A Novel Variant on the Thrombospondin Type-1 Repeat 2 Domain of ADAMTS13 in a Parturient with Suspected Hereditary Thrombotic Thrombocytopenic Purpura and Unusually High ADAMTS13 Activity

Thrombotic thrombocytopenic purpura (TTP) represents a severe form of thrombotic microangiopathy that is caused by congenital or acquired absence or severe deficiency of the plasma metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which is required for cleavage of von Willebrand factor (VWF) multimers.[1] If high molecular weight VWF is unable to degrade properly, its accumulation can result in microplatelet thrombosis that obstructs microcirculation and leads to organ damage. Red blood cell (RBC) fragments can also arise due to mechanical shearing. Accumulated abnormalities can lead to serious consequences of platelet depletion, microvascular occlusion, hemolytic anemia due to erythrocyte rupture, and concurrent tissue ischemia. The mortality of TTP can exceed 80% without prompt diagnosis and treatment. However, timely identification and intervention with plasma exchange or transfusion can dramatically reduce the mortality rates to 10 to 20%.[2]

According to the cause of ADAMTS13 deficiency, TTP can be classified as hereditary or acquired. Congenital TTP (cTTP, also named Upshaw–Schulman syndrome) is a rare inherited disorder that follows an autosomal recessive inheritance pattern. The estimated prevalence of hereditary TTP is 0.5 to 2 per 1 million population and represents about one-tenth of the incidence of acquired TTP but varies greatly in different regions. Until now, over 150 germline mutations of ADAMTS13 have been reported in cTTP.[3]

Our case is a 33-year-old female, pregnant for 8 months prior to presentation in 2023. She displayed low energy levels, drowsiness, and bleeding gums. In 2012, she underwent a cesarean section due to severe preeclampsia during a pregnancy lasting more than 6 months, but the fetus did not survive. In 2014, the patient became pregnant again; this time a live female infant was delivered via cesarean section. In 2018, the patient underwent an induced abortion at 4 months of pregnancy due to stillbirth. During the current (2023) pregnancy, the patient took oral aspirin due to a history of severe preeclampsia until 33 weeks. Blood pressure was closely monitored during pregnancy and remained within normal levels. The pregnancy history of the patient is shown in [Fig. 1]. On March 14, 2023, the patient presented to our hospital for medical attention because of significant edema, reduced fetal movement, tight lower abdomen, decreased platelet count, and blurred vision. Routine blood tests showed a low platelet count (13 × 10⁹/L), and urine analysis showed occult blood 3 + , urine protein 3 + , ultrasound analysis revealed an abnormal uterine cavity echo and blood accumulation in the lower part of the uterine cavity, as well as signs of fetal growth restriction and placental abruption grade 1. The gestational age of the fetus was also significantly lower than expected. The patient then underwent several additional blood tests, with results shown in [Table 1].

Fig. 1 The timeline for the pregnancy history of the patient. Abbreviations: NA, not available; TTP, thrombotic thrombocytopenic purpura.

Table 1 Results of routine laboratory examination

Assay and normal (expected) ranges	Proband's Results
Biological chemistry tests
Alanine aminotransferase, U/L (normal, ≤33 U/L)	10
Aspartate aminotransferase, U/L (normal, ≤32 U/L)	27
Total bilirubin, μmol/L (normal, ≤21 μmol/L)	15.2
Direct bilirubin, μmol/L (normal, ≤8.0 μmol/L)	5.6
Indirect bilirubin, μmol/L (normal, ≤12.9 μmol/L)	9.6
Creatinine, μmol/L (normal, 45–84 μmol/L)	78
Lactate dehydrogenase, U/L (normal, 135–214 U/L)	657
Thrombosis/hemostasis tests
PT, s (normal,11.5–14.5 s)	12.3
INR (normal, 0.80–1.20)	0.91
Prothrombin activity, % (normal, 75–125%)	121
APTT, s (normal, 29.0–42.0 s)	32.8
TT, s (normal,14.0–19.0 s)	18.3
Fibrinogen, g/L (normal, 2.00–4.00 g/L)	4.34
D-dimer, ug/mL FEU (normal, < 0.5 ug/mL FEU)	1.44
ADAMTS13 activity, % (normal, 65–135%)  <10%: suggests severely deficient activity or TTP.	33.0
Complete blood count and cell morphology tests
Leukocyte count, ×109/L(normal, 3.50–9.50 × 109/L)	12.28
Erythrocyte count, ×1012/L (normal, 3.80–5.10 × 1012/L)	3.03
Hemoglobin, g/L (normal, 115–150 g/L)	101.0
Mean corpuscular volume, fL (normal, 82.0–100.0 fL)	99.7
Platelet count, ×109/L (normal, 125–350 × 109/L)	13.0
Reticulocytes, % (normal, 0.5–1.5%)	3.54
Erythrocyte morphology	Schistocytes >1%
Other tests
Haptoglobin, g/L (normal, 0.36–1.95 g/L)	<0.06
Free hemoglobin, mg/L (normal, <40 mg/L)	55.20
Direct antihuman globulin test (normal and negative)	Negative
Platelet autoantibody detection	Negative
Antinuclear antibodies and extractable nuclear antigen	Normal
Hepatitis B virus	Negative
Hepatitis C virus	Negative
Treponema pallidum	Negative
Human immunodeficiency virus	Negative

Abbreviations: APTT, activated partial thromboplastin time; INR, international normalized ratio; PT, prothrombin time; TT, thrombin time.

Note: Bold values indicate values outside the expected range.

Because the proportion of schistocytes in the blood smear of the patient was about 1% ([Fig. 2]) and the PLASMIC score was 6 on admission, the clinic suspected TTP, so ADAMTS13 activity and ADAMTS13 genetic testing were immediately requested. ADAMTS13 activity was assessed using a commercial ACTIFLUOR ADAMTS13 Activity Kit (Sekisui Diagnostics, Stanford, CT) using VWF86 substrate based on fluorescence resonance energy transfer (FRET). If the ADAMTS13 activity level is <30%, ADAMTS13 inhibitor (by Bethesda assay) will be performed as a reflex test.

Fig. 2 The blood films of the proband and her family members. (a) The image shows schistocytes which were triangular and helmet shaped with sharp and irregular edges, as marked with black arrows (from proband's blood film under a microscope at three times high power field). (b) From proband's younger sister's normal blood film under a microscope at three times high power field. (c) From proband's father's normal blood film under a microscope at 3 times high power field. (d) From proband's mother's normal blood film under a microscope at 3 times high power field.

According to the 2020 ISTH guidelines,[4] patients with moderate or highly suspected TTP in the clinical evaluation should promptly be tested for ADAMTS13 activity and inhibitor or IgG antibody before treatment. When ADAMTS13 activity is less than 10%, TTP can be diagnosed. Plasma ADAMTS13 activity at 10 to 20% cannot completely exclude TTP, and clinical judgment and close follow-up are necessary. Plasma ADAMTS13 >20% can basically exclude TTP.

The ADAMTS13 activity of the patient was 33% (reference: 65–135%). Additionally, the patient had a history of severe preeclampsia, which could also cause a certain proportion (up to 1%) of schistocytes.[5] Based on the above reasons, TTP was almost excluded at first. Considering the history of adverse pregnancy and the unfavorable condition of the fetus (fetal growth was limited, fetal movement was weakened, and the fetus showed a nuchal cord), cesarean section was immediately performed on March 15, 2023 and a live baby boy was born. After cesarean section, the patient experienced transient hypertension as the blood pressure measured was 155/86 mm Hg. An oral administration of 20 mg nifedipine was then given to reduce blood pressure, and magnesium sulfate was also taken orally to prevent spasms. The results of ADAMTS13 genetic testing came out 1 week later. Unexpectedly, more than one probably deleterious coding variant were found. The subject was identified to have a heterozygous missense mutation of ADAMTS13 c.2074C > T, which results in a protein change at position 692 from arginine to cysteine (p.Arg692Cys). This mutation is rare in population databases such as ExAC and gnomAD (ExAC: 0.000008, gnomAD: 0.000021) and has been recorded in the Human Gene Mutation Database (HGMD: CM012891). In addition, the change has been classified as a pathogenic variant in the ClinVar database. VarCards is an integrated clinical and genetic database of human genome mutations that integrates SIFT, polyphen-2, MutationTaster, PROVEAN, and other programs.[6] We then used this online platform (http://www.genemed.tech/varcards/) to predict the effect of the missense mutation, which was identified as probably pathogenic. The subject also harbored a heterozygous single base deletion of ADAMTS13 c.2137delC, which results in an amino acid change at position 713 from glutamine to arginine and causes a frameshift, leading to premature termination of translation and formation of a truncated protein (p.Gln713ArgfsTer65). This mutation is a novel deletion found in the ADAMTS13 gene and neither has been reported in the general population using ExAC and gnomAD databases nor has it been reported in HGMD and other clinical databases. However, it was predicted to be a deleterious mutation by bioinformatics prediction.

Further family verification revealed that the father of the case carried the ADAMTS13 c.2074C > T heterozygous mutation, while the mother carried the ADAMTS13 c.2137delC heterozygous mutation. This evidence indicated that the proband is a compound heterozygote of ADAMTS13 c.2074C > T and c.2137delC inherited from her father and mother, respectively.

In addition, the sister of the case subject only carried ADAMTS13 c.2074C > T heterozygous mutation (inherited from the father). The ADAMTS13 activity was also determined for all three family members, and all their results were within the normal range. The detailed information for these results is presented in [Fig. 3].

Fig. 3 The mutations found in the proband and her family members. (a) Sanger sequencing peaks of the two identified mutation sites (left: ADAMTS13 c.2074C > T, right: c.2137delC). (b) Pedigrees of the proband and her family members with genotypes and ADAMTS13 activity. The proband shared the same ADAMTS13 heterozygous mutation (c.2074C > T) with her father and younger sister and shared another ADAMTS13 heterozygous mutation (c.2137delC) with her mother. Both father and mother had normal ADAMTS13 activity (ADAMTS13 activity:124% and 120%, respectively). Her younger sister also exhibited a normal ADAMTS13 activity (128%).

The two different ADAMTS13 mutations are located in the 17th and 18th exons of the ADAMTS13 gene, both of which belong to the TSR2 function domain. Mutations in the TSR2 region do not affect the correct recognition and effective hydrolysis of VWF by the enzyme but can have a negative impact on the enzyme's intracellular folding, transport, and secretion, thus greatly reducing its secretion efficiency.[7] An O-fucosylation site, which is required for ADAMTS13 secretion, is located in the tryptophan at position 757. We found a c.2137delC heterozygous mutation in the proband, which resulted in a mutation of the 713th amino acid of the protein from glutamine to arginine and a code shift (p.Gln713ArgfsTer65). The change of the protein translation in the nearby section might also affect the secretion of ADAMTS13 by influencing the O-fucosylation process of this protein.[8] [9] Taken together, we speculated that the compound heterozygous mutations in the TSR2 region might inhibit the secretion of ADAMTS13. As a result, a large amount of ADAMTS13 protein is retained in the endoplasmic reticulum and accumulates in the cells, while the ADAMTS13 level in plasma might not be supplemented in time to degrade the VWF, thus leading to a form of TTP despite the moderate ADAMTS13 activity above 20%. However, the underline pathogenic mechanism remains unclear due to the lack of function exploration. The impacts of the compound heterozygous mutations on the efficiency or secretion of ADAMTS13 would also need further clarification by cellular and animal experiments in the future.

Pregnancy is believed to trigger acute attacks of congenital and acquired TTP by inducing an increase in VWF concentration, in particular uncleaved high molecular weight VWF multimers. In a mouse model of TTP, platelet GPIbα adhesion to VWF leads to the activation of αIIbβ3 fibrinogen receptor and subsequent platelet aggregation.[10] The cause of preeclampsia in our patient is not fully understood. However, it was well recognized that it may be related to abnormal placental angiogenesis and vascular function. Placentas of pregnant women with uncontrolled congenital and acquired TTP may display extensive placental ischemia and infarcts.[11] Thus, we believe that cTTP might promote the occurrence of preeclampsia and exacerbate the symptoms of terminal organ ischemia.

Low-dose aspirin is the most rigorously studied and commonly used drug for preventing or delaying the onset of preeclampsia by inhibiting placental thromboxane A2. Preeclampsia is associated with insufficient prostacyclin (a vasodilator) and the generation of thromboxane (a vasoconstrictor that promotes platelet aggregation). A 2019 Cochrane review of 60 trials (N = 36,716 women) concluded that the use of antiplatelet agents reduced the risk of proteinuric pre-eclampsia by 18%.[12] The American College of Obstetricians and Gynecologists recommends taking 81 mg of aspirin daily from 12 to 28 weeks of pregnancy, ideally starting before 16 weeks, and continuing until delivery.[13] NICE guidelines recommend high-risk women take 75 to 150 mg of aspirin daily from 12 weeks until delivery.[14] Chronic ADAMTS13 deficiency is a related cardiovascular risk factor for which long-term aspirin therapy can be considered, but it does not inhibit platelet aggregation induced by ultralarge VWF, as may occur in TTP. So, other existing cardiovascular risk factors should be managed strictly.[15] Pretreatment with aspirin or eptifibatide reduced thrombus size by blocking the activation of αIIbβ3 or the binding of fibrinogen to αIIbβ3 in mice.[16] In addition, aspirin may reduce the risk of stroke and other thrombotic events in patients with TTP during remission.[10] The prevention of preeclampsia with low-dose aspirin and appropriate timing of cesarean delivery may be important factors for the successful delivery of this patient.

One month after cesarean section, a reexamination showed that the patient's ADAMTS13 activity was 15.7% and platelet count was 334 × 10⁹/L. We believe that the invasive operation during the cesarean section caused damage to the vascular endothelium, enhanced ADAMTS13 consumption, and increased risk of thrombus formation. We further hypothesize that due to transport barriers in the patient's secretion of ADAMTS13, the function of ADAMTS13 could not be replenished in a short period after the huge consumption, thus resulting in a decrease in ADAMTS13 activity to 15.7% after the cesarean section. However, since the cause of pregnancy was removed and active prevention and timely treatment of preeclampsia were provided and the function of the patient's ADAMTS13 enzyme to cleave VWF was not lost, platelets returned to a normal level 1 month after the cesarean section.

Headache, drowsiness, and abdominal pain are recurring features of nonovert cTTP disease, which is highly sensitive to preventive treatment. Routine preventive treatment significantly reduces the incidence of cerebrovascular events in patients with cTTP. cTTP often presents in children and during pregnancy. In childhood-onset cTTP, infection is the most common trigger, while pregnancy is the most common trigger in adult-onset cTTP. The mutations in prespacer are usually associated with the early onset of the disease and exhibit more severe symptoms after onset.[17] We previously reported a case of 26-year-old parturient with cTTP, who harbored compound heterozygous mutations in the TSR1 and Cys Rich regions of the ADAMTS13 gene, which were both prespacer mutations and interfered with ADAMTS13's correct recognition and effective hydrolysis of VWF. The measured ADAMTS13 activity was particularly low (1.1%). At the 8th month of pregnancy, she had reddish urine and ecchymosis all over her body and developed hypertension and proteinuria during the late stage of pregnancy.[18]

The patient we present in this report had constant fatigue and gingival bleeding before pregnancy, experienced blurred vision during this pregnancy, and had stillbirths during two previous pregnancies, all of which may be due to the deficiency of ADAMTS13 enzyme. Although the ADAMTS13 activity was recorded at a moderate level, which is higher than the current threshold for diagnosis of TTP according to ISTH guidelines, we still tend to regard this case as a cTTP patient combining the clinical manifestations, results of biochemical tests and genetic screening findings. Firstly, the patient represented typical symptoms which usually appear in patients of mild TTP, including bleeding, microangiopathic hemolytic anemia, mental fatigue, and disease aggravation triggered by pregnancy, a common precipitating factor for the disorder. Secondly, the laboratory results were highly consistent with TTP. The blood count test showed decreased RBC count and extremely low platelet count, and blood smear observed anomalous RBCs and fragments. Except for a slight rise of fibrinogen and D-dimer, anticipated in pregnancy, the coagulation indicators (like PT, APTT, and TT) were within normal range. The increased free hemoglobin, cooperating with the lack of haptoglobin in plasma indicated hemolysis, while there was no evidence of autoimmune hemolytic anemia (negative for Coomb's assay). In addition, a high level of serum lactic dehydrogenase (LDH) was also observed. Finally, the most important evidence was that we identified compound heterozygous mutations in the ADAMTS13 gene of the case, which we believe account for the symptoms and laboratory abnormalities of the patient. Coincidentally, Subhan et al recently reported a similar case.[19] The patient experienced nosebleeds, fatigue, and headaches starting from 20 weeks of pregnancy, which are common “nonovert” symptoms of TTP, and developed jaundice and subsequently hypertension, proteinuria, and severe thrombocytopenia at the late stage of pregnancy. The patient possessed two heterozygous mutations and additionally harbored some common variants, with ADAMTS13 activity ranging from 24 to 44%. Taken together, our case highlights that in patients with moderate ADAMTS13 activity levels, the risk of acute TTP in the context of a triggering factor such as pregnancy cannot be excluded. From a clinical perspective, we recommend that patients presenting with the above “nonovert” symptoms or with findings of a decrease in platelets should have ADAMTS13 activity testing and receive appropriate treatment as necessary. We also suggest ADAMTS13 genetic testing, even if the ADAMTS13 activity is above the accepted cut-off for diagnosis of TTP, but still reduced, when there is clinical suspicion of TTP. Detection of ADAMTS13 gene variants can prevent misdiagnosis of such cTTP pregnant women and ensure timely plasma infusion therapy for parturient and fetal life safety.

Ethical Approval

The study protocol was approved by the Tongji Hospital Ethics Committee for Research in Health. Informed consent was obtained from individuals included in this study.

^* Junkun Chen and Ning Tang contributed equally to this article.

Publication History

Article published online:
19 September 2023

• References

• 1 Sadler JE. Pathophysiology of thrombotic thrombocytopenic purpura. Blood 2017; 130 (10) 1181-1188
• 2 Hovinga JAK, Coppo P, Lämmle B, Moake JL, Miyata T, Vanhoorelbeke K. Thrombotic thrombocytopenic purpura. Nat Rev Dis Primers 2017; 3: 17020
• 3 Joly BS, Coppo P, Veyradier A. Thrombotic thrombocytopenic purpura. Blood 2017; 129 (21) 2836-2846
• 4 Zheng XL, Vesely SK, Cataland SR. et al. ISTH guidelines for the diagnosis of thrombotic thrombocytopenic purpura. J Thromb Haemost 2020; 18 (10) 2486-2495
• 5 Cunningham FG, Lowe T, Guss S, Mason R. Erythrocyte morphology in women with severe preeclampsia and eclampsia. Preliminary observations with scanning electron microscopy. Am J Obstet Gynecol 1985; 153 (04) 358-363
• 6 Li J, Shi L, Zhang K. et al. VarCards: an integrated genetic and clinical database for coding variants in the human genome. Nucleic Acids Res 2018; 46 (D1): D1039-D1048
• 7 Ercig B, Wichapong K, Reutelingsperger CPM, Vanhoorelbeke K, Voorberg J, Nicolaes GAF. Insights into 3D structure of ADAMTS13: a stepping stone towards novel therapeutic treatment of thrombotic thrombocytopenic purpura. Thromb Haemost 2018; 118 (01) 28-41
• 8 Zheng X, Chung D, Takayama TK, Majerus EM, Sadler JE, Fujikawa K. Structure of von Willebrand factor-cleaving protease (ADAMTS13), a metalloprotease involved in thrombotic thrombocytopenic purpura. J Biol Chem 2001; 276 (44) 41059-41063
• 9 Ricketts LM, Dlugosz M, Luther KB, Haltiwanger RS, Majerus EM. O-fucosylation is required for ADAMTS13 secretion. J Biol Chem 2007; 282 (23) 17014-17023
• 10 Shao B, Nusrat S, George JN, Xia L. Aspirin prophylaxis for hereditary and acquired thrombotic thrombocytopenic purpura?. Am J Hematol 2022; 97 (08) E304-E306
• 11 Pishko AM, Levine LD, Cines DB. Thrombocytopenia in pregnancy: diagnosis and approach to management. Blood Rev 2020; 40: 100638
• 12 Duley L, Meher S, Hunter KE, Seidler AL, Askie LM. Antiplatelet agents for preventing pre-eclampsia and its complications. Cochrane Database Syst Rev 2019; 2019 (10) CD004659
• 13 ACOG Committee Opinion No. ACOG Committee Opinion No. 743: low-dose aspirin use during pregnancy. Obstet Gynecol 2018; 132 (01) e44-e52
• 14 Overton E, Tobes D, Lee A. Preeclampsia diagnosis and management. Best Pract Res Clin Anaesthesiol 2022; 36 (01) 107-121
• 15 Tarasco E, Bütikofer L, Friedman KD. et al. Annual incidence and severity of acute episodes in hereditary thrombotic thrombocytopenic purpura. Blood 2021; 137 (25) 3563-3575
• 16 Shao B, Hoover C, Shi H. et al. Deletion of platelet CLEC-2 decreases GPIbα-mediated integrin αIIbβ3 activation and decreases thrombosis in TTP. Blood 2022; 139 (16) 2523-2533
• 17 Alwan F, Vendramin C, Liesner R. et al. Characterization and treatment of congenital thrombotic thrombocytopenic purpura. Blood 2019; 133 (15) 1644-1651
• 18 Chen JK, Tang N, Wang X, Huang M, Zhang C. A case of parturient with hereditary thrombotic thrombocytopenic purpura: case report of a novel variant. Semin Thromb Hemost 2022; 48 (02) 256-261
• 19 Subhan MO, de Groot R, Scully M. Unusually high ADAMTS13 activity in a patient with congenital thrombotic thrombocytopenic purpura. Br J Haematol 2023; 200 (05) 673-675