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We studied the contribution of von Willebrand factor (vWF) into blood cell adhesion to collagen-coated surfaces in whole blood of healthy volunteers. Adhesion of blood cells to collagen I was measured at shear rate of 2300 sec -1 . The interaction of platelet GPIIb/IIIa receptor with vWF was blocked with monoclonal anti-GPIIb/IIIa antibodies. The degree of cell adhesion was quantified by measuring the intensity of scattered light after 15-min perfusion: in samples with blocked GPIIb/IIIa it decreased to 0.39±0.13 V vs 0.06±0.03 V in control samples (p=0.002). Under a fluorescence microscope, intensively stained structures consisting of vWF, platelets, and leukocytes attached to the collagen surface were observed. After blockade of GPIIb/IIIa, these structures were absent. Leukocyte recruitment at high shear rates is a time-dependent process sensitive to complex interaction of vWF, leukocytes, and platelets, in which the platelet GPIIb/IIIa receptor is essential., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Engagement of integrin αIIbβ3 promotes platelet-platelet interaction and stimulates outside-in signaling that amplifies activation. Protein kinase Cδ (PKCδ) is known to play an important role in platelet activation, but its role in outside-in signaling has not been established. In the present study, we determined the role of PKCδ and its signaling pathways in integrin αIIbβ3-mediated outside-in signaling in platelets using PKCδ-deficient platelets. Platelet spreading to immobilized fibrinogen resulted in PKCδ phosphorylation, suggesting that αIIbβ3 activation caused PKCδ activation. αIIbβ3-mediated phosphorylation of Akt was significantly inhibited in PKCδ -/- platelets, indicating a role of PKCδ in outside-in signaling. αIIbβ3-mediated PKCδ phosphorylation was inhibited by proline-rich tyrosine kinase 2 (Pyk2) selective inhibitor, suggesting that Pyk2 contributes to the regulation of PKCδ phosphorylation in outside-in signaling. Additionally, Src-family kinase inhibitor PP2 inhibited integrin-mediated Pyk2 and PKCδ phosphorylation. Lastly, platelet spreading was inhibited in PKCδ -/- platelets compared to the wild-type (WT) platelets, and clot retraction from PKCδ -/- platelets was markedly delayed, indicating that PKCδ is involved in the regulation of αIIbβ3-dependent interactivities with cytoskeleton elements. Together, these results provide evidence that PKCδ plays an important role in outside-in signaling, which is regulated by Pyk2 in platelets.

Binding of integrin alphaIIbbeta3 (αiibβ3) to its ligands is a highly restricted and regulated mechanism. Any modification of the protein structure yields a dysfunctional role, especially in a redox environment. Here, we examine the effect of nitrosative stress on the αiibβ3 reconstituted into nanodiscs. Using single molecule force spectroscopy, we measured the interaction between αiibβ3 and its ligand RGD and found that in the presence of exogenous nitric oxide (NO) two force regimes are generated: a low force regime of ~100pN indicating the presence of integrin in a normal status, and a broad spectrum of high force regime (~210-450pN) suggesting the protein modification/aggregation. By high resolution atomic force microscopy imaging, we demonstrate that both NO and nitrite (a stable product formed from NO) are involved in destabilizing the transmembrane protein complex leading to release of αiibβ3 from the lipid bilayer and protein aggregation. Our experimental setup opens new ways for testing in a membrane environment the effect of radical species on integrins under clinically relevant conditions., Competing Interests: Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Immune thrombocytopenia (ITP) is an autoimmune condition primarily induced by the loss of immune tolerance to the platelet glycoproteins. Here we develop a novel flow cytometry approach to analyze integrin α IIb β 3 functioning in ITP in comparison with Glanzmann thrombasthenia (GT) (negative control) and healthy pediatric donors (positive control). Continuous flow cytometry of Fura-Red-loaded platelets from whole hirudinated blood was used for the characterization of platelet responses to conventional activators. Calcium levels and fibrinogen binding were normalized to ionomycin-induced responses. Ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Platelets from all ITP patients had significantly higher cytosolic calcium concentration in the quiescent state compared to healthy donors (15 ± 5 nM vs. 8 ± 5 nM), but calcium increases in response to all activators were normal. Clustering analysis revealed two subpopulations of ITP patients: the subgroup with high fibrinogen binding (HFB), and the subgroup with low fibrinogen binding (LFB) (8% ± 5% for LFB vs. 16% ± 3% for healthy donors in response to ADP). GT platelets had calcium mobilization (81 ± 23 nM), fibrinogen binding (5.1% ± 0.3%) and thrombus growth comparable to the LFB subgroup. Computational modeling suggested phospholipase C-dependent platelet pre-activation for the HFB subgroup and lower levels of functional integrin molecules for the LFB group., Competing Interests: The authors declare no conflict of interest.

Scope: Platelet integrin αIIbβ3 is the key mediator of atherothrombosis. Supplementation of coenzyme Q10 (CoQ10), a fat-soluble molecule that exists in various foods, exerts protective cardiovascular effects. This study aims to investigate whether and how CoQ10 acts on αIIbβ3 signaling and thrombosis, the major cause of cardiovascular diseases., Methods and Results: Using a series of platelet functional assays in vitro, it is demonstrated that CoQ10 reduces human platelet aggregation, granule secretion, platelet spreading, and clot retraction. It is further demonstrated that CoQ10 inhibits platelet integrin αIIbβ3 outside-in signaling. These inhibitory effects are mainly mediated by upregulating cAMP/PKA pathway, where CoQ10 stimulates the A 2A adenosine receptor and decreases phosphodiesterase 3A phosphorylation. Moreover, CoQ10 attenuates murine thrombus growth and vessel occlusion in a ferric chloride (FeCl 3 )-induced thrombosis model in vivo. Importantly, the randomized, double-blind, placebo-controlled clinical trial in dyslipidemic patients demonstrates that 24 weeks of CoQ10 supplementation increases platelet CoQ10 concentrations, enhances the cAMP/PKA pathway, and attenuates αIIbβ3 outside-in signaling, leading to decreased platelet aggregation and granule release., Conclusion: Through upregulating the platelet cAMP/PKA pathway, and attenuating αIIbβ3 signaling and thrombus growth, CoQ10 supplementation may play an important protective role in patients with risks of cardiovascular diseases., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: The morbidity of atrial fibrillation (AF) is 1%-2% in clinic. Radiofrequency catheter ablation (RFCA) is a type of radical interventional therapy for AF, whereas it may lead to a hypercoagulable state. This study evaluated platelet particle CD62P and platelet activation biomarker GP IIb/IIIa expressions in AF patients treated by RFCA, and aimed to analyze their relationships with the hypercoagulable state after RFCA., Patients and Methods: A total of 60 AF patients received RFCA in our hospital were enrolled. The patients were divided into group A as hypercoagulable state group and group B as non-hypercoagulable group. Healthy volunteers were selected as normal control. Serum D-Dimer, parathyroid activity index 1 (PAI-1), and tissue plasminogen activator (t-PA) content were tested by using enzyme-linked immunosorbent assay (ELISA), while peripheral CD62P and GP IIb/IIIa expressions were detected by using flow cytometry before, after, and seven days after RFCA., Results: D-Dimer and PAI-1 levels increased, while t-PA reduced in group A compared with that in group B and control (p<0.05). D-Dimer and t-PA contents gradually elevated, whereas t-PA level gradually declined in group A before, after, and seven days after RFCA (p<0.05). Serum CD62P and GP IIb/IIIa expressions in group A were significantly higher compared to that in group B and control (p<0.05). CD62P and GP IIb/IIIa levels were significantly higher seven days after RFCA compared with immediate after RFCA in group A (p<0.05). CD62P showed a positive correlation with GP IIb/IIIa in hypercoagulable state patients after RFCA (p<0.05)., Conclusions: AF patient may appear in hypercoagulable state after RFCA. CD62P and GP IIb/IIIa significantly increased and exhibited a positive correlation.

Smad4, a key transcription factor in the transforming growth factor- β signaling pathway, is involved in a variety of cell physiologic and pathologic processes. Here, we characterized megakaryocyte/platelet-specific Smad4 deficiency in mice to elucidate its effect on platelet function. We found that megakaryocyte/platelet-specific loss of Smad4 caused mild thrombocytopenia and significantly extended first occlusion time and tail bleeding time in mice. Smad4-deficient platelets showed reduced agonist-induced platelet aggregation. Further studies showed that a severe defect was seen in integrin α IIb β 3 -mediated bidirectional (inside-out and outside-in) signaling in Smad4-deficient platelets, as evidenced by reduced fibrinogen binding and α -granule secretion, suppressed platelet spreading and clot retraction. Microarray analysis showed that the expression levels of multiple genes were altered in Smad4-deficient platelets. Among these genes, spleen tyrosine kinase (Syk) and Rho-associated coiled-coil containing protein kinase 2 (ROCK2) were downregulated several times as confirmed by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Further research showed that Smad4 directly regulates ROCK2 transcription but indirectly regulates Syk. Megakaryocyte/platelet-specific Smad4 deficiency caused decreased expression levels of Syk and ROCK2 in platelets. These results suggest potential links among Smad4 deficiency, attenuated Syk, and ROCK2 expression and defective platelet activation., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

In resting platelets, the integrin αIIbβ3 is present in a low-affinity "bent" state. During platelet aggregation, intracytoplasmic signals induce conformational changes (inside-out signaling) that result in a "swung-out" conformation competent to bind ligands such as fibrinogen. The cytoskeleton plays an essential role in αIIbβ3 activation. We investigated the role of the actin interacting protein Wdr1 in αIIbβ3 activation. Wdr1-hypomorphic mice had a prolonged bleeding time (> 10 minutes) compared to that of wild-type mice (2.1 ± 0.7 minutes). Their platelets had impaired aggregation to collagen and thrombin. In a FeCl3 induced carotid artery thrombosis model, vessel occlusion in Wdr1-hypomorphic mice was prolonged significantly compared to wild-type mice (9.0 ± 10.5 minutes versus 5.8 ± 12.6 minutes (p = 0.041). Activation-induced binding of JON/A (a conformation-specific antibody to activated αIIbβ3) was significantly less in Wdr1-hypomorphic platelets at various concentrations of collagen, indicating impaired inside-out activation of αIIbβ3, despite a normal calcium response. Actin turnover, assessed by measuring F-actin and G-actin ratios during collagen- and thrombin-induced platelet aggregation, was highly impaired in Wdr1-hypomorphic platelets. Furthermore, talin failed to redistribute and translocate to the cytoskeleton following activation in Wdr1-hypomorphic platelets. These studies show that Wdr1 is essential for talin-induced activation of αIIbβ3 during platelet activation., Competing Interests: The authors have declared that no competing interests exist.

In recent years, the understanding of the molecular mechanisms involved in platelet production (megakaryopoiesis) has extremely increased, thanks to the study of genetic diseases causing inherited thrombocytopenia. Among the wide variety of transmembrane receptors covering the platelet membrane, αIIbβ3 integrin is the major one, allowing platelets to aggregate upon the occurrence of vascular breach. Platelet counts are usually normal in patients with αIIbβ3 deficiency, suggesting that its role for normal platelet production and morphology is very limited. However, recently, new clinical observations of genetic diseases provided evidence against this hypothesis, bringing new data on the role of αIIbβ3 integrin in defective megakaryopoiesis., (© 2016 médecine/sciences – Inserm.)

Platelet cold agglutinins (PCA) cause pseudothrombocytopenia, spurious thrombocytopenia due to ex vivo platelet clumping, complicating clinical diagnosis, but mechanisms and consequences of PCA are not well defined. Here, we characterised an atypical immunoglobulin (Ig)M PCA in a 37-year-old woman with lifelong bleeding and chronic moderate thrombocytopenia, that induces activation and aggregation of autologous or allogeneic platelets via interaction with platelet glycoprotein (GP)VI. Patient temperature-dependent pseudothrombocytopenia was EDTA-independent, but was prevented by integrin αIIbβ3 blockade. Unstimulated patient platelets revealed elevated levels of bound IgM, increased expression of activation markers (P-selectin and CD63), low GPVI levels and abnormally high thromboxane (TX)A2 production. Patient serum induced temperature- and αIIbβ3-dependent decrease of platelet count in allogeneic donor citrated platelet-rich plasma (PRP), but not in PRP from Glanzmann's thrombasthenia or afibrinogenaemia patients. In allogeneic platelets, patient plasma induced shape change, P-selectin and CD63 expression, (14)C-serotonin release, and TXA2 production. Activation was not inhibited by aspirin, cangrelor or blocking anti-Fc receptor (FcγRIIA) antibody, but was abrogated by inhibitors of Src and Syk, and by a soluble GPVI-Fc fusion protein. GPVI-deficient platelets were not activated by patient plasma. These data provide the first evidence for an IgM PCA causing platelet activation/aggregation via GPVI. The PCA activity persisted over a five-year follow-up period, supporting a causative role in patient chronic thrombocytopenia and bleeding.

Integrins play an essential role in hemostasis, thrombosis, and cell migration, and they transmit bidirectional signals. Transmembrane/cytoplasmic domains are hypothesized to associate in the resting integrins; whereas, ligand binding and intracellular activating signals induce transmembrane domain separation. However, how this conformational change affects integrin outside-in signaling and whether the α subunit cytoplasmic domain is important for this signaling remain elusive. Using Chinese Hamster Ovary (CHO) cells that stably expressed different integrin αIIbβ3 constructs, we discovered that an αIIb cytoplasmic domain truncation led to integrin activation but not defective outside-in signaling. In contrast, preventing transmembrane domain separation abolished both inside-out and outside-in signaling regardless of removing the αIIb cytoplasmic tail. Truncation of the αIIb cytoplasmic tail did not obviously affect adhesion-induced outside-in signaling. Our research revealed that transmembrane domain separation is a downstream conformational change after the cytoplasmic domain dissociation in inside-out activation and indispensable for ligand-induced outside-in signaling. The result implicates that the β TM helix rearrangement after dissociation is essential for integrin transmembrane signaling. Furthermore, we discovered that the PI3K/Akt pathway is not essential for cell spreading but spreading-induced Erk1/2 activation is PI3K dependent implicating requirement of the kinase for cell survival in outside-in signaling.

Objective: The essential role of platelet activation in hemostasis and thrombotic diseases focuses attention on unveiling the underlying intracellular signals of platelet activation. Disabled-2 (Dab2) has been implicated in platelet aggregation and in the control of clotting responses. However, there is not yet any in vivo study to provide direct evidence for the role of Dab2 in hemostasis and platelet activation., Approach and Results: Megakaryocyte lineage-restricted Dab2 knockout (Dab2(-/-)) mice were generated to delineate in vivo functions of Dab2 in platelets. Dab2(-/-) mice appeared normal in size with prolonged bleeding time and impaired thrombus formation. Although normal in platelet production and granule biogenesis, Dab2(-/-) platelets elicited a selective defect in platelet aggregation and spreading on fibrinogen in response to low concentrations of thrombin, but not other soluble agonists. Investigation of the role of Dab2 in thrombin signaling revealed that Dab2 has no effect on the expression of thrombin receptors and the outside-in signaling. Dab2(-/-) platelets stimulated by low concentrations of thrombin were normal in Gαq-mediated calcium mobilization and protein kinase C activation, but were defective in Gα₁₂/₁₃-mediated RhoA-ROCKII activation. The attenuated Gα₁₂/₁₃ signaling led to impaired ADP release, Akt-mammalian target of rapamycin and integrin αIIbβ3 activation, fibrinogen binding, and clot retraction. The defective responses of Dab2(-/-) platelets to low concentrations of thrombin stimulation may contribute to the impaired hemostasis and thrombosis of Dab2(-/-) mice., Conclusions: This study sheds new insight in platelet biology and represents the first report demonstrating that Dab2 is a key regulator of hemostasis and thrombosis by functional interplay with Gα₁₂/₁₃-mediated thrombin signaling., (© 2014 American Heart Association, Inc.)

Objective: Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates platelet response to multiple agonists. How this immunoreceptor tyrosine-based inhibitory motif-containing receptor inhibits G protein-coupled receptor-mediated thrombin-induced activation of platelets is unknown., Approach and Results: Here, we show that the activation of PECAM-1 inhibits fibrinogen binding to integrin αIIbβ3 and P-selectin surface expression in response to thrombin (0.1-3 U/mL) but not thrombin receptor-activating peptides SFLLRN (3×10(-7)-1×10(-5) mol/L) and GYPGQV (3×10(-6)-1×10(-4) mol/L). We hypothesized a role for PECAM-1 in reducing the tethering of thrombin to glycoprotein Ibα (GPIbα) on the platelet surface. We show that PECAM-1 signaling regulates the binding of fluorescein isothiocyanate-labeled thrombin to the platelet surface and reduces the levels of cell surface GPIbα by promoting its internalization, while concomitantly reducing the binding of platelets to von Willebrand factor under flow in vitro. PECAM-1-mediated internalization of GPIbα was reduced in the presence of both EGTA and cytochalasin D or latrunculin, but not either individually, and was reduced in mice in which tyrosines 747 and 759 of the cytoplasmic tail of β3 integrin were mutated to phenylalanine. Furthermore, PECAM-1 cross-linking led to a significant reduction in the phosphorylation of glycogen synthase kinase-3β Ser(9), but interestingly an increase in glycogen synthase kinase-3α pSer(21). PECAM-1-mediated internalization of GPIbα was reduced by inhibitors of dynamin (Dynasore) and glycogen synthase kinase-3 (CHIR99021), an effect that was enhanced in the presence of EGTA., Conclusions: PECAM-1 mediates internalization of GPIbα in platelets through dual AKT/protein kinase B/glycogen synthase kinase-3/dynamin-dependent and αIIbβ3-dependent mechanisms. These findings expand our understanding of how PECAM-1 regulates nonimmunoreceptor signaling pathways and helps to explains how PECAM-1 regulates thrombosis., (© 2014 American Heart Association, Inc.)

Objective: Kindlin-3 is a critical supporter of integrin function in platelets. Lack of expression of kindlin-3 protein in patients impairs integrin αIIbβ3-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 αIIbβ3 in platelets has not been established. Here, we evaluated the significance of the binding of kindlin-3 to integrin αIIbβ3 in platelets in supporting integrin αIIbβ3-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 αIIbβ3 on platelets similar to their wild-type counterparts. Functional analysis revealed that K3KI mice exhibited defective platelet function, including impaired integrin αIIbβ3 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 αIIbβ3 is involved in supporting integrin αIIbβ3 activation and integrin αIIbβ3-dependent responses of platelets and consequently contributes significantly to arterial thrombus formation., (© 2014 American Heart Association, Inc.)

The plasma-membrane integrin αIIbβ3 (CD41/CD61, GPIIbIIIa) is a major functional receptor in platelets during clotting. A common isoform of integrin β3, Leu33Pro is associated with enhanced platelet function and increased risk for coronary thrombosis and stroke, although these findings remain controversial. To better understand the molecular mechanisms by which this sequence variation modifies platelet function, we produced transgenic knockin mice expressing a Pro32Pro33 integrin β3. Consistent with reports utilizing human platelets, we found significantly reduced bleeding and clotting times, as well as increased in vivo thrombosis, in Pro32Pro33 homozygous mice. These alterations paralleled increases in platelet attachment and spreading onto fibrinogen resulting from enhanced integrin αIIbβ3 function. Activation with protease-activated receptor 4- activating peptide, the main thrombin signaling receptor in mice, showed no significant difference in activation of Pro32Pro33 mice as compared with controls, suggesting that inside-out signaling remains intact. However, under unstimulated conditions, the Pro32Pro33 mutation led to elevated Src phosphorylation, facilitated by increased talin interactions with the β3 cytoplasmic domain, indicating that the αIIbβ3 intracellular domains are primed for activation while the ligand-binding domain remains unchanged. Acute dosing of animals with a Src inhibitor was sufficient to rescue the clotting phenotype in knockin mice to wild-type levels. Together, our data establish that the Pro32Pro33 structural alteration modifies the function of integrin αIIbβ3, priming the integrin for outside-in signaling, ultimately leading to hypercoagulability. Furthermore, our data may support a novel approach to antiplatelet therapy by Src inhibition where hemostasis is maintained while reducing risk for cardiovascular disease.

Objective: Platelets abundantly express the membrane receptor CD36 and store its ligand thrombospondin-1 (TSP1) in the α-granules. We investigated whether released TSP1 can support platelet adhesion and thrombus formation via interaction with CD36., Approach and Results: Mouse platelets deficient in CD36 showed reduced adhesion to TSP1 and subsequent phosphatidylserine expression. Deficiency in either CD36 or TSP1 resulted in markedly increased dissolution of thrombi formed on collagen, although thrombus buildup was unchanged. In mesenteric vessels in vivo, deficiency in CD36 prolonged the time to occlusion and enhanced embolization, which was in agreement with earlier observations in TSP1-deficient mice. Thrombi formed using wild-type blood stained positively for secreted TSP1. Releasate from wild-type but not from TSP1-deficient platelets enhanced platelet activation, phosphatidylserine expression, and thrombus formation on collagen. The enhancement was dependent on CD36 because it was without effect on thrombus formation by CD36-deficient platelets., Conclusions: These results demonstrate an anchoring role of platelet-released TSP1 via CD36 in platelet adhesion and collagen-dependent thrombus stabilization. Thus, the TSP1-CD36 tandem is another platelet ligand-receptor axis contributing to the maintenance of a stable thrombus., (© 2014 American Heart Association, Inc.)

Recently we reported data suggesting that platelets could compensate for the bleeding phenotype in severe haemophilia A (HA). The aim of this study was to confirm these results in a larger population with a detailed characterisation of clinical phenotype. Patients with diagnostic severe HA (FVIII:C <1%) were scored for clinical phenotype by integrating data on age at first joint bleed, joint damage, bleeding frequency and FVIII consumption. Phenotype was defined as onset of joint bleeding-score + arthropathy-score + joint bleeding-score + (2* treatment intensity-score). After a washout period of three days, blood was collected for measurement of basal level of platelet activation, platelet reactivity, endothelial cell activation and presence of procoagulant phospholipids in plasma. Thirty-three patients with severe HA were included, 13 patients with a mild, 12 patients with an average and eight patients with a severe clinical phenotype. No relevant differences in basal level of platelet activation, platelet reactivity, endothelial cell activation and procoagulant phospholipids between all three groups were observed. The mean annual FVIII consumption per kg did not correlate with the platelet P-selectin expression and glycoprotein (GP)IIbIIIa activation on platelets. In conclusion, variability in clinical phenotype in patients with diagnostic severe HA is not related to platelet activation or reactivity, measured as platelet degranulation and platelet GPIIbIIIa opening.

von Willebrand disease type 2B (vWD-type 2B) is characterized by gain-of-function mutations in von Willebrand factor (vWF) that enhance its binding to the glycoprotein Ib-IX-V complex on platelets. Patients with vWD-type 2B have a bleeding tendency that is linked to loss of vWF multimers and/or thrombocytopenia. In this study, we uncovered evidence that platelet dysfunction is a third possible mechanism for bleeding tendency. We found that platelet aggregation, secretion, and spreading were diminished due to inhibition of integrin αIIbβ3 in platelets from mice expressing a vWD-type 2B-associated vWF (vWF/p.V1316M), platelets from a patient with the same mutation, and control platelets pretreated with recombinant vWF/p.V1316M. Impaired platelet function coincided with reduced thrombus growth. Further, αIIbβ3 activation and activation of the small GTPase Rap1 were impaired by vWF/p.V1316M following exposure to platelet agonists (thrombin, ADP, or convulxin). Conversely, thrombin- or ADP-induced Ca2+ store release, which is required for αIIbβ3 activation, was normal, indicating that vWF/p.V1316M acts downstream of Ca2+ release and upstream of Rap1. We found normal Syk phosphorylation and PLCγ2 activation following collagen receptor signaling, further implying that vWF/p.V1316M acts directly on or downstream of Ca2+ release. These data indicate that the vWD-type 2B mutation p.V1316M is associated with severe thrombocytopathy, which likely contributes to the bleeding tendency in vWD-type 2B.

The platelet protein disulfide isomerase called ERp57 mediates platelet aggregation, but its role in thrombus formation is unknown. To determine the specific role of platelet-derived ERp57 in hemostasis and thrombosis, we generated a megakaryocyte/platelet-specific knockout. Despite normal platelet counts and platelet glycoprotein expression, mice with ERp57-deficient platelets had prolonged tail-bleeding times and thrombus occlusion times with FeCl3-induced carotid artery injury. Using a mesenteric artery thrombosis model, we found decreased incorporation of ERp57-deficient platelets into a growing thrombus. Platelets lacking ERp57 have defective activation of the αIIbβ3 integrin and platelet aggregation. The defect in aggregation was corrected by the addition of exogenous ERp57, implicating surface ERp57 in platelet aggregation. Using mutants of ERp57, we demonstrate the second active site targets a platelet surface substrate to potentiate platelet aggregation. Binding of Alexa 488-labeled ERp57 to thrombin-activated and Mn(2+)-treated platelets lacking β3 was decreased substantially, suggesting a direct interaction of ERp57 with αIIbβ3. Surface expression of ERp57 protein and activity in human platelets increased with platelet activation, with protein expression occurring in a physiologically relevant time frame. In conclusion, platelet-derived ERp57 directly interacts with αIIbβ3 during activation of this receptor and is required for incorporation of platelets into a growing thrombus.

In this issue of Blood, Wang et al identify an important role for platelet-derived extracellular ERp57, a thiol isomerase enzyme, in platelet integrin regulation and recruitment into a growing thrombus.

Aims: The antiplatelet effects of clopidogrel and prasugrel have mainly been compared using different platelet reactivity tests. Further, data on the impact of both drugs on thrombin-inducible platelet activation are scarce. We therefore investigated the influence of clopidogrel and prasugrel on different flow cytometric parameters of platelet activation as well as on platelet function assessed by leukocyte-platelet interaction., Methods: Baseline, ADP and thrombin receptor-activating peptide (TRAP)-6 inducible P-selectin expression, activation of glycoprotein (GP) IIb/IIIa and monocyte-platelet aggregate (MPA) formation were determined by flow cytometry in 84 clopidogrel- and 21 prasugrel-treated patients undergoing percutaneous coronary intervention with stent implantation., Results: Baseline expressions of P-selectin and activated GPIIb/IIIa did not differ significantly between clopidogrel- and prasugrel-treated patients (both P > 0.2). After activation with ADP or TRAP-6, patients with clopidogrel therapy exhibited significantly higher platelet surface expressions of P-selectin and activated GPIIb/IIIa than prasugrel-treated patients (all P ≤ 0.001). Further, high P-selectin and high GPIIb/IIIa were significantly more frequent in clopidogrel-treated patients than in patients on prasugrel therapy (all P = 0.01). Likewise, the formation of MPA without addition of agonists did not differ significantly between patients receiving clopidogrel and patients on prasugrel therapy (P = 0.2). After activation with ADP or TRAP-6, clopidogrel-treated patients showed a significantly more pronounced formation of MPA than patients-receiving prasugrel (both P = 0.02)., Conclusions: Prasugrel reduces ADP and TRAP-6 inducible platelet activation, and leukocyte-platelet interaction more efficiently than clopidogrel., (© 2013 John Wiley & Sons Ltd.)

Thrombogenesis results from the interaction between glycoprotein receptors and their ligands, although a thrombus is affected by multiple factors such as blood flow, platelet interactions, and changes in ligand characteristics. In this study, we propose a platelet adhesion and aggregation model, focusing on the interaction between the glycoprotein receptor and its ligand. First, we conducted thrombogenesis simulations to compare physiological and pathological conditions. The results suggested that simulations of thrombogenesis differed in distribution, volume, and stability of the thrombus based on disorders of platelet adhesion, aggregation, and the activation. For example, distribution and volume were affected by the activation of GPIIb/IIIa with a GPIb/IX/V deficiency. The thrombus was also unstable, but formed from the upstream side of the injured site, with a GPIIb/IIIa deficiency. Second, we investigated thrombogenesis enhanced by the shear-induced platelet aggregation (SIPA) mechanism. The results demonstrated that the degree of SIPA decreased gradually with thrombus growth in a straight vessel. This result suggests that SIPA is a key hemostasis mechanism in an injured healthy arteriole, although it can lead to the formation of an occlusive thrombus in stenosed vessels., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Despite achievement of optimal epicardial coronary flow in the majority of patients treated for ST-segment elevation myocardial infarction (STEMI) by primary percutaneous coronary intervention (PPCI), myocardial no-reflow is a common phenomenon occurring in 5 to 50% of patients. The no-reflow phenomenon is a predictor of infarct size and an independent predictor of mortality both in the short and long term. Prevention of no-reflow is therefore a crucial step in improving prognosis of patients with STEMI. Several strategies including pharmacological and mechanical ones have been developed to improve microvascular perfusion in the setting of a myocardial infarction. Prevention starts by conservation of the microvascular reserve especially in patients at high risk of acute coronary syndromes such as diabetes patients. Optimal glycaemic control and the use of statins have been shown to reduce no-reflow in this context. Reducing ischaemic time by shortening door to balloon times, administration of intracoronary GP IIb/IIIa antagonists during PPCI and the use of manual aspiration thrombectomy have been shown to result in better myocardial perfusion and improved clinical outcome in major trials. In this review we discuss some of these major trials and studies of other therapeutic options that aim to prevent the no-reflow phenomenon.

Platelet integrin α(IIb)β(3) activation is regulated by inside-out signaling via agonist stimulation. However, when α(IIb)β(3) was exogenously expressed in cell lines such as Chinese hamster ovarian cells, physiological agonists hardly induced α(IIb)β(3) activation. To overcome this disadvantage, we characterized the functional regulation of endogenously expressed α(IIb)β(3) in a megakaryoblastic cell line, CMK, employing an initial velocity assay for PAC-1 binding. We firstly demonstrated that protease-activated receptor 1-activating peptide induced robust, but transient α(IIb)β(3) activation in CMK cells with high glycoprotein-Ib expression. Stable talin-1 or kindlin-3 knockdown cells confirmed that the protease-activated receptor 1-activating peptide-induced α(IIb)β(3) activation was dependent on talin-1 and kindlin-3 expression. In sharp contrast to exogenously expressed α(IIb)β(3) in Chinese hamster ovarian cells, transient overexpression of full-length talin (FL-talin) or talin-head domain (THD) alone did not activate α(IIb)β(3) in CMK cells, but required agonist stimulation. Similarly, kindlin-3 overexpression alone did not induce α(IIb)β(3) activation, but it significantly augmented agonist-induced α(IIb)β(3) activation. Several mutants of FL-talin and THD suggested that the head-rod interaction was critical for autoinhibition of talin-1, and the interaction between the THD and the membrane-proximal region of the β(3) cytoplasmic tail was essential for talin-mediated α(IIb)β(3) activation. In addition, THD and kindlin-3 cooperatively augmented protease-activated receptor 1-induced α(IIb)β(3) activation. We proposed that the CMK cell line is an attractive platform for investigating agonist-, talin-1-, and kindlin-3- dependent α(IIb)β(3) activation., (Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Objective: Phosphatidylserine (PS) externalization by platelets upon activation is a key event in hemostasis and thrombosis. It is currently believed that strong stimulation of platelets forms 2 subpopulations, only 1 of which expresses PS., Methods and Results: Here, we demonstrate that physiological stimulation leads to the formation of not 1 but 2 types of PS-expressing activated platelets, with dramatically different properties. One subpopulation sustained increased calcium level after activation, whereas another returned to the basal low-calcium state. High-calcium PS-positive platelets had smaller size, high surface density of fibrin(ogen), no active integrin α(IIb)β(3), depolarized mitochondrial membranes, gradually lost cytoplasmic membrane integrity, and were poorly aggregated. In contrast, the low-calcium PS-positive platelets had normal size, retained mitochondrial membrane potential and cytoplasmic membrane integrity, and combined retention of fibrin(ogen) with active α(IIb)β(3) and high proaggregatory function. Formation of low-calcium PS-positive platelets was promoted by platelet concentration increase or shaking and was decreased by integrin α(IIb)β(3) antagonists, platelet dilution, or in platelets from kindlin-3-deficient and Glanzmann thrombasthenia patients., Conclusions: Identification of a novel PS-expressing platelet subpopulation with low calcium regulated by integrin α(IIb)β(3) can be important for understanding the mechanisms of PS exposure and thrombus formation.

Background: Extracorporeal circulation (ECC) and hypothermia are routinely used in cardiac surgery to maintain stable circulatory parameters and to increase the ischaemic tolerance of the patient. However, ECC and hypothermia cause platelet activation and dysfunction possibly followed by a devastating coagulopathy. Stimulation of the adenosinediphosphate (ADP) receptor P(2)Y(12) plays a pivotal role in platelet activation. This experimental study tested P(2)Y(12) receptor blockade as an approach to protect platelets during ECC., Methods: Human blood was treated with the short-acting P(2)Y(12) blocker cangrelor (1 µM, t(1/2)<5 min) or the P(2)Y(12) inhibitor 2-MeSAMP (100 µM) and circulated in an ex vivo ECC model at normothermia (37°C) and hypothermia (28°C). Before and after circulation, markers of platelet activation and of coagulation (thrombin-antithrombin complex generation) were analysed. During hypothermic ECC in pigs, the effect of reversible P(2)Y(12) blockade on platelet function was evaluated by cangrelor infusion (0.075 µg kg(-1) min(-1))., Results: During ex vivo hypothermic ECC, P(2)Y(12) blockade inhibited platelet granule release (P<0.01), platelet-granulocyte binding (P<0.05), and platelet loss (P<0.001), whereas no effects on platelet-ECC binding, platelet CD42bα expression, glycoprotein IIb/IIIa activation, or thrombin-antithrombin complex generation were observed. During hypothermic ECC in pigs, cangrelor inhibited platelet-fibrinogen binding (P<0.05) and ADP-induced platelet aggregation (P<0.001). Platelet function was rapidly restored after termination of cangrelor infusion., Conclusions: P(2)Y(12) blockade by cangrelor prevents platelet activation during ECC and hypothermia. Owing to its short half-life, platelet inhibition can be well controlled, thus potentially reducing bleeding complications. This novel pharmacological strategy has the potential to reduce complications associated with ECC and hypothermia.

Ligand-induced ectodomain shedding of glycoprotein VI (GPVI) is a metalloproteinase-dependent event. We examined whether shear force, in the absence of GPVI ligand, was sufficient to induce shedding of GPVI. Human-citrated platelet-rich plasma or washed platelets were subjected to increasing shear rates in a cone-plate viscometer, and levels of intact and cleaved GPVI were examined by Western blot and ELISA. Pathophysiologic shear rates (3000-10 000 seconds(-1)) induced platelet aggregation and metalloproteinase-dependent appearance of soluble GPVI ectodomain, and GPVI platelet remnant. Shedding of GPVI continued after transient exposure to shear. Blockade of α(IIb)β(3), GPIbα, or intracellular signaling inhibited shear-induced platelet aggregation but minimally affected shear-induced shedding of GPVI. Shear-induced GPVI shedding also occurred in platelet-rich plasma or washed platelets isolated from a von Willebrand disease type 3 patient with no detectable VWF, implying that shear-induced activation of platelet metalloproteinases can occur in the absence of GPVI and GPIbα ligands. Significantly elevated levels of sGPVI were observed in 10 patients with stable angina pectoris, with well-defined single vessel coronary artery disease and mean intracoronary shear estimates at 2935 seconds(-1) (peak shear, 19 224 seconds(-1)). Loss of GPVI in platelets exposed to shear has potential implications for the stability of a forming thrombus at arterial shear rates.

Histones are released from dying cells and contribute to antimicrobial defense during infection. However, extracellular histones are a double-edged sword because they also damage host tissue and may cause death. We studied the interactions of histones with platelets. Histones bound to platelets, induced calcium influx, and recruited plasma adhesion proteins such as fibrinogen to induce platelet aggregation. Hereby fibrinogen cross-linked histone-bearing platelets and triggered microaggregation. Fibrinogen interactions with αIIbβ3 integrins were not required for this process but were necessary for the formation of large platelet aggregates. Infused histones associated with platelets in vivo and caused a profound thrombocytopenia within minutes after administration. Mice lacking platelets or αIIbβ3 integrins were protected from histone-induced death but not from histone-induced tissue damage. Heparin, at high concentrations, prevented histone interactions with platelets and protected mice from histone-induced thrombocytopenia, tissue damage, and death. Heparin and histones are evolutionary maintained. Histones may combine microbicidal with prothrombotic properties to fight invading microbes and maintain hemostasis after injury. Heparin may provide an innate counter mechanism to neutralize histones and diminish collateral tissue damage.

Protein kinase C-zeta (PKCζ), an atypical isoform of the PKC family of protein serine/threonine kinases, is expressed in human platelets. However, the mechanisms of its activation and the regulation of its activity in platelets are not known. We have found that under basal resting conditions, PKCζ has a high phosphorylation status at the activation loop threonine 410 (T410) and the turn motif (autophosphorylation site) threonine 560 (T560), both of which have been shown to be important for its catalytic activity. After stimulation with agonist under stirring conditions, the T410 residue was dephosphorylated in a time- and concentration-dependent manner, while the T560 phosphorylation remained unaffected. The T410 dephosphorylation could be significantly prevented by blocking the binding of fibrinogen to integrin αIIbβ3 with an antagonist, SC-57101; or by okadaic acid used at concentrations that inhibits protein serine/threonine phosphatases PP1 and PP2A in vitro. The dephosphorylation of T410 residue on PKCζ was also observed in PP1cγ null murine platelets after agonist stimulation, suggesting that other isoforms of PP1c or another phosphatase could be responsible for this dephosphorylation event. We conclude that human platelets express PKCζ, and it may be constitutively phosphorylated at the activation loop threonine 410 and the turn motif threonine 560 under basal resting conditions, which are differentially dephosphorylated by outside-in signaling. This differential dephosphorylation of PKCζ might be an important regulatory mechanism for platelet functional responses., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Activated coagulation factor XIII (FXIIIa) is a transglutaminase that crosslinks fibrin at sites of vascular injury. FXIIIa also associates with blood platelets, although its role in platelet function is unclear and requires clarification., Objectives: To evaluate the ability of FXIIIa to support platelet adhesion and spreading under conditions of physiologic flow, and to identify the underpinning receptors and signaling events., Methods and Results: Platelet adhesion to immobilized FXIIIa was measured by fluorescence microscopy, and signaling events were characterized by immunoblotting. Immobilized FXIIIa supported platelet adhesion and spreading under static conditions through mechanisms that were dually and differentially dependent on integrins α(IIb)β(3) and α(v)β(3). Platelet adhesion was independent of FXIIIa transglutaminase or protein disulfide isomerase activity. Moreover, adhesion was abolished by antibodies that prevented interaction with FXIIIa, but maintained when potential interactions with fibrinogen were blocked. Platelet adhesion to FXIIIa was reduced significantly by either the specific α(IIb)β(3) antagonist tirofiban or the selective α(v)β(3)-blocking antibody LM609, and abolished when they were used in combination. Importantly, platelet adhesion was preserved under venous and arterial flow conditions in which both integrins played essential roles. In contrast, FXIIIa stimulated the formation of filopodia and lamellipodia in adherent platelets that was mediated exclusively by α(IIb)β(3) and eliminated by the Src-family inhibitor 4-amino-5-(4-methylphenyl-7-(t-butyl)pyrazolo(3,4-d)pyrimidine, indicating a tyrosine kinase-dependent mechanism. Crucially, under conditions of arterial shear, FXIIIa accentuated platelet recruitment by von Willebrand factor and collagen., Conclusions: Our data demonstrate a potential role for FXIIIa in supporting platelet adhesion at sites of vascular damage, particularly in association with other thrombogenic matrix proteins., (© 2011 International Society on Thrombosis and Haemostasis.)

Migration of megakaryocytes (MKs) from the proliferative osteoblastic niche to the capillary-rich vascular niche is essential for proplatelet formation and platelet release. In this study, we explore the role of surface glycoprotein receptors and signaling proteins in regulating MK migration and platelet recovery after immune-induced thrombocytopenia. We show that spreading and migration of mouse primary bone marrow-derived MKs on a fibronectin matrix are abolished by the Src family kinases inhibitor PP1, the Syk kinase inhibitor R406 and the integrin alphaIIbbeta3 antagonist lotrafiban. We also demonstrate that these responses are inhibited in primary phospholipase C gamma2 (PLCgamma2)-deficient MKs. Conversely, MK spreading and migration were unaltered in the absence of the collagen receptor, the glycoprotein VI-FcRgamma-chain complex. We previously reported a correlation between a defect in MK migration and platelet recovery in the absence of platelet endothelial cell adhesion molecule-1 and the tyrosine phosphatase CD148. This correlation also holds for mice deficient in PLCgamma2. This study identifies a model in which integrin signaling via Src family kinases and Syk kinase to PLCgamma2 is required for MK spreading, migration, and platelet formation.

Whilst high pro-coagulant activity is reported in sickle cell disease (SCD), the precise role of platelets (PLTs) in SCD inflammatory and vaso-occlusive processes is unclear. Adhesion of PLTs from healthy controls (CON), SCD individuals (SCD) and SCD patients on hydroxycarbamide (SCDHC) to fibrinogen (FB) was compared using static adhesion assays. PLT adhesion molecules and intraplatelet cyclic adenosine monophosphate (icAMP) were observed by flow cytometry and enzyme-linked immunosorbent assay. SCD-PLTs demonstrated significantly greater adhesion than CON-PLTs to FB. Participation of the alpha(IIb)beta(3)-integrin in SCD-PLT adhesion was implicated by increased alpha(IIb)beta(3) activation and data showing that an alpha(IIb)beta(3)-function-inhibiting antibody significantly diminished SCD-PLT adhesion to FB. Platelet activation was potentiated by reductions in icAMP; cAMP levels were decreased in SCD-PLTs, being comparable to those of thrombin-stimulated CON-PLTs. Furthermore, SCD-PLT adhesion to FB was significantly reduced by cilostazol, an inhibitor of cAMP-hydrolyzing phosphodiesterase 3A (PDE3A). Both alpha(IIb)beta(3)-integrin activation and icAMP correlated significantly with fetal haemoglobin in SCD. Accordingly, hydroxycarbamide therapy was associated with lower PLT adhesion and higher icAMP. SCD-PLTs may be capable of adhering to proteins encountered on the inflamed vascular wall and, potentially, participate in vaso-occlusive processes. Hydroxycarbamide and, speculatively, nitric oxide donor or cyclic-nucleotide-targeted therapies may aid in the reversal of PLT adhesive properties in SCD.

The most consistent feature of Wiskott Aldrich syndrome (WAS) is profound thrombocytopenia with small platelets. The responsible gene encodes WAS protein (WASP), which functions in leucocytes as an actin filament nucleating agent -yet- actin filament nucleation proceeds normally in patient platelets regarding shape change, filopodia and lamellipodia generation. Because WASP localizes in the platelet membrane skeleton and is mobilized by alphaIIbbeta3 integrin outside-in signalling, we questioned whether its function might be linked to integrin. Agonist-induced alphaIIbbeta3 activation (PAC-1 binding) was normal for patient platelets, indicating normal integrin inside-out signalling. Inside-out signalling (fibrinogen, JON/A binding) was also normal for wasp-deficient murine platelets. However, adherence/spreading on immobilized fibrinogen was decreased for patient platelets and wasp-deficient murine platelets, indicating decreased integrin outside-in responses. Another integrin outside-in dependent response, fibrin clot retraction, involving contraction of the post-aggregation actin cytoskeleton, was also decreased for patient platelets and wasp-deficient murine platelets. Rebleeding from tail cuts was more frequent for wasp-deficient mice, suggesting decreased stabilisation of the primary platelet plug. In contrast, phosphatidylserine exposure, a pro-coagulant response, was enhanced for WASP-deficient patient and murine platelets. The collective results reveal a novel function for WASP in regulating pro-aggregatory and pro-coagulant responses downstream of integrin outside-in signalling.

Increased affinity of integrins for the extracellular matrix (activation) regulates cell adhesion and migration, extracellular matrix assembly, and mechanotransduction. Major uncertainties concern the sufficiency of talin for activation, whether conformational change without clustering leads to activation, and whether mechanical force is required for molecular extension. Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin alphaIIbbeta3. Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins. Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin alphaIIbbeta3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.

The nature of the in vivo cellular events underlying thrombus formation mediated by platelet activation remains unclear because of the absence of a modality for analysis. Lymphocyte adaptor protein (Lnk; also known as Sh2b3) is an adaptor protein that inhibits thrombopoietin-mediated signaling, and as a result, megakaryocyte and platelet counts are elevated in Lnk-/- mice. Here we describe an unanticipated role for Lnk in stabilizing thrombus formation and clarify the activities of Lnk in platelets transduced through integrin alphaIIbbeta3-mediated outside-in signaling. We equalized platelet counts in wild-type and Lnk-/- mice by using genetic depletion of Lnk and BM transplantation. Using FeCl3- or laser-induced injury and in vivo imaging that enabled observation of single platelet behavior and the multiple steps in thrombus formation, we determined that Lnk is an essential contributor to the stabilization of developing thrombi within vessels. Lnk-/- platelets exhibited a reduced ability to fully spread on fibrinogen and mediate clot retraction, reduced tyrosine phosphorylation of the beta3 integrin subunit, and reduced binding of Fyn to integrin alphaIIbbeta3. These results provide new insight into the mechanism of alphaIIbbeta3-based outside-in signaling, which appears to be coordinated in platelets by Lnk, Fyn, and integrins. Outside-in signaling modulators could represent new therapeutic targets for the prevention of cardiovascular events.

Hemopoietic cells express relatively high levels of the type I phosphoinositide (PI) 3-kinase isoforms, with p110δ and γ exhibiting specialized signaling functions in neutrophils, monocytes, mast cells, and lymphocytes. In platelets, p110β appears to be the dominant PI 3-kinase isoform regulating platelet activation, irrespective of the nature of the primary platelet activating stimulus. Based on findings with isoform-selective p110β pharmacological inhibitors and more recently with p110β-deficient platelets, p110β appears to primarily signal downstream of G(i)- and tyrosine kinase-coupled receptors. Functionally, inhibition of p110β kinase function leads to a marked defect in integrin α(IIb)β₃ adhesion and reduced platelet thrombus formation in vivo. This defect in platelet adhesive function is not associated with increased bleeding, suggesting that therapeutic targeting of p110β may represent a safe approach to reduce thrombotic complications in patients with cardiovascular disease.

The adhesion of tumor cells with platelets is important in the process of tumor metastasis. A huge work has indicated that anti-adhesion is an effective strategy for metastasis inhibition. In this article, we assess the role of platelet integrin alpha(IIb)beta(3) in adhesion of melanoma cells to platelets and the effects of heparin and modified heparins on the adhesion in vitro and in vivo. We show that platelet integrin alpha(IIb)beta(3) is involved in the interaction of human melanoma A375 cells with platelets, and the high affinity epitope resides on the alpha(IIb) subunit rather than beta(3) subunit. Heparin sulfate-like proteoglycans on tumor cell surface are implicated in the adhesion of A375 cells to integrin alpha(IIb)beta(3). We also show that RO-heparin, CR-heparin, N-2,3-DS-heparin and 2,3-O-DS-heparin can significantly inhibit A375 cells binding to the CHO cells expressing integrin alpha(IIb)beta(3) under static and flow conditions, and remarkably inhibit the adhesion of A375 cells to the immobilized platelet layers under flow conditions. We find that A375 cells and B16F10 cells are arrested in the pulmonary vessels and adhered to platelets, and the initial interaction of tumor cells with platelets in lung vessel and long-term establishment of metastatic foci can be inhibited by heparin as well as CR-heparin and N-2,3-DS-heparin. These data suggest that modified heparins can inhibit tumor cell-platelet interaction mediated by platelet integrin alpha(IIb)beta(3) and modified heparins may be a potential substitute for heparin in inhibiting tumor metastasis., ((c) 2009 UICC.)

Background and Objectives: Outside-in integrin alphaIIbbeta3 signaling involves a series of tyrosine kinase reactions that culminate in platelet spreading on fibrinogen. The aim of this study was to identify novel tyrosine phosphorylated signaling proteins downstream of alphaIIbbeta3, and explore their role in platelet signaling., Methods and Results: Utilizing proteomics to search for novel platelet proteins that contribute to outside-in signaling by the integrin alphaIIbbeta3, we identified 27 proteins, 17 of which were not previously shown to be part of a tyrosine phosphorylation-based signaling complex downstream of alphaIIbbeta3. The proteins identified include the novel immunoreceptors G6f and G6b-B, and two members of the Dok family of adapters, Dok-1 and Dok-3, which underwent increased tyrosine phosphorylation following platelet spreading on fibrinogen. Dok-3 was also inducibly phosphorylated in response to the GPVI-specific agonist collagen-related peptide (CRP) and the PAR-1 and -4 agonist thrombin, independently of the integrin alphaIIbbeta3. Tyrosine phosphorylation of Dok-1 and Dok-3 was primarily Src kinase-independent downstream of the integrin, whereas it was Src kinase-dependent downstream of GPVI. Moreover, both proteins inducibly interacted with Grb-2 and SHIP-1 in fibrinogen-spread platelets., Conclusions: This study provides new insights into the molecular mechanism regulating alphaIIbbeta3-mediated platelet spreading on fibrinogen. The novel platelet adapter Dok-3 and the structurally related Dok-1 are tyrosine phosphorylated in an Src kinase-independent manner downstream of alphaIIbbeta3 in human platelets, leading to an interaction with Grb2 and SHIP-1.

Background: We have recently reported a novel mutation in the beta3 subunit of the platelet fibrinogen receptor (alpha(IIb)beta3D723H) 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 alpha(IIb)beta3D723H or alpha(IIb)beta3D723A salt bridge mutants, but not fully activated alpha(IIb)beta3 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 alpha(IIb)beta3, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34+-derived megakaryocyte proplatelets. Mutant alpha(IIb)beta3D723H signaling was independent of Src, protein kinase C or phosphoinositide 3-kinase, but correlated with decreased RhoA activity as compared with wild-type alpha(IIb)beta3 signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO alpha(IIb)beta3D723H 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 alpha(IIb)beta3D723H 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.

Thrombotic diseases or fatalities have been reported to occasionally occur under conditions of hypergravity although the mechanism is still unclear. To investigate the effect of hypergravity on platelets that are the primary players in thrombus formation, platelet rich plasma (PRP) or washed platelets were exposed to hypergravity at 8 G for 15 minutes. No platelet aggregation was induced by 8 G alone, whereas ristocetin or collagen-induced platelet aggregation was significantly increased. The number of platelets adherent to immobilized fibrinogen and the area of platelets spreading on von Willbrand factor (VWF) matrix were increased simultaneously. Flow cytometry assay indicated that integrin alphaIIbbeta3 was partially activated in 8 G-exposed platelets, but there was no significant difference in P-selectin surface expression between platelets treated with 8 G and 1 G control. The results indicate that hypergravity leads to human platelet hyperactivity, but fails to incur essential platelet activation events, suggesting a novel mechanism for thrombotic diseases occurring under hypergravitional conditions.

The platelet integrin receptor alpha(IIb)beta(3) plays a critical role in thrombosis. We have shown previously that the octapeptide YMESRADR, corresponding to sequences 313 to 320 of the human alpha(IIb) subunit, inhibits human platelet activation and fibrinogen binding to alpha(IIb)beta(3), possibly interacting with the ligand. We investigated the effect of YMESRADR on electrically induced carotid artery thrombosis in New Zealand white rabbits. Peptide was administered via the femoral vein, starting 60 min before and continuing for 90 min after the electrical stimulation. Carotid blood flow was monitored for 90 min after the electrical stimulation. The peptide effects on platelet aggregation, in vitro and ex vivo, and on various coagulation, bleeding, and hemostatic parameters were evaluated. YMESRADR significantly inhibited rabbit platelet aggregation in vitro in a dose-dependent manner. It is important that peptide administration in vivo, at doses ranging from 3 to 15 mg/kg, prolonged the duration of the patency of the carotid artery, and no artery occlusion was observed until the end of the study (90 min after electrical stimulation). Furthermore, YMESRADR administration reduced platelet aggregation ex vivo and thrombus weight; however, these reductions reached statistical significance, compared with the control group, at the peptide doses of 12 and 15 mg/kg. YMESRADR did not affect any coagulation parameter studied and the hemostatic response observed in control animals. Thus, YMESRADR represents a novel antiplatelet agent that can inhibit thrombus formation effectively and carotid artery occlusion without causing hemorrhagic complications in a rabbit model of arterial thrombosis.

Blood clotting (formation of thrombus) plays a critical role in the evolution of a number of cardiovascular diseases. Targeted delivery of thrombolytic agents reduces the risks of hemorrhage and toxicity associated with systemic drug administration, thus offering a promising, minimally invasive approach to controlling and treating thrombosis. Platelets play a major role in the progression of thrombosis on vascular injury. Platelet integrin alphaIIbbeta3 (GP IIb/IIIa) serves as a receptor for various proteins such as fibrinogen, vWF, fibronectin and vitronectin, as well as contributing to the adhesion and aggregation of platelets in a variety of conditions. These receptor-based targeted therapies are currently under clinical studies. Integrins and RGD-based ligands for integrins are currently being investigated in imaging and drug delivery related areas of research. RGD-targeted drugs and imaging agents have been developed either by direct conjugation of the homing peptide to the drug or by conjugation of the RGD-peptide to a carrier device containing drug molecules. This review describes the role of integrin receptors in the pathophysiology of thrombosis and its use in the targeted delivery of thrombolytic agents.

Platelets play a pivotal role in atherothrombosis and therefore are primary targets of antithrombotic therapy. They release an array of agonists, such as adenosine diphosphate (ADP); adhesive molecules, such as P-selectin, thrombospondin, fibrinogen, and von Willebrand factor; coagulation factors; and growth factors. In turn, they present transmembrane receptors for a plethora of agonists and ligands. Heterodimeric glycoproteins of the integrin family bind extracellular matrix and plasma proteins; mediate adhesion, activation, spreading, and aggregation; and facilitate intercellular bidirectional signal transduction. Glycoprotein IIb/IIIa is the most abundant platelet integrin and membrane surface glycoprotein. Glycolipids, heparins, proteoglycans, tetraspanins, and a multitude of other molecules, such as tumor necrosis factor-alpha, CD40L, growth arrest-specific 6, Eph receptor tyrosine kinases, and signaling lymphocytic activation molecule receptors, have been implicated in atherothrombosis. ADP promotes platelet aggregation by binding to platelet surface receptors P2Y(1) and P2Y(12); the thienopyridines inhibit aggregation by binding covalently to P2Y(12). Thrombin, a potent initiator of platelet aggregation, activates platelets by cleaving protease-activated receptors (PARs) PAR-1 and PAR-4 and further propagates its effect by activating nearby platelets. A number of pharmacologic agents with antiplatelet actions have been developed, but the search continues for agents that strike an optimal balance between control of thrombosis and serious bleeding.

Megakaryocytes (MKs) and platelet-like particles (PLPs) have generally been obtained by culturing embryonic stem (ES) cells over feeder cells. However, using feeder cells need many labor-consuming processes and the MK and PLP fractions obtained are often contaminated by such cells and their fragments. Here we describe our new culture system for differentiating mouse ES cells to MKs and PLPs without using feeder cells. ES cells are differentiated to cells with MK-like morphology and properties, including proplatelet formation, high ploidy (>8N), and CD41 expression. The culture medium contained PLPs expressing platelet glycoproteins, CD41 and GPIb. Integrin alpha(IIb)beta(3) of PLPs can be activated by thrombin. Addition of the metalloproteinase inhibitor TAPI-2 to the culture increased the surface expression of GPIbalpha and augmented the adhesion of PLPs to immobilized von Willebrand factor through decreasing the shedding of GPIbalpha. Thus our mouse ES cells culture system is a suitable and efficient method for obtaining MKs and functional PLPs that obviates the need for feeder cells.

Integrin-linked kinase (ILK) has been implicated in the regulation of a range of fundamental biological processes such as cell survival, growth, differentiation, and adhesion. In platelets ILK associates with beta1- and beta3-containing integrins, which are of paramount importance for the function of platelets. Upon stimulation of platelets this association with the integrins is increased and ILK kinase activity is up-regulated, suggesting that ILK may be important for the coordination of platelet responses. In this study a conditional knockout mouse model was developed to examine the role of ILK in platelets. The ILK-deficient mice showed an increased bleeding time and volume, and despite normal ultrastructure the function of ILK-deficient platelets was decreased significantly. This included reduced aggregation, fibrinogen binding, and thrombus formation under arterial flow conditions. Furthermore, although early collagen stimulated signaling such as PLCgamma2 phosphorylation and calcium mobilization were unaffected in ILK-deficient platelets, a selective defect in alpha-granule, but not dense-granule, secretion was observed. These results indicate that as well as involvement in the control of integrin affinity, ILK is required for alpha-granule secretion and therefore may play a central role in the regulation of platelet function.

Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP76), an adaptor that plays a critical role in platelet activation in vitro, contains three N-terminal tyrosine residues that are essential for its function. We demonstrate that mice containing complementary tyrosine to phenylalanine mutations in Y145 (Y145F) and Y112 and Y128 (Y112/128F) differentially regulate integrin and collagen receptor signaling. We show that mutation of Y145 leads to severe impairment of glycoprotein VI (GPVI)-mediated responses while preserving outside-in integrin signaling. Platelets from Y112/128F mice, although having mild defects in GPVI signaling, exhibit defective actin reorganization after GPVI or alpha IIb beta 3 engagement. The in vivo consequences of these signaling defects correlate with the mild protection from thrombosis seen in Y112/128F mice and the near complete protection observed in Y145F mice. Using genetic complementation, we further demonstrate that all three phosphorylatable tyrosines are required within the same SLP76 molecule to support platelet activation by GPVI.

In the classical concept of platelet integrin activation, it is considered that unidirectional conformational changes of alpha(IIb)beta(3) and alpha(2)beta(1) regulate the adhesiveness of platelets for fibrin(ogen) and collagen, respectively. Here, we summarize recent evidence that these conformational changes: (i) can also occur in the reverse direction; and (ii) are not independent events. Platelet stimulation through the P2Y(12) receptors provokes only transient alpha(IIb)beta(3) activation via signaling routes involving phosphoinositide 3-kinases and Rap1b. Furthermore, alpha(IIb)beta(3) can be secondarily inactivated in platelets with prolonged high Ca(2+) rises, which expose phosphatidylserine and bind coagulation factors. Thus, platelet stimulation with strong agonists (collagen and thrombin) also results in transient integrin activation. Integrin alpha(2)beta(1) is found to be activated by a mechanism that is directly linked to alpha(IIb)beta(3) activation. Integrin alpha(2)beta(1) can adopt different activation states, depending on the trigger. Conclusively, reversibility and synchrony of platelet integrin activation are newly identified mechanisms to restrict thrombus growth and to allow optimal coagulation factor binding. Back-shifting of activated integrins towards their resting state may be a novel goal of antithrombotic medication.