Proteolysis of von Willebrand factor is decreased in acute promyelocytic leukaemia by treatment with all-trans-retinoic acid

Plasma von Willebrand factor (VWF) was investigated in five patients with acute promyelocytic leukaemia (APL) before and after administration of the differentiating agent all-trans-retinoic acid (ATRA). The purpose of the study was to see how the proteolytic state associated with APL affects VWF structure and function and whether ATRA reverses any abnormality. At the onset of APL, multimeric analysis of plasma VWF revealed a lack of the largest multimers. After ATRA, there was a progressive correction of the multimeric pattern in all cases, with transient appearance of ultralarge multimers in two cases. Proteolysis was investigated with immunopurified and reduced VWF from each patient's plasma. This was electrophoresed and probed with two monoclonal antibodies that identify the 225 kD native subunit and the three native fragments of 189, 176 and 140 kD and differentiate novel proteolytic fragments produced by different proteinases. At the onset of APL, the 225 kD native subunit was relatively decreased, with the appearance of an array of novel VWF proteolytic fragments, ranging in size from <140 to <225 kD. These novel fragments observed in patients were similar to those produced in vitro by digestion of purified VWF with plasmin or elastase. After ATRA therapy, proteolysis diminished progressively in parallel with the improvement of other haemostatic measurements, but persisted to some extent. We conclude that VWF proteolysis in APL is produced by plasmin and elastase. Changes of VWF structure and function might adversely affect haemostasis in APL. Therefore, improvement of VWF after ATRA administration might explain in part the effectiveness of this drug in reducing haemorrhagic complications.     

Diagnosis and follow-up of thrombotic thrombocytopenic purpura by means of von Willebrand factor collagen binding assay.

Thrombotic thrombocytopenic purpura (TTP) is characterized by intravascular thrombosis leading to consumption of large or unusually large von Willebrand factor (VWF) multimers. The usefulness of VWF collagen binding (VWF:CB) assay was assessed in detecting the decrease/absence of large VWF multimers or the presence of abnormally large forms in patients with TTP. Nine patients with TTP were studied during the acute phase of the disorder and the absence of large VWF multimers was demonstrated by means of the VWF:CB assay. These findings were confirmed by VWF multimer pattern analysis; VWF:CB deficiency appeared to correlate with abnormalities in large VWF multimers. The diagnostic potency of VWF:CB was especially evident when the values were expressed as VWF:CB/VWF:Ag ratio. VWF:CB was also used during the follow-up of the disorder to document improvement or restoration of large VWF multimers. VWF:CB was able to detect the absence or decrease of large VWF multimers better than VWF ristocetin cofactor (VWF:RCo); in fact, VWF:CB was defective when large VWF multimers persisted to be decreased, in contrast with what observed with VWF:RCo. In conclusion, VWF:CB is a simple test that appears to be useful, together with clinical symptoms and reduced platelet count, for the diagnosis and follow-up of TTP.     

Lack of multimer organization of von Willebrand factor in an acquired von Willebrand syndrome

We report a case of acquired von Willebrand syndrome (AVWS) in a 20-year-old-woman with systemic lupus erythematosus, in whom severe bleeding complications followed kidney biopsy. Coagulation studies demonstrated undetectable levels of ristocetin-induced platelet aggregation (RIPA), von Willebrand factor antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo), associated with significantly prolonged bleeding time; unlike type 3 von Willebrand disease (VWD), platelet VWF was reduced but not undetectable. The plasma VWF multimer pattern was characterized by the presence of only two bands, one of low molecular weight (MW) running as the protomer of plasma VWF in normals, the other of abnormally high MW without detectable intermediate multimers; this pattern resembles that of VWF present in endothelial cells. A search for an anti-VWF antibody demonstrated the presence of an inhibitor at high titre. This anti-VWF antibody did not interfere in the interaction of VWF with platelet glycoprotein (GP) Ib through the A1 domain, and did not react with the A2 domain of VWF; instead, it seemed to modify the relative representation of high and low MW VWF multimers released by normal human umbilical vein endothelial cells (HUVEC). After Azathioprine and corticosteroid treatment, the anti-VWF antibody disappeared and the patient's haemostatic profile normalized, except for the platelet VWF content which still remained decreased. We suggest that the anti-VWF antibody present in the AVWS described compromised both circulating VWF levels and their multimeric organization, inducing the maintenance of the multimer structure that VWF normally has before or in the early phase after secretion from endothelial cells.     

Reduced survival of type 2B von Willebrand factor, irrespective of large multimer representation or thrombocytopenia

Background

Type 2B von Willebrand factor (VWF) is characterized by gain of function mutations in the A1 domain inducing a greater affinity for platelet GPIb, possibly associated with the disappearance of large VWF multimers and thrombocytopenia.

Design and Methods

VWF survival was explored using 1-desamino-8-D-arginine vasopressin (DDAVP) in 18 patients with type 2B von Willebrand disease (VWD) and compared with their platelet count and large VWF multimer representation.

Results

A similarly significant shorter VWF survival, expressed as T_1/2elimination (T_1/2el), was observed in patients lacking large VWF multimers (type 2B) and in those with a normal multimer pattern (atypical type 2B) (4.47±0.41 h and 4.87±0.9 h, respectively, vs. normal 15.53±2.17 h) due mainly to a greater VWF clearance. The half-life of large VWF multimers, explored by VWF collagen binding (VWF:CB) activity, was likewise reduced. The similarly reduced VWF half-life was also confirmed by the increase in the VWF propeptide ratio (a useful tool for exploring VWF survival) which was found to be the same in type 2B and atypical type 2B patients. The post-DDAVP drop in platelet count occurred in all patients lacking large multimers but not in those with a normal multimer pattern. A correlation was always found between pre- and/or post-DDAVP thrombocytopenia and the lack of large VWF multimers in type 2B VWD while these were unrelated to the reduced VWF half-life.

Conclusions

In addition to demonstrating that a shorter VWF survival contributes to the type 2B and atypical type 2B VWD phenotype, our findings suggest that VWF clearance and proteolysis are independent phenomena.     

Intersection of mechanisms of type 2A VWD through defects in VWF multimerization, secretion, ADAMTS-13 susceptibility, and regulated storage

Type 2A VWD is characterized by the absence of large VWF multimers and decreased platelet-binding function. Historically, type 2A variants are subdivided into group 1, which have impaired assembly and secretion of VWF multimers, or group 2, which have normal secretion of VWF multimers and increased ADAMTS13 proteolysis. Type 2A VWD patients recruited through the T. S. Zimmerman Program for the Molecular and Clinical Biology of VWD study were characterized phenotypically and potential mutations identified in the VWF D2, D3, A1, and A2 domains. We examined type 2A variants and their interaction with WT-VWF through expression studies. We assessed secretion/intracellular retention, multimerization, regulated storage, and ADAMTS13 proteolysis. Whereas some variants fit into the traditional group 1 or 2 categories, others did not fall clearly into either category. We determined that loss of Weibel-Palade body formation is associated with markedly reduced secretion. Mutations involving cysteines were likely to cause abnormalities in multimer structure but not necessarily secretion. When coexpressed with wild-type VWF, type 2A variants negatively affected one or more mechanisms important for normal VWF processing. Type 2A VWD appears to result from a complex intersection of mechanisms that include: (1) intracellular retention or degradation of VWF, (2) defective multimerization, (3) loss of regulated storage, and (4) increased proteolysis by ADAMTS13.     

Impact of mutations in the von Willebrand factor A2 domain on ADAMTS13-dependent proteolysis

Classical von Willebrand disease (VWD) type 2A, the most common qualitative defect of VWD, is caused by loss of high-molecular-weight multimers (HMWMs) of von Willebrand factor (VWF). Underlying mutations cluster in the A2 domain of VWF around its cleavage site for ADAMTS13. We investigated the impact of mutations commonly found in patients with VWD type 2A on ADAMTS13-dependent proteolysis of VWF. We used recombinant human ADAMTS13 (rhuADAMTS13) to digest recombinant full-length VWF and a VWF fragment spanning the VWF A1 through A3 domains, harboring 13 different VWD type 2A mutations (C1272S, G1505E, G1505R, S1506L, M1528V, R1569del, R1597W, V1607D, G1609R, I1628T, G1629E, G1631D, and E1638K). With the exception of G1505E and I1628T, all mutations in the VWF A2 domain increased specific proteolysis of VWF independent of the expression level. Proteolytic susceptibility of mutant VWF in vitro closely correlated with the in vivo phenotype in patients. The results imply that increased VWF susceptibility for ADAMTS13 is a constitutive property of classical VWD type 2A, thus explaining the pronounced proteolytic fragments and loss of HMWM seen in multimer analysis in patients.     

ADAMTS13 activity in sickle cell disease

Sickle red blood cell (SRBC)-endothelial adhesion plays a central role in sickle cell disease (SCD)-related vaso-occlusion. As unusually large von Willebrand factor (ULVWF) multimers mediate SRBC-endothelial adhesion, we investigated the activity of ADAMTS13, the metalloprotease responsible for cleaving ULVWF multimers, in SCD. ADAMTS13 activity was determined using a quantitative immunoblotting assay. VWF:Ag and VWF:RCo were determined using commercial assays. The high-molecular-weight VWF multimer percentage was determined by employing gel electrophoresis. ADAMTS13 activity was similar among asymptomatic patients (n = 8), patients at presentation with a painful crisis (n = 23), and healthy controls. ADAMTS13/VWF:Ag ratios were lower in patients compared to healthy HbAA controls, with the lowest values at presentation with a painful crisis (P = 0.02). Division of samples in those with VWF:RCo/VWF:Ag ratios < 0.70 and those with ratios >or= 0.70 revealed significantly more samples with ratios >or= 0.70 (P = 0.01) collected during painful crises. ULVWF multimers were detected in 6 patient samples and in 1 control sample. ADAMTS13/VWF:Ag ratios were inversely related to the duration of symptoms at presentation with an acute vaso-occlusive event (r(s)-0.67, P = 0.002). Although SCD is characterized by elevated VWF:Ag levels, no severe ADAMTS13 deficiency was detected in our patients.     

Von Willebrand factor and ADAMTS13 in arterial thrombosis: a systematic review and meta-analysis

Von Willebrand Factor (VWF) plays an important role in hemostasis by mediating platelet adhesion and aggregation. Ultralarge VWF multimers are cleaved by ADAMTS13 in smaller, less procoagulant forms. An association between high VWF levels and cardiovascular disease has frequently been reported, and more recently also an association has been observed between low ADAMTS13 levels and arterial thrombosis. We reviewed the current literature and performed meta-analyses on the relationship between both VWF and ADAMTS13 with arterial thrombosis. Most studies showed an association between high VWF levels and arterial thrombosis. It remains unclear whether ADAMTS13 is a causal independent risk factor because the association between low ADAMTS13 and arterial thrombosis is so far only shown in case-control studies. Prospective studies are awaited. A causal role for ADAMTS13 is supported by mice studies of cerebral infarction where the infusion of recombinant human ADAMTS13 reduced the infarct size.     

Shear stress enhances the proteolysis of von Willebrand factor in normal plasma

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.     

von Willebrand factor and factor VIII are independently required to form stable occlusive thrombi in injured veins

von Willebrand factor (VWF) protects factor VIII (FVIII) from proteolysis and mediates the initial contact of platelets with the injured vessel wall, thus playing an important role in hemostasis and thrombosis. VWF is crucial for the formation of occlusive thrombi at arterial shear rates. However, with only a few conflicting studies published, the role of VWF in venous thrombosis is still unclear. Using gene-targeted mice, we show that in ferric chloride-injured veins platelet adhesion to subendothelium is decreased and thrombus growth is impaired in VWF(-/-) mice when compared with wild type (WT). We also observed increased embolization in the VWF(-/-) mice, which was due to lower FVIII levels in these mice as recombinant factor VIII (r-FVIII) restored thrombus stability. Despite normalization of blood clotting time and thrombus stability after r-FVIII infusion, the VWF(-/-) venules did not occlude. Transgenic platelets lacking the VWF receptor GPIbalpha extracellular domain showed decreased adhesion to injured veins. But, after a delay, all the injured venules occluded in these transgenic mice. Thus, VWF likely uses other adhesion receptors besides GPIbalpha in thrombus growth under venous shear conditions. Our studies document crucial roles for VWF and FVIII in experimental thrombosis under venous flow conditions in vivo.     

Multimeric composition of endothelial cell-derived von Willebrand factor

The multimeric composition of human endothelial cell (EC)-derived von Willebrand factor (vWF) was studied using SDS-agarose gel electrophoresis and autoradiography. Two multimers were found in lysates prepared from confluent cultures of human umbilical vein endothelial cells. The smaller multimer had a molecular weight (mol wt) of approximately 950 Kd, while the second was larger than those seen in plasma. When electrophoresis was performed using the discontinuous buffer system of Ruggeri and Zimmerman, the small multimer consisted of a single band migrating with the slowest-moving component of the corresponding plasma triplet. The large EC-vWF multimer was detected in culture media conditioned with EC monolayers for ten minutes. It remained the only multimer in media conditioned for up to three days. Calcium ionophore A23187 increased the amount of the large vWF multimer released into the culture media, but did not change its multimeric composition. The small multimer was never detected in the EC- conditioned media. These findings suggest that (1) a large, fully polymerized multimer of vWF is released from the ECs, while the small multimer probably represents a major intermediate component in the process of multimerization, and (2) plasma vWF multimers are probably generated from the large endothelial vWF after it is released into the circulation.     

von Willebrand factor and factor VIII as risk factors for arterial and venous thrombosis

Since the early 1990s attempts have been made to elucidate whether high concentrations of von Willebrand factor (VWF) and factor VIII (FVIII) in plasma are associated with an increased risk of thrombosis. Several prospective studies on the role of VWF in arterial thrombosis, mainly coronary heart disease, were performed in healthy individuals and patients with previous cardiovascular disease. Although the majority showed an association between high VWF levels and arterial thrombosis, others failed to confirm such findings. A smaller number of studies have evaluated FVIII, mainly for its association with venous thrombosis. Two prospective observations, together with several case-control studies, provided solid evidence of an association between high FVIII levels and a first or recurrent episode of venous thrombosis. On the whole, high levels of VWF and FVIII in plasma confer a moderately high risk of arterial and venous thrombosis, respectively. These findings have no therapeutic implication, but they should be taken into account in the assessment of the individual risk profile.     

Decreased plasma concentration of von Willebrand factor antigen (VWF:Ag) in patients with glycogen storage disease type Ia

Summary Despite highly increased blood lipids, patients with glycogen storage disease type Ia (GSD Ia) do not develop premature vascular complications. Since this could be due to changes of coagulation factors, coagulation tests (including von Willebrand factor (VWF) antigen (VWF:Ag) ELISA, VWF:collagen binding activity (VWF:CB) and VWF multimer analysis) were performed in 10 GSD Ia patients, single cases of other GSD types, and in both healthy and hyperlipidaemic controls. In 60% of GSD Ia patients we found abnormal results, with a decrease of VWF:Ag and multimer analysis showing reduced intensity of individual oligomers in the presence of all multimers with a normal triplet structure. We interpret these findings as an aquired ‘von Willebrand syndrome type I’ in GSD Ia. The underlying metabolic mechanism and a potential role in the protection from vascular complication still needs to be evaluated.     

Serial changes in von Willebrand factor-cleaving protease (ADAMTS13) and prognosis after acute myocardial infarction

Von Willebrand factor (VWF), a cofactor in platelet adhesion and aggregation, increases hemostasis and thrombosis. Recently, a metalloprotease that cleaves VWF multimers has been identified, namely ADAMTS13. The aim of this study was to investigate the relation between serial changes in plasma VWF and ADAMTS13 and the prognosis after acute myocardial infarction (AMI). We measured serial changes of plasma VWF and ADAMTS13 antigen levels in 92 patients with AMI and 40 control subjects. VWF levels were significantly higher in patients with AMI compared with controls (p <0.01) on admission, peaked 3 days after admission, and remained high for 14 days. In contrast, on admission, ADAMTS13 levels were significantly lower in patients with AMI compared with controls (p <0.0001), with minimum antigen levels reached after 3 days, and remained lower for 14 days. The ratio of VWF/ADAMTS13 antigen levels was higher in patients with AMI compared with controls throughout the time course. Cox hazards analysis revealed that the early increase of VWF and VWF/ADAMTS13 ratio levels and the early decrease of ADAMTS13 levels were significant predictors of future thrombotic events during the 1-year follow-up period. Kaplan-Meier analysis demonstrated that patients with major decreases of ADAMTS13 levels and high increases of VWF/ADAMTS13 levels had significantly greater probabilities for development of thrombotic events (p = 0.0104 and 0.0209, respectively). In conclusion, these findings suggest that monitoring the changes of VWF and ADAMTS13 antigen levels in the early phase might be valuable for predicting and preventing thrombosis during 1-year follow-up in patients with AMI.     

Shear stress-induced unfolding of VWF accelerates oxidation of key methionine residues in the A1A2A3 region

VWF is required for platelet adhesion to sites of vessel injury, a process vital for both hemostasis and thrombosis. Enhanced VWF secretion and oxidative stress are both hallmarks of inflammation. We recently showed that the neutrophil oxidant hypochlorous acid (HOCl) inhibits VWF proteolysis by ADAMTS13 by oxidizing VWF methionine 1606 (M1606) in the A2 domain. M1606 was readily oxidized in a substrate peptide, but required urea in multimeric plasma VWF. In the present study, we examined whether shear stress enhances VWF oxidation. With an HOCl-generating system containing myeloperoxidase (MPO) and H(2)O(2), we found that shear stress accelerated M1606 oxidation, with 56% becoming oxidized within 1 hour. Seven other methionine residues in the VWF A1A2A3 region (containing the sites for platelet and collagen binding and ADAMTS13 cleavage) were variably oxidized, one completely. Oxidized methionines accumulated preferentially in the largest VWF multimers. HOCl-oxidized VWF was hyperfunctional, agglutinating platelets at ristocetin concentrations that induced minimal agglutination using unoxidized VWF and binding more of the nanobody AU/VWFa-11, which detects a gain-of-function conformation of the A1 domain. These findings suggest that neutrophil oxidants will both render newly secreted VWF uncleavable and alter the largest plasma VWF forms such that they become hyperfunctional and resistant to proteolysis by ADAMTS13.     

Classification and characterization of hereditary types 2A, 2B, 2C, 2D, 2E, 2M, 2N, and 2U (unclassifiable) von Willebrand disease.

All variants of type 2 von Willebrand disease (VWD) patients, except 2N, show a defective von Willebrand factor (VWF) protein (on cross immunoelectrophoresis or multimeric analysis), decreased ratios for VWF:RCo/Ag and VWF:CB/Ag and prolonged bleeding time. The bleeding time is normal and FVIII:C levels are clearly lower than VWF:Ag in type 2N VWD. High resolution multimeric analysis of VWF in plasma demonstrates that proteolysis of VWF is increased in type 2A and 2B VWD with increased triplet structure of each visuable band (not present in types 2M and 2U), and that proteolysis of VWF is minimal in type 2C, 2D, and 2E variants that show aberrant multimeric structure of individual oligomers. VWD 2B differs from 2A by normal VWF in platelets, and increased ristocetine-induced platelet aggregation (RIPA). RIPA, which very likely reflects the VWF content of platelets, is normal in mild, decreased in moderate, and absent in severe type 2A VWD. RIPA is decreased or absent in 2M, 2U, 2C, and 2D, variable in 2E, and normal in 2N. VWD 2M is usually mild and characterized by decreased VWF:RCo and RIPA, a normal or near normal VWF multimeric pattern in a low resolution agarose gel. VWD 2A-like or unclassifiable (2U) is distinct from 2A and 2B and typically featured by low VWF:RCo and RIPA with the relative lack of high large VWF multimers. VWD type 2C is recessive and shows a characteristic multimeric pattern with a lack of high molecular weight multimers, the presence of one single-banded multimers instead of triplets caused by homozygosity or double hereozygosity for a mutation in the multimerization part of VWF gene. Autosomal dominant type 2D is rare and characterized by the lack of high molecular weight multimers and the presence of a characteristic intervening subband between individual oligimers due to mutation in the dimerization part of the VWF gene. In VWD type 2E, the large VWF multimers are missing and the pattern of the individual multimers shows only one clearly identifiable band, and there is no intervening band and no marked increase in the smallest oligomer. 2E appears to be less well defined, is usually autosomal dominant, and accounts for about one third of patients with 2A in a large cohort of VWD patients.     

The risk of venous and arterial thrombosis in hyperhomocysteinemic subjects may be a result of elevated factor VIII levels

In a large retrospective study of thrombophilic families, we analyzed 405 relatives of patients, hypothesizing that hyperhomocysteinemia and elevated factor VIII levels are closely related. Median factor VIII levels in hyperhomocysteinemic relatives were 169 IU/dL, compared with 136 IU/dL in normohomocysteinemic relatives (p =0.007), and were more often elevated (>150 IU/dL; p =0.006). Hyperhomocysteinemia was associated with an increased risk of venous and arterial thrombosis; relative risk (RR) 2.6 (CI 1.3-4.8) and 3.7 (CI 1.5-8.4) respectively. Relatives with elevated FVIII were also at risk; RR 2.3 (CI 1.4-4.0) for venous thrombosis and 2.3 (CI 1.0-5.1) for arterial thrombosis. After excluding all relatives with elevated factor VIII, RR for hyperhomocysteinemia and venous thrombosis dropped to 1.3 (CI 0.2-9.8) and no relatives had arterial thrombosis. We conclude that it is likely that the increased risk of venous and arterial thrombosis in hyperhomocysteinemia is mainly related to elevated FVIII levels.     

Unraveling the scissile bond: how ADAMTS13 recognizes and cleaves von Willebrand factor

von Willebrand factor (VWF) is a large adhesive glycoprotein with established functions in hemostasis. It serves as a carrier for factor VIII and acts as a vascular damage sensor by attracting platelets to sites of vessel injury. VWF size is important for this latter function, with larger multimers being more hemostatically active. Functional imbalance in multimer size can variously cause microvascular thrombosis or bleeding. The regulation of VWF multimeric size and platelet-tethering function is carried out by ADAMTS13, a plasma metalloprotease that is constitutively active. Unusually, protease activity of ADAMTS13 is controlled not by natural inhibitors but by conformational changes in its substrate, which are induced when VWF is subject to elevated rheologic shear forces. This transforms VWF from a globular to an elongated protein. This conformational transformation unfolds the VWF A2 domain and reveals cryptic exosites as well as the scissile bond. To enable VWF proteolysis, ADAMTS13 makes multiple interactions that bring the protease to the substrate and position it to engage with the cleavage site as this becomes exposed by shear. This article reviews recent literature on the interaction between these 2 multidomain proteins and provides a summary model to explain proteolytic regulation of VWF by ADAMTS13.     

Prospective study on the behaviour of the metalloprotease ADAMTS13 and of von Willebrand factor after bone marrow transplantation

Thrombotic microangiopathies (TMAs) are rare but serious complications of bone marrow transplantation (BMT). Clinical manifestations are similar to those of thrombotic thrombocytopenic purpura (TTP), but prognosis is generally poorer despite plasma exchange. The enzymatic activity of the plasma metalloprotease ADAMTS13, which cleaves ultralarge thrombogenic multimers of von Willebrand factor (VWF) derived from activated endothelial cells, is very low or undetectable in patients with classic TTP, and protease deficiency is thought to play a mechanistic role in the formation of platelet thrombi in the microcirculation. This is the first prospective study to evaluate the incidence of TMA in 46 consecutively recruited patients undergoing autologous or allogeneic BMT and explore in parallel the behaviour of ADAMTS13, VWF antigen and VWF multimer size. The incidence of post-BMT TMA was 6% (three of 46); all cases occurred after allogeneic BMT. Compared with baseline values plasma ADAMTS13 activity was significantly reduced in patients undergoing BMT, particularly after the conditioning regimen (mean values: 50 +/- 22 vs. 77 +/- 32%; P < 0.0001). In the three patients who developed TMA, ADAMTS13 decreased after conditioning, but was very low in one case only (8%). VWF antigen levels progressively increased after the conditioning regimen (228 +/- 75 vs. 178 +/- 76% at baseline, P = 0.002). The mean proportion of high-molecular weight VWF multimers did not change in the various stages of BMT, even though ultralarge multimers were transiently found in same cases with and without TMA. Hence, the measurements evaluated in this study are not clinically useful to predict the occurrence of post-BMT TMA.