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...

In a heterogeneous population of TMA patients treated with plasma exchange, ADAMTS13 activity defined two subpopulations with distinct clinical and laboratory features. These results suggest that TMA with severe ADAMTS13 deficiency is a distinct pathologic process.

Thrombotic microangiopathy (TM) is associated with abnormalities of von Willebrand factor-cleaving protease (VWCP) and other hemostatic factors. This study hypothesized that TM patients might have genetically determined thrombotic risk factors that predispose them to aberrant microvascular thrombosis. DNA samples from 30 white and 12 African American adult TM patients were analyzed for genetic alleles associated with vascular thrombosis, and plasma samples were analyzed for levels of VWCP activity. DNA was analyzed by using allele-specific polymerase chain reaction for factor V 1691A (Leiden), factor II 20 210A, methylenetetrahydrofolate reductase 667T, type 1 plasminogen activator inhibitor 4G/5G, and platelet GPIa 807T. Patients were segregated by race (white or African American) and plasma level of VWCP activity (normal or deficient). The prevalence of factor V Leiden was significantly increased among the white TM patients that had normal VWCP activity: 4 (36%) of 11 patients compared with 6 (3%) of 186 white control subjects possessed the factor V Leiden allele (P < .001; odds ratio, 17. IntroductionThrombotic microangiopathy (TM) disorders, including thrombotic thrombocytopenic purpura (TTP) and the hemolytic uremic syndrome (HUS), are characterized by widespread microvascular thrombosis leading to end-organ injury. The etiologies and pathogenic mechanisms of TM syndromes remain poorly understood. However, evidence implicates a deficiency of von Willebrand factor (VWF)-cleaving protease (VWCP) activity in the pathogenesis of TM in patients with the clinical diagnosis of TTP. 1,2 Deficiency of VWCP activity has been proposed to impair proteolytic processing of ultralarge endothelial cell-derived VWF multimers, resulting in a predisposition to platelet thrombosis. 2,3 Deficient VWCP activity provides support for a platelet-VWFdependent mechanism of microvascular thrombosis in a subset of TM patients. However, the mechanism of microvascular thrombosis in TM patients with normal levels of VWCP activity remains obscure. Patients who have TM associated with bone marrow transplantation 4 and enteropathic Escherichia coli infection, 5 as well as some patients with idiopathic TM, 6 have been reported to have normal VWCP activity. Microvascular thrombosis in these TM patients might involve a wide range of hemostatic abnormalities, and the syndromic nature of TM suggests the possibility of multiple pathogenic factors.An increasing number of genetic mutations and polymorphisms have been found to be associated with various manifestations of vascular thrombosis. The implicated mutant or polymorphic alleles affect hemostatic mechanisms by encoding variant proteins or altered regulatory sequences that have the potential to enhance thrombus formation or decrease fibrinolysis. We hypothesized that thrombosis-associated mutations or polymorphisms may contribute to the pathogenic mechanisms of TM disorders. Moreover, because certain hemostatic abnormalities, such as deficient VWCP activity, are found in some TM patients but n...