ADAMTS13 gene mutation in congenital thrombotic thrombocytopenic purpura with previously reported normal VWF cleaving protease activity

Thrombotic thrombocytopenic purpura (TTP), a potentially fatal disease, is characterized by thrombocytopenia, microangiopathic hemolysis, and, in the advanced cases, neurologic deficits. The presence of von Willebrand factor (VWF) on the platelet surface¹  and in thrombi²  suggests that VWF-platelet binding causes the development of platelet-rich thrombi in TTP.

VWF, secreted from endothelial cells as a disulfide-bonded polymeric form, undergoes conformational unfolding when exposed to high levels of shear stress such as those in the arterioles and capillaries.³  The unfolded, elongated forms of VWF exhibit a higher capacity for supporting platelet aggregation⁴  but are also more susceptible to cleavage by the plasma metalloprotease ADAMTS13.⁵  The responsiveness of large VWF multimers to shear stress may explain why VWF is uniquely capable of supporting platelet adhesion under high shear conditions and why the size of VWF is a determinant of its hemostatic effectiveness. On the other hand, cleavage of unfolded VWF by ADAMTS13 prevents the accumulation of superactive forms of VWF, a step believed to be critical for preventing VWF-platelet binding in the circulation.

A lack of ADAMTS13 activity, caused by autoimmune inhibitors or genetic mutations, is detected in patients with TTP.^{5, 6, 7}  Deficiency of ADAMTS13 explains why patients with TTP respond to plasma infusion. In patients with acquired TTP, plasma exchange is more effective because it allows the patients to receive a large amount of fresh-frozen plasma to overcome the inhibitors without imposing the risk of fluid overload. Removal of the inhibitors may also contribute to the greater efficacy of plasma exchange. Patients with congenital TTP typically respond to small amounts of plasma infusion because they do not have inhibitors of the protease.

Some investigators describe the presence of very low ADAMTS13 activity in patients without TTP.^{8, 9, 10, 11, 12, 13}  A single previous study reported normal ADAMTS13 activity in a patient who had thrombocytopenia and microangiopathic hemolysis since early infancy that was responsive to plasma infusion.¹⁴  The reasons for the discrepancy among these various studies have not been adequately addressed.

In this report, we reinvestigate the case noted above,¹⁴  previously reported to have normal levels of VWF cleaving protease activity despite the presence of features that were characteristic of congenital TTP.

The course of this case has been described previously.¹⁴  Briefly, the patient, born to parents who were first cousins of Yemenite background, developed thrombocytopenia and microangiopathic hemolysis soon after birth. She required plasma infusion (10 mL/kg) every 2 weeks to maintain normal platelet counts and hemoglobin levels. Analysis of VWF cleaving protease activity using the technique of sodium dodecyl sulfate (SDS)–agarose gel electrophoresis and immunoblotting detected approximately 50% protease activity in the patient, but normal levels in her parents.¹⁵ 

Plasma samples obtained from the patient prior to plasma transfusion and from her parents were used for analysis of VWF cleaving protease activity by using the technique of SDS–polyacrylamide gel electrophoresis and immunoblotting as previously described.⁶ 

All ADAMTS13 exons and intron-exon boundaries (except for exons 3, 4, 7, 8, 16, 19, and 25) were amplified by PCR from peripheral blood leukocyte DNA prepared from the patient and both parents as previously described.⁷  Sequencing reactions were performed by the University of Michigan DNA Sequencing Core. Approval was obtained from the Montefiore Medical Center and University of Michigan institutional review boards for these studies. Informed consent was provided according to the Declaration of Helsinki.

The VWF cleaving protease activity level was less than 0.1 U/mL in the patient and partially deficient in both parents (Figure 1). When a heat-treated plasma sample of the patient was incubated with normal control plasma, no inhibitors of the VWF cleaving activity were detected. Sequence analysis of PCR-amplified DNA products revealed that the patient was homozygous for a TT deletion near the end of exon 15. The deletion resulted in a shift in the reading frame and truncation of the predicted protein product. The parents were heterozygous for the same mutation (Figure 2).

In a case with characteristic features of hereditary TTP, or Shulman-Upshaw syndrome, we detected very low VWF cleaving protease activity (< 0.1 U/mL) in the patient and partial deficiency in her parents. DNA sequence analysis detected a novel 2–base pair deletion in the ADAMTS13 gene, confirming the genetic nature of the deficiency. The homozygous state of the patient is consistent with the known consanguinity between the parents (first cousins).

A recent report identified an inactivating mutation in the ADAMTS13 gene in all studied patients with hereditary TTP.⁷  Subsequent studies also described the presence of genetic defi-ciency in the VWF cleaving activity among patients with congenital TTP.^16,17  This patient and her parents were previously reported to have normal levels of VWF cleaving activity.¹⁴  The presence of ultra-large multimers in the patient would have suggested the possibility of resistant VWF. Since mutations of VWF that increase its susceptibility to ADAMTS13 cause type 2A von Willebrand disease,¹⁸  it is conceivable that mutations decreasing the proteolytic susceptibility of the VWF may result in the presence of ultra-large multimers. According to the shear-stress model,⁴  such mutations could cause accumulation of unfolded, superactive forms of VWF in the circulation, resulting in VWF-platelet binding and platelet thrombosis such as that observed in patients with ADAMTS13 deficiency. However, patients with such mutations are unlikely to respond to a small amount of normal plasma. Thus, the plasma responsiveness of this patient makes such an explanation unlikely. In addition, no other protease regulating the size of VWF in the normal circulation has yet been identified as a potential second cause of TTP.

The assay used in this study differs from the assay used in the previous report of this case¹⁴  in that the VWF substrate was incubated directly with the plasma sample in the presence of 0.15 M guanidine hydrochloride, not dialyzed against 1.5 M urea; the incubation lasted for one hour instead of overnight; no barium chloride was added; and cleavage of VWF was detected by measuring the dimer of the 176-kDa fragment generated from cleavage of the VWF substrate by ADAMTS13 at the bond of Tyr842 and Met843 of the mature VWF instead of SDS–agarose gel electrophoresis and immunoblotting of VWF multimers. How and whether a difference in the experimental procedures contributed to the results previously obtained in this family remain to be explored. SDS–agarose gel electrophoresis could have detected a decrease in the size of VWF multimers caused by nonspecific adsorption of the large multimers on to surface in contact with the sample mixtures or by plasmin or other proteases that were generated during the long incubation. Also, the potential contribution of barium chloride to the assay variability is unknown.

In summary, this study confirms that hereditary TTP results from genetic deficiency of ADAMTS13, with gene mutations identified in all patients studied to date at the molecular level. Furthermore, the experience in this family highlights the importance of future research to establish a standardized, validated procedure for the measurement of ADAMTS13 enzymatic activity that can be readily adapted to the clinical laboratory.

We thank Dr M. Furlan for advice and comments on the manuscript and S. Chang for technical assistance.