The present study demonstrated that the 145Met and 4R polymorphisms of GPIbα facilitate interaction with immobilized VWF under flow conditions, which is a highly adaptive physiologic response. To date, molecular mechanisms for the functional differences of the 145Thr/Met and/or VNTR polymorphisms in GPIbα have not been fully understood whereas numerous epidemiologic data have been reported. We report the first experimental data obtained using recombinant proteins to determine the functional differences of 145Thr/Met and VNTR GPIbα polymorphisms. Previously, 145Met and/or 3R/4R polymorphisms were demonstrated to be associated with an increased risk for arterial thrombosis, such as coronary artery disease or stroke (Simmonds et al, 2001; Yamada et al, 2002). Because the 145Thr/Met and VNTR polymorphisms are in linkage disequilibrium, focus on either 145Met or 3R/4R allele was likely to be sufficient to examine the association between GPIbα polymorphisms and arterial thrombosis in the epidemiological study. We reported that the frequency of either 145Met- or 4R-allele among patients with coronary artery disease was higher than that among control subjects and that the genotypes with the 145Met-allele were more frequently found in the patients with cerebrovascular disease than in control subjects (Murata et al, 1997; Sonoda et al, 2000). A large case-cohort study (Afshar-Kharghan et al, 2004) showed the relationship of the 2R/2R genotype with a lower risk of coronary heart disease in African-Americans. However, conflicting data have also been published (Hato et al, 1997; Simmonds et al, 2001). In experimental studies of these polymorphisms, Boncler et al (2002) demonstrated that the inhibitory effect of the VWF antagonist on ristocetin-induced agglutination was higher in 145Met/3R-positive platelets than in 145Met/3R-negative platelets. Ulrichts et al (2003) reported that platelets with 145Thr or recombinant GPIbα (residues 1–289) with 145Thr had a higher VWF binding affinity than 145Met. These findings are not consistent with our results although the experimental conditions of the present study differed from those of previous studies: use of ristocetin or botrocetin or use of an assay system. Other studies have also used various methods with inconsistent results (Mazzucato et al, 1996; Li et al, 2000; Jilma-Stohlawetz et al, 2003). These reports suggest that the functional analyses of GPIbα polymorphisms seem to be easily affected by several factors in relation to platelet activation or experimental conditions. Therefore, in this study, recombinant GPIbα and purified human VWF were examined under two experimental conditions to focus on the relationship between GPIbα polymorphisms and interactions with VWF. The first study, using soluble GPIbα lacking the VNTR polymorphism site, did not show the effect of the 145Thr/Met polymorphism on the major conformation because the immunoreactivity to anti-GPIbα antibodies that recognize confirmation-specific epitopes were not significantly different between these polymorphisms. The 145Thr/Met polymorphism did not affect the 125I-VWF binding in the presence of ristocetin under static conditions. Although ristocetin provides a convenient method to investigate the VWF/GPIbα interaction in vitro, it is not a physiologic substance. Thus, the second study was designed with an alternative approach, an in vitro assay for VWF/GPIbα interaction under flow conditions. Cells expressing GPIbα were prepared as a GPIbαβIX complex because expression of a full-length GPIbα alone was unstable in the cell culture system (Lopez et al, 1992). Two types of cells with naturally occurring sequences (T1R and M4R) and two types of cells with artificial or extremely rare sequences (T4R and M1R) were used to determine which polymorphism was more closely related to the VWF/GPIbα interaction. We carefully measured the GPIbα expression level on each cell because these levels were reported to affect the VWF/GPIbα interaction under flow conditions (Nishiya et al, 2000). After using FACS to obtain cells expressing similar GPIbα levels, EIA assay was performed using GUR83–35 and GUR20–5. Because these two antibodies were shown not to be influenced by the 145Thr/Met polymorphism (Table I), we used these antibodies in this assay. Perfusion analyses of the quantified cells indicated that M4R, which is a risk factor for arterial thrombosis, had a high ability to interact with VWF under a flow condition of 114/s, as compared with T1R. This flow condition of 114/s may correspond to wall shear rate for large veins in vivo (Bevan et al, 1995), where VWF-dependent platelet phenomena may not take place. Compared with platelets, however, CHO cells have 2·5- to threefold larger diameters, and the GPIbα-expressing CHO cells are approximately 20-fold higher in GPIbα density. The cell size and receptor density are likely to affect the sensitivity of cells to flow conditions. Also, we were unable to determine the order of effectiveness of the polymorphisms among the four sequences, 145Thr, 145Met, 1R, and 4R, in VWF/GPIbα interactions because T4R and M1R had a similar ability to interact with VWF. Although the synergistic effect of the 145Thr/Met and VNTR polymorphisms on GPIbα function remains unclear, the present data are compatible with previous speculations (Lopez, 1994; Murata et al, 1997) that GPIbα with 4R is longer in size and thus places the VWF-binding global domain further away from the platelet plasma membrane. Thus, VWF would be more easily accessible to the binding site on the receptor under high shear conditions. Functional polymorphisms of GPIbα might be responsible for the increased prevalence of arterial thrombosis. Our observations might explain the molecular basis for the previous epidemiologic studies. Further studies to examine the interactions between GPIbα polymorphisms and other ligands are necessary. The present data support a potentially new therapeutic approach to arterial thrombosis by targeting specific GPIbα polymorphisms.