CHO cells expressing the high affinity αIIbβ3T562N integrin demonstrate enhanced adhesion under shear

BACKGROUND: Alpha(IIb)beta3-mediated platelet adhesive interactions in the vasculature, which are dependent on the functional state of this receptor, may be sensitive to shear forces. OBJECTIVES: To evaluate the influence of the alpha(IIb)beta3 affinity state on cell attachment under flow, we compared Chinese hamster ovary cells expressing the low affinity alpha(IIb)beta3 wild-type (wt) receptor to those expressing the high affinity alpha(IIb)beta3 T562N receptor. MATERIALS AND METHODS: We designed a real-time videomicroscopy adhesion assay for von Willebrand factor (VWF) or fibrinogen under flow conditions. RESULTS: At 50 s(-1), alpha(IIb)beta3 T562N supported higher cell adhesion to fibrinogen (63.3 +/- 2.9 cells/field) than alpha(IIb)beta3 wt (38.7 +/- 2.4 cells/field, P < 0.0001). At 100 s(-1), alpha(IIb)beta3 T562N mediated cell adhesion (40.5 +/- 3.8 cells/field), while alpha(IIb)beta3 wt did not (5.3 +/- 1.4 cells/field, P < 0.001), allowing to discriminate the efficiency of each receptor. Similar findings were observed for adhesion to VWF. Complete inhibition of cell adhesion to fibrinogen was achieved with 800 microM fibrinogen gamma-chain dodecapeptide [HHLGGAKQAGDV (H12)], while Arg-Gly-Asp-Ser (RGDS) peptide (10-1000 microM) induced a dose-dependent cell detachment. These results suggest that the H12 motif allows initial attachment, in contrast to the RGDS site, which strengthens the stability of adhesion. Interestingly, compared with wt, a 10-fold lower concentration of RGDS was required to reach a similar reduction of cell adhesion mediated by alpha(IIb)beta3 T562N. CONCLUSIONS: Our data show that alpha(IIb)beta3 activation is associated with a stabilization of integrin binding to fibrinogen or VWF under shear.     

MMP-2 regulates human platelet activation by interacting with integrin αIIbβ3

Summary. Background: Human platelets contain matrix metalloproteinases (MMPs) that are secreted during platelet activation. Platelet MMPs have been implicated in the regulation of cellular activation and aggregation. Although the proaggregatory effect of MMP‐2 has been demonstrated, the functional mechanism is not clearly understood. Objectives: This work was carried out in order to elucidate the biochemical mechanism of MMP‐2‐associated platelet activation and aggregation. Methods: MMP‐2 binding to the platelet surface was analyzed by flow cytometry. The cell surface target of MMP‐2 was identified in thrombin receptor‐activating peptide‐stimulated platelets by immunoprecipitation, Western blotting and fluorescence microscopy. A recombinant hemopexin‐like domain was used to characterize the nature of MMP‐2 binding to the platelet surface. The functional significance of MMP‐2 in platelet activation was investigated by quantitative measurements of the activation markers P‐selectin (CD62P) and active α_IIbβ₃. The role of MMP‐2 in platelet aggregation was analyzed with an aggregometer. Results: ProMMP‐2 binds to integrin α_IIbβ₃ in stimulated platelets in which proMMP‐2 is converted into MMP‐2. Fibrinogen was able to replace the α_IIbβ₃‐bound MMP‐2. The molecular interaction of MMP‐2 and integrin α_IIbβ₃ was abrogated by the recombinant human hemopexin‐like domain of MMP‐2, leading to reduced cell surface expression of activation markers CD62P and active α_IIbβ₃, and resulting in suppressed platelet aggregation. Conclusion: This work clearly demonstrates that platelet activation and aggregation is regulated by MMP‐2 that specifically interacts with integrin α_IIbβ₃. The C‐terminal hemopexin‐like domain of MMP‐2 is an essential element for binding to α_IIbβ₃.     

Effect of cellular and receptor activation on the extent of integrin αIIbβ3 internalization

Summary. Previous studies by our laboratory demonstrated that internalization of fibrinogen‐bound α_IIbβ₃ correlated with both a loss of aggregation and a loss of bound fibrinogen from the platelet surface. However, these studies do not address whether cellular activation, receptor activation and/or receptor occupancy are responsible for the observed internalization of α_IIbβ₃. The present studies were designed to evaluate the roles of cellular and receptor activation states on the α_IIbβ₃ internalization process. In these studies, washed platelets were allowed to bind FITC‐D57, an antiα_IIb monoclonal antibody, and were subsequently treated with ADP, thrombin receptor activation peptide (TRAP) or antiLIBS6 monoclonal antibody. Following flow cytometric analyses for log green fluorescence, rabbit antifluorescein was added, and the samples were re‐analyzed for residual/unquenched fluorescence. Because access of the quenching antibody is limited to extracellular/surface‐associated fluorescein, protection from quenching by antifluorescein is taken as evidence of internalization. Stimulation of platelets with ADP or TRAP resulted in a significant increase in the percent internalization of α_IIbβ₃ compared to control (8.7% and 12.8% vs. 2.9%). Addition of cytochalasin E prior to stimulation resulted in a greater than 90% inhibition of both TRAP and ADP‐induced internalization, suggesting that activation‐dependent internalization is mediated by the actin cytoskeleton. To investigate whether receptor activation increases the extent of α_IIbβ₃ internalization, platelets were treated with anti‐LIBS6, which directly activates α_IIbβ₃. Stimulation with anti‐LIBS6 caused an approximate 8‐fold increase in the extent of α_IIbβ₃ internalization. To evaluate whether the activated pool of α_IIbβ₃ is preferentially internalized, platelets were incubated with PAC‐1, an antibody specific for activated α_IIbβ₃. Platelets stimulated with TRAP, demonstrated a dose‐dependent internalization of PAC‐1. However, approximately 29% of total PAC‐1 binding was internalized, irrespective of TRAP concentration, suggesting that a constant proportion of activated α_IIbβ₃ is selectively internalized in platelets. Collectively, these data suggest that α_IIbβ₃ is internalized to a greater extent in activated platelets in a cytoskeleton‐dependent manner. Furthermore, the active conformer of α_IIbβ₃ is preferentially internalized which may act as a mechanism for downregulating adhesiveness of activated platelets in the circulation.     

Platelet integrin αIIbβ3: activation mechanisms

Integrin alpha(IIb)beta(3) plays a critical role in platelet aggregation, a central response in hemostasis and thrombosis. This function of alpha(IIb)beta(3) depends upon a transition from a resting to an activated state such that it acquires the capacity to bind soluble ligands. Diverse platelet agonists alter the cytoplasmic domain of alpha(IIb)beta(3) and initiate a conformational change that traverses the transmembrane region and ultimately triggers rearrangements in the extracellular domain to permit ligand binding. The membrane-proximal regions of alpha(IIb) and beta(3) cytoplasmic tails, together with the transmembrane segments of the subunits, contact each other to form a complex which restrains the integrin in the resting state. It is unclasping of this complex that induces integrin activation. This clasping/unclasping process is influenced by multiple cytoplasmic tail binding partners. Among them, talin appears to be a critical trigger of alpha(IIb)beta(3) activation, but other binding partners, which function as activators or suppressors, are likely to act as co-regulators of integrin activation.     

A tripeptide mimetic of von Willebrand factor residues 981–983 enhances platelet adhesion to fibrinogen by signaling through integrin βIIbβ3

BACKGROUND: RGD is a major recognition sequence for ligands of platelet alpha(IIb)beta3. OBJECTIVE AND METHODS: To identify potential binding sites for alpha(IIb)beta3 apart from RGD, we screened phage display libraries by blocking the enrichment of RGD-containing phages with a GRGDS peptide and identified a novel integrin recognition tripeptide sequence, VPW. RESULTS: Platelets adhered to an immobilized cyclic VPW containing peptide in a alpha(IIb)beta3-dependent manner; platelets and alpha(IIb)beta3-expressing CHO cells adhered faster to immobilized alpha(IIb)beta3-ligands in the presence of soluble VPW. In platelets adhering to fibrinogen, VPW accelerated the activation of the tyrosine kinase Syk which controls cytoskeletal rearrangements. In alpha(IIb)beta3-expressing CHO cells, VPW induced a faster formation of stress fibers. Sequence alignment positioned VPW to V980-P981-W982 in the von Willebrand factor (vWf) A-3 domain. In blood from a vWf-deficient individual, VPW increased platelet adhesion to fibrinogen but not to collagen under flow and rescued the impaired adhesion to vWf deficient in A-3. CONCLUSION: These data reveal a VPW sequence that contributes to alpha(IIb)beta3 activation in in vitro experiments. Whether the V980-P981-W982 sequence in vWf shows similar properties under in vivo conditions remains to be established.     

Association of the antagonism of von Willebrand factor but not fibrinogen by platelet αIIbβ3 antagonists with prolongation of bleeding time

Summary.  Background:  The α _IIb β ₃ antagonists inhibit platelet aggregation and are used as antithrombotic agents for cardiothrombotic disease. The present study investigates the correlation of inhibition of fibrinogen and von Willebrand factor (VWF) binding by α _IIb β ₃ antagonists with the inhibition of platelet aggregation and prolongation of bleeding time (BT). Methods:  Inhibition of fibrinogen and VWF binding were assessed in a purified α _IIb β ₃-binding assay. As an in vitro cell-based assay, platelet aggregation and VWF-mediated adhesion studies were performed using human platelets. In vivo effects on BT were measured using a template device in dogs at the same time as an ex vivo aggregation study was performed. Results:   In vitro studies demonstrated that the antiaggregatory effects of α _IIb β ₃ antagonists correlate with their inhibition of fibrinogen binding, but not VWF. Interestingly, the effects of α _IIb β ₃ antagonists on BT could be differentiated from the inhibition of platelet aggregation. Furthermore, this differentiation was strongly correlated with the different inhibitory potencies between fibrinogen and VWF binding, as well as that between VWF-mediated adhesion and aggregation. Conclusions:  Our study provides novel evidence showing that the inhibitory effect of α _IIb β ₃ antagonists on VWF, but not fibrinogen binding, correlates with their ability to prolong BT.     

A novel functional role for the highly conserved α-subunit KVGFFKR motif distinct from integrin αIIbβ3 activation processes

BACKGROUND: The highly conserved integrin alpha-subunit membrane-proximal motif KVGFFKR plays a decisive role in modulating the activation of integrin alphaIIbbeta3. Previously, we have shown that a platelet permeable palmityl (pal)-peptide with this seven amino acid sequence can directly activate alphaIIbbeta3 leading to platelet aggregation. OBJECTIVES: To investigate further the role of the KVGFFKR motif in integrin alphaIIbbeta3 function. METHODS: We used two sequence-specific complementary model systems, palmityl pal-peptides in platelets, and mutant alphaIIbbeta3-expressing Chinese Hamster Ovary (CHO) cell lines. RESULTS: In platelets we show that the two phenylalanine amino acids in pal-KVGFFKR (pal-FF) peptide are critical for stimulating platelet aggregation. Pal-FF peptide treatment of platelets also gives rise to a tyrosine phosphorylation signal despite the presence of inhibitors of fibrinogen binding. In CHO cells, a double alanine substitution, alphaIIb(F992A, F993A)beta3, induces constitutive integrin activation but prevents actin stress fiber formation upon adhesion to fibrinogen, suggesting that alphaIIbbeta3-mediated cytoskeletal reorganization is also dependent on F992 and F993. This further highlights a critical role for the two phenylalanine residues in both of these alphaIIbbeta3-mediated processes. CONCLUSION: In addition to regulating integrin alphaIIbbeta3 activation state, the KVGFFKR motif also influences cytoskeletal reorganization. This activity is critically determined by F992 and F993 within the seven amino acid sequence.     

The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin α2β1

BACKGROUND: Activation of the platelet integrin alpha 2 beta 1 is closely regulated due to the high thrombogenicity of its ligand. As a beta 1 interacting kinase, ILK represents a candidate intracellular regulator of alpha 2 beta 1 in human platelets. OBJECTIVES: We investigated the regulation of ILK in human platelets and the role of ILK in regulating alpha 2 beta 1 activation in HEL cells, a megakaryocytic cell line. METHODS: An in-vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with beta 1-integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating alpha 2 beta 1 function assessed by overexpression studies in HEL cells. RESULTS: We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation-independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin-stimulated platelets and regulated by PI3K in collagen-stimulated cells. ILK associates with the beta 1-integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced alpha 2 beta 1-mediated adhesion whereas overexpression of kinase-dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of alpha 2 beta 1. CONCLUSIONS: Our findings that ILK regulates alpha 2 beta 1 in HEL cells, is activated in platelets and associates with beta 1-integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets.     

α IIbβ3 priming and clustering by orally active and intravenous integrin antagonists

BACKGROUND: Drugs that block platelet-platelet and platelet-fibrin interactions via the alpha(IIb)beta(3) (glycoprotein IIb/IIIa) receptor are used daily in patients undergoing percutaneous coronary interventions. Along with expected increases in spontaneous bleeding, clinical trials have revealed a surprising increase in thrombosis when these drugs are used without other anticoagulants. A better understanding of their mechanisms can minimize these risks. OBJECTIVES: This study tested the hypothesis that interventions designed to block fibrinogen binding inevitably leave the alpha(IIb)beta(3) receptor in an activated state. It compared the effects on platelet function and alpha(IIb)beta(3) conformation of the orally active compounds orbofiban and roxifiban, the i.v. agents eptifibatide and tirofiban, and echistatin, an arginine-glycine-aspartate (RGD) disintegrin. METHODS: The integrin antagonist concentrations required to saturate platelets and to block platelet-platelet and platelet-fibrin interactions were determined by flow cytometry, aggregometry, and clot-based adhesion assays, respectively. Analytical ultracentrifugation measured each antagonist's effects on the solution structure of alpha(IIb)beta(3). Fluorescence anisotropy provided equilibrium and kinetic data for integrin:antagonist interactions. RESULTS: Both orally active drugs bound more tightly and inhibited platelet aggregation and adhesion to fibrin more effectively than echistatin. Analytical ultracentrifugation yielded this order for perturbing alpha(IIb)beta(3) conformation (priming) and promoting oligomerization (clustering): echistatin > eptifibatide > orbofiban > tirofiban > roxifiban. Roxifiban was also most effective at disrupting the rapidly forming/slowly dissociating alpha(IIb)beta(3):echistatin complex. CONCLUSIONS: Our results suggest that the same molecular mechanisms that enable glycoprotein IIb/IIIa inhibitors to bind tightly to the alpha(IIb)beta(3) receptor and block fibrinogen binding contribute to their ability to perturb the resting integrin's conformation, thus limiting the safety and efficacy of both oral and i.v. integrin antagonists.     

A mechanism to safeguard platelet adhesion under high shear flow: von Willebrand factor–glycoprotein Ib and integrin α2β1–collagen interactions make complementary, collagen-type-specific contributions to adhesion

BACKGROUND: Blood vessels contain different types of collagen, with types I and III being the major components of vascular collagen. Platelet adhesion under high shear stress has been suggested to depend on the binding of von Willebrand factor (VWF) to collagen. OBJECTIVE: We analyzed the collagen type specificity for the interaction with VWF and high shear stress platelet adhesion. METHODS: VWF binding to different types of immobilized collagen and effects of antibodies against glycoprotein Ib (gpIb) and integrin alpha(2)beta(1) on platelet adhesion to type I and III collagens under high shear were analyzed. RESULTS: VWF showed high-affinity, selective binding to human and bovine type III collagens, but weak or no affinity for types I, II, IV and V under static conditions. Anti-integrin alpha(2)beta(1) markedly inhibited adhesion to type I collagen, but did not affect that to type III collagen. Anti-gpIb antibody significantly inhibited adhesion to type III collagen. Adding both antibodies abrogated the adhesion to either type I or III collagen. CONCLUSIONS: Both the gpIb-VWF interaction and the integrin alpha(2)beta(1)-collagen interaction contribute to platelet adhesion to collagen under high shear stress, and integrin alpha(II)beta(1) makes a greater contribution to adhesion to type I collagen because less VWF is bound to it.     

CIB1 deficiency results in impaired thrombosis: the potential role of CIB1 in outside-in signaling through integrin αIIbβ3

Summary.  Background: Agonist-induced inside-out signaling activates platelet integrin α_IIbβ₃, rendering it to bind plasma fibrinogen (Fg). Fg binding induces outside-in signaling that culminates in platelet aggregation, leading to physiological hemostasis and pathological thrombosis. How outside-in signaling through α_IIbβ₃ regulates hemostasis and thrombosis is not well understood. We have previously shown that CIB1 is involved in regulating α_IIbβ₃ function. Objective: To determine the in vivo role of CIB1 in the process of hemostasis and thrombosis. Methods and Results: Genetic ablation of Cib1 significantly increased mouse tail bleeding time. Greater than 50% of the Cib1 null mice showed a rebleeding phenotype. Time taken for complete occlusion of carotid artery upon 10% FeCl₃-induced injury was significantly delayed in the absence of Cib1. This was also associated with unstable thrombus formation. The inside-out signaling appears normal as ADP-, collagen- and PAR4 peptide-induced aggregation and fibrinogen binding was unaffected. The absence of Cib1 also affected the ability of platelets to spread on immobilized Fg, but not filopodia formation. Spreading could be restored in Cib1 null platelets by the addition of exogenous ADP. Outside-in signaling-dependent tyrosine phosphorylation of the integrin β₃ subunit was significantly reduced in the absence of Cib1 as determined by Western blot analysis. Conclusion: Using gene knockout mice, we show for the first time that lack of Cib1 results in impaired thrombosis. CIB1 regulates these processes by affecting platelet spreading, but not platelet filopodia formation. These in vivo and in vitro results clearly show that CIB1 is a key regulator of thrombosis.     

Involvement of the β3 E749ATSTFTN756 region in stabilizing integrin αIIbβ3-ligand interaction

Summary. Platelet integrin α_IIbβ₃ must be activated via intracellular mechanisms before it binds soluble ligands, and it is thought to be activated at its extracellular site by surface‐bound ligands. Integrin activation is associated with rearrangement of the cytoskeleton and phosphorylation of proteins that become localized in focal contacts. In these processes, the cytoplasmic tail of the β‐subunit plays a central role. We introduced peptides homologous to the E⁷⁴⁹ATSTFTN⁷⁵⁶ domain (E–N peptide) and the T⁷⁵⁵NITYRGT⁷⁶² domain (T–T peptide) of β₃ in streptolysin O‐permeabilized platelets and analyzed the initial interaction with soluble fibronectin, fibrinogen and PAC‐1 after stimulation with thrombin. E–N peptide left the initial binding of fibronectin intact but interfered with stable receptor occupancy. E–N peptide also inhibited fibrinogen binding, thereby reducing the formation of large aggregates. Strikingly, E–N peptide did not disturb the binding of PAC‐1, which is known to reflect activation of the integrin. E–N peptide also inhibited tyrosine phosphorylation of focal adhesion kinase, a response known to be dependent on α_IIbβ₃. T–T peptide did not affect these processes. In a model for outside‐in integrin activation, E–N peptide disrupted the binding of CHO cells expressing α_IIbβ₃ to surface‐bound ligand. Again, T–T peptide had no effect. We conclude that the E⁷⁴⁹ATSTFTN⁷⁵⁶ region of the β₃‐tail stabilizes the binding of soluble and surface‐bound ligand to integrin α_IIbβ₃ via a mechanism that involves the phosphorylation of FAK.     

von Willebrand factor mediates platelet spreading through glycoprotein Ib and αIIbβ3 in the presence of botrocetin and ristocetin, respectively

BACKGROUND: von Willebrand factor (VWF) plays a critical role in the process of hemostasis by mediating flow-dependent adhesion and spreading of platelets on exposed extracellular matrix proteins following vascular injury. To accomplish this, VWF binds to two distinct platelet receptors: glycoprotein (GP)Ib-IX-V and integrin alpha(IIb)beta3. OBJECTIVE: To evaluate the ability of GPIb and alpha(IIb)beta3 to mediate platelet adhesion and lamellipodia formation on immobilized VWF in the presence of the biochemical modulators, ristocetin and botrocetin. RESULTS: In the presence of botrocetin and inhibitors of adenosine diphosphate (ADP) and thromboxane A2 (TxA2), VWF is able to support formation of lamellipodia through a GPIb-dependent mechanism that is independent of alpha(IIb)beta3 and PI3-kinase. Lamellipodia formation under these conditions is incomplete. In marked contrast, in the presence of ristocetin, VWF stimulates formation of fully spread lamellipodia through a pathway that is dependent upon alpha(IIb)beta3 and PI3-kinase. Furthermore, alpha(IIb)beta3 also supports platelet spreading on VWF alone, but only in the absence of inhibitors of ADP and TxA2. The localization of filamentous actin and the Arp2/3 complex in platelets on VWF in the presence of botrocetin and ristocetin are distinct, yielding disparate lamellipodium kinetic signatures. Interestingly, botrocetin significantly enhances platelet adhesion to VWF under flow in whole blood in an alpha(IIb)beta3-independent manner, while ristocetin augments washed platelet adhesion and spreading to VWF under flow in an alpha(IIb)beta3-dependent manner. CONCLUSIONS: These observations demonstrate that VWF is able to induce lamellipodia formation through distinct receptors, and has important consequences for investigation of the role of VWF-GPIb interactions in the context of platelet regulation.     

Differential regulation of adapter proteins Dok2 and Dok1 in platelets, leading to an association of Dok2 with integrin αIIbβ3

BACKGROUND: We previously demonstrated that Dok2 is rapidly phosphorylated on tyrosine residues in platelets in response to thrombin, the immunoreceptor tyrosine-based activation motif-coupled collagen receptor glycoprotein (GP) VI, and by integrin alphaIIbbeta3. OBJECTIVES AND METHODS: In this study we further delineate the regulation of phosphorylation of Dok2 and compare this to the related adapter Dok1. RESULTS: We demonstrate expression of Dok1 in platelets and the unexpected observation that the adapter protein undergoes tyrosine phosphorylation in response to thrombin but not to GPVI or integrin alphaIIbbeta3. Furthermore, Dok1 phosphorylation is transient, peaking at 30 s and returning to basal by 5 min, whereas Dok2 phosphorylation is delayed but sustained. Dok2 phosphorylation, but not that of Dok1, is inhibited by Src kinase inhibitors and by chelation of intracellular calcium. Further, phosphorylation of Dok2 by thrombin and integrin alphaIIbbeta3 in mouse platelets is independent of Syk and phospholipase Cgamma2. Additionally, Dok2 coimmunoprecipitates with integrin alphaIIbbeta3 downstream of Src kinases. CONCLUSIONS: These results demonstrate differential modes of regulation of Dok1 and Dok2 in platelets. Further, they raise the interesting possibility that Dok2 plays an important role in integrin outside-in signaling through a physical and functional interaction with integrin alphaIIbbeta3.     

Rap1b is critical for glycoprotein VI-mediated but not ADP receptor-mediated α2β1 activation

Summary. Background: The platelet α₂β₁ integrin functions as both an adhesion and signaling receptor upon exposure to collagen. Recent studies have indicated that α₂β₁ function can be activated via inside‐out signaling, similar to the prototypical platelet integrin α_IIbβ₃. However, signaling molecules that regulate α₂β₁ activation in platelets are not well defined. A strong candidate molecule is the small GTPase Rap1b, the dominant platelet isoform of Rap1, which regulates α_IIbβ₃ activation. Objectives: We hypothesized that Rap1b positively regulates α₂β₁ during agonist‐induced platelet activation. Methods: To test whether Rap1b activates α₂β₁ downstream of glycoprotein (GP)VI or other platelet receptors, we stimulated platelets purified from Rap1b^?/? or wild‐type mice with diverse agonists and measured α₂β₁ activation using fluorescein isothiocyanate‐labeled monomeric collagen. We also examined the role of Rap1b in outside‐in signaling pathways by analyzing adhesion and spreading of Rap1b^?/? or wild‐type platelets on monomeric, immobilized collagen. Finally, we monitored the activation status of related Rap GTPases to detect changes in signaling pathways potentially associated with Rap1b‐mediated events. Results: Rap1b^?/? platelets displayed comparable ADP‐induced or thrombin‐induced α₂β₁ activation as wild‐type platelets, but reduced convulxin‐dependent α₂β₁ activation. Rap1b^?/? platelets exhibited increased spreading on immobilized collagen but similar adhesion to immobilized collagen compared to wild‐type platelets. Rap1b^?/? platelets also showed Rap1a and Rap2 activation upon agonist stimulation, possibly revealing functional compensation among Rap family members. Conclusions: Rap1b is required for maximal GPVI‐induced but not ADP‐induced activation of α₂β₁ in murine platelets.     

Three novel β-propeller mutations causing Glanzmann thrombasthenia result in production of normally stable pro-αIIb, but variably impaired progression of pro-αIIbβ3 from endoplasmic reticulum to Golgi

BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation in response to most physiological agonists and caused by either a lack or dysfunction of the platelet integrin alpha(IIb)beta3 (glycoprotein IIb/IIIa). OBJECTIVES: To determine the molecular basis of GT and characterize the mutations by in vitro expression studies. PATIENTS: We studied three unrelated patients from southern India whose diagnosis was consistent with GT. RESULTS: Immunoprecipitation of the cell lysates and immunoblotting showed no detectable mature alpha(IIb) in the G128S mutant, in contrast to 6% and 33% of the normal amount of mature alpha(IIb) in the S287L and G357S mutants, respectively. Pulse-chase analysis demonstrated pro-alpha(IIb) in the mutants comparable with the normal pro-alpha(IIb), but no conversion to mature alpha(IIb) in the G128S mutant, and only trace conversion to mature alpha(IIb) in the S287L and G357S mutants. The disappearance of pro-alpha(IIb) in the three mutants was similar to that in cells expressing normal alpha(IIb)beta3 or alpha(IIb) only. All three mutants demonstrated pro-alpha(IIb)beta3 complexes and co-localized with an ER marker by immunofluorescence. The G128S mutant showed no co-localization with a Golgi marker, and the other two mutants showed minimal and moderate co-localization with the Golgi marker. CONCLUSIONS: These three beta-propeller mutations do not affect the production of pro-alpha(IIb), its ability to complex with beta3, or its stability, but do cause variable defects in transport of pro-alpha(IIb)beta3 complexes from the endoplasmic reticulum to the Golgi.     

Association of polymorphisms of platelet membrane integrins αIIbβ3 (HPA-1b/PlA2) and α2β1 (α2807TT) with premature myocardial infarction

Conflicting results of an association of the human platelet antigen 1b (HPA-1b/PlA2), localized on the beta-subunit of the integrin alpha(IIb)beta3, and the alpha(2)807TT genotype of the integrin alpha2beta1 with coronary atherosclerosis and myocardial infarction have been reported. Both platelet receptor polymorphisms were genotyped in 3261 patients who had undergone coronary angiography, including 1175 survivors of a myocardial infarction, 1211 individuals with coronary artery disease but no history of myocardial infarction, and 571 control patients without angiographic coronary artery disease, and in 793 blood donors. In a case-control design, the prevalence of HPA-1b and alpha(2)807TT genotypes did not differ significantly between the patient groups with coronary artery disease or myocardial infarction and patient controls or blood donors. By contrast, using a multivariate case-only design, it was found that the median age of onset of myocardial infarction was 5.2 years earlier (P = 0.006) in carriers of the HPA-1b allele and 6.3 years earlier (P = 0.006) in carriers of the alpha(2)807TT genotype in the 264 survivors of myocardial infarction of recent onset with one- or two-vessel coronary artery disease. A significant interaction with the conventional risk factors hypercholesterolemia, smoking, diabetes, hypertension, and hyperfibrinogenemia was excluded. Human platelet antigen 1b and alpha(2)807TT are associated with premature myocardial infarction but not with coronary artery disease, suggesting a role of distinct integrin genotypes for increased platelet thrombogenicity. This association requires confirmation in follow-up studies.     

Formation of platelet strings and microthrombi in the presence of ADAMTS-13 inhibitor does not require P-selectin or β3 integrin

BACKGROUND: Ultra-large von Willebrand factor (ULVWF) and the receptor P-selectin are released from endothelial Weibel-Palade bodies during injury or inflammation. VWF mediates platelet adhesion and P-selectin promotes leukocyte rolling. ADAMTS-13 limits the duration of platelet adhesion by cleaving the ULVWF. In the absence of ADAMTS-13, long VWF filaments decorated with platelets form. Recent in vitro studies suggested that P-selectin might anchor these platelet strings to endothelium, but whether the same mechanism exists in vivo remains to be elucidated. METHODS: We address the role of P-selectin and beta(3) integrin in platelet string formation in vivo using intravital microscopy by infusing inhibitory ADAMTS-13 antibody in P-selectin-/- and beta(3)-deficient mice and activating the endothelium by injecting histamine. RESULTS: We show that inhibition of ADAMTS-13 combined with endothelial activation leads to similar extents of platelet string formation in wild-type, P-selectin- and integrin beta(3)-deficient mice. Further, in venules the platelet strings can coalesce into VWF-platelet aggregates. This process utilizes neither the platelet beta(3) integrin nor P-selectin. We also show in vitro that platelets can act as a bridge between the VWF fibers and that VWF can self-associate even in areas devoid of platelets. CONCLUSIONS: The formation or retention of the platelet strings does not require P-selectin or the endothelial VWF receptor alpha(v)beta(3). Furthermore, in the presence of low ADAMTS-13 activity, VWF-dependent and alpha(IIb)beta(3)-independent platelet clustering occurs in veins, as has been shown at high arterial shear rates. Our study further supports the importance of regulation of VWF multimer size upon secretion from Weibel-Palade bodies.     

Activation of integrin αIIbβ3 in the glycoprotein Ib-high population of a megakaryocytic cell line, CMK, by inside-out signaling

Summary. Affinity/avidity state of integrin α_IIbβ₃ is regulated by intracellular inside‐out signaling. Although several megakaryocytic cell lines have been established, soluble ligand binding to α_IIbβ₃ expressed in these cells by cellular agonists has not been demonstrated. We have re‐examined agonist‐induced α_IIbβ₃ activation on megakaryocytic cell lines with a marker of the late stage of megakaryocytic differentiation, glycoprotein Ib (GPIb). Activation of α_IIbβ₃ was assessed by PAC1 and soluble fibrinogen binding to the cells. We found that α_IIbβ₃ expressed in CMK cells with high GPIb expression was activated by a phorbor ester, phorbol myristate acetate (PMA). Although the population of the GPIb^high cells was <0.5% of the total cells, incubation with a nucleoside analog, ribavirin, efficiently increased the PMA‐reactive GPIb^high cells. Not only PMA but also a calcium ionophore, A23187, induced α_IIbβ₃ activation, and PMA and A23187 had an additive effect on α_IIbβ₃ activation. Ligand binding to the activated α_IIbβ₃ in the GPIb^high CMK cells is totally abolished by an α_IIbβ₃‐specific antagonist, and inhibited by wortmannin, cytochalasin‐D and prostaglandin E₁, and the effects of these inhibitors on α_IIbβ₃ activation in the GPIb^high CMK cells were compatible with those in platelets. We have also demonstrated that the ribavirin‐treated CMK cells express PKC‐α, ‐β, ‐δ and ‐θ, and suggested that PKC‐α and/or ‐β appear to be responsible for PMA‐induced activation of α_IIbβ₃ in CMK cells.     

Thrombin induces GPIb-IX-mediated fibrin binding to αIIbβ3 in a reconstituted Chinese hamster ovary cell model

BACKGROUND: The interaction of thrombin with platelet glycoprotein (GP) Ib-IX-V has been recently suggested to induce fibrin-dependent platelet aggregation associated with signaling events. The approaches used to avoid the protease-activated receptor (PAR) thrombin receptors in platelets have provided controversial conclusions regarding the precise mechanism and molecules involved in the response. OBJECTIVES: In the present study, we developed a cellular model to investigate the functional consequences following the binding of thrombin to GPIb-IX. METHODS: We used Chinese hamster ovary (CHO) cells stably expressing human alpha(IIb)beta(3) and/or GPIb-IX complexes (CHO-alpha(IIb)beta(3)-IbIX cells) to analyze the effect of thrombin on the binding of polymerizing fibrin by using fluorescein isothiocyanate-fibrinogen as precursor. RESULTS: Thrombin induces, in a dose-dependent manner, the binding of polymerizing fibrin to CHO-alpha(IIb)beta(3)-IbIX cells. This response is not observed in cells expressing only one of the receptors, and it can be blocked by monoclonal antibodies against alpha(IIb)beta(3) and GPIbalpha. We show that the reaction is not due to simple cell trapping by the fibrin clot, and provide data supporting a role of a signaling pathway in which the 14-3-3zeta adaptor and calcium-calmodulin-dependent events are involved. CONCLUSIONS: The present data support a significant role of GPIb-IX and alpha(IIb)beta(3) receptors in an alternative fibrin-mediated pathway of platelet activation induced by thrombin.