Case report 401
Spontaneous hemarthrosis in combined Glanzmann thrombasthenia and type 2N von Willebrand disease Elena Pontaraa, Paolo Greseleb, Maria Grazia Cattinia, Viviana Daidonea, Giovanni Barbona, Antonio Girolamic, Ezio Zanona and Alessandra Casonatoa Glanzmann thrombasthenia is a rare autosomal recessive inherited bleeding disorder characterized by the lack of platelet aggregation, caused by deficiencies and/or abnormalities of platelet GPIIb-IIIa receptor. We report a case of Glanzmann thrombasthenia combined with type 2N von Willebrand disease (VWD), a variant characterized by an impaired capacity of FVIII to bind von Willebrand factor (VWF), which results in an autosomally transmitted reduction in circulating FVIII levels. Glanzmann thrombasthenia stems from compound T1214C and G1234A mutations in the ITGA2B gene; the type 2N VWD is due to a heterozygous G2561A mutation in the VWF gene (R854Q). The haemostatic phenotype of a 48-year-old female patient was unusually characterized by a severe chronic arthropathy with loss of cartilage and the presence of subchondrial cysts involving both ankles. The arthropathy was quantified with the compatible MRI scoring system (currently used to assess arthropathy in haemophilia), reaching almost the highest score. These haemorrhagic complications are very rare in Glanzmann thrombasthenia and resemble those seen in severe haemophilia; for such, a
Introduction Glanzmann’s thrombasthenia is a rare inherited platelet function disorder, associated with a normal or near normal platelet count. It is caused by a deficient or abnormal glycoprotein IIb-IIIa (GPIIb-IIIa), the platelet receptor for fibrinogen and other adhesive proteins, which is involved in platelet plug formation. Two main subgroups of Glanzmann’s thrombasthenia have been described: one associated with a purely quantitative GPIIb-IIIa defect and the other with an abnormal function [1–3]. von Willebrand disease (VWD) is the most common inherited bleeding disorder; it is caused by reduced levels or abnormalities of von Willebrand factor (VWF), a large multimeric glycoprotein involved in platelet adhesion and aggregation at the site of vessel wall injury [4]. VWF binds extracellular matrix, platelet GPIb and platelet GPIIb-IIIa. VWF also acts as a carrier of circulating factor VIII, ensuring the latter’s stability and guaranteeing the required concentration of FVIII at the site of vascular injury. Depending on the nature of the VWF defect, VWD is classified as type 1 in the case of a quantitative VWF defect or type 2 when there is an abnormal VWF function 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
reason we decided to explore the patient’s FVIII and VWF parameters. Our findings suggest that the type 2N R854Q mutation, which is normally asymptomatic at the heterozygous level, may be expressed in the presence of a combined impairment of primary haemostasis. Blood Coagul Fibrinolysis 25:401–404 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Blood Coagulation and Fibrinolysis 2014, 25:401–404 Keywords: Glanzmann thromboasthenia, type 2N von Willebrand disease, von Willebrand factor, von Willebrand factor:FVIIIB a Department of Cardiologic, Thoracic and Vascular Sciences, University of Padua Medical School, Padua, bDepartment of Internal Medicine, University of Perugia, Perugia and cDepartment of Medicine, University of Padua Medical School, Padua, Italy
Correspondence to Alessandra Casonato, Department of Cardiologic, Thoracic and Vascular Sciences, via Ospedale Civile 105, Padova, Italy Tel: +39 049 8213012; fax: +39 049 657391; e-mail: [email protected] Received 5 September 2013 Revised 30 October 2013 Accepted 4 December 2013
[5]. Among patients with type 2 VWD, some have type 2N, which is a variant characterized by a VWF with a defective FVIII carrier function [6,7]. With the exception of type 2N, all forms of VWD involve mucocutaneous bleedings (especially epistaxis, gingival bleeding, easy bruising and menorrhagia) that typically become promptly manifest after a trauma; the bleeding symptoms in type 2N resemble haemophilia A, with haemarthrosis and intramuscular haematomas. In Glanzmann’s thrombasthenia, the related bleeding symptoms are very similar to those of VWD, due to an impaired primary haemostasis [8]. Here, we report on a case of combined Glanzmann’s thrombasthenia and type 2N VWD, focusing on the finding of spontaneous haemarthrosis, which is uncommon in Glanzmann’s thrombasthenia and never seen in cases heterozygous for the type 2N VWF defect.
Materials and methods All individuals involved were studied after obtaining their written informed consent in accordance with the Helsinki Declaration and the study was approved by our institutional review board. DOI:10.1097/MBC.0000000000000067
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
402 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 4
Haemostatic tests
Basic haemostatic analyses, plasma VWF antigen (VWF:Ag), VWF collagen binding (VWF:CB), VWF ristocetin cofactor (VWF:RCo), ristocetin-induced platelet aggregation (RIPA), factor FVIII (FVIII) and capacity of VWF to bind FVIII (VWF:FVIIIB) were conducted as described elsewhere [9]. Anti-FVIII was sought using the Bethesda method. The search for anti-VWF antibodies was done with a home-made ELISA method, using coated purified human VWF and anti-IgG-HRP (Dako, Glostrup, Denmark) as a second antibody. The measure of a2-antiplasmin activity was performed by means of a chromogenic method (Siemens, Marburg, Germany). Genetic analysis
Genomic DNA was extracted from peripheral blood leukocytes using the QIAampDNA Blood Mini kit (Qiagen, Hilden, Germany). Exons 18, 19 and 20 of the VWF gene were amplified and sequenced as previously reported [10]. All PCR reactions were obtained using the AmpliTaq Gold (Applied Biosystems, AB, Foster City, California, USA) and a thermal cycler GeneAmpPCR System 2700 (AB). The Big Dye Terminator Sequencing kit v.2.5 (Perkin Elmer, Wellesley, Massachusetts, USA) and an ABI 3100 Genetic Analyser (AB) were used for DNA sequencing. Identification of GPIIb-IIIa gene mutations was performed by Professor M. Margaglione (Foggia, Italy).
Results Case report
The proband is a 48-year-old woman with a lifelong history of bleeding. She has suffered from epistaxis, gingivorrhagia, easy bruising, menorrhagia, bleeding after tooth extraction, haemorrhage after surgery and haemarthrosis. Her parents were not blood-related and nobody in her family had a bleeding tendency. The patient scored 18 with the Condensed MCMDM-1 Bleeding Questionnaire for VWD [11], as opposed to no more than 4 in normal individuals. When she was admitted to our Center, she presented with a chronic bilateral ankle arthropathy. Standard rheumatic tests (i.e. reactive protein C, ESR, RT and ANA) were found to be normal. MRI showed numerous sites of ankle joint damage, with moderate effusion of the tibioastragalar joint and hemosiderin deposits, loss of cartilage and subchondrial cysts. The right ankle showed an uneven loss of cartilage height associated with subchondrial erosion at the calcaneocuboid joint. Left knee demonstrated oedema and hyperhemia of the spongeous tissue. There was no evidence of any significant alterations in the right knee. The Compatible MRI scoring system for assessing haemophilic arthropathy was used to measure arthropathy of the patient with Glanzmann’s thrombasthenia [12,13]. Using the P-scale, based on a method that measures the
most severe joint abnormality, the patient with Glanzmann’s thrombasthenia achieved the highest score for arthropathy in the left ankle [10] on a scale of 0–10 and 9 for the right ankle (Table 1), indicating that both joints were severely damaged.
Haemostatic and genetic data
The patient’s main haemostatic findings are summarized in Table 2. There was evidence of prolonged filtroaggregation (PFA-100) and failure of ADP, collagen and adrenalin to induce platelet aggregation, but ristocetininduced platelet aggregation (RIPA) was normal. Platelet surface glycoprotein analysis by flow cytometry revealed a severe aIIbb3 deficiency (Table 3). The integrin alphaIIb subunit gene (ITGA2B) sequencing showed that the patient was a compound heterozygote for the T1214C and G1234A mutations that induce the I374T and G381R substitutions, respectively; these mutations account for patient’s Glanzmann’s thrombasthenia. The other haemostatic parameters investigated were normal, except for a slight reduction in VWF:FVIIIB, in absolute terms (54.5 vs. normal 65–150 U/dl), and a greater reduction in VWF:FVIIIB/VWF:Ag ratio (VWF:FVIIIB ratio) (0.62 vs. normal >0.75) (Table 2). No anti-FVIII and anti-VWF antibodies were detected, and normal were the levels of fibrinogen, FIX, FXI, FXIII and a2-antiplasmin activity. Despite the normal aPTT and FVIII values, the lower VWF:FVIIIB and VWF:FVIIIB ratio prompted us to surmise that the patient was carrying a type 2N VWF defect at the heterozygous level [14]. To verify this hypothesis, we analysed the exons 18–19–20 of the VWF gene, encoding for the FVIII-binding domain of VWF. We found that, at heterozygous level, the patient carried the G2561A mutation in exon 20, which induces the substitution of an arginine by a glutamine at position 854 of VWF. This is the most common mutation associated with type 2N VWD [14]. No other mutations were searched for in the remaining VWF gene.
Discussion A number of bleeding symptoms are reported in patients with Glanzmann’s thrombasthenia, having purpura, epistaxis, gingival haemorrhage, menorrhagia as almost constant features, although it is rare or very rare to observe haemarthrosis. Here, we report on a patient with Glanzmann’s thrombasthenia, characterized by spontaneous MRI-based score for arthropathy in our patient with Glanzmann thrombasthenia
Table 1 Joints
Ankle (left) Ankle (right) Knee (left) Knee (right)
Score 10 9 4 0
Most severe joint abnormality Full-thickness loss of joint cartilage Partial loss of cartilage thickness Synovial hypertrophy –
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
I374T and G381R
Flow cytometric analysis of the main antigens expressed on the platelet surface of our patient with Glanzmann thrombasthenia
Table 3
Collagen-epinephrine cartridges. b Platelet aggregation induced by ADP, collagen and adrenalin. c Ristocetin-induced platelet aggregation at 1.2 mg/ml ristocetin. d Integrin alphaIIb subunit gene.
Parameters
a
R854Q 0.62 >0.75 100 60–130 58.5 60–84 105 70–100
32 30–40
156 150–350
>300 94–193
Absent Present
74.9 60–160
87.6 60–160
104 60–130
54.5 65–150
VWF gene mutation VWF.RCo (U/dl) RIPA %c PT %
aPTT (s)
Platelet count (103/ml)
PFA100 (s)a
Platelet aggregationb
Patient Normal range
Table 2
Patient’s pertinent haemostatic and genetic findings
FVIII (U/dl)
VWF:Ag (U/dl)
VWF:CB (U/dl)
VWF:FVIIIB (U/dl)
VWF:FVIIIB/VWF:Ag ratio
ITGA2B gene mutationsd
Glanzmann’s thrombasthenia and type 2N VWD Pontara et al. 403
CD41(SZ22)(GPIIb/IIIa)/CD42a CD41(P2)/CD42a CD41(A2A9/6)/CD42a CD61 (SZ21)(GPIIIa)/CD42a CD61 (SAP) /CD42a Binding PAC1 (MFI) con ADP 10 mmol/l
Results
Normal values
0.076 0.061 0.046 0.07 0.076 2.3
1 0.33 0.15 0.026 0.72 0.08 0.91 0.13 0.63 0.047 12.7 2.5
haemarthrosis, associated with a type 2N VWF defect at heterozygous level. The spectrum of haemorrhagic symptoms in Glanzmann’s thrombasthenia was analysed in 177 patients with Glanzmann’s thrombasthenia and showed that only 3% of the patients (five out of 177) suffered from haemarthrosis [8]. One of them suffered from trauma-induced haemarthrosis, although the circumstances of the other four were not described [8]. Another report [15] described cysts in periarticular joint of a patient with recurrent haemarthrotic episodes. More recently, a Japanese patient with Glanzmann’s thrombasthenia was described who suffered from haemarthrosis of the left knee, which developed after an outdoor sports trauma, but no bone lesions on MRI [16]. Joint disease is one of the most common complications of haemophilia, affecting up to 90% of the severe forms. Haemophilic arthropathy is caused by recurrent episodes of bleeding into the joint, which correlate with the severity of the FVIII or FIX defect; such episodes can be prevented by replacement therapy. The precise mechanisms of haemophilic arthropathy are unknown, but it is well known that synovial changes precede cartilage changes over the course of haemophilic arthropathy. The patient with Glanzmann’s thrombasthenia described here showed a typical haemophilic arthropathy, characterized by loss of cartilage and the presence of cysts and subchondrial erosions that were apparently not the consequence of traumas. The arthropathy was severe enough to make it difficult for the patient to walk. Her haemostatic picture appeared to be the result of a compound haemostatic defect, that is Glanzmann’s thrombasthenia, associated with an impaired primary haemostasis and type 2N VWD, which damages the capacity of VWF to bind FVIII and the coagulation cascade. Patients homozygous or compound heterozygous for type 2N VWD are characterized by a phenotype very similar to that of haemophilia A, while heterozygotes for type 2N VWD (and those associated with R854Q mutations as in our patient) are asymptomatic. Heterozygotes for the R854Q mutation also have an almost normal haemostatic laboratory, with the exception of the VWF:FVIIIB test, and the VWF:FVIIIB ratio in particular [14,17]. The association
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
404 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 4
between Glanzmann’s thrombasthenia and VWD has already been reported, taking into account that VWD is the most common inherited bleeding disorder [18,19], which involved quantitative VWF defects (type 1 and type 3 VWD). Spontaneous bleeding is uncommon in Glanzmann’s thrombasthenia, and in this disorder, the most serious haemorrhagic episodes are due to trauma or an excessive physiological bleeding, such as menorhagies. Haemarthrosis was spontaneous in our patient and the arthropathic picture seemed to be consistent with recurrent episodes. An extensive analysis of FVIII and VWF (currently not performed in patients with Glanzmann’s thrombasthenia) in our patient was prompted by the finding of her severe arthropathy. We were seeking a decrease in FVIII levels or the presence of a FVIII inhibitor or an anti-VWF antibody, or both. In the end, we found that the patient’s VWF had only a lower FVIII binding capacity. It remains to be clarified why such a mildly impaired FVIII defect or FVIII–VWF interaction could cause a bleeding complication typical of severe forms of haemophilia A or B. It may be that the association of Glanzmann’s thrombasthenia with type 2N VWD causes the appearance or the worsening of bleeding symptoms typical of the latter condition at homozygous level. In the light of our results, it might be reasonable to consider that other reported patients with Glanzmann’s thrombasthenia with recurrent haemarthrosis might also have been carrying a type 2N VWF defect or a mild haemophilia [8,15]. In this case, the search for FVIII and VWF abnormalities should be performed.
References 1
2
3
4
5
6
7
8 9
10
11
12
The lesson that we can learn is that, when the severity or nature of Glanzmann’s thrombasthenia patients’ bleeding symptoms cannot be justified on the basis of the platelet GPIIb-IIIa defect, we have to seek another, associated haemostatic abnormality.
13
Acknowledgements
15
We thank Dr F Sartorello for her contribution to the genetic tests, and Dr G Saggiorato for the searching of anti-FVIII antibodies.
14
16
17
This work was supported by a grant from the Ministero dell’Universita’ e della Ricerca Scientifica e Tecnologica (MURST, 60%, 2010).
18
Conflicts of interest
19
The authors declare no conflict of interests.
Glanzmann E. Hereditare Hamorrhagische Thromboasthenic. Ein Beitrag zur Pathologie der Blutplattchen. [Inherited haemorrhagic thrombasthenia]. Jarbuch der Kinderheikunde 1918; 88:1–42. Larrieu MJ, Caen JP, Meyer DO, Vainer H, Sultan Y, Bernard J. Congenital bleeding disorders with long bleeding time and normal platelet count. II. Von Willebrand’s disease (report of thirty-seven patients). Am J Med 1968; 45:354–372. Phillips DR, Jenkins CS, Luscher EF, Larrieu M. Molecular differences of exposed surface proteins on thrombasthenic platelet plasma membranes. Nature 1975; 257:599–600. Ruggeri ZM. Structure of von Willebrand factor and its function in platelet adhesion and thrombus formation. Best Pract Res Clin Haematol 2001; 14:257–279. Sadler JE, Budde U, Eikenboom JC, Favaloro EJ, Hill FG, Holmberg L, et al., Working Party on von Willebrand Disease Classification. Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand factor. J Thromb Haemost 2006; 4:2103–2114. Mazurier C, Dieval J, Jorieux S, Delobel J, Goudemand M. A novel von Willebrand factor (vWF) defect in patient with factor VIII (FVIII) deficiency but with normal levels and multimeric patterns of both plasma and platelet vWF. Characterization of abnormal vWF/FVIII interaction. Blood 1990; 75:20–26. Casonato A, Pontara E, Sartorello F, Gemmati D, Cattini MG, Girolami A. Combined haemophilia A and type 2N von Willebrand disease: defect of both factor VIII level and FVIII binding capacity of von Willebrand factor. Haematologica 2001; 86:1110–1111. George JN, Caen JP, Nurden AT. Glanzmann’s thromboasthenia: the spectrum of clinical disease. Blood 1990; 75:1383–1395. Casonato A, Pontara E, Zerbinati P, Zucchetto A, Girolami A. The evaluation of FVIII binding activity of von Willebrand factor by means of an ELISA method: significance and clinical implications. Am J Clin Pathol 1998; 109:347–352. Casonato A, Sartorello F, Cattini MG, Pontara E, Soldera C, Bertomoro A, et al. An Arg760Cys in the consensus sequence of von Willebrand factor propeptide cleavage site is responsible for a new von Willebrand disease variant. Blood 2003; 101:151–156. Bowman M, Mundell G, Grabell J, Hopman WM, Rapson D, Lillicrap D, et al. Generation and validation of the Condensed MCMDM-1VWD Bleeding Questionnaire for von Willebrand disease. J Thromb Haemost 2008; 6:2062–2066. Lundin B, Babyn P, Doria AS, Kilcoyne R, Ljung R, Miller S, et al. Compatible scales for progressive and additive MRI assessments of haemophilic arthropathy. Haemophilia 2005; 6:658–663. Doria AS, Babyn S, Lundin B, Kilcoyne RF, Miller S, Rivard GE, et al. Reliability and construct validity of the compatible MRI scoring system for evaluation of haemophilic knees and ankles of haemophilic children. Expert MRI working group of the International Prophylaxis Study Group. Haemophilia 2006; 12:503–513. Casonato A, Pontara E, Sartorello F, Cattini MG, Perutelli P, Bertomoro A, et al. Identifying carriers of type 2N von Willebrand disease: procedures and significance. Clin Appl Thromb Hemost 2007; 13:194–200. Klofkorn R, Lightsey A. Hemarthrosis associated with Glanzmann’s thromboasthenia. Arthritis Rheum 1979; 22:1390–1393. Urakawa H, Nishida Y, Tsukushi S, Katsumi A, Ishiguro N. Glanzmann thromboasthenia detected because of knee hemarthrosis: a case report. J Ped Orthop 2010; 19:521–523. Casonato A, Daidone V, Barbon G, Pontara E, Di Pasquale I, Gallinaro L, et al. A common ancestor more than 10 000 years old for patients with R854Q-related type 2N von Willebrand’s disease in Italy. Haematologica 2013; 98:147–152. Ahmad F, Kannan M, Kishor K, Saxena R. Coinheritance of severe von Willebrand disease with Glanzmann thromboasthenia. Clin Appl Thromb/ Haemost 2010; 16:529–532. Nounou R, Spence D. Glanzmann’s thromboasthenia with von Willebrand’s disease. J Clin Pathol 1993; 46:1134–1136.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Spontaneous hemarthrosis in combined Glanzmann thrombasthenia and type 2N von Willebrand disease Elena Pontaraa, Paolo Greseleb, Maria Grazia Cattinia, Viviana Daidonea, Giovanni Barbona, Antonio Girolamic, Ezio Zanona and Alessandra Casonatoa Glanzmann thrombasthenia is a rare autosomal recessive inherited bleeding disorder characterized by the lack of platelet aggregation, caused by deficiencies and/or abnormalities of platelet GPIIb-IIIa receptor. We report a case of Glanzmann thrombasthenia combined with type 2N von Willebrand disease (VWD), a variant characterized by an impaired capacity of FVIII to bind von Willebrand factor (VWF), which results in an autosomally transmitted reduction in circulating FVIII levels. Glanzmann thrombasthenia stems from compound T1214C and G1234A mutations in the ITGA2B gene; the type 2N VWD is due to a heterozygous G2561A mutation in the VWF gene (R854Q). The haemostatic phenotype of a 48-year-old female patient was unusually characterized by a severe chronic arthropathy with loss of cartilage and the presence of subchondrial cysts involving both ankles. The arthropathy was quantified with the compatible MRI scoring system (currently used to assess arthropathy in haemophilia), reaching almost the highest score. These haemorrhagic complications are very rare in Glanzmann thrombasthenia and resemble those seen in severe haemophilia; for such, a
Introduction Glanzmann’s thrombasthenia is a rare inherited platelet function disorder, associated with a normal or near normal platelet count. It is caused by a deficient or abnormal glycoprotein IIb-IIIa (GPIIb-IIIa), the platelet receptor for fibrinogen and other adhesive proteins, which is involved in platelet plug formation. Two main subgroups of Glanzmann’s thrombasthenia have been described: one associated with a purely quantitative GPIIb-IIIa defect and the other with an abnormal function [1–3]. von Willebrand disease (VWD) is the most common inherited bleeding disorder; it is caused by reduced levels or abnormalities of von Willebrand factor (VWF), a large multimeric glycoprotein involved in platelet adhesion and aggregation at the site of vessel wall injury [4]. VWF binds extracellular matrix, platelet GPIb and platelet GPIIb-IIIa. VWF also acts as a carrier of circulating factor VIII, ensuring the latter’s stability and guaranteeing the required concentration of FVIII at the site of vascular injury. Depending on the nature of the VWF defect, VWD is classified as type 1 in the case of a quantitative VWF defect or type 2 when there is an abnormal VWF function 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
reason we decided to explore the patient’s FVIII and VWF parameters. Our findings suggest that the type 2N R854Q mutation, which is normally asymptomatic at the heterozygous level, may be expressed in the presence of a combined impairment of primary haemostasis. Blood Coagul Fibrinolysis 25:401–404 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Blood Coagulation and Fibrinolysis 2014, 25:401–404 Keywords: Glanzmann thromboasthenia, type 2N von Willebrand disease, von Willebrand factor, von Willebrand factor:FVIIIB a Department of Cardiologic, Thoracic and Vascular Sciences, University of Padua Medical School, Padua, bDepartment of Internal Medicine, University of Perugia, Perugia and cDepartment of Medicine, University of Padua Medical School, Padua, Italy
Correspondence to Alessandra Casonato, Department of Cardiologic, Thoracic and Vascular Sciences, via Ospedale Civile 105, Padova, Italy Tel: +39 049 8213012; fax: +39 049 657391; e-mail: [email protected] Received 5 September 2013 Revised 30 October 2013 Accepted 4 December 2013
[5]. Among patients with type 2 VWD, some have type 2N, which is a variant characterized by a VWF with a defective FVIII carrier function [6,7]. With the exception of type 2N, all forms of VWD involve mucocutaneous bleedings (especially epistaxis, gingival bleeding, easy bruising and menorrhagia) that typically become promptly manifest after a trauma; the bleeding symptoms in type 2N resemble haemophilia A, with haemarthrosis and intramuscular haematomas. In Glanzmann’s thrombasthenia, the related bleeding symptoms are very similar to those of VWD, due to an impaired primary haemostasis [8]. Here, we report on a case of combined Glanzmann’s thrombasthenia and type 2N VWD, focusing on the finding of spontaneous haemarthrosis, which is uncommon in Glanzmann’s thrombasthenia and never seen in cases heterozygous for the type 2N VWF defect.
Materials and methods All individuals involved were studied after obtaining their written informed consent in accordance with the Helsinki Declaration and the study was approved by our institutional review board. DOI:10.1097/MBC.0000000000000067
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
402 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 4
Haemostatic tests
Basic haemostatic analyses, plasma VWF antigen (VWF:Ag), VWF collagen binding (VWF:CB), VWF ristocetin cofactor (VWF:RCo), ristocetin-induced platelet aggregation (RIPA), factor FVIII (FVIII) and capacity of VWF to bind FVIII (VWF:FVIIIB) were conducted as described elsewhere [9]. Anti-FVIII was sought using the Bethesda method. The search for anti-VWF antibodies was done with a home-made ELISA method, using coated purified human VWF and anti-IgG-HRP (Dako, Glostrup, Denmark) as a second antibody. The measure of a2-antiplasmin activity was performed by means of a chromogenic method (Siemens, Marburg, Germany). Genetic analysis
Genomic DNA was extracted from peripheral blood leukocytes using the QIAampDNA Blood Mini kit (Qiagen, Hilden, Germany). Exons 18, 19 and 20 of the VWF gene were amplified and sequenced as previously reported [10]. All PCR reactions were obtained using the AmpliTaq Gold (Applied Biosystems, AB, Foster City, California, USA) and a thermal cycler GeneAmpPCR System 2700 (AB). The Big Dye Terminator Sequencing kit v.2.5 (Perkin Elmer, Wellesley, Massachusetts, USA) and an ABI 3100 Genetic Analyser (AB) were used for DNA sequencing. Identification of GPIIb-IIIa gene mutations was performed by Professor M. Margaglione (Foggia, Italy).
Results Case report
The proband is a 48-year-old woman with a lifelong history of bleeding. She has suffered from epistaxis, gingivorrhagia, easy bruising, menorrhagia, bleeding after tooth extraction, haemorrhage after surgery and haemarthrosis. Her parents were not blood-related and nobody in her family had a bleeding tendency. The patient scored 18 with the Condensed MCMDM-1 Bleeding Questionnaire for VWD [11], as opposed to no more than 4 in normal individuals. When she was admitted to our Center, she presented with a chronic bilateral ankle arthropathy. Standard rheumatic tests (i.e. reactive protein C, ESR, RT and ANA) were found to be normal. MRI showed numerous sites of ankle joint damage, with moderate effusion of the tibioastragalar joint and hemosiderin deposits, loss of cartilage and subchondrial cysts. The right ankle showed an uneven loss of cartilage height associated with subchondrial erosion at the calcaneocuboid joint. Left knee demonstrated oedema and hyperhemia of the spongeous tissue. There was no evidence of any significant alterations in the right knee. The Compatible MRI scoring system for assessing haemophilic arthropathy was used to measure arthropathy of the patient with Glanzmann’s thrombasthenia [12,13]. Using the P-scale, based on a method that measures the
most severe joint abnormality, the patient with Glanzmann’s thrombasthenia achieved the highest score for arthropathy in the left ankle [10] on a scale of 0–10 and 9 for the right ankle (Table 1), indicating that both joints were severely damaged.
Haemostatic and genetic data
The patient’s main haemostatic findings are summarized in Table 2. There was evidence of prolonged filtroaggregation (PFA-100) and failure of ADP, collagen and adrenalin to induce platelet aggregation, but ristocetininduced platelet aggregation (RIPA) was normal. Platelet surface glycoprotein analysis by flow cytometry revealed a severe aIIbb3 deficiency (Table 3). The integrin alphaIIb subunit gene (ITGA2B) sequencing showed that the patient was a compound heterozygote for the T1214C and G1234A mutations that induce the I374T and G381R substitutions, respectively; these mutations account for patient’s Glanzmann’s thrombasthenia. The other haemostatic parameters investigated were normal, except for a slight reduction in VWF:FVIIIB, in absolute terms (54.5 vs. normal 65–150 U/dl), and a greater reduction in VWF:FVIIIB/VWF:Ag ratio (VWF:FVIIIB ratio) (0.62 vs. normal >0.75) (Table 2). No anti-FVIII and anti-VWF antibodies were detected, and normal were the levels of fibrinogen, FIX, FXI, FXIII and a2-antiplasmin activity. Despite the normal aPTT and FVIII values, the lower VWF:FVIIIB and VWF:FVIIIB ratio prompted us to surmise that the patient was carrying a type 2N VWF defect at the heterozygous level [14]. To verify this hypothesis, we analysed the exons 18–19–20 of the VWF gene, encoding for the FVIII-binding domain of VWF. We found that, at heterozygous level, the patient carried the G2561A mutation in exon 20, which induces the substitution of an arginine by a glutamine at position 854 of VWF. This is the most common mutation associated with type 2N VWD [14]. No other mutations were searched for in the remaining VWF gene.
Discussion A number of bleeding symptoms are reported in patients with Glanzmann’s thrombasthenia, having purpura, epistaxis, gingival haemorrhage, menorrhagia as almost constant features, although it is rare or very rare to observe haemarthrosis. Here, we report on a patient with Glanzmann’s thrombasthenia, characterized by spontaneous MRI-based score for arthropathy in our patient with Glanzmann thrombasthenia
Table 1 Joints
Ankle (left) Ankle (right) Knee (left) Knee (right)
Score 10 9 4 0
Most severe joint abnormality Full-thickness loss of joint cartilage Partial loss of cartilage thickness Synovial hypertrophy –
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
I374T and G381R
Flow cytometric analysis of the main antigens expressed on the platelet surface of our patient with Glanzmann thrombasthenia
Table 3
Collagen-epinephrine cartridges. b Platelet aggregation induced by ADP, collagen and adrenalin. c Ristocetin-induced platelet aggregation at 1.2 mg/ml ristocetin. d Integrin alphaIIb subunit gene.
Parameters
a
R854Q 0.62 >0.75 100 60–130 58.5 60–84 105 70–100
32 30–40
156 150–350
>300 94–193
Absent Present
74.9 60–160
87.6 60–160
104 60–130
54.5 65–150
VWF gene mutation VWF.RCo (U/dl) RIPA %c PT %
aPTT (s)
Platelet count (103/ml)
PFA100 (s)a
Platelet aggregationb
Patient Normal range
Table 2
Patient’s pertinent haemostatic and genetic findings
FVIII (U/dl)
VWF:Ag (U/dl)
VWF:CB (U/dl)
VWF:FVIIIB (U/dl)
VWF:FVIIIB/VWF:Ag ratio
ITGA2B gene mutationsd
Glanzmann’s thrombasthenia and type 2N VWD Pontara et al. 403
CD41(SZ22)(GPIIb/IIIa)/CD42a CD41(P2)/CD42a CD41(A2A9/6)/CD42a CD61 (SZ21)(GPIIIa)/CD42a CD61 (SAP) /CD42a Binding PAC1 (MFI) con ADP 10 mmol/l
Results
Normal values
0.076 0.061 0.046 0.07 0.076 2.3
1 0.33 0.15 0.026 0.72 0.08 0.91 0.13 0.63 0.047 12.7 2.5
haemarthrosis, associated with a type 2N VWF defect at heterozygous level. The spectrum of haemorrhagic symptoms in Glanzmann’s thrombasthenia was analysed in 177 patients with Glanzmann’s thrombasthenia and showed that only 3% of the patients (five out of 177) suffered from haemarthrosis [8]. One of them suffered from trauma-induced haemarthrosis, although the circumstances of the other four were not described [8]. Another report [15] described cysts in periarticular joint of a patient with recurrent haemarthrotic episodes. More recently, a Japanese patient with Glanzmann’s thrombasthenia was described who suffered from haemarthrosis of the left knee, which developed after an outdoor sports trauma, but no bone lesions on MRI [16]. Joint disease is one of the most common complications of haemophilia, affecting up to 90% of the severe forms. Haemophilic arthropathy is caused by recurrent episodes of bleeding into the joint, which correlate with the severity of the FVIII or FIX defect; such episodes can be prevented by replacement therapy. The precise mechanisms of haemophilic arthropathy are unknown, but it is well known that synovial changes precede cartilage changes over the course of haemophilic arthropathy. The patient with Glanzmann’s thrombasthenia described here showed a typical haemophilic arthropathy, characterized by loss of cartilage and the presence of cysts and subchondrial erosions that were apparently not the consequence of traumas. The arthropathy was severe enough to make it difficult for the patient to walk. Her haemostatic picture appeared to be the result of a compound haemostatic defect, that is Glanzmann’s thrombasthenia, associated with an impaired primary haemostasis and type 2N VWD, which damages the capacity of VWF to bind FVIII and the coagulation cascade. Patients homozygous or compound heterozygous for type 2N VWD are characterized by a phenotype very similar to that of haemophilia A, while heterozygotes for type 2N VWD (and those associated with R854Q mutations as in our patient) are asymptomatic. Heterozygotes for the R854Q mutation also have an almost normal haemostatic laboratory, with the exception of the VWF:FVIIIB test, and the VWF:FVIIIB ratio in particular [14,17]. The association
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
404 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 4
between Glanzmann’s thrombasthenia and VWD has already been reported, taking into account that VWD is the most common inherited bleeding disorder [18,19], which involved quantitative VWF defects (type 1 and type 3 VWD). Spontaneous bleeding is uncommon in Glanzmann’s thrombasthenia, and in this disorder, the most serious haemorrhagic episodes are due to trauma or an excessive physiological bleeding, such as menorhagies. Haemarthrosis was spontaneous in our patient and the arthropathic picture seemed to be consistent with recurrent episodes. An extensive analysis of FVIII and VWF (currently not performed in patients with Glanzmann’s thrombasthenia) in our patient was prompted by the finding of her severe arthropathy. We were seeking a decrease in FVIII levels or the presence of a FVIII inhibitor or an anti-VWF antibody, or both. In the end, we found that the patient’s VWF had only a lower FVIII binding capacity. It remains to be clarified why such a mildly impaired FVIII defect or FVIII–VWF interaction could cause a bleeding complication typical of severe forms of haemophilia A or B. It may be that the association of Glanzmann’s thrombasthenia with type 2N VWD causes the appearance or the worsening of bleeding symptoms typical of the latter condition at homozygous level. In the light of our results, it might be reasonable to consider that other reported patients with Glanzmann’s thrombasthenia with recurrent haemarthrosis might also have been carrying a type 2N VWF defect or a mild haemophilia [8,15]. In this case, the search for FVIII and VWF abnormalities should be performed.
References 1
2
3
4
5
6
7
8 9
10
11
12
The lesson that we can learn is that, when the severity or nature of Glanzmann’s thrombasthenia patients’ bleeding symptoms cannot be justified on the basis of the platelet GPIIb-IIIa defect, we have to seek another, associated haemostatic abnormality.
13
Acknowledgements
15
We thank Dr F Sartorello for her contribution to the genetic tests, and Dr G Saggiorato for the searching of anti-FVIII antibodies.
14
16
17
This work was supported by a grant from the Ministero dell’Universita’ e della Ricerca Scientifica e Tecnologica (MURST, 60%, 2010).
18
Conflicts of interest
19
The authors declare no conflict of interests.
Glanzmann E. Hereditare Hamorrhagische Thromboasthenic. Ein Beitrag zur Pathologie der Blutplattchen. [Inherited haemorrhagic thrombasthenia]. Jarbuch der Kinderheikunde 1918; 88:1–42. Larrieu MJ, Caen JP, Meyer DO, Vainer H, Sultan Y, Bernard J. Congenital bleeding disorders with long bleeding time and normal platelet count. II. Von Willebrand’s disease (report of thirty-seven patients). Am J Med 1968; 45:354–372. Phillips DR, Jenkins CS, Luscher EF, Larrieu M. Molecular differences of exposed surface proteins on thrombasthenic platelet plasma membranes. Nature 1975; 257:599–600. Ruggeri ZM. Structure of von Willebrand factor and its function in platelet adhesion and thrombus formation. Best Pract Res Clin Haematol 2001; 14:257–279. Sadler JE, Budde U, Eikenboom JC, Favaloro EJ, Hill FG, Holmberg L, et al., Working Party on von Willebrand Disease Classification. Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand factor. J Thromb Haemost 2006; 4:2103–2114. Mazurier C, Dieval J, Jorieux S, Delobel J, Goudemand M. A novel von Willebrand factor (vWF) defect in patient with factor VIII (FVIII) deficiency but with normal levels and multimeric patterns of both plasma and platelet vWF. Characterization of abnormal vWF/FVIII interaction. Blood 1990; 75:20–26. Casonato A, Pontara E, Sartorello F, Gemmati D, Cattini MG, Girolami A. Combined haemophilia A and type 2N von Willebrand disease: defect of both factor VIII level and FVIII binding capacity of von Willebrand factor. Haematologica 2001; 86:1110–1111. George JN, Caen JP, Nurden AT. Glanzmann’s thromboasthenia: the spectrum of clinical disease. Blood 1990; 75:1383–1395. Casonato A, Pontara E, Zerbinati P, Zucchetto A, Girolami A. The evaluation of FVIII binding activity of von Willebrand factor by means of an ELISA method: significance and clinical implications. Am J Clin Pathol 1998; 109:347–352. Casonato A, Sartorello F, Cattini MG, Pontara E, Soldera C, Bertomoro A, et al. An Arg760Cys in the consensus sequence of von Willebrand factor propeptide cleavage site is responsible for a new von Willebrand disease variant. Blood 2003; 101:151–156. Bowman M, Mundell G, Grabell J, Hopman WM, Rapson D, Lillicrap D, et al. Generation and validation of the Condensed MCMDM-1VWD Bleeding Questionnaire for von Willebrand disease. J Thromb Haemost 2008; 6:2062–2066. Lundin B, Babyn P, Doria AS, Kilcoyne R, Ljung R, Miller S, et al. Compatible scales for progressive and additive MRI assessments of haemophilic arthropathy. Haemophilia 2005; 6:658–663. Doria AS, Babyn S, Lundin B, Kilcoyne RF, Miller S, Rivard GE, et al. Reliability and construct validity of the compatible MRI scoring system for evaluation of haemophilic knees and ankles of haemophilic children. Expert MRI working group of the International Prophylaxis Study Group. Haemophilia 2006; 12:503–513. Casonato A, Pontara E, Sartorello F, Cattini MG, Perutelli P, Bertomoro A, et al. Identifying carriers of type 2N von Willebrand disease: procedures and significance. Clin Appl Thromb Hemost 2007; 13:194–200. Klofkorn R, Lightsey A. Hemarthrosis associated with Glanzmann’s thromboasthenia. Arthritis Rheum 1979; 22:1390–1393. Urakawa H, Nishida Y, Tsukushi S, Katsumi A, Ishiguro N. Glanzmann thromboasthenia detected because of knee hemarthrosis: a case report. J Ped Orthop 2010; 19:521–523. Casonato A, Daidone V, Barbon G, Pontara E, Di Pasquale I, Gallinaro L, et al. A common ancestor more than 10 000 years old for patients with R854Q-related type 2N von Willebrand’s disease in Italy. Haematologica 2013; 98:147–152. Ahmad F, Kannan M, Kishor K, Saxena R. Coinheritance of severe von Willebrand disease with Glanzmann thromboasthenia. Clin Appl Thromb/ Haemost 2010; 16:529–532. Nounou R, Spence D. Glanzmann’s thromboasthenia with von Willebrand’s disease. J Clin Pathol 1993; 46:1134–1136.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
