Scientific Report 2007

Molecular and Experimental Medicine

Division of Blood Cell and Vascular Biology

Molecular Genetics of Hemostasis and Thrombosis

T.J. Kunicki, Y. Cheli, D. Jensen,* P. Marchese, D. Habart, T. Wiltshire,* M. Cooke,* J.A. Fernandez, J. Ware,** Z.M. Ruggeri, S. Kanaji, B. Jacquelin, M. Chang,*** D.J. Nugent,*** E. Trifiro, K. Furihata, F.M. Pulcinelli****

* Genomics Institute of the Novartis Research Foundation, San Diego, California
** University of Arkansas for Medical Sciences, Little Rock, Arkansas
*** Children's Hospital of Orange County, Orange, California
**** University "La Sapienza," Rome, Italy

Klf4 as a Modifier Gene of Hemostasis

Glycoprotein VI (GPVI) is an important collagen receptor that is expressed by megakaryocytes and platelets. In mice bioengineered to lack the gene for GPVI, the expected abnormal platelet responses to collagen occur in vitro, but a dichotomous bleeding phenotype is evident in vivo. Tail-bleeding times are either prolonged or normal and parallel the ability of the mice to form thrombi in an in vivo model of injury of the carotid artery. The prolonged bleeding time is inherited as an autosomal recessive trait.

We mapped this phenotype to a dominant locus on chromosome 4, and we found a strain-related difference in Klf4, the gene for the transcription factor Krüppel-like factor 4 (Klf4), that correlates precisely with the in vivo bleeding phenotype. A cytosine-adenosine repeat polymorphism in the promoter region results in increased transcriptional activity of the 129X1/SvJ haplotype, designated Klf4B, relative to that of the C57BL/6J haplotype, designated Klf4A. Evidence from several laboratories has established that Klf4 is an important transcription regulator in cells that are largely responsible for the composition of the extracellular matrix, including endothelial cells, smooth muscle cells, and fibroblasts. Thus, Klf4 is a legitimate candidate as a modifier gene that regulates the in vivo bleeding phenotype of mice deficient in GPVI.

Coregulation of ITGA2 Expression

In humans, expression of the integrin α₂β₁ is controlled predominantly by variations in the transcription of ITGA2, the integrin subunit gene, that coincide with a single-nucleotide polymorphism (C-52T) and a unique cytosine-adenine repeat polymorphism in the 5′ regulatory region. This site is involved in coregulation of gene expression, and the longer alleles are specifically bound by members of a transcriptional coregulator complex composed of poly-ADP-ribosyl polymerase 1, topoisomerase IIβ, DNA-dependent protein kinase, Ku80, and Ku70. The association of this complex leads to enhanced histone 3 acetylation and a transcriptionally favorable chromatin structure. The binding of this coactivator complex to the specific-length cytosine-adenine repeat sequence contributes to haplotype-dependent variability of cell α₂β₁ content.

Transcriptional and Epigenetic Regulation of the Integrin Collagen Receptor Locus

An integrin collagen receptor locus on human chromosome 5q11.2 includes the integrin genes ITGA1 and ITGA2 and the cell-cycle regulation gene PELO embedded within ITGA1 intron 1. ITGA1 transcription is controlled by a CArG box bound by serum response factor; transcriptional regulation of PELO is similar to that of ITGA2 and is controlled by Sp1. During thrombopoietin-induced in vitro differentiation of primary human cord blood mononuclear cells into megakaryocytes, rapid, progressive CpG methylation of ITGA1, but not PELO or ITGA2, occurs. Thus, selective CpG methylation of the ITGA1 promoter is a specific feature of α₁β₁ regulation that coincides with the initiation of megakaryocyte differentiation.

Functional Comparison of Platelet GP6 Haplotypes

Two major haplotypes, GP6a and GP6b, genes for GPVI in humans, influence collagen-related platelet function. The ectodomains of the membrane glycoproteins GPVIa and GPVIb differ at 3 residues, S219P, K237E, and T249A, but these substitutions have no effect on binding of the receptors to type I collagen, collagen-related peptide, or convulxin. Two additional substitutions are located in the cytoplasmic domain, Q317L and H322N. The presence of leucine at position 317 increases the relative binding of GPVIb to calmodulin in vitro, and platelets from donors who are GP6b/b have significantly lower activation-dependent proteolysis of endogenous GPVI than do platelets from donors who are GP6a/a.

Thus, the cytoplasmic domain of GPVIb impairs the activation-induced proteolysis of GPVI through the increased binding of the glycoprotein to calmodulin. Our data are consistent with the theory that differences in GPVIa and GPVIb binding to the regulatory protein calmodulin modulate GPVI proteolysis and shedding. Because the soluble form of GPVI attenuates thrombosis in several in vitro and in vivo models, this difference in haplotypes can have an impact on risk for bleeding or thrombosis.

Publications

Kato, K., Furihata, K., Cheli, Y., Radis-Baptista, G., Kunicki, T.J. Effect of multimer size and a natural dimorphism on the binding of convulxin to platelet glycoprotein (GP)VI. J. Thromb. Haemost. 4:1107, 2006.