Background-Thrombotic thrombocytopenic purpura is a potentially fatal disease characterized by widespread platelet thrombi in the microcirculation. In the normal circulation, von Willebrand factor is cleaved by a plasma protease. We explored the hypothesis that a deficiency of this protease predisposes patients with thrombotic thrombocytopenic purpura to platelet thrombosis.

von Willebrand factor (vWF) in the circulation is subjected to proteolysis. In a recent study, we reported that normal plasma contains a protease activity that cleaves vWF in a shear-dependent manner, causing a decrease in its multimer size while generating dimers of the 140-kD and the 176-kD fragments indistinguishable from those found in normal plasma. In this study, the plasma protease has been partially purified and characterized and the role of vWF conformation in its cleavage by the protease has been further investigated. Guanidine HCl caused unfolding of vWF in a concentration-dependent manner, resulting in a shift in its fluorescence emission maxima to longer wavelengths. A dramatic increase in its proteolytic susceptibility was seen at 1.1 to 1.2 mol/L guanidine HCl, a concentration causing only a 3- to 4-nm shift in vWF emission maxima. Although vWF molecules refolded as guanidine HCl was removed by dialysis, the refolding was accompanied only by a partial recovery of the proteolytic resistance. The plasma protease, partially purified by approximately 900 folds by Sephacryl S- 300 HR gel filtration, Matrex gel orange A dye affinity chromatography, and Q Sepharose anion exchange, had a molecular mass of approximately 200 kD and was inhibited by EDTA, EGTA, or 1,10-phenanthroline. The inhibition by EGTA or EDTA could be reversed by Ca2+ but not by mg2+. It was not inhibited by a panel of synthetic and natural protease inhibitors or adsorbed by gelatin-agarose, and it was present in plasmas deficient in proteins involved in coagulation and anticoagulation. The vWF fragments generated by the protease, as mapped by peptide-specific antibodies VP-1 and LJ-7745, were in distinguishable from the natural fragments but distinct from those produced by plasmin. High molecular weight endothelial vWF, after exposure to guanidine HCLI or high shear stress, was cleaved by the protease to smaller forms. These results support the model that endothelial secreted vWF is converted to multimers by a novel plasma metalloproteinase. Although native vWF exists in a conformation relatively resistant to cleavage, an alteration in the conformation by shear stress can lead to enhanced proteolytic susceptibility. This model may explain the decrease in vWF multimer sizes in various clinical conditions.

Thrombotic thrombocytopenic purpura (TTP) is a disorder with characteristic von Willebrand factor (VWF)-rich microthrombi affecting the arterioles and capillaries of multiple organs. The disorder frequently leads to early death unless the patients are treated with plasma exchange or infusion. Studies in the last decade have provided ample evidence to support that TTP is caused by deficiency of a plasma metalloprotease, ADAMTS13. When exposed to high shear stress in the microcirculation, VWF and platelets are prone to form aggregates. This propensity of VWF and platelet to form microvascular thrombosis is mitigated by ADAMTS13, which cleaves VWF before it is activated by shear stress to cause platelet aggregation in the circulation. Deficiency of ADAMTS13, due to autoimmune inhibitors in patients with acquired TTP and mutations of the ADAMTS13 gene in hereditary cases, leads to VWF-platelet aggregation and microvascular thrombosis of TTP. In this review, we discuss the current knowledge on the pathogenesis, diagnosis and management of TTP, address the ongoing controversies, and indicate the directions of future investigations.

While von Willebrand factor (vWF) is secreted from endothelial cells as a very large polymer, it circulates as a series of multimers that are reducible to a 225-kD polypeptide and three proteolytic fragments of 189, 176, and 140 kD. Cleavage at the Tyr-842/Met-843 bond of the vWF polypeptide creates the 140- and 176-kD fragments. In the process of understanding vWF multimer formation, the role of shear stress in vWF proteolysis was investigated in this study. A shear-rate-dependent loss of the largest multimers was observed when normal plasma was perfused through long capillary tubings achieving shear rates normally encountered in the circulation. The shear-dependent vWF change was not observed when purified vWF or normal plasma containing calcium chelator EGTA or EDTA was perfused. As the large multimers decreased, an increase in the smaller multimers, including 200- and 350-kD bands, was detected. Elution and immunoblotting studies with peptide-specific antibodies LJ-7745 and VP-1 showed that the 200-kD band was a dimer of the 140-kD fragment, whereas the 350-kD band was a dimer of the 176-kD fragment. When analyzed after disulfide bonds were reduced, sheared plasma showed an increase in the 176- and 140-kD fragments, but not the 189-kD fragment. Finally, shearing of purified vWF enhanced its proteolytic cleavage when it was subsequently incubated with the cryosupernatant fraction of normal plasma or with cathepsin G, a leukocyte granule serine protease. These results show that shear stress is capable of enhancing the susceptibility of vWF to proteolytic cleavage. It promotes vWF proteolysis in normal plasma at a site that generates the 140-kD/176-kD fragments, leading to a decrease in multimer size. Shear stress might be involved in modulating the size of vWF in the circulation.

Hemolytic uremic syndrome (HUS) usually occurs after infection with Shiga toxin-producing bacteria. Thrombotic thrombocytopenic purpura, a disorder with similar clinical manifestations, is associated with deficient activity of a circulating metalloprotease that cleaves von Willebrand factor at the Tyr842-Met843 peptide bond in a shear stress-dependent manner. We analyzed von Willebrand factor-cleaving metalloprotease activity and the status of von Willebrand factor in 16 children who developed HUS after Escherichia coli O157:H7 infection and in 29 infected children who did not develop this complication. Von Willebrand factor-cleaving metalloprotease activity was normal in all subjects, but von Willebrand factor size was decreased in the plasma of each of 16 patients with HUS. The decrease in circulating von Willebrand factor size correlated with the severity of thrombocytopenia and was proportional to an increase in von Willebrand factor proteolytic fragments in plasma. Immunohistochemical studies of the kidneys in four additional patients who died of HUS demonstrated glomerular thrombi in three patients, and arterial and arteriolar thrombi in one patient. The glomerular thrombi contained fibrin but little or no von Willebrand factor. A decrease in large von Willebrand factor multimers, presumably caused by enhanced proteolysis from abnormal shear stress in the microcirculation, is common in HUS. HUS, characterized by acute renal failure, hemolysis with schistocytes on blood smears, and thrombocytopenia, is accompanied by thrombotic microangiopathy of the kidneys and of other organs (1). The syndrome covers a diverse spectrum of microangiopathic disorders (2-4), but most cases occur after infection with Shiga toxin-producing bacteria, such as Escherichia coli O157:H7 (5) or Shigella dysenteriae serotype 1 (6).TTP, a disorder with some clinical, laboratory, and histopathologic similarities to HUS, has been associated with abnormal homeostasis of von Willebrand factor, a protein that is secreted from endothelial cells as a disulfide-linked polymer of a polypeptide with 2050 amino acid residues. Circulating von Willebrand factor is normally cleaved between Tyr842 and Met843 (7) in a shear stress-dependent manner (8) by a plasma metalloprotease (9, 10), generating a series of multimers. Without this metalloprotease activity, von Willebrand factor, when unfolded by shear stress (11), has increased platelet-aggregating activity (12). It is postulated that this increased activity facilitates the formation of arteriolar and capillary platelet thrombi in TTP. Indeed, acquired TTP has been associated with deficient von Willebrand factor-cleaving metalloprotease activity caused by inhibitory antibodies (12, 13).

Abstract. Thrombotic thrombocytopenic purpura (TTP) and the hemolytic uremic syndrome (HUS) are both characterized by thrombocytopenia, microangiopathic hemolysis, and organ dysfunction. Other disorders occasionally present with similar manifestations. Recent studies have demonstrated that deficiency in the von Willebrand factor cleaving protease AD-AMTS13, due to genetic mutations or autoimmune inhibitors, causes TTP. Molecular cloning of ADAMTS13 elucidates the structure of the protease, raising the prospect for advances in diagnosis and treatment of the disease. Assay of ADAMTS13 activity distinguishes TTP from HUS and other types of thrombotic microangiopathy (TMA); therefore, the term TTP/HUS should be avoided because it obscures the known or potential differences among the various types of TMA.

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Thrombotic microangiopathy, which includes thrombotic thrombocytopenic purpura (TTP), shiga-toxin-associated hemolytic uremic syndrome (Stx-HUS) and atypical HUS, is characterized by the development of hyaline thrombi in the microvasculature resulting in thrombocytopenia, microangiopathic hemolysis, and organ dysfunction. Renal failure is a predominant complication of both Stx-HUS and atypical HUS, whereas neurological complications are more prominent in TTP. Other disorders such as lupus or bone marrow transplantations may occasionally present with features of thrombotic microangiopathy. Recent studies have found autoimmune inhibitors or genetic mutations of a von Willebrand factor (VWF) cleaving metalloprotease ADAMTS13 in patients with TTP. In approximately 30-50% of patients with atypical HUS, mutations have been detected in complement factor H, membrane cofactor protein (CD46), or factor I. All three proteins are involved in the regulation of complement activation. Additionally, autoantibodies of factor H have been described in patients without genetic mutations. These advances illustrate that dysregulation of VWF homeostasis or complement activation owing to genetic or autoimmune mechanisms may lead to the syndrome of thrombotic microangiopathy.