Compression force sensing regulates integrin ?IIb?3 adhesive function on diabetic platelets.
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ABSTRACT:
SUBMITTER: Ju L
PROVIDER: S-EPMC5852038 | biostudies-literature | 2018 Mar
REPOSITORIES: biostudies-literature
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Project description:The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin ?IIb?3, raising the possibility that it participates in integrin ?IIb?3 outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin ?IIb?3-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P2 accumulation. Although agonist-induced integrin ?IIb?3 affinity regulation was unaltered in Dok-2(-/-) platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin ?IIb?3 adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays.
| S-EPMC3931064 | biostudies-literature
Project description:Using direct injection mass spectrometry (DIMS) we discovered that deoxyribose-1-phosphate (dRP) is released by platelets upon activation. Interestingly, the addition of exogenous dRP to human platelets significantly increased platelet aggregation and integrin ?IIb?3 activation in response to thrombin. In parallel, genetically modified platelets with double genetic deletion of thymidine phosphorylase and uridine phosphorylase were characterised by reduced release of dRP, impaired aggregation and decreased integrin ?IIb?3 activation in response to thrombin. In vitro platelet adhesion onto fibrinogen and collagen under physiological flow conditions was potentiated by treatment of human platelets with exogenous dRP and impaired in transgenic platelets with reduced dRP release. Human and mouse platelets responded to dRP treatment with a sizeable increase in reactive oxygen species (ROS) generation and the pre-treament with the antioxidant apocynin abolished the effect of dRP on aggregation and integrin activation. Experiments directly assessing the activation of the small G protein Rap1b and protein kinase C suggested that dRP increases the basal levels of activity of these two pivotal platelet-activating pathways in a redox-dependent manner. Taken together, we present evidence that dRP is a novel autocrine amplifier of platelet activity, which acts on platelet redox levels and modulates integrin ?IIb?3.
| S-EPMC6082634 | biostudies-literature
Project description:BackgroundBidirectional integrin ?IIb?3 signaling is essential for platelet activation. The platelet adaptor protein Disabled-2 (Dab2) is a key regulator of integrin signaling and is phosphorylated at serine 24 in eukaryotic cells. However, the mechanistic insight and function of Dab2-serine 24 phosphorylation (Dab2-pSer24) in platelet biology are barely understood. This study aimed to define whether and how Dab2 is phosphorylated at Ser24 during platelet activation and to investigate the effect of Dab2-pSer24 on platelet function.ResultsAn antibody with confirmed specificity for Dab2-pSer24 was generated. By using this antibody as a tool, we showed that protein kinase C (PKC)-mediated Dab2-pSer24 was a conservative signaling event when human platelets were activated by the platelet agonists such as thrombin, collagen, ADP, 12-O-tetradecanoylphorbol-13-acetate, and the thromboxane A2 activator U46619. The agonists-stimulated Dab2-pSer24 was attenuated by pretreatment of platelets with the RGDS peptide which inhibits integrin outside-in signaling by competitive binding of integrin ?IIb with fibrinogen. Direct activation of platelet integrin outside-in signaling by combined treatment of platelets with manganese dichloride and fibrinogen or by spreading of platelets on fibrinogen also resulted in Dab2-pSer24. These findings implicate that Dab2-pSer24 was associated with the outside-in signaling of integrin. Further analysis revealed that Dab2-pSer24 was downstream of Src-PKC-axis and phospholipase D1 underlying the integrin ?IIb?3 outside-in signaling. A membrane penetrating peptide R11-Ser24 which contained 11 repeats of arginine linked to the Dab2-Ser24 phosphorylation site and its flanking sequences (RRRRRRRRRRR19APKAPSKKEKK29) and the R11-S24A peptide with Ser24Ala mutation were designed to elucidate the functions of Dab2-pSer24. R11-Ser24 but not R11-S24A inhibited agonists-stimulated Dab2-pSer24 and consequently suppressed platelet spreading on fibrinogen, with no effect on platelet aggregation and fibrinogen binding. Notably, Ser24 and the previously reported Ser723 phosphorylation (Dab2-pSer723) occurred exclusively in a single Dab2 molecule and resulted in distinctive subcellular distribution and function of Dab2. Dab2-pSer723 was mainly distributed in the cytosol of activated platelets and associated with integrin inside-out signaling, while Dab2-pSer24 was mainly distributed in the membrane fraction of activated platelets and associated with integrin outside-in signaling.ConclusionsThese findings demonstrate for the first time that Dab2-pSer24 is conservative in integrin ?IIb?3 outside-in signaling during platelet activation and plays a novel role in the control of cytoskeleton reorganization and platelet spreading on fibrinogen.
| S-EPMC7869483 | biostudies-literature
Project description:Tight regulation of integrin affinity is critical for hemostasis. A final step of integrin activation is talin binding to 2 sites within the integrin ? cytoplasmic domain. Binding of talin to a membrane-distal NPxY sequence facilitates a second, weaker interaction of talin with an integrin membrane-proximal region (MPR) that is critical for integrin activation. To test the functional significance of these distinct interactions on platelet function in vivo, we generated knock-in mice expressing talin1 mutants with impaired capacity to interact with the ?3 integrin MPR (L325R) or NPLY sequence (W359A). Both talin1(L325R) and talin1(W359A) mice were protected from experimental thrombosis. Talin1(L325R) mice, but not talin(W359A) mice, exhibited a severe bleeding phenotype. Activation of ?IIb?3 was completely blocked in talin1(L325R) platelets, whereas activation was reduced by approximately 50% in talin1(W359A) platelets. Quantitative biochemical measurements detected talin1(W359A) binding to ?3 integrin, albeit with a 2.9-fold lower affinity than wild-type talin1. The rate of ?IIb?3 activation was slower in talin1(W359A) platelets, which consequently delayed aggregation under static conditions and reduced thrombus formation under physiological flow conditions. Together our data indicate that reduction of talin-?3 integrin binding affinity results in decelerated ?IIb?3 integrin activation and protection from arterial thrombosis without pathological bleeding.
| S-EPMC3999757 | biostudies-literature
Project description:Thromboembolic events (TEE) underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs) functionalized with bitistatin (Bit), a disintegrin that specifically binds to the ?IIb?3 integrin, platelet fibrinogen receptor (PFR) on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP-Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, ?IIb?3 receptor antagonist, specifically blocked F-NDP-Bit-PFR complex formation. Moreover, F-NDP-Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP). Our results suggest that engineered F-NDP-Bit particles could serve as noninvasive, "real-time" optical diagnostics for clots present in blood vessels.
| S-EPMC5440039 | biostudies-literature
Project description:Binding sites on glycoprotein (GP) IIb/IIIa exposed by 0.5 unit/ml alpha-thrombin are insensitive to prostaglandin I2 (PGI2), in contrast with sites exposed by ADP or platelet-activating factor. Here we show that the thrombin receptor agonist peptide (TRAP) (SFLLRN; 15 microM) opens almost the same number of GPIIb/IIIa molecules as 0.5 unit/ml alpha-thrombin (64840 +/- 8920 compared with 81050 +/- 6030 molecules of fibronectin bound/platelet), but these sites rapidly close on addition of PGI2. To investigate whether alpha-thrombin and TRAP initiate different signalling pathways, we measured phospholipase C (PLC)-mediated control of GPIIb/IIIa and its sensitivity to cyclic AMP. Optimal concentrations of alpha-thrombin and TRAP activated PLC maximally, but TRAP induced only about 50% protein kinase C PKC) activation after 10 min stimulation compared with alpha-thrombin. These concentrations also suppressed PGI2-induced cyclic AMP accumulation, with alpha-thrombin inducing complete inhibition and TRAP about 10% less. Direct activation of PKC by phorbol 12-myristate 13-acetate confirmed earlier observations that PGI2-induced cyclic AMP accumulation is partly inhibited via PKC. Applying different concentration of alpha-thrombin, TRAP or a combination of alpha-thrombin and the thrombin receptor inhibitory peptide (TRIP) (Mpr-F-Cha-Cha-RKPNDK-NH2; 800 microM) (Mpr, 3-mercaptopropionic acid; Cha, cyclohexylalanine), we show that the different means of stimulating the thrombin receptor all suppressed PGI2-induced cyclic AMP accumulation via (i) activation of PKC and (ii) activation of the heterotrimeric G-protein, Gi. We conclude that complete inhibition of cyclic AMP accumulation requires activation of both PKC and Gi, as observed with 0.5 unit/ml alpha-thrombin. Although TRAP almost fully exposes GPIIb/IIIa, its activation of PKC is incomplete, enabling PGI2 to raise cyclic AMP concentration from 1.4 +/- 0.7 to 4.1 +/- 1.3 nmol/10(11) platelets (P < 0.005) which is sufficient to close exposed GPIIb/IIIa molecules.
| S-EPMC1135775 | biostudies-other
Project description:OBJECTIVE:Kindlin-3 is a critical supporter of integrin function in platelets. Lack of expression of kindlin-3 protein in patients impairs integrin ?IIb?3-mediated platelet aggregation. Although kindlin-3 has been categorized as an integrin-binding partner, the functional significance of the direct interaction of kindlin-3 with integrin ?IIb?3 in platelets has not been established. Here, we evaluated the significance of the binding of kindlin-3 to integrin ?IIb?3 in platelets in supporting integrin ?IIb?3-mediated platelet functions. APPROACH AND RESULTS:We generated a strain of kindlin-3 knockin (K3KI) mice that express a kindlin-3 mutant that carries an integrin-interaction defective substitution. K3KI mice could survive normally and express integrin ?IIb?3 on platelets similar to their wild-type counterparts. Functional analysis revealed that K3KI mice exhibited defective platelet function, including impaired integrin ?IIb?3 activation, suppressed platelet spreading and platelet aggregation, prolonged tail bleeding time, and absence of platelet-mediated clot retraction. In addition, whole blood drawn from K3KI mice showed resistance to in vitro thrombus formation and, as a consequence, K3KI mice were protected from in vivo arterial thrombosis. CONCLUSIONS:These observations demonstrate that the direct binding of kindlin-3 to integrin ?IIb?3 is involved in supporting integrin ?IIb?3 activation and integrin ?IIb?3-dependent responses of platelets and consequently contributes significantly to arterial thrombus formation.
| S-EPMC4167429 | biostudies-literature
Project description:It is currently believed that inactive tyrosine kinase c-Src in platelets binds to the cytoplasmic tail of the ?3 integrin subunit via its SH3 domain. Although a recent NMR study supports this contention, it is likely that such binding would be precluded in inactive c-Src because an auto-inhibitory linker physically occludes the ?3 tail binding site. Accordingly, we have re-examined c-Src binding to ?3 by immunoprecipitation as well as NMR spectroscopy. In unstimulated platelets, we detected little to no interaction between c-Src and ?3. Following platelet activation, however, c-Src was co-immunoprecipitated with ?3 in a time-dependent manner and underwent progressive activation as well. We then measured chemical shift perturbations in the (15)N-labeled SH3 domain induced by the C-terminal ?3 tail peptide NITYRGT and found that the peptide interacted with the SH3 domain RT-loop and surrounding residues. A control peptide whose last three residues where replaced with those of the ?1 cytoplasmic tail induced only small chemical shift perturbations on the opposite face of the SH3 domain. Next, to mimic inactive c-Src, we found that the canonical polyproline peptide RPLPPLP prevented binding of the ?3 peptide to the RT- loop. Under these conditions, the ?3 peptide induced chemical shift perturbations similar to the negative control. We conclude that the primary interaction of c-Src with the ?3 tail occurs in its activated state and at a site that overlaps with PPII binding site in its SH3 domain. Interactions of inactive c-Src with ?3 are weak and insensitive to ?3 tail mutations.
| S-EPMC4505490 | biostudies-literature
Project description:Adaptor proteins play a critical role in the assembly of signalling complexes after engagement of platelet receptors by agonists such as collagen, ADP and thrombin. Recently, using proteomics, the Dok (downstream of tyrosine kinase) adapter proteins were identified in human and mouse platelets. In vitro studies suggest that Dok-1 binds to platelet integrin ?3, but the underlying effects of Dok-1 on ?IIb?3 signalling, platelet activation and thrombosis remain to be elucidated. In the present study, using Dok-1-deficient (Dok-1-/-) mice, we determined the phenotypic role of Dok-1 in ?IIb?3 signalling. We found that platelets from Dok-1-/- mice displayed normal aggregation, activation of ?IIb?3 (assessed by binding of JON/A), P-selectin surface expression (assessed by anti-CD62P), and soluble fibrinogen binding. These findings indicate that Dok-1 does not affect "inside-out" platelet signalling. Compared with platelets from wild-type (WT) mice, platelets from Dok-1-/- mice exhibited increased clot retraction (p <?0.05 vs WT), increased PLC?2 phosphorylation, and enhanced spreading on fibrinogen after thrombin stimulation (p <?0.01 vs WT), demonstrating that Dok-1 negatively regulates ?IIb?3 "outside-in" signalling. Finally, we found that Dok-1-/- mice exhibited significantly shortened bleeding times and accelerated carotid artery thrombosis in response to photochemical injury (p <?0.05 vs WT mice). We conclude that Dok-1 modulates thrombosis and haemostasis by negatively regulating ?IIb?3 outside-in signalling.
| S-EPMC4854776 | biostudies-literature
Project description:Integrin-linked kinase (ILK) is an important signaling regulator that assembles into the heteroternary complex with adaptor proteins PINCH and parvin (termed the IPP complex). We recently reported that ILK is important for integrin activation in a Chinese hamster ovary (CHO) cell system. We previously established parental CHO cells expressing a constitutively active chimeric integrin (?IIb?6B?3) and mutant CHO cells expressing inactive ?IIb?6B?3 due to ILK deficiency. In this study, we further investigated the underlying mechanisms for ILK-dependent integrin activation. ILK-deficient mutant cells had trace levels of PINCH and ?-parvin, and transfection of ILK cDNA into the mutant cells increased not only ILK but also PINCH and ?-parvin, resulting in the restoration of ?IIb?6B?3 activation. In the parental cells expressing active ?IIb?6B?3, ILK, PINCH, and ?-parvin were co-immunoprecipitated, indicating the formation of the IPP complex. Moreover, short interfering RNA (siRNA) experiments targeting PINCH-1 or both ?- and ?-parvin mRNA in the parent cells impaired the ?IIb?6B?3 activation as well as the expression of the other components of the IPP complex. In addition, ILK mutants possessing defects in either PINCH or parvin binding failed to restore ?IIb?6B?3 activation in the mutant cells. Kindlin-2 siRNA in the parental cells impaired ?IIb?6B?3 activation without disturbing the expression of ILK. For CHO cells stably expressing wild-type ?IIb?3 that is an inactive form, overexpression of a talin head domain (THD) induced ?IIb?3 activation and the THD-induced ?IIb?3 activation was impaired by ILK siRNA through a significant reduction in the expression of the IPP complex. In contrast, overexpression of all IPP components in the ?IIb?3-expressing CHO cells further augmented THD-induced ?IIb?3 activation, whereas they did not induce ?IIb?3 activation without THD. These data suggest that the IPP complex rather than ILK plays an important role and supports integrin activation probably through stabilization of the active conformation.
| S-EPMC3871693 | biostudies-literature