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.     

Nitric oxide inhibits von Willebrand factor‐mediated platelet adhesion and spreading through regulation of integrin αIIbβ3 and myosin light chain

Summary. Background: von Willebrand factor (VWF)‐mediated platelet adhesion and spreading at sites of vascular injury is a critical step in hemostasis. This process requires two individual receptors: glycoprotein Ib (GPIb)‐V‐IX and integrin α_IIbβ₃. However, little is known about the negative regulation of these events. Objectives: To examine if the endogenous platelet inhibitor nitric oxide (NO) has differential effects on adhesion, spreading and aggregation induced by immobilized VWF. Results: S‐nitrosoglutathione (GSNO) inhibited platelet aggregation on immobilized VWF under static and flow conditions, but had no effect on platelet adhesion. Primary signaling events underpinning the actions of NO required cyclic GMP but not protein kinase A. Dissecting the roles of GPIb and integrin α_IIbβ₃ demonstrated that NO targeted α_IIbβ₃‐mediated aggregation and spreading, but did not significantly influence GPIb‐mediated adhesion. To understand the relationship between the effects of NO on adhesion and subsequent aggregation, we evaluated the activation of α_IIbβ₃ on adherent platelets. NO reduced the phosphorylation of extracellular stimuli‐responsive kinase (ERK) and p38, required for integrin activation resulting in reduced binding of the activated α_IIbβ₃‐specific antibody PAC‐1 on adherent platelets. Detailed analysis of platelet spreading initiated by VWF demonstrated key roles for integrin α_IIbβ₃ and myosin light chain (MLC) phosphorylation. NO targeted both of these pathways by directly modulating integrin affinity and activating MLC phosphatase. Conclusion: These data demonstrate that initial activation‐independent platelet adhesion to VWF via GPIb is resistant to NO, however, NO inhibits GPIb‐mediated activation of α_IIbβ₃ and MLC leading to reduced platelet spreading and aggregation.     

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.     

Integrin α2β1 induces phosphorylation-dependent and phosphorylation-independent activation of phospholipase Cγ2 in platelets: role of Src kinase and Rac GTPase

Summary. Background: Platelet adhesion promoted by integrin  α₂β₁ induces integrin  α_IIbβ₃ activation through the phospholipase C (PLC)‐dependent stimulation of the small GTPase Rap1b. Objective: To analyze the mechanism of PLC activation downstream of α₂β₁ that is required for regulation of Rap1b and α_IIbβ₃. Methods: Human and murine platelets were allowed to adhere to immobilized type I monomeric collagen through α₂β₁. Tyrosine phosphorylation of PLCγ2, PLC activation, accumulation of GTP‐bound Rap1b and fibrinogen binding were measured and compared. Results: Integrin  α₂β₁ recruitment induced an evident PLC activation that was concomitant with robust tyrosine phosphorylation of PLCγ2, and was suppressed in platelets from PLCγ2‐knockout mice. Moreover, PLCγ2^?/? platelets were unable to accumulate active Rap1b and to activate α_IIbβ₃ upon adhesion through α₂β₁. Inhibition of Src kinases completely prevented tyrosine phosphorylation of PLCγ2 in adherent platelets, but did not affect its activation, and both Rap1b and α_IIbβ₃ stimulation occurred normally. Importantly, α_IIbβ₃‐induced phosphorylation and activation of PLCγ2, as well as accumulation of active Rap1b, were totally suppressed by Src inhibition. Integrin  α₂β₁ recruitment triggered the Src kinase‐independent activation of the small GTPase Rac1, and activation of Rac1 was not required for PLCγ2 phosphorylation. However, when phosphorylation of PLCγ2 was blocked by the Src kinase inhibitor PP2, prevention of Rac1 activation significantly reduced PLCγ2 activation, GTP‐Rap1b accumulation, and α_IIbβ₃ stimulation. Conclusions: Src kinases and the Rac GTPases mediate independent pathways for PLCγ2 activation downstream of α₂β₁.     

Glycoprotein Ib–IX-mediated activation of integrin αIIbβ3: effects of receptor clustering and von Willebrand factor adhesion

Summary. The interaction between the platelet glycoprotein (GP) Ib–IX complex and von Willebrand factor (VWF) initiates both hemostasis and pathological thrombosis. This interaction is not only the first adhesive event of platelets at sites of vessel injury, but also facilitates fibrinogen binding to α_IIbβ₃, which subsequently results in platelet aggregation. Since it has been suggested that GP Ib–IX clustering may promote platelet activation, we investigated the effect of such clustering on both VWF–GP Ib–IX and fibrinogen–α_IIbβ₃ bonds using optical tweezers. In our system, fusion of tandem repeats of FK506‐binding protein (FKBP) to the cytoplasmic tail of the GP IX subunit of the GP Ib–IX complex allowed subsequent receptor clustering within the plasma membrane by the bivalent, cell‐permeant small molecule ligand AP20187. We measured binding forces between polystyrene beads coated with either plasma‐derived VWF or the VWF A1 domain and GP Ib–IX(FKBP)₂, and those between fibrinogen‐coated beads and α_IIbβ₃ expressed on Chinese hamster ovary cells. The minimal detachment force between GP Ib–IX(FKBP)₂ and A1 or plasma‐derived VWF doubled after AP20187 was added. The binding force between immobilized fibrinogen and α_IIbβ₃ was not changed by the clustering agent; however, the strength of single fibrinogen–α_IIbβ₃ bonds increased significantly after ligation of GP Ib–IX(FKBP)₂ by A1. These results demonstrate that GP Ib–IX clustering increases the overall strength of its interaction with VWF. Furthermore, signals from GP Ib–IX can activate α_IIbβ₃, thereby increasing the strength of its interaction with fibrinogen.     

Overexpression of the partially activated αIIbβ3D723H integrin salt bridge mutant downregulates RhoA activity and induces microtubule-dependent proplatelet–like extensions in Chinese hamster ovary cells

Summary. Background: We have recently reported a novel mutation in the β₃ subunit of the platelet fibrinogen receptor (α_IIbβ₃D723H) identified in a patient with dominantly inherited macrothrombocytopenia, and we have shown that this mutation promotes a new phenotype in Chinese hamster ovary (CHO) cells, characterized by fibrinogen‐dependent, microtubule‐driven proplatelet‐like cell extensions. Results: Here we demonstrate that the partially activated α_IIbβ₃D723H or α_IIbβ₃D723A salt bridge mutants, but not fully activated α_IIbβ₃ mutants, cause this phenotype. Time‐lapse videomicroscopy clearly differentiated these stable microtubule‐driven and nocodazole‐sensitive extensions from common dynamic actin‐driven pseudopodia. In addition, overexpression of a mitochondrial marker confirmed their functional role in organelle transport. Comparative immunofluorescence analysis of the subcellular localization of α_IIbβ₃, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34⁺‐derived megakaryocyte proplatelets. Mutant α_IIbβ₃D723H signaling was independent of Src, protein kinase C or phosphoinositide 3‐kinase, but correlated with decreased RhoA activity as compared with wild‐type α_IIbβ₃ signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO α_IIbβ₃D723H cells prevented protrusion formation on fibrinogen. Most interestingly, RhoA/ROCK inhibition was necessary, but not sufficient, and integrin activity was additionally required to induce CHO cell extension formation. Conclusions: CHO α_IIbβ₃D723H cell protrusions and megakaryocyte proplatelets, like neuronal cell neurites, result from a common integrin‐dependent signaling pathway, promoting strongly decreased RhoA activity and leading to microtubule‐driven formation of cytoplasmic extensions.     

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.     

An αIIbβ3 antagonist prevents thrombosis without causing Fc receptor γ‐chain IIa‐mediated thrombocytopenia

Essentials

• FcγRIIa‐mediated thrombocytopenia is associated with drug‐dependent antibodies (DDAbs).
• We investigated the correlation between α_IIbβ₃ binding epitopes and induction of DDAbs.
• An FcγRIIa‐transgenic mouse model was used to evaluate thrombocytopenia among anti‐thrombotics.
• An antithrombotic with binding motif toward α_IIbβ‐propeller domain has less bleeding tendency.

Summary

Background

Thrombocytopenia, a common side effect of Arg‐Gly‐Asp‐mimetic antiplatelet drugs, is associated with drug‐dependent antibodies (DDAbs) that recognize conformation‐altered integrin α_IIbβ₃.

Objective

To explore the correlation between α_IIbβ₃ binding epitopes and induction of DDAb binding to conformation‐altered α_IIbβ₃, we examined whether two purified disintegrins, TMV‐2 and TMV‐7, with distinct binding motifs have different effects on induction of α_IIbβ₃ conformational change and platelet aggregation in the presence of AP2, an IgG₁ inhibitory mAb raised against α_IIbβ₃.

Methods

We investigated the possible mechanisms of intrinsic platelet activation of TMV‐2 and TMV‐7 in the presence of AP2 by examining the signal cascade, tail bleeding time and immune thrombocytopenia in Fc receptor γ‐chain IIa (FcγRIIa) transgenic mice.

Results

TMV‐7 has a binding motif that recognizes the α_IIb β‐propeller domain of α_IIbβ₃, unlike that of TMV‐2. TMV‐7 neither primed the platelets to bind ligand, nor caused a conformational change of α_IIbβ₃ as identified with the ligand‐induced binding site mAb AP5. In contrast to eptifibatide and TMV‐2, cotreatment of TMV‐7 with AP2 did not induce FcγRIIa‐mediated platelet aggregation and the downstream activation cascade. Both TMV‐2 and TMV‐7 efficaciously prevented occlusive thrombosis in vivo. Notably, both eptifibatide and TMV‐2 caused severe thrombocytopenia mediated by FcγRIIa, prolonged tail bleeding time in vivo, and repressed human whole blood coagulation indexes, whereas TMV‐7 did not impair hemostatic capacity.

Conclusions

TMV‐7 shows antiplatelet and antithrombotic activities resulting from a mechanism different from that of all other tested α_IIbβ₃ antagonists, and may offer advantages as a therapeutic agent with a better safety profile.     

A mutation in the β3 cytoplasmic tail causes variant Glanzmann thrombasthenia by abrogating transition of αIIbβ3 to an active state

Summary. Background: The cytoplasmic tails of α_IIb and β₃ regulate essential α_IIbβ₃ functions. We previously described a variant Glanzmann thrombasthenia mutation in the β₃ cytoplasmic tail, IVS14: ?3C>G, which causes a frameshift with an extension of β₃ by 40 residues. Objectives: The aim of this study was to characterize the mechanism by which the mutation abrogates transition of α_IIbβ₃ from a resting state to an active state. Methods: We expressed the natural mutation, termed 742ins, and three artificial mutations in baby hamster kidney (BHK) cells along with wild‐type (WT) α_IIb as follows: β₃‐742stop, a truncated mutant to evaluate the effect of deleted residues; β₃‐749stop, a truncated mutant that preserves the NPLY conserved sequence; and β₃‐749ins, in which the aberrant tail begins after the conserved sequence. Flow cytometry was used to determine ligand binding to BHK cells. Results and conclusions: Surface expression of α_IIbβ₃ of all four mutants was at least 60% of WT expression, but there was almost no binding of soluble fibrinogen following activation with activating antibodies (anti‐ligand‐induced‐binding‐site 6 [antiLIBS6] or PT25‐2). Activation of the α_IIbβ₃ mutants was only achieved when both PT25‐2 and antiLIBS6 were used together or following treatment with dithiothreitol. These data suggest that the ectodomain of the four mutants is tightly locked in a resting conformation but can be forced to become active by strong stimuli. These data and those of others indicate that the middle part of the β₃ tail is important for maintaining α_IIbβ₃ in a resting conformation.     

Triple heterozygosity in the integrin αIIb subunit in a patient with Glanzmann's thrombasthenia

Summary. We report triple heterozygosity in the integrin α_IIb subunit in a 5‐year‐old Canadian girl with Glanzmann's thrombasthenia. The patient has a severe bleeding history possibly aggravated by low VWF suggestive of associated type 1 von Willebrand's disease. Platelet aggregation was absent or severely reduced for all physiologic agonists. Flow cytometry showed an ～ 4% residual surface expression of α_IIbβ₃. Western blotting confirmed a low platelet expression of both subunits. PCR‐SSCP and direct sequencing showed no abnormalities in the β₃ gene, but revealed a G→A transition at a splice site [IVS 19 (+1)] of exon 19 in the α_IIb gene. Of maternal inheritance, the splice site mutation was associated with intermediate levels of α_IIbβ₃ in carriers. Unexpectedly, two G→A transitions were detected in exon 29 of the α_IIb gene and led to V⁹⁵¹→M and A⁹⁵⁸→T amino acid substitutions. Family studies using restriction enzymes showed that both exon 29 mutations were paternal in origin and cosegregated across three generations. Transient expression in which mutated α_IIb was cotransfected with wild‐type β₃ in COS‐7 cells showed that V⁹⁵¹→M gave a much reduced surface expression of α_IIbβ₃ and a block in the maturation of pro‐α_IIb. In contrast, the A⁹⁵⁸ substitution appeared to be a novel polymorphism. Our studies highlight an unusual mixture of defects giving rise to severe bleeding in a child and describe the first pathological missense mutation affecting a C‐terminal residue of the calf‐2 domain of α_IIb.     

RGD‐ligand mimetic antagonists of integrin αIIbβ3 paradoxically enhance GPVI‐induced human platelet activation

Summary. Background: The integrin α_IIbβ₃ is the major mediator of platelet aggregation and has, therefore, become an important target of antithrombotic therapy. Antagonists of α_IIbβ₃, for example abciximab, tirofiban and eptifibatide, are used in the treatment of acute coronary syndromes. However, in addition to effective blockade of the integrin, binding of can induce conformational changes in the integrin and can also induce integrin clustering. This class effect of RGD‐ligand mimetics might, therefore, underlie paradoxical platelet activation and thrombosis previously reported. Objectives: To examine the components of signaling pathways and functional responses in platelets that may underlie this phenomenon of paradoxical platelet activation. Methods: We assessed the effect of lotrafiban, and other α_IIbβ₃ antagonists including the clinically used drug tirofiban, on tyrosine phosphorylation of key signaling proteins in platelets by immunoblotting and also platelet functional outputs such as cytosolic calcium responses, phosphatidylserine exposure (pro‐coagulant activity) and dense granule release. Results: In all cases, no effect of α_IIbβ₃ antagonists were observed on their own, but these integrin antagonists did lead to a marked potentiation of glycoprotein VI (GPVI)‐associated FcR γ‐chain phosphorylation, activation of Src family kinases and Syk kinase. This correlated with increased dense granule secretion, cytosolic calcium response and exposure of phosphatidylserine on the platelet surface. P2Y₁₂ antagonism abolished the potentiated phosphatidylserine exposure and dense granule secretion but not the cytosolic calcium response. Conclusions: These data provide a mechanism for enhancement of platelet activity by α_IIbβ₃ inhibitors, but also reveal a potentially important signaling pathway operating from the integrin to GPVI signaling.     

CD40 ligand shedding is regulated by interaction between matrix metalloproteinase‐2 and platelet integrin αIIbβ3

Summary. Background: CD40 ligand (CD40L, CD154) in the circulatory system is mainly contained in platelets, and surface‐expressed CD40L on activated platelets is subsequently cleaved by proteolytic activity to generate soluble CD40L (sCD40L). However, the enzyme responsible for the shedding of CD40L in activated platelets has not been clearly identified yet. We have recently found that molecular interaction of matrix metalloproteinase‐2 (MMP‐2) with integrin α_IIbβ₃ is required for the enhancement of platelet activation. Objectives: To elucidate the biochemical mechanism of MMP‐2‐associated sCD40L release. Methods: Localization of MMP‐2 and CD40L in platelets was analyzed by flow cytometry and fluorescence microscopy. The release of sCD40L from activated platelets was measured by enzyme‐linked immunosorbent assay. MMP‐2 binding to α_IIbβ₃ was analyzed by immunoprecipitation and western blotting. Recombinant hemopexin‐like domain and MMP‐2‐specific inhibitor were used to characterize the nature of MMP‐2 binding and catalytic activity. Results: It was revealed that interaction of MMP‐2 with α_IIbβ₃ is required for effective production of sCD40L in activated human platelets. Platelet activation and release of sCD40L were significantly affected by inhibition of platelet‐derived MMP‐2 activity or by inhibition of binding between the enzyme and the integrin. It was also found in platelet‐rich plasma that MMP‐2 activity is responsible for generating sCD40L. Conclusions: The results presented here strongly suggest that MMP‐2 interacts with α_IIbβ₃ to regulate the shedding of CD40L exposed on the surfaces of activated human platelets.     

A role for adhesion and degranulation‐promoting adapter protein in collagen‐induced platelet activation mediated via integrin α2β1

Summary. Background: Collagen‐induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α₂β₁. Adhesion and degranulation‐promoting adapter protein (ADAP) regulates α_IIbβ₃ in platelets and α_Lβ₂ in T cells, and is phosphorylated in GPVI‐deficient platelets activated by collagen. Objectives: To determine whether ADAP plays a role in collagen‐induced platelet activation and in the regulation and function of α₂β₁. Methods: Using ADAP^?/? mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation. Results and Conclusions: Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP^?/? platelets. However, aggregation and signaling induced by collagen‐related peptide (CRP), a GPVI‐selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α₂β₁‐selective ligand GFOGER and to a peptide (III‐04), which supports adhesion that is dependent on both GPVI and α₂β₁, was reduced in ADAP^?/? platelets. An impedance‐based label‐free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non‐fluorescent differential‐interference contrast microscopy, which revealed reduced filpodia formation in ADAP^?/? platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen‐binding integrin α₂β₁. In addition, we found that ADAP^?/? mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild‐type animals. This may reflect increased removal of platelets from the circulation.     

What is vinculin needed for in platelets?

Summary. Background: Vinculin links integrins to the cell cytoskeleton by virtue of its binding to proteins such as talin and F‐actin. It has been implicated in the transmission of mechanical forces from the extracellular matrix to the cytoskeleton of migrating cells. Vinculin’s function in platelets is unknown. Objective: To determine whether vinculin is required for the functions of platelets and their major integrin, α_IIbβ₃. Methods: The murine vinculin gene (Vcl) was deleted in the megakaryocyte/platelet lineage by breeding Vcl fl/fl mice with Pf4–Cre mice. Platelet and integrin functions were studied in vivo and ex vivo. Results: Vinculin was undetectable in platelets from Vcl fl/fl Cre⁺ mice, as determined by immunoblotting and fluorescence microscopy. Vinculin‐deficient megakaryocytes exhibited increased membrane tethers in response to mechanical pulling on α_IIbβ₃ with laser tweezers, suggesting that vinculin helps to maintain membrane cytoskeleton integrity. Surprisingly, vinculin‐deficient platelets displayed normal agonist‐induced fibrinogen binding to α_IIbβ₃, aggregation, spreading, actin polymerization/organization, clot retraction and the ability to form a procoagulant surface. Furthermore, vinculin‐deficient platelets adhered to immobilized fibrinogen or collagen normally, under both static and flow conditions. Tail bleeding times were prolonged in 59% of vinculin‐deficient mice. However, these mice exhibited no spontaneous bleeding and they formed occlusive platelet thrombi comparable to those in wild‐type littermates in response to carotid artery injury with FeCl₃. Conclusion: Despite promoting membrane cytoskeleton integrity when mechanical force is applied to α_IIbβ₃, vinculin is not required for the traditional functions of α_IIbβ₃ or the platelet actin cytoskeleton.     

Phorbol ester‐evoked Ca2+signaling in human platelets is via autocrine activation of P2X1 receptors,not a novel non‐capacitative Ca2+entry

Summary. Background: Platelets are reported to possess a protein kinase C (PKC)‐dependent non‐capacitative Ca²⁺entry (NCCE) pathway. The phorbol ester, phorbol, 12‐myristate, 13‐acetate (PMA) has been suggested to stimulate platelet NCCE. Recently we demonstrated important roles in store‐operated Ca²⁺entry in human platelets for Na⁺/Ca²⁺ exchangers (NCXs) and autocrine signaling between platelets after dense granule secretion. As PMA evokes dense granule secretion, we have investigated the role of NCXs and autocrine signaling in PMA‐evoked Ca²⁺entry. Objectives: To investigate the roles of NCXs and dense granule secretion in PMA‐evoked Ca²⁺signaling in human platelets. Methods: Fura‐2‐ or sodium‐binding benzofuran isophthalate (SBFI)‐loaded platelets were used to monitor cytosolic Ca²⁺or Na⁺ concentrations. Dense granule secretion was monitored as ATP release using luciferin–luciferase. Results: The NCX inhibitors KB‐R7943 or SN‐6, and removal of extracellular Na⁺, significantly reduced PMA‐evoked Ca²⁺entry. PMA‐evoked dense granule secretion was almost abolished by pretreatment with the PKC inhibitor Ro‐31‐8220 and significantly slowed by KB‐R7943. The P_2X1 antagonists Ro‐0437626 or MRS‐2159, or desensitization of P_2X1 receptors by prior treatment with α,β‐Methylene‐ATP or omitting apyrase from the medium, reduced PMA‐evoked Ca²⁺entry. Ro‐0437626 or chelation of extracellular Ca²⁺ slowed but did not abolish PMA‐evoked ATP release, indicating that PMA‐evoked dense granule secretion does not require P_2X1 receptor activation but is accelerated by P_2X1‐mediated Ca²⁺entry. The presence of NCX3 in human platelets was demonstrated by Western blotting. Conclusion: PMA‐evoked Ca²⁺entry results from an NCX3‐dependent dense granule secretion and subsequent P_2X1 receptor activation by secreted ATP, rather than activation of a novel NCCE pathway.     

Platelet‐based coagulation: different populations,different functions

Summary. Platelets in a thrombus interact with (anti)coagulation factors and support blood coagulation. In the concept of cell‐based control of coagulation, three different roles of platelets can be distinguished: control of thrombin generation, support of fibrin formation, and regulation of fibrin clot retraction. Here, we postulate that different populations of platelets with distinct surface properties are involved in these coagulant functions. Platelets with elevated Ca²⁺ and exposed phosphatidylserine control thrombin and fibrin generation, while platelets with activated α_IIbβ₃ regulate clot retraction. We review how coagulation factor binding depends on the platelet activation state. Furthermore, we discuss the ligands, platelet receptors and downstream intracellular signaling pathways implicated in these coagulant functions. These insights lead to an adapted model of platelet‐based coagulation.     

Signaling role of CD36 in platelet activation and thrombus formation on immobilized thrombospondin or oxidized low‐density lipoprotein

Summary. Background and Objective: Platelets abundantly express glycoprotein CD36 with thrombospondin‐1 (TSP1) and oxidized low‐density lipoprotein (oxLDL) as proposed ligands. How these agents promote platelet activation is still poorly understood. Methods and Results: Both TSP1 and oxLDL caused limited activation of platelets in suspension. However, immobilized TSP1 and oxLDL, but not LDL, strongly supported platelet adhesion and spreading with a major role of CD36. Platelet spreading was accompanied by potent Ca²⁺ rises, and resulted in exposure of P‐selectin and integrin activation, all in a CD36‐dependent manner with additional contributions of α_IIbβ₃ and ADP receptor stimulation. Signaling responses via CD36 involved activation of the protein tyrosine kinase Syk. In whole blood perfusion, co‐coating of TSP1 or oxLDL with collagen enhanced thrombus formation at high‐shear flow conditions, with increased expression on platelets of activated α_IIbβ₃, P‐selectin and phosphatidylserine, again in a CD36‐dependent way. Conclusions: Immobilized TSP1 and oxLDL activate platelets partly via CD36 through a Syk kinase‐dependent Ca²⁺ signaling mechanism, which enhances collagen‐dependent thrombus formation under flow. These findings provide novel insight into the role of CD36 in hemostasis.     

Platelet activation by dimeric β2-glycoprotein I requires signaling via both glycoprotein Ibα and apolipoprotein E receptor 2'

Summary. Background: Dimerization of β₂‐glycoprotein I (β₂‐GPI) by autoantibodies is thought to trigger the clinical manifestations observed in the antiphospholipid syndrome. Arterial thrombosis, a frequently occurring clinical manifestation of the antiphospholipid syndrome, is a process in which platelets play a crucial role. Previous work has shown that binding of dimeric β₂‐GPI to the platelet receptors apolipoprotein E receptor 2′ (ApoER2′) and glycoprotein Ibα (GPIbα) mediates increased platelet activation in an in vitro thrombosis model. Objective: The individual roles of ApoER2′ and GPIbα in mediating platelet activation by dimeric β₂‐GPI has hitherto been unclear. In this study, we have determined the roles of either receptor in platelet activation by dimeric β₂‐GPI. Methods: Platelet activation by dimeric β₂‐GPI was studied under conditions of flow. Intracellular signaling induced by dimeric β₂‐GPI was subsequently analyzed by means of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and western blot analysis. Results: The increase in platelet deposition onto a fibronectin surface under conditions of flow by dimeric β₂‐GPI was completely abolished by inhibition of the interaction of dimeric β₂‐GPI with either GPIbα or ApoER2′. Upon platelet stimulation with dimeric β₂‐GPI, GPIbα translocated to the cytoskeleton via the scaffold protein 14‐3‐3ζ. Concomitantly, ApoER2′ dissociated from the adapter protein Disabled1, presumably through phosphorylation of the cytoplasmic tail. Inhibition of one process could not inhibit the other. Conclusion: We show that dimeric β₂‐GPI signals via two distinct pathways in platelets, both of which are required for platelet activation. Abrogation of either signal results in loss of activation.     

Bleeding tendency and impaired platelet function in a patient carrying a heterozygous mutation in the thromboxane A2 receptor

Summary. Background: Thromboxane A₂ receptor (TXA₂R) abnormality appears to dominantly disturb platelet function. Objectives: To reveal a molecular genetic defect in a patient with TXA₂R abnormality and investigate the mechanism for the impaired response to TXA₂. Patient: The proband (OSP‐2, PT) was a 7‐year‐old Japanese girl, suffering from repeated mucocutaneous bleeding. Methods and results: U46619 (2.5 and 10 μm )‐induced platelet aggregation was remarkably impaired in the proband and her father. Immunoblots showed that TXA₂R expression levels in their platelets were approximately 50% of controls, and nucleotide sequence analysis revealed that they were heterozygous for a novel mutation, c.167dupG in the TXA₂R cDNA. Expression studies using Chinese hamster ovary (CHO) cells indicated that the mutation is responsible for the expression defect in TXA₂R. We then examined α_IIbβ₃ activation by employing an initial velocity analysis and revealed that U46619 failed to induce a sustained α_IIbβ₃ and Rap1B activation in the proband. In addition, platelet secretion as monitored by P‐selectin expression was markedly impaired in response to U46619 but not to ADP. The interaction between secreted ADP and P2Y₁₂ has been shown to play a critical role in the sustained α_IIbβ₃ activation (Kamae et al. J Thromb Haemost 2006; 4 : 1379). As expected, small amounts of exogenous ADP (0.5 μm ) partially restored the sustained α_IIbβ₃ activation induced by U46619. Conclusion: Our present data strongly suggest that the impaired platelet activation in response to U46619 in the heterozygous subject for the TXA₂R mutation is, at least in part, as a result of the decrease in ADP secretion.