Ser-752-->Pro mutation in the cytoplasmic domain of integrin beta 3 subunit and defective activation of platelet integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) in a variant of Glanzmann thrombasthenia.

Integrins are membrane receptors which mediate cell-cell or cell-matrix adhesion. Integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) acts as a fibrinogen receptor of platelets and mediates platelet aggregation. Platelet activation is required for alpha IIb beta 3 to shift from noncompetent to competent for binding soluble fibrinogen. The steps involved in this transition are poorly understood. We have studied a variant of Glanzmann thrombasthenia, a congenital bleeding disorder characterized by absence of platelet aggregation and fibrinogen binding. The patient's platelets did not bind fibrinogen after platelet activation by ADP or thrombin, though his platelets contained alpha IIb beta 3. However, isolated alpha IIb beta 3 was able to bind to an Arg-Gly-Asp-Ser affinity column, and binding of soluble fibrinogen to the patient's platelets could be triggered by modulators of alpha IIb beta 3 conformation such as the Arg-Gly-Asp-Ser peptide and alpha-chymotrypsin. These data suggested that a functional Arg-Gly-Asp binding site was present within alpha IIb beta 3 and that the patient's defect was not secondary to a blockade of alpha IIb beta 3 in a noncompetent conformational state. This was evocative of a defect in the coupling between platelet activation and alpha IIb beta 3 up-regulation. We therefore sequenced the cytoplasmic domain of beta 3, following polymerase chain reaction (PCR) on platelet RNA, and found a T-->C mutation at nucleotide 2259, corresponding to a Ser-752-->Pro substitution. This mutation is likely to be responsible for the uncoupling of alpha IIb beta 3 from cellular activation because (i) it is not a polymorphism, (ii) it is the only mutation in the entire alpha IIb beta 3 sequence, and (iii) genetic analysis of the family showed that absence of the Pro-752 beta 3 allele was associated with the normal phenotype. Our data thus identify the C-terminal portion of the cytoplasmic domain of beta 3 as an intrinsic element in the coupling between alpha IIb beta 3 and platelet activation.     

Platelet glycoprotein IIb-IIIa (alpha IIb beta 3 integrin) confers fibrinogen- and activation-dependent aggregation on heterologous cells

To analyze molecular mechanisms of platelet aggregation, we have studied the aggregation of Chinese hamster ovary (CHO) cells expressing between 1 and 4 x 10(5) recombinant human glycoprotein (GP) IIb-IIIa molecules per cell (A5 cells). These cells aggregated as measured by the disappearance of single cells during rotary agitation. Aggregation was dependent on the presence of extracellular fibrinogen (approximately 500 nmol/L) and divalent cations, and required prior activation of the GPIIb-IIIa. A synthetic peptide (GRGDSP) and monoclonal anti-GPIIb-IIIa antibody (2G12) that block platelet aggregation also blocked aggregation of these cells. Parent CHO cells or those expressing recombinant GPIIb-IIIa containing a point mutation that causes variant thrombasthenia both failed to aggregate when stimulated in the presence of fibrinogen. These data show that GPIIb-IIIa is the only unique platelet surface component required for aggregation.     

Ligand density dramatically affects integrin alpha IIb beta 3-mediated platelet signaling and spreading

The impact of ligand density on integrin-mediated cell adhesion and outside-in signaling is not well understood. Using total internal reflection fluorescent microscopy, conformation-specific antibodies, and Ca(2+) flux measurements, we found that the surface density of fibrinogen affects alpha II b beta 3-mediated platelet signaling, adhesion, and spreading. Adhesion to fibrinogen immobilized at low density leads to rapid increases in cytosolic Ca(2+) and sequential formation of filopodia and lamellipodia. In contrast, adhesion to high-density fibrinogen results in transient or no increases in Ca(2+) and simultaneous formation of filopodia and lamellipodia. alpha II b beta 3 receptors at the basal surface of platelets engage fibrinogen in a ringlike pattern at the cell edges under both conditions. This engagement is, however, more dynamic and easily reversed on high-density fibrinogen. Src and Rac activity and actin polymerization are important for adhesion to low-density fibrinogen, whereas PKC/PI3 kinases contribute to platelet spreading on high-density fibrinogen. We conclude that 2 fundamentally different signaling mechanisms can be initiated by a single integrin receptor interacting with the same ligand when it is immobilized at different densities.     

Essential role for phosphoinositide 3-kinase in shear-dependent signaling between platelet glycoprotein Ib/V/IX and integrin alpha(IIb)beta(3).

Platelet adhesion and aggregation at sites of vascular injury are critically dependent on the interaction between von Willebrand factor (VWF) and 2 major platelet adhesion receptors, glycoprotein (GP) Ib/V/IX and integrin alpha(IIb)beta(3). GP Ib/V/IX binding to VWF mediates platelet tethering and translocation, whereas activation of integrin alpha(IIb)beta(3) promotes cell arrest. To date, the signaling pathways used by the VWF-GP Ib/V/IX interaction to promote activation of integrin alpha(IIb)beta(3), particularly under shear, have remained poorly defined. In this study, the potential involvement of type 1 phosphoinositide (PI) 3-kinases in this process was investigated. Results show that platelet adhesion and spreading on immobilized VWF results in a specific increase in the PI 3-kinase lipid product, PtdIns(3,4)P(2). Under static conditions, inhibiting PI 3-kinase with LY294002 or wortmannin did not prevent platelet adhesion, integrin alpha(IIb)beta(3) activation, or platelet spreading although it significantly delayed the onset of these events. In contrast, PI 3-kinase inhibition under shear dramatically reduced both platelet adhesion and spreading. Real-time analysis of intracellular calcium demonstrated that under static conditions inhibiting PI 3-kinase delayed the onset of intracellular fluxes in adherent platelets, but did not affect the final magnitude of the calcium response. However, under shear, inhibiting PI 3-kinase dramatically reduced intracellular calcium mobilization and integrin alpha(IIb)beta(3) activation, resulting in impaired thrombus growth. The studies demonstrate a shear-dependent role for PI 3-kinase in promoting platelet adhesion on immobilized VWF. Under static conditions, platelets appear to mobilize intracellular calcium through both PI 3-kinase-dependent and -independent mechanisms, whereas under shear PI 3-kinase is indispensable for VWF-induced calcium release.     

Inhibition of platelet integrin alpha(IIb)beta(3) by peptides that interfere with protein kinases and the beta(3) tail

alpha-Thrombin stimulation of human platelets initiates inside-out signaling to integrin alpha(IIb)beta(3) (glycoprotein IIb/IIIa), resulting in the exposure of ligand binding sites. In the present study, the regulation of alpha(IIb)beta(3) via protein kinases was investigated in platelets permeabilized with streptolysin O by introducing peptides that interfere with these enzymes and with possible regulatory domains in the cytosolic tail of the beta(3) subunit. Compared with intact platelets, the permeabilized platelets preserved >80% of the aggregation, secretion, and alpha(IIb)beta(3) ligand binding capacity. The peptide YIYGSFK, a substrate for Src kinases, inhibited alpha-thrombin-induced ligand binding to alpha(IIb)beta(3), but a reversed peptide with Y-->F substitutions (KFSGFIF) had no effect. Ligand binding to alpha(IIb)beta(3) was also inhibited by the peptide RKRCLRRL, which binds irreversibly to the catalytic domain of protein kinase C. Peptides corresponding to parts of the protein C inhibitor and beta(2)-glycoprotein I were used as negative controls and failed to interfere with ligand binding. Possible target domains for protein kinases are present in the cytoplasmic tail of the beta(3) subunit. The LLITIHDR peptide, matching the membrane-proximal domain of beta(3) (residues 717 to 724), had no effect, but NNPLYKEA (residues 743 to 750), EATSTFTN (residues 749 to 756), and TNITYRGT (residues 755 to 762), which mimicked overlapping domains of the carboxy-terminal part of beta(3), reduced alpha-thrombin-induced ligand binding by 60+/-4%, 97+/-1%, and 97+/-2% (n=3) at 500 micromol/L peptide, respectively. These observations indicate that Src kinases and protein kinase C take part in inside-out signaling to integrin alpha(IIb)beta(3) and identify target domains in beta(3) that contribute to the regulation of this integrin.     

A Ser752-->Pro substitution in the cytoplasmic domain of beta3 in a Glanzmann thrombasthenia variant fails to prevent interactions between the alphaIIbbeta3 integrin and the platelet granule pool of fibrinogen

A Glanzmann thrombasthenia variant with a beta3 Ser752-->Pro cytoplasmic domain substitution has platelets that fail to aggregate or bind soluble fibrinogen (Fg) after activation. Despite this, Fg is normally present in the alpha-granules. We have used immunoelectron microscopy to examine the reactivity of Fg with the different pools of alphaIIbbeta3 in the patient's platelets. Immunogold labelling was performed on cryosections using an anti-ligand-induced binding site (LIBS) monoclonal antibody (mAb), which binds to alphaIIbbeta3 only when Fg is bound, or a mixture of two anti-receptor-induced binding site (RIBS) mAbs that specifically recognize receptor-bound Fg. Labelling of the alpha-granule membrane and channels of the surface-connected canalicular system in unstimulated platelets confirmed that the mutated alphaIIbbeta3 retains the capacity to transport Fg. When the patient's platelets were stimulated with ADP in the presence of Fg, as expected there was a much-decreased activation of surface-exposed alphaIIbbeta3. However, thrombin-induced activation was associated with both secretion and a rapid increase in the labelling of internal membrane systems by anti-RIBS and anti-LIBS mAbs, with mobilization of the internal Fg pool. Yet labelling on the surface of the patient's platelets was transient. Our studies implied that alphaIIbbeta3 in platelets may bind fibrinogen in different activation states and that this patient specifically lacked high-affinity binding.     

Hematopoietic reconstitution of SLP-76 corrects hemostasis and platelet signaling through alpha IIb beta 3 and collagen receptors

Mice deficient in the hematopoietic cell-specific adapter protein SLP-76 demonstrate a failure of T cell development and fetal hemorrhage. Although SLP-76-deficient platelets manifest defective collagen receptor signaling, this alone may not explain the observed bleeding diathesis. Because alpha IIb beta 3, the platelet fibrinogen receptor, is required for normal hemostasis, we explored a potential role for SLP-76 in alpha IIb beta 3 signaling. Interaction of soluble or immobilized fibrinogen with normal human or murine platelets triggers rapid tyrosine phosphorylation of SLP-76. Moreover, platelet adhesion to fibrinogen stimulates actin rearrangements, filopodial and lamellipodial extension, and localization of tyrosine phosphorylated proteins to the cell periphery. In contrast, SLP-76-deficient murine platelets bind fibrinogen normally, but spread poorly and exhibit reduced levels of phosphotyrosine. The in vivo bleeding diathesis as well as the defects in platelet responses to fibrinogen and collagen are reversed by retroviral transduction of SLP-76 into bone marrow derived from SLP-76-deficient mice. These studies establish that SLP-76 functions downstream of alpha IIb beta 3 and collagen receptors in platelets. Furthermore, expression of SLP-76 in hematopoietic cells, including platelets, plays a necessary role in hemostasis.     

Adenosine diphosphate (ADP)-induced thromboxane A(2) generation in human platelets requires coordinated signaling through integrin alpha(IIb)beta(3) and ADP receptors.

Adenosine diphosphate (ADP) is a platelet agonist that causes platelet shape change and aggregation as well as generation of thromboxane A(2), another platelet agonist, through its effects on P2Y1, P2Y12, and P2X1 receptors. It is now reported that both 2-propylthio-D-beta gamma-dichloromethylene adenosine 5'-triphosphate (AR-C67085), a P2Y12 receptor-selective antagonist, and adenosine-2'-phosphate-5'-phosphate (A2P5P), a P2Y1 receptor-selective antagonist, inhibited ADP-induced thromboxane A(2) generation in a concentration-dependent manner, indicating that coactivation of the P2Y12 and P2Y1 receptors is essential for this event. SC49992, a fibrinogen receptor antagonist, blocked ADP-induced platelet aggregation and thromboxane A(2) production in a concentration-dependent manner. Similarly, P2 receptor antagonists or SC49992 blocked ADP-induced arachidonic acid liberation. Whereas SC49992 blocked arachidonic acid-induced platelet aggregation, it failed to inhibit thromboxane A(2) generation induced by arachidonic acid. Thus, ADP-induced arachidonic acid liberation, but not subsequent conversion to thromboxane A(2), requires outside-in signaling through the fibrinogen receptor. The Fab fragment of ligand-induced binding site-6 (LIBS6) antibody, which induces a fibrinogen-binding site on the integrin alpha(IIb)beta(3), caused both platelet aggregation and thromboxane A(2) generation. Inhibitors of phosphoinositide 3-kinase, Syk, Src kinases, or protein tyrosine phosphatases inhibited platelet aggregation but not thromboxane A(2) generation, indicating that these signaling molecules have no significant role in phospholipase A(2) activation. In the presence of P2 receptor antagonists A2P5P or AR-C67085, LIBS6 failed to generate thromboxane A(2), suggesting that inside-out signaling through ADP receptors is necessary for this event. It was concluded that both outside-in signaling from the fibrinogen receptor and inside-out signaling from the P2Y1 and P2Y12 receptors are necessary for phospholipase A(2) activation, resulting in arachidonic acid liberation and thromboxane A(2) generation.     

Solution structures of the cytoplasmic tail complex from platelet integrin alpha IIb- and beta 3-subunits

Integrin adhesion receptors constitute a cell-signaling system whereby interactions in the small cytoplasmic domains of the heterodimeric alpha- and beta-subunits provoke major functional alterations in the large extracellular domains. With two-dimensional NMR spectroscopy, we examined two synthetic peptides [alphaIIb((987)MWKVGFFKRNR) and beta3((716)KLLITIHDRKEFAKFEEERARAKWD)] encompassing the membrane-proximal regions of the cytoplasmic domain motifs from the platelet integrin complex alphaIotaIotabbeta3. These membrane-proximal regions contain two conserved motifs, represented by (989)KVGFFKR in the alphaIIb-subunit, and (716)KLLITIHDR in the beta3-subunit. The dimer interaction consists of two adjacent helices with residues V990 and F993 of the alphaIotaIotab-subunit heavily implicated in the dimer interfacial region, as is I719 of beta3. These residues are situated within the conserved motifs of their respective proteins. Further structural analysis of this unique peptide heterodimer suggests that two distinct conformers are present. The major structural difference between the two conformers is a bend in the beta3-peptide between D723 and A728, whereas the helical character in the other regions remains intact. Earlier mutational analysis has shown that a salt bridge between the side chains of alphaIotaIotab(R955) and beta3(D723) is formed. When this ion pair was modeled into both conformers, increased nuclear Overhauser effect violations suggested that the more bent structure was less able to accommodate this interaction. These results provide a molecular level rationalization for previously reported biochemical studies, as well as a basis for an atomic level understanding of the intermolecular interactions that regulate integrin activity.     

A Leu262Pro mutation in the integrin beta(3) subunit results in an alpha(IIb)-beta(3) complex that binds fibrin but not fibrinogen

Platelet retraction of a fibrin clot is mediated by the platelet fibrinogen receptor, alpha(IIb)beta(3). In certain forms of the inherited platelet disorder, Glanzmann thrombasthenia (GT), mutant alpha(IIb)beta(3) may interact normally with fibrin yet fail to support fibrinogen-dependent aggregation. We describe a patient (LD) with such a form of GT. Platelets from LD supported normal clot retraction but failed to bind fibrinogen. Platelet analysis using flow cytometry and immunoblotting showed reduced but clearly detectable alpha(IIb)beta(3), findings consistent with type II GT. Genotyping of LD revealed 2 novel beta(3) mutations: a deletion of nucleotides 867 to 868, resulting in a premature stop codon at amino acid residue 267, and a T883C missense mutation, resulting in a leucine (Leu) 262-to-proline (Pro) substitution. Leu262 is highly conserved among beta integrin subunits and lies within an intrachain loop implicated in subunit association. Leu262Probeta(3) cotransfected with wild-type alpha(IIb) into COS-7 cells showed delayed intracellular maturation and reduced surface expression of easily dissociable complexes. In human embryonic kidney 293 cells, Leu262Probeta(3) formed a complex with endogenous a(v) and retracted fibrin clots similarly to wild-type beta(3). The same cells, however, were unable to bind immobilized fibrinogen. The molecular requirements for alpha(IIb)beta(3) to interact with fibrin compared with fibrinogen, therefore, appear to differ. The region surrounding beta(3) Leu262 may maintain beta(3) in a fibrinogen-binding, competent form, but it appears not to be required for receptor interactions with fibrin.     

Endocytosis of fibrinogen into megakaryocyte and platelet alpha- granules is mediated by alpha IIb beta 3 (glycoprotein IIb-IIIa) [published erratum appears in Blood 1993 Nov 1;82(9):2936]

Recently, we showed that platelet alpha-granule fibrinogen is derived entirely from endocytic uptake and not from megakaryocyte synthesis, as previously thought. In this present report, we identify the receptor that mediates endocytosis. We have found that barbourin, a unique disintegrin that is a specific antagonist of alpha IIb beta 3, inhibits the endocytic uptake of fibrinogen into alpha-granules. Continuous intravenous infusion of barbourin (200 micrograms/h) into guinea pigs blocked collagen-induced platelet aggregation as well as endocytosis of biotinylated fibrinogen into megakaryocytes; however, endocytosis of biotinylated albumin by megakaryocytes was not affected. Thus, we have shown that endocytosis of fibrinogen into megakaryocyte and platelet alpha-granules is receptor-mediated, and that alpha IIb beta 3 is the primary receptor.     

Fibrinogen binding to the integrin alpha(IIb)beta(3) modulates store-mediated calcium entry in human platelets

Effects of the occupation of integrin alpha(IIb)beta(3) by fibrinogen on Ca(++) signaling in fura-2-loaded human platelets were investigated. Adding fibrinogen to washed platelet suspensions inhibited increases in cytosolic [Ca(++)] concentrations ([Ca(++)](i)) evoked by adenosine diphosphate (ADP) and thrombin in a concentration-dependent manner in the presence of external Ca(++) but not in the absence of external Ca(++) or in the presence of the nonselective cation channel blocker SKF96365, indicating selective inhibition of Ca(++) entry. Fibrinogen also inhibited store-mediated Ca(++) entry (SMCE) activated after Ca(++) store depletion using thapsigargin. The inhibitory effect of fibrinogen was reversed if fibrinogen binding to alpha(IIb)beta(3) was blocked using RDGS or abciximab and was absent in platelets from patients homozygous for Glanzmann thrombasthenia. Fibrinogen was without effect on SMCE once activated. Activation of SMCE in platelets occurs through conformational coupling between the intracellular stores and the plasma membrane and requires remodeling of the actin cytoskeleton. Fibrinogen inhibited actin polymerization evoked by ADP or thapsigargin in control cells and in cells loaded with the Ca(++) chelator dimethyl BAPTA. It also inhibited the translocation of the tyrosine kinase p60(src) to the cytoskeleton. These results indicate that the binding of fibrinogen to integrin alpha(IIb)beta(3) inhibits the activation of SMCE in platelets by a mechanism that may involve modulation of the reorganization of the actin cytoskeleton and the cytoskeletal association of p60(src). This action may be important in intrinsic negative feedback to prevent the further activation of platelets subjected.     

Platelet HDL(3) binding sites are not related to integrin alpha(IIb)beta(3) (GPIIb-IIIa)

Early studies considered that fibrinogen receptor (glycoprotein [GP] IIb-IIIa or platelet integrin alpha(IIb)beta(3)) is the binding site for low-density lipoprotein (LDL) and high-density lipoprotein type 3 (HDL(3)). Recent data, however, do not support the hypothesis that the binding of LDL to human intact resting platelets is related to integrin alpha(IIb)beta(3). In this study we present evidence that platelet integrin alpha(IIb)beta(3) is also not involved in the interaction of HDL(3) and human intact resting platelets. Firstly, specific ligands for platelet integrin alpha(IIb)beta(3), such as fibrinogen, vitronectin, von Willebrand factor and fibronectin, were unable to inhibit the binding of HDL(3) to intact resting platelets. Secondly, the HDL(3) binding characteristics (K(d) and B(max) values), the activation of protein kinase C (PKC) and the inhibition of thrombin-induced inositoltriphosphate (IP(3)) formation and calcium (Ca(2+)) mobilization mediated by HDL(3) particles were similar in platelets from control subjects and patients with type I and type II Glanzmann's thrombasthenia, which are characterized by total and partial lack of GPIIb-IIIa and fibrinogen, respectively. In contrast, nitrosylation of tyrosine residues of HDL(3) by tetranitromethane fully abolished both the ability of particles to interact with its specific binding sites and the functional effects. Thirdly, polyclonal antibodies against the GPIIb-IIIa complex (edu-3 and 5B12), human antiserums against platelet alloantigens (anti-Bak(a/B) and anti-PL(A1/2)), anti-integrin subunits (anti-alpha(V) and anti-beta(3)), and a wide panel of monoclonal antibodies (mAbs) against well-known epitopes of GPIIb (M3, M4, M5, M6, M8 and M95-2b) and GPIIIa (P23-7, P33, P37, P40, and P97) did not affect the binding of HDL(3) particles to human intact resting platelets. Overall results show that neither the GPIIb-IIIa complex nor GPIIb or GPIIIa individually are the membrane binding proteins for HDL(3)on intact resting platelets.     

The Ig-ITIM superfamily member PECAM-1 regulates the "outside-in" signaling properties of integrin alpha(IIb)beta3 in platelets

Previous studies have implicated the immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM) superfamily member platelet endothelial cell adhesion molecule-1 (PECAM-1) in the regulation of integrin function. While PECAM-1 has been demonstrated to play a role as an inhibitory coreceptor of immunoreceptor tyrosine-based activation motif (ITAM)-associated Fcgamma receptor IIa (FcgammaRIIa) and glycoprotein VI (GPVI)/FcR gamma-chain signaling pathways in platelets, its physiologic role in integrin alpha(IIb)beta3-mediated platelet function is unclear. In this study, we investigate the functional importance of PECAM-1 in murine platelets. Using PECAM-1-deficient mice, we show that the platelets have impaired "outside-in" integrin alpha(IIb)beta3 signaling with impaired platelet spreading on fibrinogen, failure to retract fibrin clots in vitro, and reduced tyrosine phosphorylation of focal adhesion kinase p125 (125FAK) following integrin alpha(IIb)beta3-mediated platelet aggregation. This functional integrin alpha(IIb)beta3 defect could not be attributed to altered expression of integrin alpha(IIb)beta3. PECAM-1-/- platelets displayed normal platelet alpha granule secretion, normal platelet aggregation to protease-activated receptor-4 (PAR-4), adenosine diphosphate (ADP), and calcium ionophore, and static platelet adhesion. In addition, PECAM-1-/- platelets displayed normal "inside-out" integrin alpha(IIb)beta3 signaling properties as demonstrated by normal agonist-induced binding of soluble fluoroscein isothiocyanate (FITC)-fibrinogen, JON/A antibody binding, and increases in cytosolic-free calcium and inositol (1,4,5)P3 triphosphate (IP3) levels. This study provides direct evidence that PECAM-1 is essential for normal integrin alpha(IIb)beta3-mediated platelet function and that disruption of PECAM-1 induced a moderate "outsidein" integrin alpha(IIb)beta3 signaling defect.     

RGT, a synthetic peptide corresponding to the integrin beta 3 cytoplasmic C-terminal sequence, selectively inhibits outside-in signaling in human platelets by disrupting the interaction of integrin alpha IIb beta 3 with Src kinase

Mutational analysis has established that the cytoplasmic tail of the integrin β3 subunit binds c-Src (termed as Src in this study) and is critical for bidirectional integrin signaling. Here we show in washed human platelets that a cell-permeable, myristoylated RGT peptide (myr-RGT) corresponding to the integrin β3 C-terminal sequence dose-dependently inhibited stable platelet adhesion and spreading on immobilized fibrinogen, and fibrin clot retraction as well. Myr-RGT also inhibited the aggregation-dependent platelet secretion and secretion-dependent second wave of platelet aggregation induced by adenosine diphosphate, ristocetin, or thrombin. Thus, myr-RGT inhibited integrin outside-in signaling. In contrast, myr-RGT had no inhibitory effect on adenosine diphosphate-induced soluble fibrinogen binding to platelets that is dependent on integrin inside-out signaling. Furthermore, the RGT peptide induced dissociation of Src from integrin β3 and dose-dependently inhibited the purified recombinant β3 cytoplasmic domain binding to Src-SH3. In addition, phosphorylation of the β3 cytoplasmic tyrosines, Y⁷⁴⁷ and Y⁷⁵⁹, was inhibited by myr-RGT. These data indicate an important role for β3-Src interaction in outside-in signaling. Thus, in intact human platelets, disruption of the association of Src with β3 and selective blockade of integrin IIbβ3 outside-in signaling by myr-RGT suggest a potential new antithrombotic strategy.     

Platelet-erythrocyte interactions enhance alpha(IIb)beta(3) integrin receptor activation and P-selectin expression during platelet recruitment: down-regulation by aspirin ex vivo

Activated platelets release biologically active compounds, which then recruit additional platelets into an evolving thrombus. We studied activation of alpha(IIb)beta(3) and exposure of P-selectin on platelets recruited by releasates obtained from collagen-treated platelets and evaluated modifications in prothrombotic effects of releasates induced by platelet-erythrocyte interactions and aspirin treatment. Releasates from collagen-stimulated platelets induced alpha(IIb)beta(3) activation and P-selectin exposure (monitored by flow cytometry using fluorescein isothiocyanate-PAC-1 and phycoerythrin-CD62 antibodies). These responses were markedly amplified by releasates from combined platelet-erythrocyte suspensions. This finding demonstrates a novel mechanism(s) by which erythrocytes intensify platelet aggregability and mediate increased platelet recruitment. Because P-selectin and alpha(IIb)beta(3) are potential sites for platelet-leukocyte interactions, erythrocytes may also modulate leukocyte recruitment. Following aspirin ingestion both the recruiting capacity of platelet releasates and erythrocyte-induced amplification of platelet recruitment were down-regulated. These events represent an additional antithrombotic property of aspirin. We also examined the possibility that arachidonic acid, or eicosanoids derived therefrom, can induce a prothrombotic activity of erythrocytes. The TXA(2)-analog U46 619 and free arachidonate, but not PGI(2) or 12-HETE, induced increases in cytosolic Ca(++) and promoted phosphatidylserine (PS) exposure on a subpopulation of erythrocytes. PS exposure and increases in erythrocyte [Ca(++)](i) are associated with enhanced procoagulant activity, increased endothelial adhesion, and reduced erythrocyte deformability. Our findings, therefore, suggest that TXA(2) and arachidonic acid, derived from activated platelets, induce a prothrombotic phenotype on erythrocytes in proximity. We conclude that by these mechanisms, erythrocytes can actively contribute to platelet-driven thrombogenesis and microvascular occlusion.     

The role of platelet collagen receptor (glycoprotein Ia/IIa; integrin alpha2 beta1) polymorphisms in thrombotic disease

Differences in rates of platelet activation induced by extracellular matrix components such as collagens markedly influence normal hemostasis and the pathologic outcome of thrombosis. Thus, platelet collagen receptors, the integrin alpha2beta1, glycoprotein VI, and the glycoprotein Ib complex, represent unexploited targets of pharmacologic control. Polymorphisms of these receptors are now understood as factors that potentially contribute to thrombotic risk. There is substantial evidence that the GPIbalpha variable number of tandem repeats A or B alleles, the -5C allele of GPIbalpha, and the integrin alpha2 allele 1 (T807) each contribute to risk for and morbidity from thrombotic disease. The extent of their individual contributions is disputed. More well-designed, large, prospective, genetic and epidemiologic studies are needed to clarify the role of these and other platelet receptor polymorphisms, and additional in vitro studies are needed to provide a sound biologic explanation for the outcomes of clinical correlations.     

Sequence 274-368 in the beta 3-subunit of the integrin alpha IIb beta 3 provides a ligand recognition and binding domain for the gamma-chain of fibrinogen that is independent of platelet activation

Several bacterial-expressed recombinant fragments encompassing the extracellular part of the beta 3 subunit of the integrin alpha IIb beta 3 were shown to recognize and bind soluble and immobilized forms of fibrinogen. Two of them, designated as rIII-11 (beta 3 274-368) and rIII-13 (beta 3 274-403), did not contain the established RGD-ligand binding sequence. In fact, they interacted, in a Ca(2+)-independent manner, with the C-terminal part of the fibrinogen gamma chain. Both beta 3 fragments blocked the participation of fibrinogen in the induction of platelet aggregation induced by adenosine diphosphate. Fragment rIII-13 was recognized by the anti-beta 3 monoclonal antibody B2A. This antibody, which possesses an epitope exposed on both resting and activated platelets, inhibited fibrinogen binding as well as platelet adhesion and aggregation. In conclusion, the results demonstrated that the 274-368 sequence of the beta 3 subunit of integrin alpha IIb beta 3 constitutes a fibrinogen ligand binding domain, distinct from the RGD-binding site, that is required for both platelet adhesion and aggregation.     

Missense mutations in the beta(3) subunit have a different impact on the expression and function between alpha(IIb)beta(3) and alpha(v)beta(3).

Alpha(IIb)beta(3) and alpha(v)beta(3) belong to the beta(3) integrin subfamily. Although the beta(3) subunit is a key regulator for the biosynthesis of beta(3) integrins, it remains obscure whether missense mutations in beta(3) may induce the same defects in both alpha(IIb)beta(3) and alpha(v)beta(3). In this study, it is revealed that thrombasthenic platelets with a His280Pro mutation in beta(3), which is prevalent in Japanese patients with Glanzmann thrombasthenia, did contain significant amounts of alpha(v)beta(3) (about 50% of control) using sensitive enzyme-linked immunosorbent assay. Expression studies showed that the His280Probeta(3) mutation impaired alpha(IIb)beta(3) expression but not alpha(v)beta(3) expression in 293 cells. To extend these findings, the effects of several beta(3) missense mutations leading to an impaired alpha(IIb)beta(3) expression on alpha(v)beta(3) function as well as expression was examined: Leu117Trp, Ser162Leu, Arg216Gln, Cys374Tyr, and a newly created Arg216Gln/Leu292Ser mutation. Leu117Trp and Cys374Tyr beta(3) mutations did impair alpha(v)beta(3) expression, while Ser162Leu, Arg216Gln, and Arg216Gln/Leu292Ser mutations did not. With regard to ligand binding function, Ser162Leu mutation induced especially distinct effects between 2 beta(3) integrins: it markedly impaired ligand binding to alpha(IIb)beta(3) but not to alpha(v)beta(3) at all. These data clearly demonstrate that the biosynthesis and the ligand binding function of alpha(IIb)beta(3) and those of alpha(v)beta(3) are regulated in part by different mechanisms. Present data would be a clue to elucidate the regulatory mechanism of expression and function of beta(3) integrins.