The activation state of alphavbeta 3 regulates platelet and lymphocyte adhesion to intact and thrombin-cleaved osteopontin

Cleavage of osteopontin by thrombin has been reported to enhance cell adhesion. We asked whether thrombin could regulate the alpha(v)beta(3)-mediated adhesion of platelets and B lymphocytes to this substrate. Although there was no difference in the extent or the avidity of thrombin- and ADP-stimulated platelet adhesion to intact or thrombin-cleaved human osteopontin, both the extent and avidity of phorbol ester-stimulated B cell adhesion to thrombin-cleaved osteopontin was significantly increased. Thus, these data suggest that the ability of alpha(v)beta(3) to recognize osteopontin can be differentially regulated in a cell-specific manner. To localize the alpha(v)beta(3) binding site on osteopontin, we measured cell adhesion to the two thrombin cleavage products of osteopontin and to a series of nested RGD-containing osteopontin peptides cross-linked to albumin. Whereas ADP-stimulated platelets adhered to the amino-terminal but not the carboxyl-terminal osteopontin fragment and to the osteopontin peptide RGDSVVYGLR, phorbol ester-stimulated B cells did not adhere to this peptide, although they did so in the presence of 1 mm Mn(2+). Thus, our data confirm that thrombin cleavage enhances the accessibility of the binding motif for alpha(v)beta(3) on osteopontin, but this enhancement is also a function of the activation state of alpha(v)beta(3). Moreover, they indicate that the sequence RGDSVVYGLR contains sufficient information to specify activation-dependent alpha(v)beta(3)-mediated platelet and lymphocyte adhesion.     

Activation-dependent adhesion of human platelets to Cyr61 and Fisp12/mouse connective tissue growth factor is mediated through integrin alpha(IIb)beta(3).

Cyr61 and connective tissue growth factor (CTGF), members of a newly identified family of extracellular matrix-associated signaling molecules, are found to mediate cell adhesion, promote cell migration and enhance growth factor-induced cell proliferation in vitro, and induce angiogenesis in vivo. We previously showed that vascular endothelial cell adhesion and migration to Cyr61 and Fisp12 (mouse CTGF) are mediated through integrin alpha(v)beta(3). Both Cyr61 and Fisp12/mCTGF are present in normal blood vessel walls, and it has been demonstrated that CTGF is overexpressed in advanced atherosclerotic lesions. In the present study, we examined whether Cyr61 and Fisp12/mCTGF could serve as substrates for platelet adhesion. Agonist (ADP, thrombin, or U46619)-stimulated but not resting platelets adhered to both Cyr61 and Fisp12/mCTGF, and this process was completely inhibited by prostaglandin I(2), which prevents platelet activation. The specificity of Cyr61- and Fisp12/mCTGF-mediated platelet adhesion was demonstrated by specific inhibition of this process with polyclonal anti-Cyr61 and anti-Fisp12/mCTGF antibodies, respectively. The adhesion of ADP-activated platelets to both proteins was divalent cation-dependent and was blocked by RGDS, HHLGGAKQAGDV, or echistatin, but not by RGES. Furthermore, this process was specifically inhibited by the monoclonal antibody AP-2 (anti-alpha(IIb)beta(3)), but not by LM609 (anti-alpha(v)beta(3)), indicating that the interaction is mediated through integrin alpha(IIb)beta(3). In a solid phase binding assay, activated alpha(IIb)beta(3), purified by RGD affinity chromatography, bound to immobilized Cyr61 and Fisp12/mCTGF in a dose-dependent and RGD-inhibitable manner. In contrast, unactivated alpha(IIb)beta(3) failed to bind to either protein. Collectively, these findings identify Cyr61 and Fisp12/mCTGF as two novel activation-dependent adhesive ligands for the integrin alpha(IIb)beta(3) on human platelets, and implicate a functional role for these proteins in hemostasis and thrombosis.     

The platelet cytoskeleton regulates the affinity of the integrin alpha(IIb)beta(3) for fibrinogen.

Agonist-generated inside-out signals enable the platelet integrin alpha(IIb)beta(3) to bind soluble ligands such as fibrinogen. We found that inhibiting actin polymerization in unstimulated platelets with cytochalasin D or latrunculin A mimics the effects of platelet agonists by inducing fibrinogen binding to alpha(IIb)beta(3). By contrast, stabilizing actin filaments with jasplakinolide prevented cytochalasin D-, latrunculin A-, and ADP-induced fibrinogen binding. Cytochalasin D- and latrunculin A-induced fibrinogen was inhibited by ADP scavengers, suggesting that subthreshold concentrations of ADP provided the stimulus for the actin filament turnover required to see cytochalasin D and latrunculin A effects. Gelsolin, which severs actin filaments, is activated by calcium, whereas the actin disassembly factor cofilin is inhibited by serine phosphorylation. Consistent with a role for these factors in regulating alpha(IIb)beta(3) function, cytochalasin D- and latrunculin A-induced fibrinogen binding was inhibited by the intracellular calcium chelators 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethyl ester and EGTA acetoxymethyl ester and the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A. Our results suggest that the actin cytoskeleton in unstimulated platelets constrains alpha(IIb)beta(3) in a low affinity state. We propose that agonist-stimulated increases in platelet cytosolic calcium initiate actin filament turnover. Increased actin filament turnover then relieves cytoskeletal constraints on alpha(IIb)beta(3), allowing it to assume the high affinity conformation required for soluble ligand binding.     

Integrin alpha IIb beta 3-dependent calcium signals regulate platelet-fibrinogen interactions under flow. Involvement of phospholipase C gamma 2

Platelet adhesion to fibrinogen is important for platelet aggregation and thrombus growth. In this study we have examined the mechanisms regulating platelet adhesion on immobilized fibrinogen under static and shear conditions. We demonstrate that integrin alpha IIb beta 3 engagement of immobilized fibrinogen is sufficient to induce an oscillatory calcium response, necessary for lamellipodial formation and platelet spreading. Released ADP increases the proportion of platelets exhibiting a cytosolic calcium response but is not essential for calcium signaling or lamellipodial extension. Pretreating platelets with the Src kinase inhibitor PP2, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (APB-2), or the phospholipase C (PLC) inhibitor U73122 abolished calcium signaling and platelet spreading, suggesting a major role for Src kinase-regulated PLC isoforms in these processes. Analysis of PLC gamma 2-/- mouse platelets revealed a major role for this isoform in regulating cytosolic calcium flux and platelet spreading on fibrinogen. Under flow conditions, platelets derived from PLC gamma 2-/- mice formed less stable adhesive interactions with fibrinogen, particularly in the presence of ADP antagonists. Our studies define an important role for PLC gamma 2 in integrin alpha IIb beta 3-dependent calcium flux, necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore, they establish an important cooperative signaling role for PLC gamma 2 and ADP in regulating platelet adhesion efficiency on fibrinogen.     

Affinity modulation of the alpha IIb beta 3 integrin (platelet GPIIb-IIIa) is an intrinsic property of the receptor.

To analyze the basis of affinity modulation of integrin function, we studied cloned stable Chinese hamster ovary cell lines expressing recombinant integrins of the beta 3 family (alpha IIb beta 3 and alpha v beta 3). Antigenic and peptide recognition specificities of the recombinant receptors resembled those of the native receptors found in platelets or endothelial cells. The alpha IIb beta 3-expressing cell line (A5) bound RGD peptides and immobilized fibrinogen (Fg) but not soluble fibrinogen or the activation-specific monoclonal anti-alpha IIb beta 3 (PAC1), indicating that it was in the affinity state found on resting platelets. Several platelet agonists failed to alter the affinity state of ("activate") recombinant alpha IIb beta 3. The binding of soluble Fg and PAC1, however, was stimulated in both platelets and A5 cells by addition of IgG papain-digestion products (Fab) fragments of certain beta 3-specific monoclonal antibodies. These antibodies stimulated PAC1 binding to platelets fixed under conditions rendering them unresponsive to other agonists. Addition of these antibodies to detergent-solubilized alpha IIb beta 3 also stimulated specific Fg binding. These data demonstrate that certain anti-beta 3 antibodies activate alpha IIb beta 3 by acting directly on the receptor, possibly by altering its conformation. Furthermore, they indicate that the activation state of alpha IIb beta 3 is a property of the receptor itself rather than of the surrounding cell membrane microenvironment.     

Selective recognition of adhesive sites in surface-bound fibrinogen by glycoprotein IIb-IIIa on nonactivated platelets

We demonstrate that unstimulated platelets attach to immobilized fibrinogen in a selective process mediated by the membrane glycoprotein (GP) complex IIb-IIIa (alpha IIb beta 3). The initial attachment, independent of platelet activation, is followed by spreading and irreversible adhesion even in the presence of activation inhibitors. Using fibrinogen fragments derived from plasmin digestion, we found that unstimulated platelets do not attach to immobilized fragment E, which contains an Arg-Gly-Asp sequence at A alpha 95-97, and adhere to fragments X and D, both containing the gamma 400-411 dodecapeptide adhesion sequence, less efficiently than to intact fibrinogen. Thus, the carboxyl terminus of the A alpha chain, missing in the "early" fragment X used in these studies, appears to be involved in the interaction of fibrinogen with unstimulated platelets. In contrast, activated platelets adhere to immobilized fibrinogen and fragments X, D, and E in a time-dependent and equivalent manner. Although activated platelets adhere to immobilized vitronectin, fibronectin, and von Willebrand factor through GP IIb-IIIa, unstimulated platelets fail to adhere to vitronectin and have only a limited capacity to adhere to fibronectin and von Willebrand factor. These results demonstrate that GP IIb-IIIa on unstimulated platelets displays a recognition specificity for attachment to immobilized adhesive proteins that is distinct from that seen following platelet activation. Thus, unstimulated platelets selectively interact with fibrinogen, and the initial attachment is followed by spreading and irreversible adhesion in the absence of exogenous agonists. This process may be regulated by plasmin cleavage of the fibrinogen A alpha chain and may play an important role during normal hemostasis and during the pathological development of thrombotic vascular occlusions.     

Substrate specificity of alpha(v)beta(3) integrin-mediated cell migration and phosphatidylinositol 3-kinase/AKT pathway activation

The alpha(v)beta(3) integrin has been shown to bind several ligands, including osteopontin and vitronectin. Its role in modulating cell migration and downstream signaling pathways in response to specific extracellular matrix ligands has been investigated in this study. Highly invasive prostate cancer PC3 cells that constitutively express alpha(v)beta(3) adhere and migrate on osteopontin and vitronectin in an alpha(v)beta(3)-dependent manner. However, exogenous expression of alpha(v)beta(3) in noninvasive prostate cancer LNCaP (beta(3)-LNCaP) cells mediates adhesion and migration on vitronectin but not on osteopontin. Activation of alpha(v)beta(3) by epidermal growth factor stimulation is required to mediate adhesion to osteopontin but is not sufficient to support migration on this substrate. We show that alpha(v)beta(3)-mediated cell migration requires activation of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB/AKT) pathway since wortmannin, a PI 3-kinase inhibitor, prevents PC3 cell migration on both osteopontin and vitronectin; furthermore, alpha(v)beta(3) engagement by osteopontin and vitronectin activates the PI 3-kinase/AKT pathway. Migration of beta(3)-LNCaP cells on vitronectin also occurs through activation of the PI 3-kinase pathway; however, AKT phosphorylation is not increased upon engagement by osteopontin. Furthermore, phosphorylation of focal adhesion kinase (FAK), known to support cell migration in beta(3)-LNCaP cells, is detected on both substrates. Thus, in PC3 cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin and osteopontin; in beta(3)-LNCaP cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin, whereas adhesion to osteopontin does not support alpha(v)beta(3)-mediated cell migration and PI 3-kinase/AKT pathway activation. We conclude therefore that alpha(v)beta(3) exists in multiple functional states that can bind either selectively vitronectin or both vitronectin and osteopontin and that can differentially activate cell migration and intracellular signaling pathways in a ligand-specific manner.     

Activation of individual alphaIIbbeta3 integrin molecules by disruption of transmembrane domain interactions in the absence of clustering

We used laser tweezers-based force spectroscopy to measure the binding strength between fibrinogen molecules covalently bound to latex beads and either wild-type alphaIIbbeta3 molecules or alphaIIbbeta3 molecules containing the transmembrane domain mutations beta3 G708N or alphaIIb G972N expressed on Chinese hamster ovary cells. As we demonstrated previously for alphaIIbbeta3 on agonist-stimulated platelets and for purified alphaIIbbeta3 molecules incubated with Mn(2+), two regimes of rupture forces were present when wild-type alphaIIbbeta3 was activated by the monoclonal antibody PT25-2: rupture forces of 20-60 pN with an exponentially decreasing probability of detection and rupture forces in the range of 60-150 pN with a maximum at approximately 70-80 pN. Both rupture force regimes were specific for fibrinogen binding to the activated conformation of alphaIIbbeta3 because they were inhibited by alphaIIbbeta3-specific antagonists. Identical rupture force regimes were present constitutively when cells expressing the alphaIIb and beta3 transmembrane domain mutants were studied, confirming that these mutations induced an active alphaIIbbeta3 conformation. Moreover, there were no significant differences in the yield strength of the low-to-moderate and strong force regimes when alphaIIbbeta3 was activated by PT25-2 or the transmembrane domain mutations, implying that there was no fundamental difference in the way these forms of activated alphaIIbbeta3 interacted with fibrinogen. Thus, the two-step pathway of the interaction of alphaIIbbeta3 with fibrinogen we have identified appears to be a fundamental property of the high-affinity state of alphaIIbbeta3 and is identical regardless of whether this affinity state is achieved by intracellular, extracellular, or membrane-associated events.     

RhoA sustains integrin alpha IIbbeta 3 adhesion contacts under high shear

The small GTPase RhoA modulates the adhesive nature of many cell types; however, despite high levels of expression in platelets, there is currently limited evidence for an important role for this small GTPase in regulating platelet adhesion processes. In this study, we have examined the role of RhoA in regulating the adhesive function of the major platelet integrin, alpha(IIb)beta(3). Our studies demonstrate that activation of RhoA occurs as a general feature of platelet activation in response to soluble agonists (thrombin, ADP, collagen), immobilized matrices (von Willebrand factor (vWf), fibrinogen) and high shear stress. Blocking the ligand binding function of integrin alpha(IIb)beta(3), by pretreating platelets with c7E3 Fab, demonstrated the existence of integrin alpha(IIb)beta(3)-dependent and -independent mechanisms regulating RhoA activation. Inhibition of RhoA (C3 exoenzyme) or its downstream effector Rho kinase had no effect on integrin alpha(IIb)beta(3) activation induced by soluble agonists or adhesive substrates, however, both inhibitors reduced shear-dependent platelet adhesion on immobilized vWf and shear-induced platelet aggregation in suspension. Detailed analysis of the sequential adhesive steps required for stable platelet adhesion on a vWf matrix under shear conditions revealed that RhoA did not regulate platelet tethering to vWf or the initial formation of integrin alpha(IIb)beta(3) adhesion contacts but played a major role in sustaining stable platelet-matrix interactions. These studies define a critical role for RhoA in regulating the stability of integrin alpha(IIb)beta(3) adhesion contacts under conditions of high shear stress.     

Activated platelets form protected zones of adhesion on fibrinogen and fibronectin-coated surfaces

Leukocytes form zones of close apposition when they adhere to ligand- coated surfaces. Because plasma proteins are excluded from these contact zones, we have termed them protected zones of adhesion. To determine whether platelets form similar protected zones of adhesion, gel-filtered platelets stimulated with thrombin or ADP were allowed to adhere to fibrinogen- or fibronectin-coated surfaces. The protein- coated surfaces with platelets attached were stained with either fluorochrome-conjugated goat anti-human fibrinogen or anti-human fibronectin antibodies, or with rhodamine-conjugated polyethylene glycol polymers. Fluorescence microscopy revealed that F(ab')2 anti- fibrinogen (100 kD) did not penetrate into the contact zones between stimulated platelets and the underlying fibrinogen-coated surface, while Fab antifibrinogen (50 kD) and 10 kD polyethylene glycol readily penetrated and stained the substrate beneath the platelets. Thrombin- or ADP-stimulated platelets also formed protected zones of adhesion on fibronectin-coated surfaces. F(ab')2 anti-fibronectin and 10 kD polyethylene glycol were excluded from these adhesion zones, indicating that they are much less permeable than those formed by platelets on fibrinogen-coated surfaces. The permeability properties of protected zones of adhesion formed by stimulated platelets on surfaces coated with both fibrinogen and fibronectin were similar to the zones of adhesion formed on fibronectin alone. mAb 7E3, directed against the alpha IIb beta 3 integrin blocked the formation of protected adhesion zones between thrombin-stimulated platelets and fibrinogen or fibronectin coated surfaces. mAb C13 is directed against the alpha 5 beta 1 integrin on platelets. Stimulated platelets treated with this mAb formed protected zones of adhesion on surfaces coated with fibronectin. These protected zones were impermeable to F(ab')2 antifibronectin but were permeable to 10 kD polyethylene glycol. These results show that activated platelets form protected zones of adhesion and that the size of molecules excluded from these zones depends upon the composition of the matrix proteins to which the platelets adhere. They also show that formation of protected zones of adhesion by platelets requires alpha IIb beta 3 integrins while the permeability properties of these zones of adhesion are regulated by both alpha IIb beta 3 and alpha 5 beta 1 integrins.     

Immobilised echistatin promotes platelet adhesion and protein tyrosine phosphorylation

Echistatin, a 5000-Da disintegrin, is a strong competitive inhibitor of platelet alpha(IIb)beta(3) binding to fibrinogen. In addition to its antiplatelet activity, echistatin also exhibits activating properties by inducing a switch of alpha(IIb)beta(3) conformation towards an active state. However, soluble echistatin, which is a monomeric ligand, provides only receptor affinity modulation, but it is unable to activate integrin-dependent intracellular signals. Since proteins may exhibit a multivalent functionality as a result of their absorption to a substrate, in this study we evaluated whether immobilised echistatin is able to stimulate platelet adhesion and signalling. The immobilisation process led to an increase of echistatin affinity for integrin(s) expressed on resting platelets. Unlike the soluble form, immobilised echistatin bound at comparable extent either unstimulated or ADP-activated platelets. Furthermore, echistatin presented in this manner was effective in stimulating integrin-dependent protein tyrosine phosphorylation. Platelets adhering to immobilised echistatin showed a pattern of total tyrosine phosphorylated proteins resembling that of fibrinogen-attached platelets. In particular, solid-phase echistatin induced a strong phosphorylation of tyrosine kinases pp72(syk) and pp125(FAK). Inhibitors of platelet signalling, such as apyrase, prostaglandin E(1), cytochalasin D and bisindolylmaleimide, while not affecting platelet adhesion to immobilised echistatin, abolished pp125(FAK) phosphorylation. This suggests that signals activating protein kinase C function, dense granule secretion and cytoskeleton assembly might be involved in echistatin-induced pp125(FAK) phosphorylation.     

Relationships between Rap1b, affinity modulation of integrin alpha IIbbeta 3, and the actin cytoskeleton

The affinity of integrin alpha(IIb)beta(3) for fibrinogen is controlled by inside-out signals that are triggered by agonists like thrombin. Agonist treatment of platelets also activates Rap1b, a small GTPase known to promote integrin-dependent adhesion of other cells. Therefore, we investigated the role of Rap1b in alpha(IIb)beta(3) function by viral transduction of GFP-Rap1 chimeras into murine megakaryocytes, which exhibit inside-out signaling similar to platelets. Expression of constitutively active GFP-Rap1b (V12) had no effect on unstimulated megakaryocytes, but it greatly augmented fibrinogen binding to alpha(IIb)beta(3) induced by a PAR4 thrombin receptor agonist (p < 0.01). The Rap1b effect was cell-autonomous and was prevented by pre-treating cells with cytochalasin D or latrunculin A to inhibit actin polymerization. Rap1b-dependent fibrinogen binding to megakaryocytes was blocked by POW-2, a novel monovalent antibody Fab fragment specific for high affinity murine alpha(IIb)beta(3). In contrast to GFP-Rap1b (V12), expression of GFP-Rap1GAP, which deactivates endogenous Rap1, inhibited agonist-induced fibrinogen binding (p < 0.01), as did dominant-negative GFP-Rap1b (N17) (p < 0.05). None of these treatments affected surface expression of alpha(IIb)beta(3). These studies establish that Rap1b can augment agonist-induced ligand binding to alpha(IIb)beta(3) through effects on integrin affinity, possibly by modulating alpha(IIb)beta(3) interactions with the actin cytoskeleton.     

Savignygrin, a platelet aggregation inhibitor from the soft tick Ornithodoros savignyi, presents the RGD integrin recognition motif on the Kunitz-BPTI fold

Savignygrin, a platelet aggregation inhibitor that possesses the RGD integrin recognition motif, has been purified from the soft tick Ornithodoros savignyi. Two isoforms with similar biological activities differ because of R52G and N60G in their amino acid sequences, indicating a recent gene duplication event. Platelet aggregation induced by ADP (IC50, 130 nm), collagen, the thrombin receptor-activating peptide, and epinephrine was inhibited, although platelets were activated and underwent a shape change. The binding of alpha-CD41 (P2) to platelets, the binding of purified alpha(IIb)beta3 to fibrinogen, and the adhesion of platelets to fibrinogen was inhibited, indicating a targeting of the fibrinogen receptor. In contrast, the adhesion of osteosarcoma cells that express the integrin alpha(v)beta3 to vitronectin or fibrinogen was not inhibited, indicating the specificity of savignygrin toward alpha(IIb)beta3. Savignygrin shows sequence identity to disagregin, a platelet aggregation inhibitor from the tick Ornithodoros moubata that lacks an RGD motif. The cysteine arrangement of savignygrin is similar to that of the bovine pancreatic trypsin inhibitor family of serine protease inhibitors. A homology model based on the structure of the tick anticoagulant peptide indicates that the RGD motif is presented on the substrate-binding loop of the canonical BPTI inhibitors. However, savignygrin did not inhibit the serine proteases fXa, plasmin, thrombin, or trypsin. This is the first report of a platelet aggregation inhibitor that presents the RGD motif using the Kunitz-BPTI protein fold.     

Histamine effects on endothelial cell fibronectin interaction studied by atomic force microscopy

Atomic force microscopy was used to investigate the cellular response to histamine, one of the major inflammatory mediators that cause endothelial hyperpermeability and vascular leakage. AFM probes were labeled with fibronectin and used to measure binding strength between alpha5beta1 integrin and fibronectin by quantifying the force required to break single fibronectin-integrin bonds. The cytoskeletal changes, binding probability, and adhesion force before and after histamine treatment on endothelial cells were monitored. Cell topography measurements indicated that histamine induces cell shrinkage. Local cell stiffness and binding probability increased twofold after histamine treatment. The force necessary to rupture single alpha5beta1-fibronectin bond increased from 34.0 +/- 0.5 pN in control cells to 39 +/- 1 pN after histamine treatment. Experiments were also conducted to confirm the specificity of the alpha5beta1-fibronectin interaction. In the presence of soluble GRGDdSP the probability of adhesion events decreased >50% whereas the adhesion force between alpha5beta1 and fibronectin remained unchanged. These data indicate that extracellular matrix-integrin interactions play an important role in the endothelial cell response to changes of external chemical mediators. These changes can be recorded as direct measurements on live endothelial cells by using atomic force microscopy.     

A novel anti-platelet monoclonal antibody (3C7) specific for the complex of integrin alpha IIb beta3 inhibits platelet aggregation and adhesion

Activation or ligand binding induces conformational changes in alpha IIb beta3, resulting in exposure of neoepitopes named ligand-induced binding sites. We reported here a novel monoclonal antibody developed by using Chinese hamster ovary (CHO) cells expressing an activated alpha IIb beta3 mutant (CHO alpha IIb beta3Delta717) as the immunogen. This IgG 2b kappa named 3C7 was specific for the complex of alpha IIb beta3 as demonstrated by flow cytometry, immunoprecipitation, and EDTA chelating. The binding of 3C7 to platelets increased significantly when platelets were activated by ADP/thrombin or occupied by RGDS peptides, fibrinogen, or PAC-1, suggesting that 3C7 was an anti-ligand-induced binding site antibody. The antibody failed to bind to the CHO cells expressing another alpha IIb beta3 mutant (beta3Y178A) suggesting that the Cys177-Cys184 loop of beta3 was likely the epitope for 3C7. 3C7 inhibited platelet aggregation, which was initiated by ADP or thrombin in a dose-dependent manner (IC50s of 5.6 and 0.05 microg/ml, respectively). The antibody also inhibited platelet adhesion to immobilized fibrinogen but not to fibronectin or collagen. These findings suggested that 3C7 was a potent antagonist of integrin alpha IIb beta3 and a potential anti-thrombotic agent.     

Regulation of outside-in signaling in platelets by integrin-associated protein kinase C beta

Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin alpha(IIb)beta(3) in platelets, but the responsible PKC isoforms and mechanisms are unknown. Alpha(IIb)beta(3) interacts directly with tyrosine kinases c-Src and Syk. Therefore, we asked whether alpha(IIb)beta(3) might also interact with PKC. Of the several PKC isoforms expressed in platelets, only PKC beta co-immunoprecipitated with alpha(IIb)beta(3) in response to the interaction of platelets with soluble or immobilized fibrinogen. PKC beta recruitment to alpha(IIb)beta(3) was accompanied by a 9-fold increase in PKC activity in alpha(IIb)beta(3) immunoprecipitates. RACK1, an intracellular adapter for activated PKC beta, also co-immunoprecipitated with alpha(IIb)beta(3), but in this case, the interaction was constitutive. Broad spectrum PKC inhibitors blocked both PKC beta recruitment to alpha(IIb)beta(3) and the spread of platelets on fibrinogen. Similarly, mouse platelets that are genetically deficient in PKC beta spread poorly on fibrinogen, despite normal agonist-induced fibrinogen binding. In a Chinese hamster ovary cell model system, adhesion to fibrinogen caused green fluorescent protein-PKC beta I to associate with alpha(IIb)beta(3) and to co-localize with it at lamellipodial edges. These responses, as well as Chinese hamster ovary cell migration on fibrinogen, were blocked by the deletion of the beta(3) cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to beta(3). These studies demonstrate that the interaction of alpha(IIb)beta(3) with activated PKC beta is regulated by integrin occupancy and can be mediated by RACK1 and that the interaction is required for platelet spreading triggered through alpha(IIb)beta(3). Furthermore, the studies extend the concept of alpha(IIb)beta(3) as a scaffold for multiple protein kinases that regulate the platelet actin cytoskeleton.     

Agonist-induced platelet adhesion: use of the method in patients with myocardial infarction

A new method of agonist-induced platelet adhesion has been developed for the evaluation of platelet activity. Platelet adhesion to plastic was stimulated by low doses of ADP, epinephrine and stable thromboxane analogue U46619. These inducers cause more than 3-fold increase of platelet adhesion in concentrations by 5-10 times lower than those necessary for stimulation of platelet aggregation in Born aggregometer. The method was applied for evaluation of platelet adhesive activity in patients with myocardial infarction. A dramatic increase of agonists-induced platelet adhesion was registered in patients with acute infarction, most significantly expressed when epinephrine was used as platelet agonist.     

Ca2+ ionophore A23187 and thrombin inhibit the pertussis-toxin-induced ADP-ribosylation of the alpha-subunit of the inhibitory guanine-nucleotide-binding protein and other proteins in human platelets

Inhibitory guanine-nucleotide-binding proteins (Gi proteins) are substrates for pertussis toxin and the decreased pertussis-toxin-dependent ADP ribosylation of Gi proteins upon prior specific hormonal stimulation of cells is thought to reflect the receptor-mediated activation of Gi proteins, leading to their subsequent dissociation into alpha i and beta/gamma subunits. In the present study, the effect of various platelet stimuli on the subsequent pertussis-toxin-dependent ADP ribosylation of the alpha subunit of Gi (Gi alpha) in saponized platelets and platelet membranes were studied. Stimulation of intact platelets with the Ca(2+)-ionophore A23187 or thrombin, but not phorbol 12,13-dibutyrate, decreased the subsequent pertussis-toxin-dependent ADP ribosylation of Gi alpha in saponin-permeabilized platelets in a time-dependent and dose-dependent manner. Thrombin was more effective than A23187. Parallel measurements of Ca2+ mobilization and pertussis-toxin-dependent ADP ribosylation of Gi alpha in platelets showed that Ca2+ mobilization could only partly account for the decrease in pertussis-toxin-dependent ADP ribosylation in platelets stimulated by thrombin. When the ADP-ribosylation reaction was carried out in platelet membranes, a decrease in ADP ribosylation was still observed after stimulation of platelets with thrombin, but not with A23187. In addition to Gi alpha, two other proteins were found to be ADP ribosylated by pertussis toxin; their ADP ribosylation was also decreased after A23187 and thrombin stimulation of platelets. The results indicate that Ca2+ mobilization can decrease the pertussis-toxin-dependent ADP ribosylation of Gi alpha in saponized platelets; the decrease of pertussis-toxin-dependent ADP ribosylation of Gi alpha after thrombin stimulation of platelets can only, in part, be explained by Ca2+ mobilization and involves additional mechanisms; the decrease in pertussis-toxin-dependent ADP ribosylation after A23187 and thrombin stimulation is not confined to G1 alpha and involves other proteins. We conclude that the decrease in pertussis-toxin-dependent ADP ribosylation of Gi in thrombin-stimulated platelets might not be solely caused by a specific structural change, such as dissociation of Gi. It is likely that A23187 and thrombin stimulation of platelets generates substances which interfere with the ADP-ribosylating activity of pertussis toxin.     

Preferential binding of platelets to monocytes over neutrophils under flow

This study was undertaken to systematically investigate the binding kinetics of platelet recruitment by monocytes relative to neutrophils in bulk suspensions subjected to shear as well as the molecular requirements of leukocyte-platelet binding. Hydrodynamic shear-induced collisions augment the proportion of monocytes with adherent platelets more drastically than that of neutrophils with bound platelets. These heterotypic interactions are further potentiated by platelet activation with thrombin or to a lesser extent by monocyte stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Monocyte-platelet heteroaggregation increases with increasing shear rate and shear exposure time. Platelet P-selectin binding to monocyte P-selectin-glycoprotein-ligand-1 is solely responsible for maximal platelet adhesion to unstimulated monocytes in shear flow. However, the enhanced platelet binding to fMLP-treated monocytes involves a sequential two-step process, wherein P-selectin-PSGL-1 interactions are stabilized by CD18-integrin involvement. Blocking platelet alpha(IIb)beta(3) or monocyte beta(1)-integrin function had no effect. This study underscores the preferential recruitment of platelets by monocytes relative to neutrophils in shear flow, and demonstrates that the shear environment of the vasculature coupled to the state of cell activation modulates the dynamics and molecular constituents mediating monocyte-platelet adhesion.     

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.