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Phosphatidylinositol 3,4,5-trisphosphate regulates Ca2+ entry via Btk in platelets and megakaryocytes without increasing phospholipase C activity

2000; Springer Nature; Volume: 19; Issue: 12 Linguagem: Inglês

10.1093/emboj/19.12.2793

1460-2075

Jean‐Max Pasquet, Lynn Quek, Christiaan Stevens, Régis Bobe, Michael Huber, Vincent Duronio, Gerald Krystal, Steve P. Watson,

Blood disorders and treatments

Article15 June 2000free access Phosphatidylinositol 3,4,5-trisphosphate regulates Ca2+ entry via Btk in platelets and megakaryocytes without increasing phospholipase C activity Jean-Max Pasquet Jean-Max Pasquet Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT UK Search for more papers by this author Lynn Quek Lynn Quek Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT UK Search for more papers by this author Christiaan Stevens Christiaan Stevens Jack Bell Research Centre, Vancouver, BC, V6H 3Z6 Canada Search for more papers by this author Régis Bobe Régis Bobe Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT UK Search for more papers by this author Michael Huber Michael Huber Terry Fox Laboratory, BC Cancer Agency, 601 West Avenue, Vancouver, BC, V5Z IL3 Canada Search for more papers by this author Vincent Duronio Vincent Duronio Jack Bell Research Centre, Vancouver, BC, V6H 3Z6 Canada Search for more papers by this author Gerald Krystal Gerald Krystal Terry Fox Laboratory, BC Cancer Agency, 601 West Avenue, Vancouver, BC, V5Z IL3 Canada Search for more papers by this author Steve P. Watson Corresponding Author Steve P. Watson Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT UK Search for more papers by this author Jean-Max Pasquet Jean-Max Pasquet Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT UK Search for more papers by this author Lynn Quek Lynn Quek Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT UK Search for more papers by this author Christiaan Stevens Christiaan Stevens Jack Bell Research Centre, Vancouver, BC, V6H 3Z6 Canada Search for more papers by this author Régis Bobe Régis Bobe Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT UK Search for more papers by this author Michael Huber Michael Huber Terry Fox Laboratory, BC Cancer Agency, 601 West Avenue, Vancouver, BC, V5Z IL3 Canada Search for more papers by this author Vincent Duronio Vincent Duronio Jack Bell Research Centre, Vancouver, BC, V6H 3Z6 Canada Search for more papers by this author Gerald Krystal Gerald Krystal Terry Fox Laboratory, BC Cancer Agency, 601 West Avenue, Vancouver, BC, V5Z IL3 Canada Search for more papers by this author Steve P. Watson Corresponding Author Steve P. Watson Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT UK Search for more papers by this author Author Information Jean-Max Pasquet1, Lynn Quek1, Christiaan Stevens2, Régis Bobe1, Michael Huber3, Vincent Duronio2, Gerald Krystal3 and Steve P. Watson 1 1Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT UK 2Jack Bell Research Centre, Vancouver, BC, V6H 3Z6 Canada 3Terry Fox Laboratory, BC Cancer Agency, 601 West Avenue, Vancouver, BC, V5Z IL3 Canada *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:2793-2802https://doi.org/10.1093/emboj/19.12.2793 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The role of phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P3) and Btk in signalling by the collagen receptor glycoprotein VI was investigated. PI3,4,5P3 was increased in platelets from mice deficient in the SH2 domain-containing inositol 5-phosphatase (SHIP), in response to collagen related peptide (CRP). Tyrosine phosphorylation and activation of phospholipase Cγ2 (PLCγ2) were unaltered in SHIP−/− platelets, whereas Btk was heavily tyrosine phosphorylated under basal conditions and maximally phosphorylated by low concentrations of CRP. There was an increase in basal Ca2+, maximal expression of P-selectin, and potentiation of Ca2+ and aminophospholipid exposure to CRP in SHIP−/− platelets in the presence of Ca2+ (1 mM). Microinjection of PI3,4,5P3 into megakaryocytes caused a 3-fold increase in Ca2+ in response to CRP, which was absent in X-linked immunodeficiency (Xid) mice, which have a mutation in the PH domain of Btk. There was a corresponding partial reduction in the sustained level of intracellular Ca2+ in response to CRP in Xid mice but no change in PLC activity. These results demonstrate a novel pathway of Ca2+ entry that involves PI3,4,5P3 and Btk, and which is independent of increased PLC activity. Introduction Collagen activates platelets through a tyrosine kinase-based signalling pathway that shares many features with signalling by immune receptors (Watson and Gibbins, 1998). Activation of this pathway is mediated through the surface receptor glycoprotein VI (GPVI), which is associated with the Fc receptor γ-chain in the membrane (Gibbins et al., 1997; Tsuji et al., 1997; Kehrel et al., 1998). GPVI was recently cloned and shown to have two extracellular immunoglobulin domains, and to have close homology to the Fcα receptor (Clemetson et al., 1999). Crosslinking of GPVI leads to tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) in the Fc receptor γ-chain via the Src family kinases Fyn and Lyn (Melford et al., 1997; Ezumi et al., 1998; Briddon and Watson, 1999). This leads to binding of the protein tyrosine kinase Syk to the phosphorylated ITAM, resulting in autophosphorylation and activation. Syk plays a pivotal role in the regulation of phospholipase Cγ2 (PLCγ2), and also of the majority of other proteins that are phosphorylated downstream and/or recruited into intracellular signalling complexes upon activation of GPVI (Watson and Gibbins, 1998). A number of the proteins that lie downstream of Syk have important roles in the regulation of PLCγ2. This includes the two adapters LAT and SLP-76, which are required for tyrosine phosphorylation and activation of the phospholipase (Gross et al., 1999a; Pasquet et al., 1999b). The p85/110 kDa heterodimeric form of phosphatidylinositol 3-kinase (PI3-kinase) is also required for activation of PLCγ2 (Pasquet et al., 1999a). This action is mediated partially through the binding of PLCγ2 to the PI3-kinase product phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P3), probably via its N-terminal PH domain by analogy to the regulation of PLCγ1 (Falasca et al., 1998). This interaction is believed to support the recruitment of PLCγ2 to the membrane (Gratacap et al., 1998; Pasquet et al., 1999a). PI3,4,5P3 is also required for the activation of a second protein with a PH domain that is involved in the regulation of PLCγ2, the tyrosine kinase Btk. Platelets deficient in Btk, isolated from donors with the X-linked immunodeficiency agammaglobulinaemia (XLA) (Tsukada et al., 1993), exhibit impaired tyrosine phosphorylation of PLCγ2 and activation upon stimulation by collagen and collagen-related peptide (CRP; Quek et al., 1998). Several other proteins expressed in platelets have PH domains and are regulated by 3-phosphorylated lipids, including the GTP exchange factor Vav (Cichowski, 1996) and protein kinase B (Franke et al., 1997). The significance of the majority of these proteins in the regulation of PLCγ2 and other events contributing to platelet activation by GPVI is not known. The PI3-kinase pathway generates at least two second messenger lipids, PI3,4,5P3 and phosphatidylinositol 3,4-bisphosphate (PI3,4P2), which regulate a distinct set of intracellular proteins through interaction with their PH domains. PI3,4,5P3 is formed by the action of class I PI3-kinases, which includes the p85/110 kDa heterodimer described above, on phosphatidylinositol 4,5-bisphosphate. PI3,4P2 is generated either by dephosphorylation of PI3,4,5P3 (by a lipid 5-phosphatase) or through de novo synthesis by the action of a class II PI3-kinase on phosphatidylinositol 4-phosphate (Fruman et al., 1998). Platelets express at least one form of lipid 5′-phosphatase, the SH2 domain-containing inositol 5-phosphatase (SHIP) (Giurato et al., 1997). SHIP has been shown to be tyrosine phosphorylated and to undergo translocation to the cytoskeleton following outside-in signalling by the integrin GPIIb-IIIa (αIIbβ3) in human platelets (Giurato et al., 1997). The role of SHIP in platelet activation by GPVI is not known. In order to study the relative contribution of the two lipid messengers PI3,4,5P3 and PI3,4P2 to platelet activation, we used murine platelets deficient in the lipid 5-phosphatase, SHIP (i.e. from SHIP−/− mice). An elevation of PI3,4,5P3 in the SHIP−/− platelets was associated with constitutive tyrosine phosphorylation of Btk and increased basal Ca2+ in the presence of extracellular Ca2+ ([Ca2+]e), whereas PLC activity in response to GPVI activation was unchanged. Studies on X-linked immunodeficiency (Xid) mice, which express a mutant form of Btk unable to bind PI3,4,5P3, revealed a critical role for this kinase in Ca2+ influx, which was dependent on PI3,4,5P3. This work demonstrates a novel pathway of Ca2+ entry regulated through PI3,4,5P3 and Btk but independent of an increase in PLC activity. Results SHIP is tyrosine phosphorylated in CRP-stimulated platelets In order to investigate a possible role for SHIP in GPVI signalling in human platelets, platelets were stimulated by the GPVI-selective agonist CRP (Morton et al., 1995; Asselin et al., 1997) and the 5′-phosphatase immunoprecipitated using a specific antibody. CRP-stimulated a concentration and time-dependent increase in tyrosine phosphorylation of SHIP (Figure 1). Maximal phosphorylation was seen at a concentration of 1 μg/ml CRP after 60 s. This corresponds with the time course and concentration dependency of tyrosine phosphorylation of a large number of other proteins in CRP-stimulated platelets including Syk and PLCγ2. Several tyrosine-phosphorylated proteins were present in the SHIP immunoprecipitate and their level of tyrosine phosphorylation increased up to 300 s (Figure 1B). A tyrosine-phosphorylated band of 130 kDa migrating just below SHIP was not recognized by the SHIP antibody. Two other tyrosine-phosphorylated bands of 55 and 65 kDa were detected at 60 s and seen clearly at 300 s. A weakly phosphorylated band at 90 kDa was also detected. The identity of these bands is not known, although several could be isoforms of SHIP (Damen et al., 1998). These results demonstrate that SHIP is tyrosine phosphorylated and assembled into intracellular signalling complexes in CRP-stimulated platelets. Similar results were seen in murine platelets (not shown). Figure 1.SHIP is tyrosine phosphorylated in CRP-stimulated human platelets. Indomethacin-preincubated washed platelets (5 × 108/ml) were stimulated in an aggregometer cuvette with stirring in the presence of 1 mM EGTA at 37°C under the following conditions: (A) various concentrations of CRP for 2 min or (B) CRP (1 μg/ml) for varying times; (C) CRP (1 μg/ml), thrombin (1 unit/ml) and A23187 (3 μM) for 2 min in the presence of (D) BAPTA-AM (20 μM); and (E) PP1 (10 μM). The reaction was stopped by addition of ice-cold lysis buffer. Anti-SHIP immunoprecipitates were run on SDS–PAGE and transferred to PVDF membranes. Membranes were probed for phosphotyrosine using mAb 4G10 antibody (upper panel) and SHIP (lower panel). Molecular weight markers are indicated on the left. SHIP is shown by an arrow and major associated proteins by asterisks on the right. Results are representative of three to five experiments. Download figure Download PowerPoint The G protein-coupled receptor agonist thrombin and the Ca2+ ionophore A23187 stimulate a similar level of SHIP tyrosine phosphorylation and increase in association with intracellular proteins to that induced by CRP. These experiments were performed under conditions in which activation of the integrin αIIbβ3 was prevented by inclusion of EGTA (Figure 1). Tyrosine phosphorylation of SHIP induced either by CRP or thrombin was substantially inhibited by the chelator of intracellular Ca2+ ([Ca2+]i) BAPTA (loaded as BAPTA-AM) (Figure 1D). Src kinase inhibition by PP1 fully abolished CRP-induced SHIP tyrosine phosphorylation whereas thrombin still induced significant phosphorylation (Figure 1E). These results demonstrate that CRP and thrombin stimulate tyrosine phosphorylation of SHIP downstream of PLC through a Ca2+-dependent mechanism. Phosphatidylinositol 3,4,5-trisphosphate is increased in SHIP−/− platelets in response to CRP CRP has been reported to stimulate the rapid formation of [32P]PI3,4,5P3 in human platelets, peaking at 60 s after stimulation (Pasquet et al., 1999a). As shown in Figure 2A, CRP also induces formation of [32P]PI3,4,5P3 in mouse platelets (Figure 2A). In SHIP−/− platelets, the basal level of [32P]PI3,4,5P3 was slightly elevated and the response to CRP increased ∼5-fold relative to SHIP+/+ platelets, whereas the level of [32P]PI3,4P2 was reduced by ∼60% (Figure 2A and B). In comparison, the level of [32P]phosphatidylinositol 4,5-bisphosphate was similar in SHIP+/+ and SHIP−/− platelets (not shown). These results demonstrate a role for SHIP in the metabolism of PI3,4,5P3 and reveal that PI3,4P2 is formed, at least partially, by metabolism of PI3,4,5P3. The SHIP-independent increase in PI3,4P2 could be derived from either PI3,4,5P3 by the action of another lipid 5′-phosphatase, such as SHIP2 (Pesesse et al., 1998) (it is not known whether SHIP2 is expressed in platelets), or from phosphatidylinositol 4-phosphate by the action of a class II PI3-kinase (Zhang et al., 1998). Figure 2.Phospholipid metabolism in SHIP−/− platelet mice in response to CRP. Platelets (4 × 108/ml) were labelled with 1 mCi/ml [32P]orthophosphate (A–C) or loaded with Fura-2-AM (D). Stimulation was performed at 37°C for 2 min with: 0.1 μg/ml CRP (A) and (B); 0.1 and 10 μg/ml CRP and 0.001 and 0.1 U/ml thrombin (C); and 5 μg/ml CRP (D). In (A–C), the reaction was stopped by addition of 50 μl of 5 N HCl. After phospholipid extraction and deacylation as described in Materials and methods, samples were run on HPLC. Inositol lipids were identified by comparison with standards. PI3,4,5P3 (A); PI3,4P2 (B); phosphatidic acid (PA) (C); phosphatidic acid formation was analysed after phospholipid extraction by TLC and quantification by scintillation counting. (D) Ca2+: Ca2+ was measured in Fura-2-loaded platelets by recording fluorescence at 510 nm following excitation at 340 and 380 nM. Results in (A) and (B) are expressed as a mean ± range. Similar results were obtained in two separate experiments. Results in (C) are from one experiment carried out in triplicate and is representative of three. Results in (D) are from six experiments and are shown as mean ± SE. Download figure Download PowerPoint Phospholipase C activity is not changed in SHIP−/− platelets In order to determine whether PLC activity is altered in the SHIP−/− platelets, we measured the 1,2-diacylglycerol metabolite, phosphatidic acid, and the major protein kinase C substrate, pleckstrin. Although phosphatidic acid can be derived by phospholipase D activity, the level of this enzyme in platelets is low and the product would not be significantly radiolabelled under the non-equilibrium conditions used (Huang et al., 1991). The level of [32P]phosphatidic acid was similar in SHIP+/+ and SHIP−/− platelets under basal conditions and in response to stimulation by low and high concentrations of CRP and thrombin (Figure 2C). Similar results were seen for [32P]phosphorylation of pleckstrin (not shown). These results demonstrate that PLC activation is not enhanced in response to CRP and thrombin in the SHIP−/− platelets. A further indirect marker of PLC activity is the mobilization of Ca2+. This was measured in Fura-2-loaded platelets in Ca2+-free medium containing EGTA (estimated [Ca2+]e <1 nM). The basal level of Ca2+ and increases in response to CRP were similar in SHIP+/+ and SHIP−/− platelets consistent with a lack of change in PLC activity (not shown). In contrast, there was a 2-fold increase in the basal concentration of Ca2+ in the presence of 1 mM [Ca2+]e in the SHIP−/− platelets relative to controls (Table I and Figure 2D). [Ca2+]i was also increased in response to CRP in the SHIP−/− platelets in the presence of [Ca2+]e, although this was primarily the result of the increase in the basal level (Table I and Figure 2D). This suggests a role of 3-phosphorylated lipids in regulating Ca2+ influx in platelets. Table 1. Change in intracellular Ca2+ induced by CRP in SHIP−/− mice platelets SHIP genotype Basal CRP +/+ 98 ± 9 (n = 6) 365 ± 26 (n = 5) −/− 177 ± 11 (n = 8)* 500 ± 31 (n = 6)* Fura-2 loaded platelets (108/ml) were stimulated in a spectrofluorimeter cuvette at 37°C under stirring in the presence of 1 mM extracellular Ca2+. Baseline was recorded for 30 s and then CRP (10 μg/ml) was added. The asterisk indicates that p <0.05 when values are compared with the control data. Btk is hyperphosphorylated in SHIP−/− platelets The general pattern of tyrosine phosphorylation in control and SHIP−/− platelets in response to CRP was similar, with the exception of a broad 75 kDa band that comigrates with Syk, Btk and SLP-76 (not shown). An increase in phosphorylation of this band was also seen in non-stimulated SHIP−/− platelets. Phosphorylation of individual proteins implicated in the regulation of PLCγ2, notably those with a molecular weight of 70–76 kDa, was investigated in the SHIP−/− mice following immunoprecipitation using specific antibodies. CRP stimulated tyrosine phosphorylation of Btk, SLP-76 and Syk in SHIP+/+ platelets over the concentration range 0.1–10 μg/ml (Figure 3). Tyrosine phosphorylation of Syk, LAT and PLCγ2 was not significantly modified in the SHIP−/− platelets compared with SHIP+/+ cells (Figure 3). However, there was a slight increase in tyrosine phosphorylation of SLP-76 and a marked increase in phosphorylation of Btk in the SHIP−/− platelets (Figure 3). Btk was hyperphosphorylated in unstimulated platelets and maximally phosphorylated by the lowest concentration of CRP (0.01 μg/ml) that was used (Figure 3). Btk is therefore responsible for the increase in tyrosine phosphorylation of the 75 kDa band observed in whole-cell lysates. Figure 3.BTK is hyperphosphorylated in SHIP−/− mice platelets. Washed platelets (5 × 108/ml) were stimulated by the indicated CRP concentration for 2 min in the presence of 1 mM EGTA and the reaction was stopped by addition of ice-cold lysis buffer. After immunoprecipitation, samples were run on SDS–PAGE and transferred to PVDF membrane. Membranes were probed for phosphotyrosine using mAb 4G10 (upper panel) and the immunoprecipitated proteins (lower panel). Results are representative of three experiments. Download figure Download PowerPoint The increase in tyrosine phosphorylation of Btk in the presence of an elevated level of PI3,4,5P3 is consistent with observations in other cells. PI3,4,5P3 binds to the PH domain of Btk supporting its association to the membrane where it is phosphorylated by a Src family kinase (Mahajan et al., 1995; Afar et al., 1996; Rawlings et al., 1996; Li et al., 1997; Scharenberg et al., 1998). Btk activated in this way undergoes autophosphorylation and stimulates phosphorylation of cellular substrates. Measurement of P-selectin expression and procoagulant activity in SHIP−/− mice The functional consequence of the increase in PI3,4,5P3 in response to CRP was investigated by measurement of P-selectin expression and aminophospholipid exposure (procoagulant activity). P-selectin is a marker of α-granule secretion, which can be monitored by flow cytometry using a specific antibody. The exposure of aminophospholipids can also be measured by flow cytometry using annexin V, which binds with high affinity to phosphatidylserine. Aminophospholipid exposure is only seen in Ca2+-containing medium because of the high intracellular levels of the cation required for this response (Dachary-Prigent et al., 1993). Expression of P-selectin is not dependent on [Ca2+]e, but is increased in its presence (compare Figure 4A and B). Figure 4.Ca2+-dependent potentiation of P-selectin expression and procoagulant activity in SHIP−/− mice platelets. Washed platelets (5 × 108/ml) were stimulated with the indicated CRP concentration for 2 min in the presence of 1 mM EGTA (A) or 1 mM Ca2+ (B) and (C). Aliquots were incubated with P-selectin antibody and FITC-conjugated secondary antibodies for 10 min (A and B) or 10 μl of annexin-V–FITC (C). Results are presented as a dot-plot, showing fluorescence versus size (FL1/FSC), or expressed as a percentage of total cells responding (lower panel). Results are from one experiment that is representative of five separate experiments. Download figure Download PowerPoint A significantly greater number of cells (∼20%) show expression of P-selectin in SHIP−/− platelets in the absence of [Ca2+]e (free Ca2+ 3-fold) and duration (>10 min) of the Ca2+ response to CRP in control cells in the presence of 200 μM [Ca2+]e (Figure 5). In contrast, there was no potentiation of the Ca2+ response to CRP following microinjection of PI3,4,5P3 into Xid megakaryocytes, demonstrating a critical role for the PH domain of Btk in this response (Figure 5). These results provide evidence for a pathway of regulation of Ca2+ influx regulated through PI3,4,5P3 and Btk. Figure 5.PI3,4,5P3 potentiates Ca2+ influx in response to CRP in control but not Xid megakaryocytes. Megakaryocytes from Balb/c (A and B) or Xid (C and D) mice were microinjected with Fura-2 (A and C) or Fura-2 and PI3,4,5P3 (B and D) and the changes in [Ca2+]i has been determined in response to CRP 4 μg/ml. [Ca2+]e was 200 μM. Time 0 was set when CRP was added to the megakaryocytes, and the calcium response was then calculated at each 30 s after CRP addition up to 150 s. Each curve represents results of eight to 10 cells from a minimum of four mice. Download figure Download PowerPoint The physiological significance of this pathway can be addressed by comparison of the increase in [Ca2+]i in control and Xid platelets and megakaryocytes in response to CRP. It is possible to perform this study because activation of PLCγ2 in response to GPVI is not altered significantly in Xid mice in response to CRP. This is illustrated by the similar degree of phosphorylation of pleckstrin, formation of phosphatidic acid and tyrosine phosphorylation of PLCγ2 in response to CRP in control and Xid platelets (Figure 6). This contrasts with studies on XLA human platelets where a marked inhibition of PLCγ2 phosphorylation and activation is seen (Quek et al., 1998). Figure 6.PLC activity is unchanged but the sustained phase of Ca2+ entry is reduced in Xid platelets in response to CRP. (A) Pleckstrin phosphorylation. 32P-labelled platelets were stimulated by CRP for 2 min and the reaction was stopped by addition of sample buffer. Following electrophoresis, the gel was dried and pleckstrin phosphorylation was analysed by autoradiography. (B) Phosphatidic acid. 32P-labelled platelets were stimulated by CRP for 2 min and the reaction was stopped by addition of chloroform:methanol (1:2) and phosphatidic acid separated by thin layer chromatography as described in Materials and methods. Following autoradiography, spots were scraped and counted by Cerenkov. Results are the mean of three experiments. (C) Control and Xid platelets were stimulated with CRP (2 μg/ml) and then PLCγ2 was immunoprecipitated. PLCγ2 tyrosine phosphorylation was detected in the 4G10 blot (upper panel) and PLCγ2 loading was detected by reprobing (lower panel). (D) Platelets from control and Xid mice were loaded with Fura-2 as described in the Materials and methods. The baseline was recorded for 30 s and platelets were stimulated with CRP (indicated by an arrow) in the presence of [Ca2+]e (1 mM). The concentration of [Ca2+]i is calculated using the ratio of fluorescence, using a Kd of 224 nM. The trace is representative of 10 experiments from five animals. Download figure Download PowerPoint In the presence of [Ca2+]e, CRP stimulated a rapid initial peak in [Ca2+]i that decayed to a plateau in control platelets and megakaryocytes (Figure 6D). A similar initial peak in [Ca2+]i was observed in platelets and megakaryocytes from Xid mice in response to CRP, whereas the plateau was reduced by 20–40% in both cases (Figures 5 and 6). The latter supports a role for Btk in the sustained entry of Ca2+. The plateau was abolished in the absence of [Ca2+]e (not shown) demonstrating the presence of another mechanism of Ca2+ influx, which is presumably regulated by capacitative Ca2+ entry. Discussion In the present study, the role of the inositol 5′-phosphatase SHIP and the PH domain of the tyrosine kinase Btk have been investigated in platelets through respective studies on SHIP−/− and Xid mice stimulated by the collagen receptor GPVI. In response to CRP, the level of PI3,4,5P3 is elevated, whereas that of PI3,4P2 is reduced in SHIP−/− platelets demonstrating a major role for SHIP in the metabolism of PI3,4,5P3. These changes were associated with near maximal tyrosine phosphorylation of Btk under basal conditions, whereas PLC activity was unchanged. There was also an increase of basal Ca2+ in the SHIP−/− platelets in the presence of [Ca2+]e, and potentiation of Ca2+ influx in response to CRP, although this appeared to be primarily the result of the increase in basal Ca2+. The increase in phosphorylation of Btk is likely to be mediated by recruitment to the plasma membrane through interaction with PI3,4,5P3 and subsequent phosphorylation by a Src family kinase, probably Lyn (Mahajan et al., 1995; Afar et al., 1996; Rawlings et al., 1996; Li et al., 1997; Scharenberg et al., 1998). The increase of [Ca2+]i in SHIP−/− platelets provides a molecular explanation for the dramatic expression of P-selectin under basal conditions, and potentiation of aminophospholipid exposure by CRP in Ca2+-containing buffer. A lower level of [Ca2+]i is required for expression of P-selectin, accounting for the qualitative difference in results for the two responses. These results are similar to observations in bone marrow-derived mast cells and chicken DT-40 deficient in SHIP. An increase in Ca2+ influx was observed upon agonist stimulation that was not associated with a change in PLCγ activity as demonstrated by measurement of IP3 (Bolland et al., 1998; Huber et al., 1998b). In contrast to these studies, however, the major effect in the SHIP−/− platelets was on the basal level of Ca2+ in the presence of [Ca2+]e. This suggests that the 3-phosphorylated lipid-dependent pathway of Ca2+ influx is near maximal under basal conditions in the SHIP−/− platelets. This is consistent with the observation that tyrosine phosphorylation of Btk is also near maximal under basal c