Abstract

Germline mutations in the X-linked transcription factor, GATA-1, cause several hematopoietic disorders resulting in anemia and/or macrothrombocytopenia. Features of the platelet ultrastructural pathology, including hypo- and agranular megathrombocytes (Mtc) in all mutations, tubular membrane sheets often in parallel association in megakaryocytes and Mtc of patients with the V205M and G208S variations, platelets sequestered in Mtc and platelets in platelets in Mtc (as many as five in one Mtc) in the V205M, G208S and R216Q disorders, together with platelets attached to platelets attached to platelets forming large Mtc in the V205M and G208S mutations have helped to explain how Mtc are formed and why they fail to separate from proplatelets of megakaryocytes and enter the circulation as single cells. However, the findings do not explain why patients with GATA-1 mutations often have serious bleeding problems. Major receptors for hemostasis and thrombosis are present on GATA-1 Mtc, although, in some cases, slightly reduced in frequency. Yet, while the cells bind to and spread fully on glass and plastic, they adhere poorly to vWF or collagen coated glass slides under flow conditions, aggregate less well than normal platelets when stirred with collagen or ristocetin in an aggregometer and fail to retract clots. The findings suggest that internal activation mechanisms for the surface receptors are defective. The present study has solubilized GATA-1 Mtc from two males with the G208S mutation and normal platelets in sodium dodecyl sulfate/ EDTA in the presence of protease inhibitors and analyzed their cytoskeletal proteins on reduced, one dimensional polyacrylamide gels using a Mini-Slab Gel Apparatus. Gels were electrophoresed by the method of Laemmli. Proteins were stained with colloidal Coomassie G250 blue. GATA-1 Mtc cytoskeletal proteins were the same as those from the normal subjects, except for the absence of Talin. Western blot analysis to prove the missing protein is Talin was carried out. Normal platelet and GATA-1 Mtc cytoskeletal proteins were transferred to nitrocellulose sheets. Identification and localization of the missing protein was accomplished using a mouse monoclonal antibody against human Talin and a secondary anti-mouse IgG peroxidase conjugate. The Western blot analysis confirmed that the missing Mtc protein is Talin. Recent studies have shown that binding of the 235–245 kD actin-binding protein Talin to the β subunit cytoplasmic tails of integrins is the final step of integrin activation. The absence of Talin in GATA-1 Mtc may result in failure of full integrin activation accounting for functional failure in hemostasis.

Disclosures: No relevant conflicts of interest to declare.

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