtype of von Willebrand disease (5, 14), the most common bleeding disorder seen in the hematology clinic.Proteolytic processing of UL-VWF by ADAMTS13 appears to occur at least at two different sites: one is on the endothelial cells where UL-VWF is newly released from Weibel-Palade bodies upon stimulation (15,16), and the other may occur in solution (or blood) (3, 10). The cleavage of the cell membraneanchored UL-VWF by ADAMTS13 occurs very rapidly and requires low (15-17) or almost no shear stress (18, 19). However, the cleavage of UL-VWF on the endothelial cell membrane does not appear to be sufficient to reduce the VWF multimer sizes that have been observed in plasma. This is inferred from our study on cultured endothelial cells. The soluble VWF cleaved from the endothelial cell membrane by ADAMTS13 remains ultralarge in size, and the multimer distribution is not different from that released from Weibel-Palade...

Summary Decrease of plasma activity of ADAMTS13, a metalloenzyme that cleaves von Willebrand factor (VWF) and prevents adhesion and aggregation of platelets, has been reported early after onset of systemic inflammation resulting from infections and after severe trauma. Here, we determined whether trauma-induced systemic (sterile) inflammation would be associated with a reduction of plasma ADAMTS13 activity in pediatric patients and its association with disease severity and outcome. Pediatric patients (n=106) with severe trauma at a level 1 pediatric trauma center between 2014 and 2016 were prospectively enrolled. Blood samples were collected upon arrival and at 24 hours and analyzed for plasma levels of ADAMTS13 activity, VWF antigen, collagen binding activity, human neutrophil peptides (HNP) 1-3, coagulation abnormalities, endothelial glycocalyx damage, and clinical outcome. Plasma samples were also collected for similar measurements from 52 healthy pediatric controls patients who underwent elective minor surgery. The median patient age was 9 years with 81% sustaining blunt trauma. The median injury severity score was 22 and the mortality rate was 11%. Plasma levels of ADAMTS13 activity were significantly lower and plasma levels of VWF antigen and HNP1-3 proteins were significantly higher for pediatric trauma patients on admission and at 24 hours when compared to controls. Finally, the lowest plasma ADAMTS13 activity was found in patients who died from their injuries. We conclude that relative plasma deficiency of ADAMTS13 activity may be associated with more severe traumatic injury, significant endothelial glycocalyx damage, coagulation abnormalities and mortality after severe trauma in pediatric patients.

Deficiency of ADAMTS13 results in thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective treatment to date. We show in the present study that an administration of a self-inactivating lentiviral vector encoding human full-length ADAMTS13 and a variant truncated after the spacer domain (MDTCS) in mice by in utero injection at embryonic days 8 and 14 resulted in detectable plasma proteolytic activity (~5–70%), which persisted for the length of the study (up to 24 weeks). Intravascular injection via a vitelline vein at E14 was associated with significantly lower rate of fetal loss than intra-amniotic injection, suggesting that the administration of vector at E14 may be a preferred gestational age for vector delivery. The mice expressing ADAMTS13 and MDTCS exhibited reduced sizes of von Willebrand factor compared to the Adamts13−/− mice expressing eGFP. Moreover, the mice expressing both ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time as compared to the Adamts13−/− expressing eGFP. The data demonstrate the successful correction of the prothrombotic phenotypes in Adamts13−/− mice by a single in utero injection of lentiviral vectors encoding human ADAMTS13 genes, providing the basis for developing a gene therapy for hereditary TTP in humans.

Purpose of review ADAMTS13 is a zinc-containing metalloprotease that cleaves von Willebrand factor (VWF). Deficiency of plasma ADAMTS13 activity is accountable for a potentially fatal blood disorder thrombotic thrombocytopenic purpura (TTP). Understanding of ADAMTS13–VWF interaction is essential for developing novel treatments to this disorder. Recent findings Despite the proteolytic activity of ADAMTS13 being restricted to the metalloprotease domain, the ancillary proximal C-terminal domains including the disintegrin domain, first TSP-1 repeat, cysteine-rich region, and spacer domain are all required for cleavage of VWF and its analogs. Recent studies have added to our understandings of the role of the specific regions in the disintegrin domain, the cysteine-rich domain, and the spacer domain responsible for its interaction with VWF. Additionally, regulative functions of the distal portion of ADAMTS13 including the TSP-1 2–8 repeats and the CUB domains have been proposed. Finally, fine mapping of anti-ADAMTS13 antibody epitopes have provided further insight into the essential structural elements in ADAMTS13 for VWF binding and the mechanism of autoantibody-mediated TTP. Summary Significant progress has been made in our understandings of the structure–function relationship of ADAMTS13 in the past decade. To further investigate ADAMTS13–VWF interactions for medical applications, these interactions must be studied under physiological conditions in vivo.