Table of contents :

• quantitative alterations
• thrombocytopenia (< 100,000 / mL) => hemorrhages.
• deficitary thrombocytopoiesis
• bone marrow hypoplasia or aplasia
• congenital bone marrow hypoplasia or aplasia
• intrinsic aetiology : congenital thrombocytopenias represent a very small percentage of the thrombocytopenias that are seen by hematologists and oncologists. Even when chemotherapy-induced thrombocytopenia and overt infections are excluded and only isolated thrombocytopenia is considered, at least 95% of these cases in both children and adults will be primarily autoimmune thrombocytopenia (ITP) or drug-induced thrombocytopenia. However, more and more routine platelet counts are obtained allowing the identification of asymptomatic or mildly symptomatic children and adults, which increases the number of isolated thrombocytopenias referred to specialists. Even in adults, a small percentage of cases represent a type of congenital thrombocytopenia (CTP), and a number of cases of CTP have been misdiagnosed as ITP and the patients subjected to splenectomy and/or cyclophosphamide, among other inappropriate therapies^ref. Recent developments, especially in identification of molecular defects, have highlighted the manifestations of the congenital cases of nonimmune thrombocytopenia and improved the ability to identify and distinguish among them^{ref1, ref2}. Traditionally, the CTPs are divided into categories based on accessible information. Since blood smears are universally available, platelet size (as estimated on smear) is often considered to be the first point of triage. However, at least three additional triage points may be identified by history prior to review of the smear that would suggest CTP. One is a family history of "ITP." The second is the absence of an increase in the platelet count in response to ITP treatments. The third is the presence of certain "associated features" (see below) that are identified along with the thrombocytopenia. Some of these features, in the context of isolated thrombocytopenia, would suggest very specific diagnoses, e.g. thrombocytopenia with absent radii (TAR). Depending upon the specificity of the finding and the availability of specific testing, a suspected entity can then be confirmed or rejected by further testing, i.e., functional or molecular study, as needed.

Reasons to suspect hereditary thrombocytopenia :
• family history of thrombocytopenia, especially parent-child or maternal uncle-nephew. The most obvious feature suggesting CTP, while nonspecific, is the presence of a family history of thrombocytopenia. This should immediately raise suspicion of CTP, rather than ITP, especially if more than two family members are involved and/or the family members are closely related, especially parent and child or maternal uncle and nephew. Many types of CTP have an autosomal dominant inheritance, but several are X-linked or autosomal recessive, which means that the affected child or adult may be the propositus/index case. A family history of "ITP" has been reported, for example, in familial autoimmune disease in which the family history may be more consistent with systemic lupus (SLE). Based on anecdotal experience, the infrequent cases of familial ITP often appear coincidental, e.g., two cousins. In the past, cases of familial CTP have been considered (and reported) to be ITP because of the presence of platelet-associated antibodies using tests now known to have low specificity. While current testing that is antigen-specific (involves a specific platelet glycoprotein such as GPIIb/IIIa) has greater predictive value than tests in which the whole platelet is the target, it remains to be demonstrated that this testing will distinguish ITP from CTP, especially in those subsets in which there may be an autoimmune component
• lack of platelet response to autoimmune thrombocytopenia (AITP) therapies including IVIG, IV anti-D, steroids, and splenectomy and also immune modulating treatments, e.g., azathioprine, rituximab. In cases where it is necessary to distinguish CTP from ITP, the most definitive finding available by history or clinically, prior to specific laboratory investigation for CTP including review of the smear where diagnostic, is the "failure" to respond to ITP-specific therapy. This includes not only IVIG and IV anti-D, but also splenectomy and steroids. It also potentially includes other immune-modulating therapies such as azathioprine, cyclophosphamide, and anti-CD20. While the lack of response in patients with CTP to these treatments is a universally accepted criterion based on anecdotal experience, an exact definition of lack of response has not been established or verified. For example, there are no well-defined response thresholds, i.e., how high does the platelet count have to go after treatment with IVIG to diagnose ITP and exclude CTP? Arbitrarily, a peak platelet response to treatment > 30,000/µl increase from baseline would suggest that the case in question is not CTP. Conversely, a < 10,000/µl peak increase is compatible with CTP, although it just as compatible with a diagnosis of "refractory" ITP. Numbers in between might favor ITP but are even more ambiguous. A confusing feature is that some children with Wiskott-Aldrich syndrome and velocardiofacial syndrome (also known as DiGeorge syndrome) respond to corticosteroids, IVIG, and/or splenectomy either because there is an immunological component to the thrombocytopenia or because impeding the clearance mechanism for aberrant platelets helps to offset impaired platelet production. Since "spontaneous" fluctuation in the platelet count may occur (for example as a result of a viral infection), the assessment of two treatment responses is probably more helpful as a diagnostic criterion.
• diagnostic features on smear such as abnormal size of platelets (small, large, or giant); absence of platelet alpha granules (gray platelets); Döhle-like bodies (MYH9); or microcytosis (XLT-T). The fourth clinically available parameter involves the review of the peripheral blood smear. Newer automatic blood cell analyzers are superior to the previous generation in their recognition of platelets, especially large platelets, so that size measurements appear to be more reliable than they had been in the past. However, particularly for very small or very large platelets, and for very low platelet counts, the accuracy of the MPV obtained in standard laboratories remains to be determined and may vary from case to case, so that visual inspection of the smear remains the "gold standard" for platelet size in clinical practice. If platelets appear very large (the size of red cells or even larger), this would be compatible with Bernard-Soulier syndrome or the MYH9 defects, e.g., May-Hegglin syndrome. Döhle-like bodies in neutrophils also suggest May-Hegglin syndrome. The presence of very small platelets is most consistent with Wiskott-Aldrich, whether the complete syndrome or the XLT form. Platelet clumping may suggest von Willebrand type IIb, although pseudothrombocytopenia would need to be excluded either by using citrate as the anticoagulant or by making smears directly from a drop of blood. Microcytosis suggests the XLT-T form involving a mutation in the DNA face of GATA-1^ref
• bleeding out of proportion to the platelet count
• onset at birth
• associated features such as absent radii, mental retardation, renal failure, high tone hearing loss, cataracts, or the development of leukemia. Certain features, if identified in patients with persistent thrombocytopenia, suggest specific types of CTP, which can be classified in several different ways depending upon the other clinical features of the case and the laboratory techniques available to the physician. In certain types of CTP, the uniformity of the presence and heterogeneity in the associated features are still uncertain because < 10 cases have been described. These clinical features that might occur either in the patient or in a family member include (but are not limited to) presentation at birth, absence of radii, high-tone hearing loss, renal failure, bifid uvula, right-sided aortic arch, neonatal hematochezia, eczema, frequent (severe) infections, platelet dysfunction, and a family history of leukemia. Frequent infections may overlap with immune thrombocytopenias secondary to hypogammaglobulinemia
• persistence of a stable level of thrombocytopenia for years
Classification schemes for hereditary thrombocytopenias :
• location of molecular defect, i.e., can the diagnosis be confirmed as an MYH9 defect?
• mechanism of thrombocytopenia: poor production or accelerated destruction
• mode of inheritance: autosomal dominant or X-linked
• platelet size on smear: very large, normal, or small; other findings, e.g., microcytosis of RBC, Döhle-like bodies in neutrophils
• associated features including clinical and laboratory findings:
• findings on exam or by history, e.g., absent radii, renal
• failure, hearing loss
• laboratory abnormalities, e.g., flow cytometry for platelet glycoprotein expression, platelet function testing, assessment of the von Willebrand factor multimer composition
Classification of congenital thrombocytopenias including gene defect, inheritance, and associated features :

inherited thrombocytopenias		gene  (localization)	inherited  pattern	clinical and laboratory features
Wiskott-Aldrich syndrome (WAS)		WAS (Xp11)	X-L	Severe immunodeficiency.  Defective WAS protein.  Small platelets. Eczema
X-linked thrombocytopenia (XLT)		WAS (Xp11) Exon2	X-L	Mild immunodeficiency. Defective WAS protein. Small platelets
familial platelet disorder with  predisposition to acute  myelogenous leukemia (FPD/AML)		CBFA2 (21q22)	AD	Propensity to develop myelodysplastic  syndrome or acute myelogenous leukemia.  Normal platelet size. Dysfunctional platelets
amegakaryocytic thrombocytopenia (CAMT)		c-Mpl / TPO-R (1p34)	AR	Hypomegakaryocytic thrombocytopenia  evolving into bone marrow aplasia.  Normal platelet size
amegakaryocytic thrombocytopenia  with radio-ulnar synostosis (CTRUS)		HOXA11 (7p15-14)	AD	Reduced-absent megakaryocytes.  Radio-ulnar synostosis ± other malformations.  Possible sensorineural hearing loss.  Normal platelet size.
thrombocytopenia-absenti radii (TAR) syndrome		n.d.	AR	Thrombocytopenia usually severe in the first  year of life. Reduced megakaryocytes.  Bilateral radial aplasia ± other malformations.  Normal platelet size
Bernard-Soulier syndrome (BSS)		GPIba (17p13)  GPIbß (22q11)  GPIX (3q21)	AD	Defective GPIb/IX/V.  Homozygous subjects: defective ristocetin-  induced platelet agglutination (RIPA). Giant platelets.  Heterozygous subjects: mild thrombocyto-  penia, normal ristocetin-induced platelet  agglutination. Large platelets.
velocardiofacial syndrome (VCFS) / DiGeorge syndrome		1q22, 10 p4	AD	Right heart defect. Palate defect. T cell  immune deficiency. Evans syndrome.  Large platelets
von Willebrand disease type 2B (VW2B)		VW2B	AD	Platelet clumping. Abnormal (hyperreactive)  ristocetin-induced platelet agglutination
platelet type von Willebrand disease (PltVWF)		GP1ba (17p13)	AD	Dysmegakaryocytopoiesis. Large platelets.
benign Mediterranean  macrothrombocytopenia		n.d.	AD	Dysmegakaryocytopoiesis. Large platelets.
X-linked thrombocytopenia and  dyserythropoiesis with or  without anemia  X-linked thrombocytopenia-  thalassemia (XLTT)		GATA-1 (Xp11)	X-L	Anemia (mild to nil), unbalanced globin chain  synthesis resembling ß-thalassemia,  peripheral red cell hemolysis,  dysmegakaryocytopoiesis, splenomegaly.  Large platelets.
MYH9-related disease	May-Hegglin anomaly (MHA)	MYH9 (22q12-13)	AD	Neutrophil inclusions + giant platelets
	Sebastian syndrome (SBS)			neutrophil inclusions + giant platelets.
	Fechtner syndrome (FTNS)			neutrophil inclusions + hearing loss +  cataract + nephritis + giant platelets
	Epstein syndrome (EPTS)			hearing loss +  cataract + nephritis + giant platelets.
gray platelet syndrome (GPS)		n.d.	AD	pale platelets on blood films due to reduced-  absent -granules. Large platelets

Specific inherited thrombocytopenias :
• congenital amegakaryocytic thrombocytopenia (CAMT)

Aetiology : mutation in the THPO / c-mpl. In the absence of a signal from thrombopoietin (TPO), megakaryocytes do not proliferate.
Symptoms & signs : severe thrombocytopenia that is often recognized on day 1 of life or at least within the first month. CAMT is often initially confused with fetal and neonatal alloimmune thrombocytopenia, but the neonate fails to improve and responds only to platelet transfusion. Eventually a "diagnostic" bone marrow biopsy may be required which can be technically difficult in a neonate. 10-30% of cases have orthopedic or neurologic abnormalities. Intracranial hemorrhage is not rare (5 of 24 cases in one survey), and treatment other than platelet transfusions is largely ineffective. 50% of the 24 cases in the same survey progressed to aplastic pancytopenia within the first 5 years of life; 1 case of leukemia was also seen. In children with this disorder, aplastic anemia develops within 1-5 years after birth because of stem-cell exhaustion, consistent with the effects of TPO on HSCs
Therapy : while certain cytokines (IL-3, GM-CSF, IL-11) may have limited efficacy in individual patients, none are consistently effective and their use may result in substantial toxicity. TPO, or a thrombopoietic agent, seems unlikely to be of use because the underlying defect is a mutation in c-mpl. Platelet transfusions are administered for very low counts and as prophylaxis in patients who have had major bleeds. Matching strategies are generally pursued only in the context of developing refractoriness to leukocyte-reduced random donor units. The only effective treatment thus far has been allogeneic HSCT. An approach to gene therapy is being pursued in which insertion of a dimerized artificial TPO receptor is intended to convey a growth advantage to stem cells that take up and express the vector, allowing them to eventually replace the marrow.
• thrombocytopenia-absenti radii (TAR) syndrome is suggested by the finding of isolated, severe neonatal thrombocytopenia (similar to CAMT) along with characteristic physical anomalies (associated features). These are not limited to absent radii but also include other orthopedic abnormalities, e.g. of the ulna, humerus, and tibia^ref. For example, an isolated abnormality of the radii was only identified in 4 of 54 cases of TAR in a survey, whereas abnormalities of the ulna and knees occured in well over 50% of patients. Patients with TAR have a high incidence of serious bleeding including intracranial hemorrhage and gastrointestinal (GI) bleeding. However, unlike those with CAMT, patients with TAR tend to improve and their platelet counts increase with time. The dogma is that these patients will achieve normal platelet counts within 1 year of birth^ref. However, milder thrombocytopenia often persists and the platelets may fall again during adulthood. Signaling via the TPO receptor is abnormal, but the defect in the signaling pathway has not been defined
• thrombocytopenia absent corpus callosum, characterized by refractory thrombocytopenia, agenesis of the corpus callosum, hypoplastic cerebellum, abnormal facies (microcephaly, broad nasal root with upturned nose, small upper lip, and micrognathia), and developmental delay
• AMT with radial-ulnar synostosis (CTRUS) is a rare entity (3 cases reported) that behaves like TAR, including initially severe thrombocytopenia with subsequent improvement^ref. Initially, the forearm appears normal or subtle abnormalities may be detected. The diagnosis of CTRUS is made later when pronation-supination of the forearm is discovered to be restricted. Hox 11a was reported to be abnormal in the initial cases but at least 1 other case did not have this abnormality. A study of cases of TAR did not identify mutations in this or other Hox genes
• microcytosis and X-linked inheritance : there are 2 partially overlapping forms of familial thrombocytopenia: those with microcytic anemia, i.e., the XLT-T syndrome (X-linked thrombocytopenia– thalassemia (XLT-T)), and those that include dyserythropoiesis. Patients with both types of familial thrombocytopenia have large platelets that, in combination with the microcytic erythrocytes, distinguish them from XLT/WASp. The second type of XLT and anemia could be considered to be a form of myelodysplasia (MDS). These cases reflect the importance of GATA-1 in thrombopoiesis and erythropoiesis. The recent evaluation of a second family with the XLT–ß-thalassemia mutation suggests that anemia derived from mutations of GATA-1 that affect the binding site for FOG (Friend of GATA) is more severe than that derived from the one known mutation interfering with binding of GATA-1 to DNA.4 Fanconi's anemia may also present as thrombocytopenia with mild anemia. Furthermore, the other anomalies characteristic of Fanconi anemia (abnormal thumbs, failure to thrive, renal anomalies, etc.) may not be present or immediately apparent at the time of presentation
• Wiskott-Aldrich syndrome (WAS)or the XLT form of WAS classically have marked or severe thrombocytopenia and smaller than normal platelets. The only other entity in which such distinctly small platelets are found is in patients with TORCH (toxo-rubella-cytomegalovirus-herpes) infections, e.g. HHV-5 / CMV. In addition to severe thrombocytopenia, WAS is an important congenital immunodeficiency syndrome that includes an inability to make anti-polysaccharide antibodies, resulting in a predilection to pneumococcal sepsis. Eczema is common, although its relationship to the underlying defect is unclear. The platelets at birth may not appear small, possibly because of lack of splenic maturity, and newborns and very young infants may present with thrombocytopenia, milk allergy, and hematochezia. The XLT form seems to involve defects primarily in exon 2 of the WAS gene; these patients have minimal immunodeficiency. WAS/XLT is unusual in that the thrombocytopenia responds to splenectomy^ref, apparently for the same reasons that hereditary spherocytic anemia does: the defective platelets are no longer destroyed efficiently. The possibility that they make anti-platelet antibodies as part of the immunodeficiency state is also possible although unlikely in other than exceptional cases. Even when the thrombocytopenia is moderately severe, the risk of hemorrhage may be high because the platelet mass is low relative to the count and the platelets may also be dysfunctional. Treatment of the patient to increase the platelet count beyond what can be accomplished by platelet transfusion involves either splenectomy or allogeneic HSCT. In XLT or in patients with WAS for whom a HSCT cannot be performed, splenectomy is appropriate^ref. However, in WAS and even in XLT, there may be an increased risk of overwhelming postsplenectomy sepsis. Determining an adequate response to pneumococcal vaccine, either Pneumovax or Prevnar, is important, and careful antibiotic prophylaxis as well as monitoring antibody levels is mandatory to minimize the risk of postsplenectomy sepsis and IVIG may need to be given monthly. There is approximately a 10% cumulative incidence of lymphoma in either form, WAS or XLT, in those who have not undergone transplantation, which is very close to the mortality from allogeneic HSCT
• velocardiofacial syndrome (VCFS) / DiGeorge syndrome: another form of thrombocytopenia, with similarities to XLT, and the first of the large platelet syndromes to be discussed, is VCF^ref. VCF, like WAS/XLT, involves a variable clinical immunodeficiency but the thrombocytopenia is generally mild. Rightsided heart disease, neonatal hypocalcemia, cleft palate, neuro-psychologic issues, and not only ITP but also Evans syndrome suggest the possibility of VCF. VCF is associated with mutations in chromosome 1q22 and 10p4 and molecular diagnosis is available. Although several gene defects have been identified, the mutation is yet to be identified in others with a similar clinical syndrome. In VCF, as in WAS/XLT, two forms of thrombocytopenia are seen: one autoimmune and one hereditary. The autoimmune thrombocytopenia in VCF may respond to ITP therapies but is often severe and likely to be chronic. Evans syndrome may recur. Milder forms of VCF without prominent heart disease or hypocalcemia are often not identified until adolescence or adulthood for the following reasons:
• the immunodeficiency may be sufficiently subtle to be missed.
• the thrombocytopenia may be asymptomatic and therefore avert detection.
• the "heart disease" may be clinically silent, i.e., a right-sided aortic arch.
• either the oropharyngeal findings are subtle, such as a bifid uvula, or else a cleft palate was repaired in the first year of life and subsequently forgotten.
The nonimmune component of the thrombocytopenia may be linked to the Bernard-Soulier gene, resulting in marked macrothrombocytopenia; platelet function has not been well characterized but clinically has not seemed to be a major issue. The thrombocytopenia may have an autoimmune component. Underlying behavioral abnormalities that are associated with VCF may be initiated or worsened by corticosteroids administered to treat presumed autoimmune thrombocytopenia
• MYH9-related diseases : the most common forms of CTP are accompanied by macrothrombocytopenia, and among these the most common are a group now known collectively as the MYH9-related diseases^ref. What had been separate but overlapping syndromes (May-Hegglin anomaly (MHA), Fechtner syndrome (FTNS), Epstein syndrome (EPTS), and Sebastian syndrome (SBS)) have been shown in several laboratories to involve mutations of the gene that codes for myosin IIA. Myosin IIB is normally expressed in cell types other than platelets and neutrophils, which only express myosin IIA. While not yet precisely defined, there is consensus that the associated features of these overlapping syndromes involve the site of the mutation within the MYH9 gene. These associated features include leukocyte inclusions, renal failure, hearing loss, and cataracts. In the commonest form, May-Hegglin, Döhle-like bodies may be seen in neutrophils in addition to the very large (giant) platelets identified on peripheral smear. The platelet count varies and can be < 20,000/µL; however, the very large platelets often lead to the reporting of falsely low counts. Platelet function is generally preserved, and cases of these syndromes are frequently identified in asymptomatic patients.
• familial thrombocytopenia-leukemia (Tel-AML1) syndrome is important to identify because it is the thrombocytopenia most linked to malignancy. Fortunately, it has a somewhat unique presentation. The thrombocytopenia is usually mild, approximately 80–100,000/µL, but nonetheless signs and symptoms of bleeding are common. This is consistent with clinically significant platelet dysfunction, similar to the XLT form of WAS and Bernard-Soulier syndrome. Inheritance is autosomal dominant. Platelet function testing, where possible, reveals a storage pool disorder. Approximately half of the patients may go on to develop a malignancy, 2/3 of which are myeloid leukemia and 1/3 solid tumors. Recently, mutations in the transcription factor CBFA2 have been identified in two families with this syndrome
• Bernard-Soulier syndrome (BSS) should be considered when a patient presents with macrothrombocytopenia and bleeding out of proportion to the platelet count in the absence of other clinical or hematologic abnormalities. Bernard-Soulier syndrome is a result of the absence of the GPIb-V-IX complex on the platelet surface. This can occur either in the homozygous or heterozygous form, although clinically there may be some overlap in the clinical manifestations. In homozygotes, the platelets are comparable in size to those seen in the MYH9 syndromes. Automated platelet counts are often inaccurate because the platelets are so large. Epistaxis is relatively common. Diagnosis in the routine laboratory can be strongly suspected by lack of aggregation (agglutination) of platelets by "high dose" ristocetin in homozygotes. With this test result, the primary differential diagnosis is von Willebrand disease, which would be highly unlikely if the levels of von Willebrand factor (VWF) and the pattern of the VWF multimers were normal. Heterozygotes may be more difficult to identify. Flow cytometry to quantify platelet glycoproteins is confirmatory in homozygotes and diagnostic for heterozygotes if GPIb-V-IX is absent or greatly reduced in number. Milder variants, e.g. "Bolzano," have been described in which greater than expected amounts of the platelet glycoproteins are expressed
• Mediterranean macrothrombocytopenia is a disorder without associated features in which there is a defect in the demarcatory membrane system by which megakaryocytes divide up their cytoplasm into platelets. Fewer larger platelets are made and released, but it is thought that the overall platelet mass remains approximately normal
• von Willebrand disease 2B (VW2B) is an autosomal dominant disorder caused by the production of an abnormal VWF molecule with a propensity to form ultra-large multimers that bind more avidly to platelets than normal vWF and may promote platelet clumping: a gain of function mutation. Pseudothrombocytopenia may occur as a result of in vitro clumping. Affected individuals have no associated findings. The platelet count tends to fluctuate especially with stress and hormonal changes such as those associated with pregnancy. Postpartum hemorrhage may occur as the levels of VWF fall. Bleeding at times of menses may be heavy and other mucous membrane bleeding is seen. Desmopressin acetate (DDAVP) may result in more marked thrombocytopenia by increasing the level of the overly avid VWF multimers exacerbating platelet agglutination. The platelets may be normal in size or large. Assessment of multimer size distinguishes Type IIb vWF from the less common platelet type von Willebrand disease in which mutations in GPIb are responsible for the enhanced interaction with von Willebrand factor molecule
• Paris-Trousseau syndrome^{ref1, ref2}
• Paris-Trousseau-type thrombocytopenia (TCTP)
• Paris-Trousseau-type congenital dysmegakaryopoietic thrombocytopenia
• Jacobsen’s syndrome (JBS)
• thrombocytopenia 2 (THC2)
• gray platelet syndrome
• Montreal platelet syndrome
Therapy : management of patients with CTP is not well defined. There is no uniform approach to the treatment of patients with CTP who are bleeding or are to undergo surgery. The available agents include DDAVP; antifibrinolytics such as e-aminocaproic acid especially for mouth or nose bleeding; hormonal therapy to lessen menses; platelet transfusion; HSCT for severe cases, i.e., CAMT or homozygous Bernard-Soulier; and rFVIIa, which in the field of platelet disorders has been used most often with effect in Glanzmann thrombasthenia. The use of leukocyte-reduced platelets has lessened sensitization to platelets and development of refractoriness to platelet transfusion, but platelet transfusion should still be reserved for cases of true need. The dose of rFVIIa remains to be defined as only very anecdotal use has been reported. It may be in the range of 20–40 units/kg instead of the 90 units/kg used for hemophiliacs with inhibitors. Whether thrombopoietic agents will have a role in the future in at least some of these entities seems probable but entirely unexplored.
• extrinsic aetiology
• infective aetiology
• rubella virus
• HHV-5 / CMV
• HBV
• Toxoplasma gondii
• drugs used by mother during pregnancy (chlorothiazide, ethanol)
• acquired bone marrow hypoplasia or aplasia
• acquired idiopathic amegakaryocytic thrombocytopenia
• secondary amegakaryocytic thrombocytopenia
• infections
• drugs
• bone marrow aplasia
• autoimmune amegakaryocytic thrombocytopenia
• uneffective thrombocytopoiesis :
• avitaminoses :
• vitamin B₁₂deficit
• folate deficit
• Fe²⁺ deficit
• leukemias
• paroxismal nocturnal hemoglobinuria (PNH)
• congenital hereditary thrombocytopenia
• Wiskott-Aldrich syndrome (WAS)
• alterations in control factors :
• decreased TPO :
• congenital thrombocytopenia
• acquired thrombocytopenia
• HIV-1 (correlates with viral loads and CD4 lymphocytopenia)
• nephropathies
• hemodyalisis
• unstable regulation of thrombocytopoiesis :
• cyclic thrombocytopenia or tidal platelet dysgenesia
• purpura foeminarum cyclica
• Garcia's disease / cyclic thrombocytopenia (congenital in males)^ref
• increased peripheral platelet destruction
• immune-mediated peripheral thrombocytopenias
• autoantibodies
• autoimmune thrombocytopenic purpura (ATP) / idiopathic (or immunologic) thrombocytopenic purpura (ITP) / primary thrombocytopenic purpura / essential thrombocytopenia / Werlhof disease / morbus maculosus Werlhofii
• type II heparin-induced thrombocytopenia (HIT)
• alloantibodies
• in newborns : mother IgGs against human leukocyte antigens (HLA) or human platelet antigens (HPA-1a / PL^A1) inherited from father
• post-transfusion purpura (PTP) : adsorption of HPA or Ag-Ab complexes on platelets of the recipient, expecially in PL^A1- individuals which are recipient for PL^A1+ platelets. Cross-reactive allo-Ab
• drug-induced immune-mediated peripheral thrombocytopenias
• secondary non immune-mediated peripheral thrombocytopenias
• congenital non immune-mediated peripheral thrombocytopenias
• immature newborn
• newborn from mother affected by preeclampsia
• erythroblastosis fetalis or neonatorum / congenital or hemolytic anemia of newborn / hemolytic disease of the newborn (HDN)
• toxoplasmosis, other [viruses], rubella virus, HHV-5 / CMV, HSV (TORCH complex) syndrome
• cavernous hemangiomatosis / Kasabach-Merritt syndrome / hemangioma-thrombocytopenia syndrome : a blood disorder usually occurring in the first few months of life in which severe thrombocytopenia and other evidence of intravascular coagulation are accompanied by rapidly expanding hemangiomas of the trunk, extremities, and abdominal viscera, sometimes associated with bleeding and anemia. Bleeding is thought to be due to trapping and destruction of platelets within the tumor and depletion of circulating clotting factors
• acquired
• DIC
• thrombotic microangiopathies : thromboses in terminal arterioles and capillaries
• secondary
• neoplasms
• pregnancy : in 5% of pregnancies, usually asymptomatic, self-limiting after delivery and not affecting the newborn
• infections
• hemolytic uremic syndrome (HUS) / Gasser syndrome
• autoimmune reactions (not against platelets)
• weeks to months after exposure to :
• mitomycin C
• azathioprine
• inhibitors of the Ca²⁺-activated phosphatase, calcineurin (cyclosporine or tacrolimus [FK 506])
• quinine
• combinations of chemotherapeutic agents
• total-body irradiation
• allogeneic HSCT, kidney, liver, heart, or lung transplant^ref.
The microvascular thrombi may be either predominantly renal or systemic in these heterogenous entities
• primary
• thrombotic thrombocytopenic purpura (TTP) / Moschcowitz's disease

Epidemiology : annual incidence in the USA estimated at 3.7^ref to 11 cases per million people^ref. The change in diagnostic criteria (only microangiopathic hemolytic anemia and thrombocytopenia, without an apparent alternative cause) has resulted in an increase by a factor of 7 in the number of patients treated for TTP^ref
Aetiology :

ADAMTS13 plasma activity < 5% of normal		clinical presentation
ADAMTS13 mutations (Upshaw-Schulman syndrome (USS)) : 577C>T (R193W) 1342C>G (Q448E) 2017A>T (I673F) 2723G>A (C908Y) 3367C>T (R1123C) 1342C>G (Q448E) 414+1G>A : splice 686+1G>A : splice 1244+2T>G : splice	disease presentation in infancy/childhood	familial TTPref chronic relapsing TTP episodes at about 3-week intervalsref1, ref2, ref3
	disease presentation delayed
autoantibodies against ADAMTS13 (acquired idiopathic ("out-of-the-blue") TTP) occurs in adults and older childrenref1, ref2	transient	single episode TTP
	recurrent	recurrent (intermittent) TTP in 11–36% of patients following successful treatment
	thienopyridine-associated (a small fraction of patients treated for arterial thrombosis with the platelet adenosine diphosphate receptor-inhibiting thienopyridine drugs, ticlopidine or clopidogrel develop TTP within a few weeks after the initiation or therapyref)	ticlopidine/clopidogrel-TTP
ADAMTS13 transient production or survival (?) defect		acquired idiopathic TTP (?)
late in pregnancy or immediately after delivery (autoantibodies may also be present)		pregnancy-associated TTP

Pathogenesis : in 1982, "unusually large" (UL) VWF multimers found in plasma samples taken repeatedly from 4 patients with chronic relapsing TTP were proposed as the systemic agglutinating agents^ref. The patients described were believed to have defective ULVWF multimeric "processing"^ref. Convincing evidence for ULVWF multimers as the cause of platelet clumping in TTP has accumulated in the subsequent 22 years^ref. Monomers of VWF (280,000 daltons) are linked by disulfide bonds into multimers with varying molecular masses that range into the millions of daltons. Multimers of VWF are constructed within megakaryocytes and endothelial cells, and stored within platelet a-granules and endothelial cell Weibel-Palade bodies. Most plasma VWF multimers are derived from endothelial cells. Both endothelial cells and platelets produce VWF multimers larger than the multimers in normal plasma. These ULVWF multimers bind more efficiently than the largest plasma VWF multimers to the glycoprotein (GP) Ib components of platelet GPIb-IX-V receptors^{ref1, ref2}. The initial attachment of ULVWF multimers to GPIb receptors, and subsequently to activated platelet GPIIb-IIIa complexes, induces platelet adhesion and aggregation in vitro in the presence of elevated levels of fluid shear stress^ref. After retrograde secretion by endothelial cells, ULVWF multimers become entangled in subendothelial collagen, thereby maximizing the VWF-mediated adhesion of blood platelets to any subendothelium exposed by vascular damage and endothelial cell desquamation. An efficient "processing activity"^{ref1, ref2} in normal plasma prevents the highly adhesive ULVWF multimers that are also secreted antegrade into the vessel lumen from persisting in the bloodstream. The ULVWF "processing activity" is now known to be a specific VWF-cleaving metalloprotease present in normal plasma^{ref1, ref2, ref3}. The enzyme degrades ULVWF multimers by cleaving 842Tyr-843Met peptide bonds in susceptible A2 domains of VWF monomeric subunits. The VWF-cleaving metalloprotease is number 13 in a family of 19 distinct ADAMTS-type enzymes identified to date. The ADAMTS enzymes are numbered 1–20, but ADAMTS5 and ADAMTS11 have been found to be identical. ADAMTS13 is a disintegrin and metalloprotease with eight thrombospondin-1-like domains composed of an amino-terminal reprolysin-type metalloprotease domain followed by a disintegrin domain; a thrombospondin-1-like domain; a cysteine-rich domain containing an arginine-glycine-aspartate (RGD) sequence and an adjacent spacer portion; 7 additional thrombospondin-1-like domains; and 2 similar CUB domains at the carboxyl-terminal end of the molecule. CUB domains, found only in ADAMTS13 among the ADAMTS enzymes, contain peptide sequences present in Complement subcomponents C1r/C1s; embryonic sea Urchin protein egf; and BMP-1.

ADAMTS13 is a Zn²⁺- and Ca²⁺-requiring 190,000 dalton glycosylated protein that is encoded on chromosome 9q34 and produced predominantly in the liver. ADAMTS13 activity is inhibited in vitro by ethylenediaminetetraacetic acid (EDTA) and, therefore, functional assays of the enzyme are usually performed using plasma anticoagulated with citrate^{ref1, ref2, ref3, ref4, ref5, ref6}. Plasma anticoagulated with heparin, D-phenylalanylprolylarginyl chloromethyl ketone (PPACK), hirudin and other direct thrombin inhibitors, or even serum, may also be satisfactory for testing. ULVWF multimers are cleaved by ADAMTS13 as they are secreted in long "strings" from stimulated endothelial cells^{ref1, ref2}. The ULVWF multimeric strings may be anchored in the endothelial cell membrane to P-selectin molecules that are secreted concurrently with the ULVWF multimers from Weibel-Palade bodies^ref. Endothelial cells are stimulated to secrete ULVWF multimers by histamine, Shiga toxin, TNF-a, IL-8 and IL-6 in complex with IL-6 receptor^ref. CUB domains, as well as one or more of the thrombospondin-1-like domains, may be involved in binding the ADAMTS13 to ULVWF multimers as they are secreted by the endothelial cells. Specifically, ADAMTS13 enzymes may attach under flowing conditions to accessible A3 domains in the monomeric subunits of ULVWF multimers^ref, and then cleave Tyr 842–843 Met peptide bonds in adjacent A2 domains (Figure 2B). Partial unfolding of emerging ULVWF multimers by fluid shear stress may increase the efficiency of ADAMTS13 attachment to, or cleavage of, ULVWF multimers^ref. ADAMTS13 activity in normal and TTP plasma :
In normal individuals, ADAMTS13 enzyme molecules from the plasma attach to, and then cleave, unusually large von Willebrand factor (ULVWF) multimers that are secreted in long "strings" from stimulated endothelial cells.

The ULVWF multimeric strings may be anchored in the endothelial cell membrane to P-selectin molecules that are secreted concurrently with the ULVWF multimers from Weibel-Palade bodies. P-selectin molecules are retained in the endothelial cell membrane by a transmembrane portion. Each ADAMTS13 molecule may dock via one or both of its C-terminal CUB domains, possibly along with one or more thrombospondin-1-like domains, to exposed A3 domains in ULVWF monomeric subunits. The attached ADAMTS13 molecules then cleave Tyr 842–843 Met peptide bonds in the adjacent A2 domains of ULVWF monomeric subunits. The smaller VWF forms that circulate after cleavage do not induce the adhesion and aggregation of platelets during normal blood flow.

Absent or severely reduced activity of ADAMTS13 in patients with TTP prevents the timely cleavage of ULVWF multimers secreted by endothelial cells. Uncleaved ULVWF multimers induce the adhesion and subsequent aggregation of platelets in flowing blood. Congenital deficiencies of ADAMTS13 activity caused by gene mutations or acquired defects of ADAMTS13 caused by autoantibodies result in TTP.
Failure to degrade ULVWF multimers has long been suspected to cause the familial and acquired idiopathic types of TTP or predispose an individual to these disorders^{ref1, ref2} (Figure 2C). Critical experiments verifying this concept were reported in 1997–1998. In 1997, Furlan et al^ref described 4 patients with chronic relapsing TTP who had a deficiency of VWF-cleaving protease activity in plasma. Because no inhibitor of the enzyme was detected, the deficiency was ascribed to an abnormality in the production, survival or function of the protease. The following year, elegant papers by Furlan et al^ref and Tsai and Lian^ref elucidated the pathogenesis of the more common acquired idiopathic type of TTP. The acquired idiopathic patients had little if any plasma VWF-cleaving protease activity during acute episodes; however, the activity returned to normal upon recovery. An IgG autoantibody produced transiently against the enzyme accounted for the lack of protease activity during the acquired idiopathic TTP episodes^ref.

Patients with familial, chronic relapsing TTP almost always have ULVWF multimers in their plasma^{ref1, ref2} because they chronically have < 5% of normal plasma ADAMTS13—unless they have recently received plasma infusions. ULVWF multimers are also detectable in some patient plasma samples during acute episodes of acquired idiopathic TTP, but not after recovery^ref, due to transient inhibition of ADAMTS13 to < about 5% of normal only during acute TTP episodes^{ref1, ref2, ref3, ref4, ref5}. Severe deficiency of ADAMTS13 activity in TTP patient plasma often correlates with a failure to cleave ULVWF multimers as they emerge in long strings from the surface of stimulated endothelial cells^ref. ULVWF multimeric strings are anchored to the endothelial cells^{ref1, ref2} via P-selectin molecules, which have transmembrane domains and are secreted along with ULVWF multimers from Weibel-Palade bodies^ref. P-selectin molecules are predominantly retained in the endothelial cell membrane. Passing platelets adhere via their GPIb receptors to the long ULVWF multimeric strings anchored to P-selectin^ref. (Platelets do not adhere to the smaller VWF forms that circulate after cleavage of ULVWF multimers, perhaps because the binding site for platelet GPIb in VWF A1 domains is not exposed on smaller VWF forms^ref) Many additional platelets subsequently aggregate under flowing conditions, probably via their activated IIb-IIIa complexes, onto the ULVWF multimeric strings to form large, potentially occlusive, platelet thrombi. ULVWF multimeric strings are capable of detaching from endothelial cells in the absence of ADAMTS-13 activity, the presence of fluid shear stress, and the increasing torque generated by ULVWF-platelet adherence^{ref1, ref2}. The detached ULVWF-platelet strings may "embolize" to microvessels downstream and contribute to organ ischemia. The formation of ULVWF-platelet thrombi and emboli may account for the detection of ULVWF multimers in the plasma of familial TTP patients chronically and of acquired idiopathic TTP patients during acute episodes^{ref1, ref2, ref3}. Increased VWF antigen has been found by flow cytometry on platelets during episodes of familial or acquired TTP, and abundant VWF antigen (but not fibrinogen) has been observed by immunohistochemistry on platelet occlusive lesions in TTP^ref. The absent or severely reduced plasma ADAMTS13 activity in familial TTP^{ref1, ref2, ref3} is usually a consequence of homozygous (or double heterozygous) mutations in both of the ADAMTS13 alleles located at chromosome 9q34^ref. Mutations in familial TTP have been detected all along the gene, in regions encoding different domains^ref. In severe familial deficiency of ADAMTS13 activity, episodes of TTP usually commence in infancy or childhood. In others, however, overt TTP episodes do not develop for years (e.g., during a first pregnancy), if ever. These latter observations suggest that :
• in vivo ADAMTS13 activity on ULVWF multimers emerging from stimulated endothelial cells may exceed the estimates of plasma enzyme activity using in vitro non-physiologic assays
• accentuated secretion of ULVWF multimers by endothelial cells induced by estrogen or pro-inflammatory cytokines^ref is required to provoke TTP episodes in some patients with severe plasma ADAMTS13 deficiency.
Many patients with acquired idiopathic TTP also usually have absent or severely reduced plasma ADAMTS13 activity only during an initial episode or later recurrence^{ref1, ref2, ref3}.
Plasma ADAMTS13 activity in healthy adults ranges from about 50% to 178%. Activity is often reduced below normal in liver disease, disseminated malignancies, chronic metabolic and inflammatory conditions, pregnancy, and newborns^ref. With the exception of those peri-partum women who develop overt TTP, the ADAMTS13 activity in these conditions is not reduced to the extremely low values (< 5% of normal) found in most patients with familial or acquired idiopathic TTP. Neither bone marrow transplantation-associated thrombotic microangiopathy nor "classical" HUS is usually associated with absent or severely reduced plasma ADAMTS13 activity^{ref1, ref2}. The explanation for VWF abnormalities in the plasma of a few chemotherapy/transplant-associated thrombotic microangiopathy patients is not known.
A complete lack of ADAMTS13 in mice was a prothrombotic state, but it alone was not sufficient to cause TTP-like symptoms. The phenotypic differences of ADAMTS13 deficiencies between humans and mice may reflect differences in hemostatic system functioning in these species. Alternatively, factors in addition to ADAMTS13 deficiency may be necessary for development of TTP^ref. Genetic defects or environmental factors may stimulate endothelial activation or damage via TTP triggers, such as oxidative stress^ref, infection^ref, antiendothelial cell antibodies^ref, or complement dysfunction^{ref1, ref2}
Symptoms & signs : occlusive ischemia of the brain (=> neurologic abnormalities) or the gastrointestinal tract is common, and acute renal failure may occur; fever. Most TTP patients with congenital ADAMTS13 deficiency had their first acute episode in the newborn period or early infancy. Only a number of exceptional cases remain asymptomatic until adulthood^ref. Patients with identical ADAMTS13 genotypes, but different symptoms, have also been described^{ref1, ref2}, suggesting that the etiology of TTP cannot be explained by a single defect in ADAMTS13. Secondary triggering factors may promote the pathogenic platelet thrombus formation that results in TTP. The value of ADAMTS13 measurements for establishing the diagnosis of TTP and determining the indication for plasma exchange treatment remains uncertain. Although a severe deficiency of ADAMTS13 activity (< 5%) may be an abnormality that is specific for TTP^ref, the presenting symptoms of patients with severe ADAMTS13 deficiency are often not severe and not distinguishable from many other common illnesses. Patients can have the characteristic presenting features and clinical course of TTP without severe ADAMTS13 deficiency or even with normal ADAMTS13 activity (> 50%)^ref, and also patients can have severe ADAMTS deficiency for many years with no illness^ref. Patients with TTP and severe ADAMTS13 deficiency are heterogeneous, with remarkably variable presenting features^{ref1, ref2}. 9 of the 22 patients with severe ADAMTS13 deficiency (< 5%) in the Oklahoma TTP-HUS Registry had no neurologic abnormalities, not even mild symptoms such as headache and confusion. Nonspecific symptoms, such as weakness, abdominal pain, nausea, vomiting, and diarrhea, were common presenting complaints. Some patients had had symptoms for 3 weeks before their diagnosis; the median duration of symptoms prior to diagnosis was 6 days. The presenting symptoms of these patients were similar to patients diagnosed with idiopathic TTP-HUS but who did not have severe ADAMTS13 deficiency^ref.
Presenting features of 22 consecutive patients with severe deficiency (< 5%) of ADAMTS13 activity (data are presented on 22 consecutive patients from the Oklahoma TTP-HUS Registry, November 13, 1995-December 31, 2003, who had a severe deficiency of ADAMTS13 activity at the time of their initial presentation. This experience represents an extension of our previously published data^ref. The number of major presenting symptoms exceeds 22 because some patients had multiple major symptoms. The duration of symptoms describes the patient’s history of the initial onset of symptoms until diagnosis of TTP-HUS. Severe neurologic abnormalities included coma, stroke, seizure, or fluctuating focal signs; mild neurologic abnormalities included headache, blurred vision, ataxia, or mental status changes with transient confusion. Acute renal failure and renal insufficiency have been previously defined.3 Lactate dehydrogenase (LDH) values were adjusted to an upper limit of normal value of 200 U/L. Age, duration of symptoms, and laboratory data are median values. Laboratory data are the most abnormal values at the time of diagnosis of TTP-HUS ± 7 days, to account for transient effects of transfusion and potential worsening after diagnosis) :
• age : 39 years (range, 19–71)
• gender : 18 (82%) female
• race : 10 (45%) African-American
• obesity : 12 (55%) BMI >= 30 kg/m²
• presenting symptoms:
• neurologic abnormalities : 13 (59%)
• nausea, vomiting, diarrhea : 6 (27%)
• abdominal pain : 6 (27%)
• weakness : 4 (18%)
• chest pain : 3 (14%)
• hematuria : 2 (9%)
• menorrhagia : 1 (5%)
• purpura : 1 (5%)
• flank pain : 1 (5%)
• duration of symptoms 6 days (1–21)
• neurologic abnormalities:
• severe : 10 (45%)
• mild : 3 (14%)
• none : 9 (41%)
• renal function:
• acute renal failure 1 (5%)
• renal insufficiency 8 (36%)
• normal 13 (59%)
• platelet count : 9000/µL (range, 4000–27,000/µL)
• hematocrit : 21% (range, 13%–30%)
• LDH : 1431 U/L (range, 436–3909 U/L)
Patients with severe illnesses that can mimic the presenting clinical features of TTP, such as sepsis and preeclampsia, may have low but detectable levels of ADAMTS13^{ref1, ref2, ref3}. The clinical importance of less severe ADAMTS13 deficiencies is unknown. Furthermore, because of the long half-life of ADAMTS13 in plasma, estimated to be 2.6 days^ref, transfusion of red cells and platelets before the diagnosis of TTP is considered can increase plasma ADAMTS13 activity. Initial studies reported that a severe deficiency of ADAMTS13 distinguished syndromes described as TTP from the related syndromes described as HUS^ref. Although this distinction is valid in many patients, overlap of these syndromes can occur. Acute renal failure, the characteristic clinical feature that is often used to designate syndromes as HUS, may rarely occur in adults with acquired severe ADAMTS13 deficiency. Children with congenital TTP caused by mutations of the ADAMTS13 gene may have severe renal failure^{ref1, ref2}. Also children with typical HUS caused by E coli 0157:H7 infection may rarely have severe ADAMTS13 deficiency^ref.
Laboratory examinations :
• azotemia
• profound thrombocytopenia (with increased marrow megakaryocytes) < 20,000/µL
• schistocytes^ref >= 2 in a microscopic field with a magnification of 100 suggests microangiopathic hemolysis^ref, although in some cases, schistocytes are less frequent The RBC fragmentation occurs, presumably, as blood flows through turbulent areas of the microcirculation partially occluded by platelet aggregates. Schistocytes appear on the peripheral blood smear (> 1% of total RBCs) as an indication of microangiopathic hemolytic anemia
• serum levels of LDH are extremely elevated as a consequence of hemolysis and the leakage of LDH from ischemic or necrotic tissue cells
• normal serum creatinine levels; transient high levels may occur in one third of patients, and acute renal failure occurs infrequently^ref
• bone marrow aspirate : transient dyserythropoietic changes^ref
• clotting studies are usually normal : rarely, disseminated intravascular coagulation may be present in patients with TTP, presumably the result of tissue ischemia
• by 2001, ADAMTS13 had been purified and cloned by several groups (George JN, Sadler JE, Lämmle B. Platelets: thrombotic thrombocytopenic purpura. Hematology (Am Soc Hematol Educ Program). 2002:315–334). VWF is synthesized primarily by vascular endothelial cells. In the endoplasmic reticulum, VWF dimer formation occurs through disulfide bond formation near the C-termini. The pro-VWF dimers transit to the Golgi apparatus, where the pro-sequence is cleaved and multimers form by N-terminal disulfide bond formation. ULVWF multimers, disulfide-bonded at both N- and C-terminal domains, are formed in their mature form and stored in Weibel-Palade bodies or secreted into the plasma. ULVWF multimers are highly active in collagen binding and platelet aggregation. Under normal physiological conditions, ULVWF multimers depolymerize into smaller multimers ranging in size from 500 to 20,000 kDa. Depolymerization is catalyzed by a plasma metalloprotease, ADAMTS13, which specifically cleaves the peptide bond between Y1605 and M1606 in the A2 domain of VWF. Functional deficiency of ADAMTS13, caused by genetic mutation, inhibitory autoantibodies, or other etiologies, leads to the accumulation of ULVWF multimers in plasma. These multimers promote microvascular thrombi of platelets that result in platelet consumption and hemolysis. Once microvascular thrombi are formed in the brain or kidney, patients may suffer neurological dysfunction or renal failure. The ADAMTS13 precursor consists of 1427 amino acids and contains a signal peptide, a short propeptide, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin type I repeat (TSP1), a Cys-rich domain, a spacer domain, 7 additional TSP1 repeats, and 2 CUB domains (George JN, Sadler JE, Lämmle B. Platelets: thrombotic thrombocytopenic purpura. Hematology (Am Soc Hematol Educ Program). 2002:315–334). The metalloprotease domain of ADAMTS13 is necessary but not sufficient for VWF cleavage, which appears to require other domains as well. A mutant with a common SNP leading to the P475S substitution in the Cys-rich domain, found in Japanese populations with an allelic frequency of about 5%, showed reduced VWF cleavage, indicating a significant role for the Cys-rich domain^ref. The analysis of sequentially truncated C-terminal mutants suggests that both the Cys-rich and spacer domains are indispensable for VWF-cleaving activity^{ref1, ref2}. In addition, epitope analysis of ADAMTS13 autoantibodies in acquired TTP has shown that the Cys-rich and spacer domains usually are involved in antibody reactivity^{ref1, ref2}. Taken together, the Cys-rich and spacer domains seem to be essential for the VWF-cleaving activity of ADAMTS13. Although removal of the propeptide sequence in ADAM family members is usually essential for their enzymatic activity, pro-ADAMTS13 exhibited normal proteolytic activity toward VWF, indicating that propeptide removal is not required for the activity of ADAMTS13^ref. As of 2002, 5 main assays for ADAMTS13 activity were in use to study ADAMTS13 activity in thrombotic microangiopathies and other pathophysiological conditions. However, more accurate and simpler methods were needed :

	substrate	denaturant	incubation  time	detection method	indication of VWF proteolysis
Furlan et alref	purified VWF	1.5 mol/L  urea	overnight	SDS-agarose gel  electrophoresis,  immunoblotting	decreased  multimer size
Tsairef	purified VWF	0.15 mol/L  guanidine HCl	1 hour	SDS-polyacrylamide  gel electrophoresis,  immunoblotting	generation of  dimer of 176 kDa  fragment
Obert et alref	recombinant VWF	1.5 mol/L  urea	overnight	2-site immuno-  radiometric assay	decreased VWF  antigen
Gerritsen et alref	VWF in EDTA-  treated and  dialyzed plasma	1.5 mol/L  urea	2 hour	residual collagen  binding	decreased  collagen binding
Böhm et alref	purified VWF	1.5 mol/L  urea	overnight	residual ristocetin  cofactor activity	decrease of  ristocetin induced  platelet aggregation
Kokame et alref	recombinant VWF  fragment, VWF73	none	20–60 min	SDS-polyacrylamide  gel electrophoresis,  immunoblotting	generation of  proteolytic fragment
Whitelock et alref	VWF A2 domain	none	2 hour	ELISA	decrease of  intact A2 domain
Zhou and Tsairef	recombinant VWF  fragment, VWF73	none	3 hour	enzyme immunoassay	decrease of VWF73

• the first assay method was developed by Furlan et al^ref. The substrate for this method was protease-free plasma VWF. The plasma samples are first diluted, then activated by barium chloride, mixed with substrate and dialyzed overnight against low ionic strength buffer, pH 8.0, containing 1.5 M urea. The reaction products were separated by sodium dodecyl sulfate (SDS)–1.4% agarose gel electrophoresis followed by immunoblotting. The resolution of the ladders of the degraded VWF multimers was excellent and reproducible, but the method required several days to complete.
• the second method was reported by Tsai^ref. Plasma samples were incubated with guanidine HCl-treated protease-free VWF for one hour. Next, the products were separated by SDS–polyacrylamide gel electrophoresis, followed by immunoblotting for dimers of C-terminal 176-kDa fragments of VWF. An advantage of this method is that dimer formation directly reflects the cleavage of the scissile bonds.
• Obert et al reported an elegant and high-throughput method^ref, in which the plasma samples were incubated with recombinant VWF overnight and the degraded VWF fragments were detected by two-site immunoradiometric assay in microtiter plates. This test can be performed in the setting of a hospital laboratory and the cumbersome immunoblot technique is not required. In 102 healthy volunteers, the range (± 2 SD) of ADAMTS13 activity was 50–178%, where the mean was 114%.
• Gerritsen et al reported a functional assay based on the preferential binding of high molecular weight forms of VWF to collagen^ref. Plasma that was treated with EDTA and dialyzed against the buffer was used as substrate. Since EDTA is a chelator of divalent cations, EDTA treatment of plasma abolishes the VWF-cleaving activity. Depolymerized forms of VWF showed impaired binding to microtiter plates coated with collagen type III, and collagen-bound VWF was quantified using a specific anti-VWF antibody. In 177 healthy volunteers, ADAMTS13 activity was widely distributed, ranging from 40% to 170%.
• Böhm et al used the ristocetin-induced platelet aggregation activity to quantitate the residual VWF multimers for their ADAMTS13 assay^ref. This assay is based on the positive correlation between VWF multimer size and ristocetin cofactor activity of platelet aggregation. The method requires no special laboratory equipment or expertise. In 80 healthy volunteers, the ADAMTS13 activity ranged from 52% to 134%.
To evaluate the various assays for ADAMTS13 activity, a pilot multicenter comparison of the four different assays—the quantitative immunoblotting of VWF (Furlan’s method), the two-site immunoradiometric assay (Obert’s method), the residual collagen binding assay (Gerritsen’s method), and the residual ristocetin cofactor assay (Böhm’s method)—has been performed^ref. The test consisted of 30 plasma samples from patients with hereditary and acquired TTP and other conditions associated with ADAMTS13 levels ranging from < 3% to > 100%. All assays identified plasma with severe ADAMTS13 deficiency (< 5%), notwithstanding some exceptions observed using the collagen-binding assay, suggesting that all were useful to screen for suspected TTP. For samples with normal to moderately reduced activity, results were less concordant.
Specific minimum recombinant substrate for ADAMTS13 activity : coagulation proteases such as thrombin or factor Xa that are classified as "serine proteases" have a restricted substrate specificity recognizing short amino acid sequences on the N-terminal side of the scissile bonds. Based on these sequences, peptidyl substrates specific for many of these proteases were developed and utilized for activity assays. ADAMTS13 is a metalloprotease rather than a serine protease, and it cleaves a peptide bond between Y1605 and M1606 in the A2 domain of VWF. The development of peptidyl substrates for ADAMTS13 has been attempted but not achieved, suggesting that cleavage depends not only on specific residues in the close vicinity of the scissile bond, but also on more remote sequences in VWF. We utilized a recombinant protein approach to develop an ADAMTS13 substrate, using the amino acid sequence of the A2 domain of VWF that contains the scissile bond^ref. To identify the minimal region of VWF required for cleavage by ADAMTS13, we prepared 5 recombinant proteins, each containing a region of the A2 domain of VWF (Figure 3A). 2 criteria were set:
• the region must contain the cleavage site by ADAMTS13
• it must not contain Cys residues that often interfere with the proper folding of recombinant proteins.
The longest region that meets the criteria was D1459 to R1668 of VWF. For purification and detection, the protein was flanked with N-terminal glutathione-S-transferase (GST) and C-terminal 6xHis (H) tags. This was designated GST-D1459R1668-H (substrate A). We also prepared 4 shorter recombinant proteins (substrates B-E), as shown below :

(A) Structures of substrates A–E. The domain structure of human preproVWF is depicted. Five recombinant proteins flanked with GST- and H-tags were expressed for the ADAMTS13 assay. Substrates D and E were designated as GST-VWF73-H and GST-VWF64-H, respectively.
(B) Cleavage of recombinant proteins. The recombinant proteins were incubated with normal plasma at 37°C for 1 hour. The reaction products were run by SDS-polyacrylamide gel electrophoresis and the products were detected by anti-GST antibodies. The product bands are indicated by asterisks. The nonspecific bands of approximately 50 kDa are plasma albumin.
(C) Amino acid sequence of VWF73. Nine amino acid residues indispensable for cleavage are underlined.
(D) Effect of urea and ion concentration on cleavage. GST-VWF73-H was incubated with plasma for the indicated time in reaction buffer (5 mM Tris-HCl, 10 mM BaCl2, pH 8.0) or in the same buffer supplemented with 1.5 M urea or 150 mM NaCl. The products are marked by an asterisk.
(E) Cleavage of GST-VWF73-H by patients’ plasma. GST-VWF73-H was incubated with serially diluted normal plasma (0%–100%) or with plasma from patients with Upshaw-Schulman syndrome (USS), ticlopidine-associated thrombotic thrombocytopenic purpura (Tc-TTP), idiopathic TTP, or hemolytic uremic syndrome (HUS), and the products were detected by anti-GST antibodies^ref.
5 recombinant proteins were expressed in E coli and purified by affinity chromatography. The purified proteins were incubated with plasma for 1 hour at 37°C and the reaction mixture was electrophoresed on an SDS-polyacrylamide gel. The cleaved products were then detected by Western blot using anti-GST antibodies. 4 out of 5 recombinant proteins were successfully cleaved with plasma ADAMTS13; however, substrate E (the shortest) was not (Figure B). Therefore, the minimum substrate for ADAMTS13 was substrate D, which contains the region from D1596 to R1668 (73 amino acid residues). We designated substrates D and E as GST-VWF73-H and GST-VWF64-H, respectively. The amino acid sequence of substrate D is shown in Figure 3C. As described, substrate E was resistant to cleavage. Therefore, the region of nine amino acid residues (EAPDLVLQR) underlined in Figure 3C was likely essential for cleavage by ADAMTS13. Mass spectrometry analysis of both substrates, VWF73-H and VWF64-H, showed that their estimated molecular weights were exact, indicating no modification. To understand the substrate specificity of ADAMTS13, we determined the solution structure of ¹H and ¹⁵N double-labeled substrates VWF73-H and VWF64-H by nuclear magnetic resonance (NMR). The results indicated an extended structure for both peptides, suggesting an induced-fit substrate recognition mechanism. It was previously reported that ADAMTS13 cleaves VWF in vitro in the presence of urea and low ionic strength conditions. In hypotonic buffer containing 5 mM Tris-HCl and 10 mM BaCl₂, GST-VWF73-H was cleaved by plasma in a time-dependent manner (Figure D). The supplementation of 1.5 M urea or 150 mM NaCl reduced the cleavage, indicating low ADAMTS13 activity under these conditions. GST-VWF73-H was cleaved in a dose-dependent manner with high sensitivity. This assay was able to detect as little as 3% activity (Figure E)^ref. GST-VWF73-H was not cleaved by either plasma from patients with congenital deficiency of ADAMTS13 activity due to ADAMTS13 mutations (designated as Upshaw-Schulman syndrome [USS]) or plasma from patients with acquired TTP associated with ticlopidine (Tc-TTP). ADAMTS13 activity was observed in 4 patients with idiopathic TTP, but no activity was found in 5 other patients. All patients with hemolytic uremic syndrome (HUS) exhibited activity. Most patients with acquired TTP have autoantibodies that inhibit ADAMTS13 activity in their plasma. GST-VWF73-H was also useful for the detection of autoantibody when normal plasma was incubated with plasma from the patients with acquired TTP^ref. Another attempt at developing recombinant substrates has been reported in which the E coli–expressed A2 domain was used and proved to be a substrate for ADAMTS13^ref. There are several advantages to our method. First, ours is a direct assay for measuring ADAMTS13 product generation, which should provide a more accurate analysis than indirect methods that measure substrate depletion. Second, the bacteria-expressed protein avoids the need to make protease-free VWF. The recombinant protein is simple to prepare: we can produce 1 mg of pure GST-VWF73-H, sufficient for > 2000 assays, from 100 mL of E coli culture in 2 days. Third, the bacterial expression system is easily modified. For example, a mouse version of GST-VWF73-H was expressed to measure the mouse ADAMTS13 activity^ref. Fourth, having the substrate tagged with 2 different molecules makes it easy to modify the detection of product. For example, Zhou and Tsai used GST-VWF73-H to develop an ELISA system to measure ADAMTS13 activity in a 96-well format^ref. Finally, VWF73 will be used as the lead compound to develop more convenient and rational substrates for ADAMTS13 activity assays. One potential disadvantage of this method is that GST-VWF73-H is not a natural substrate. A recent report showed that ADAMTS13 may recognize the A3 domain of VWF^ref. Therefore, if the defects of the enzyme in patients with TTP affect the ADAMTS13 binding site for the A3 domain, cleavage of GST-VWF73-H will not reflect these defects. TTP is a life-threatening disease. If untreated, mortality may exceed 90%, but plasma exchange therapy significantly reduces mortality. Therefore, measurement of ADAMTS13 activity could be useful for early diagnosis, which is essential for successful treatment of an acute episode. For example, in July 2003, a Japanese pharmaceutical company began ADAMTS13 monitoring, using GST-VWF73-H, for the early diagnosis of patients with suspected TTP associated with ticlopidine. This approach may be effective in preventing the morbidity caused by drug-associated TTP. In addition, periodical ADAMTS13 measurements may be important for monitoring the therapeutic efficacy of plasma exchange or plasma infusion. Low levels of ADAMTS13 activity have been observed occasionally in several conditions, including heparin-induced thrombocytopenia, severe sepsis, and HUS^ref. But ADAMTS13 activity in these conditions is consistently detectable, and severe ADAMTS13 deficiency appears to be specific for TTP^{ref1, ref2}. Consequently, ADAMTS13 assays may help in the differential diagnosis of thrombocytopenia, especially when accompanied by microangiopathic changes. In particular, the discrimination of TTP from HUS can be an urgent issue for treatment. Therefore, an assay for ADAMTS13 activity with a narrow normal range is needed. Some groups currently developing more rapid and quantitative methods for determining ADAMTS13 activity. One of the goals is to develop a bed-side assay for ADAMTS13 activity, for the diagnosis of congenital or autoimmune TTP.
In current clinical practice, thrombocytopenia, schistocytosis, and an impressively elevated serum LDH value are sufficient to suggest the diagnosis^ref. The evaluation of patients with suspected TTP is difficult because the diagnostic criteria—(1) thrombocytopenia, (2) microangiopathic hemolytic anemia, and (3) no other clinically apparent etiology—are not specific^ref.
Moschcowitz's pentad: thrombocytopenia, microangiopathic hemolytic anemia (MAHA), fluctuating neurologic signs, renal failure, and fever (Moschcowitz E. Hyalin thrombosis of the terminal arterioles and capillaries: a hitherto undescribed disease. Proc N Y Pathol Soc. 1924;24: 21-24)
Evaluation of women during pregnancy is especially difficult. Although pregnancy is associated with TTP especially near term or post partum^{ref1, ref2}, signs characteristic of TTP may also occur in patients with severe preeclampsia, eclampsia, and the HELLP syndrome (hemolysis, elevated liver-enzyme levels, and a low platelet count)^ref.
Familial clustering is not sufficient for unambiguously diagnosing hereditary ADAMTS13 deficiency and congenital TTP, since hereditary predisposition to development of anti-ADAMTS13 IgG has been found^ref.
Differential diagnosis : malignant hypertension, severe preeclampsia, sepsis, and disseminated cancer
Therapy :
• plasma exchange : in 1977, Byrnes and Khurana^ref reported that relapses of TTP could be prevented or reversed by the infusion of only a few units of fresh frozen plasma or its cryoprecipitate-poor fraction (cryosupernatant), without concurrent plasmapheresis. It was shown in 1985 that the processing of ULVWF multimers was restored in patients with familial, chronic relapsing TTP by transfusing fresh-frozen plasma or cryosupernatant^ref. Tsai and Lian^ref and Furlan et al^ref demonstrated in 1998 that these plasma products, as well as solvent/detergent-treated plasma^ref, contain active ADAMTS13. The infusion about every 3 weeks of normal ADAMTS13 into familial TTP patients producing defective ADAMTS13 molecules^{ref1, ref2} is sufficient to prevent TTP episodes. The efficacy of plasma exchange for some categories of patients (e.g., those who have undergone allogeneic HSCT^{ref1, ref2}) is uncertain or unlikely. In adults who have thrombotic thrombocytopenic purpura after a prodrome of bloody diarrhea or acute, immune-mediated drug toxicity, evidence of any benefit of plasma-exchange treatment is limited to case series^{ref1, ref2}
• the plasma t_1/2 of infused ADAMTS13 activity is about 2 days, so perhaps ADAMTS13 molecules dock and cleave one secreted ULVWF multimeric string after another over a longer time period^{ref1, ref2, ref3}. The sequence of ADAMTS13 has been determined, and it has been partially purified^{ref1, ref2} and produced in recombinant active form^ref for ultimate therapeutic use.
• acetylsalicylic acid : platelet activation leading to microvascular thrombi is the main pathologic feature of TTP. Consequently, it is logical to complement plasma exchange and corticosteroids with inhibitors of platelet function. For this reason, aspirin was part of the standard treatment protocols in the 2 largest studies of plasma exchange^{ref1, ref2}. There have been reports of sudden deterioration and death among patients with TTP during recovery thrombocytosis because they were not taking platelet inhibitors^ref. Antiplatelet agents are usually not recommended for patients with TTP when bleeding is observed or when they also have severe thrombocytopenia, but it might be advisable for these patients to start treatment with aspirin once platelet counts begin to rise again in response to plasma exchange. Moreover, maintenance treatment with inhibitors of platelet function may prevent relapses of TTP^ref. However, a large case series^ref, published at the same time as the Canadian clinical trial, reported outcomes among patients who underwent plasma exchange without the use of acetylsalicylic acid that were similar to the outcomes among patients in the Canadian trial. A more recent randomized trial^ref reported a nonsignificant reduction in mortality when aspirin and dipyridamole were given in addition to plasma exchange and corticosteroids, as compared with plasma exchange and corticosteroids alone. Low-dose aspirin is recommended by the British Committee for Standards in Haematology for patients with platelet counts > 50,000/ml^ref
• gene therapy : because plasma ADAMTS13 of only about 5% is often sufficient to prevent or truncate TTP episodes^{ref1, ref2}, it may eventually extend remissions in familial, chronic relapsing TTP
• see also therapy for autoimmune TTP
Prognosis : although the value of ADAMTS13 measurements for establishing the diagnosis of TTP is uncertain, demonstration of severe ADAMTS13 deficiency and documentation of the inhibitor titer may help to anticipate the clinical course of patients with acquired TTP. 4 case series^{ref1, ref2, ref3, ref4} have reported lower mortality among patients diagnosed with TTP who had severe ADAMTS13 deficiency. In the 2 studies^{ref1, ref2} that distinguished patients who had idiopathic TTP from patients who had potential etiologies and associated conditions that could have contributed to their illness, the mortality rates were less in the patients with idiopathic TTP. These reports suggested that the high rates of mortality among patients who did not have severe ADAMTS13 deficiency may have been caused by other serious conditions, such as systemic infections. Among the 4 case series reporting mortality in patients with severe ADAMTS13 deficiency^{ref1, ref2, ref3, ref4}, overall mortality was 13%. Among the 3 case series reporting relapses in patients with severe ADAMTS13 deficiency, the frequencies of relapse were 10 of 44 (23%),15 6 of 16 (38%)^ref, and 7 of 16 patients (44%)^ref : data from 5 case series describing the occurrence of death and relapse among patients distinguished by severe deficiency or lack of severe deficiency of ADAMTS13 activity.
 

case series ADAMTS13 deficient (no. of patients)		ADAMTS13 deficient			ADAMTS13 not deficient
		total no.  patients	death  (no.)	relapse  (no.)	total no. patients	death  (no.)	relapse  (no.)
Veyradier et alref (63 of 111 patients who were described as having TTP rather than hemolytic uremic syndrome (HUS). Deaths were not reported. Patients were distinguished as "sporadic" or "intermittent." For this table, sporadic was assumed to be a single episode while intermittent was assumed to indicate the occurrence of relapses)		44	-	10	19	-	0
Mori et alref (18 of 27 patients who were described as having TTP rather than HUS. Relapses were not reported)		12	2	-	6	4	-
Raife et alref (107 consecutive patients described as having thrombotic microangiopathy; syndromes resembling TTP or HUS were not distinguished. Relapses were not reported)		50	4	-	57	10	-
Zheng et alref (all 37 patients were described as having TTP. Patients who were described as idiopathic had no apparent pre-existing illness. Patients described as non-idiopathic had had hematopoietic stem cell transplantation, were pregnant or postpartum, had systemic lupus erythematosis, or had taken FK506, mitomycin C, or clopidogrel.)	idiopathic (20)	16	3	6	4	0	1
	non-idiopathic (17)	0	-	-	17	10	1
Vesely et alref (185 patients who had ADAMTS13 measurements at the time of their initial presentation, representing 90% of the 206 patients enrolled in the Oklahoma TTP-HUS Registry, November 13, 1995-December 31, 2003. Data on 142 of these patients, up to June 30, 2002, have been previously published.3 Patients defined as idiopathic ha