A novel histidine tyrosine phosphatase, TULA-2, associates with Syk and negatively regulates GPVI signaling in platelets.

T-cell ubiquitin ligand-2 (TULA-2) is a recently discovered histidine tyrosine phosphatase thought to be ubiquitously expressed. In this work, we have investigated whether TULA-2 has a key role in platelet glycoprotein VI (GPVI) signaling. This study indicates that TULA-2 is expressed in human and murine platelets and is able to associate with Syk and dephosphorylate it. Ablation of TULA-2 resulted in hyperphosphorylation of Syk and its downstream effector phospholipase C-γ2 as well as enhanced GPVI-mediated platelet functional responses. In addition, shorter bleeding times and a prothrombotic phenotype were observed in mice lacking TULA-2. We therefore propose that TULA-2 is the primary tyrosine phosphatase mediating the dephosphorylation of Syk and thus functions as a negative regulator of GPVI signaling in platelets.

Determination of the substrate specificity of protein-tyrosine phosphatase TULA-2 and identification of Syk as a TULA-2 substrate.

TULA-1 (UBASH3A/STS-2) and TULA-2 (p70/STS-1) represent a novel class of protein-tyrosine phosphatases. Previous studies suggest that TULA-2 is sequence-selective toward phosphotyrosyl (Tyr(P)) peptides. In this work the substrate specificity of TULA-1 and -2 was systematically evaluated by screening a combinatorial Tyr(P) peptide library. Although TULA-1 showed no detectable activity toward any of the Tyr(P) peptides in the library, TULA-2 recognizes two distinct classes of Tyr(P) substrates. On the N-terminal side of Tyr(P), the class I substrates contain a proline at the Tyr(P)-1 position, a hydrophilic residue at the Tyr(P)-2 position, and aromatic hydrophobic residues at positions Tyr(P)-3 and beyond. The class II substrates typically contain two or more acidic residues, especially at Tyr(P)-1 to Tyr(P)-3 positions, and aromatic hydrophobic residues at other positions. At the C-terminal side of Tyr(P), TULA-2 generally prefers acidic and aromatic residues. The library screening results were confirmed by kinetic analysis of representative peptides selected from the library as well as Tyr(P) peptides derived from various Tyr(P) proteins. TULA-2 is highly active toward peptides corresponding to the Tyr(P)-323 and Tyr(P)-352 sites of Syk, and the Tyr(P)-397 site of focal adhesion kinase and has lower activity toward other Tyr(P) sites in these proteins. In glycoprotein VI-stimulated platelets, knock-out of the TULA-2 gene significantly increased the phosphorylation level of Syk at Tyr-323 and Tyr-352 sites and to a lesser degree at the Tyr-525/526 sites. These results suggest that Syk is a bona fide TULA-2 substrate in platelets.

Cbl-b is a novel physiologic regulator of glycoprotein VI-dependent platelet activation.

Cbl-b, a member of the Cbl family of E3 ubiquitin ligases, plays an important role in the activation of lymphocytes. However, its function in platelets remains unknown. We show that Cbl-b is expressed in human platelets along with c-Cbl, but in contrast to c-Cbl, it is not tyrosine-phosphorylated upon glycoprotein VI (GPVI) stimulation. Cbl-b, unlike c-Cbl, is not required for Syk ubiquitylation downstream of GPVI activation. Phospholipase Cgamma2 (PLCgamma2) and Bruton's tyrosine kinase (BTK) are constituently associated with Cbl-b. Cbl-b-deficient (Cbl-b(-/-)) platelets display an inhibition in the concentration-response curve for GPVI-specific agonist-induced aggregation, secretion, and Ca(2+) mobilization. A parallel inhibition is found for activation of PLCgamma2 and BTK. However, Syk activation is not affected by the absence of Cbl-b, indicating that Cbl-b acts downstream of Syk but upstream of BTK and PLCgamma2. When Cbl-b(-/-) mice were tested in the ferric chloride thrombosis model, occlusion time was increased and clot stability was reduced compared with wild type controls. These data indicate that Cbl-b plays a positive modulatory role in GPVI-dependent platelet signaling, which translates to an important regulatory role in hemostasis and thrombosis in vivo.

Lyn, PKC-delta, SHIP-1 interactions regulate GPVI-mediated platelet-dense granule secretion.

Protein kinase C-delta (PKC-delta) is expressed in platelets and activated downstream of protease-activated receptors (PARs) and glycoprotein VI (GPVI) receptors. We have previously shown that PKC-delta positively regulates PAR-mediated dense granule secretion, whereas it negatively regulates GPVI-mediated dense granule secretion. We further investigated the mechanism of such differential regulation of dense granule release by PKC-delta in platelets. SH2 domain-containing inositol phosphatase-1 (SHIP-1) is phosphorylated on Y1020, a marker for its activation, upon stimulation of human platelets with PAR agonists SFLLRN and AYPGKF or GPVI agonist convulxin. GPVI-mediated SHIP-1 phosphorylation occurred rapidly at 15 seconds, whereas PAR-mediated phosphorylation was delayed, occurring at 1 minute. Lyn and SHIP-1, but not SHIP-2 or Shc, preferentially associated with PKC-delta on stimulation of platelets with a GPVI agonist, but not with a PAR agonist. In PKC-delta-null murine platelets, convulxin-induced SHIP-1 phosphorylation was inhibited. Furthermore, in Lyn null murine platelets, GPVI-mediated phosphorylations on Y-1020 of SHIP-1 and Y311 of PKC-delta were inhibited. In murine platelets lacking Lyn or SHIP-1, GPVI-mediated dense granule secretions are potentiated, whereas PAR-mediated dense granule secretions are inhibited. Therefore, we conclude that Lyn-mediated phosphorylations of PKC-delta and SHIP-1 and their associations negatively regulate GPVI-mediated dense granule secretion in platelets.

Role of phosphoinositide 3-kinase beta in glycoprotein VI-mediated Akt activation in platelets.

Glycoprotein (GP) VI is a critical platelet collagen receptor. Phosphoinositide 3-kinase (PI3K) plays an important role in GPVI-mediated platelet activation, yet the major PI3K isoforms involved in this process have not been identified. In addition, stimulation of GPVI results in the activation of Akt, a downstream effector of PI3K. Thus, we investigated the contribution of PI3K isoforms to GPVI-mediated platelet activation and Akt activation. A protein kinase C inhibitor GF 109203X or a P2Y(12) receptor antagonist AR-C69931MX partly reduced GPVI-induced Akt phosphorylation. Platelets from mice dosed with clopidogrel also showed partial Akt phosphorylation, indicating that GPVI-mediated Akt phosphorylation is regulated by both secretion-dependent and -independent pathways. In addition, GPVI-induced Akt phosphorylation in the presence of ADP antagonists was completely inhibited by PI3K inhibitor LY294002 and PI3Kbeta inhibitor TGX-221 indicating an essential role of PI3Kbeta in Akt activation directly downstream of GPVI. Moreover, GPVI-mediated platelet aggregation, secretion, and intracellular Ca(2+) mobilization were significantly inhibited by TGX-221, and less strongly inhibited by PI3Kalpha inhibitor PIK75, but were not affected by PI3Kgamma inhibitor AS252424 and PI3Kdelta inhibitor IC87114. Consistently, GPVI-induced integrin alpha(IIb)beta(3) activation of PI3Kgamma(-/-) and PI3Kdelta(-/-) platelets also showed no significant difference compared with wild-type platelets. These results demonstrate that GPVI-induced Akt activation in platelets is dependent in part on G(i) stimulation through P2Y(12) receptor activation by secreted ADP. In addition, a significant portion of GPVI-dependent, ADP-independent Akt activation also exists, and PI3Kbeta plays an essential role in GPVI-mediated platelet aggregation and Akt activation.

Curcumin inhibits GPVI-mediated platelet activation by interfering with the kinase activity of Syk and the subsequent activation of PLCgamma2.

Turmeric (Curcuma longa), a herbal remedy and culinary spice, has been used in traditional Indian culture for millennia. An active ingredient found in turmeric is curcumin (diferuloylmethane). In the current study, we investigated the antiplatelet properties of this naturally occurring compound. Curcumin inhibited human platelet aggregation and dense granule secretion induced by GPVI agonist convulxin in a concentration-dependent manner. At 50 microM, it effectively inhibited the maximal extent of aggregation and dense granule secretion to as much as 75%. It also dramatically inhibited the activation-dependent tyrosine phosphorylation of Y753 and Y759 on PLCgamma2, but did not affect the phosphorylation of Y145 residue on the cytosolic adaptor protein SLP-76. Interestingly, curcumin had no significant effect on the phosphorylation of Y525/Y526 present on the activation loop of Syk (spleen tyrosine kinase), but had a significant inhibitory effect on in vitro Syk kinase activity. Moreover, the inhibitory action of curcumin is not due to an inhibition of thromboxane generation because all our studies were performed using aspirin-treated platelets. We conclude that curcumin inhibits platelet activation induced by GPVI agonists through interfering with the kinase activity of Syk and the subsequent activation of PLCgamma2.

Regulation of plasmin-induced protease-activated receptor 4 activation in platelets.

Plasmin, a major extracellular protease, activates platelets through PAR4 receptors. Plasmin-induced full aggregation is achieved at lower concentrations (0.1 U/mL) in murine platelets as compared to human platelets (1 U/mL). In COS7 cells expressing the murine PAR4 (mPAR4) receptor, 1 U/mL plasmin caused a higher intracellular calcium mobilization than in cells expressing the human PAR4 (hPAR4) receptor. This difference was reversed when the tethered ligand sequences of mPAR4 and hPAR4 were interchanged through site-directed mutagenesis. We further investigated whether PAR3 expressed in murine platelets serves as a co-receptor for PAR4 activation by plasmin. In COS7 cells, co-expressing mPAR3 and mPAR4, plamsin produced a smaller intracellular calcium mobilization compared to cells expressing mPAR4 alone, suggesting that PAR3 might inhibit plasmin-induced PAR4 stimulation. Consistent with these results, PAR3 null murine platelets also showed a greater plasmin-induced calcium mobilization and aggregation compared to wild-type murine platelets. In conclusion, murine platelets are more sensitive to activation by plasmin than human platelets due to differences in the primary sequence of PAR4. In contrast to thrombin-dependent activation of platelets, wherein PAR3 acts as a co-receptor, mPAR3 inhibits plasmin-induced PAR4 activation.

Evaluation of [3-(1-methyl-1H-indol-3-yl-methylene)-2-oxo-2, 3-dihydro-1H-indole-5-sulfonamide] (OXSI-2), as a Syk-selective inhibitor in platelets.

In the present study, we characterized OXSI-2 [3-(1-Methyl-1H-indol-3-yl-methylene)-2-oxo-2, 3-dihydro-1H-indole-5-sulfonamide], a putative inhibitor of Syk, and determined its specificity and selectivity in platelets. We found that OXSI-2 completely abolished convulxin-induced platelet functional responses. In order to determine whether OXSI-2 inhibited Src family kinase-mediated platelet responses, we evaluated its effect on Src family kinase (SFK)-mediated signaling events in platelets, viz. Lyn-mediated phosphorylation of Y352 on Syk, LAT-Y191 phosphorylation by Syk, and protease-activated receptor (PAR)-mediated phosphorylation of ERK. In the present work, we report that convulxin mediated Syk tyrosine 352 phosphorylation is not inhibited by OXSI-2, whereas piceatannol and PP2 abolished it. Syk-mediated Y191 LAT phosphorylation is abolished by all the three inhibitors. AYPGKF-induced phosphorylation of ERK was marginally inhibited by OXSI-2, whereas treatment with PP2 and piceatannol completely abolished it. However, PAR-mediated thromboxane generation (an event mediated by ERK) was potentiated by OXSI-2 whereas PP2 and piceatannol brought thromboxane to basal levels. Protein kinase C (PKC) inhibitors are known to potentiate PAR-mediated thromboxane generation in platelets. In contrast, OXSI-2, unlike PKC inhibitors, did not inhibit secretion. Therefore, we conclude that OXSI-2 is not a Syk-selective inhibitor in platelets because of its unexplained non-specific effects.

Expression of enzymatically-active phospholipase Cgamma2 in E. coli.

Phospholipase C-gamma-2 (PLCgamma2) activation is a key signaling event for many cell functions. In order to delineate the pathways that lead to PLCgamma2 activation, we devised a quick method for obtaining sufficient PLCgamma2. We obtained the full-length cDNA for human PLCgamma2 and expressed it in E. coli using the expression vector pT5T. To enhance the protein expression, tandem AGG-AGG arginine codons at the amino acid positions 1204-1205 were replaced by CGG-CGG arginine codons. The protein expression was detected in a Western blot analysis by both anti-PLCgamma2 antibodies and the antibodies that are raised against the tripeptide epitope (Glu-Glu-Phe) tag that are genetically-engineered to its carboxyl terminal. Crude lysates that were prepared from bacteria that express PLCgamma2 were found to catalyze the hydrolysis of phosphatidylinositol 4,5 bisphosphate. Similar to previous reports on PLCgamma2 that is isolated from mammalian tissue, the recombinant enzyme was Ca2+ dependent with optimal activity at 1-10 microM Ca2+.

RhoA downstream of G(q) and G(12/13) pathways regulates protease-activated receptor-mediated dense granule release in platelets.

Platelet secretion is an important physiological event in hemostasis. The protease-activated receptors, PAR 1 and PAR 4, and the thromboxane receptor activate the G(12/13) pathways, in addition to the G(q) pathways. Here, we investigated the contribution of G(12/13) pathways to platelet dense granule release. 2MeSADP, which does not activate G(12/13) pathways, does not cause dense granule release in aspirin-treated platelets. However, supplementing 2MeSADP with YFLLRNP (60muM), as selective activator of G(12/13) pathways, resulted in dense granule release. Similarly, supplementing PLC activation with G(12/13) stimulation also leads to dense granule release. These results demonstrate that supplemental signaling from G(12/13) is required for G(q)-mediated dense granule release and that ADP fails to cause dense granule release because the platelet P2Y receptors, although activate PLC, do not activate G(12/13) pathways. When RhoA, downstream signaling molecule in G(12/13) pathways, is blocked, PAR-mediated dense granule release is inhibited. Furthermore, ADP activated RhoA downstream of G(q) and upstream of PLC. Finally, RhoA regulated PKCdelta T505 phosphorylation, suggesting that RhoA pathways contribute to platelet secretion through PKCdelta activation. We conclude that G(12/13) pathways, through RhoA, regulate dense granule release and fibrinogen receptor activation in platelets.

Hematopoietic lineage cell specific protein 1 (HS1) is a functionally important signaling molecule in platelet activation.

Collagen activates platelets through an intracellular signaling cascade downstream of glycoprotein VI (GPVI). We have investigated the contribution of hematopoietic lineage cell-specific protein 1 (HS1) downstream of GPVI in platelet activation. Stimulation of GPVI leads to tyrosine phosphorylation of HS1, which is blocked by Src-family kinase inhibitors. Coimmunoprecipitation experiments revealed that HS1 associates with Syk and phosphatidylinositol 3-kinases. HS1-null mice displayed increased bleeding times and increased time to occlusion in the FeCl(3) in vivo thrombosis model compared with their wild-type littermates. In addition, aggregation and secretion responses were diminished in HS1-null mouse platelets after stimulation of GPVI and protease-activated receptor 4 (PAR-4) agonists compared with wild-type littermate mouse platelets. Finally, Akt phosphorylation was diminished after GPVI or PAR-4 stimulation in platelets from HS1-null mice compared with their wild-type littermates. These results demonstrate that phosphorylation of the HS1 protein occurs downstream of GPVI stimulation and that HS1 plays a significant functional role in platelet activation downstream of GPVI and PARs.

Rapid ubiquitination of Syk following GPVI activation in platelets.

Spleen tyrosine kinase (Syk) activation is a key intermediate step in the activation of platelets by the physiologic agonist collagen. We have found that Syk is rapidly ubiquitinated upon activation of platelets by collagen, collagen-related peptide (CRP), and convulxin. The Src family kinase inhibitors prevented Syk phosphorylation and its ubiquitination, indicating that the process is downstream of Src kinases. The ubiquitination of Syk did not cause degradation of the protein as evidenced by the lack of effect of proteasomal and lysosomal inhibitors. We separated ubiquitinated Syk from its nonubiquitinated counterpart and used an in vitro kinase assay to compare their activities. We found that the ubiquitinated Syk appeared to be about 5-fold more active. Using a phosphospecific antibody to Syk (Tyr525/Tyr526) that measures activated Syk, we found that most (60%-75%) of the active Syk is in the ubiquitinated fraction. This result explains the apparent high specific activity of ubiquitinated Syk. In c-Cbl-deficient mice, Syk is not ubiquitinated, implicating c-Cbl as the E3 ligase involved in Syk ubiquitination. Furthermore, Syk is not dephosphorylated in these mice. We propose that c-Cbl plays a regulatory role in glycoprotein VI (GPVI)/Fc receptor gamma (FcRgamma)-chain-dependent platelet activation through its interaction with Syk.

Glycoprotein VI-mediated platelet fibrinogen receptor activation occurs through calcium-sensitive and PKC-sensitive pathways without a requirement for secreted ADP.

Collagen activates platelets by transducing signals through glycoprotein VI (GPVI). It is not clear whether collagen can directly activate fibrinogen receptors on the adherent platelets without a role for positive feedback agonists. We investigated the contribution of secondary G protein signaling to the mechanism of GPVI-stimulated platelet aggregation using the GPVI-selective agonists, convulxin and collagen-related peptide (CRP) as well as collagen. Adenosine diphosphate (ADP) scavengers or ADP receptor antagonists shifted the concentration-response curve slightly to the right at low concentrations of convulxin, whereas platelet aggregation at higher concentrations of convulxin was unaffected by these agents. ADP receptor antagonists shifted the concentration-response curve of collagen- or CRP-induced platelet aggregation to the right at all the concentrations. Protein kinase C inhibitor, Ro 31-8220, or a calcium chelator 5,5'-dimethyl-BAPTA shifted the concentration-response curve of convulxin-induced platelet aggregation to the right. In addition, pretreatment with both Ro 31-8220 and dimethyl-BAPTA resulted in total inhibition of convulxin-mediated aggregation. Blockade of either the calcium- or protein kinase C-regulated pathway leads to inhibition of fibrinogen receptor activation on platelets adherent to collagen, but inhibition of both pathways leads to abolished fibrinogen receptor activation. We conclude that collagen-induced activation of fibrinogen receptor on adherent platelets through GPVI signaling occurs without any significant role for secreted ADP or thromboxane A(2). Furthermore, protein kinase C- and calcium-regulated pathways independently contribute to GPVI-mediated platelet aggregation.

Activation of phospholipase Cgamma2 by tyrosine phosphorylation.

Phospholipase Cgamma2 (PLCgamma2) has been implicated in collagen-induced signal transduction in platelets and antigen-dependent signaling in B-lymphocytes. It has been suggested that tyrosine kinases activate PLCgamma2. We expressed the full-length cDNA for human PLCgamma2 in bacteria and purified the recombinant enzyme. The recombinant enzyme was Ca(2+)-dependent with optimal activity in the range of 1 to 10 microM Ca(2+). In vitro phosphorylation experiments with recombinant PLCgamma2 and recombinant Lck, Fyn, and Lyn tyrosine kinases showed that phosphorylation of PLCgamma2 led to activation of the recombinant enzyme. Using site-directed mutagenesis, we investigated the role of specific tyrosine residues in activation of PLCgamma2. A mutant form of PLCgamma2, in which all three tyrosines at positions 743, 753, and 759 in the SH2-SH3 linker region were replaced by phenylalanines, exhibited decreased Lck-induced phosphorylation and completely abolished the Lck-dependent activation of PLCgamma2. Individual mutations of these tyrosine residues demonstrated that tyrosines 753 and 759, but not 743, were responsible for Lck-induced activation of PLCgamma2. To confirm these results, we procured a phosphospecific antibody to a peptide containing phosphorylated tyrosines corresponding to residues 753 and 759. This antibody recognized phosphorylated wild-type PLCgamma2 on Western blots but did not interact with unphosphorylated PLCgamma2 or with PLCgamma2 containing mutated tyrosine residues at 753 and 759. Using this antibody, we showed in intact platelets that collagen, a PLCgamma2-dependent agonist, induces phosphorylation of PLCgamma2 at Y753 and Y759. These studies demonstrate the importance of these two tyrosine residues in regulating the activity of PLCgamma2.

Protein kinase C- and calcium-regulated pathways independently synergize with Gi pathways in agonist-induced fibrinogen receptor activation.

Platelet fibrinogen receptor activation is a critical step in platelet plug formation. The fibrinogen receptor (integrin alphaIIbbeta3) is activated by agonist-mediated G(q) stimulation and resultant phospholipase C activation. We investigated the role of downstream signalling events from phospholipase C, namely the activation of protein kinase C (PKC) and rise in intracellular calcium, in agonist-induced fibrinogen receptor activation using Ro 31-8220 (a PKC inhibitor) or dimethyl BAPTA [5,5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N', N'-tetra-acetic acid], a high-affinity calcium chelator. All the experiments were performed with human platelets treated with aspirin, to avoid positive feedback from thromboxane A2. In the presence of Ro 31-8220, platelet aggregation caused by U46619 was completely inhibited while no effect or partial inhibition was seen with ADP and the thrombin-receptor-activating peptide SFLLRN, respectively. In the presence of intracellular dimethyl BAPTA, ADP- and U46619-induced aggregation and anti-alphaIIbbeta3 antibody PAC-1 binding were completely abolished. However, similar to the effects of Ro 31-8220, dimethyl BAPTA only partially inhibited SFLLRN-induced aggregation, and was accompanied by diminished dense-granule secretion. When either PKC activation or intracellular calcium release was abrogated, aggregation and fibrinogen receptor activation with U46619 or SFLLRN was partially restored by additional selective activation of the G(i) signalling pathway. In contrast, when both PKC activity and intracellular calcium increase were simultaneously inhibited, the complete inhibition of aggregation that occurred in response to either U46619 or SFLLRN could not be restored with concomitant G(i) signalling. We conclude that, while the PKC- and calcium-regulated signalling pathways are capable of inducing activating fibrinogen receptor independently and that each can synergize with G(i) signalling to cause irreversible fibrinogen receptor activation, both pathways act synergistically to effect irreversible fibrinogen receptor activation.

Tec kinases mediate sustained calcium influx via site-specific tyrosine phosphorylation of the phospholipase Cgamma Src homology 2-Src homology 3 linker.

Tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of non-receptor tyrosine kinases can phosphorylate PLCgamma in vitro, the specific kinase(s) controlling BCR-dependent PLCgamma activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLCgamma2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCgamma2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLCgamma2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btk-deficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr(753) and Tyr(759). Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCgamma2 carboxyl-terminal sites, Tyr(1197) and Tyr(1217), was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLCgamma2 SH2-SH3 linker.

Dynamic regulation of microtubule coils in ADP-induced platelet shape change by p160ROCK (Rho-kinase).

Platelet shape change is an extremely rapid process mediated by both the calcium-sensitive and p160ROCK pathways. The present study examines how different features of shape change studied by scanning electron microscopy clearly correlate to changes in the pattern of light absorbance measured in an aggregometer. Platelets change shape from the initial 'disc' form by producing: membrane 'blebs', sphere formation (cell-rounding), filopodia extension, and surface membrane folding. The presentation of these features was dramatically slower in the absence of intracellular calcium mobilization. In the presence of the p160ROCK-inhibitor, Y-27632, shape change was initially normal but platelets rapidly transformed back to smooth discs with extended filopodia. The reappearance of the disc shape is reflected by an increase in the amplitude of oscillations in the aggregometer shape change tracing. The kinetics of actin/cytoskeleton association correlated with filopodia formation but not with disc to sphere transformation. Changes in the level of tubulin polymerization correlated with changes from disc to sphere morphology. These experiments are consistent with a role for a RhoA/Rho kinase-regulated pathway in the maintenance of a spherical platelet shape after agonist-dependent activation. Continued disruption of the cytoskeletal microtubule ring, appears to be a Rhokinase-dependent event involved in the transformation of discoid platelets into spheres.