(A) BFP micrograph. A micropipette-aspirated RBC with a probe bead attached to the apex (left) was aligned against a platelet held by an opposing micropipette (right). (B) BFP functionalization. The …
(A) Schematic of a force-clamp experiment mode. Position (horizontal axis) of the probe bead attached to the apex of the RBC aspirated by the fixed micropipette (left) goes through indicated stages …
The unfolding length can be estimated directly (indicated) or calculated from the difference between the two best-fit contour lengths (318.6–279.3 = 39.3 nm). Both give comparable results.
(A–C) Schematics of GPIbα on the platelet membrane (A), highlighting the folded (−) and unfolded (+) LRRD (B) and MSD (C). Different regions and binding sites for VWF-A1 and mAbs are indicated. (D–G)…
(A,B) Nonparametric kernel density estimation using the software R with the given kernel ('gaussian' or 'epanechnikov') and bandwidth selected by Sheather and Jones (1991). Both estimates identify …
(A–D) Frequency (A,B) and force (C,D) of LRRD (A,C) or MSD (B,D) unfolding events occurred in the ramping phase induced by pulling via A1WT (blue) or A1R1450E (red) with indicated preset clamped …
Statistics and cooperativity evaluation of the GPIbα domains unfolding.
(A) Decision rules for and statistical summary of GPIbα domain unfolding in force-clamp experiment mode. Criteria for deciding whether or not (+ or −) and which (LRRD, MSD, or both) GPIbα domain(s) was (were) unfolded are based on BFP profile signatures and the unfolding lengths. YES = observed, NO = not observed. NA = not applicable. (B) Related to Figure 3F–H. Evaluation of LRRD and MSD unfolding cooperativity. All probabilities were calculated from occurrence data in (A). Observed joint probabilities were compared to their predicted counterparts based on the assumption that LRRD and MSD unfolded independently. For example, in 'WT A1 vs. Platelet' under 25 pN: The probability of LRRD unfolding is P(LRRD) = 3.4% + 6.9% + 2.76% = 13.06%. The probability of MSD unfolding is P(MSD) = 7.6% + 13.8% + 6.9% + 2.76% = 31.06%. The probability of MSD ramped unfolding is P(MSD, ramp) = 7.6% + 2.76% = 10.36%. The probability of MSD clamped unfolding is P(MSD, clamp) = 13.8% + 6.9% = 20.7%.
Frequencies of LRRD unfolding events occurred in the ramping phase induced by pulling GC via A1WT (blue) or A1R1450E (red) with 10, 25, 40 and 60 pN preset clamped forces.
(A–C) Mean ± s.e.m. of MSD time-to-unfold (tu, A) and GPIbα bond lifetimes (tb, B,C) with A1WT (blue) or A1R1450E (red) were measured in the clamping phase at different forces in the absence (−) or …
MSD unfolding rates (ku) and the fraction (w1) and off-rates (k1, k2) of GPIbα dissociating from A1WT or A1R1450E under different forces.
w1 represents the fraction of binding events that dissociate with the off-rate k1. The fraction of events that dissociate with the off-rate of k2 is simply calculated as w2 = 1-w1. NA = not applicable.
(A) Time-to-unfold (tu) distributions of A1WT MSD clamped unfolding with (red) and without (black) a preceding LRRD unfolding at 25 pN clamped force. The unfolding rate ku was calculated from the …
(A) Representative epi-fluorescence pseudo-colored images of intraplatelet Ca2+ of null (top row), α- (middle row), and β- (bottom row) types at indicated times. (B) Representative time courses of …
(A,B) Concurrent measurement of single-platelet Ca2+ flux and kinetics of single GPIb bonds with anti-GPIbα (A) and anti-GPIbβ (B). Top: representative epi-fluorescence pseudo-colored images of …
(A) Binary classifications of threshold performance outcomes on tmax–ΔImax data. (B) The receiver operating characteristic (ROC) plot: the true positive rate (i.e., sensitivity) against the false …
(A–D) Force-ramp fBFP experiment mode. Individual ΔImax values and their mean ± s.e.m. (A,B, points), Ca2+ types (C,D, stacked bars, left ordinate), and mean ± s.e.m. of pre-Ca2+largest rupture …
(A–D) Individual ΔImax values and their mean ± s.e.m. (points, left ordinate) in platelets triggered by A1 (A,C) or WM23 (B,D) binding, which were segregated into groups with (+) or without (−) …
The number of lifetime events (A), their average lifetimes (B), MSD unfolding occurrence frequency (C), expected number of MSD unfolding events (D), and calculated probabilities of observing at …
For a circulating platelet distal to an injured site without physical contact, its GPIb signaling will not be triggered (A). Upon interacting with VWF, platelets tether and translocate on the …
This animation (produced by Adobe Flash; 12 fps) illustrates the resemblance between platelet translocation on the sub-endothelium (a collagen network covered with VWF on the surface, upper panel) …
The video consists of two parts in series. Part I is an animation (produced by Adobe Flash; 12 fps), and part II is a video recording of a representative fluorescence BFP experiment (recorded by a …
Similar to Video 2, this video consists of two parts in series. In part I, the synchronized BFP illustration (upper panel), A1–GPIbα molecular interaction (middle panel) and 'Force vs. Time' signal …