von Willebrand factor-cleaving protease (ADAMTS13) activity in normal non-pregnant women, pregnant and post-delivery women.

ADAMTS13 dysfunction has been involved in the pathogenesis of Thrombotic Thrombocytopenic Purpura. This disorder occurs more frequently in women and, in 13% of them, is associated with pregnancy. However, there is little information on the protease behaviour in normal pregnancy. We studied von Willebrand factor and ADAMTS13 activity changes in normal non-pregnant, pregnant and post-delivery women. Fifty-five non-pregnant women, normal blood bank donors, who were not taking contraceptive pills were included as controls. A prospective cross-sectional study of 270 normal pregnant and post-delivery women was carried out. ADAMTS13 activity decreased progressively as from the period of 12-16 weeks up to the end of early puerperium (mean 52%, range 22-89, p < 0.0001), to increase slightly thereafter. Nulliparous presented mildly lower levels of ADAMTS13 activity than parous women (65% vs. 83 %, p = 0.0003), and primigravidae than multigravidae between 6-11 weeks up to 17-23 weeks of pregnancy (69% vs. 80%, p = 0.005). Although in all women the protease levels were the same by blood groups, the O blood group non-pregnant women showed a higher mean of ADAMTS13 activity than those non-O (78% vs. 69%, p = 0.064). Our results suggest that the changing levels of protease activity during pregnancy and puerperium, induced by unidentified mechanisms, could render the peripartum time more vulnerable to developed thrombotic microangiopathies.

Control of von Willebrand factor multimer size by a fibronectin-related substance.

Fraction (F) II and FIII obtained by heparin-Sepharose after digestion of partially purified fibronectin (FN) with cathepsin D and F3, obtained like FIII but from untreated FN, exerted activity (arFN) on unfolded purified von Willebrand factor (vWF) that controls vWF multimer size. Our aim was to evaluate the arFN of F from commercial FN, commercial 30 kDa (with heparin affinity), 45 kDa (gelatin affinity) and 70 kDa FN fragments (gelatin and heparin affinity) and whole FN. The arFN was detected in FII, FIII, F2, F3, 30 kDa, 45 kDa and 70 kDa fragments. The least contaminated sample was the 30 kDa commercial fragment. Characterization studies of this sample revealed two bands: a blurred band of approximately 60 kDa and a sharp major band of 32 +/- 6 kDa. The 32 +/- 6 kDa band fragment failed to produce arFN because it was stronger than in F2 and FIII band fragments at the same position and with the same arFN. Our data suggest that a fragment of approximately 60 kDa that co-purified with FN, with affinity to heparin and gelatin, has the arFN that controls vWF multimer size.

Von Willebrand factor cleaving protease activity in the physiopathology of microangiopathic disorders.

The von Willebrand factor cleaving protease (VWFCP) modulates the von Willebrand factor (VWF) multimeric size in normal plasma. VWFCP activity levels are decreased in different physiological and pathologic situations. Different techniques have been developed to unfold the purified VWF (perfusion at high shear rate, dialysis against urea in nitrocellulose filters), to detect the VWFCP activity on it (multimeric analysis of VWF, collagen binding to VWF assay) and to use the patient plasma both as the source of the enzyme and substrate. In this paper we compared the above mentioned methods with new ones: normal plasma dialyzed on membranes instead of purified VWF, dialysis of the samples against urea in tubing instead of nitrocellulose filters, and sonicated plasma to remove the endogenous VWF. The perfusion assay and detection by multimeric analysis showed a limit of detection (25%) of VWFCP activity. Dialysis against urea in both supports and detection by multimeric analysis, showed a better limit of detection (3%), but the recovery of the samples was not as efficient in nitrocellulose filters as it was in tubing. The detection by collagen binding to VWF has more advantages because it allows to analyze more samples than the multimeric analysis does in the same assay. The dialysis of plasma by membranes to obtain the source of exogenous VWF requires no complex equipment. The method, which uses patient plasma as the source of the enzyme and substrate, was inapplicable in our experience because the values could not be interpolated in the reference curve.

Von Willebrand factor cleaving protease activity in the physiopathology of microangiopathic disorders

The von Willebrand factor cleaving protease (VWFCP) modulates the von Willebrand factor (VWF) multimeric size in normal plasma. VWFCP activity levels are decreased in different physiological and pathologic situations. Different techniques have been developed to unfold the purified VWF (perfusion at high shear rate, dialysis against urea in nitrocellulose filters), to detect the VWFCP activity on it (multimeric analysis of VWF, collagen binding to VWF assay) and to use the patient plasma both as the source of the enzyme and substrate. In this paper we compared the above mentioned methods with new ones: normal plasma dialyzed on membranes instead of purified VWF, dialysis of the samples against urea in tubing instead of nitrocellulose filters, and sonicated plasma to remove the endogenous VWF. The perfusion assay and detection by multimeric analysis showed a limit of detection (25%) of VWFCP activity. Dialysis against urea in both supports and detection by multimeric analysis, showed a better limit of detection (3%), but the recovery of the samples was not as efficient in nitrocellulose filters as it was in tubing. The detection by collagen binding to VWF has more advantages because it allows to analyze more samples than the multimeric analysis does in the same assay. The dialysis of plasma by membranes to obtain the source of exogenous VWF requires no complex equipment. The method, which uses patient plasma as the source of the enzyme and substrate, was inapplicable in our experience because the values could not be interpolated in the reference curve

Von Willebrand factor cleaving protease activity in the physiopathology of microangiopathic disorders.

The von Willebrand factor cleaving protease (VWFCP) modulates the von Willebrand factor (VWF) multimeric size in normal plasma. VWFCP activity levels are decreased in different physiological and pathologic situations. Different techniques have been developed to unfold the purified VWF (perfusion at high shear rate, dialysis against urea in nitrocellulose filters), to detect the VWFCP activity on it (multimeric analysis of VWF, collagen binding to VWF assay) and to use the patient plasma both as the source of the enzyme and substrate. In this paper we compared the above mentioned methods with new ones: normal plasma dialyzed on membranes instead of purified VWF, dialysis of the samples against urea in tubing instead of nitrocellulose filters, and sonicated plasma to remove the endogenous VWF. The perfusion assay and detection by multimeric analysis showed a limit of detection (25

) of VWFCP activity. Dialysis against urea in both supports and detection by multimeric analysis, showed a better limit of detection (3

), but the recovery of the samples was not as efficient in nitrocellulose filters as it was in tubing. The detection by collagen binding to VWF has more advantages because it allows to analyze more samples than the multimeric analysis does in the same assay. The dialysis of plasma by membranes to obtain the source of exogenous VWF requires no complex equipment. The method, which uses patient plasma as the source of the enzyme and substrate, was inapplicable in our experience because the values could not be interpolated in the reference curve.

Von Willebrand factor cleaving protease activity in the physiopathology of microangiopathic disorders

The von Willebrand factor cleaving protease (VWFCP) modulates the von Willebrand factor (VWF) multimeric size in normal plasma. VWFCP activity levels are decreased in different physiological and pathologic situations. Different techniques have been developed to unfold the purified VWF (perfusion at high shear rate, dialysis against urea in nitrocellulose filters), to detect the VWFCP activity on it (multimeric analysis of VWF, collagen binding to VWF assay) and to use the patient plasma both as the source of the enzyme and substrate. In this paper we compared the above mentioned methods with new ones: normal plasma dialyzed on membranes instead of purified VWF, dialysis of the samples against urea in tubing instead of nitrocellulose filters, and sonicated plasma to remove the endogenous VWF. The perfusion assay and detection by multimeric analysis showed a limit of detection (25%) of VWFCP activity. Dialysis against urea in both supports and detection by multimeric analysis, showed a better limit of detection (3%), but the recovery of the samples was not as efficient in nitrocellulose filters as it was in tubing. The detection by collagen binding to VWF has more advantages because it allows to analyze more samples than the multimeric analysis does in the same assay. The dialysis of plasma by membranes to obtain the source of exogenous VWF requires no complex equipment. The method, which uses patient plasma as the source of the enzyme and substrate, was inapplicable in our experience because the values could not be interpolated in the reference curve (AU)

Requirement of a plasma fraction for the loss of large Von Willebrand factor multimers induced by high wall shear rate

The perfusion of serum, citrated whole blood and citrated plasma, through a simple tube system resulted in a significant loss of large von Willebrand factor (vWf) multimers, without decrease in antigen levels. Maximum loss of large multimers was observed at a shear rate of 15,000 s(-1) for 15 min. Heparin, aprotinin, soybean trypsin inhibitor, phenylmethylsulphonylfluoride, N-ethylmaleimide, leupeptin or calpain inhibitor peptide could not prevent the loss of large vWf multimers in citrated plasma. The addition of EDTA calcium salt partially prevented it, and it was totally prevented by EDTA without calcium. Perfusion of purified vWf did not induce the loss of large multimers, but this did happen after the addition of either whole serum or a plasma fraction. The activity of this plasma fraction disappeared at pH < 6.8. Besides, we have found that the binding to subendothelium of purified vWf diluted in dialized serum was lower at pH 7.2 than at pH 6.0. Chromatographic studies demonstrated that the loss of large vWf multimers, induced by high shear rates, involves a plasma substance(s) of molecular weight larger than 200 kD; calpain and granulocyte or cysteine proteases do not seem to be this plasma substance(s).

Requirement of a plasma fraction for the loss of large Von Willebrand factor multimers induced by high wall shear rate

The perfusion of serum, citrated whole blood and citrated plasma, through a simple tube system resulted in a significant loss of large von Willebrand factor (vWf) multimers, without decrease in antigen levels. Maximum loss of large multimers was observed at a shear rate of 15,000 s(-1) for 15 min. Heparin, aprotinin, soybean trypsin inhibitor, phenylmethylsulphonylfluoride, N-ethylmaleimide, leupeptin or calpain inhibitor peptide could not prevent the loss of large vWf multimers in citrated plasma. The addition of EDTA calcium salt partially prevented it, and it was totally prevented by EDTA without calcium. Perfusion of purified vWf did not induce the loss of large multimers, but this did happen after the addition of either whole serum or a plasma fraction. The activity of this plasma fraction disappeared at pH < 6.8. Besides, we have found that the binding to subendothelium of purified vWf diluted in dialized serum was lower at pH 7.2 than at pH 6.0. Chromatographic studies demonstrated that the loss of large vWf multimers, induced by high shear rates, involves a plasma substance(s) of molecular weight larger than 200 kD; calpain and granulocyte or cysteine proteases do not seem to be this plasma substance(s). (AU)