Es. There was no difference between women who were (n = 6) or

Es. There was no difference between women who were (n = 6) or were not (n = 23) taking oral contraception.1934-21-0 platelet Accumulation is not Correlated to Hematocrit or Platelet Count in the Normal RangeThere was a weak negative correlation between hematocrit and SC at each shear rate although this dependence was not statistically significant (data not shown). Similarly, LagT and VPLTFigure 3. Platelet accumulation as function of shear rate. Platelets were labeled with a PE/Cy5 labeled mouse antihuman CD41a antibody and their accumulation was measured over the course of a 5 min flow assay. The top row shows the raw images and the bottom row shows the binary images following image processing at wall shear rates of 150 s21 (A, A’), 300 s21 (B, B’), 750 s21 (C, C’) and 1500 s21 (D, D’). Scale bar = 100 mm. doi:10.1371/journal.pone.0054680.gVariability in Microfluidic Flow BIBS39 AssaysFigure 5. Distribution of platelet surface coverage (SC) in cohort of normal donors. Histogram of percent surface coverage (SC) for 150 s21 (A), 300 s21 (B), 750 s21 (C), and 1500 s21 (D) for the fifty normal donors. doi:10.1371/journal.pone.0054680.gFigure 4. Surface coverage, rate and lag time of platelet accumulation as a function of wall shear rate. Recalcified citrated whole blood was perfused over type I collagen for 5 min and platelet accumulation was monitored as a function of wall shear rate and time (n = 50). Platelet accumulation was characterized by (A) percent surface 18325633 coverage (SC) after 5 min, (B) the rate of platelet accumulation expressed as percent surface coverage per second (VPLT), and (C) the lag time to 1 surface coverage (LagT). Lines with ** denotes a p,0.01 for the Mann-Whitney U-test. doi:10.1371/journal.pone.0054680.gwere not correlated to hematocrit. There was no correlation between platelet count on any of the metrics of platelet accumulation. The hematocrit and platelet count for every individual in the cohort was within the normal ranges (Table 1).Platelet Accumulation and Platelet Receptor GenotypeIn each donor we measured variants for three genes ?ITGA2, GP6 and GP1BA ?that encode for platelet adhesion receptors ?a2b1, GPVI, and GB1b ?to determine if the presence of specific alleles affected platelet accumulation in the MFA. These genetic variants were selected based on their biological significance andFigure 6. The effect of VWF plasma levels on platelet accumulation. Platelet surface coverage increases with increasing VWF plasma levels at all wall shear rates. For each shear rate, bars represent the average platelet surface coverage in each quartile of VWF levels in a cohort of normal donors (n = 50). doi:10.1371/journal.pone.0054680.gVariability in Microfluidic Flow AssaysTable 3. Spearman correlation coefficient between VWF levels and platelet surface coverage (SC), lag time (LagT) and platelet accumulation velocity (VPLT).Wall Shear Rate (s21) SC 150 300 750 1500 0.51** 0.38** 0.40** 0.32*LagT 0.08 0.14 20.42* 20.VPLT 0.55** 0.30* 0.52** 0.56**p,0.05; **p,0.01. doi:10.1371/journal.pone.0054680.tprevious reports of association with platelet or clinical bleeding and thrombotic phenotypes (Table S1). Individuals with the AA genotype of the GP6 gene had higher SC than individuals with the AG allele (Fig. 8). LagT was not different between the two GP6 genotypes, but VPLT was significantly (p,0.05) higher for the AA genotype at 150 s21. There was no difference in VWF levels between the two genotypes and no significant interaction (p = 0.64).Es. There was no difference between women who were (n = 6) or were not (n = 23) taking oral contraception.Platelet Accumulation is not Correlated to Hematocrit or Platelet Count in the Normal RangeThere was a weak negative correlation between hematocrit and SC at each shear rate although this dependence was not statistically significant (data not shown). Similarly, LagT and VPLTFigure 3. Platelet accumulation as function of shear rate. Platelets were labeled with a PE/Cy5 labeled mouse antihuman CD41a antibody and their accumulation was measured over the course of a 5 min flow assay. The top row shows the raw images and the bottom row shows the binary images following image processing at wall shear rates of 150 s21 (A, A’), 300 s21 (B, B’), 750 s21 (C, C’) and 1500 s21 (D, D’). Scale bar = 100 mm. doi:10.1371/journal.pone.0054680.gVariability in Microfluidic Flow AssaysFigure 5. Distribution of platelet surface coverage (SC) in cohort of normal donors. Histogram of percent surface coverage (SC) for 150 s21 (A), 300 s21 (B), 750 s21 (C), and 1500 s21 (D) for the fifty normal donors. doi:10.1371/journal.pone.0054680.gFigure 4. Surface coverage, rate and lag time of platelet accumulation as a function of wall shear rate. Recalcified citrated whole blood was perfused over type I collagen for 5 min and platelet accumulation was monitored as a function of wall shear rate and time (n = 50). Platelet accumulation was characterized by (A) percent surface 18325633 coverage (SC) after 5 min, (B) the rate of platelet accumulation expressed as percent surface coverage per second (VPLT), and (C) the lag time to 1 surface coverage (LagT). Lines with ** denotes a p,0.01 for the Mann-Whitney U-test. doi:10.1371/journal.pone.0054680.gwere not correlated to hematocrit. There was no correlation between platelet count on any of the metrics of platelet accumulation. The hematocrit and platelet count for every individual in the cohort was within the normal ranges (Table 1).Platelet Accumulation and Platelet Receptor GenotypeIn each donor we measured variants for three genes ?ITGA2, GP6 and GP1BA ?that encode for platelet adhesion receptors ?a2b1, GPVI, and GB1b ?to determine if the presence of specific alleles affected platelet accumulation in the MFA. These genetic variants were selected based on their biological significance andFigure 6. The effect of VWF plasma levels on platelet accumulation. Platelet surface coverage increases with increasing VWF plasma levels at all wall shear rates. For each shear rate, bars represent the average platelet surface coverage in each quartile of VWF levels in a cohort of normal donors (n = 50). doi:10.1371/journal.pone.0054680.gVariability in Microfluidic Flow AssaysTable 3. Spearman correlation coefficient between VWF levels and platelet surface coverage (SC), lag time (LagT) and platelet accumulation velocity (VPLT).Wall Shear Rate (s21) SC 150 300 750 1500 0.51** 0.38** 0.40** 0.32*LagT 0.08 0.14 20.42* 20.VPLT 0.55** 0.30* 0.52** 0.56**p,0.05; **p,0.01. doi:10.1371/journal.pone.0054680.tprevious reports of association with platelet or clinical bleeding and thrombotic phenotypes (Table S1). Individuals with the AA genotype of the GP6 gene had higher SC than individuals with the AG allele (Fig. 8). LagT was not different between the two GP6 genotypes, but VPLT was significantly (p,0.05) higher for the AA genotype at 150 s21. There was no difference in VWF levels between the two genotypes and no significant interaction (p = 0.64).