Defects in Glanzmann thrombasthenia and LAD-III (LAD-1/v) syndrome: the role of integrin β1 and β3 in platelet adhesion to collagen

Blood ◽  
2012 ◽  
Vol 119 (2) ◽  
pp. 583-586 ◽  
Author(s):  
Edith van de Vijver ◽  
Iris M. De Cuyper ◽  
Anja J. Gerrits ◽  
Arthur J. Verhoeven ◽  
Karl Seeger ◽  
...  
Keyword(s):  
Leukocyte Adhesion ◽  
Thrombus Formation ◽  
Diagnostic Methods ◽  
Severe Bleeding ◽  
Bleeding Tendency ◽  
Aggregation Assay ◽  
Glanzmann Thrombasthenia ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Receptor

Abstract Patients with Glanzmann thrombasthenia or Leukocyte Adhesion Deficiency-III syndrome (LAD-III or LAD-1/variant) present with increased bleeding tendency because of the lack or dysfunction of the fibrinogen receptor GPIIb/IIIa (integrin αIIbβ3), respectively. Although the bleeding disorder is more severe in LAD-III patients, classic aggregometry or perfusion of Glanzmann or LAD-III platelets over collagen-coated slides under physiologic shear rate does not discriminate between these 2 conditions. However, in a novel flow cytometry-based aggregation assay, Glanzmann platelets were still capable of forming small aggregates upon collagen stimulation, whereas LAD-III platelets were not. These aggregates required functional GPIa/IIa (integrin α2β1) instead of integrin αIIbβ3, thus explaining the clinically more severe bleeding manifestations in LAD-III patients, in which all platelet integrins are functionally defective. These findings provide genetic evidence for the differential requirements of platelet integrins in thrombus formation and demonstrate that correct integrin function assessment can be achieved with a combination of diagnostic methods.

scholarly journals A Short Half-Life αIIbβ3 Antagonist ANTP266 Reduces Thrombus Formation

2018 ◽  
Vol 19 (8) ◽  
pp. 2306 ◽  
Author(s):  
Tong-Dan Liu ◽  
Shen-Hong Ren ◽  
Xue Ding ◽  
Zhou-Ling Xie ◽  
Yi Kong
Keyword(s):  
Half Life ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Bleeding Risk ◽  
Effective Dosage ◽  
Enzyme Linked Immunosorbent Assay ◽  
Severe Bleeding ◽  
Antithrombotic Agent ◽  
Integrin Αiibβ3 ◽  
Plasma Half Life

Integrin αIIbβ3 plays a pivotal role in platelet aggregation. Three αIIbβ3 antagonists have been approved by the Food and Drug Administration (FDA) for the treatment of cardiovascular diseases. Unfortunately, all of these three drugs can cause the side effect of severe bleeding. Therefore, developing a new αIIbβ3 antagonist with low bleeding was needed. In the present study, we screened compounds by using a fibrinogen/integrin αIIbβ3 enzyme-linked immunosorbent assay (ELISA), and a novel αIIbβ3 antagonist ANTP266 was attained. The antithrombotic effects of ANTP266 were estimated by using two animal models, the bleeding risk was estimated by using a mice tail cutting assay, and the plasma half-life time was tested by LC-MS/MS. The results showed that ANTP266 potently decreased thrombosis formation, while not prolonging bleeding time at its effective dosage. The bleeding of ANTP266 reduced rapidly as time went on from 5 to 60 min, but tirofiban produced high bleeding continuously. The plasma half-life of ANTP266 in rats was 10.8 min. Taken together, ANTP266 is an effective antithrombotic agent with a low bleeding risk. The shorter bleeding time benefits from its short plasma half-life. ANTP266 could be a candidate for developing the αIIbβ3 antagonist of rapid elimination for a patient undergoing percutaneous coronary intervention.

scholarly journals The renal microcirculation in chronic kidney disease: novel diagnostic methods and therapeutic perspectives

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shulin Li ◽  
Fei Wang ◽  
Dong Sun
Keyword(s):  
Chronic Kidney Disease ◽  
Kidney Disease ◽  
Renal Fibrosis ◽  
Molecular Mechanisms ◽  
Leukocyte Adhesion ◽  
Diagnostic Methods ◽  
Mesenchymal Transition ◽  
Microvascular Injury ◽  
Renal Microcirculation

AbstractChronic kidney disease (CKD) affects 8–16% of the population worldwide and is characterized by fibrotic processes. Understanding the cellular and molecular mechanisms underpinning renal fibrosis is critical to the development of new therapeutics. Microvascular injury is considered an important contributor to renal progressive diseases. Vascular endothelium plays a significant role in responding to physical and chemical signals by generating factors that help maintain normal vascular tone, inhibit leukocyte adhesion and platelet aggregation, and suppress smooth muscle cell proliferation. Loss of the rich capillary network results in endothelial dysfunction, hypoxia, and inflammatory and oxidative effects and further leads to the imbalance of pro- and antiangiogenic factors, endothelial cell apoptosis and endothelial-mesenchymal transition. New techniques, including both invasive and noninvasive techniques, offer multiple methods to observe and monitor renal microcirculation and guide targeted therapeutic strategies. A better understanding of the role of endothelium in CKD will help in the development of effective interventions for renal microcirculation improvement. This review focuses on the role of microvascular injury in CKD, the methods to detect microvessels and the novel treatments to ameliorate renal fibrosis.

Cadherin-6 Forms a Functional Adhesion Complex with Alpha-Catenin and Beta-Catenin in Platelets and Is Required for Platelet Aggregation and Thrombus Formation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2232-2232 ◽  
Author(s):  
Michele Mumaw ◽  
Maria de la Fuente ◽  
Carolyn Aldana ◽  
Wei Li ◽  
Marvin T Nieman
Keyword(s):  
Platelet Aggregation ◽  
Adhesion Molecules ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Bleeding Complications ◽  
Severe Bleeding ◽  
Beta Catenin ◽  
Adhesion Complex

Abstract The regulation of hemostasis and thrombus formation is a tightly controlled event that has catastrophic consequences when it is deregulated. One of the hallmarks of the thrombus is aggregated platelets. Upon platelet stimulation, adhesion molecules become activated and mediate multiple cell-cell interactions. Therapeutically, blocking platelet adhesion is a proven method for preventing pathological arterial thrombus formation. However, targeting the primary adhesion receptor, integrin αIIbβ3, results in severe bleeding complications. Therefore, identifying novel proteins or uncovering novel functions for known proteins in platelets is a necessary first step to facilitate the development of safer anti-platelet therapeutics. We have identified that the cell adhesion molecule cadherin-6 forms a functional adhesion complex with α-catenin and β-catenin in platelets. The goal of our project was to determine the mechanism of cadherin-6 mediated adhesion in platelets. Our initial experiments demonstated that cadherin-6 and β-catenin co-localize at the plasma membrane in platelets using confocal immunofluorescence microscopy. We determined that α-catenin and β-catenin co-immunoprecipitate with cadherin-6 from platelet lysates. To examine the functional role of cadherin-6 on platelet aggregation we used a cadherin-6 blocking antibody (10 μg/ml). Blocking cadherin-6 inhibited mouse platelet aggregation induced by PAR4 peptide. We next determined the role of cadherin-6 in vivo by examining carotid artery thrombosis after 7.5% FeCl3 treatment. C57Bl6 mice were injected with cadherin-6 antibody IV and labeled with rhodamine 6G by jugular vein injection. Thrombus formation was imaged in real time by fluorescent intravital microscopy. Blocking cadherin-6 prevented thrombosis for the duration of the experiment (30 min). To verify that the effects that we observed were specific to cadherin-6 expressed on platelets, we isolated platelets from donor mice and treated with cadherin-6 antibody or control IgG ex vivo. The treated platelets were perfused into recipient mice that were irradiated with 11 Gy to make the animals thrombocytopenic. The cadherin-6 antibody treated platelets formed an occlusion at 26.4 ± 3.6 min vs. 13.7 ± 2.0 min for the IgG (p=0.03). Importantly, the cadherin-6 antibody did not affect platelet counts compared to IgG controls 2.97 ± 0.40 (×108) vs. 3.02 ± 0.20 (×108). These combined studies show that caderhin-6 forms a complex with the necessary proteins required to mediate adhesion in platelets. Our results demonstrate that platelet cadherin-6 has a physiologically important role during platelet activation and thrombus formation in vivo. In summary, we have identified a novel adhesion complex in platelets that may provide a mechanism to limit platelet aggregation therapeutically. On going studies will determine the regulation of the cadherin-6/catenin complex and how cadherin-6 cooperates with other platelet adhesion molecules. Disclosures No relevant conflicts of interest to declare.

scholarly journals JAM-A protects from thrombosis by suppressing integrin αIIbβ3-dependent outside-in signaling in platelets

Blood ◽  
2012 ◽  
Vol 119 (14) ◽  
pp. 3352-3360 ◽  
Author(s):  
Meghna U. Naik ◽  
Timothy J. Stalker ◽  
Lawrence F. Brass ◽  
Ulhas P. Naik
Keyword(s):  
Platelet Activation ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Receptor Activation ◽  
Clot Retraction ◽  
Integrin Αiibβ3 ◽  
Genetic Ablation ◽  
Fibrinogen Receptor ◽  
Inside Out

Abstract Mounting evidence suggests that agonist-initiated signaling in platelets is closely regulated to avoid excessive responses to injury. A variety of physiologic agonists induce a cascade of signaling events termed as inside-out signaling that culminate in exposure of high-affinity binding sites on integrin αIIbβ3. Once platelet activation has occurred, integrin αIIbβ3 stabilizes thrombus formation by providing agonist-independent “outside-in” signals mediated in part by contractile signaling. Junctional adhesion molecule A (JAM-A), a member of the cortical thymocyte marker of the Xenopus (CTX) family, was initially identified as a receptor for a platelet stimulatory mAb. Here we show that JAM-A in resting platelets functions as an endogenous inhibitor of platelet function. Genetic ablation of Jam-A in mice enhances thrombotic function of platelets in vivo. The absence of Jam-A results in increase in platelet aggregation ex vivo. This gain of function is not because of enhanced inside-out signaling because granular secretion, Thromboxane A2 (TxA2) generation, as well as fibrinogen receptor activation, are normal in the absence of Jam-A. Interestingly, integrin outside-in signaling such as platelet spreading and clot retraction is augmented in Jam-A–deficient platelets. We conclude that JAM-A normally limits platelet accumulation by inhibiting integrin outside-in signaling thus preventing premature platelet activation.

Natural history and early diagnosis of LAD-1/variant syndrome

Blood ◽  
2006 ◽  
Vol 109 (8) ◽  
pp. 3529-3537 ◽  
Author(s):  
Taco W. Kuijpers ◽  
Robin van Bruggen ◽  
Nanne Kamerbeek ◽  
Anton T. J. Tool ◽  
Gonul Hicsonmez ◽  
...  
Keyword(s):  
Clinical Symptoms ◽  
Leukocyte Adhesion ◽  
Cell Types ◽  
Small Gtpases ◽  
Severe Bleeding ◽  
Genetic Linkage Analysis ◽  
Bleeding Tendency ◽  
Disease Entity ◽  
Primary Defect ◽  
Rap1 Activation

Abstract The syndrome of leukocyte adhesion deficiency (LAD) combined with a severe Glanzmann-type bleeding disorder has been recognized as a separate disease entity. The variability in clinical and cell biological terms has remained largely unclear. We present data on 9 cases from 7 unrelated families, with 3 patients being actively followed for more than 12 years. The disease entity, designated LAD-1/variant syndrome, presents early in life and consists of nonpussing infections from bacterial and fungal origin, as well as a severe bleeding tendency. This is compatible with 2 major blood cell types contributing to the clinical symptoms (ie, granulocytes and platelets). In granulocytes of the patients, we found adhesion and chemotaxis defects, as well as a defect in NADPH oxidase activity triggered by unopsonized zymosan. This last test can be used as a screening test for the syndrome. Many proteins and genes involved in adhesion and signaling, including small GTPases such as Rap1 and Rap2 as well as the major Rap activity-regulating molecules, were normally present. Moreover, Rap1 activation was intact in patients' blood cells. Defining the primary defect awaits genetic linkage analysis, which may be greatly helped by a more precise understanding and awareness of the disease combined with the early identification of affected patients.

The Critical Role of Kindlin-3 in Intiation of Physiological Thrombus Formation ~ Analysis of Kindlin-3 Deficient Patient

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1062-1062
Author(s):  
Hisashi Kato ◽  
Shigenori Honda ◽  
Hirokazu Kashiwagi ◽  
Nobuko Nishiura ◽  
Keigo Akuta ◽  
...  
Keyword(s):  
Cell Line ◽  
Nonsense Mutation ◽  
Thrombus Formation ◽  
Severe Bleeding ◽  
Sequencing Analysis ◽  
Aggregate Formation ◽  
Hel Cells ◽  
Healthy Control ◽  
Inside Out

Background: Kindlin-3 which is expressed mainly in hematopoietic cells, is essential for platelet fibrinogen receptor integrin αIIbβ3 (GPIIb-IIIa) activation and kindlin-3 deficiency causes severe bleeding problems. αIIbβ3 activation is tightly regulated by inside-out signaling, and the direct interaction of talin and kindlin-3 with β3-cytoplasmic tail following CalDAG-GEFI-induced Rap1 activation is critical for αIIbβ3 activation. We have reported that immediate and sustained αIIbβ3 activation by inside-out signaling is important for thrombus formation under physiological conditions (Blood 2016). However, the details of inside-out signaling are not still fully understood. Recently we identified patient with kindlin-3 deficiency as the first Japanese patient with kindlin-3 deficiency caused by novel p.W277X nonsense mutation. To clarify the role of kindlin-3 and the molecular mechanism of inside-out signaling, we analyzed single platelet behavior adhered on collagen under physiological flow conditions, and established kindlin-3 deficient human erythroleukemia HEL cell line. Case: The patient was 8-month old female, born to Japanese consanguineous parents. She has been suffering from bleeding tendency shortly after birth. Her peripheral blood showed slightly decreased platelet count (95x103/L), anemia (Hb 6.9 g/L), and elevated leukocyte count of 37.2x106/L with 1.0% blast. Although the surface expressions of glycoproteins were comparable to healthy control, her platelet aggregations and αIIbβ3 activation were strongly impaired in all agonist stimulations due to defective kindlin-3 expression. The sequencing analysis revealed the homozygous novel nonsense mutation, p.W277X (c.918G>A) in kindlin-3. Methods: Human platelets were obtained from healthy control subjects (CT) and patients with kindlin-3 deficiency and Glanzmann thrombasthenia (GT). Whole blood was perfused at a shear rate 1,250s-1 on collagen surface and shear-induced in vitro thrombus formation during 10 minutes was observed. To evaluate the single platelet behavior after the initial attachment on collagen, each single platelet was analyzed by CellTracker software (Pccinini F. et al. Bioinformatics 2015). To further determine the role of kindlin-3 in inside-out signaling, Kindlin-3 was knocked out by CRISPR-Cas9 system and established kindlin-3 deficient HEL cell line. Results: First, we compared shear-induced thrombus formation between CT, GT, and kindlin-3 deficiency. In contrast to the stable and large platelet aggregate formation in CT after 10 minutes blood perfusion, almost no aggregate was observed in both GT and Kindlin-3 deficiency. In kindlin-3 deficiency, the initial platelet attachment on collagen seemed comparable to that of CT and GT. However, the single adherent platelets looked unstable. Next, we determined the behavior of initially attached platelets. Between CT, GT, and kindlin-3 deficiency, the numbers of platelets attached on collagen during 10 seconds were comparable. Between the initially attached platelets, 31.25% of CT platelets formed stable adhesion followed by platelet aggregation. Similar to CT, 33% of GT platelets adhered stably. However, these GT platelets did not proceed to aggregate formation due to αIIbβ3 deficiency. In contrast to GT, kindlin-3 deficient platelets showed increased detachment, only 9% of initially attached platelets stably adhered. These results suggest that kindlin-3 is indispensable for platelet initial adhesion to collagen by integrin α2β1 activation and explains severe bleeding symptoms in kindlin-3 deficiency than GT. To further investigate how kindlin-3 contributes in initial platelet adhesion to collagen, we established kindlin-3 deficient HEL cell line. In contrast to parental HEL cells, PMA stimulation did not induce αIIbβ3 activation in kindlin-3 deficient HEL cells suggesting impaired inside-out signaling. The introduction of wild type kindlin-3 cDNA, but not W227X mutant, rescued αIIbβ3 activation. Conclusion: The detailed analyses by tracking single adherent platelet on collagen confirmed the role of kindlin-3 in initial step of physiological thrombus formation. Newly established kindlin-3 deficient HEL cell line is useful for further exploration of the role of kindlin-3 and inside-out signaling in platelets and expected contribution for development of new antiplatelet therapy. Disclosures Tomiyama: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Chugai: Honoraria; Kyowa-Kirin: Honoraria.

Mice Lacking the Signaling Molecule, CalDAG-GEFI, Represent a Mouse Model for Leukocyte Adhesion Deficiency Type III.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 674-674
Author(s):  
Wolfgang Bergmeier ◽  
Tobias Goerge ◽  
Hong-Wei Wang ◽  
Stephen M. Cifuni ◽  
Jill R. Crittenden ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Leukocyte Adhesion ◽  
Thrombus Formation ◽  
Neutrophil Infiltration ◽  
Neutrophil Function ◽  
Calcium Flux ◽  
Leukocyte Adhesion Deficiency ◽  
Normal Surface

Abstract Defective inside-out activation of β 1, β 2, and β 3 integrins in platelets and leukocytes is a main characteristic of patients with leukocyte adhesion deficiency (LAD)-III syndrome. We have recently shown that CalDAG-GEFI, a member of the CalDAG-GEF/RasGRP family of intracellular signaling molecules that catalyzes the exchange of GTP for GDP bound to Rap1, plays a key role for the activation of α IIbβ 3 in murine platelets. Here we studied the role of CalDAG-GEFI for neutrophil function as well as the activation of β 1 integrins in platelets. Neutrophils from CalDAG-GEFI−/ − mice showed normal surface expression of key adhesion receptors such as L-selectin, PSGL-1, or β 1/β 2 integrins. Calcium flux, degranulation, and oxygen radical formation were similar in wild-type (WT) and mutant cells. In contrast, β 2 integrin-mediated adhesion to fibrinogen was significantly reduced in cells lacking CalDAG-GEFI when compared to controls. In vivo, CalDAG-GEFI-deficient neutrophils showed normal rolling along stimulated venules, while firm adhesion was almost completely inhibited. A similar defect in firm adhesion was observed in WT mice pre-treated with blocking antibodies against β 2 integrins. To determine the role of CalDAG-GEFI in neutrophil emigration, inflammation was induced in the peritoneum or the skin. In both models, neutrophil infiltration was significantly reduced in CalDAG-GEFI−/ − mice when compared to controls. We further demonstrate that CalDAG-GEFI regulates the activation of β 1 integrins in platelets and that CalDAG-GEFI-deficiency leads to a complete inhibition of arterial thrombus formation in mice. Due to its central role in the activation of β 1, β 2, and β 3 integrins, we propose CalDAG-GEFI as a candidate gene defective in LAD-III patients.

scholarly journals β-arrestin-1 participates in thrombosis and regulates integrin αIIbβ3 signalling without affecting P2Y receptors desensitisation and function

2012 ◽  
Vol 107 (04) ◽  
pp. 735-748 ◽  
Author(s):  
Mathieu Schaff ◽  
Nicolas Receveur ◽  
Catherine Bourdon ◽  
Philippe Ohlmann ◽  
François Lanza ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
P2y Receptors ◽  
Integrin Αiibβ3 ◽  
Akt Phosphorylation ◽  
Fibrinogen Binding ◽  
And Function ◽  
G Protein Coupled ◽  
Receptor Desensitisation

Summaryβ-arrestin-1 (β-arr1) and β-arrestin-2 (β-arr2) are cytosolic proteins well-known to participate in G protein-coupled receptor desensitisation and signalling. We used genetically-inactivated mice to evaluate the role of β-arr1 or β-arr2 in platelet function, P2Y receptor desensitisation, haemostasis and thrombosis. Platelet aggregation, soluble fibrinogen binding and P-selectin exposure induced by various agonists were near normal in β-arr1−/− and β-arr2−/− platelets. In addition, deficiency in β-arr1 or β-arr2 was not critical for P2Y receptors desensitisation. A functional redundancy between β-arr1 and β-arr2 may explain these unchanged platelet responses. Interestingly, β-arr1−/− but not β-arr2−/− mice were protected against laser- and FeCl3-induced thrombosis. The tail bleeding times, number of rebleeds and volume of blood loss were unchanged in β-arr1−/− and β-arr2−/− mice, suggesting no defect in haemostasis. β-arr1−/− platelet activation upon adhesion to immobilised fibrinogen was inhibited, as attested by a 37 ± 5% (n = 3, p<0.0001) decrease in filopodia extension, suggesting defective signalling through integrin αIIbβ3. β-arr1 appeared to be located downstream of Src family kinases and to regulate αIIbβ3 signalling by increasing Akt phosphorylation. Overall, this study supports a role for β-arr1 in promoting thrombus formation, in part through its participation in αIIbβ3 signalling, and no role of β-arr1 and β-arr2 in agonist-induced platelet activation and P2Y receptors desensitisation.

scholarly journals Control of thrombus embolization and fibronectin internalization by integrin αIIbβ3 engagement of the fibrinogen γ chain

Blood ◽  
2003 ◽  
Vol 102 (10) ◽  
pp. 3609-3614 ◽  
Author(s):  
Heyu Ni ◽  
Jessie M. Papalia ◽  
Jay L. Degen ◽  
Denisa D. Wagner
Keyword(s):  
Binding Site ◽  
Vessel Wall ◽  
Thrombus Formation ◽  
Wild Type ◽  
Integrin Αiibβ3 ◽  
Platelet Receptor ◽  
Β3 Integrin ◽  
Dependent Mechanism ◽  
Stable Incorporation

AbstractFibrin(ogen) deficiency (Fg-/-) was shown previously to be compatible with rapid thrombus growth within injured arterioles, but platelet fibronectin content was increased and newly formed thrombi were unstable. To further define the role of fibrin(ogen) in thrombus formation and stabilization, platelet biology was examined in mice expressing a form of fibrinogen that clots normally but lacks the γ chain C-terminal binding site for αIIbβ3 (FgγΔ5). Thrombus growth within the arterioles of FgγΔ5 mice appeared faster than in wild-type mice despite a far greater emboli formation. Unlike Fg-/- mice, the emboli were relatively small and released from the top of thrombi, rather than by fracture at the vessel wall. The fibronectin content in FgγΔ5 platelets was also dramatically increased through a β3 integrin-dependent mechanism. The following has been concluded: (1) Fibrin formation contributes to, but is not sufficient for, the stabilization of arterial thrombi. Platelet receptor engagement of the C-terminal of the Fg γ chain contributes to the stable incorporation of platelets into thrombi. (2) Alternative ligands to fibrinogen can support efficient thrombus growth. (3) Fibrinogen is internalized through αIIbβ3 engagement of the fibrinogen γ chain element, and this interaction secondarily controls the fibronectin content of platelets. (Blood. 2003;102: 3609-3614)

scholarly journals Platelet Integrin αIIbβ3 Activation Kinetics in Inherited Platelet Functional Disorders ∼ the Role of ADP Receptor P2Y12, Caldag-GEFI and Kindlin-3 αIIbβ3 Activation By inside-out Signaling

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1136-1136
Author(s):  
Hisashi Kato ◽  
Nobuko Nishiura ◽  
Keigo Akuta ◽  
Hirokazu Kashiwagi ◽  
Koichi Kokame ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Thrombus Formation ◽  
Severe Bleeding ◽  
Activation Kinetics ◽  
Healthy Control ◽  
Initial Activation ◽  
Adp Receptor ◽  
Inside Out

Abstract Background and objectives Activation of platelet fibrinogen receptor integrin αIIbβ3 (GPIIb-IIIa) by inside-out signaling is essential for platelet aggregate formation. In αIIbβ3 activation, it has been established that CalDAG-GEFI-induced Rap1 activation is necessary to induce the direct interaction of β3 cytoplasmic tail with talin and kindlin-3. Agonist induced platelet and αIIbβ3 activation are generally assessed using platelet aggregation assay and flow cytometric analysis of monoclonal antibody specific for activated αIIbβ3, PAC-1, binding. However, we found that the results of PAC-1 binding assay were not associated with severity of bleeding symptoms in patient with CalDAG-GEFI (Blood 2016) or ADP receptor P2Y12 (J Thromb Haemost 2005) deficiency. To further determine the role of molecules in inside-out signaling on αIIbβ3 activation and the impact on hemostasis, we studied the αIIbβ3 activation kinetics in patient's platelets deficient for αIIbβ3, P2Y12, CalDAG-GEFI, or kindlin-3 using the αIIbβ3 velocity assay (Exp Hematol 2013). Methods Human platelets were obtained from healthy control subjects and patients with inherited platelet disorders (Glanzmann thrombasthenia, P2Y12 deficiency, CalDAG-GEFI deficiency, and kindlin-3 deficiency). Agonist induced platelet and αIIbβ3 activation were assessed by light transmission aggregometer and PAC-1 binding on flow cytometry. In addition to the conventional PAC-1 binding assay which is simultaneous incubation of stimulated platelets with FITC-PAC-1 for 20 minutes, the time course of αIIbβ3 activation was determined by αIIbβ3 velocity assay. In brief, to measure the activation state of αIIbβ3 at the time of interest (0, 30, 60, 120 and 300 seconds), FITC-labeled PAC-1 was added after the PAR1-AP stimulation. After 30 seconds incubation with FITC-PAC-1, the bound FITC-PAC-1 was immediately assessed by flow cytometry. To determine the thrombus formation under the physiological condition, shear-induced thrombus formation on collagen coated surface was observed. Results In the patient with kindlin-3 deficiency, strongly impaired platelet aggregation and αIIbβ3 activation determined by conventional PAC-1 binding were correlated to the patients severe bleeding problems appeared from early infancy. The αIIbβ3 activation kinetics of kindling-3 deficient platelets showed no PAC-1 binding during 300 seconds after the thrombin receptor agonist PAR1-AP stimulation like platelets of Granzmann thrombasthenia, which suggests the essential role of Kindlin-3 in αIIbβ3 activation. In contrast, in spite of the strongly impaired PAC-1 binding in conventional assay for P2Y12 deficient platelets, the 67 year-old patient showed only minor bleeding symptoms and no transfusion was required during delivery. The αIIbβ3 activation observed in P2Y12 deficiency was transient and the level of initial activation at 30 seconds was the same as healthy control platelets. Under the flow conditions, P2Y12 deficient platelets could form thrombus albeit unstable and fragile. In the 16-yaer-old patient with CalDAG-GEFI deficiency who has been suffering from severe nose bleed and menorrhagia, her platelet aggregation and conventional PAC-1 binding were only modestly affected. The time course of αIIbβ3 activation showed delayed activation. Although αIIbβ3 activation in CalDAG-GEFI deficient platelets reached to similar to that of control at 300 seconds, the initial activation at 30 seconds was significantly decreased to only 19% of control. Her platelets could adhere on collagen under the flow condition. However, almost no aggregate formation was observed which explains her severe bleeding symptoms. Conclusion The analyses of αIIbβ3 activation kinetics in platelets with inherited platelet disorder revealed the importance of immediate and sustained αIIbβ3 activation for hemostasis in physiological conditions. In addition to well established platelet functional assay, the analysis of αIIbβ3 activation kinetics contributes to understand the patient's bleeding symptoms and also clarifies the role of each molecule in inside-out αIIbβ3 activation, CalDAG-GEFI for the immediate activation, P2Y12 for the maintenance of activation, and Kindlin-3 at the last step of αIIbβ3 activation. Disclosures Kanakura: Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Tomiyama:Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis Pharma Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sysmex Corporation: Consultancy; Chugai Pharmaceutical Co., Ltd.: Honoraria.

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