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.     

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.     

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.     

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.     

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.     

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.     

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.     

Cytoplasmic regions of the β3 subunit of integrin αIIbβ3 involved in platelet adhesion on fibrinogen under flow conditions

Summary.  Platelet adhesion to surface-bound fibrinogen depends on integrin α_IIbβ₃. In the present study, we investigated the role of the regions ⁷⁴⁹EATSTFT⁷⁵⁶N and ⁷⁵⁵TNITYRG⁷⁶²T of the β₃ cytoplasmic tail in the regulation of platelet adhesion under flow conditions, by introducing peptide mimetics in platelets. Introduction of peptide EATSTFTN (E–N) increased surface coverage by 35%, an effect caused by 25% more adhesion. In contrast, peptide TNITYRGT (T–T) decreased surface coverage by 16%, as a result of 25% less adhesion. An S→P substitution in the E–N peptide, thereby mimicking a mutation in Glanzmann's thrombasthenia, abolished the effect of E–N. A suboptimal concentration of cytochalasin D is known to enhance ligand binding to α_IIbβ₃ in platelet suspensions. Under flow, cytochalasin D (1 µmol L⁻¹) induced 50% more platelet adhesion, with a strong reduction in platelet spreading. Both peptides opposed the increase in adhesion by cytochalasin D and partly (E–N) and completely (T–T) restored platelet spreading. Thus, the ⁷⁴⁹EATSTFT⁷⁵⁶N and ⁷⁵⁵TNITYRG⁷⁶²T regions of β₃ contribute to the regulation of αIIbβ3 anchorage to the cytoskeleton and platelet spreading to an adhesive surface.     

Immunologic and structural analysis of eight novel domain-deletion β3 integrin peptides designed for detection of HPA-1 antibodies

Summary. Background: The single‐nucleotide polymorphism (SNP) rs5918 in the ITGB3 gene defines the human platelet antigen‐1 (HPA‐1) system encoding a Leu (HPA‐1a) or Pro (HPA‐1b) at position 33. HPA‐1 antibodies are clinically the most relevant in the Caucasoid population, but detection currently requires α_IIbβ₃ integrin from the platelets of HPA‐genotyped donors.Objectives: We set out to define the β₃ integrin domains required for HPA‐1a antibody binding and produce recombinant soluble β₃ peptides for HPA‐1 antibody detection.Methods: We designed two sets (1a and 1b) of four soluble β₃ domain‐deletion peptides (ΔSDL, ΔβA, PSIHybrid, PSI), informed by crystallography studies and computer modeling. The footprints of three human HPA‐1a‐specific phage antibodies were defined by analyzing binding patterns to the β₃ peptides and canine platelets, and models of antibody–antigen interfaces were derived. Specificity and sensitivity for HPA‐1a detection were assessed using sera from 140 cases of fetomaternal alloimmune thrombocytopenia (FMAIT). Results: Fusion of recombinant proteins to calmodulin resulted in high‐level expression in Drosophila S2 cells of all eight β₃ peptides. Testing of FMAIT samples indicated that ΔβA‐Leu33 is the superior peptide for HPA‐1a antibody detection, with 96% sensitivity and 95% specificity. The existence of type I and II categories of HPA‐1a antibodies was confirmed by the study of HPA‐1a phage antibody footprints and the reactivity pattern of clinical samples with the four β₃‐Leu33 peptides, but there was no correlation between antibody category and clinical severity of FMAIT. Conclusions: Soluble recombinant β₃ peptides can be used for detection of clinical HPA‐1a antibodies.     

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.     

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.     

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.     

Role of P-selectin, β2-integrins, and Src tyrosine kinases in mouse neutrophil–platelet adhesion

Summary. Background: The initial interaction of human polymorphonuclear leukocytes (PMN) with activated human platelets is mediated by P‐selectin and its leukocyte ligand PSGL‐1; subsequently the interaction is strengthened by activation of αMβ2 via protein tyrosine phosphorylation mediated by Src kinases and binding of activated αMβ2 to its platelet counterreceptor(s). Objectives: Because mouse models are being used to define the role of PMN–platelet interactions in thrombosis and the response to vascular injury, we investigated the molecular determinants responsible for the interaction of murine PMNs with activated murine platelets. Methods: Mouse platelets were labeled with the green fluorescent dye BCECF and then activated with thrombin and fixed with 1% paraformaldehyde. Mouse PMNs were labeled with the red fluorescent dye hydroethidine and then stirred with the fixed platelets. After stopping the reaction with paraformaldehyde, formation of mixed cell conjugates was analyzed by flow cytometry. Results: In time course experiments, 90 ± 1.9% of PMNs formed mixed conjugates with platelets after 2 min and the mean (± SEM) number of platelets per positive PMN was 8.4 ± 1.5. A monoclonal antibody to P‐selectin reduced the percentage of PMNs with attached platelets to 16 ± 2.4% (P = 0.001), and only 8 ± 5% of PMNs interacted with platelets from P‐selectin?/? mice. In contrast, monoclonal antibodies to PSGL‐1, β₂‐integrin, and αIIbβ3 had much less or no effect on the production of mixed cell aggregates. To better identify a secondary contribution of β₂‐integrins, P‐selectin interactions were disrupted by briefly adding 5 mm EGTA to already‐formed mixed cell aggregates. Brief EGTA treatment alone reduced the percentage of PMNs with attached platelets to 70 ± 3.5% (P = 0.004 vs. no treatment), but did not modify the number of platelets per positive PMN (9.5 ± 1.7). The combination of brief EGTA treatment and a monoclonal antibody to β₂‐integrin lowered the percentage of PMN with attached platelets to 50 ± 7% and reduced the number of platelets attached per positive PMN to 3.6 ± 0.7 (P = 0.03 vs. brief EGTA treatment only). Brief EGTA treatment did not modify the effect of the other antibodies. When the incubation was stopped with EGTA the Src inhibitors PP1 and PP2 reduced PMN–platelet adhesion, while the inactive analog PP3 was ineffective. Conclusions: These results confirm that P‐selectin plays a prominent role in mediating the initial interactions between mouse PMN and platelets, and provide support for additional contributions from β₂‐integrins and Src family kinases.     

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.     

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.     

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.     

β3-adrenoceptors and intestinal motility

Summary— Early substantial evidence of the low susceptibility to β-adrenoceptor antagonists of non α-adrenergic responses reducing gut motility and tone was reluctantly accepted as indicating a third β-receptor subtype different from the β₁ and β₂. This applied likewise to lipolysis until new selective “lipolytic” β-agonists poorly effective at established β-receptors were introduced. Shortly afterwards these “lipolytic” as well as certain newer and even more selective β-adrenoceptor agonists were shown to be potent inhibitors of intestinal motility. The latter are the “gut-specific” phenylethanolaminotetralins whose availability as pure isomers attested to the stringent stereochemical requirements for selectivity at non-β₁, non-β₂ β-adrenoceptors. Acceptance of the functionally based concept of a β₃-adrenoceptor was boosted on structural grounds by molecular biology studies. Sequence analysis indicated the existence in humans and rodents of genes coding for a third subtype of β-receptor that, when expressed in transfected heterologous cells, had a pharmacological profile distinct from the previously established subtypes. Finally, aryloxypropanolaminotetralins have been prepared as the first selective antagonists of β₃-adrenoceptors, thus providing unambiguous conclusive evidence of the distinctive functional features of those abundant in the rat colon. The therapeutic potential in gastroenterology of the newer compounds targetable on the β₃-adrenoceptor is suggested by their potent intestinal action in vivo in animal models without any of the cardiovascular or other unwanted effects of conventional β₃-adrenoceptor agonists and antagonists, and by the clinically confirmed importance of β-adrenergic control of motor function throughout the alimentary canal. However, open questions include the incidence of species-related differences in β₃-adrenoceptors, and as yet there are no data on gastrointestinal functions in humans under the influence of drugs designed to act selectively at these receptors.     

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.     

The new platelet alloantigen Caba: a single point mutation Gln716His on the α2 integrin

BACKGROUND: Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloimmunization against fetal platelet (PLT) antigens, inherited from the father and absent from maternal PLTs. STUDY DESIGN AND METHODS: A 29‐year‐old mother gave birth to a severely thrombocytopenic newborn (16 × 10⁹ PLTs/L) leading to PLT transfusion therapy associated with intravenous immunoglobulins. The outcome was uneventful. Maternal serum showed a specific positive reaction with the antigen‐capture assay (monoclonal antibody [MoAb]‐specific immobilization of PLT antigens) only when it was tested with the paternal PLTs and a panel of MoAbs against glycoprotein (GP)Ia‐IIa (α₂β₁ integrin) suggesting the implication of a new PLT antigen. RESULTS: Nucleotide sequence analysis of GPIa cDNA of the father and newborn showed a nucleotide substitution at position 2235 (2235G > T according to the international nomenclature). This substitution induces a Q716H amino acid change in the GPIa mature protein, located outside the I domain involved in cell adhesion for collagen. In vitro analysis of recombinant Chinese hamster ovary (CHO) cells expressing wild‐type or mutant (Q716H) human GPIa allowed us to demonstrate that this single amino acid substitution is responsible and sufficient for inducing Cab^a antigen expression. Adhesion of CHO cells to collagen was not modified by the Cab polymorphism, nor by the maternal anti‐Cab^a alloantibodies, indicating that the mutation does not affect the function of integrin α₂β₁. In a Caucasian population study, none of the 104 unrelated blood donors was found to be Cab^a(+). CONCLUSION: We describe here a new PLT alloantigen Cab^a involved in a severe case of FNAIT. Laboratory investigation for the “common” PLT alloantigens is no longer sufficient to evaluate neonatal alloimmune thrombocytopenia in suspected cases.     

Triiodothyronine and amiodarone effects on β3-adrenoceptor density and lipolytic response to the β3-adrenergic agonist BRL 37344 in rat white adipocytes

Summary— The β-adrenergic effects of catecholamines are potentiated by thyroid hormones in adipose tissue. Amiodarone (AM) is structurally similar to thyroid hormones and was used to explore the mechanism of the triiodothyronine (T₃) effect on β-adrenergic receptors (β-ARs) in adipose tissue. AM decreases the expression of some T₃ sensitive genes in various tissues and antagonizes the effect of T₃ on its nuclear receptors. In this study, the T₃, AM and AM + T₃ effects on the β1- and β₃-AR density were assessed on rat white adipocytes by radioligand binding using [³H]CGP 12177 after characterization of these subtypes by displacement of [³H]CGP 12177 binding by isoproterenol, BRL 37344 and noradrenaline. BRL 37344 was used to study β₃-AR lipolysis. White adipocytes from hyperthyroid rats had increased responsiveness (E_max × 2) and sensitivity (+ 38%) to BRL 37344, while those given AM alone had decreased values. Moreover, AM antagonized the T₃ effect on lipolysis. The β₁-binding characteristics (receptor density [B_max]: 45 ± 4 fmol/mg of proteins; dissociation factor [K_d]: 0.96 ± 0.10 nM) were not modified by either compound. Finally, T₃ significantly increased β₃-AR density (587 ± 69 versus 363 ± 25 fmol/mg of proteins) and K_d (38 ± 2 versus 23 ± 3 nM), while AM alone had no effect and did not antagonize the T₃ effect on β₃-AR number. In conclusion, the hyperthyroid state in the rat potentiated the lipolytic response of white adipocytes to a specific β₃-agonist and increased the β₃-AR density without changing in β₁-AR number and affinity. Furthermore, the lack of antagonism between AM and T₃ on β₃-AR expression suggests that T₃ does not work directly on the β₃-AR gene. Moreover, AM induced a functional tissular hypothyroid-like effect and its antilipolytic effect probably occurred at a postreceptor level.