Threonine phosphorylation of the beta 3 integrin cytoplasmic tail, at a site recognized by PDK1 and Akt/PKB in vitro, regulates Shc binding

The mechanism of outside-in signaling by integrins parallels that for growth factor receptors. In both pathways, phosphorylation of a cytoplasmic segment on tyrosine generates a docking site for proteins containing Src homology 2 (SH2) and phosphotyrosine binding domains. We recently observed that phosphorylation of a threonine (Thr-753), six amino acids proximal to tyrosine 759 in beta(3) of the platelet specific integrin alpha(IIb)beta(3), inhibits outside-in signaling through this receptor. We hypothesized that the presence of phosphothreonine 753 either renders beta(3) a poor substrate for tyrosine kinases or inhibits the docking capabilities of the tyrosyl-phosphorylated form of beta(3.) The first alternative was tested by comparing the phosphorylation of beta(3) model peptides by the tyrosine kinase pp60(c-src) and we found that the presence of a phosphate group on a residue corresponding to Thr-753 did not detectably alter the kinetics of tyrosine phosphorylation. However, the presence of phosphate on this threonine inhibited the binding of Shc to tyrosyl-phosphorylated beta(3) peptide. The inhibitory effect of the phosphate group could be mimicked by substituting an aspartic acid for Thr-753, suggesting that a negative charge at this position modulates the binding of Shc and possibly other phosphotyrosine binding domain- and SH2-containing proteins. A survey of several protein kinases revealed that Thr-753 was avidly phosphorylated by PDK1 and Akt/PKB in vitro. These observations suggest that activation of PDK1 and/or Akt/PKB in platelets may modulate the binding activity and/or specificity of beta(3) for signaling molecules.     

Integrin tyrosine phosphorylation in platelet signaling.

The beta 3 integrin cytoplasmic tyrosine (ICY) motif of alpha IIb beta 3 becomes tyrosine phosphorylated during platelet aggregation, causing Shc and myosin to interact with the beta-integrin cytoplasmic domain. Platelets from mice lacking beta 3 ICY motif tyrosines formed defective aggregates and poorly retracted clots, establishing integrin tyrosine phosphorylation as a key mediator of beta 3-integrin signals.     

Fibrin clot retraction by human platelets correlates with alpha(IIb)beta(3) integrin-dependent protein tyrosine dephosphorylation

We have analyzed tyrosine phosphorylation associated with retraction of the fibrin clot by washed platelets in purified fibrinogen. Retraction was dependent on integrin alpha(IIb)beta(3), based on absence of retraction of alpha(IIb)beta(3)-deficient thrombasthenic platelets. However, only a subset of alpha(IIb)beta(3)-blocking antibodies or peptides were able to inhibit retraction, suggesting a differential engagement of alpha(IIb)beta(3) in fibrin clot retraction versus aggregation. Immunoblotting demonstrated a phosphorylated protein pattern comparable with aggregation at early time points. However, as opposed to aggregation, tyrosine phosphorylation decreased rapidly in parallel to retraction (up to 60 min). Dephosphorylation was alpha(IIb)beta(3)-dependent, since it was blocked by alpha(IIb)beta(3)-specific inhibitors and was absent in thrombasthenic platelets. Inhibition of platelet clot retraction by phenyl-arsine oxide and peroxovanadate, suggested a role for tyrosine phosphatases. Cytochalasin D and E (5 microm) blocked fibrin clot retraction and tyrosine dephosphorylation, suggesting regulation by actin cytoskeleton assembly. Tyrosine phosphatase activities were found associated with clot retraction using the "in-gel" tyrosine phosphatase assay; however, none were alpha(IIb)beta(3)-dependent. An 85-kDa protein and to a lesser degree "Src" showed the closest dose-dependent correlation between inhibition of tyrosine dephosphorylation and inhibition of retraction. We thus postulate that alpha(IIb)beta(3) engagement in fibrin clot retraction drives, in an actin cytoskeleton-dependent manner, the interaction of tyrosine phosphatases and of the tyrosine-phosphorylated substrates 85-kDa protein and Src, the dephosphorylation of which regulates the force generation and/or transmission required for full contraction of the fibrin matrix.     

The Pro33 isoform of integrin beta3 enhances outside-in signaling in human platelets by regulating the activation of serine/threonine phosphatases

Integrin beta(3) is polymorphic at residue 33 (Leu(33) or Pro(33)), and the Pro(33)-positive platelets display enhanced aggregation, P-selectin secretion, and shorter bleeding times. Because outside-in signaling is critical for platelet function, we hypothesized that the Pro(33) variant provides a more efficient signaling than the Leu(33) isoform. When compared with Pro(33)-negative platelets, Pro(33)-positive platelets demonstrated significantly greater serine/threonine phosphorylation of extracellular signal-regulated kinase (ERK2) and myosin light chain (MLC) but not cytoplasmic phospholipase A2 upon thrombin-induced aggregation. Tyrosine phosphorylation of integrin beta(3) and the adaptor protein Shc was no different in the fibrinogen-engaged platelets from both genotypes. The addition of Integrilin (alpha(IIb)beta(3)-fibrinogen blocker) or okadaic acid (serine/threonine phosphatase inhibitor) dramatically enhanced ERK2 and MLC phosphorylation in the Pro(33)-negative platelets when compared with Pro(33)-positive platelets, suggesting that integrin engagement during platelet aggregation activates serine/threonine phosphatases. The phosphatase activity of myosin phosphatase (MP) that dephosphorylates MLC is inactivated by phosphorylation of the myosin binding subunit of MP at Thr(696), and aggregating Pro(33)-positive platelets exhibited an increased Thr(696) phosphorylation of MP. These studies highlight a role for the dephosphorylation events via the serine/threonine phosphatases during the integrin outside-in signaling mechanism, and the Leu(33) --> Pro polymorphism regulates this process. Furthermore, these findings support a mechanism whereby the reported enhanced alpha granule secretion in the Pro(33)-positive platelets could be mediated by an increased phosphorylation of MLC, which in turn is caused by an increased phosphorylation and subsequent inactivation of myosin phosphatase.     

Platelet endothelial aggregation receptor 1 (PEAR1), a novel epidermal growth factor repeat-containing transmembrane receptor, participates in platelet contact-induced activation

The present study was designed to identify novel membrane proteins that signal during platelet aggregation. Because one putative mechanism for signaling by a membrane protein involves phosphorylation, we used oligonucleotide-based microarray analyses and mass spectrometric proteomics techniques to specifically discover membrane proteins and also identify those proteins that become phosphorylated on tyrosine, threonine, or serine residues upon platelet aggregation. Surprisingly, both techniques converged to identify a novel membrane protein we have termed PEAR1 (platelet endothelial aggregation receptor 1). Sequence analysis of PEAR1 predicts a type-1 membrane protein, 15 extracellular epidermal growth factor-like repeats, and multiple cytoplasmic tyrosines. Analysis of the tissue distribution of PEAR1 showed that it was most highly expressed in platelets and endothelial cells. Upon platelet aggregation induced by physiological agonists, PEAR1 became phosphorylated on tyrosine (Tyr-925), and serine (Ser-953 and Ser-1029) residues. PEAR1 tyrosine phosphorylation was blocked by eptifibatide, an alpha(IIb)beta(3) antagonist, which inhibits platelet aggregation. Immune clustering of PEAR1 resulted in PEAR1 phosphorylation. Aggregation-induced PEAR1 tyrosine phosphorylation lead to the subsequent association with the ShcB adaptor protein. Platelet proximity induced by centrifugation also induced PEAR1 tyrosine phosphorylation, a reaction not inhibited by eptifibatide. These data suggest that PEAR1 is a novel platelet receptor that signals secondary to alpha(IIb)beta(3)-mediated platelet-platelet contacts.     

Distinct contributions of glycoprotein VI and alpha(2)beta(1) integrin to the induction of platelet protein tyrosine phosphorylation and aggregation

Platelet activation by collagen depends principally on two receptors, alpha(2)beta(1) integrin (GPIa-IIa) and GPVI. During this activation, the nonreceptor protein tyrosine kinase pp72(syk) is rapidly phosphorylated, but the precise contribution of alpha(2)beta(1) integrin and GPVI to signaling for this phosphorylation is not clear. We have recently found that proteolysis of platelet alpha(2)beta(1) integrin by the snake venom metalloproteinase, jararhagin, results in inhibition of collagen-induced platelet aggregation and pp72(syk) phosphorylation. In order to verify whether the treatment of platelets with jararhagin had any effect on GPVI signaling, in this study we stimulated platelets treated with either jararhagin or anti-alpha(2)beta(1) antibody with two GPVI agonists, an antibody to GPVI and convulxin. Platelet shape change and phosphorylation of pp72(syk) by both GPVI agonists was preserved, as was the structure and function of GPVI shown by (125)I-labeled convulxin binding to immunoprecipitated GPVI from jararhagin-treated platelets. In contrast, defective platelet aggregation in response to GPVI agonists occurred in both jararhagin-treated and alpha(2)beta(1)-blocked platelets. This apparent cosignaling role of alpha(2)beta(1) integrin for platelet aggregation suggests the possibility of a topographical association of this integrin with GPVI. We found that both platelet alpha(2)beta(1) integrin and GPVI coimmunoprecipitated with alpha(IIb)beta(3) integrin. Since platelet aggregation requires activation of alpha(IIb)beta(3) integrin, defective aggregation in the absence of alpha(2)beta(1) suggests that this receptor may provide a signaling link between GPVI and alpha(IIb)beta(3). Our study therefore demonstrates that platelet signaling leading to pp72(syk) phosphorylation initiated with GPVI engagement by either convulxin or GPVI antibody does not depend on alpha(2)beta(1) integrin. However, alpha(IIb)beta(3) integrin may, in this model, require functional alpha(2)beta(1) integrin for its activation.     

Pathological shear stress stimulates the tyrosine phosphorylation of alpha-actinin associated with the glycoprotein Ib-IX complex.

Shear-induced platelet responses are triggered by VWF binding to the platelet GpIb-IX complex, and there is evidence that this ligand-receptor coupling stimulates transmembranous signaling through the cytoplasmic tail of glycoprotein (Gp) Ib alpha. To investigate the mechanism by which signaling is effected, new molecular interactions involving GpIb-IX that develop in response to pathological shearing stress were examined in intact human platelets. Exposure to shear, but not alpha-thrombin, results in the co-immunoprecipitation of the actin cross-linking protein alpha-actinin with the GpIb-IX complex. Blockers of VWF binding to GpIb alpha or actin polymerization inhibit the association of alpha-actinin with the GpIb-IX complex, but the association of alpha-actinin with the GpIb-IX complex is not affected by inhibiting VWF binding to platelet integrin alpha IIb beta 3 (GpIIb-IIIa). alpha-Actinin becomes tyrosine phosphorylated in response to pathological shear stress, and phosphorylated alpha-actinin associates with GpIb-IX. In resting platelets, class IA heterodimeric phosphatidylinositol 3-kinase (PI 3-K) and protein kinase N (PKN) associate with nonphosphorylated alpha-actinin. Shear stress causes PI 3-K to disassociate from alpha-actinin, while it stimulates PKN binding to alpha-actinin. These results demonstrate that shear-induced VWF binding to GpIb alpha causes enhanced binding of cytoskeletal alpha-actinin to GpIb-IX and suggest that alpha-actinin, perhaps through tyrosine phosphorylation, serves as an adapter for a signaling complex that could regulate VWF-induced platelet aggregation.     

Essential role of protein kinase C delta in platelet signaling, alpha IIb beta 3 activation, and thromboxane A2 release

The protein kinase C (PKC) family is an essential signaling mediator in platelet activation and aggregation. However, the relative importance of the major platelet PKC isoforms and their downstream effectors in platelet signaling and function remain unclear. Using isolated human platelets, we report that PKCdelta, but not PKCalpha or PKCbeta, is required for collagen-induced phospholipase C-dependent signaling, activation of alpha(IIb)beta(3), and platelet aggregation. Analysis of PKCdelta phosphorylation and translocation to the membrane following activation by both collagen and thrombin indicates that it is positively regulated by alpha(IIb)beta(3) outside-in signaling. Moreover, PKCdelta triggers activation of the mitogen-activated protein kinase-kinase (MEK)/extracellular-signal regulated kinase (ERK) and the p38 MAPK signaling. This leads to the subsequent release of thromboxane A(2), which is essential for collagen-induced but not thrombin-induced platelet activation and aggregation. This study adds new insight to the role of PKCs in platelet function, where PKCdelta signaling, via the MEK/ERK and p38 MAPK pathways, is required for the secretion of thromboxane A(2).     

SHIP1 and Lyn Kinase Negatively Regulate Integrin alpha IIb beta 3 signaling in platelets

Integrin alpha(IIb)beta(3) plays a critical role in platelet function, promoting a broad range of functional responses including platelet adhesion, spreading, aggregation, clot retraction, and platelet procoagulant function. Signaling events operating downstream of this receptor (outside-in signaling) are important for these responses; however the mechanisms negatively regulating integrin alpha(IIb)beta(3) signaling remain ill-defined. We demonstrate here a major role for the Src homology 2 domain-containing inositol 5-phosphatase (SHIP1) and Src family kinase, Lyn, in this process. Our studies on murine SHIP1 knockout platelets have defined a major role for this enzyme in regulating integrin alpha(IIb)beta(3)-dependent phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) accumulation, necessary for a cytosolic calcium response and platelet spreading. SHIP1 phosphorylation and PtdIns(3,4,5)P(3) metabolism is partially regulated through Lyn kinase, resulting in an enhanced calcium flux and spreading response in Lyn-deficient mouse platelets. Analysis of platelet adhesion dynamics under physiological blood flow conditions revealed an important role for SHIP1 in regulating platelet adhesion on fibrinogen. Specifically, SHIP1-dependent PtdIns(3,4,5)P(3) metabolism down-regulates the stability of integrin alpha(IIb)beta(3)-fibrinogen adhesive bonds, leading to a decrease in the proportion of platelets forming shear-resistant adhesion contacts. These studies define a major role for SHIP1 and Lyn as negative regulators of integrin alpha(IIb)beta(3) adhesive and signaling function.     

Regulation of the pp72syk protein tyrosine kinase by platelet integrin alpha IIb beta 3.

pp72syk is essential for development and function of several hematopoietic cells, and it becomes activated through tandem SH2 interaction with ITAM motifs in immune response receptors. Since Syk is also activated through integrins, which do not contain ITAMs, a CHO cell model system was used to study Syk activation by the platelet integrin, alpha IIb beta 3. As in platelets, Syk underwent tyrosine phosphorylation and activation during CHO cell adhesion to alpha IIb beta 3 ligands, including fibrinogen. This involved Syk autophosphorylation and the tyrosine kinase activity of Src, and it exhibited two novel features. Firstly, unlike alpha IIb beta 3-mediated activation of pp125FAK, Syk activation could be triggered by the binding of soluble fibrinogen and abolished by truncation of the alpha IIb or beta 3 cytoplasmic tail, and it was resistant to inhibition by cytochalasin D. Secondly, it did not require phosphorylated ITAMs since it was unaffected by disruption of an ITAM-interaction motif in the SH2(C) domain of Syk or by simultaneous overexpression of the tandem SH2 domains. These studies demonstrate that Syk is a proximal component in alpha IIb beta 3 signaling and is regulated as a consequence of intimate functional relationships with the alpha IIb beta 3 cytoplasmic tails and with Src or a closely related kinase. Furthermore, there are fundamental differences in the activation of Syk by alpha IIb beta 3 and immune response receptors, suggesting a unique role for integrins in Syk function.     

Orthovanadate decreases the leptin content in isolated mouse fat pads via proteasome activation

In a physiological milieu platelets continue to be exposed to agonists long after clot formation. We studied the regulation of postaggregation events consequent on protease-activated receptor (PAR) 1 ligation with either thrombin or the thrombin receptor-activating peptide (TRAP). Stimulation with TRAP (20 microM) but not with thrombin (1 U/ml) for 15 min evoked platelet disaggregation by about 30% and downregulation of high-affinity fibrinogen binding sites on integrin alpha(IIb)beta(3) to nearly prestimulation levels. Concurrently, only TRAP disorganized the actin-based cytoskeleton, with decrease in the cytoskeletal content of focal contact-associated proteins like integrin alpha(IIb)beta(3), Src, and focal adhesion kinase (FAK). While protein tyrosine kinases were activated during the initial period of platelet aggregation with either agonist, stimulation of protein tyrosine phosphatases determined the successive phase of reduced phosphotyrosine content. SHP-1, an abundant protein tyrosine phosphatase in the platelets, was tyrosine phosphorylated on challenge of PAR-1 and coprecipitated with two unidentified tyrosine phosphorylated proteins of 140 and 60 kDa; in addition, SHP-1 tyrosine phosphorylation (which is associated with enhanced phosphatase activity) was sustained until 15 min. Activity of calpain was upregulated following incubation with thrombin and not with TRAP. Collectively, these data suggest that signaling pathways elicited by PAR-1 agonists thrombin and TRAP are markedly different, which could have important implications on late platelet responses.     

Adhesive ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK

Tyrosine phosphorylation of multiple platelet proteins is stimulated by thrombin and other agonists that cause platelet aggregation and secretion. The phosphorylation of a subset of these proteins, including a protein tyrosine kinase, pp125FAK, is dependent on the platelet aggregation that follows fibrinogen binding to integrin alpha IIb beta 3. In this report, we examined whether fibrinogen binding, per se, triggers a process of tyrosine phosphorylation in the absence of exogenous agonists. Binding of soluble fibrinogen was induced with Fab fragments of an anti-beta 3 antibody (anti-LIBS6) that directly exposes the fibrinogen binding site in alpha IIb beta3. Proteins of 50-68 KD and 140 kD became phosphorylated on tyrosine residues in a fibrinogen- dependent manner. This response did not require prostaglandin synthesis, an increase in cytosolic free calcium, platelet aggregation or granule secretion, nor was it associated with tyrosine phosphorylation of pp125FAK. Tyrosine phosphorylation of the 50-68-kD and 140-kD proteins was also observed when (a) fibrinogen binding was stimulated by agonists such as epinephrine, ADP, or thrombin instead of by anti-LIBS6; (b) fragment X, a dimeric plasmin-derived fragment of fibrinogen was used instead of fibrinogen; or (c) alpha IIb beta 3 complexes were cross-linked by antibodies, even in the absence of fibrinogen. In contrast, no tyrosine phosphorylation was observed when the ligand consisted of monomeric cell recognition peptides derived from fibrinogen (RGDS or gamma 400-411). Fibrinogen-dependent tyrosine phosphorylation was inhibited by cytochalasin D. These studies demonstrate that fibrinogen binding to alpha IIb beta 3 initiates a process of tyrosine phosphorylation that precedes platelet aggregation and the phosphorylation of pp125FAK. This reaction may depend on the oligomerization of integrin receptors and on the state of actin polymerization, organizational processes that may juxtapose tyrosine kinases with their substrates.     

The P2Y(12) receptor induces platelet aggregation through weak activation of the alpha(IIb)beta(3) integrin--a phosphoinositide 3-kinase-dependent mechanism

High concentrations of adenosine-5'-diphosphate ADP are able to induce partial aggregation without shape change of P2Y(1) receptor-deficient mouse platelets through activation of the P2Y(12) receptor. In the present work we studied the transduction pathways selectively involved in this phenomenon. Flow cytometric analyses using R-phycoerythrin-conjugated JON/A antibody (JON/A-PE), an antibody which recognizes activated mouse alpha(IIb)beta(3) integrin, revealed a low level activation of alpha(IIb)beta(3) in P2Y(1) receptor-deficient platelets in response to 100 microM ADP or 1 microM 2MeS-ADP. Adrenaline induced no such activation but strongly potentiated the effect of ADP in a dose-dependent manner. Global phosphorylation of (32)P-labeled platelets showed that P2Y(12)-mediated aggregation was not accompanied by an increase in the phosphorylation of myosin light chain (P(20)) or pleckstrin (P(47)) and was not affected by the protein kinase C (PKC) inhibitor staurosporine. On the other hand, two unrelated phosphoinositide 3-kinase inhibitors, wortmannin and LY294002, inhibited this aggregation. Our results indicate that (i) the P2Y(12) receptor is able to trigger a P2Y(1) receptor-independent inside-out signal leading to alpha(IIb)beta(3) integrin activation and platelet aggregation, (ii) ADP and adrenaline use different signaling pathways which synergize to activate the alpha(IIb)beta(3) integrin, and (iii) the transduction pathway triggered by the P2Y(12) receptor is independent of PKC but dependent on phosphoinositide 3-kinase.     

Kinetic regulation of beta 3 integrin tyrosine phosphorylation

Tyrosine phosphorylation of beta(3) integrins is a permissive stage in the activation of alpha(IIb)beta(3) and alpha(v)beta(3) in platelets and leukocytes, respectively. In this study we demonstrated direct phosphorylation of beta(3) integrins as a result of interaction with soluble monomeric ligand, and we characterized the differential kinetics of beta(3) phosphorylation as a consequence of alpha subunit pairing. We found that beta(3) phosphorylation is initiated by RGD peptide binding in a dose-dependent and saturable fashion with alpha(IIb)beta(3) becoming phosphorylated and dephosphorylated more rapidly than alpha(v)beta(3). Site mapping of phosphate incorporation reveals significant phosphorylation at Tyr-747 in both beta(3) integrin species with incorporation at Tyr-759 found at significant levels only in alpha(IIb)beta(3). Mutation of cytoplasmic beta(3) tyrosine residues in a transfection model prevents cell adhesion via these integrins. These data demonstrate that recognition of ligand is sufficient to induce beta(3) tyrosine phosphorylation and suggests that this event is regulated by the alpha subunit pairing of beta(3).     

Tyrosine dephosphorylation, but not phosphorylation, of p130Cas is dependent on integrin alpha IIb beta 3-mediated aggregation in platelets: implication of p130Cas involvement in pathways unrelated to cytoskeletal reorganization

The newly described adapter molecule p130 Crk-associated substrate (Cas) has been reported to contribute to cytoskeletal organization through assembly of actin filaments and to be pivotal in embryonic development and in oncogene-mediated transformation. We characterized the regulation of Cas tyrosine phosphorylation in highly differentiated, anucleate platelets. Phospholipase C-activating receptor agonists, including collagen, thrombin receptor-activating peptide (TRAP), and U46619 (a thromboxane A2 analogue), and A23187 (a Ca2+ ionophore) induced rapid Cas tyrosine phosphorylation in platelets. 12-O-Tetradecanoylphorbol 13-acetate and 1-oleoyl-2-acetyl-sn-glycerol, protein kinase C (PKC) activators, also induced Cas tyrosine phosphorylation, albeit sluggishly. Cas tyrosine phosphorylation induced by collagen or TRAP was transient in aggregating platelets; Cas became dephosphorylated in a manner dependent on integrin alpha IIb beta 3-mediated aggregation. While BAPTA-AM (an intracellular Ca2+ chelator) inhibited Cas phosphorylation induced by collagen or TRAP, Ro31-8220 (a PKC inhibitor) rather prolonged it. Under the conditions, this PKC inhibitor suppressed platelet aggregation but not intracellular Ca2+ mobilization. In contrast to Cas involvement in focal adhesions in other cells, platelet Cas phosphorylation preceded the activation of focal adhesion kinase (FAK), and blockage of alpha IIb beta 3-mediated platelet aggregation with a GRGDS peptide resulted in prolongation of stimulation-dependent Cas tyrosine phosphorylation but in suppression of FAK tyrosine phosphorylation. Furthermore, TRAP-induced Cas phosphorylation was insensitive to cytochalasin D, an actin polymerization inhibitor. The failure of FAK to associate with Cas in immunoprecipitation studies also suggests that Cas tyrosine phosphorylation is independent of FAK activation. Of the signaling molecules investigated in this study, Src seemed to associate with Cas. Finally, Cas existed mainly in cytosol and membrane cytoskeleton fractions in the resting state, and remained unchanged during platelet aggregation, when FAK translocated to the cytoskeletal fraction. Our findings on platelet Cas suggest that (i) rapid Cas tyrosine phosphorylation occurs following phosphoinositide turnover by receptor-mediated agonists and may be mediated by intracellular Ca2+ mobilization; (ii) PKC activation, by itself, may elicit sluggish Cas phosphorylation; (iii) Cas tyrosine dephosphorylation, but not phosphorylation, is dependent on integrin alpha IIb beta 3-mediated aggregation; and (iv) Cas is not involved in cytoskeletal reorganization. Anucleate platelets seem to provide a unique model system to fully elucidate the functional role(s) of Cas.     

Costimulation of Gi- and G12/G13-mediated signaling pathways induces integrin alpha IIbbeta 3 activation in platelets

Platelet activation is a complex process induced by a variety of stimuli, which act in concert to ensure the rapid formation of a platelet plug at places of vascular injury. We show here that fibrillar collagen, which initiates platelet activation at the damaged vessel wall, activates only a small fraction of platelets in suspension directly, whereas the majority of platelets becomes activated by mediators released from collagen-activated platelets. In Galpha(q)-deficient platelets that do not respond with activation of integrin alpha(IIb)beta(3) to a variety of mediators like thromboxane A2 (TXA2), thrombin, or ADP, collagen at high concentrations was able to induce aggregation, an effect that could be blocked by antagonists of the TXA2 or P2Y12 receptors. The activation of TXA2 or P2Y12 receptors alone, which in Galpha(q)-deficient platelets couple to G12/G13 and Gi, respectively, did not induce platelet integrin activation or aggregation. However, concomitant activation of both receptors resulted in irreversible integrin alpha(IIb)beta3-mediated aggregation of Galpha(q)-deficient platelets. Thus, the activation of G12/G13- and Gi-mediated signaling pathways is sufficient to induce integrin alpha(IIb)beta3 activation. Although G(q)-mediated signaling plays an important role in platelet activation, it is not strictly required for the activation of integrin alpha(IIb)beta3. This indicates that the efficient induction of platelet aggregation through G-protein-coupled receptors is an integrated response mediated by various converging G-protein-mediated signaling pathways involving G(q) and G(i) as well as G12/G13.     

Direct demonstration of involvement of the adaptor protein ShcA in the regulation of Ca2+-induced platelet aggregation

Platelet aggregation is mediated by conformational change of integrin alpha(IIb)beta(3). Tyrosine-phosphorylation of cytoplasmic domain of beta(3) upon platelet activation has been demonstrated to play a critical role in this process. Recently, the adaptor protein ShcA has been shown to bind to the tyrosine-phosphorylated beta(3), while it remains open whether ShcA plays any role in platelet aggregation. Here, we show that ShcA bound to tyrosine-phosphorylated beta(3)-tail peptide through its phosphotyrosine-binding domain in vitro. Then, we examined the involvement of ShcA in platelet aggregation by a previously established in vitro assay using platelets permeabilized with streptolysin-O, where exogenous addition of platelet cytosol is required for reconstitution of the Ca(2+)-induced aggregation. When ShcA was specifically depleted with anti-ShcA antibody from the cytosol, this ShcA-depleted cytosol lost the aggregation-supporting activity, which was rescued by addition of purified recombinant ShcA. Thus, ShcA is essential for the Ca(2+)-induced platelet aggregation.     

Calcium integrin-binding protein activates platelet integrin alpha IIbbeta 3.

alpha(IIb)beta(3), a platelet-specific integrin, plays a critical role in platelet aggregation. The affinity of alpha(IIb)beta(3) for its ligands such as fibrinogen and von Willebrand factor is tightly regulated in an uncharacterized intracellular process termed inside-out signaling. Calcium integrin-binding protein (CIB) has been identified as a protein interacting with the cytoplasmic tail of the alpha(IIb) subunit of alpha(IIb)beta(3), but its physiological role has not been defined. In the present study, I demonstrate that CIB activates alpha(IIb)beta(3) both in vitro and in vivo. CIB interacts directly with the alpha(IIb) cytoplasmic tail, thereby increasing the affinity of alpha(IIb)beta(3) for fibrinogen in an in vitro fibrinogen-binding assay. The interaction of CIB with the alpha(IIb) cytoplasmic tail is enhanced in a Ca(2+)-dependent manner. A physiological agonist, ADP, stimulates platelets, activating alpha(IIb)beta(3). When the interaction of CIB with the alpha(IIb) cytoplasmic tail is blocked in native platelets by a permeable competing peptide, alpha(IIb)beta(3) activation is not detected even in the presence of ADP. This result indicates that direct interaction of CIB with the alpha(IIb) cytoplasmic tail converts alpha(IIb)beta(3) from a resting to an active conformation. This suggests that CIB plays an important role in one of the pathways that modulate the affinity of alpha(IIb)beta(3) for its ligand.     

Distinct roles for Rap1b protein in platelet secretion and integrin αIIbβ3 outside-in signaling

Rap1b is activated by platelet agonists and plays a critical role in integrin α(IIb)β(3) inside-out signaling and platelet aggregation. Here we show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that α(IIb)β(3) outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b-deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b-deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y(12) or TXA(2) receptor deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b-deficient platelets compared with wild-type controls. The defects in clot retraction and spreading on fibrinogen of Rap1b-deficient platelets were not rescued by addition of MnCl(2), which elicits α(IIb)β(3) outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin α(IIb)β(3) outside-in signaling.     

Thrombin-stimulated phosphatidylinositol 3-kinase activity in platelets is associated with activation of PYK2 tyrosine kinase: activation of both enzymes is aggregation independent

In this study, we investigated the activation of a new member of the focal adhesion kinase family of tyrosine kinases, the proline-rich tyrosine kinase, or PYK2, in platelets. We show that PYK2 is tyrosine phosphorylated and its activity is increased during early stages of platelet aggregation. This activation coincided with increased association of phosphatidylinositol (PI) 3-kinase and PYK2, as determined by both anti-PI 3-kinase and anti-PYK2 immunoprecipitates. However, under basal conditions, some association of PYK2 and PI 3-kinase was consistently observed, even though little or no tyrosine phosphorylated PYK2 could be detected. In addition, both increased PI 3-kinase activity and increased PYK2 activity could be detected in immunoprecipitates following thrombin stimulation. All of these events were unaffected by blocking platelet aggregation with arginine-glycine-aspartate-serine (RGDS) peptide, which interferes with binding of the platelet integrin alpha(IIb)beta(3) to fibrinogen. Neither was the activation of the PYK2 kinase activity affected by blocking PI 3-kinase activity. These results support a model in which PYK2 is associated with PI 3-kinase in unstimulated platelets and following activation of platelets, there is an increase in tyrosine phosphorylation of PYK2, increased PYK2 activity, and increased association of PYK2 with PI 3-kinase, which may contribute to the increase in PI 3-kinase activity. All of these were found to be early events independent of subsequent platelet aggregation.