Figure 6
Figure 6. Coated platelets in HIT. (A) P-selectin expression, Annexin V binding, and FX expression on human platelets. Data are expressed as the ratio of MFI of antibody or Annexin V binding after stimulation with KKO plus PF4 to MFI after stimulation with RTO plus PF4. Mean ± 1 SEM is shown. Significance was determined using two-tailed Student t tests. N = 30, each done in duplicate. The dotted line represents the value when there is no difference in activation marker expression between PF4 plus KKO or RTO, with P values shown relative to that of the control. (B) Relative level of P-selectin expression, Annexin V binding, and FX expression on platelets after stimulation with PF4 plus KKO in monocyte-depleted samples (black) and PRP (gray) to whole blood. N = 17 each. The dotted line represents the value when there is no difference in activation marker expression compared with whole blood stimulated with PF4 plus KKO, with P values shown relative to that of the control. (C) P-selectin expression, Annexin V, and FX binding to platelets after blocking of FcγRIIA, FcγRI, FcγRIII, and FcγRIIA plus FcγRI in whole blood samples. N = 10, each done in duplicate. (D) Same as (C) after blocking of individual FcγRs in isolated monocytes as in microfluidic experiments in Figure 4. N = 10, each done in duplicate. Results in (C-D) are expressed as the MFI ratio of sample stimulated with PF4 plus KKO after blocking the FcγRs with specific mAb to the sample incubated with isotype control. The dotted line represents the value where there is no effect of blocking the FcγRs. (E) P-selectin expression, and binding of Annexin V and FX using human platelets stimulated with PF4 and KKO in the absence of monocytes (ie, PRP), or repletion with transgenic mouse monocytes expressing FcγRIIA (ie, FcγRIIA+), and mouse monocytes that normally do not express FcγRIIA (ie, WT). The results are expressed as in (A). The significance for each parameter was calculated by comparing samples stimulated in the presence of either source monocytes to PRP alone. N = 5, each in duplicate. Wilcoxon Signed-Rank tests and one sample t tests were used to determine the significance of inhibition when compared with 1, no inhibition. n.s., not statistically significant between blocking FcγRIIA and blocking FcγRIIA plus FcγRI.

Coated platelets in HIT. (A) P-selectin expression, Annexin V binding, and FX expression on human platelets. Data are expressed as the ratio of MFI of antibody or Annexin V binding after stimulation with KKO plus PF4 to MFI after stimulation with RTO plus PF4. Mean ± 1 SEM is shown. Significance was determined using two-tailed Student t tests. N = 30, each done in duplicate. The dotted line represents the value when there is no difference in activation marker expression between PF4 plus KKO or RTO, with P values shown relative to that of the control. (B) Relative level of P-selectin expression, Annexin V binding, and FX expression on platelets after stimulation with PF4 plus KKO in monocyte-depleted samples (black) and PRP (gray) to whole blood. N = 17 each. The dotted line represents the value when there is no difference in activation marker expression compared with whole blood stimulated with PF4 plus KKO, with P values shown relative to that of the control. (C) P-selectin expression, Annexin V, and FX binding to platelets after blocking of FcγRIIA, FcγRI, FcγRIII, and FcγRIIA plus FcγRI in whole blood samples. N = 10, each done in duplicate. (D) Same as (C) after blocking of individual FcγRs in isolated monocytes as in microfluidic experiments in Figure 4. N = 10, each done in duplicate. Results in (C-D) are expressed as the MFI ratio of sample stimulated with PF4 plus KKO after blocking the FcγRs with specific mAb to the sample incubated with isotype control. The dotted line represents the value where there is no effect of blocking the FcγRs. (E) P-selectin expression, and binding of Annexin V and FX using human platelets stimulated with PF4 and KKO in the absence of monocytes (ie, PRP), or repletion with transgenic mouse monocytes expressing FcγRIIA (ie, FcγRIIA+), and mouse monocytes that normally do not express FcγRIIA (ie, WT). The results are expressed as in (A). The significance for each parameter was calculated by comparing samples stimulated in the presence of either source monocytes to PRP alone. N = 5, each in duplicate. Wilcoxon Signed-Rank tests and one sample t tests were used to determine the significance of inhibition when compared with 1, no inhibition. n.s., not statistically significant between blocking FcγRIIA and blocking FcγRIIA plus FcγRI.

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