Congenital or immunomediated deficiencies of the metalloprotease that cleaves physiologically von Willebrand factor (vWF) reduce or abolish the degradation of ultralarge vWF multimers that cause the formation of intravascular platelet thrombi in patients with thrombotic thrombocytopenic purpura (TTP). There is little knowledge on the behavior of the protease in other physiological and pathologic conditions. Such knowledge is important to evaluate the specificity of low protease plasma levels in the diagnosis of TTP. Using an enzyme immunoassay, the protease was measured in 177 control subjects of different ages, in 26 full-term newborns, and in 69 women during normal pregnancy. Because TTP is often associated with multiorgan involvement and acute phase reactions, clinical models of these pathologic conditions were also investigated, including decompensated liver cirrhosis (n ‫؍‬ 42), chronic uremia (n ‫؍‬ 63), acute inflammatory states (n ‫؍‬ 15), and the preoperative and postoperative states (n ‫؍‬ 24). Protease levels were lower in healthy persons older than 65 than in younger persons. They were low in newborns but became normal within 6 months, and they were lower in the last 2 trimesters of pregnancy than in the first. Protease levels were also low in patients with cirrhosis, uremia, and acute inflammation, and they fell in the postoperative period. There was an inverse relation between low protease and high plasma levels of vWF antigen and collagen-binding activity. In conclusion, low plasma levels of the vWF cleaving protease are not a specific beacon of TTP because the protease is also low in several physiological and pathologic conditions. (Blood. 2001;98:2730-2735)

Type 2B von Willebrand disease (VWD2B)is caused by an abnormal von Willebrand factor (VWF) with increased affinity for the platelet receptor glycoprotein Ib-␣ (GPIb-␣) that may result in moderate to severe thrombocytopenia. We evaluated the prevalence and clinical and molecular predictors of thrombocytopenia in a cohort of 67 VWD2B patients from 38 unrelated families characterized by VWF mutations. Platelet count, mean platelet volume, and morphologic evaluations of blood smear were obtained at baseline and during physiologic (pregnancy) or pathologic (infections, surgeries) stress conditions. Thrombocytopenia was found in 20 patients (30%) at baseline and in 38 (57%) after stress conditions, whereas platelet counts were always normal in 16 patients (24%) from 5 families carrying the P1266L/Q or R1308L mutations. VWF in its GPIb-␣-binding conformation (VWFGPIb-␣/BC) was higher than normal in all except the 16 cases without thrombocytopenia (values up to 6-fold higher than controls). The risk of bleeding was higher in patients with thrombocytopenia (adjusted hazard ratio ‫؍‬ 4.57; 95% confidence interval, 1.17-17.90) and in those with the highest tertile of bleeding severity score (5.66; 95% confidence interval, 1.03-31.07). Prediction of possible thrombocytopenia in VWD2B by measuring VWF-GPIb-␣/BC is important because a low platelet count is an independent risk factor for bleeding. (Blood. 2009;113: 526-534)

Background From 20 to 50% of patients who survive an acute episode of the acquired form of thrombotic thrombocytopenic purpura relapse but clinical and laboratory markers of recurrence are not well established. Design and MethodsIn 109 patients enrolled in an international registry we evaluated, in the frame of a retrospective cohort study, the predictive role of the metalloprotease ADAMTS13 as measured in plasma during remission. Anti-ADAMTS13 antibodies and von Willebrand factor were also evaluated in a smaller number of the same patients. ResultsMedian values of ADAMTS13 activity and antigen were significantly lower in patients with recurrent thrombotic thrombocytopenic purpura than in those with no recurrence (activity: 12% vs. 41%; p=0.007; antigen: 36% vs. 58%; p=0.003). A severe deficiency of ADAMTS13 activity (10% or less) was associated with a higher likelihood of recurrence (odds ratio 2.9; 95% confidence interval 1.3 to 6.8; p=0.01). Anti-ADAMTS13 antibodies were also more prevalent in patients with recurrent thrombotic thrombocytopenic purpura (odds ratio 3.1; 95% confidence interval 1.4 to 7.3; p=0.006). The presence during remission of both severe ADAMTS13 deficiency and anti-ADAMTS13 antibodies increased the likelihood of recurrence 3.6 times (95% confidence interval 1.4 to 9.0; p=0.006). The presence of ultralarge von Willebrand factor multimers and of associated diseases or conditions did not increase recurrence. ConclusionsSurvivors of an acute episode of acquired thrombotic thrombocytopenic purpura with severely reduced levels of ADAMTS13 and/or with anti-ADAMTS13 antibodies during remission have an approximately three-fold greater likelihood of developing another episode of thrombotic thrombocytopenic purpura than patients with higher protease activity and no antibody.Key words: thrombotic thrombocytopenic purpura, ADAMTS13, von Willebrand factor, risk factors, recurrence.Citation: Peyvandi F, Lavoretano S, Palla R, Feys H B., Vanhoorelbeke K, Battaglioli T, Valsecchi C, Canciani MT, Fabris F, Zver S, Réti M, Mikovic D, Karimi M, Giuffrida G, Laurenti L, antibodies as markers for recurrence of acquired thrombotic thrombocytopenic purpura during remission. Haematologica 2008 Feb; 93(2):232-239. DOI: 10.3324/haematol.11739 ©2008 Ferrata Storti Foundation. This is an open-access paper. ADAMTS13 and anti-ADAMTS13 antibodies as markers for recurrence of acquired thrombotic thrombocytopenic purpura during remission IntroductionAcquired thrombotic thrombocytopenic purpura (TTP) is a rare but severe disease characterized by microangiopathic hemolytic anemia and consumptive thrombocytopenia leading to disseminated microvascular thrombosis that causes variable signs and symptoms of organ ischemia and damage.1 The pathophysiology of TTP has become clearer after it was demonstrated that the plasma metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin 1 repeats) cleaves the platelet-adhesive plasma protein von Willebrand factor (VWF) at the peptide bond Tyr1605-Met1...

These studies were designed with the purpose of providing clinico-pharmacological information relevant to the use of DDAVP in the management of mild haemophilia and von Willebrand's disease (VWD). In healthy subjects, intravenous DDAVP produced its maximal response at a dose of 0.3 micrograms/kg. The extent of the increase in factor VIII coagulant activity (VIII:C) and factor VIII related antigen (VIIIR:Ag) induced by this dose was not significantly different from that observed with the same dose in haemophiliacs and VWD patients. In these, the bleeding time was not shortened. DDAVP given intranasally was followed by a two-fold increase of VIII:C. This route of administration might be adopted to provide an emergency aid in bleeding patients and to yield higher VIII:C levels in blood donors. In healthy subjects, the half-disappearance time of autologous VIII:C after increase induced by i.v. DDAVP is similar to that observed in patients with VWD treated in the same conditions, whereas the response appears to be more prolonged in haemophiliacs. This study shows that the consistency of the VIII:C response tends to decrease when repeated doses are given to healthy subjects. Repeatedly-treated haemophiliacs and VWD patients showed varied patterns, ranging from no change of the response to its early abolishment.

for the Italian Registry of Recurrent and Familial HUS/TTP Whether measurement of ADAMTS13 activity may enable physicians to distinguish thrombotic thrombocytopenic purpura (TTP) from hemolytic uremic syndrome (HUS) is still a controversial issue. Our aim was to clarify whether patients with normal or deficient ADAMTS13 activity could be distinguished in terms of disease manifestations and multimeric patterns of plasma von Willebrand factor (VWF). ADAMTS13 activity, VWF antigen, and multimeric pattern were evaluated in patients with recurrent and familial TTP (n ‫؍‬ 20) and HUS (n ‫؍‬ 29). Results of the collagen-binding assay of ADAMTS13 activity were confirmed in selected samples by testing the capacity of plasma to cleave recombinant VWF A1-A2-A3. Most patients with TTP had complete or partial deficiency of ADAMTS13 activity during the acute phase, and in some the defect persisted at remission. However, complete ADAMTS13 deficiency was also found in 5 of 9 patients with HUS during the acute phase and in 5 patients during remission. HUS patients with ADAMTS13 deficiency could not be distinguished clinically from those with normal ADAMTS13. In a subgroup of patients with TTP or HUS, the ADAMTS13 defect was inherited, as documented by half-normal levels of ADAMTS13 in their asymptomatic parents, consistent with the heterozygous carrier state. In patients with TTP and HUS there was indirect evidence of increased VWF fragmentation, and this occurred also in patients with ADAMTS13 deficiency. In conclusion, deficient ADAMTS13 activity does not distinguish TTP from HUS, at least in the recurrent and familial forms, and it is not the only determinant of VWF abnormalities in these conditions. (Blood. 2002;100:778-785)

SummaryThe plasma metalloprotease ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 motif 13) cleaves prothrombotic ultralarge multimers of the platelet-adhesive protein von Willebrand factor (ULVWF) into less active multimers that promote haemostasis in injured blood vessels. When the enzyme is dysfunctional or undetectable, circulating ULVWF may cause massive intravascular aggregation of platelets and thrombotic thrombocytopenic purpura. This study compared ADAMTS13 antigen and activity in a large set of plasmas collected from subjects with various conditions of health and disease, most of which were associated with an increased thrombotic tendency. Pathological conditions were liver cirrhosis (n ¼ 90), inflammatory bowel disease (n ¼ 44) and cardiac surgery (n ¼ 30). Healthy conditions were pregnancy (n ¼ 42), oral contraceptive intake (n ¼ 33) and the neonatal state (n ¼ 41). Normal individuals of different ages were taken as controls (n ¼ 132). The antigen assay showed less variability than the collagen binding-based activity assay. Antigen values correlated well with activity in normal individuals, but were discrepant to various degrees in neonates, pregnancies of later maternal age and cardiac surgery. No discrepancies were noted in liver cirrhosis and inflammatory bowel disease, which were both associated with low-plasma levels of ADAMTS13. The parallel measurement of ADAMTS13 activity and antigen provides a new tool for understanding the behaviour of the VWF cleaving protease in health and disease.

SummaryThe congenital or acquired deficiency of the von Willebrand factor (VWF) cleaving protease, ADAMTS-13 has been specifically associated with a diagnosis of thrombotic thrombocytopenic purpura (TTP), a microangiopathy characterized by the formation of occlusive platelet thrombi. The mechanisms of TTP were investigated in 100 patients diagnosed on the basis of the presence of at least three of the following: thrombocytopenia, haemolytic anaemia, elevated serum levels of lactate dehydrogenase and neurological symptoms. Plasma levels of ADAMTS-13 were severely reduced (<10% of normal) in 48%, moderately reduced (between 10% and 46%) in 24% and normal (>46%) in 28%. A neutralizing antibody was the cause of the deficiency in 38% of the cases, with a higher prevalence of this mechanism (87%) in the 48 patients with severely reduced ADAMTS-13. Double heterozygosity for a 29 base pair (bp) deletion and a nucleotide insertion and homozygosity for a 6 bp deletion in the ADAMTS13 gene were identified only in two patients born from consanguineous marriages. In conclusion, this study indicated that ADAMTS-13 was normal in nearly one-third of patients with TTP and that ADAMTS-13 deficiency was not associated with the presence of neutralizing antibodies in more than half of the patients.

Key Points VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios define the pathophysiological mechanisms that play a role in VWD and various VWF mutations. A high VWFpp/VWF:Ag ratio indicates increased clearance of VWF and a high FVIII:C/VWF:Ag ratio decreased synthesis of VWF.