Platelet Glycoprotein VI Haplotypes and The Generation Of Microparticles In Health and Disease

Platelets are well known for their role in hemostasis, thrombosis, and wound repair. However, there is growing evidence for established platelet paradigms being relevant to other biological processes, such as inflammation and cancer. As one example, the platelet-specific collagen receptor, glycoprotein (GP) VI, has been shown to exacerbate rheumatoid arthritis in mouse models of the disease and also support experimental metastasis. GPVI exists as a membrane surface receptor that once engaged is capable of eliciting activation-dependent intraplatelet signals. An outcome of GPVI engagement is the release of platelet-derived microparticles (arbitrarily defined as less than 1 μm) and, in the case of rheumatoid arthritis, the microparticle further augments the inflammatory cascade. Beyond this example, platelet-derived microparticles are being recognized as relevant in a wide range of proinflammatory and prothrombotic pathophysiologic states. Characterization of the human GPVI gene has revealed genetic haplotypes corresponding to altered in vitro platelet response. A growing body of evidence has also linked these haplotypes to pathologic responses in the acute setting, such as myocardial infarction and stroke. The GPVI haplotypes, referred to as GP6a and GP6b, differ in 5 codons resulting in 3 polymorphic amino acids in the extracellular portion of the GPVI (S219P, K237E, and T249A) and 2 polymorphic amino acids in the cytoplasmic tail (Q317L and H322N). While GPVI haplotypes have been linked to cardiovascular disease outcome, a gap exists in determining whether the same polymorphisms influence platelet microparticle production and whether microparticle levels might vary in disease states. We have performed a detailed flow cytometry analysis of GPVI-dependent platelet-derived microparticles both in vivo and following agonist stimulation in vitro using blood from normal donors. A correlation exists between GPVI haplotypes and circulating levels of platelet-specific (αIIb) microparticles. Comparing homozygous GP6a (n=34) and heterozygous GP6a/b (n=17) individuals reveals an approximate 2-fold difference in the number of platelet microparticles that is statistically significant (p= 0.005). The GPVI dependent in vitro release is also haplotype specific following addition of type I fibrillar collagen. However, in a human condition of chronic inflammation, such as rheumatoid arthritis, haplotype-dependent changes in platelet-derived microparticle are not observed and reveal a maximum level of microparticle release occurring in the chronic inflammatory state. The results demonstrate that common polymorphisms within the human GPVI gene impact the level of circulating platelet-specific microparticles in normal individuals and in a state of chronic inflammation. The physiologic contribution of GPVI in disease states must consider the representative haplotype and levels of GPVI-dependent microparticles.