Tyrosine Phosphorylation In Platelets Research Articles

GPIbα-selective activation of platelets induces platelet signaling events comparable to GPVI activation events

Platelet glycoprotein (GP)Ib-IX-V, which binds von Willebrand factor (VWF), and GPVI, which binds collagen, form an adhesion-signaling complex on platelets and mediate platelet adhesion in flowing blood. Platelet activation following engagement of GPIb-IX-V/GPVI by VWF/collagen is critical for initiation and development of a protective thrombus across a site of damaged or exposed endothelium. We examined platelet aggregation and signaling following selective engagement of platelet GPIbα (the major ligand-binding subunit of GPIb-IX-V) by a multivalent surface-expressed GPIbα-binding VWF-A1 domain on COS-7 cells. COS-7 cells expressing the VWF-A1 domain containing an R543W mutation (a gain-of-function mutation found in Type 2B von Willebrand's Disease) were used as a selective agonist for GPIb-IX-V. When incubated in a cell-to-platelet ratio of up to 1 : 1200, VWF-A1/R543W cells caused rapid, spontaneous aggregation of washed platelets that was GPIbα- and αIIbβ3-dependent (blocked by inhibitory anti-VWF-A1, anti-GPIbα and anti-αIIbβ3 antibodies). Platelet aggregation was also sensitive to inhibitors of Src, phosphoinositide 3-kinase (PI3-kinase) or Syk, confirming a role for these proteins in GPIbα-mediated signal transduction. Platelet tyrosine phosphorylation patterns and specific tyrosine phosphorylation of Syk after GPIbα engagement by VWF-A1/R543W was comparable to that induced by engagement of GPVI by collagen or collagen-related peptide (CRP). These data indicate signaling events triggered by specific ligation of GPIbα can lead to robust platelet activation and help define GPIb-IX-V as both an adhesion and signaling receptor on platelets.

The Cytokine Signal Inhibitor Lnk Promotes αIIbβ3 Integrin Outside-In Signaling through β3 Tyrosine Phosphorylation in Platelets.

Lnk is an SH2 domain-containing adapter protein that inhibits cytokine signaling. Lnk−/− mice exhibit a marked increase in numbers of hematopoietic stem cells, megakaryocytes and platelets, presumably due to the lack of negative regulation in thrombopoietin-mediated signals by Lnk. We previously reported that Lnk might play an unanticipated role in platelet integrin αIIbβ3 outside-in signaling. Lnk−/− platelets exhibited defects in full spreading on fibrinogen, clot retraction and formation of thrombi on collagen under flow conditions while they showed normal inside-out signaling (Blood , 106 (11):115a, 2005). However the mechanism(s) in which Lnk participates in αIIbβ3 outside-in signaling had not been elucidated. Here we report that in normal platelets Lnk forms a complex with c-Src, Syk, Fyn and adhesion and degranulation promoting adaptor protein (ADAP) but not SLP-76 in a manner dependent on αIIbβ3 ligation and Src kinase activation. c-Src-, but not Syk-, mediated tyrosine phosphorylation of C-terminus in Lnk appeared to be indispensable for the complex formation and Lnk-mediated function. Furthermore we have shown that Lnk is required for the association of Fyn to αIIbβ3 and for β3 subunit tyrosine phosphorylation while activation of non-receptor tyrosine kinases (c-Src and Syk) in proximity to αIIbβ3 is independent of Lnk. Thus, these results provide new insights into Lnk function and the mechanism by which Lnk contributes to integrin signaling in the adhesion responses of platelets.

Urtica dioica extract reduces platelet hyperaggregability in type 2 diabetes mellitus by inhibition of oxidant production, Ca<sup>2+</sup> mobilization and protein tyrosine phosphorylation

Platelet hyperaggregability is involved in the pathogenesis of type 2 diabetes mellitus. Thrombin-evoked platelet aggregation includes the activation of several intracellular pathways, including endogenous generation of reactive oxygen species (ROS), Ca2+ mobilization and protein tyrosine phosphorylation. Here we show that crude aqueous extract from Urtica dioica reduces thrombin-evoked aggregation in platelets from healthy donors and diabetics, in a concentration-dependent manner. U. dioica extract showed a potent antioxidant activity and prevented thrombin-evoked ROS generation in platelets from healthy and diabetic donors. Treatment with U. dioica extract reduced Ca2+ entry induced by thrombin or selective depletion of the two Ca2+ stores in platelets (without altering Ca2+ release), reduced protein tyrosine phosphorylation and reversed the abnormal Ca2+ mobilization and tyrosine phosphorylation in platelets from diabetics. We conclude that extract from U. dioica shows antiaggregant actions and might be used for the treatment and/or prevention of cardiovascular complications associated with type 2 diabetes mellitus.

Flavonoids inhibit the platelet TxA2 signalling pathway and antagonize TxA2 receptors (TP) in platelets and smooth muscle cells

Flavonoids may affect platelet function by several mechanisms, including antagonism of TxA(2) receptors (TP). These TP are present in many tissues and modulate different signalling cascades. We explored whether flavonoids affect platelet TP signalling, and if they bind to TP expressed in other cell types. Platelets were treated with flavonoids, or other selected inhibitors, and then stimulated with U46619. Similar assays were performed in aspirinized platelets activated with thrombin. Effects on calcium release were analysed by fluorometry and changes in whole protein tyrosine phosphorylation and activation of ERK 1/2 by Western blot analysis. The binding of flavonoids to TP in platelets, human myometrium and TPalpha- and TPbeta-transfected HEK 293T cells was explored using binding assays and the TP antagonist (3)H-SQ29548. Apigenin, genistein, luteolin and quercetin impaired U46619-induced calcium mobilization in a concentration-dependent manner (IC(50) 10-30 microm). These flavonoids caused a significant impairment of U46619-induced platelet tyrosine phosphorylation and of ERK 1/2 activation. By contrast, in aspirin-treated platelets all these flavonoids, except quercetin, displayed minor effects on thrombin-induced calcium mobilization, ERK 1/2 and total tyrosine phosphorylation. Finally, apigenin, genistein and luteolin inhibited by >50% (3)H-SQ29548 binding to different cell types. These data further suggest that flavonoids may inhibit platelet function by binding to TP and by subsequent abrogation of downstream signalling. Binding of these compounds to TP occurs in human myometrium and in TP-transfected HEK 293T cells and suggests that antagonism of TP might mediate the effects of flavonoids in different tissues.

Effect of homocysteine on platelet activation induced by collagen

Objective The present study investigated the effects of homocysteine on platelet activation induced by collagen and the downstream signaling pathways potentially involved in these effects. Methods Washed human platelets were incubated with homocysteine and collagen type I. The effects of homocysteine on platelet aggregation and adhesion and the tyrosine phosphorylation of total platelet proteins, Src kinase, and phospholipase-Cγ2 (PLCγ2) were studied. Results Homocysteine (10 to 100 μM) increased collagen-induced aggregation of washed platelets. Upon homocysteine (50 to 100 μM) treatment, platelet deposition to collagen-coated surface was significantly augmented under the low shear rate model (100/s) but not under the high shear rate model (1600/s). Collagen-stimulated total protein tyrosine phosphorylation in platelets was further enhanced by incubation with homocysteine. This effect was almost abrogated by genistein. Homocysteine potentiated collagen-stimulated tyrosine phosphorylation of the Src kinase and PLCγ2, which was partly decreased by integrin β 1 blocking antibody. Conclusion Homocysteine (at 10 to 100 μM) potentiates collagen type I induced-platelet activation through signaling components of glycoprotein VI and integrin α 2β 1 pathway. Our results suggested that upregulation of tyrosine phosphorylation of proteins such as Src and PLCγ2 is involved in the downstream signaling events of homocysteine stimulation in human platelets.

Role of Erythrocytes in Signal Transduction Mechanisms of Recruiting Platelets: Effects on Platelet Tyrosine Phosphorylation and Cytoskeletal Reorganization.

Platelet activation induces the release of granular components and metabolic products. This platelet releasate is a complex physiological agonist that upon interaction with other platelets induces platelet recruitment and thrombus growth. The agonistic potency of the platelet releasate is increased when platelets are stimulated in the presence of intact erythrocytes (RBCs) (Santos et al J. Clin Invest 1991; 87: 571; Valles et al Blood 1991; 78:154). The result is enhanced recruitment, α IIbβ3 receptor activation and P-selectin exposure in recruiting platelets (Valles et al Blood 2002; 99:3978). Receptor-mediated platelet activation initiates mechanisms of signal transduction that culminate in platelet functional responses. The biochemical mechanisms regulating the effects of cell-releasates in promoting platelet recruitment have not yet been elucidated. The aims of this study are: a) to characterize the effects of releasates from collagen-stimulated platelets on protein tyrosine phosphorylation (PTP), cytoskeletal reorganization and translocation to cytoskeleton of proteins in platelets being tested for recruitment; and b) to study the effects of platelet-erythrocyte interactions in these processes. Washed platelets (WP) or WP+RBCs (Htc. 40%) were stimulated with fibrillar collagen (1μg/mL) and rapidly centrifuged to obtain a cell-free collagen-free releasate within 1 min. An aliquot of this releasate was used as platelet agonist and the proaggregatory response (recruitment) was monitored by optical aggregometry as in the references above. For kinetic studies the reaction was halted at 15–180 sec., and PTP, cytoskeletal reorganization and cytoskeletal-associated tyrosine phosphorylated substrates were evaluated (Santos et al Circulation 2000; 102:1924–1930). Our results demonstrate for the first time that the platelet releasate induces PTP in the platelets tested for recruitment. This effect is remarkably amplified with releasates from platelet-erythrocyte mixtures especially at earlier time points. Of interest is the strong FAK phosphorylation induced by this releasate, a kinase thought to play a role in αIIbβ3 clustering and focal adhesion formation. The greater potency of the platelet-erythrocyte releasate was also evident in cytoskeletal reorganization (actin, ABP, talin). The platelet-erythrocyte releasate induced more PTP and cytoskeletal reorganization than 20 m M ADP. Cytoskeletal reorganization and translocation of tyrosine phosphorylated substrates to the cytoskeleton induced by the platelet-erythrocyte releasate were quicker than those induced by 1 U/ml thrombin and were of comparable final intensity. Interestingly, erythrocytes markedly enhanced translocation of FAK kinase and αIIbβ3 to the platelet cytoskeleton. The data reveal new biochemical mechanisms regulating platelet recruitment and demonstrate the remarkable role of erythrocytes in the signal transduction mechanisms of platelets during the recruitment process, which is a limiting step in mural thrombus formation. (Grant FIS 03/0270).

EDTA inhibits collagen-induced ATP+ADP secretion and tyrosine phosphorylation in platelets independently of Mg 2+ chelation and decrease in pH

The effects of Mg 2+ and EDTA on collagen-induced platelet dense granule secretion and protein tyrosine phosphorylation were studied in the presence and absence of various inhibitors of autocrine agonists that are released from secretory granules. Addition of EDTA to gel-filtered platelets in Tyrode's solution caused a decrease in pH of 0.7-0.9 pH units, due to chelation of Mg 2+ , and a marked inhibition of cATP+ADP secretion induced by collagen (25 w g/ml). Lowering pH of the platelet suspension to the same extent by HCl had no effect on secretion which also was the same in the absence of exogenous Mg 2+ as in its presence. Similarly, secretion induced by collagen per se , i.e., isolated from autocrine agonists by inhibitors, which was about 50% less than with autocrine agonists, was not affected by the presence or absence of exogenous Mg 2+ . The level of tyrosine phosphorylation of several proteins in resting platelets was higher in the absence of Mg 2+ than in its presence, but the onset of collagen-induced phosphorylation and dephosphorylation was more rapid in the absence of Mg 2+ . Tyrosine phosphorylation of p38 was specifically enhanced in the presence of inhibitors of autocrine phosphorylation and even more enhanced in the absence of Mg 2+ . EDTA inhibited the protein phosphorylation to the same extent in the presence as in the absence of exogenous Mg 2+ . These results show that EDTA inhibits collagen-induced dense granule secretion neither through chelation of Mg 2+ nor lowering of pH, and thus probably through other effects on the platelet by the EDTA2- species that are reflected in protein tyrosine phosphorylation signalling.

Thrombopoietin stimulates cortactin translocation to the cytoskeleton independently of tyrosine phosphorylation

Cortactin is an F-actin-binding protein expressed in platelets. During aggregation by thrombin, cortactin associates with Src, is tyrosine phosphorylated, and then translocates to the cytoskeleton. It is also found to associate with Syk during platelet shape change. Since cortactin undergoes tyrosine phosphorylation in platelets activated by thrombopoietin (TPO) that exhibit neither shape change nor aggregation, we investigated whether it could also relocalize to the detergent-insoluble fraction. We demonstrate that cortactin was present as a tyrosine-phosphorylated protein and co-localized with Syk in the Triton X-100-insoluble fraction of TPO-activated platelets. TPO stimulated Syk activation and association with cortactin. Conversely, cortactin associated with the kinases, Syk and Src. Cortactin tyrosine phosphorylation was blocked by Syk kinase inhibitor, piceatannol or Src family kinase inhibitor, PP2, suggesting that it depends on these two kinases. However, piceatannol or PP2 did not prevent cortactin translocation to the detergent-insoluble fraction. These data suggest that tyrosine phosphorylation is not required for cortactin translocation to the detergent-insoluble compartment. Furthermore, TPO activates, through its receptor c-Mpl, a signalling pathway to the cytoskeleton.

Thrombopoietin stimulates cortactin translocation to the cytoskeleton independently of tyrosine phosphorylation.

Cortactin is an F-actin-binding protein expressed in platelets. During aggregation by thrombin, cortactin associates with Src, is tyrosine phosphorylated, and then translocates to the cytoskeleton. It is also found to associate with Syk during platelet shape change. Since cortactin undergoes tyrosine phosphorylation in platelets activated by thrombopoietin (TPO) that exhibit neither shape change nor aggregation, we investigated whether it could also relocalize to the detergent-insoluble fraction. We demonstrate that cortactin was present as a tyrosine-phosphorylated protein and co-localized with Syk in the Triton X-100-insoluble fraction of TPO-activated platelets. TPO stimulated Syk activation and association with cortactin. Conversely, cortactin associated with the kinases, Syk and Src. Cortactin tyrosine phosphorylation was blocked by Syk kinase inhibitor, piceatannol or Src family kinase inhibitor, PP2, suggesting that it depends on these two kinases. However, piceatannol or PP2 did not prevent cortactin translocation to the detergent-insoluble fraction. These data suggest that tyrosine phosphorylation is not required for cortactin translocation to the detergent-insoluble compartment. Furthermore, TPO activates, through its receptor c-Mpl, a signalling pathway to the cytoskeleton.

Collagen, Convulxin, and Thrombin Stimulate Aggregation-independent Tyrosine Phosphorylation of CD31 in Platelets: EVIDENCE FOR THE INVOLVEMENT OF Src FAMILY KINASES

Platelet endothelial cell adhesion molecule-1 (CD31) is a 130-kDa glycoprotein receptor present on the surface of platelets, neutrophils, monocytes, certain T-lymphocytes, and vascular endothelial cells. CD31 is involved in adhesion and signal transduction and is implicated in the regulation of a number of cellular processes. These include transendothelial migration of leukocytes, integrin regulation, and T-cell function, although its function in platelets remains unclear. In this study, we demonstrate the ability of the platelet agonists collagen, convulxin, and thrombin to induce tyrosine phosphorylation of CD31. Furthermore, we show that this event is independent of platelet aggregation and secretion and is accompanied by an increase in surface expression of CD31. A kinase capable of phosphorylating CD31 was detected in CD31 immunoprecipitates, and its activity was increased following activation of platelets. CD31 tyrosine phosphorylation was reduced or abolished by the Src family kinase inhibitor PP2, suggesting a role for these enzymes. In accordance with this, each of the Src family members expressed in platelets, namely Fyn, Lyn, Src, Yes, and Hck, was shown to co-immunoprecipitate with CD31. The involvement of Src family kinases in this process was confirmed through the study of mouse platelets deficient in Fyn.

Evidence against a direct role of the integrin alpha2beta1 in collagen-induced tyrosine phosphorylation in human platelets.

In the present study we have investigated whether the collagen receptor alpha2beta1 (GPIa-IIa; GP, glycoprotein) regulates protein tyrosine phosphorylation in platelets directly through activation of tyrosine kinases or indirectly through modification of the response to GPVI. The interaction of collagen with alpha2beta1 was inhibited in two distinct ways, using the metalloprotease jararhagin, which cleaves the beta1 subunit, or the antibody P1E6 which competes with binding of collagen to the integrin. The two inhibitors caused a shift to the right in the collagen concentration response curves for protein tyrosine phosphorylation and platelet activation consistent with a causal relationship between the two events. There was no change in the overall pattern of tyrosine phosphorylation in response to high concentrations of collagen in the presence of alpha2beta1 blockade demonstrating that the integrin is not required for this event. In contrast, jararhagin and P1E6 had a small, almost negligible inhibitory effect against responses to the GPVI-selective agonist collagen-related peptide (CRP) and the G protein-coupled receptor agonist thrombin. Crosslinking of alpha2beta1 in solution or by adhesion to a monolayer using a variety of antibodies to either subunit of the integrin did not induce detectable protein tyrosine phosphorylation in whole cell lysates. The snake venom toxin trimucytin-stimulated a similar pattern of tyrosine phosphorylation to that induced by crosslinking of GPVI which was maintained in the presence of jararhagin. Trimucytin may therefore induce activation via GPVI rather than alpha2beta1 as previously thought. These observations show that the integrin alpha2beta1 is not required for regulation of tyrosine phosphorylation by collagen.

Regulation and Function of WASp in Platelets by the Collagen Receptor, Glycoprotein VI

Wiskott Aldrich syndrome (WAS) is an X-linked recessive disorder associated with abnormalities in platelets and lymphocytes giving rise to thrombocytopenia and immunodeficiency. WAS is caused by a mutation in the gene encoding the cytoskeletal protein (WASp). Despite its importance, the role of WASp in platelet function is not established. WASp was recently shown to undergo tyrosine phosphorylation in platelets after activation by collagen, suggesting that it may play a selective role in activation by the adhesion molecule. In the present study, we show that WASp is heavily tyrosine phosphorylated by a collagen-related peptide (CRP) that binds to the collagen receptor glycoprotein (GP) VI, but not to the integrin 2β1. Tyrosine phosphorylation of WASp was blocked by Src family kinase inhibitors and reduced by treatment with wortmannin and in patients with X-linked agammaglobulinemia (XLA), a condition caused by a lack of functional expression of Btk. This indicates that Src kinases, phosphatidylinositol 3-kinase (PI 3-kinase), and Btk all contribute to the regulation of tyrosine phosphorylation of WASp. The functional importance of WASp was investigated in 2 WAS brothers who show no detectable expression of WASp. Platelet aggregation and secretion from dense granules induced by CRP and thrombin was slightly enhanced in the WAS platelets relative to controls. Furthermore, there was no apparent difference in morphology in WAS platelets after stimulation by these agonists. These observations suggest that WASp does not play a critical role in intracellular signaling downstream of tyrosine kinase-linked and G protein-coupled receptors in platelets.

Regulation and Function of WASp in Platelets by the Collagen Receptor, Glycoprotein VI

AbstractWiskott Aldrich syndrome (WAS) is an X-linked recessive disorder associated with abnormalities in platelets and lymphocytes giving rise to thrombocytopenia and immunodeficiency. WAS is caused by a mutation in the gene encoding the cytoskeletal protein (WASp). Despite its importance, the role of WASp in platelet function is not established. WASp was recently shown to undergo tyrosine phosphorylation in platelets after activation by collagen, suggesting that it may play a selective role in activation by the adhesion molecule. In the present study, we show that WASp is heavily tyrosine phosphorylated by a collagen-related peptide (CRP) that binds to the collagen receptor glycoprotein (GP) VI, but not to the integrin α2β1. Tyrosine phosphorylation of WASp was blocked by Src family kinase inhibitors and reduced by treatment with wortmannin and in patients with X-linked agammaglobulinemia (XLA), a condition caused by a lack of functional expression of Btk. This indicates that Src kinases, phosphatidylinositol 3-kinase (PI 3-kinase), and Btk all contribute to the regulation of tyrosine phosphorylation of WASp. The functional importance of WASp was investigated in 2 WAS brothers who show no detectable expression of WASp. Platelet aggregation and secretion from dense granules induced by CRP and thrombin was slightly enhanced in the WAS platelets relative to controls. Furthermore, there was no apparent difference in morphology in WAS platelets after stimulation by these agonists. These observations suggest that WASp does not play a critical role in intracellular signaling downstream of tyrosine kinase-linked and G protein-coupled receptors in platelets.

Erythropoietin Improves Signaling through Tyrosine Phosphorylation in Platelets from Uremic Patients

Erythropoietin has shown to be effective in the correction of the hemostatic defect present in uremic patients. We have investigated the possible effect of recombinant human erythropoietin (rHuEPO) on the signaling processes occurring in platelets. Platelet suspensions were obtained from hemodialyzed patients before and after at least one month of initiating treatment with rHuEPO. Aliquots of non-activated or thrombin-activated platelets were treated to obtain platelet lysates or processed to extract platelet cytoskeleton. Samples were resolved by 8% SDS-polyacrylamide gel electrophoresis followed by Western blotting. After thrombin activation, proteins p120, p85, p78, p75, pp62, pp60, p59, p58, p56, p54 and p52 associated with the Triton-insoluble cytoskeletal fraction appeared phosphorylated in control profiles. In profiles from platelets obtained from uremic patients before treatment with rHuEPO, only proteins p58 and p56 appeared clearly and p54 was slightly phosphorylated. However, in platelets from the same patients under rHuEPO treatment, thrombin-induced phosphorylation improved to levels even above those observed in control profiles. Specially, the band at 54KDa appeared consistently more phosphorylated in all the patients under rHuEPO treatment. Although it is accepted that part of the hemostatic effect of erythropoietin is mediated by an increase in hematocrit, our study suggests that it enhances platelet signaling in uremic platelets which may explain the improvement of platelet response to activating stimulus before clinically noticeable elevation of hematocrit.

Protein kinase C-δ activation and tyrosine phosphorylation in platelets

Several protein kinase C (PKC) isoforms are expressed in human platelets. We report that PKC-δ is tyrosine phosphorylated within 30 s of platelet activation by thrombin. This correlated with a 2–3-fold increase in the kinase activity of PKC-δ relative to unstimulated platelets. The tyrosine phosphorylated PKC-δ isoform was associated with the platelet particulate (100 000× g insoluble) fraction. α IIbβ 3 integrin mediated platelet adhesion to fibrinogen did not significantly affect PKC-δ activity. Tyrosine phosphorylation of PKC-δ was similarly not detected in fibrinogen adherent platelet lysates. Treatment of the platelets with mAb 7E3 prior to the addition of thrombin blocked aggregation having no effect on the thrombin induced PKC-δ activation. We conclude that PKC-δ is activated in platelets by an α IIbβ 3 independent pathway.

A role for Bruton's tyrosine kinase (Btk) in platelet activation by collagen.

Bruton's tyrosine kinase (Btk) is essential for normal B-cell receptor signalling. The lack of expression of functional Btk in humans leads to the B-cell deficiency X-linked agammaglobulinaemia (XLA). We report here that Btk is also important for signalling via the collagen receptor glycoprotein VI (GPVI) in platelets. GPVI is coupled to the Fc receptor gamma chain (FcRgamma). The FcRgamma-chain contains a consensus sequence known as the immune-receptor tyrosine-based activation motif (ITAM). Tyrosine phosphorylation of the ITAM upon GPVI stimulation is the initial step in the regulation of phospholipase C gamma2 (PLCgamma2) isoforms via the tyrosine kinase p72(Syk) (Syk) in platelets. Here we show that collagen and a collagen-related peptide (CRP), which binds to GPVI but does not bind to the integrin alpha2beta1, induced Btk tyrosine phosphorylation in platelets. Aggregation, dense granule secretion and calcium mobilisation were significantly diminished but not completely abolished in platelets from XLA patients in response to collagen and CRP. These effects were associated with a reduction in tyrosine phosphorylation of PLCgamma2. In contrast, aggregation and secretion stimulated by thrombin in Btk-deficient platelets were not significantly altered. Our results demonstrate that Btk is important for collagen signalling via GPVI, but is not essential for thrombin-mediated platelet activation.

Comparison between the loss of platelet membrane asymmetry, microvesiculation and the tyrosine phosphorylation of proteins

Platelet activation by agents such as the Ca 2+-ionophore A23187 or Ca 2+-ATPase inhibitors leads to the generation of a procoagulant surface and the formation of microparticles. These responses are late events of platelet activation and readily detected by flow cytometry using annexin V-FITC as an aminophospholipid probe. One Ca 2+-ATPase inhibitor, 2,5-di-(tertbutyl)-1,4-benzohydroquinone induced aminophospholipid exposure without microparticle formation. Previous work has shown that microparticle formation is strictly linked to the activation of calpain, a thiol-protease that modifies the platelet cytoskeleton and some signal transduction enzymes. We now report how the detection of platelet tyrosine phosphorylation by western-blotting clearly shows that when platelet activation and aminophospholipid exposure are accompanied by microparticle formation there is a decrease in the tyrosine phosphorylation of proteins.

Regulation of Platelet Plasma Membrane Ca2+-ATPase by cAMP-dependent and Tyrosine Phosphorylation

As a consequence of its central role in the regulation of calcium metabolism in the platelet, the plasma membrane Ca2+-ATPase (PMCA) was assessed for cAMP-dependent and tyrosine phosphorylation. Addition of forskolin or prostaglandin E1, agents known to elevate platelet cAMP and calcium efflux, to platelets pre-labeled with [32P]PO4 resulted in the direct phosphorylation of platelet PMCA. Similarly, addition of the catalytic subunit of protein kinase A to platelet plasma membranes resulted in a 1.4-fold stimulation of activity. Thus, the previously reported inhibition of platelet activation by elevated intracellular cAMP may be accomplished in part by stimulation of PMCA, likely resulting in a decrease in intracellular calcium. Treatment with thrombin evoked tyrosine phosphorylation of platelet PMCA, while PMCA from resting platelets exhibited little tyrosine phosphorylation. Phosphorylation of platelet plasma membranes by pp60(src) resulted in 75% inhibition of PMCA activity within 15 min. Similarly, membranes isolated from thrombin-treated platelets exhibited 40% lower PMCA activity than those from resting platelets. Phosphorylation of erythrocyte ghosts and purified PMCA by pp60(src) also resulted in up to 75% inhibition of Ca2+-ATPase activity, and inhibition was correlated with tyrosine phosphorylation. Sequencing of a peptide obtained after 32P labeling of purified erythrocyte PMCA in vitro showed that tyrosine 1176 of PMCA4b is phosphorylated by pp60(src). These results indicate that tyrosine phosphorylation of platelet PMCA may serve as positive feedback to inhibit PMCA and increase intracellular calcium during platelet activation.

Crkl is constitutively tyrosine phosphorylated in platelets from chronic myelogenous leukemia patients and inducibly phosphorylated in normal platelets stimulated by thrombopoietin

Platelet functions such as aggregation and clot retraction are often abnormal in chronic mylogenous leukemia (CML) patients. However, the molecular mechanisms of these altered functions are unknown. As expression of the p210bcr-abl oncogene product, a constitutively active tyrosine kinase, is known to have an essential role in the pathogenesis of CML and tyrosine phosphorylation is intimately involved in various aspects of platelet activation, we examined the pattern of protein tyrosine phosphorylation in platelets from 15 CML patients by immunoblotting with a monoclonal antiphosphotyrosine antibody (4G10). Before and after stimulation with thrombin, the only consistent difference between normal and CML platelets was the presence of a tyrosine phosphorylated protein with a relative molecular weight of 39 kD. This tyrosine phosphorylated protein was identified as crid, an SH2, SH3 containing adapter protein. Thus, as previously demonstrated for neutrophils from CML patients, tyrosine phosphorylation of p39crkl persists in mature platelets. No tyrosine phosphorylation of crid was detected following stimulation with thrombin in normal platelets. However, crkl became incorporated into the Triton X-100 insoluble residue following thrombin stimulation in a manner dependent on platelet aggregation. Further, we found that crkl is an endogenous substrate for calpain, a protease that may be involved in postaggregation signaling processes. This suggests that crkl may be involved in the reorganization of the cytoskeleton during normal platelet aggregation and its tyrosine phosphorylation in CML platelets may contribute to the abnormal platelet function in CML patients. Finally, we found that thrombopoietin induces tyrosine phosphorylation of crk1 in normal platelets and FDCP cells genetically engineered to express human c-Mpl. This suggests that crk1 can be phosphorylated by a kinase other than p210bcr-abl and that crk1 may have a role in signaling by thrombopoietin.

Crkl Is Constitutively Tyrosine Phosphorylated in Platelets From Chronic Myelogenous Leukemia Patients and Inducibly Phosphorylated in Normal Platelets Stimulated by Thrombopoietin

AbstractPlatelet functions such as aggregation and clot retraction are often abnormal in chronic mylogenous leukemia (CML) patients. However, the molecular mechanisms of these altered functions are unknown. As expression of the p210bcr-abl oncogene product, a constitutively active tyrosine kinase, is known to have an essential role in the pathogenesis of CML and tyrosine phosphorylation is intimately involved in various aspects of platelet activation, we examined the pattern of protein tyrosine phosphorylation in platelets from 15 CML patients by immunoblotting with a monoclonal antiphosphotyrosine antibody (4G10). Before and after stimulation with thrombin, the only consistent difference between normal and CML platelets was the presence of a tyrosine phosphorylated protein with a relative molecular weight of 39 kD. This tyrosine phosphorylated protein was identified as crkl, an SH2, SH3 containing adapter protein. Thus, as previously demonstrated for neutrophils from CML patients, tyrosine phosphorylation of p39crkl persists in mature platelets. No tyrosine phosphorylation of crkl was detected following stimulation with thrombin in normal platelets. However, crkl became incorporated into the Triton X-100 insoluble residue following thrombin stimulation in a manner dependent on platelet aggregation. Further, we found that crkl is an endogenous substrate for calpain, a protease that may be involved in postaggregation signaling processes. This suggests that crkl may be involved in the reorganization of the cytoskeleton during normal platelet aggregation and its tyrosine phosphorylation in CML platelets may contribute to the abnormal platelet function in CML patients. Finally, we found that thrombopoietin induces tyrosine phosphorylation of crkl in normal platelets and FDCP cells genetically engineered to express human c-Mpl. This suggests that crkl can be phosphorylated by a kinase other than p210bcr-abl and that crkl may have a role in signaling by thrombopoietin.