News and Publications

Multiple Alleles of the Integrin ₂ Gene and Inherited Differences in the Surface Density of the Human Platelet Collagen Receptor ₂ß₁

T.J. Kunicki, M. Kritzik, D.S. Annis, D.J. Nugent*

* Children's Hospital of Orange County, Orange, CA

The integrin ₂ß₁ is a receptor for collagen that plays a fundamental role in adhesion of blood platelets to the extracellular matrix. We previously reported that levels of platelet ₂ß₁ among randomly selected persons can vary up to 10-fold and that this variation correlates with differences in adhesiveness to type I or type III collagen. These extreme differences were unique to ₂ß₁ and were not observed with 2 other platelet integrins that share the ß₁ subunit, namely, the fibronectin receptor, ₅ß₁, and the laminin receptor, ₆ß₁. The pI and electrophoretic properties of samples of ₂ protein isolated from donors with high receptor density were indistinguishable from those of samples isolated from donors with low receptor density.

Because of these findings, we deemed it likely that differences in expression levels would correlate with genetic differences related to the ₂ subunit. When we compared the complete cDNA sequence of ₂ from 6 donors with various densities of the platelet receptor, we found absolutely no differences in the deduced amino acid sequence. However, 2 linked, dimorphic codon differences within the cDNA sequence of ₂ stood out and correlated with receptor density. The first is a TTT vs TTC difference at codon Phe224; the second, an ACA vs ACG difference at codon Thr246.

Southern blot hybridization with specific antisense DNA probes indicated that the gene frequencies of each allele in a random donor population (n = 85) were 0.606 (TTC . . . ACG) and 0.394 (TTT . . . ACA). The correlation between the alleles TTT . . . ACA (codons 224--246) and high receptor density (n = 30; P < .002) was significant; the complimentary alleles TTC . . . ACG were associated with low receptor density. Heterozygous subjects have intermediate levels of ₂ß₁, and familial studies confirmed that these allelic polymorphisms are inherited characteristics. These findings established that the level of platelet ₂ß₁ is an inherited trait and that these silent alleles within the coding sequence of the ₂ gene are linked to the genetic basis for variation in receptor density.

Further Characterization of Integrin ₂ Gene Alleles Associated With Differences in the Surface Density of Platelet ₂ß₁

M. Kritzik, B. Savage, D.J. Nugent,* Sentot Santoso,** Z.M. Ruggeri, T.J. Kunicki

* Children's Hospital of Orange County, Orange, CA
** Institute for Clinical Immunology and Transfusion Medicine, Giessen, Germany

Three allelic differences in the gene for the ₂ integrin subunit are associated with the level of expression of ₂ß₁ on the surface of platelets. We previously defined 2 linked silent polymorphisms in the ₂ gene coding region at nucleotides 807 (cytosine or thymine) and 873 (guanine or adenine). We subsequently identified 1 more rare nucleotide polymorphism in the coding region at nucleotide 837 (thymine or cytosine) and 4 additional linked polymorphisms within the introns that flank these coding sequences. Moreover, we determined that the alloantigenic Br polymorphism, which resides in a distal coding region at nucleotide 1648, is also linked to the 837 polymorphism.

Thus, 3 ₂ gene alleles, defined by 8 nucleotide polymorphisms, have now been discovered (Fig. 1). Allele 1 (807T/837T/873A/Br^b) is associated with increased levels of ₂ß₁; allele 2 (807C/837T/873G/Br^b) and allele 3 (807C/837C/873G/Br^a) are associated with lower levels of ₂ß₁. The gene frequencies of each allele in a random donor population (n = 85) were 0.394 for allele 1, 0.529 for allele 2, and 0.076 for allele 3.

Using real-time epifluorescence microscopy, we also showed that the rate of platelet attachment to type I collagen in whole blood under conditions of high shear rate (>1500 sec^-1), within the initial 3 minutes of attachment, is proportional to the density of ₂ß₁ receptors on platelets (Fig. 2). The density of platelet ₂ß₁ has an important effect on platelet adhesion to collagen in whole blood and therefore on platelet function in vivo, contributing to an increased risk of thrombosis or to bleeding in relevant disease states.

PUBLICATIONS

Kritzik M., Savage B., Nugent D.J., Santoso S., Ruggeri Z.M., Kunicki T.J. Nucleotide polymorphisms in the ₂ gene define multiple alleles which are associated with differences in platelet ₂ß₁. Blood 92:2382, 1998.

Santoso, S., Kroll, H., Kunicki, T.J., Haberbosch, W., Gardemann, A. Association of the platelet glycoprotein IA C₈₀₇T gene polymorphism with coronary artery disease and myocardial infarction in low risk patients. Lancet, in press.