Platelets play a major role in hemostasis and congenital defects in platelet function are associated with bleeding manifestations. The goal of this project is to define the biochemical mechanisms underlying the platelet dysfunction in patients with congenital platelet function defects and thereby enhance our understanding of the normal platelet activation mechanisms which is vital to the development of newer therapeutic strategies for both bleeding and thrombotic disorders. The vast majority of patients with congenital platelet defects are generally characterized by impaired aggregation responses and dense granule secretion on activation; most of them have normal dense granule stores. These patients are lumped into a loosely defined group called """"""""platelet secretion defects"""""""" or """"""""activation defects."""""""" In them the underlying biochemical and molecular mechanisms are totally unknown. The hypothesis to be tested is that these patients have defects in the signal transduction mechanisms. This project focuses on specific patients in whom the applicant has demonstrated hitherto undescribed deficiencies in two major proteins involved in signal transduction mechanisms, namely, a) phospholipase C (Aim 1), and b) GTP-binding protein Gaq (Aim 2). These proteins play fundamental roles in cellular signaling mechanisms in a wide array of cells. Detailed studies in two patients suggest an unique abnormality in PLC activation with decreased PLC-b2 expression.
In Aim 1 Dr. Rao will characterize the molecular defect in PLC-b2 by cloning, sequencing and expressing PLC-b2 CDNA. The mutant PLC-b2 will be studied.
In Aim 2, the applicant will characterize the molecular defect in a patient identified with impaired G-protein-function associated with abnormal Ca2+ mobilization, arachidonate release and a hitherto undescribed selective deficiency in platelet Gaq subunit.
In Aim 3, Dr. Rao will study neutrophils and monocytes from these patients to determine if they share the defect in signal transduction mechanisms and define the impact on leucocyte function. This project represents application of state-of-the-art techniques to define the molecular mechanisms of platelet dysfunction in a group of patients who are very poorly characterized at present and contribute an untapped sour of new information. These studies will provide new information on the role of two major proteins, PLC-b2 and Gaq, in platelet signal transduction mechanisms.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL056724-03
Application #
6165067
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1998-03-01
Project End
2002-02-28
Budget Start
2000-03-01
Budget End
2001-02-28
Support Year
3
Fiscal Year
2000
Total Cost
$274,661
Indirect Cost
Name
Temple University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Jalagadugula, Gauthami; Mao, Guangfen; Kaur, Gurpreet et al. (2011) Platelet protein kinase C-theta deficiency with human RUNX1 mutation: PRKCQ is a transcriptional target of RUNX1. Arterioscler Thromb Vasc Biol 31:921-7
Aneja, K; Jalagadugula, G; Mao, G et al. (2011) Mechanism of platelet factor 4 (PF4) deficiency with RUNX1 haplodeficiency: RUNX1 is a transcriptional regulator of PF4. J Thromb Haemost 9:383-91
Kaur, Gurpreet; Jalagadugula, Gauthami; Mao, Guangfen et al. (2010) RUNX1/core binding factor A2 regulates platelet 12-lipoxygenase gene (ALOX12): studies in human RUNX1 haplodeficiency. Blood 115:3128-35
Jalagadugula, Gauthami; Mao, Guangfen; Kaur, Gurpreet et al. (2010) Regulation of platelet myosin light chain (MYL9) by RUNX1: implications for thrombocytopenia and platelet dysfunction in RUNX1 haplodeficiency. Blood 116:6037-45
Jalagadugula, Gauthami; Dhanasekaran, Danny N; Rao, A Koneti (2008) Phorbol 12-myristate 13-acetate (PMA) responsive sequence in Galphaq promoter during megakaryocytic differentiation. Regulation by EGR-1 and MAP kinase pathway. Thromb Haemost 100:821-8
Sun, L; Gorospe, J R; Hoffman, E P et al. (2007) Decreased platelet expression of myosin regulatory light chain polypeptide (MYL9) and other genes with platelet dysfunction and CBFA2/RUNX1 mutation: insights from platelet expression profiling. J Thromb Haemost 5:146-54
Sun, Liansheng; Mao, Guangfen; Kunapuli, Satya P et al. (2007) Alternative splice variants of phospholipase C-beta2 are expressed in platelets: effect on Galphaq-dependent activation and localization. Platelets 18:217-23
Jalagadugula, G; Dhanasekaran, D N; Kim, S et al. (2006) Early growth response transcription factor EGR-1 regulates Galphaq gene in megakaryocytic cells. J Thromb Haemost 4:2678-86
Rao, A Koneti (2004) Molecular and biochemical basis for the platelet dysfunction in myeloproliferative disorders. Semin Hematol 41:6-9
Sun, Liansheng; Mao, Guangfen; Rao, A Koneti (2004) Association of CBFA2 mutation with decreased platelet PKC-theta and impaired receptor-mediated activation of GPIIb-IIIa and pleckstrin phosphorylation: proteins regulated by CBFA2 play a role in GPIIb-IIIa activation. Blood 103:948-54

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