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Keywords:

  • aggregation;
  • bleeding time;
  • platelet secretion;
  • Rac1 GTPase;
  • Rac inhibitor

Summary.

  1. Top of page
  2. Summary.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Background: Rac1 GTPase, a member of the Ras-related Rho GTPase family, is the major Rac isoform present in platelets and has been shown to be involved in cell actin cytoskeleton reorganization and adhesion. Agonists that induce platelet secretion and aggregation also activate Rac1 GTPase, raising the possibility that Rac1 GTPase may be involved in regulation of platelet function. Objectives: To rigorously define the role of Rac1 in platelet regulation. Methods: We have used a dual approach of gene targeting in mice and pharmacologic inhibition of Rac1 by NSC23766, a rationally designed specific small molecule inhibitor, to study the role of Rac1 in platelet function. Results: Platelets from mice as well as human platelets treated with NSC23766 exhibited a significant decrease in: (i) active Rac1 species and phosphorylation of the Rac effector, p21-activated kinase; (ii) expression of P-selectin and secretion of adenosine triphosphate induced by thrombin or U46619; and (iii) aggregation induced by adenosine 5′-diphosphate, collagen, thrombin and U46619, a stable analog of thromboxane A2. NSC23766 did not alter the cAMP or cGMP levels in platelets. Consistent with the requirement of Rac1 for normal platelet function, the bleeding times in Rac1–/– mice or mice given NSC23766 were significantly prolonged. Conclusions: Our data show that deficiency or inhibition of Rac1 GTPase blocks platelet secretion. The inhibition of secretion, at least in part, is responsible for diminished platelet aggregation and prolonged bleeding times observed in Rac1 knockout or Rac1 inhibitor-treated mice.


Introduction

  1. Top of page
  2. Summary.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Platelet activation is a multistep process involving several biochemical signaling pathways. The roles of adenosine 5′-diphosphate (ADP) secreted from the platelets, synthesis and release of thomboxane A2 (TXA2), rise in cytosolic calcium, activation of protein kinase C and phosphoinositide-3 kinase (PI3 K) by diverse agonists in platelet activation are well established [1–6]. In addition, the ability of classic heterotrimeric G proteins as well as Ras-related small molecular weight proteins such as Rap1b to act as important signal transducers in the regulation of platelet activation is also well appreciated [7–10]. Recent emerging evidence suggests that Rac GTPases, particularly Rac1 GTPase and Rac2 GTPase, of the Rho family of small GTPases are involved in the regulation of various hematopoietic cell functions, including hematopoietic stem cell proliferation, engraftment and bone marrow retention, neutrophil chemotaxis and superoxide production, macrophage phagocytosis, and B-cell and T-cell immunologic responses [11–14].

In platelets, activation of Rac1 GTPase has been implicated in actin polymerization [15], lamellipodia formation [16–19], and the stability of platelet aggregates under shear stress [18]. Diverse platelet agonists such as ADP, collagen, thrombin and U46619, a TXA2 analog, all have been reported to induce activation of Rac1 GTPase via their specific receptors and downstream signaling cascades, leading to stimulation of phospholipase C (PLC) and calcium mobilization [20]. The activated Rac1, in turn, has been shown to stimulate PLC-γ2 [21] as well as PI3 K [3]. Moreover, activation of the G protein-coupled receptors by agonists such as ADP, TXA2 and thrombin has been shown to activate PI3 K [4], which may also lead to activation of Rac1 GTPase. The bidirectional positive feedback loops for the activation of Rac1 GTPase by PLC-γ2 and PI3 K and activation of PLC-γ2 and PI3 K by Rac1 GTPase suggest that Rac1 GTPase is involved in the regulation of agonist-elicited signals leading to platelet activation.

One key step of platelet aggregation is secretion of α-granules and dense granules that release P-selectin and ADP, respectively. Given the suggested role of Rac1 in exocytosis in a number of other cell types [22,23], we hypothesize that Rac1 GTPase plays a critical role in platelet secretion and aggregation. In the present studies, we utilized a dual approach of gene targeting and pharmacologic blockage of Rac1 to investigate the effect of Rac1 GTPase deficiency or inhibition on: (i) activation of platelet Rac1 GTPase and its effector molecule p21-activated kinase (PAK); (ii) expression of P-selectin, secretion of adenosine triphosphate (ATP) and platelet aggregation in vitro; and (iii) murine tail bleeding times, as a measure of in vivo platelet function. We report for the first time that Rac1 GTPase is involved in the regulation of platelet secretion and aggregation. Our results, combined with previous studies, strongly suggest that pharmacologic targeting of Rac1 represents a novel approach for developing future antiplatelet agents.

Materials and methods

  1. Top of page
  2. Summary.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Materials

Chemicals and reagents were purchased either from Sigma-Aldrich (St Louis, MO, USA) or from specifically noted sources. Collagen and U46619 were obtained from Chrono-Log Corporation (Havertown, PA, USA) and Calbiochem (San Diego, CA, USA) respectively. NSC23766 was rationally designed [24] and synthesized in our laboratory as reported previously [11].

Mouse maintenance, blood collection and preparation of washed mice platelet suspensions

The Mx-cre;Rac1loxP/loxP mice were generated previously [11,12]. All experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committees at the Children’s Hospital Research Foundation, Cincinnati, OH and at Ohio University. Inducible deletion of Rac1 GTPase from the platelets in the mice was achieved by four or five i.p. injections of poly(I:C) as previously described [11,12]. Blood was drawn by cardiac puncture from anesthetized wild-type (WT) and Rac1-deficient mice into a syringe containing 160 μL of ACD (2.5% trisodium citrate, 2% dextrose, 1.5% citric acid), and transferred into tubes containing 500 μL of HEPES-buffered Tyrode’s solution. Platelets were isolated, immediately after adding 5 mm EDTA, by centrifugation at 90 × g for 10 min. The remainder of the blood was diluted to 1.5 mL with HEPES-buffered Tyrode’s solution (pH 6.5) and centrifuged at 90 × g for 10 min to recover additional platelets. Platelets were washed three times in the presence of apyrase (0.4 U mL–1), an enzyme that hydrolyzes ADP, and 2.0% EGTA, with HEPES-buffered Tyrode’s solution (pH 6.5), and finally resuspended in HEPES-buffered Tyrode’s solution without calcium (pH 7.4).

Collection of blood and preparation of washed human platelet suspensions

Blood from normal healthy human volunteers, who reported being free of medication for 2 weeks, was collected according to the Institutional Review Board approved protocols. Blood was collected into acid/citrate/dextrose solution at a ratio of 1:6 (v/v). Platelet-rich plasma (PRP) was obtained by centrifugation of blood at 120 × g for 15 min at room temperature. The PRP was centrifuged at 1100 × g for 10 min at room temperature, and the platelet pellet was resuspended in HEPES/Tyrode’s solution without calcium (pH 6.5). Apyrase (0.4 U mL–1) was added to the platelet suspensions. Two per cent EGTA was added (1:9 v/v) to platelet suspensions just before centrifugation at 1100 × g for 10 min. This washing procedure was repeated three times. Platelets were finally resuspended in HEPES/Tyrode’s solution without calcium (pH 7.4), and counted in Hemavet 950FS (Drew Scientific, Oxford, CT, USA). The platelet count was adjusted to 3 × 108 mL–1 for aggregation studies.

Assessment of P-selectin expression, secretion of ATP and platelet aggregation

The PRP was incubated with 1 mm aspirin at 37°C for 30 min, and platelets were then isolated by centrifugation, washed twice, and finally resuspended in HEPES-buffered Tyrode’s solution without calcium (pH 7.4), containing 0.2% bovine serum albumin and apyrase (0.05 U mL–1). Washed platelets (1–1.5 × 106) were incubated with 10 μL of fluorescein isothiocyanate-conjugated anti-CD62P (P-selectin) antibody solution for 30 min at 37°C without stirring. Expression of P-selectin on platelet surfaces was quantified by flow cytometry (FACSCalibur, Becton-Dickinson, San Jose, CA, USA) and the cellquest software program [25]. Secretion of ATP from the dense granules was assessed by a luminescence method using a luciferin/luciferase kit from Chrono-Log Corporation. Platelet aggregation was monitored by a standard optical density method [26] using an Aggregometer from Chrono-Log Corporation.

Rac1, RhoA and Cdc42 GTPase and PAK assays

Platelets were lyzed with lysis buffer [50 mm Tris–HCl (pH 7.2), 500 mm NaCl, 10 mm MgCl2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 5 μg of leupeptin and aprotinin per millilitre, 0.1 mm phenylmethanesulfonyl fluoride (PMSF)] for 60 min. The lysates were centrifuged at 18 000 × g at 4°C for 10 min. Equal volumes of the lysates were incubated with glutathione-S-transferase–PAK bound to glutathione–sepharose 4B beads for 60 min at 4°C. The beads were washed three times with a washing buffer [50 mm Tris–HCl (pH 7.2), 150 mm NaCl, 10 mm MgCl2, 1% Triton X-100, 5 μg of leupeptin and aprotinin per millilitre, 0.1 mm PMSF]. The Rac1, RhoA and Cdc42 proteins bound to the beads were quantitatively detected by Western blotting using anti-Rac1, anti-RhoA and anti-Cdc42 antibodies.

For PAK activation assays, washed platelets were lyzed in a buffer containing 0.5% Triton X-100, 50 mm Tris–HCl (pH 7.5), 0.1 mm EDTA, 150 mm NaCl, 50 mm NaF and 0.2 mm Na3VO4, 1 mm dithiothreitol, 0.4 mm PMSF and complete protease inhibitor, and incubated in ice for 30 min. The platelet lysates were incubated with protein A/G–sepharose (Santa Cruz, Santa Cruz, CA, USA) and anti-PAK antibody (Cell Signaling, Danvers, CA, USA) for 1 h at 4°C, and analyzed for PAK expression in immunoprecipitates and supernatants by immunoblotting with specific antibodies against phospho-PAK and total PAK (Cell Signaling) or β-actin as a loading control (Sigma, St Louis, MO, USA) as previously described [11].

Assessment of platelet cAMP and cGMP levels

Washed platelets were incubated with NSC23766 for varying time periods at 37°C. Platelet cAMP and cGMP levels were measured with minor modifications of the procedure detailed previously [27]. The reactions were terminated by adding an equal volume of ice-cold 12% trichloroacetic acid [28]. The samples were centrifuged in an Eppendorf microcentrifuge for 5 min. The supernatants were collected and washed three times with 5 mL of water-saturated diethyl ether. The platelet cAMP and cGMP levels were quantified using cAMP and cGMP enzyme-immunoassay kits from Cayman Chemical, Ann Arbor, MI, USA.

Tail bleeding time measurement

Mice were anesthetized with and kept under a constant flow of 2.5% isoflurane and 0.35% oxygen. The tip of the tail at 2 mm diameter was cut and immediately immersed in saline at 37°C. The bleeding time was defined as the time needed for the cessation of a visible blood stream for 1 min [29]. Monitoring of the bleeding times was stopped at 10 min by cauterizing the tails to prevent excessive loss of blood [10].

Statistical analysis

Data are expressed as means ± SD or SE (as described in the figure legends). Statistical significance between the bleeding times of the WT, Rac1–/– and NSC23766-treated WT mice was analyzed by anova.

Results

  1. Top of page
  2. Summary.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Gene targeting and pharmacologic inhibition of Rac1 GTPase in mouse or human platelets

To investigate the effect of deficiency of Rac1 GTPase on platelet function, we generated Rac1 conditional knockout mice carrying Rac1–/– alleles in the blood lineages, as conventional Rac1 deletion causes early embryonic lethality [30]. We utilized a tissue-specific, interferon-inducible deletion approach [11,12] to generate Rac1–/– hematopoietic cells, including Rac1–/– platelets, in the Mx-cre;Rac1loxP/loxP mice. The platelets derived from the Mx-cre;Rac1loxP/loxP mice 20 days after poly(I:C) treatment clearly lacked Rac1 GTPase expression (Fig. 1A).

image

Figure 1.  Gene targeting of Rac1 GTPase and inhibition of Rac GTPase by NSC23766. (A) Expression of Rac1 in platelet lysates from the wild-type (WT) and genetically targeted mice was probed by Western blotting Rac1. β-Actin expression was used as a loading control. Platelets from the WT mice but not from the Rac1–/– mice expressed Rac1 GTPase. (B) Washed human platelets were incubated with thrombin (0.1 U mL–1) for 2 min, and the activities of Rac1, Cdc42 and RhoA were quantified. A 3-min preincubation of platelets with 10 and 30 μm NSC23766 inhibited Rac1-GTP formation but not that of RhoA or Cdc42. Total Rac1, RhoA and Cdc42 as well as β-actin are shown as loading controls. (C) Collagen-induced (3.0 μg mL–1) phosphorylation of p21-activated kinase (PAK) is significantly diminished in the Rac1–/– mice platelets as compared to the platelets from WT mice. (D) A 3-min preincubation of washed human platelets with NSC23766 (30 μm) inhibited collagen-induced (0.75 μg mL–1) phosphorylation of PAK. All incubations of platelets with inducers and/or inhibitors were carried out in the absence of added calcium. Activation of Rac1 and phosphorylation of PAK were determined as described in Materials and methods.

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In parallel with the gene-targeting approach in mice, we tested the effects of pharmacologic inhibition of Rac GTPases by NSC23766, a rationally designed small molecule inhibitor that specifically interferes with Rac interaction with upstream guanine nucleotide exchange factors, on Rac1 activity in human platelets. As shown in Fig. 1B, a 3-min preincubation of washed human platelets with NSC23766 inhibited thrombin-induced activation of Rac1 GTPase in a concentration-dependent manner. Thrombin-induced platelet activation has also been shown to induce activation of Cdc42 and RhoA. We investigated the possibility that NSC23766, in addition to blocking activation of Rac GTPases, may also be inhibiting RhoA and or Cdc42 activity in platelets. The data in Fig. 1B show that up to 30 μm NSC23766 did not inhibit activation of RhoA GTPase, and only slightly (∼15%) inhibited activation of Cdc42. These data, taken together with our earlier findings that NSC23766 inhibits activation of Rac GTPases by interfering with specific Rac–guanine nucleotide exchange factor (GEF) interactions and does not affect activation of RhoA and Cdc42 by their respective GEFs [24], demonstrate that NSC23766 is a Rac-specific inhibitor.

Collagen-induced activation of Rac1 GTPase has been linked with phosphorylation of its effector, PAK1, in platelets [19]. We therefore investigated the possibility that genetic deficiency or pharmacologic inhibition of Rac would block collagen-induced phosphorylation of PAK1. The data in Fig. 1C,D clearly show that collagen-induced phosphorylation of PAK1 is inhibited in platelets from Rac1–/– mice as well as in human platelets preincubated with 30 μm NSC23766 for 3 min. The findings that inhibition of Rac1 GTPase by NSC23766 mimics the deficiency of Rac1 GTPase and blocks Rac1 downstream signaling support our dual approach of gene targeting and pharmacologic inhibition of Rac1 in studying the role of Rac1 in platelet function.

Gene targeting or pharmacologic inhibition of Rac1 GTPase blocks platelet secretion

Rac1 GTPase has been reported to be involved in exocytosis, particularly the secretion of human growth hormone from bovine chromaffin cells [22] and insulin secretion from INS-1 β-cells [23]. Rac2 GTPase has been implicated in neutrophil granule exocytosis [13]. However, the role of Rac GTPases in platelet secretion has not been examined so far. We investigated the possibility that Rac1, in addition to regulating lamellipodia formation and adhesion, may also affect platelet secretion. We quantified the expression of P-selectin from α-granules and the secretion of ATP from dense granules in platelets from Rac1-deficient mice as well as in human platelets preincubated with NSC23766. As shown in Fig. 2A, thrombin (0.1 U mL–1) induced expression of P-selectin on Rac1–/– platelets was decreased by more than 60% as compared to the WT platelets. In addition, the Rac1–/– platelets exhibited a 76% decrease in U46619-induced (1.0 μm) ATP secretion (Fig. 2A). Mimicking the Rac1 gene-targeting effects, a 3-min preincubation of washed human platelets with NSC23766 inhibited the thrombin-induced (0.1 U mL–1) P-selectin expression and the U46619-induced (1.0 μm) secretion of ATP in a concentration-dependent manner (Fig. 2B). Thus, deficiency or inhibition of Rac1 GTPase blocks platelet secretion from both the α-granules and the dense granules. These results implicate Rac1 activity as a regulator of platelet secretion.

image

Figure 2.  Deficiency or inhibition of Rac1 diminished expression of P-selectin from α-granules and secretion of adenosine triphosphate (ATP) from dense granules. (A) Thrombin-induced (0.1 U mL–1) expression of P-selectin and U46619-induced (1.0 μm) secretion of ATP is diminished in platelets from Rac1–/– mice as compared to platelets from wild-type (WT) mice. (B) Thrombin-induced (0.1 U mL–1) expression of P-selectin and U46619-induced (1.0 μm) secretion of ATP is inhibited by NSC23766 in a concentration-dependent manner. P-selectin was quantified by flow cytometry in aspirin-treated washed platelets, without added calcium, containing 0.2% bovine serum albumin and apyrase (0.05 U mL–1) according to Quinton et al. (J Thromb Haemost 2004; 2: 978–84), as detailed in Materials and methods. Secretion of ATP from platelets was quantified by a luminescence method using the luciferin/luciferase kit from the Chrono-Log Corporation (Havertown, PA, USA). The luciferin/luciferase reagent was added to platelets 1 min prior to addition of U46619. Results are reported as means ± SD for WT and Rac1–/– platelets (n = 6, P < 0.001 for both P-selectin expression and ATP secretion) and for NSC23766-treated platelets (n = 4, P < 0.009 for P-selectin expression and P < 0.003 for ATP secretion).

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Gene targeting or pharmacologic blockade of Rac1 GTPase inhibits platelet aggregation induced by diverse agonists

Adenosine 5′-diphosphate secreted from platelets contributes to irreversible or maximal platelet aggregation induced by diverse agonists [1,2,6]. The diminished platelet secretion because of the deficiency or inhibition of Rac1 GTPase (Fig. 2) may lead to defective platelet aggregation. We tested this possibility by investigating the aggregation responses induced by ADP, collagen, thrombin and U46619, a stable analog of TXA2, in WT and Rac1–/– platelets. A decreased aggregation response was observed in the Rac1–/– platelets as compared with the WT platelets when each of these agonists was applied (Fig. 3A). Consistent with the diminished aggregation responses observed in Rac1–/– platelets, pharmacologic inhibition of Rac1 GTPase by NSC23766 also inhibited human platelet aggregation responses induced by ADP, collagen, thrombin or U46619 in a concentration-dependent manner (Fig. 3B).

image

Figure 3.  Deficiency or inhibition of Rac1 inhibited platelet aggregation induced by diverse agonists. (A) Platelet aggregation induced by adenosine 5′ diphosphate (ADP) (1.0 μm), collagen (2.0 μg mL–1), thrombin (0.1 U mL–1) and U46619 (1.0 μm) is diminished in platelets from Rac1–/– mice as compared to platelets from wild-type mice. (B) Addition of NSC23766 at the indicated concentrations to platelets 3 min prior to stimulation inhibited ADP-induced (3.0 μm), U46619-induced (1.0 μm), thrombin-induced (0.1 U mL–1) and collagen-induced (0.75 μg mL–1) aggregation in a concentration-dependent manner. Incubation of platelets with inducers was carried out in the absence of added calcium. Platelet aggregation was monitored by a standard optical density method using an Aggregometer from Chrono-Log-Corporation. The aggregation tracings are representative of three to five independent experiments.

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The time course of thrombin-induced secretion and aggregation in the presence or absence of NSC23766 links inhibition of aggregation to blockade of granular secretion

We further investigated the possibility that NSC23766 inhibits platelet aggregation by blocking granular secretion by assessing the time course of secretion and aggregation induced by varying concentrations of thrombin. The data in Fig. 4 show that NSC23766 (30 μm) blocked both the primary and secondary secretion of ATP from dense granules as well as aggregation induced by thrombin. A gradual increase in thrombin concentrations reversed the inhibitory effect of NSC23766 on primary and secondary secretion from dense granules as well as aggregation (Fig. 4). Inhibition of ADP-induced aggregation and secretion by NSC23766 was also reversed by a gradual increase in ADP concentrations (data not shown). The finding that the time course of inhibition of secretion parallels the time course of the inhibition of aggregation, taken together with the concentration-dependent inhibitory effects of NSC23766 on platelet secretion (Fig. 2) and aggregation (Fig. 3) induced by diverse agonists, suggests that NSC23766 inhibits platelet aggregation by blocking granular secretion. Moreover, the fact that inhibition of Rac1 GTPase mimics the platelet aggregation defects observed in Rac1–/– platelets under diverse stimulatory conditions further suggests that Rac1 inhibits platelet aggregation by regulating secretion induced by diverse agonists.

image

Figure 4.  Kinetics of thrombin-induced secretion and aggregation in the presence or absence of NSC23766 links inhibition of aggregation to blockade of secretion. Washed human platelets, without added calcium, were incubated with 30 μm NSC23766 for 3 min prior to addition of varying concentrations of thrombin, and platelet aggregation and secretion were monitored simultaneously. Secretion of adenosine triphosphate from platelets was quantified by a luminescence method using the luciferin/luciferase kit from the Chrono-Log Corporation (Havertown, PA, USA). The luciferin/luciferase reagent was added to platelets 1 min prior to addition of thrombin. The aggregation and secretion tracings are representative of four independent experiments.

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Inhibition of Rac1 GTPase does not alter platelet cAMP or cGMP levels

Cyclic nucleotides are known inhibitors of platelet activation [27], and have also been shown to inhibit Rac1 GTPase [31]. ADP and prostaglandin E1 (PGE1) lower and elevate platelet cAMP levels via their specific receptors by, respectively, inhibiting or stimulating adenylyl cyclase [27]. Lowering of cAMP levels promotes platelet activation, whereas elevation of cAMP inhibits it. We investigated the possibility that NSC23766 may be inhibiting platelet activation by elevating cAMP levels or by preventing the drop in basal cAMP levels induced by agonist (e.g. ADP). As shown in Fig. 5, incubations of washed human platelets with ADP (10 μm) lowered the basal cAMP level by 25% from 4.31 to 3.25 pmol. PGE1 increased platelet cAMP levels by 4-fold to 17.87 pmol, and ADP significantly lowered the PGE1-elevated cAMP level to 7.19 pmol. However, addition of NSC23766 (30 μm) to platelets neither affected the basal cAMP levels nor prevented the ADP-induced decrease or the PGE1-induced rise in platelet cAMP levels (Fig. 5). These data demonstrate that NSC23766 does not inhibit platelet activation by altering platelet cAMP levels.

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Figure 5.  NSC23766 did not alter the basal or adenosine 5′ diphosphate (ADP)- and prostaglandin E1 (PGE1)-modified platelet cAMP levels. Washed human platelets were incubated with NSC23766 (30 μm) for 3 min prior to addition of ADP (10 μm) or PGE1 (0.5 μm). The reactions were terminated at 5 min after addition of ADP or PGE1 by adding equal volumes of ice-cold 12% trichloroacetic acid, and the platelet cAMP levels were quantified using enzyme-immunoassay kits from Cayman Chemicals, Ann Arbor, MI. Addition of ADP lowered platelet cAMP levels by 25% (P < 0.05), whereas addition of PGE1 (0.5 μm) increased cAMP levels 4-fold (P < 0.005) in washed human platelets. However, addition of NSC23766 (30 μm) did not affect cAMP levels in the absence (P > 0.1) or presence of either ADP (P > 0.2) or PGE1 (P > 0.3). The results are reported as means ± SD (n = 4).

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Rac1 has recently been shown to activate transmembrane guanylyl cyclase via PAK, leading to an increase in cGMP [32]. The transmembrane guanylyl cyclase has not been shown to be involved in platelet activation. However, soluble guanylyl cyclase-mediated generation of cGMP appears to have both stimulatory, at low concentrations, and inhibitory, at high concentrations, effects on platelet activation [33]. Thrombin-induced secretion, particularly at a low concentration (0.02 U mL–1), has been shown to increase platelet cGMP levels [34]. Because the role of the increase in cGMP in platelet activation remains unclear [35,36], we investigated the effects of NSC23766 on the basal and thrombin-stimulated platelet cGMP levels. We found that thrombin increased basal cGMP level from 328 ± 38 fmol/108 platelets to 434 ± 66 fmol/108 platelets. However, a 3-min preincubation of platelets with NSC23766 (30 μm) did not alter cGMP levels in the absence (358 ± 104 fmol/108 platelets) or presence (429 ± 78 fmol/108 platelets) of thrombin. These results suggest that the antiplatelet activity of NSC23766 is not mediated by changes in cGMP levels.

Gene targeting or pharmacologic inhibition of Rac1 prolongs tail bleeding time in mice

Platelet aggregation is essential for primary hemostatic plug formation to arrest bleeding. To associate the possible in vivo role of Rac1 GTPase with platelet function, we investigated whether deficiency or inhibition of Rac1 GTPase would affect the tail bleeding times in mice. As shown in Fig. 6A, the tail bleeding times in Rac1–/– mice (>514 s) were prolonged as compared to those of WT mice (∼137 s). The effects of inhibition of Rac1 GTPase by NSC23766 on bleeding times were monitored by assessing the tail bleeding time 30 min after i.p. administration of NSC23766 (2.5 mg kg–1) to the WT mice. The data in Fig. 6A show that NSC23766 prolonged the bleeding time 3-fold. To confirm that the prolonged bleeding observed in mice given NSC23766 was due to defective platelet aggregation, we investigated the effect of i.p. administration of NSC23766 (2.5 mg kg–1) on platelet aggregation. The data in Fig. 6B show that U46619-induced (3.0 μm) aggregation is inhibited in mice given NSC23766. These findings that deficiency of Rac1 GTPase or inhibition of Rac1 prolongs the tail bleeding times in mice and inhibits ex vivo platelet aggregation suggest that diminished platelet function because of deficiency or inhibition of Rac1 GTPase can result in an altered hemostatic response.

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Figure 6.  Deficiency or inhibition of Rac1 GTPase prolonged tail bleeding time and blocked ex vivo platelet aggregation. (A) The tail bleeding times were assessed in wild-type (WT) (n = 18) and Rac1–/– (n = 8) mice or in WT mice after 30 min of i.p. administration of 2.5 mg kg–1 NSC23766 (n = 7), as detailed in Materials and methods. Each bar graph is the mean ± SE of the bleeding times in 7–18 mice. The differences in the bleeding times between the WT and the Rac1–/– mice and the NSC23766-treated mice were statistically significant (P < 0.0001) by anova. (B) U46619-induced aggregation in citrated platelet-rich plasma from the mice administered saline or NSC23677 (2.5 mg kg–1). The aggregation tracings are representative of three independent experiments.

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Discussion

  1. Top of page
  2. Summary.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Rho family small GTPases serve as signal transducers of diverse extracellular stimuli such as growth factor and cytokine receptors, integrins, and G-protein-coupled receptors [37–39]. They have been implicated in multiple cell functions, including actin reorganization, microtubule dynamics, endocytosis and exocytosis. Conceptually, many of these cell functions are essential for the physiologic or pathologic role played by blood platelets. The current study was undertaken to determine the possible role of Rac GTPases of the Rho family in the regulation of platelet function by using a gene-targeting approach in mice coupled with pharmacologic inhibition of Rac GTPases in mice and in human platelets. The data in this report provide evidence that Rac1 GTPase is involved in the regulation of platelet secretion and aggregation induced by diverse agonists.

The role of Rac1 GTPase in cell exocytosis has been investigated by others using a dominant negative Rac1 mutant (Rac-N17), expression of Rho guanine nucleotide dissociation inhibitor (GDI), and overexpression of a Rac1 effector, PAK, in model cell lines [22]. Using this approach, Rac1 GTPase has been shown to be activated by agonists that induce secretion from bovine chromaffin cells, and activated Rac1 GTPase, in turn, has been reported to modulate secretion by regulating cytoskeletal organization [22]. This and other reports [23] suggest that Rac1 GTPase may play a role in secretory processes. However, platelets do not lend themselves to such molecular manipulations. So far, no evidence is available to associate Rac with platelet secretory processes. As inducers of platelet secretion also activate Rac1 GTPase, we investigated the effects of gene targeting of Rac1 GTPase as well as pharmacologic inhibition of Rac1 GTPase on platelet secretion responses. Our observations that platelets from Rac1–/– mice as well as human platelets preincubated with NSC23766 inhibited agonist-induced secretion from both α-granules and dense granules (Fig. 2) suggest that Rac1 GTPase plays an important role in platelet secretory responses.

It is now well established that ADP secreted from platelets plays a central role in inducing irreversible or maximal platelet aggregation induced by diverse agonists, including collagen, low concentrations of thrombin, or TXA2 [1,2,6]. Our findings that aggregation responses induced by ADP, collagen, thrombin and U46619 are impaired in both Rac1–/– platelets (Fig. 3A) and in human platelets preincubated with NSC23766 (Fig. 3B) suggest that Rac1 is required for platelet aggregation. It is possible that deficiency or inhibition of Rac1 GTPase, at least in part, inhibits platelet aggregation by blocking the platelet secretion. This possibility is further strengthened by our findings that the time course of inhibition of primary and secondary secretion by NSC23766 parallels the time course of inhibition of aggregation (Fig. 4). Recently, McCarty et al. [18] have reported that thrombin and ADP induce normal aggregation responses in platelets from Rac1-deficient mice. The discrepancy between their report and our findings that platelets from Rac1-deficient mice exhibited diminished aggregation responses to both thrombin and ADP may be due to the differences in the experimental conditions. McCarty et al. [18] examined thrombin-induced aggregation in the presence of apyrase and indomethacin to prevent contribution of the secreted mediators namely, ADP and TXA2, of platelet activation. The presence of these two inhibitors may have masked the differences in the secretory and consequently the aggregation responses in the WT and the Rac-deficient platelets. Our studies investigated ADP-induced aggregation in citrated PRP, whereas they examined ADP-induced aggregation in heparinized PRP. It is possible that the use of heparin instead of citrate abolished the differences in the ADP-induced aggregation responses between the WT and Rac-deficient platelets. Our findings that both gene targeting and pharmacologic inhibition of Rac1 diminished platelet secretion, taken together with other reports suggesting a role for Rac1 in secretion from other cells (e.g. bovine chromaffin and INS-1 β-cells [22,23]), supports our conclusion that Rac1 regulates platelet secretion. Secretion of ADP from platelets increases the aggregation response; hence, regulation of secretion by Rac1 would indirectly regulate platelet aggregation.

Platelet activation agonists such as thrombin or collagen stimulate multiple signaling pathways, including PLC-mediated hydrolysis of PI(4,5)P2 and generation of PIP3 by PI3 K, which induce activation of Rac1 GTPase and actin polymerization, leading to lamellipodia formation. Activated Rac1, in turn, stimulates PLC and PI3 K and thereby establishes a positive feedback loop for activation of platelets. Absence of this positive feedback because of deficiency or inhibition of Rac1 GTPase should affect all platelet responses, including secretion and aggregation mediated via the PLC and PI3 K signaling pathways. McCarty et al. [18] have reported that Rac1–/–Rac2–/– platelets exhibit diminished lamellipodia formation when exposed to thrombin. We investigated the role of Rac1 in actin polymerization using platelets from Rac1-deficient mice or platelets preincubated with NSC23766, and observed that platelets exhibited significantly reduced actin reorganization in response to thrombin (data not shown). These results confirm the report by McCarty et al. [18] that Rac1 GTPase is involved in the platelet morphologic response through regulation of the actin cytoskeleton. Our findings that deficiency or inhibition of Rac1 GTPase inhibits cytoskeletal reorganization as well as granular secretion suggest that the contribution of Rac1 GTPase to platelet activation extends beyond platelet spreading and lamellipodia formation, and includes the secretory response.

Recently, Rac1 GTPase has been shown to activate the transmembrane guanylyl cyclase [32]. However, its role in activation of soluble guanylyl cyclase is not known at this time. In platelets, activation of soluble guanylyl cyclase and consequent generation of cGMP has been shown to both stimulate [34] and inhibit [36] platelet activation, whereas activation or inhibition of adenylyl cyclase and the consequent increase or decrease in basal cAMP levels has been shown to inhibit or facilitate, respectively, platelet activation. Moreover, both cAMP and cGMP have been shown to inhibit activation of Rac1 GTPase [31]. These reports suggest that the interplay between cyclic nucleotides and Rac1 GTPase plays an important role in the regulation of platelet function and raise the possibility, although we have shown previously that NSC23766 inhibits activation of Rac GTPases by interfering with specific Rac–GEF interactions [24], that NSC23766 may be inhibiting platelet activation by altering cyclic nucleotides levels. Our findings that NSC23766 did not alter either cAMP (Fig. 5) or cGMP levels in basal or agonist-stimulated platelets suggest that the Rac-specific small molecule inhibitor of Rac–GEF interaction achieves its antiplatelet actions independently of cAMP or cGMP levels. The nature of the GEF involved in Rac activation in platelets has not been reported so far, and will be interesting to explore in future studies. However, Rac1–GEF Tiam1 has been reported to be involved in the activation of Rac1 in endothelial cells [40] as well as in fibroblast cells [41]. We have shown previously that NSC23766 inhibits binding of Tiam1 to Rac1 GTPase [42]. This raises the possibility that Rac1 activation in platelets may involve Tiam1 or a closely related molecule.

The data in Figs 2 and 3 show that gene targeting or inhibiting Rac activity by NSC23766 blocks platelet activation in vitro, and suggest that Rac plays an important role in platelet function and may be targeted to downregulate platelet activation. We tested this possibility by investigating the tail bleeding times, as a measure of in vivo platelet function, in Rac1–/– mice and in mice given NSC23766 by i.p. injection. Our findings that deficiency or inhibition of Rac1 GTPase prolonged the tail bleeding times (Fig. 6) demonstrate that Rac activity is critical for the integrity of platelet function in vitro and in vivo. These findings, taken together with a recently published report showing that Rac1 GTPase is essential for stable platelet aggregate formation [18], suggest that diminished platelet function because of inhibition of Rac1 GTPase results in a defective hemostatic response. The possibility that deficiency or inhibition of Rac1 GTPase may also affect the tail bleeding times because of loss of endothelial integrity cannot be ruled out at this time. However, the Rac1 genetic deletion was achieved by the Mx-cre mouse strain, which should not express the cre recombinase in either endothelial cells or smooth muscle cells upon poly(I:C) induction [11].

In summary, multiple inducers of platelet activation stimulate Rac1 and its effector PAK1 [16,17,43]. Deficiency of Rac1 GTPase or inhibition of Rac GTPase by NSC23766, a rationally designed specific inhibitor of the Rac–GEF interaction, inhibits activation of Rac1 and PAK1, and blocks platelet secretion and aggregation induced by diverse agonists. In addition, deficiency or inhibition of Rac1 GTPase results in defective platelet function and consequently a defective hemostatic response in mice. Collectively, these data suggest that Rac1 plays an important role in the regulation of platelet secretion and aggregation, and may serve as a novel antiplatelet target.

Acknowledgements

  1. Top of page
  2. Summary.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

We thank J. Thuma, J. Brown and D. Pratt of the Ohio University College of Osteopathic Medicine for excellent technical assistance and graphic support for this paper.

Disclosure of Conflict of Interests

  1. Top of page
  2. Summary.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

This work was supported by NIH grants GM60523 and GM53943 (to Y. Zheng), DK62752 (to D. A. Williams), and grants from the American Heart Association Ohio Valley Affiliate and the RSAC at OUCOM (to H. Akbar). J. A. Cancelas is a recipient of an award from the National Blood Foundation (USA). J. Kim, K. Funk, X. Shang, L. Chen and J. F. Johnson state that they have no conflict of interest.

References

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  2. Summary.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
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