Bipin Kulkarni

Glanzmann's thrombasthenia: updated

Glanzmanns thrombasthenia is an autosomal recessive disorder, rare in a global context, but a relatively more common platelet function defect in communities where consanguineous marriages are more frequent. On clinical grounds alone, it cannot be distinguished from other congenital platelet function defects. Epistaxis, gum bleeding, menorrhagia are the common clinical manifestations, whereas large muscle hematoma or hemarthrosis seldom occur in these patients. Essential diagnostic features are a normal platelet count and morphology, a greatly prolonged bleeding time, absence of platelet aggregation in response to ADP, collagen, epinephrine, thrombin and to all aggregating agents which ultimately depend on fibrinogen binding to platelets for this effect, flow cytometry, studies of GPIIb-IIIa receptors on the platelet membrane surface using monoclonal antibodies. The present review describes some of the uncommon features of the disorders and the currently available options which the treating physicians should be aware of during the management of these patients. Although by definition all patients with Glanzmanns thrombasthenia have a virtually complete failure of platelet aggregation, a number of variant forms of GT have been described in which the glycoproteins are present in normal or near normal amounts but are functionally defective. Understanding the pathophysiology of the disorder by the treating physicians is of utmost importance. Presence of high affinity platelet receptors resulting in thrombasthennia-like phenotype may require an antagonistic treatment atypical of classical GT management. It has now been established that different genetic mutations of either GPIIb or IIIa genes results in such a heterogeneity of thrombasthenia phenotype. Glanzmanns thrombasthenia is a paradigm for treating coronary artery disease patients with GPIIb-IIIa antibody and inhibitors. By using these medicines we create a temporary GT-like situation. Hence, understanding this disease is of utmost importance to the practicing cardiologist. As mutations for different variant forms of GT become known, our understanding of how GPIIb-IIIa molecules can be activated to act as a receptor for fibrinogen molecules will be increased. Such understanding undoubtedly will help us to devise better drugs with GPIIb-IIIa inhibitors. Molecular biology techniques have enabled us to equivocally detect heterozygote carriers who are clinically asymptomatic. However, there may be several laboratories in the developing world, which have no access to molecular biology techniques. Development of more robust techniques of quantitation of platelet receptors has enabled an accurate diagnosis of heterozygote carriers or an unborn fetus in the second trimester. The importance of the GPIIb-IIIa polymorphisms in carrier and prenatal diagnosis has not been properly studied. Nowadays the less direct method of PLA1 typing (determination of the levels of platelet antigen) of the foetal platelets as early as 16 weeks of intrauterine life can be used for prenatal diagnosis of GT.

Contribution of natural anticoagulant and fibrinolytic factors in modulating the clinical severity of haemophilia patients

The role of natural anticoagulants, fibrinolytic cascade factors and common prothrombotic gene polymorphisms in modulating disease severity were studied in 35 ‘clinically mild’ and 37 ‘clinically severe’ haemophilia patients with severe factor VIII or IX deficiency (<0·01 IU/ml). Strong association of deficiencies of proteins C and S, antithrombin III, tissue factor pathway inhibitor and tissue plasminogen activator, together with factor V Leiden and endothelial protein C receptor 23 bp insertion polymorphisms were observed in the ‘clinically milder’ group as compared with the ‘clinically severe’ group. These results indicate a synergistic modulation of bleeding tendency in haemophilia patients by factors in the anticoagulant and fibrinolytic systems.

Development of inhibitors in patients with haemophilia from India

Four hundred and seven patients (352 haemophilia A and 55 haemophilia B) were investigated for the presence of factor VIII and IX inhibitors. Twenty‐four out of 292 severe and two out of 36 moderate haemophilia A patients showed the presence of inhibitors. The mean age at development of inhibitors was 17.7 years (range 6–52 years). In 12 patients the inhibitors were detected due to suboptimal response to factor replacement therapy (symptomatic) and in the remaining 14 patients the inhibitors were detected during the routine screening of the patients’ samples for inhibitors. They had, however, responded well to the usual doses of factor concentrates and there was no suspicion in these patients that they had developed an inhibitor (asymptomatic). There were two families in which the inhibitors were detected in more than one family member. The level of inhibitors in symptomatic patients ranged from 2.2 Bethesda units (BU) mL–1 to 460.6 BU mL–1, and in asymptomatic patients it ranged from 0.8 BU mL–1 to 3.2 BU mL–1. The inhibitors persisted in all patients except one, who developed an inhibitor postoperatively for a brief period of 3 months. All these patients were followed up from first factor exposure and were tested for inhibitors at least twice a year. The mean number of exposure days before they developed inhibitors was 47.5 exposure days (range 17–98 exposure days). No inhibitors appeared after more than 100 exposure days in any of the patients. When 50 consecutive patients were investigated for intron 22 inversions of the factor VIII gene, 17 patients were found to be positive for inversions (10 proximal inversion; seven distal inversion) out of whom four patients developed inhibitors, three patients belonging to the same family. Out of 35 haemophilia B patients, only one patient developed an inhibitor. The overall prevalence of inhibitors was thus 8.2%, which is similar to the reports from western countries, prior to the introduction of highly purified factor concentrate therapy.

JAK2 Mutations Across a Spectrum of Venous Thrombosis Cases

The JAK2(V617F)mutation is recurrent in polycythemia vera and essential thrombocythemia, which are myeloproliferative neoplasms frequently associated with arterial and venous thromboembolism. It has also been reported as a marker for occult myeloproliferative disorder (MPD) in patients with splanchnic venous thrombosis. Limited data are available regarding the prevalence of the JAK2(V617F) mutation in patients with thrombosis outside the splanchnic region. For the study, 321 cases of venous thrombosis in the splanchnic and nonsplanchnic regions (cerebral venous thrombosis [CVT], 70; deep venous thrombosis [DVT], 36; Budd-Chiari syndrome [BCS], 137; portal venous thrombosis [PVT], 78) were studied for the presence of JAK2 mutations. The prevalence values for the JAK2 mutation were 3% (1/36), 8.8% (12/137), 5% (4/78), and 3% (2/70) in DVT, BCS, PVT, and CVT, respectively; 19 (5.9%) of 321 cases were positive for the JAK2 mutation. Of 111 healthy subjects screened for this mutation, none were found to be carriers. Determination of the JAK2(V617F) mutation may be useful to identify patients who should be carefully observed for the development of overt MPDs. The significance of screening for this mutation in nonsplanchnic thrombosis cases needs to be analyzed in a larger series.

Influence of CYP2C9 and VKORC1 gene polymorphisms on warfarin dosage, over anticoagulation and other adverse outcomes in Indian population.

The aim of this study was to determine the frequencies of SNPs in the vitamin K epoxide reductase complex subunit 1 (VKORC1) and cytochrome P450 2C9 (CYP2C9) genes and their effect on warfarin dose requirement, over anticoagulation and other adverse outcomes in Indian population. A total of 145 warfarin treated patients for various clinical conditions were screened for VKORC1 and CYP2C9 gene polymorphisms by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. We found that homozygous VKORC1-1639 AA and CYP2C9 (*)3/(*)3 polymorphisms showed 100% association with risk of over anticoagulation and other adverse events. Carriers of two heterozygous variant genotypes also showed significant association with risk of over anticoagulation and bleeding. Single variant carrier patients were found to require low warfarin dose as compared to wild type (CYP2C9(*)1/(*)1 and VKORC1- 1639 GG) patients. The major impact of VKORC1 and CYP2C9 genotypes was observed in the first month of anticoagulation. A drastic variation from other Asian countries was observed in Indian population with regard to the distribution of different VKORC1 -1639 genotypes. Our results suggest that both VKORC1 and CYP2C9 genotypes showed significant impact on warfarin dose requirement, over anticoagulation in the first month of anticoagulation and number of bleeding episodes. The variation in therapeutic dosage of warfarin and the associated adverse events across different populations is due to the wide differences in the frequency of these warfarin sensitive alleles.

Human platelet alloantigen polymorphism in Glanzmann's thrombasthenia and its impact on the severity of the disease.

Summary. Glanzmanns thrombasthenia (GT) is an autosomal recessive disorder of platelets caused by the deficiency or abnormality of platelet receptors. Several platelet alloantigen systems reside on glycoprotein (GP) IIb and GPIIIa, of which the human platelet antigen 1 (HPA‐1) system is important. Studies have shown that, in the normal population, the HPA‐1b phenotype results in increased platelet aggregation and increased fibrinogen binding, increasing the risk of myocardial infarction. GT produces severe bleeding, but in a subset of patients has a relatively milder course. Forty‐one GT patients and 100 healthy control subjects were genotyped for platelet alloantigens HPA‐1 to HPA‐6, using PCR–ASA (polymerase chain reaction–allele‐specific amplification), and for GPIIb‐IIIa expression and fibrinogen binding using flow cytometric techniques. Platelet alloantigen distributions were similar in the patient and control groups. With the exception of the two HPA‐1b/1b homozygous patients (> 10%), 25 GT patients had less than 5% aggregation to 6 µmol/l ADP, and 16 patients showed between 5% and 10% aggregation to 6 µmol/l ADP. Seven out of 37 patients with HPA‐1a/1a phenotype showed 1–5% fibrinogen binding and GPIIb–IIIa receptors. The two HPA‐1b/1b patients showed 34·6% and 32% fibrinogen binding and > 10% GPIIb–IIIa receptors. This study determined the platelet alloantigen distribution in a large cohort of unrelated GT patients from western India. GT patients homozygous for HPA‐1b/1b had higher levels of platelet aggregation and fibrinogen binding as well as a milder course, as evidenced by infrequent epistaxis and no transfusion requirement to date.

Correlation of thromboelastographic patterns with clinical presentation and rationale for use of antifibrinolytics in severe haemophilia patients.

Summary.  Thromboelastography (TEG) assesses the global pattern of blood coagulation in the whole blood. Present day management of haemophilia is based on replacement therapy with lost factor by parenteral administration of factor concentrates. It is very well known that interaction of cellular components in the blood also affect the thrombin generation which in turn might produce varied TEG patterns that might reflect the clinical severity in haemophilia patients. We evaluated 66 severe haemophilia A and B patients (as assessed by one‐stage assay) by TEG and correlated the varied TEG patterns with the clinical severity in the patients. Four distinct TEG patterns were observed; Group A consisted of eight patients with hypercoagulable patterns while group B consisted of two patients showing hyperfibrinolytic patterns. Group C comprised of 17 patients whose TEG tracings did not show the initiation of clot formation at all while group D comprising of 39 patients showed varied clot initiation times ranging from almost normal pattern to a highly prolonged split times. Groups A and D patients were relatively milder clinically while groups B and C were clinically severe as assessed by the number of bleeding episodes, the frequency of transfusion and joint deformity. Subsequently we also evaluated the in vitro efficacy of the antifibrinolytic drug, EACA in normalizing the TEG patterns in 12 patients (group C) who did not show the initiation of clot formation in the TEG tracings to see the contribution of fibrinolysis in producing such patterns. The use of EACA in vitro in this group improved the TEG profile of these patients. In conclusion, the classification of severe haemophilia patients based on TEG patterns correlated well with the clinical severity and the ex vivo use of antifibrinolytics like EACA are effective in improving the TEG profile of all patients who had an abnormal TEG pattern without any clot initiation.

Rare coagulation factor deficiencies: a countrywide screening data from India.

As compared to haemophilia, although the clinical features and the management strategies for rare coagulation factor deficiencies are discussed, little is known about them. This study was undertaken to assess the distribution, clinical presentation and treatment of patients with rare coagulation factor deficiency disorders in a cross‐sectional population of India. Blood samples and other clinical details from patients suspected of rare coagulation factor deficiencies were collected by the Haemophilia Treatment Centers across India and were diagnosed at National Institute of Immunohaematology, Mumbai. A total of 321 cases of rare clotting factor deficiencies were diagnosed, of which 88% were severe, 10% moderate and 2% mild. Commonest deficiency encountered was factor XIII (FXIII) (30%) followed by FX (15.6%), FVII (15%), fibrinogen (12.1%), FXI (9%), combined V and VIII deficiency (5.6%) and congenital multiple vitamin K‐dependent coagulation factor deficiency (MCFD, 2.1%). Major representation of these deficiencies was from Southern and Western India (82%). Mucocutaneous bleeding was the commonest clinical presentation (59%); intracranial (IC) haemorrhage was seen in 18% of the patients; menorrhagia was an important clinical pointer in women in the reproductive age group (78%); 8% of the severe cases had no history of bleeding and 73% of the FXIII deficiency cases had umbilical stump bleeding. The major therapeutic products used was fresh frozen plasma (64%), cryoprecipitate (15%), whole blood (15%), antifibrinolytics (5%) and recombinant FVIIa (1%). A distinct pattern in the distribution of rare clotting factor deficiencies was observed which was based on multiple factors that include ethnicity and the available diagnostic facilities in different regions of this vast country.

Platelet function tests using platelet aggregometry: need for repetition of the test for diagnosis of defective platelet function.

Four hundred and ninety seven patients were referred to our center for platelet aggregation studies because of spontaneous mucocutaneous bleeds. All these patients had normal complete blood count, platelet count and peripheral smears except in ten patients of Bernard Soulier Syndrome where platelet count was marginally reduced in the presence of giant platelets. Two hundred and eighty patients were found to have normal platelet aggregation to ADP, collagen, ristocetin and arachidonic acid. Out of the remaining 217 patients, 62 patients were diagnosed to have Glanzmanns thrombasthenia, 10 Bernard Soulier Syndrome, 6 storage pool deficiencies, 7 cyclooxygenase deficiencies and 72 von Willebrand disease. In all the patients with GT and BSS, diagnosis was confirmed with flow cytometry using multiple monoclonal antibodies to GPIIb-IIIa and GPIb-IX. There were sixty patients where initial platelet aggregation studies showed reduced (<30%) aggregation to either ADP, collagen, ristocetin or arachidonic acid in its various combination, however in 12 such patients (20%) the platelet aggregation studies were normal on repetition. All our platelet aggregation studies were done only after assuring that the patient is not taking any medicine for at least 7 – 10 days which may affect the platelet function tests. The present study shows that single atypically abnormal platelet aggregation studies should always be repeated. Finally in 48/217 patients (22%) some aggregation abnormality to one or more of the agonists persisted, although we could not categorize these patients into any clear-cut platelet disorder. None of these 48 patients platelet associated immunoglobulin was increased by flow cytometry. It is possible that large number of patients from that disorder will finally prove to be some form of platelet secretory defect. In north India similar group of defect in a large number of patients have been reported as isolated PF3 abnormality or thrombasthenic thrombopathy.

An improved, semi quantitative clot based assay for factor XIII

haemophilia care. Moreover, the need for weight reduction is becoming increasingly important as the prevalence of overweight and obesity increases in the haemophilia population. A report of a recent nationwide survey in the Netherlands described obesity as a new disaster for haemophilia patients. In our previous study, we found that the prevalence of obesity in our adult (‡20 years old) haemophilia patients in Mississippi was 36% and there was a prevalence (21%) of obese young haemophiliacs (aged 2–19.9 years) comparable to the national average ( 21%) [4]. In conclusion, the growing risk of obesity in haemophilia is associated with a much higher cost for prophylactic treatment. It is thus imperative that preventive measures are taken to identify haemophilia patients at risk for obesity and to institute appropriate intervention at an early stage to help reduce weight in such patients. Such an approach will ultimately have a significant economic impact in reducing the burden of treating this expensive disease.