Identification of Rac Isoforms Present in Platelets
—To study the role of Rac proteins in regulating the organization of the platelet actin cytoskeleton, we first examined which Rac proteins are expressed in murine and human platelets. Previous reports have shown that Rac1 is ubiquitously expressed, whereas Rac2 is a hematopoietic cell-specific GTPase (26
). Rac3 is thought to play an important role in neural development and has been shown to have a specific localization in the developing mouse brain (27
). In an attempt to identify which Rac isoforms are present in platelets, we first examined a serial analysis of gene expression (SAGE) library that had been prepared from primary murine megakaryocytes, the platelet precursor cell.3
This revealed the presence of 57 Rac1, 4 Rac2, and 0 Rac3 sequence tags out of a total of 53,046, indicating that Rac1 was likely to be the predominant isoform and that Rac3 was very unlikely to be present, consistent with its restricted distribution.
Specific antibodies were then used to verify which Rac isoforms are present at the protein level in murine and human platelets. By using specific antibodies against all three family members, which do not exhibit cross-reactivity with the other isoforms (data not shown), immunoblot analyses established that murine and human platelets express Rac1 but have no detectable Rac2 expression at the protein level (). Wild-type and Rac2-/-
thymocyte lysates were run alongside as a positive control. Furthermore, there were no compensatory changes in the levels of Rac1 or Rac 2 in Rac2-/-
platelets, respectively. A specific antibody to Rac3, raised against the 15 carboxyl-terminal residues (27
), failed to detect expression of Rac3 in human or murine platelets (data not shown). These data confirm that Rac1 is the predominant isoform in human and murine platelets. They further indicate that neither Rac2 nor Rac3 is likely to be present in platelets, although the small number of SAGE tags for Rac2 raises the possibility that it may be expressed at a very low level, and for this reason later experiments were performed on mice deficient in Rac1, Rac2, and both of these Rac isoforms.
FIGURE 1. Rac isoforms present in murine and human platelets (plt). Equal amounts of human and murine platelet lysates were analyzed for Rac expression using noncross-reactive Rac1 (A) or Rac2 (B) antibodies. Lysates from wild-type (WT) and Rac2-/-thymus were included (more ...)
Regulation of Rac Activity in Platelets—With a view to studying the role of Rac1, we measured the activation state of the small GTPase in stimulated human and murine platelets by precipitation with a glutathione S-transferase fusion protein encompassing the CRIB domain of PAK, which has the binding region for the GTP-bound but not GDP-bound state. Stimulation of human platelet suspensions with the G protein-coupled agonist thrombin caused a marked increase in GTP-bound Rac1, which was not significantly altered in the presence of the ADP scavenger apyrase, thereby demonstrating that thrombin stimulation can lead to an increase in Rac activation independent of ADP (). Stimulation with ADP also caused a smaller increase in active Rac1 (1.33 ± 0.06-fold increase as assessed by densitometry; mean ± S.E.; n = 3), which was abrogated in the presence of apyrase (1.02 ± 0.03-fold). Similar trends were observed for murine platelets (). Thrombin stimulated a robust increase in GTP-bound Rac1 in the absence and presence of apyrase, whereas the smaller, ADP-dependent increase (1.23 ± 0.09-fold) was eliminated in the presence of apyrase (0.99 ± 0.04-fold).
FIGURE 2. Effects of agonists on Rac1 activation in platelets both in suspension and on a fibrinogen surface. Equal numbers of washed human (A) or murine (B) platelets (5 × 108/ml) were stimulated with thrombin (thr; 1 unit/ml) or ADP (10μM) in (more ...)
It is well established that platelets undergo a dramatic change in morphology upon binding to immobilized adhesive proteins. Thus, experiments were designed to determine whether Rac1 was activated during spreading on immobilized surfaces. For these studies, purified platelets were placed on fibrinogen- or BSA-coated dishes in the presence of ADP inhibitors for 10 min before lysis. As shown in , we were unable to detect an increase in Rac1 activation following spreading of human or mouse platelets on fibrinogen (1.04 ± 0.02- and 1.05 ± 0.04-fold increase in GTP-bound Rac1 relative to BSA sample, respectively). A similar result was observed when spreading was allowed to proceed between 1 and 45 min (data not shown). In contrast, stimulation of fibrinogen-bound platelets with thrombin caused a sustained level of Rac1 activation ().
The Role of Rac in Platelet Actin Assembly
—Agonist-induced platelet activation is associated with an increase in cellular F-actin levels. We measured F-actin levels in human and mouse platelets using a modified version of the FITC-phalloidin binding assay (24
) after stimulation with vehicle, thrombin, or ADP. As shown in , thrombin stimulation caused a 1.8- and 2.6-fold increase in F-actin content above resting levels in human and murine platelets, respectively, in good agreement with previous findings (28
). A slightly smaller increase in the levels of F-actin was observed in response to ADP stimulation in both species.
FIGURE 3. Quantitation of F-actin in human and murine platelets responding to various stimuli. Purified human and wild-type (WT) or Rac1-/-Rac2-/-murine platelets were activated with 1 unit/ml thrombin or 10 μM ADP for 60 s. Samples were then fixed with (more ...)
The availability of mice lacking Rac1, Rac2, or both isoforms allowed us to examine the functional role of Rac in promoting platelet actin assembly. Thrombin stimulated a similar increase in the level of F-actin in platelets from Rac1-/-Rac2-/-mice to that in controls (). In sharp contrast, the stimulation of actin polymerization by ADP was abrogated in Rac1-/-Rac2-/-platelets. Similar results were obtained when increases in cellular F-actin content was quantified via flow cytometry (thrombin stimulation, 2.04 ± 0.08-fold versus 2.20 ± 0.28-fold increase in F-actin; ADP stimulation, 1.52 ± 0.03-fold versus 0.96 ± 0.05-fold increase in F-actin for wild-type and Rac1-/-Rac2-/-platelets, respectively; mean ± S.E.; n = 3). Similar results to those recorded for Rac1-/-Rac2-/- platelets were also observed in Rac1-deficient platelets, whereas thrombin- and ADP-induced changes in F-actin formation were indistinguishable between control and Rac2-/- platelets (data not shown). Furthermore, we found that collagen induced an increase in F-actin content in a Rac1/Rac2-dependent manner. However, we found that collagen-induced F-actin level increases were largely dependent upon the action of secondary mediators, therefore demonstrating that Rac1/Rac2 is required for the secondary effects of ADP to induced F-actin changes following collagen stimulation (data not shown). Taken together, these data demonstrate that Rac1 plays the major role in ADP-induced actin polymerization in platelets. The similar level of formation of F-actin in response to thrombin in the absence of Rac1 and Rac2 demonstrates the presence of an alternative pathway of assembly of F-actin (see “Discussion?”).
We next aimed to investigate the role of Rac in integrin-mediated F-actin assembly by measuring the temporal exposure of actin filament barbed ends in platelets spreading on fibrinogen. This was achieved by monitoring the incorporation of Cy3-labeled monomeric actin into permeabilized platelets, which directly correlates with the level of barbed-end exposure (24
). Our data demonstrate that wild-type and Rac1-/-
platelets incorporate equivalent levels of Cy3-labeled monomeric actin as they spread on immobilized fibrinogen (supplemental Fig. S1). In contrast, incorporation of monomeric Cy3-labeled actin into platelets was abrogated in the presence of cytochalasin D, a potent actin-polymerization inhibitor (data not shown). Taken together, these data demonstrate that αIIb
integrin engagement of immobilized fibrinogen induces barbed-end exposure (as measured by the incorporation of monomeric actin) in wild-type platelets and that this process is independent of Rac1/Rac2.
The Role of Rac in Platelet Aggregation
—The role of Rac GTPases in the process of agonist-induced platelet shape change and aggregation was examined by using a Born aggregometer. This apparatus measures light transmission through a platelet suspension, with an increase in optical density correlating to platelet spheration (shape change) and a decrease indicative of platelet aggregation. As shown in , the magnitude and time course of both shape change and aggregation in response to intermediate concentrations of thrombin (0.1 units/ml) or ADP (10 μM) was similar for wild-type and Rac1-/-
mouse platelets. We obtained similar results when platelets were treated with submaximal concentrations of thrombin (0.04 units/ml) or ADP (1 μM) (data not shown). It is noteworthy that aggregation to thrombin is not altered in the absence of Rac1/Rac2, even though this response is dependent on hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C (5
). Similar results to those recorded for Rac1-/-
platelets were also observed for Rac1-deficient and Rac2-deficient platelets (data not shown).
FIGURE 4. Platelets from Rac1/Rac2-deficient mice have normal aggregation responses. A, washed platelets (2 × 108/ml) from wild-type (WT) and Rac1-/-Rac2-/-mice were stimulated with 0.1 unit/ml thrombin and the change in optical density indicative of aggregation (more ...) The Role of Rac in Platelet Spreading on Fibrinogen
—Ensuing experiments aimed to examine the role of Rac in mediating platelet spreading. A reproducible series of morphological changes occurs when platelets contact a fibrinogen-coated surface in the presence of inhibitors of ADP and thromboxanes. However, there exists a marked difference between the patterns of spreading observed for human versus
murine platelets. Human platelets bind and fully spread on immobilized fibrinogen in the absence of external agonists (). Moreover, stimulation with either thrombin or ADP has a minimal effect on the extent of human platelet spreading (). In contrast, wild-type murine platelets exhibit only partial formation of a lamellae-like structure on fibrinogen under nonstimulated conditions (). However, stimulation of murine platelets with either thrombin or ADP greatly enhances the degree of lamellipodia formation, resulting in a dramatic increase in the final platelet surface area (). In both species, spreading is completely inhibited in the presence of the actin-polymerization inhibitor cytochalasin D (), consistent with previous work (22
FIGURE 5. Spreading of human and murine platelets on fibrinogen. Purified human and wild-type (WT) or Rac1-/-Rac2-/-murine platelets (2 × 107/ml) were placed on coverslips coated with fibrinogen for 45 min and imaged using DIC microscopy. Platelets were (more ...)
TABLE ONE Effects of external stimulation on platelet surface area following adhesion to fibrinogen Purified human and wild-type (WT) and Rac1-/-Rac2-/- murine platelets (2 × 107/ml) were placed on coverslips coated with fibrinogen for 45 min. Platelets (more ...)
Lamellipodial actin assembly has been reported to be regulated by Rac in permeabilized platelets using a dominant negative form of the GTPase (5
). The mutant mouse platelets were used to address the roles of Rac1 and Rac2 in mediating spreading in platelets. Upon exposure to immobilized fibrinogen, Rac1-/-
platelets underwent a similar level of adhesion and changes in morphology, extending filopodia and forming partial lamellae-like structures, to that observed for wild-type mouse platelets (). More specifically, quantitative image analysis revealed that Rac1-/-
platelets formed equivalent number of filopodia as observed for wild-type platelets, and these filopodia were of similar thickness and length (). However, upon thrombin stimulation and exposure to immobilized fibrinogen a dramatic inhibition of lamellipodia formation was observed in Rac1-/-
platelets. More importantly, these cells retain the ability to form pronounced filopodia, which were significantly thicker and longer than those observed in the absence of thrombin ( and ). In sharp contrast, ADP-induced morphological changes were absent in Rac1-/-
platelets, with the platelets being indistinguishable from non-stimulated levels of spreading on fibrinogen. Similar results to those recorded for Rac1-/-
platelets were also observed in Rac1-deficient platelets, although there was no apparent difference in the spreading of Rac2-deficient platelets compared with wild-type cells in the presence of thrombin or ADP stimulation (data not shown).
TABLE TWO Quantitative analysis of platelet morphology following adhesion to fibrinogen Purified wild-type (WT) and Rac1-/-Rac2-/- murine platelets (2 × 107/ml) were placed on coverslips coated with fibrinogen for 45 min. Platelets were treated with apyrase (more ...)
To investigate further the kinetics of filopodia and lamellipodia formation in the absence of the two Rac isoforms, we monitored spreading of wild-type and Rac1-/-Rac2-/-platelets on fibrinogen using time-lapse video microscopy (). Upon initial contact with the surface, both control and Rac1/Rac2-deficient platelets undergo rounding, before generating short, dynamic filopodia and limited formation of lamellae-like structures. However, in the presence of thrombin, platelets rapidly generate sheet-like lamellipodial membrane that proceed to fill in the gaps between the filopodia (supplemental video 1), resulting in a 60-70% increase in surface area, as shown in . In contrast, thrombin-stimulated Rac1/Rac2-deficient platelets extend elongated filopodia, but fail to generate lamellipodia (supplemental video 2), and so have a corresponding reduction in surface area (). Furthermore, the ADP-dependent increase in surface area observed in wild-type platelets is eliminated in the absence of Rac1 and Rac2 (). Similar results were observed for Rac1-/-platelets, whereas Rac2-/-platelet spreading was indistinguishable from control platelets (data not shown). Taken together, these studies demonstrate an important role for Rac1, but not Rac2, in G protein receptor-coupled agonist-induced lamellipodia formation in platelets. However, Rac1 and Rac2 are not required for the limited spreading on fibrinogen that is observed in the presence of inhibitors of thromboxanes and ADP.
FIGURE 6. Real time imaging of platelet spreading on fibrinogen. Purified murine platelets (2 × 107/ml) were exposed to a fibrinogen-coated surface in the presence of apyrase (2 units/ml) and indomethacin (10 μM) and observed in real time using (more ...) The Role of Rac in Arp2/3 Complex Redistribution in Spread Murine Platelets
—Recent reports have identified the Arp2/3 complex as a major regulator of platelet actin dynamics, being incorporated into the actin cytoskeleton and redistributed to the edge of actin-rich lamellae in platelets following spreading on glass (31
). To explore whether Rac plays a role in this process, we used double-label immunofluorescence to compare the location of Arp2/3 with that of filamentous actin in murine platelet spreading on fibrinogen. Under nonstimulated conditions, a speckled cytoplasmic staining of Arp2/3 was observed in both wild-type and Rac1-/-
platelets (). However, upon thrombin stimulation, the Arp2/3 complex localizes uniformly around the rim of spread wild-type platelets and in the center of activated platelets, in agreement with previous observations (14
). A similar distribution was observed in human platelets on fibrinogen (data not shown). In the absence of Rac1 (not shown) or Rac1 and Rac2, however, the Arp2/3 complex was localized to the tip and to the base of the filopodial protrusions, whereas in some cases, it was also present on the shaft of the filopodia. Higher magnification of the filopodia from the Rac1/Rac2-deficient platelets, as denoted by arrows
in , emphasize the relationship between Arp2/3 and actin filaments ().
FIGURE 7. Location of Arp2/3 in spread platelets.
A, purified wild-type (WT) and Rac1/ 2-/-murine platelets (2 × 107/ml) were exposed to a fibrinogen-coated surface in the presence of apyrase (2 units/ml) and indomethacin (10 μM) with or without (more ...) Role of Rac in Platelet Spreading on Collagen and Laminin
—Recent reports have demonstrated that Rac is activated during platelet spreading on the extracellular matrix protein collagen in the absence of inhibitors of the secondary mediators, ADP and thromboxanes (20
). In platelet suspensions, activation of Rac by collagen has been shown previously to be entirely dependent on the release of ADP and thromboxanes, thereby revealing that engagement of GPVI or integrin α2
by collagen is unable to promote detectable activation of the small G protein Rac (18
). This observation was confirmed on murine platelets that had been allowed to spread on immobilized collagen in the presence of inhibitors of the actions of thromboxanes and ADP (), whereas in the absence of inhibitors, an increase in Rac activation by collagen was observed (data not shown). Furthermore, pre-stimulation of collagen-bound platelets with thrombin caused a sustained level of Rac activation (). Thrombin was also observed to stimulate activation of Rac on a laminin surface, which supports adhesion through integrin α6
, although the extracellular matrix protein was unable to activate Rac on its own ().
FIGURE 8. Spreading of murine platelets on collagen and laminin. A, effects of platelet spreading on collagen and laminin on Rac activation. Washed murine platelets (5 × 108/ml) were added to BSA-, collagen (Coll)-, or laminin (LM)-coated dishes in the (more ...)
We next examined whether Rac is required for spreading on collagen and laminin. In contrast to observations on fibrinogen, murine platelets bind and fully spread on immobilized collagen or laminin in the absence of external agonists (). Further stimulation with either thrombin or ADP had minimal effect on the extent of wild-type murine platelet spreading on collagen or laminin (). Most strikingly, lamellipodia formation was abolished for both Rac1- and Rac1/Rac2-deficient platelets on collagen or laminin, although a small degree of filopodia formation remained. Filopodia elongation and thickening was markedly enhanced when Rac-deficient platelets were treated with thrombin, in a similar way to that seen on fibrinogen, whereas ADP treatment had no effect upon the platelet morphology (). Taken together, our data demonstrate that Rac1 plays a critical role in cytoskeletal reorganization downstream of engagement of receptors for collagen and laminin.
TABLE THREE Effects of external stimulation on platelet surface area following adhesion to collagen and laminin Purified human and murine platelets (2 × 107/ml) were placed on coverslips coated with collagen or laminin for 45 minutes. Platelets were treated (more ...)
Most interestingly, the weak aggregation response to a low concentration of collagen (0.6 μg/ml) was significantly reduced in the absence of Rac1/Rac2, whereas the response to higher concentrations (3
μg/ml) was not altered (). More importantly, Rac1/Rac2-deficient platelets failed to aggregate in response to low concentrations of the GPVI-specific agonist collagen-related peptide (). Therefore, this prompted us to probe the possible role of Rac in mediating the ability of GPVI to activate platelets. Our data demonstrate that tyrosine phosphorylation of Syk and phospholipase Cλ2, two proteins that play a central role in GPVI signaling, was not altered in response to CRP in Rac1/Rac2-deficient platelets (not shown). Furthermore, equivalent increases in intracellular calcium, as measured using the calcium reporter Oregon Green 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid 1-AM, was observed for both Rac1/Rac2-deficient and wild-type platelets in response to CRP stimulation (not shown). Similar results were observed in the absence of Rac1, although there was no defect in response in the absence of Rac2 (not shown). It is noteworthy that all experiments examining the effects of CRP stimulation were performed in the presence of inhibitors of the positive feedback signals arising from ADP and thromboxanes. These results therefore demonstrate an important role for Rac in mediating aggregation downstream of GPVI, although this does not appear to be due to an impairment in activation of phospholipase C.
Investigation of the Role of Rac in Platelet Adhesion and Thrombus Formation under Flow in Vitro
—We sought to investigate further the functional role of Rac on platelet adhesion to and platelet aggregation on collagen and VWF/thrombin under shear conditions using an in vitro
flow-based assay. We perfused whole blood from wild-type, Rac1-/-
, and Rac1-/-
mice over either a collagen-coated or VWF/thrombin-coated surface at 1000 s-1
for 4 min. Subsequently, samples were fixed, and adherent platelets were imaged by either DIC or confocal icroscopy. Thrombin was co-immobilized with VWF to enhance thrombus formation, whereas immobilized thrombin alone did not support platelet recruitment (data not shown). Blood from control mice exhibited robust formation of densely packed platelets on both collagen and VWF/thrombin (). In marked contrast, Rac1-/-
platelets attached along the length of the collagen fibers, but for the most part did not support platelet aggregate formation (). Similarly, thrombus formation of Rac1-/-
platelets on VWF/thrombin was markedly reduced (). Quantitation of surface area coverage by platelets revealed a significant decrease for Rac1-/-
platelets as compared with wild-type on both surfaces (). Furthermore, the analysis of the three-dimensional structure of the aggregate using confocal microscopy revealed that the mean height of the aggregate was reduced from 7.87 ± 0.41 μm2
in control platelets to 2.34 ± 0.12 μm2
platelets on collagen (n
= 20). These results are consistent with the notion that Rac1-/-
platelets formed a single layer on the collagen fibers in contrast to the multilayer platelet thrombi observed in control blood. In support of these findings, platelet treatment with an αIIb
antagonist, which has been shown previously to produce a single layer of platelets bound to collagen (33
), resulted in a 2.07 ± 0.42 μm2
mean thrombus height. Similar results to those recorded for Rac1-/-
platelets were also observed in Rac1-deficient platelets, whereas Rac2-/-
platelet aggregate formation was indistinguishable from that of control platelets (data not shown).
FIGURE 9. Role of Rac in platelet adhesion and aggregate stability on collagen under flow. A, mouse blood anticoagulated with D-phenyl-alanyl-1-prolyl-1 arginine chloromethyl ketone and heparin was perfused through a collagen-coated microslide at a shear rate of (more ...)
TABLE FOUR Role of Rac1/Rac2 on platelet adhesion to collagen under flow Murine whole blood specimens were incubated for 10 min at 37 °C prior to being perfused for 4 min over either a collagen-coated or VWF/thrombin-coated surface at 1000 s[H11002]1. Platelet (more ...)
In an attempt to elucidate the mechanisms resulting in the decreased Rac1-/-Rac2-/-thrombus size, we monitored DIOC6-labeled platelet accumulation onto collagen in real time via fluorescent microscopy. As shown in , a layer of single platelets is initially deposited onto the collagen-coated surface as blood from wild-type mice is perfused. As time increases, these collagen-bound platelets capture free-flowing platelets, resulting in an increase in thrombus size as evidenced by an increase in the fluorescence level of the thrombus because of the accumulation of fluorescently labeled cells. Similarly, when Rac1-/-Rac2-/- murine blood was perfused over collagen, an initial layer of platelets was observed to bind, with no significant difference in the time required for thrombus initiation. However, subsequent Rac1-/-Rac2-/- platelets that were recruited by the collagen-bound cells failed to resist the shear stress and were eventually stripped off and carried away downstream, as exemplified by the arrow at 20 and 30 s, respectively, in . Thus, Rac1-/-Rac2-/- platelet-embolus formation resulted in a single layer of platelet deposition on the collagen surface, due to the inability of these platelets to support shear-resistant platelet-platelet interactions. A similar process of embolization was observed when Rac1-/-Rac2-/- platelets bound to VWF/thrombin-coated surfaces under flow (not shown). Rac1-/- platelets were observed to form emboli to the same degree as Rac1-/-Rac2-/- platelets, whereas Rac2-/- thrombus formation was similar to that of control platelets (data not shown). Taken together, our data demonstrate that Rac1, but not Rac2, is required for stable thrombus formation under shear flow on both collagen and VWF/thrombin surfaces.
Role of Rac in Thrombus Formation in Vivo
—We next wished to investigate the role of Rac in platelet accumulation at sites of injury in a more physiologically relevant environment. We therefore used real time fluorescence and bright field microscopy to examine the dynamic profile of platelet accumulation in arterioles in the mouse cremaster micro-circulation after laser-induced injury (34
). In this model of mild injury, platelet accumulation following endothelial damage occurs in a specific temporal pattern (). A kinetics curve was constructed based on the median value of the integrated fluorescent intensity over a period of time (). In wild-type mice, during the initial phase of this dynamic process, platelets rapidly accumulated until a maximum thrombus size (741 ± 224; arbitrary units of fluorescence) was obtained (). Subsequently, a loss of platelets occurred, leading to a diminishing in size over the course of several minutes until the platelet content was stabilized within the thrombi, leaving a flattened mural thrombus at the end of the recording period. This final stage was identified as a plateau in the kinetics curve. In distinct contrast, an atypical pattern of thrombus formation was observed in Rac1-deficient mice (). More specifically, a decrease in platelet accumulation was observed at all time points for Rac1-/-
mice (), and a significant reduction in the peak thrombus size (173 ± 52) was recorded. Atypical patterns of thrombus formation in Rac1-/-
mice included numerous peaks indicative of many emboli detaching from the main thrombus body (data not shown), a “flatter” kinetics profile indicative of poor platelet accumulation, and a lack of a plateau phase at the end suggesting no mural thrombus remained attached to the vessel wall.
FIGURE 10. Role of Rac in thrombus formation in vivo. A, thrombus formation over time in wild-type (top) and Rac1-/- (bottom) mice. Platelets were labeled in vivo with Alexa 488-conjugated goat anti-rat antibody bound to rat anti-CD41 antibody. The Alexa 488 fluorochrome (more ...)