Differential Dynamics of Platelet Contact and Spreading

Abstract

Platelet spreading is critical for hemostatic plug formation and thrombosis. However, the detailed dynamics of platelet spreading as a function of receptor-ligand adhesive interactions has not been thoroughly investigated. Using reflection interference contrast microscopy, we found that both adhesive interactions and PAR4 activation affect the dynamics of platelet membrane contact formation during spreading. The initial growth of close contact area during spreading was controlled by the combination of different immobilized ligands or PAR4 activation on fibrinogen, whereas the growth of the total area of spreading was independent of adhesion type and PAR4 signaling. We found that filopodia extend to their maximal length and then contract over time; and that filopodial protrusion and expansion were affected by PAR4 signaling. Upon PAR4 activation, the integrin α_IIbβ₃ mediated close contact to fibrinogen substrata and led to the formation of ringlike patterns in the platelet contact zone. A systematic study of platelet spreading of GPVI-, α₂-, or β₃-deficient platelets on collagen or fibrinogen suggests the integrin α₂ is indispensable for spreading on collagen. The platelet collagen receptors GPVI and α₂ regulate integrin α_IIbβ₃-mediated platelet spreading on fibrinogen. This work elucidates quantitatively how receptor-ligand adhesion and biochemical signals synergistically control platelet spreading.

Introduction

As a result of vascular trauma, exposure of subendothelial collagen induces platelets to form a hemostatic plug. This plug is the result of sequential adhesion, activation, and aggregation of individual platelets. Platelet spreading, the process by which adherent platelets first flatten at sites of vascular injury and increase their contact area by deformation of the plasma membrane, has been recognized as a crucial step for hemostasis and thrombosis (1).

Platelet adhesion to collagen at medium to high rates of shear (wall shear rates of 50–40,000 s⁻¹ (2,3)) begins with a molecular interaction between platelet glycoprotein Ibα (GPIbα) and circulating von Willebrand factor (vWF) that becomes immobilized on exposed collagen. GPIbα-vWF interactions allow platelet translocation or rolling on exposed collagen but is not sufficient for stable arrest. Firm platelet adhesion to collagen is believed to require the participation of two major platelet collagen receptors, glycoprotein VI (GPVI) and integrin α₂β₁ (4). GPVI plays the central role in the interaction between platelets and collagen as not only a surface adhesion receptor but also the major agonist for the initial activation and granule release (5–9) whereas α₂β₁ requires inside-out activation via G-protein-coupled receptor signaling.

Integrin α_IIbβ₃ typically displays bidirectional signaling (4,10). Signals induced by an agonist lead to an increase in the affinity of α_IIbβ₃ to extracellular ligands, which, in turn, triggers outside-in signaling within the cell initiated by the receptor ligation. A major early consequence of α_IIbβ₃ outside-in signaling is platelet spreading. Jirousková et al. (11) showed that the kinetics of filopodia and lamellipodia formation and the increase in cytosolic Ca²⁺ were affected by the amount of immobilized fibrinogen in α_IIbβ₃-mediated platelet spreading. An actin nodule (a localized high concentration of actin), which appears before lamellipodia formation, was recently identified as necessary for platelet spreading on fibrinogen (12). However, an important gap still exists in our understanding as to how a platelet regulates its proximity on fibrinogen or collagen to seal the breach in the vascular system.

Cell spreading can be quantified by measuring the distance between the membrane and a planar transparent substrate. Reflection interference contrast microscopy (RICM), which visualizes the contact zones between the basal cell membrane and a substrate in a time-resolved manner, is suitable for studying the membrane dynamics of cell spreading on a ligand-immobilized surface (13–15). Reininger et al. (2) utilized RICM to visualize the formation of adhesion points on platelet membrane and generation of platelet-derived microparticles mediated by GPIbα-vWF interactions under flow. Previously, we showed using RICM that the spreading of n-formyl-L-methionyl-L-leucyl-L-phenylalanine-stimulated neutrophils is anisotropic and directional (16). Experiments on cells of different types, but not platelets, reported by Cuvelier et al. (17) suggested the possibility of a universal relation of cell spreading between contact radius and the time in the early stage of cell spreading.

Here we applied this technique to elucidate the membrane dynamics of platelets during spreading, using genetically modified mice to examine the molecular basis of spreading. We hypothesized that ligation of adhesion receptors such as GPVI, α₂β₁, or α_IIbβ₃, as well as the activation through protease-activated receptor 4 (PAR4), can differentially modulate platelet spreading. To examine the relative importance of each of these stimuli, we investigated the dynamic reorganization of the platelet membrane and the proximity of the membrane to a collagen or fibrinogen surface using wild-type (WT), GPVI^−/−, α₂^−/−, or β₃^−/− mouse platelets. We found that the formation of close contact zones on platelet membrane is differentially regulated by combined adhesions of a platelet to collagen and fibrinogen together or PAR4 signaling on fibrinogen. However, the expansion of the total membrane area during platelet membrane during spreading is not affected by the type of stimulus. Understanding the membrane dynamics of platelet spreading helps to provide new insights into how platelets integrate and convert adhesive and biochemical signals into the morphological regulation of plasma membrane to stabilize hemostatic response.

Materials and Methods

Platelet spreading analysis and confocal microscopy are described in the Supporting Material. Mouse handling and procedures were in strict accordance with University of Pennsylvania and Institutional Animal Care and Use Committee protocols.

Mouse platelet preparation

Spreading dynamics of platelets was examined with platelets derived from GPVI, α₂-, or β₃-deficient mice as well as WT mice. After anesthesia (40 mg/kg pentobarbital i.p.), mouse blood was drawn via the inferior vena cava using 93 μM PPACK (Calbiochem, Gibbstown, NJ) as the anticoagulant. The blood was diluted with an equal volume of Tyrode's buffer (4 mM HEPES, pH 7.4, 135 mM NaCl, 2.7 mM KCl, 3.3 mM NaH₂PO₄, 2.4 mM MgCl₂, 0.1% glucose, and 0.1% BSA) and centrifuged at 129× g for 7 min to obtain platelet-rich plasma (PRP). Platelet count was normalized to 2.5 × 10⁸ platelets/ml using Tyrode's buffer. Washed platelets (WP) were obtained by an additional centrifugation at 341× g for 7 min and resuspended in Tyrode's buffer to the same volume as that for PRP.

Platelet spreading

Glass coverslips (No. 1.5; Corning, Corning, NY) were coated with 100 μg/ml bovine type I collagen (PureCol; Inamed Biomaterials, Fremont, CA) or human fibrinogen (Enzyme Research, South Bend, IN) overnight at 4°C. In some experiments, the mixture of collagen and fibrinogen was immobilized. After that, the functionalized slides were rinsed to remove excess protein. Platelets were spread on the surfaces and visualized by RICM as described below. To investigate mouse PAR4 signaling in the absence of active thrombin (which was blocked by 93 μM PPACK), 0.2 mM PAR4 agonist AYPGKF (BACHEM, Torrance, CA) was added to platelets.

Reflection interference contrast microscopy

Platelet spreading was observed in reflection interference contrast microscopy (RICM) mode through an inverted microscope (Axiovert 200; Carl Zeiss, Gottingen, Germany) equipped with an antiflex 63×/1.3 NA oil immersion objective and an appropriate polarizer and analyzer. A platelet was illuminated through the objective by monochromatic light (546 nm) generated by passing the light from a 100 W mercury lamp through an interference filter. Images passed by an analyzer were recorded with a charge-coupled device camera (Retiga Exi Fast Cooled Mono 12-bit camera, 32-0082B-128; QIMAGING, Surrey, British Columbia, Canada) and IPLab software (BD Biosciences, Franklin Lakes, NJ). Images were taken every 1 s; the time was set to 1 s when the cell formed the first contact as determined by a change of interference intensity.

Although there was usually no significant lag time between the first contact and beginning of the active membrane expansion, the lag time was included in the measurements if it existed. The principle of RICM and its application to quantitative analysis of dynamics of adhesion of cells have been described elsewhere (13,15–19). Briefly, monochromatic light is incident on the cell that is bound to a glass substrate in a transparent buffer. The incident light is reflected from the glass-buffer interface and again from the buffer-cell interface. These two reflected rays interfere and give rise to an interference pattern. An interfering pattern of alternating dark and bright patches is obtained that reflects the height distribution of the lower surface of the cell membrane (Fig. 1 A). In a trinary image obtained by the RICM image analysis, the solid zones correspond to close contact areas where the membrane is close to the substrate (∼40 nm).

Figure 1.

Dynamic spreading of murine platelets on collagen in vitro. (A) Time-resolved RICM of early stages of mouse WT platelets spreading on a collagen-coated substrate at 37°C. The images were taken at 1 fps up to 7 min after the first contact. The scale bar represents 3 μm. (B) Grayscale-shaded trinary images converted from panel A that contain the solid patches showing close contacts (∼40 nm; cell-to-substrate distance), open zones corresponding to the cell spreading area close to the substrate (40–110 nm). The uniform shaded area represents a background. (C) The theoretical relation between intensity and cell-to-substrate distance. Based on the relation, close contact was defined as the cell area when the cell-to-substrate distance is <40 nm. (D) A representative trinary image showing close contact patches (solid) and whole spreading area (open). A typical area growth profile of a mouse WT platelet on immobilized collagen plotted in a linear scale (E) or a log-log scale (F). (Open squares) Whole spreading area. (Solid squares) Close contact area. A, Area; t, time in seconds; b, area growth exponent; and c, y axis intercept.

As stated previously, 40 nm is arbitrarily chosen to represent a threshold of adherent zone because it is a little less than half-way to the maximum, which occurs at 110 nm (16,20). The areas in open representation represent a whole spreading area where the basal membrane is located in the range of 40–110 nm above the glass slide. As shown previously (21), this technique allowed us to evaluate height fluctuations with 10–20-nm vertical resolution. Note that this procedure is not expected to produce absolute heights, but relative separations. The uniform shaded areas represent a background (Fig. 1, B–D). The contact area is shown as solid patches, and the whole spreading area as open patches, in trinary images. The cell-to-substrate distance were measured using an algorithm based on our previous work (16). The detailed procedure is provided in the Supporting Material.

Results

Establishment of RICM system to investigate platelet spreading on immobilized ligands

Our study of the early stages of platelet spreading focused on the changes in whole spreading and close contact areas and local membrane protrusion over time by observing morphological changes and by measuring the regions of platelet contact with ligand-coated substrates. We found, by using time-lapse RICM imaging, that individual platelets generally spread in a rapid and spatially isotropic manner with increasing area of membrane until ∼2 min after the cells formed initial contacts (detected as dark or bright patches) with the surface. After initial fast spreading, the spreading area increased slowly while presumably forming stable adhesions (Fig. 1; and see Movie S1 in the Supporting Material). A double logarithmic plot of the changes of spreading area over time (Fig. 1 F) showed that the whole and close area growth of spreading can be divided into two distinctive phases that generally followed power laws, A_i (t) ∼ t^bi, where b_i is the area growth exponent in the i^th phase. The slope of data in a log-log plot becomes an exponent that is often used to identify the dynamic scaling of spreading (16), independent of the data's magnitude.

The close contact area as well as whole spreading area initially followed a fast continuous phase and then growth of both areas slowed to a late spreading phase. Thus, b_i is a simple yet comprehensive metric to indicate how membrane area grows over time. This value allows us to compare platelet initial response to different biochemistries. Overall, the dynamics of close contact area as well as whole spreading area and the corresponding membrane height changes in the RICM system allow us to study how a platelet regulates membrane morphological dynamics over time after the mechanical ligation of adhesion receptors and biochemical stimulation through G-protein coupled receptors.

Initial growth of close contact area in platelet spreading is affected by the combination of immobilized ligands and PAR4 activation

To investigate how the ligation of platelet adhesion receptors or the stimulation of a platelet through PAR4 signaling affects the area growth of a platelet spreading over time, time-lapsed RICM was performed with WP or PRP isolated from WT mice on an immobilized collagen and/or fibrinogen without or with 0.2 mM AYPGKF (Fig. 2; see Movie S2). A key question is how activation of the thrombin signaling pathway can act as a surrogate for specific adhesion, thus addressing how adhesion and signaling are interconnected to drive platelet spreading. Thrombin-induced mouse platelet activation is mediated by G-protein-coupled PAR4. Thus, the activation via PAR4 signaling is crucial to platelet clot formation and stability. We note that the dynamics of spreading area over time, when platelets were introduced in PRP, was very similar with those when WP were used (Fig. 2, A–C). This indicates that plasma proteins in PRP did not significantly affect the spreading kinetics of WT platelets in our studies.

Figure 2.

Growth of platelet spreading area on collagen, fibrinogen, or a mixture of collagen and fibrinogen. Dynamic changes of spreading area of mouse WT platelets on immobilized collagen (A), fibrinogen (B), or collagen and fibrinogen (C) without or with a PAR4 agonist peptide AYPGKF at 37°C. (Circles) Whole spreading area. (Squares) Close contact area over time. Data were expressed as the mean ± SE. (D) Area growth exponents of close contact area in the early spreading regime (b_c1) on different ligands. Data were expressed as the mean ± SE. A one-way between subjects ANOVA was conducted to compare the effect of ligands on b_c1 in the spreading of platelets in PRP (bar graphs in cyan color) on collagen, fibrinogen, and a mixture of collagen and fibrinogen. There was a significant effect of immobilized ligands on b_c1 at the p < 0.05 level for the three conditions (F(2, 46) = 5.3527, p = 0.0081). Post hoc comparisons using the Newman-Keuls multiple comparison test indicated that the mixture of collagen and fibrinogen condition was significantly different than collagen and fibrinogen condition. However, the collagen condition did not significantly differ from the fibrinogen condition.

Although different immobilized ligands were used without or with PAR4 activation, both whole spreading area and close contact area generally increased rapidly as soon as platelets in suspension were in contact with functional substrates and later slowed. We found that the temporal growth of whole spreading area of a platelet was independent of immobilized ligands or cellular activation through PAR4. However, the area growth exponent in the fast spreading phase of close contact area, b_c1, significantly increased on substrates coated with collagen and fibrinogen together compared to those with either collagen or fibrinogen only (Fig. 2 D). For instance, platelets in PRP expand their close contact zones on a mixture of collagen and fibrinogen with the average exponent of 2.30 (SE = 0.57, n = 13), on collagen with 1.28 (SE = 0.12, n = 21), and on fibrinogen with 0.65 (SE = 0.29, n = 15). However, the difference in b_c1 between collagen and fibrinogen is not statistically significant. Details of statistical analyses are given in the legend of Fig. 2 D. These results suggest that the initial growth of close contact area in the platelet spreading is affected by the combination of the different receptor-ligand interactions, but not the type of adhesive ligands.

In addition, PAR4 activating peptide increased the growth rate of close contacts on immobilized fibrinogen. The average b_c1 of WP spreading significantly increased from 0.82 (SE = 0.12, n = 17) to 2.63 (SE = 0.34, n = 17) when 0.2 mM AYPGKF was added. An increase in b_c1 with the PAR4 activation, from 0.65 (SE = 0.29, n = 15) to 2.01 (SE = 0.34, n = 37), was also observed when PRP was used (Fig. 2 D). However, when platelets spread on immobilized collagen or a mixture of collagen and fibrinogen, the exponents were not significantly altered with PAR4 stimulation. Details of initial growth exponents of mouse WT platelets in spreading are summarized in Table S1 in the Supporting Material.

The results suggest that PAR4 signaling can augment the initial growth of close contact area in platelet spreading on fibrinogen, but not on collagen, presumably because platelets carry multiple collagen receptors that can access different signaling pathways. On fibrinogen, PAR4 activation in platelets led to a correspondingly increased close contact and whole spreading area over time (see Fig. S1). In contrast, the growth rate of whole spreading area is not limited by either interactions of platelet receptors with ligands or PAR4 signaling (Fig. 2, A–C, and see Table S1). Overall, these observations suggest that the formation of close contacts and whole membrane expansion during spreading are differentially regulated in platelets.

Temporal dynamics of filopodia and lamellipodia formation is affected by PAR4 signaling

We further investigated how the stimulation of mouse WT platelets via PAR4 engagement affected the dynamics of local membrane protrusion during spreading on fibrinogen. As observed by time-lapse RICM, platelet adhesion, and spreading on fibrinogen began within 4 min after WP were added to the substrates. In contrast, platelet adhesion to fibrinogen with the addition of 0.2 mM AYPGKF began within seconds of platelet addition and started spreading (data not shown). To analyze local morphologic changes of a platelet membrane in contact with the substrate during spreading, RICM micrographs were recorded in high temporal resolution of 1 frame/s after the first second of initial contact. This analysis revealed significant differences in the kinetics of filopodia and lamellipodia formation on fibrinogen with and without the addition of the AYPGKF peptide (Fig. 3, A and B).

Figure 3.

Morphological changes of the platelet membrane during spreading on fibrinogen. Time-lapse RICM micrographs of platelets spreading on immobilized fibrinogen were recorded for 8 min after the first contact of platelets with the substrates. Individual platelets were analyzed for the appearance, length, positioning, and filopodial height over time. A representative RICM micrograph of a platelet spread on fibrinogen without (A) or with (B) 0.2 mM AYPGKF at 37°C. The scale bar represents 3 μm. (C) The number of filopodia over time during the platelet spreading on fibrinogen without (○) or with (●) 0.2 mM AYPGKF. Data were expressed as the mean ± SE of a total of 12 platelets from two independent experiments in each condition. (D) The appearance of new filopodia and onset of lamellipodia formation, counting the time platelets first contacted the substrates in the RICM as t = 1 s. (Box plots) Median and the 25th and 75th percentiles of the time to the onset and end of filopodia formation and the onset of lamellipodia formation in each condition. A total of 12 platelets at each condition were analyzed from two independent experiments. Distribution of lengths and angles of filopodia in platelet spreading on fibrinogen without (E) or with (F) 0.2 mM AYPGKF. The representative polar coordinate map of filopodia in each condition was obtained from monitoring a platelet for 3 min after the first adhesive contact of the platelet with a substrate. Each open circle represents the means of both lengths and angles of a filopodium from the onset of each filopodium formation to 3 min and error bars represent the SEs in radial and polar directions, respectively. The filopodial length was defined as the shortest distance from the tip of a filopodium to the platelet body. Normalized length of a filopodium in the spreading of platelets on fibrinogen without (G) or with (H) 0.2 mM AYPGKF. The length of filopodia was analyzed every 10 s from the onset and the end of filopodia formation. Time and filopodial length were normalized with a maximal value. (I) A representative intensity map of a spread platelet on fibrinogen without adding AYPGKF. The color bar represents the intensity between the basal membrane and a fibrinogen-coated substrate. (J) The intensity map of filopodia of platelets spreading on fibrinogen with 0.2 mM AYPGKF. The interference intensity of filopodia was analyzed over the entire length of filopodia. A total of 26 filopodia in five platelets were analyzed from two independent experiments and the color maps represent the mean and error (SD), respectively.

Filopodia started to form as soon as a platelet was in contact with a fibrinogen-coated surface both without and with AYPGKF. With PAR4 activation, the number of filopodia increased up to a maximum (1.83 ± 0.76; mean ± SE) in 2 min but decreased to 0 within 4 min, whereas in the absence of PAR4 activation the numbers of filopodia increased with time and reached a plateau (5.27 ± 0.62) within 4 min (Fig. 3 C). In addition, lamellipodia formed with filopodia simultaneously on platelets adhering to fibrinogen (denoted by a red arrow in Fig. 3 D, median = 30 s, n = 12) and later the formation of new filopodia ceased (denoted by a blue line in Fig. 3 D, median = 210 s) upon the PAR4 stimulation. In contrast, platelets adhering to fibrinogen without the PAR4 stimulation continued to form new filopodia for duration of experiments (denoted by a blue arrow in Fig. 3 D). These results indicate that platelet activation through PAR4 allows platelets to spread lamellipodially rather than filopodially.

To investigate how PAR4 stimulation in platelets spreading on fibrinogen affects the length and angle of the filopodia over time, we tracked the position of filopodia every 10 s after platelets first contacted fibrinogen-coated surfaces. Straight lines from tips of filopodia to the centroid of the platelets were drawn and the length of a filopodium was defined as the shortest distance from the tip of a filopodium to the boundary of the platelet body. Fig. 3, E and F, showed the locations of all filopodia (mean ± SE of length and angle in a two-dimensional polar coordinate system) in a representative platelet for 3 min after the platelet was in close contact with fibrinogen without or with PAR4 activation, respectively. Filopodia were spatially isotropic with the average length of 0.69 μm (SD = 0.27, n = 37) and although the frequency of extension was reduced by PAR4 activation, the spatial orientation of filopodial extension was not significantly altered by the addition of AYPGKF. These data indicate that the spatial dynamics of filopodia protrusion is isotropic and that the orientation is not dependent on the activation of platelets via PAR4 engagement.

Because new filopodia extend and retract over time, we examined the temporal dynamics of filopodial length by normalizing the length of filopodia every 10 s with a maximum in the lifetime of filopodia (Fig. 3 G; see Movie S3). Interestingly, filopodia protruded to a maximal length as they formed and they shortened their extension over time. The same dynamics of extension and retraction was also observed on fibrinogen with the addition of AYPGKF (Fig. 3 H).

We used interference intensity from RICM images to investigate how the height of filopodia varied over the length of the membrane protrusion. The closer the cell membrane is to a substrate, the darker the corresponding area in RICM images. As shown in Fig. 3 I, the average intensity of a filopodium over a fibrinogen-coated substrate was significantly lower than that of the platelet body, suggesting that the filopodia formed a more intimate contact with the substrates than the platelet body. Furthermore, we found that the intensity of the tip of filopodia is the lowest over the entire length of filopodia; this intimate contact extended to the entire outer half of the filopodia (Fig. 3 J). Overall, these results indicate that platelets form close contact with fibrinogen by extending filopodia from the body dynamically in the early stage of spreading.

The density of immobilized fibrinogen affects the membrane proximity to the substrate in platelet spreading

The spreading of mouse WT platelets on low- and high-density immobilized fibrinogen was observed to investigate the effect of the ligand density on the platelet morphologic change and its proximity to a substrate over time (Fig. 4; see Movie S4). A quantity of 1 μg/ml fibrinogen was used to prepare surfaces with low-density fibrinogen and 100 μg/mL was used to make surfaces of high-density fibrinogen. WP was added to observation chambers containing 0.2 mM AYPGKF. The decrease in immobilized fibrinogen concentration led to delayed onset of lamellipodia formation, resulting in sequential formation of filopodia and lamellipodia compared with the rapid lamellipodial spreading of platelets on high-density fibrinogen (Fig. 4, A and B).

Figure 4.

Effect of fibrinogen surface density on platelet membrane dynamics, height, and area growth. Time-lapse RICM micrographs of platelets in spreading on low-density (A) or high-density (B) fibrinogen with 0.2 mM AYPGKF. (C–H) A representative RICM micrograph, overall intensity map, and reconstructed intensity map (of a boxed section in Fig. 4, C or F) of a platelet spreading on low-density (C–E) or high-density (F–H) fibrinogen. The scale bar represents 3 μm. Note that the y axes in panels E and H are not to scale. (I) Area growth dynamics of platelets on different density of fibrinogen. (Open squares and circles) Whole spreading area and close contact area of platelets on low-density fibrinogen, respectively. (Shaded marks) Area of platelets spread on high-density fibrinogen.

These observations are consistent with previous work on human platelets observed by using total internal reflection fluorescence microscopy (11). Interestingly, low-density fibrinogen substrates supported more intimate contact between the platelet basal membrane and the substrate than the high-density fibrinogen (Fig. 4, C–H). This result agrees with a previous observation that most fibrinogen molecules are oriented horizontally on the surface when immobilized at low density and vertically at high density (22). However, the growth rates of close contact (b_c1 = 2.77 on low-density fibrinogen versus 2.63 on high-density fibrinogen) and whole spreading area (b_w1 = 0.69 on low-density fibrinogen versus 0.49 on high-density fibrinogen) in the fast spreading regime were not significantly affected by the fibrinogen density on substrates (Fig. 4 I). Overall, these results show that the amounts of fibrinogen affects the proximity of membrane to the substrate and local membrane protrusion dynamics, but not the kinetics of close contact and membrane expansion of platelets during spreading.

Integrin α_IIb forms ringlike patterns around the center of a platelet during spreading on fibrinogen

As shown in Fig. 4, C and F, RICM imaging revealed that platelets produced dark ringlike patterns inside platelet bodies when they spread on fibrinogen. However, this pattern was not observed in the platelet spreading on collagen (Fig. 1 A). We hypothesized that integrin α_IIbβ₃ interaction with fibrinogen induced the close contact by forming the distinctive ringlike patterns. Time-lapse confocal imaging was used to determine whether the integrin α_IIbβ₃ is involved in the formation of the ringlike patterns that are in close contact with fibrinogen during spreading. Platelets labeled with fluorescent and nonfunctional blocking antibody against integrin α_IIb domain were placed on a fibrinogen-coated slide to monitor the spatiotemporal dynamics of α_IIb during spreading.

We found that fluorescently labeled α_IIb domains were located in the periphery of a platelet when the cell adhered to the substrate (Fig. 5; see Movie S5). However, the domains moved toward the center of the platelet and formed a ringlike pattern as the platelet spread. In contrast, platelets from β₃-deficient mice did not show ringlike patterns on fibrinogen (see Movie S6), suggesting that the ringlike pattern at the basal surface of platelets during spreading on fibrinogen is caused by integrin α_IIbβ₃ interactions.

Figure 5.

Platelet integrin α_IIb dynamics during spreading on fibrinogen. Time-lapse confocal imaging was used to observe the redistribution of integrin α_IIb on fibrinogen. Fluorescent images (left column) were converted into pseudo-colormap images (right column) to show the intensity of fluorescence on integrin α_IIb on the platelet membrane over time.

Cross-regulation of collagen receptors, GPVI, and integrin α₂β₁ with a fibrinogen receptor, α_IIbβ₃ in platelet spreading

To study how defects in a platelet adhesion receptor expression affect the spreading of platelets, we observed the spreading of platelets isolated from β₃, GPVI, or α₂-deficient mice on immobilized collagen or/and fibrinogen without or with 0.2 mM AYPGKF (see Movie S6, Movie S7, and Movie S8). In addition to using WP, PRP was used to examine the effect of plasma proteins on the spreading of platelets. WP from WT and GPVI, α₂-, or β₃-deficient mice did not adhere to the substrates coated with 100 μg/ml acid-soluble collagen (Fig. 6 A). However, platelets adhered well to the substrates coated with the same concentration of fibrillar collagen (data not shown), implying that the acid-soluble collagen is less active than fibrillar collagen in the stimulation of platelets to induce cell adhesion.

Figure 6.

Comprehensive map of platelet spreading. Platelets isolated from WT (blue), GPVI−/− (yellow), α₂^−/− (red), or β₃^−/− (green) mice were seeded on collagen (A, D, G, and J), fibrinogen (B, E, H, and K) or a mixture of collagen and fibrinogen (C, F, I, and L). WP or PRP was used without or with 0.2 mM AYPGKF.

However, it was necessary to use 100 μg/ml acid-soluble collagen for the reminder of experiments because an increase in the concentration of acid-soluble collagen or use of fibrillar collagen to immobilize collagen on a glass slide caused a translucent surface that prevented clear RICM imaging. As expected, β₃^−/− deficiency in platelets significantly inhibited the adhesion of platelets to fibrinogen or a mixture of collagen and fibrinogen (Fig. 6, B and C). Interestingly, deficiency in either GPVI or α₂ in platelets increased b_c1 on fibrinogen (0.82 ± 0.12 in WT (n = 17); 1.42 ± 0.17 in GPVI^−/− (n = 60), (p < 0.005); 1.55 ± 0.26 in α₂^−/− (n = 13), (p < 0.05)) (Fig. 6 B), even though α₂^−/− platelets displayed a delay in spreading. On a mixture of collagen with fibrinogen, α₂-deficient platelets showed a significant decrease in whole spreading area in the late spreading regime (17.40 ± 2.69 μm² in WT (n = 5); 14.35 ± 1.02 μm² in GPVI^−/− (n = 18, not statistically significant); 2.15 ± 1.17 μm² in α₂^−/− (n = 2), (p < 0.005) at 6 min) and a significant increase in b_c1 after an extensive delay (Fig. 6 C). These data suggest that the deficiency in GPVI or α₂ significantly inhibits integrin α_IIbβ₃ outside-in signaling-mediated platelet spreading on fibrinogen, and the inhibition is more severe in α₂-deficient platelets than GPVI-deficient platelets.

As shown in Fig. 6 D, the activation of WP through PAR4 stimulation enabled GPVI^−/− and α₂^−/− platelets as well as WT platelets to adhere to immobilized collagen with a similar b_c1 (1.33 ± 0.25 in WT (n = 18); 1.36 ± 0.24 in GPVI^−/− (n = 76); 0.83 ± 0.27 in α₂^−/− (n = 18)). However, α₂-deficient platelets showed a significant decrease in whole spreading area on collagen compared to WT or GPVI-deficient platelets (3.29 ± 0.32 μm² in α₂^−/− (p < 0.05); 7.52 ± 0.85 μm² in WT; 8.09 ± 0.42 μm² in GPVI^−/− at 6 min). This suggests that α₂β₁-collagen binding under PAR4 activation is more critical to membrane spreading than GPVI-collagen binding. In addition, the PAR4 stimulation in platelets restored b_c1 to that seen on fibrinogen-coated substrates (Fig. 6, E and F), suggesting that integrin α_IIbβ₃ interaction with fibrinogen is a dominant factor in the membrane spreading of platelets on fibrinogen. We found that β₃-deficient platelets adhered to immobilized fibrinogen with the PAR4 activation, implying that other platelet receptors might be involved in the spreading on fibrinogen (Fig. 6, E and K).

When PRP was placed on collagen-coated surfaces, we found that platelets from WT or GPVI- or β₃-deficient mice adhered to the substrates (Fig. 6 G) whereas WP did not adhere. This result suggests that plasma proteins in PRP help platelets adhere to immobilized collagen. However, α₂-deficient platelets failed to adhere to immobilized collagen whereas GPVI-deficient platelets adhered to the substrates with a severe defect in whole spreading area (1.34 ± 0.24 μm² in GPVI^−/− (n = 3), (p < 0.0001) vs. 12.35 ± 1.48 μm² in WT (n = 21) at 6 min) and a significant increase in b_c1. PAR4 stimulation in α₂-deficient platelets restored the whole membrane expansion on collagen but b_c1 was not recovered (3.08 ± 1.16 in α₂^−/− (n = 3) vs. 1.05 ± 0.13 in WT (n = 28)) (Fig. 6 J).

In contrast, b_c1 in GPVI-deficient platelets (1.13 ± 0.31 in n = 14) was very similar with that of WT platelets when platelets were stimulated with AYPGKF. Overall, these results indicate that the integrin α₂β₁ plays a major role in spreading on collagen. The inhibition of the growth of close contact area in the fast spreading regime in GPVI- or α₂-deficient platelets was also observed when platelets in PRP were placed on immobilized fibrinogen (b_c1 = 0.65 ± 0.29 in WT (n = 15); 1.71 ± 0.25 in GPVI^−/− (n = 10), (p < 0.05); 1.65 ± 0.40 in α₂^−/− (n = 4), (p < 0.05)) (Fig. 6 H). The response of platelets in PRP is similar with that in WP (Fig. 6 B), suggesting that the deficiency in GPVI or α₂ in platelets affects the adhesion and spreading of platelets on fibrinogen. The PAR4 stimulation in β₃-deficient platelets recovered the spreading of the platelets on fibrinogen (Fig. 6, E and K) but the b_c1 (0.90 ± 0.10 (n = 60)) was significantly altered (p < 0.05) compared with the value in WT platelets (2.01 ± 0.34) (Fig. 6 K). Therefore, this result indicates that the significant increase in the initial growth rate of close contact area in WT platelets observed with the addition of PAR4 agonist (Fig. 2, B and D) was caused by the platelet integrin α_IIbβ₃ interaction with fibrinogen. Overall, these observations demonstrate that integrin α₂ is indispensable to platelet spreading on collagen. In addition, collagen receptors, GPVI, and integrin α₂ are involved in integrin α_IIbβ₃-mediated platelet spreading on fibrinogen.

Discussion

In this work, we studied how a platelet controls the dynamics of membrane proximity on substrates coated with different ligands using an array of molecular modified platelets. By utilizing real-time RICM imaging and detailed analysis, we demonstrated that the growth of close contact of membrane is differentially regulated by the growth of whole membrane expansion in the platelet spreading.

A concise summary of our results:

• 1.The combined response of a platelet on a mixture of collagen and fibrinogen or under PAR4 activation on fibrinogen increases the growth of close contact areas in the fast spreading regime.
• 2.The growth of whole spreading area is independent of type of ligand and PAR4 activation.
• 3.Platelet filopodia changes their lateral and vertical distribution dynamically during spreading and PAR4 activation in platelets during spreading induces fast growth of lamellipodia.
• 4.Fibrinogen density affected the closeness of the platelet membrane to the surface, but not the kinetics of membrane contact and expansion areas.
• 5.Integrin α₂ is indispensable for spreading on collagen, and GPVI and integrin α₂ in platelets regulate the α_IIbβ₃-mediated platelet spreading on fibrinogen.

We have cataloged a wide variety of platelet responses with a simple yet meaningful metric, the exponent of areal increase for both total and close contacts. We have shown that the growth of close contact of platelet membrane is independent of single ligand-type and density, as proposed by Cuvelier et al. (17). However, combined adhesions of platelets on collagen and fibrinogen together increases the growth of close contact areas, which is characterized by a growth law such as A ∼ t^2.5, which is different than suggested by Cuvelier et al. (17). The synergistic interaction of collagen and fibrinogen increases the rate of spreading beyond that seen for a single receptor, and the universal power-law of cell spreading is not valid in this case.

In addition, PAR4 activation significantly increases the growth of close contacts during the spreading of platelets on fibrinogen. However, the kinetics of whole membrane expansion exhibits the same growth rate regardless of different ligands and PAR4 signaling. Note that the observed A ∼ t growth law in whole membrane expansion agrees with the universal power-law. These findings imply that the increase in the contact zone is governed by the combination of specific adhesive interactions and signaling pathways (through specific agonists), whereas overall membrane expansion is controlled by collective cellular structural constraints. The fact that the deficiency in platelet adhesion receptors altered the kinetics of formation of close contact confirms the dependency of close contact in spreading on receptor-ligand interactions. We postulate that platelet flattening by increasing membrane proximity to a complex environment including collagen, fibrinogen, and agonists together is relevant to its physiological role in repairing an injured vessel wall in the face of arterial flow.

We demonstrated that when platelets spread on fibrinogen-coated surfaces only, dark circular rings form near the periphery of the cells initially and migrate toward the center of platelets over time. These ringlike patterns are also observed when we tracked the integrin α_IIb subunit on fibrinogen substrates, suggesting that adhesive interactions of α_IIbβ₃ with fibrinogen cause the ring formation of close contact with the greatest proximity during spreading. Although previous work showed a similar redistribution of α_IIbβ₃ molecules by receptor cross-linking (11,23–25), our study shows, to our knowledge for the first time, that the distinctive ringlike contact patterns of platelets were correlated to the spatiotemporal dynamics of α_IIbβ₃ on fibrinogen during spreading.

Filopodia are spikelike plasma membrane protrusions that are filled with tight parallel bundles of filamentous actin (26). In contrast, lamellipodia are sheetlike protrusions that are filled with a branched network of actin. We found that filopodia dynamics in platelet spreading is affected by PAR4 activation but their length and positioning are not affected by the cellular activation. In addition, their length reached a maximum and then decreased over time, and the most intimate contact between the filopodia and substrate is at the filopodial tips. Interestingly, Zidovska and Sackmann (21) recently showed that the tips of macrophage filopodia also touch the surface of the substrate while spreading.

We speculate that platelets might use their protrusions to sense their local environment, thus serving as antennae during spreading. Whereas fibrinogen surface density affected the filopodia dynamics and platelet membrane height on the substrate, the fibrinogen density did not alter the spreading kinetics, which is similar to that previously reported for the spreading of HeLa cells on fibronectin (17). Thus, on platelets, some minimal amount of fibrinogen is necessary for proper spreading. Interference patterns may be perturbed by reflections from the upper membrane even though the intensities are generally converted to the corresponding object-to-substrate distances. The disturbed reflections from multiple membranes are shown as repetitive adhesion fringes (27) or white fringes in extended lamellipodia (13,20). Recently, dual-wavelength RICM was introduced, which overcomes this limitation and thus enables us to determine absolute heights (28,29). Because we did not employ dual wavelength RICM, we interpret our data semiquantitatively (16,20), which still allows us to distinguish between close contact and whole spreading areas of platelet membrane, where close contact is <40 nm from the surface.

We demonstrate that GPVI^−/− or α₂^−/− platelets showed spreading defects on fibrinogen even though integrin α_IIbβ₃ (a dominant platelet receptor against fibrinogen) seems to present normally on the platelet membrane. Our findings support a model of collagen receptor-α_IIbβ₃ activation in which the ligation of a major collagen receptor, GPVI or α₂β₁, is sufficient to activate α_IIbβ₃ (4,30) and therefore, the collagen receptor deficiency in platelets results in altering spreading dynamics on fibrinogen through outside-in signaling. Interestingly, platelets with a β₃ integrin subunit could spread on fibrinogen in the presence of a PAR4 agonist, which suggests that other adhesion molecules serve as receptors for fibrinogen, and that PAR4 ligation can act as a surrogate for the activity of an adhesion receptor (indicating adhesion receptors play a role in activating vital signaling pathways needed for the rapid formation of close contacts). In addition to the integrin α_IIbβ₃, α_Vβ₃, and α₂β₁ (or VLA-2), there are α₅β₁ (or VLA-5) and α₆β₁ (or VLA-6) on the platelet surface (10). In addition, close contact patches are distinctively observed at the edges of β₃^−/− platelets on fibrinogen whereas α₂ domain in WT platelets formed ringlike contact zones at the cell center. Therefore, further investigation is necessary to determine that adhesion receptors are involved in the formation of close contact with fibrinogen in the absence of β₃^−/− in platelets.

In conclusion, real-time analysis in the early stages of platelet spreading using RICM revealed a differential regulation of close contact and whole membrane expansion on immobilized proteins and under PAR4 activation. We believe our study not only leads to new approaches to obtain more detailed information about the proximity of platelets on various adhesive ligands but may also provide important quantitative insights for the understanding of hemostasis under static conditions and under flow.