A simple, novel and robust test to diagnose type I Glanzmann thrombasthenia.

Glanzmann thrombasthenia (GT) is an inherited bleeding disorder due to either absence or dysfunction of fibrinogen binding receptors, i.e either GPIIb (GPIIβ) or GPIIIa (GPIIIα) on platelet membrane. The complete fibrinogen receptor, i.e. GPIIβGPIII which binds fibrinogen on activated platelets involves association of these two glycoproteins. Absence of either or both of these receptors can, therefore, cause platelet dysfunction. In the endoplasmic reticulum, pro GPIIβ forms heterodimer with GPIIIα, a pre-requisite for surface expression. In the Golgi, proGPIIβ is cleaved into heavy and light chains, linked through a disulfide bridge (mature-GPIIβ). The membrane glycoprotein GPIIβ consists of four major domains, β-propeller, thigh, calf-1 and calf-2 domains. The main contact site with GPIIβ is located in the β-propeller, while calf-1 and calf-2 domains contribute minor interfaces. In type I disease no GPIIIα; GPIIβ; receptors are reproduced or only a very low number, can be detected on the platelet surface, while in type II GT their number is measurable. Due to the high rate of consanguineous marriages, GT is the commonest platelet function defect in places like India, Arab countries and Israel. In many parts of the world, the availability of a quick and robust test to detect Glanzmann thrombasthenia could be useful, and platelet aggregometry and flow cytometry are not only costly techniques, but also need experienced technicians who are in short supply. We used a bifunctional bioengineered disintegrin/alkaline phosphatase hybrid protein ErAPv to evaluate 14 consecutive cases of Glanzmann thrombasthenia and 20 normal healthy controls. Disintegrins have high affinity and selectivity for integrins which selectively bind and inhibit integrin function. The eristostatin (Er) portion selectively binds to the GPIIβGPIIIα glycoprotein on the surface of the platelet membrane while the APv portions allow the development of a dot in a one-step method. Platelet aggregation was performed by using agonists ristocetin (1.25 mg mL), ADP (6 μM), collagen (4 μg mL), epinephrine (4 μM) and arachidonic acid (0.75 mM). Flow cytometry analysis of platelet membrane surface receptors was carried out using FITC labeled antiGPIIβGPIIIα, antiβ3, anti-GPIβ, anti-GPIX (BD Biosciences, Pharmingen, San Diego, USA), antiGPIIβ and antifibrinogen antibodies (Dako, Glostrup, Denmark). For dot blot analysis, 3 microliters of washed platelets (2×10 mL) were blotted onto a polyvinilydine difluoride [PVDF] membrane and left to dry. The PVDF strips were blocked with 5% nonfat milk in Tris-buffered saline, pH 8.2 (20mM Tris, 0.9% NaCl, 20mM NaN3) or blocking solution for 2 hrs. at room temperature with mild agitation. ErAPv protein was added (40 μL/mL of blocking solution) and further incubated for 2 hrs. Membranes were washed 3×5 mins. with Tris-buffered saline with mild agitation to remove the unbound protein. NBT/BCIP alkaline phosphatase substrate (1:1.25) (Roche Diagnostics, Mannheim, Germany) was added until dots developed. A clinical severe type II GT homozygous for 1619 del C was taken as positive control (case 12). A substrate blank was also included as negative control. Out of the 14 GT patients, 12 were type I and 2 were type II variants. Nine were born out of first degree consanguineous marriages. Five patients did not have any history of transfusion, while others had history of minimal transfusion. Fourteen patients showed classical features of GT, i.e. severely reduced or no aggregation with all the agonists except reduced or normal aggregation with ristocetin. The GPIIβGPIIIα content in 12 of the 14 patients was found to be <1% of normal. Two patients had a normal αIIββ3α content of 56% and 58% respectively, suggesting that these patients have a variant type of thrombasthenia with the GPIIbβGPIIIα receptors