N-Acetylcysteine for thrombotic thrombocytopenic purpura: is a von Willebrand factor-inhibitory dose feasible in vivo?

To examine whether enhanced in vivo proteolysis of von Willebrand factor (vWF) would account for the reported loss of larger multimers in acute thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS), we studied eight patients with acute TTP/HUS whose blood samples were collected into an anticoagulant containing a cocktail of protease inhibitors to impede in vitro proteolysis. In all, enhanced proteolytic degradation of vWF was expressed as a relative decrease in the intact 225-Kd subunit of vWF and a relative increase in the 176-Kd fragment. However, instead of the loss of larger forms of normal multimers reported by other investigators, the plasma of all but one of our patients (whether they had TTP or HUS) contained a set of larger than normal (supranormal) multimers. Hence, although proteolytic fragmentation of vWF was enhanced during acute TTP/HUS, this phenomenon was not associated with the loss of larger multimers. In the five patients who survived the acute disease and underwent plasma exchange (three with HUS and two with chronic relapsing TTP), subunits and fragments returned to normal values, and supranormal multimers were no longer detected in plasma. In conclusion, even though vWF proteolysis is enhanced in acute TTP/HUS, it does not lead to loss of larger multimers.

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Increased Fragmentation of von Willebrand Factor, Due to Abnormal Cleavage of the Subunit, Parallels Disease Activity in Recurrent Hemolytic Uremic Syndrome and Thrombotic Thrombocytopenic Purpura and Discloses Predisposition in Families

Blood ◽

10.1182/blood.v94.2.610 ◽

1999 ◽

Vol 94 (2) ◽

pp. 610-620 ◽

Cited By ~ 36

Author(s):

Miriam Galbusera ◽

Marina Noris ◽

Chiara Rossi ◽

Silvia Orisio ◽

Jessica Caprioli ◽

...

Keyword(s):

Hemolytic Uremic Syndrome ◽

Thrombotic Thrombocytopenic Purpura ◽

Acute Phase ◽

Von Willebrand Factor ◽

Thrombocytopenic Purpura ◽

Uremic Syndrome ◽

Sporadic Cases ◽

Von Willebrand ◽

Willebrand Factor

Abstract We investigated here the changes in von Willebrand factor (vWF) multimers in recurrent, sporadic and familial forms of hemolytic uremic syndrome (HUS)/thrombotic thrombocytopenic purpura (TTP) to see whether they are actually proteolyzed in vivo in these patients. Molecular determinants of fragments in vWF were also characterized to identify possible sites of cleavage of the subunit. Unusually large vWF multimers were found in blood of 8 of 10 patients with recurrent HUS/TTP, both in the acute phase and in remission, but never in familial and sporadic cases. Instead, all of the groups showed evidence of enhanced fragmentation of vWF multimers during the acute phase. Increased fragmentation was also shown by decrease in native 225-kD vWF subunit. In recurrent and sporadic HUS/TTP, enhanced fragmentation normalized at remission, but the abnormality persisted in familial HUS/TTP patients. The latter findings suggest that patients with familial HUS/TTP may have a congenital abnormality in vWF processing. Analysis with specific monoclonal antibodies showed the presence of the normal vWF fragments with apparent molecular mass of 189, 176, and 140 kD in all patients; however, in 6 recurrent and in 5 familial cases, novel fragments that differed in size from normal ones were found. The size of these abnormal fragments differed from one patient to another and none of them was ever found in normal plasma. These results documented, for the first time in HUS/TTP, an abnormal cleavage of the vWF subunit that might account for the increased fragmentation observed in these patients.

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Increased Fragmentation of von Willebrand Factor, Due to Abnormal Cleavage of the Subunit, Parallels Disease Activity in Recurrent Hemolytic Uremic Syndrome and Thrombotic Thrombocytopenic Purpura and Discloses Predisposition in Families

Blood ◽

10.1182/blood.v94.2.610.414k22_610_620 ◽

1999 ◽

Vol 94 (2) ◽

pp. 610-620 ◽

Cited By ~ 1

Author(s):

Miriam Galbusera ◽

Marina Noris ◽

Chiara Rossi ◽

Silvia Orisio ◽

Jessica Caprioli ◽

...

Keyword(s):

Hemolytic Uremic Syndrome ◽

Thrombotic Thrombocytopenic Purpura ◽

Acute Phase ◽

Von Willebrand Factor ◽

Thrombocytopenic Purpura ◽

Uremic Syndrome ◽

Sporadic Cases ◽

Von Willebrand ◽

Willebrand Factor

We investigated here the changes in von Willebrand factor (vWF) multimers in recurrent, sporadic and familial forms of hemolytic uremic syndrome (HUS)/thrombotic thrombocytopenic purpura (TTP) to see whether they are actually proteolyzed in vivo in these patients. Molecular determinants of fragments in vWF were also characterized to identify possible sites of cleavage of the subunit. Unusually large vWF multimers were found in blood of 8 of 10 patients with recurrent HUS/TTP, both in the acute phase and in remission, but never in familial and sporadic cases. Instead, all of the groups showed evidence of enhanced fragmentation of vWF multimers during the acute phase. Increased fragmentation was also shown by decrease in native 225-kD vWF subunit. In recurrent and sporadic HUS/TTP, enhanced fragmentation normalized at remission, but the abnormality persisted in familial HUS/TTP patients. The latter findings suggest that patients with familial HUS/TTP may have a congenital abnormality in vWF processing. Analysis with specific monoclonal antibodies showed the presence of the normal vWF fragments with apparent molecular mass of 189, 176, and 140 kD in all patients; however, in 6 recurrent and in 5 familial cases, novel fragments that differed in size from normal ones were found. The size of these abnormal fragments differed from one patient to another and none of them was ever found in normal plasma. These results documented, for the first time in HUS/TTP, an abnormal cleavage of the vWF subunit that might account for the increased fragmentation observed in these patients.

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In-vitro and in-vivo consequences of mutations in the von Willebrand factor cleaving protease ADAMTS13 in thrombotic thrombocytopenic purpura

Thrombosis and Haemostasis ◽

10.1160/th06-05-0236 ◽

2006 ◽

Vol 96 (10) ◽

pp. 454-464 ◽

Cited By ~ 51

Author(s):

Roberta Donadelli ◽

Federica Banterla ◽

Miriam Galbusera ◽

Cristina Capoferri ◽

Sara Bucchioni ◽

...

Keyword(s):

Thrombotic Thrombocytopenic Purpura ◽

Von Willebrand Factor ◽

Protease Activity ◽

Mammalian Cells ◽

Expression System ◽

Thrombocytopenic Purpura ◽

Von Willebrand ◽

Willebrand Factor

SummaryThrombotic thrombocytopenic purpura (TTP) is a disease characterized by microvascular thrombosis, often associated with deficiency of the von Willebrand factor (VWF) cleaving protease ADAMTS13.We investigated the spectrum of ADAMTS13 gene mutations in patients with TTP and congenital ADAMTS13 deficiency to establish the consequences on ADAMTS13 processing and activity. We describe five missense (V88M, G1239V, R1060W, R1123C and R1219W), 1 nonsense (W1016Stop) and 1 insertion (82_83insT) mutations. In two patients no mutation was identified despite undetectable protease activity. Expression in HEK293 mammalian cells (V88M, G1239V, R1123C and R1219W) documented that three missense mutants were not secreted, whereas theV88M was secreted at low levels and with reduced activity. We also provide evidence that impaired secretion of ADAMTS13 mutants observed in vitro translates into severely reduced ADAMTS13 antigen levels in patients in vivo. To evaluate whether the small amounts of mutant protease present in the circulation of patients had VWF cleaving activity, WT and mutant rADAMTS13 were stably expressed in Drosophila S2 cells under the influence of the Drosophila BiP protein signal sequence, which allows protein secretion. Drosophila expression system showed a 40–60% protease activity in the mutants. Several single nucleotide polymorphisms (SNPs) within exons and intron boundaries were found in patients, suggesting that the interplay of SNPs could at least in part account for ADAMTS13 functional abnormalities in patients without mutations. In conclusion, defective secretion and impaired activity of the mutants concur to determine an almost complete deficiency of ADAMTS13 activity in patients with a homozygous or two heterozygous ADAMTS13 mutations.

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Enhanced proteolysis of plasma von Willebrand factor in thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome

Blood ◽

10.1182/blood.v74.3.978.978 ◽

1989 ◽

Vol 74 (3) ◽

pp. 978-983 ◽

Cited By ~ 5

Author(s):

PM Mannucci ◽

R Lombardi ◽

A Lattuada ◽

P Ruggenenti ◽

GL Vigano ◽

...

Keyword(s):

Hemolytic Uremic Syndrome ◽

Thrombotic Thrombocytopenic Purpura ◽

Von Willebrand Factor ◽

Relative Increase ◽

Thrombocytopenic Purpura ◽

Uremic Syndrome ◽

Von Willebrand ◽

Willebrand Factor

Abstract To examine whether enhanced in vivo proteolysis of von Willebrand factor (vWF) would account for the reported loss of larger multimers in acute thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS), we studied eight patients with acute TTP/HUS whose blood samples were collected into an anticoagulant containing a cocktail of protease inhibitors to impede in vitro proteolysis. In all, enhanced proteolytic degradation of vWF was expressed as a relative decrease in the intact 225-Kd subunit of vWF and a relative increase in the 176-Kd fragment. However, instead of the loss of larger forms of normal multimers reported by other investigators, the plasma of all but one of our patients (whether they had TTP or HUS) contained a set of larger than normal (supranormal) multimers. Hence, although proteolytic fragmentation of vWF was enhanced during acute TTP/HUS, this phenomenon was not associated with the loss of larger multimers. In the five patients who survived the acute disease and underwent plasma exchange (three with HUS and two with chronic relapsing TTP), subunits and fragments returned to normal values, and supranormal multimers were no longer detected in plasma. In conclusion, even though vWF proteolysis is enhanced in acute TTP/HUS, it does not lead to loss of larger multimers.

Download Full-text