OBJECTIVETo detect the plasma activity of von Willebrand factor-cleaving protease (ADAMTS13) in the patients with prothrombotic status, and explore the effect and significance of ADAMTS13 in the prothrombotic status. The correlation of ADAMTS13 with von Willebrand factor (vWF), thrombospondin 1 (TSP1), C-reactive protein etc, and blood pressure was simultaneously analyzed．

METHODSThe activity of ADAMTS13 in patient groups (atherosclerosis, diabetes, acute promyelocytic leukemia, cancer and sepsis, a total of 260 cases) and in control group 50 cases were evaluated by residue collagen binding assay（R-CBA）, the protein levels of TSP1 and vWF were measured by ELISA kits； the correlation of ADAMTS13 activity with CRP, creatinine, and blood pressure was analyzed with statistical soft ware.

RESULTSThe activity of plasma ADAMTS13 in patient group was significantly lower than that in normal control group(P<0.05). And the protein levels of TSP1 and vWF in the patients with prothrombotic status were higher than those in the normal controls(P<0.05). Analysis of the correlation showed that the ADAMTS13 activity correlated negatively with the levels of TSP1 protein, blood sugar, blood pressure, D-dimer, creatinine,and CRP levels (P<0.05), however, the ADAMTS13 activity did not significantly correlate with the levels of serum lipids, blood type, platelet number and hemoglobin level(P>0.05).

CONCLUSIONThe plasma ADAMTS13 activity is decreased in the patients with prothrombotic status, suggesting that the decreased ADAMTS13 activity may participate in the occurrence of prothrombotic status, and the dectection of plasma ADAMTS13 activity may help the diagnosis of pro-thrombotic disease.

ADAMTS13 Protein ; Factor Analysis, Statistical ; Fibrin Fibrinogen Degradation Products ; Humans ; Sepsis ; Thrombosis ; von Willebrand Factor

This study was purposed to investigate the changes of von Willebrand factor cleaving protease (ADAMTS13) activity and vWF antigen level in patients with acute myelogenous leukemia (AML) before and after treatment and evaluate their clinical significance. Seventy-three AML patients were enrolled in this study, the sodium citrate anticoagulated plasma was collected before and after their induction chemotherapy. Fluorescence resonance energy transfer substrate vWF73 (FRETS-vWF73) assay was established to detect the plasma ADAMTS13 activity while vWF antigen level was measured by ELISA. The results showed that the ADAMTS13 activity in newly diagnosed patients with AML before induction therapy was obviously lower than that in normal controls (63.3 ± 25.5)% vs (105.1 ± 37.7)(P < 0.01), while the vWF antigen level was higher than that in normal controls (226.6 ± 127.0)% vs (111.4 ± 39.7)% (P < 0.01). After standard induction chemotherapy, the ADAMTS13 activity of AML patients in complete remission period was higher than that in AML patients before therapy (P < 0.01), and was not significant difference with that in normal controls; the vWF antigen was significantly lower than that in AML patients before therapy (P < 0.01), but it still was higher than that in controls (P < 0.05). The ADAMTS13 activity in newly diagnosed AML patients complicated with infection before therapy was obviously lower than that in AML patients without infection (52.2 ± 20.6)% vs (73.9 ± 24.7)% (P < 0.01), while the vWF antigen level was significantly higher than that in AML patients without infection (262.2 ± 135.7)% vs (193.8 ± 110.2)% (P < 0.05). The ADAMTS13 activity in AML patients with disseminated intravascular coagulation (DIC) was significantly lower than that in AML patients without DIC (42.0 ± 14.5)% vs (73.4 ± 22.7)% (P < 0.01), while the vWF antigen level was obviously higher that in AML patients without DIC (274.2 ± 140.0)% vs (204.7 ± 115.5)% (P < 0.01). It is concluded that the ADAMTS13 activity in newly diagnosed AML patients befor induction therapy has been confiremed to be lower and the vWF antigen level to be higher, especially in AML patients with infection or DIC. The ADAMTS13 and vWF antigen may play a role in the pathogenesis of AML and the formation of infection and DIC.
ADAM Proteins ; blood ; ADAMTS13 Protein ; Disseminated Intravascular Coagulation ; Humans ; Leukemia, Myeloid, Acute ; blood ; von Willebrand Factor ; analysis

INTRODUCTION: ADAMTS13 (a disintegrin-like and metalloproteinase with a thrombospondin Type 1 motif, member 13) plays a fundamental role in the regulation of haemostasis and thrombosis. Its deficiency leads to an accumulation of ultra-large von Willebrand multimers, inducing spontaneous platelet aggregation, thrombosis in the microvasculature, and thrombotic thrombocytopenic purpura (TTP), a condition with 90% mortality when left untreated. Prompt quantification of ADAMTS13 antigen, activity and autoantibody plays a crucial role in the diagnosis and management of TTP and can help differentiate it from other thrombotic microangiopathies (TMAs). Reference ranges for ADAMTS13 are generally derived from Caucasian patients. Given that polymorphism in the ADAMTS13 gene can be associated with variable ADAMTS13 levels, we aimed to establish the first reference range in Singapore and provide a crucial laboratory test for institutions here and elsewhere. METHODS: 150 healthy voluntary donors (75 men, 75 women) aged 21-60 years, with an ethnic mix mirroring Singapore's population profile, were recruited. ADAMTS13 antigen, activity and autoantibody levels were measured using the fluorescence resonance energy transfer-vWF73 and enzyme-linked immunosorbent assay methodologies. RESULTS: Levels (activity 0.65-1.79 IU/mL, antigen 0.36-1.17 IU/mL, autoantibody 1.4-12.5 U/mL) were not statistically different between the genders and various age groups. CONCLUSION TTP and TMAs are encountered in a wide range of specialties. The availability of new assays in Singapore will aid clinicians in the timely management of these conditions. Standardising reference ranges established for Singapore against World Health Organization standards allows harmonisation of measurements between laboratories and for future research collaborations.
ADAMTS13 Protein/analysis* ; Adult ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Male ; Purpura, Thrombotic Thrombocytopenic/diagnosis* ; Reference Values ; Singapore

OBJECTIVETo construct and identify the monoclonal antibodies against von willebrand factor cleaving protease(ADAMTS13), and to study their biological function.

METHODSBALB/c mice were immunized by purified recombinant ADAMTS13 truncated eukaryotic protein (ADAMTS13-T7). Murine anti-human ADAMTS13 monoclonal antibodies (McAbs) were constructed by standard hybridoma technology and identified by ELISA. The recognition of McAbs with full-length recombinant ADAMTS13 protein was identified by Western blot. In function assay, the influence of McAbs on the proteolysis of vWF by ADAMTS13 was observed.

RESULTSA group of 6 murine anti-ADAMTS13 McAbs was obtained with the clone number 1G11, 2F11, 6G3, 9E1, 10A8 and 10B4. In ELISA, the highest titers of 1G11 and 2F11 were observed, both of which showed a higher affinity to ADAMTS13-T7 than full-length ADAMTS13. The Western blot demonstrated that the 6 McAbs all could recognize ADAMTS13, among which 1G11 and 2F11 showed stronger reaction with ADAMTS13. In addition, under the denatured conditions, 1G11 and 2F11 could inhibit hydrolysis of vWF by ADAMTS13, and that was stronger with the increasing of McAbs concentration.

CONCLUSIONMcAbs against ADAMTS13 have been gained, two of which are inhibitory antibodies.

ADAMTS13 Protein ; Animals ; Antibodies, Monoclonal ; analysis ; immunology ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Hybridomas ; Mice ; Mice, Inbred BALB C ; von Willebrand Factor

BACKGROUNDvon Willebrand factor (vWF) mediates the initial capture of platelets to vascular subendothelium and is essential for platelet aggregation under high fluid shear stress as in arterial stenosis. On release from endothelial cells, vWF is rapidly cleaved by ADAMTS13/vWF-cleaving protease (vWF-CP). We investigated the clinical significance of changes in plasma vWF and vWF-CP activities in chronic renal disease.

METHODSPlasma vWF and vWF-CP activities were measured using enzyme-linked immunosorbent assay (ELISA) and residual collagen binding assay respectively in patients with lupus nephritis (n = 31), primary nephritic syndrome (n = 25), diabetic nephropathy (n = 45), chronic glomerulonephritis (n = 38) and 40 normal controls. The relation of their levels with pathological and renal status was analyzed.

RESULTSIn all diseased patients the levels of vWF were significantly higher and vWF-CP activity significantly lower than the controls (both P < 0.01). vWF in the four subgroups did not correlate with the stage of disease but correlated negatively with vWF-CP activity. vWF-CP activity was not changed two weeks after renal transplantation. Renal biopsy demonstrated that the vWF level in stage IV was higher than in stages II and III while vWF-CP activity was lower in patients with lupus nephritis. After eight-week treatment, the vWF level significantly decreased and the vWF-CP activity significantly increased in systemic lupus erythema, disease activity index < 9, but not with index = 9. Even though the vWF-CP activity was significantly lower in membranous nephropathy than in minimal change disease, mesangial proliferative glomerulonephritis or IgA glomerulonephritis, the vWF level was not significantly different.

CONCLUSIONSThe alterations of plasma vWF and vWF-CP activities were associated with different renal pathologies. Injury to endothelial cells and autoantibodies against vWF-CP activity may result in higher vWF level and lower vWF-CP activity in chronic renal disease and thus a mechanism for worsening of chronic renal disease and thrombosis.

ADAM Proteins ; blood ; ADAMTS13 Protein ; Adolescent ; Adult ; Aged ; Chronic Disease ; Female ; Humans ; Kidney Diseases ; blood ; Kidney Transplantation ; Lupus Nephritis ; blood ; Male ; Middle Aged ; von Willebrand Factor ; analysis

Von Willebrand factor (vWF) is a glycoprotein with a crucial role in the formation of platelet thrombi, and ADAMTS13 is the main enzyme responsible for vWF cleavage. Both are important in the relationship between diabetic nephropathy, hypercoagulability, and cardiovascular disease. This study evaluated a potential relationship between vitamin D (vitD) levels, vWF, ADAMTS13 activity, and inflammation in diabetic patients on chronic hemodialysis (HD). Blood samples from 52 diabetic patients on chronic HD were obtained to determine vitD levels, vWF, and ADAMTS13 activity, and inflammatory markers. HD patients were grouped according to 25-hydroxyvitamin D [25(OH) VitD]<25 nmol/L (n=16) or >25 nmol/L (n=36). vWF antigen and vWF activity were elevated in both groups, with an average of 214.3±82.6% and 175.8±72.6%, respectively. Average ADAMTS13 activity was within the normal range in both groups. Blood samples from the vitD <25 nmol/L group showed a positive correlation between c-reactive protein (CRP) and vWF levels (P=0.023; r=0.564; 95% confidence interval=0.095-0.828), with a negative correlation between HbA1c and 25(OH) VitD (P=0.015; r=-0.337; 95% confidence interval=-0.337-0.19). Diabetic patients on chronic HD had elevated vWF levels and activity with no significant change in ADAMTS13 activity. The correlation between CRP and vWF levels in the 25(OH) VitD<25 nmol/L group suggests inflammatory-related endothelial dysfunction in these patients.
ADAMTS13 Protein/*metabolism ; Aged ; C-Reactive Protein/analysis ; Diabetes Mellitus, Type 2/complications/*diagnosis/metabolism ; Female ; Hemoglobin A, Glycosylated/analysis ; Humans ; Male ; Middle Aged ; Renal Dialysis ; Renal Insufficiency, Chronic/complications/*diagnosis/metabolism ; Vitamin D/*analogs & derivatives/blood ; von Willebrand Factor/*metabolism