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.

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.

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.

Effect of Dipyridamole on Spontaneous Platelet Aggregation in Whole Blood Decreases with the Time After Venepuncture: Evidence for the Role of ADP

1987 ◽  
Vol 58 (02) ◽  
pp. 744-748 ◽  
Author(s):  
A R Saniabadi ◽  
G D O Lowe ◽  
J C Barbenel ◽  
C D Forbes
Keyword(s):  
Platelet Aggregation ◽  
Correlation Coefficient ◽  
Whole Blood ◽  
Blood Platelet ◽  
Inhibitory Effect ◽  
Time Interval ◽  
Adp Receptor ◽  
Spontaneous Platelet Aggregation

SummarySpontaneous platelet aggregation (SPA) was studied in human whole blood at 3, 5, 10, 20, 30, 40 and 60 minutes after venepuncture. Using a whole blood platelet counter, SPA was quantified by measuring the fall in single platelet count upon rollermixing aliquots of citrated blood at 37° C. The extent of SPA increased with the time after venepuncture, with a correlation coefficient of 0.819. The inhibitory effect of dipyridamole (Dipy) on SPA was studied: (a) 10 μM at each time interval; (b) 0.5-100 μM at 3 and 30 minutes and (c) 15 μM in combination with 100 μM adenosine, 8 μM 2-chloroadenosine (2ClAd, an ADP receptor blocker) and 50 μM aspirin. There was a rapid decrease in the inhibitory effect of Dipy with the time after venepuncture; the correlation coefficient was -0.533. At all the concentrations studied, Dipy was more effective at 3 minutes than at 30 minutes after venepuncture. A combination of Dipy with adenosine, 2ClAd or aspirin was a more effective inhibitor of SPA than either drug alone. However, when 15 μM Dipy and 10 μM Ad were added together, the inhibitory effect of Dipy was not increased significantly, suggesting that Dipy inhibits platelet aggregation independent of Ad. The increase in SPA with the time after venepuncture was abolished when blood was taken directly into the anticoagulant containing 5 μM 2ClAd. It is suggested that ADP released from the red blood cells is responsible for the increased platelet aggregability with the time after venepuncture and makes a serious contribution to the artifacts of in vitro platelet function studies.

Collagen-Induced Platelet Aggregation: -Evidence Against the Essential Role of Platelet Adenosine Diphosphate

1979 ◽  
Vol 42 (04) ◽  
pp. 1193-1206 ◽  
Author(s):  
Barbara Nunn
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Essential Role ◽  
Atp Release ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
High Doses ◽  
Induced Aggregation

SummaryThe hypothesis that platelet ADP is responsible for collagen-induced aggregation has been re-examined. It was found that the concentration of ADP obtaining in human PRP at the onset of aggregation was not sufficient to account for that aggregation. Furthermore, the time-course of collagen-induced release in human PRP was the same as that in sheep PRP where ADP does not cause release. These findings are not consistent with claims that ADP alone perpetuates a collagen-initiated release-aggregation-release sequence. The effects of high doses of collagen, which released 4-5 μM ADP, were not inhibited by 500 pM adenosine, a concentration that greatly reduced the effect of 300 μM ADP. Collagen caused aggregation in ADP-refractory PRP and in platelet suspensions unresponsive to 1 mM ADP. Thus human platelets can aggregate in response to collagen under circumstances in which they cannot respond to ADP. Apyrase inhibited aggregation and ATP release in platelet suspensions but not in human PRP. Evidence is presented that the means currently used to examine the role of ADP in aggregation require investigation.

scholarly journals Antithrombotic Effect of the Ethanol Extract of Angelica gigas Nakai (AGE 232)

Life ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 939
Author(s):  
Pia Loreto Werlinger Bravo ◽  
Hui Jin ◽  
Hyunwoo Park ◽  
Min Sang Kim ◽  
Hirofumi Matsui ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Umbilical Vein ◽  
Ethanol Extract ◽  
Developed Countries ◽  
Severe Bleeding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Angelica Gigas ◽  
Angelica Gigas Nakai

Cardiovascular diseases, such as stroke, are the most common causes of death in developed countries. Ischemic stroke accounts for 85% of the total cases and is caused by abnormal thrombus formation in the vessels, causing deficient blood and oxygen supply to the brain. Prophylactic treatments include the prevention of thrombus formation, of which the most used is acetylsalicylic acid (ASA); however, it is associated with a high incidence of side effects. Angelica gigas Nakai (AG) is a natural herb used to improve blood circulation via anti-platelet aggregation, one of the key processes involved in thrombus formation. We examined the antithrombotic effects of AGE 232, the ethanol extract of A. gigas Nakai. AGE 232 showed a significant reduction in death or paralysis in mice caused by collagen/epinephrine-induced thromboembolism in a dose-dependent manner and inhibition of collagen-induced human platelet aggregation in a concentration-dependent manner. Additionally, AGE 232-treated mice did not show severe bleeding in the gut compared to ASA-treated mice. AGE 232 resulted in a decrease in the number of neutrophils attached to the human umbilical vein endothelial cells (HUVECs) and lower inhibition of COX-1 in response to bleeding and damage to blood vessels, a major side effect of ASA. Therefore, AGE 232 can prevent thrombus formation and stroke.

scholarly journals Role of GRK6 in the Regulation of Platelet Activation through Selective G Protein-Coupled Receptor (GPCR) Desensitization

2020 ◽  
Vol 21 (11) ◽  
pp. 3932 ◽  
Author(s):  
Preeti Kumari Chaudhary ◽  
Sanggu Kim ◽  
Youngheun Jee ◽  
Seung-Hun Lee ◽  
Kyung-Mee Park ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
G Protein ◽  
Platelet Activation ◽  
Functional Role ◽  
Thrombus Formation ◽  
Receptor Activation ◽  
G Protein Coupled ◽  
Gpcr Desensitization ◽  
Stimulation Of

Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A2, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and α-granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6−/− platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6−/− platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate Gq-coupled 5HT2A and Gz-coupled α2A adrenergic receptors, respectively, was not affected in GRK6−/− platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6−/− platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y1, P2Y12, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKCδ) phosphorylation were significantly potentiated in GRK6−/− platelets. Finally, GRK6−/− mice exhibited an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery and shorter tail bleeding times, indicating that GRK6−/− mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.

Platelet Release Reaction in vivo in Patients with Ischemic Heart Disease (IHD) After Exercise and its Prevention with Dipyridamole

1979 ◽  
Author(s):  
H. Yamazaki ◽  
T. Motomiya ◽  
T. Sano
Keyword(s):  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Statistical Significance ◽  
Isometric Exercise ◽  
Voluntary Contraction ◽  
Anti Platelet Drug ◽  
Healthy Control ◽  
Von Willebrand ◽  
Willebrand Factor

Although an interaction between platelets and arteriosclerotic vessel wall is thought to be important in thrombus formation, a little information was obtained in clinical subjects. We have reported that platelet aggregation Increased in patients with IHD after exercise. To analyse the mechanism of this phenomenon, changes in platelet sensitivity to ADP aggregation, plasma von Willebrand factor and beta-thromboglobulin level were measured in 30 IHD and 30 healthy controls before and Immediately after an isometric exercise (handgrip of 50% voluntary contraction for 2 min). Platelet sensitivity and vWF were determined by original methods detecting microscopically the highest dilution of serially two-fold diluted ADP or test plasma mixed with ristocetin to give platelet aggregation. Beta-TG was measured by RIA Kit. An effect of anti-platelet drug was also observed in IHD. The patients with IHD were administered with placebo or dipyridamole (400 mg/day for 4 weeks) in a crossover single blind fashion. Under placebo, platelet sensitivity to aggregation, vWF and beta-TG increased immediately after the exercise with a statistical significance in IHD. In the healthy control and IHD under dipyridamole, these increases were not observed. The phenomenon may suggest that platelets circulating in sclerotic vessels tend to release and are enhanced in reactivity with smaller stimuli than those in healthy. Such changes might be prevented with dipyridamole.

Platelet JNK1 is involved in secretion and thrombus formation

Blood ◽  
2010 ◽  
Vol 115 (20) ◽  
pp. 4083-4092 ◽  
Author(s):  
Frédéric Adam ◽  
Alexandre Kauskot ◽  
Paquita Nurden ◽  
Eric Sulpice ◽  
Marc F. Hoylaerts ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Blood Perfusion ◽  
Collagen Matrix ◽  
In Vivo Model ◽  
Wild Type ◽  
Platelet Secretion

Abstract The role of c-Jun NH2-terminal kinase 1 (JNK1) in hemostasis and thrombosis remains unclear. We show here, with JNK1-deficient (JNK1−/−) mice, that JNK1 plays an important role in platelet biology and thrombus formation. In tail-bleeding assays, JNK1−/− mice exhibited longer bleeding times than wild-type mice (396 ± 39 seconds vs 245 ± 32 seconds). We also carried out in vitro whole-blood perfusion assays on a collagen matrix under arterial shear conditions. Thrombus formation was significantly reduced for JNK1−/− platelets (51%). In an in vivo model of thrombosis induced by photochemical injury to cecum vessels, occlusion times were 4.3 times longer in JNK1−/− arterioles than in wild-type arterioles. Moreover, in vitro studies carried out in platelet aggregation conditions demonstrated that, at low doses of agonists, platelet secretion was impaired in JNK1−/− platelets, leading to altered integrin αIIbβ3 activation and reduced platelet aggregation, via a mechanism involving protein kinase C. JNK1 thus appears to be essential for platelet secretion in vitro, consistent with its role in thrombus growth in vivo. Finally, we showed that ERK2 and another isoform of JNK affect platelet aggregation through 2 pathways, one dependent and another independent of JNK1.

Identification of p.W246L As a Novel Mutation in the GP1BA Gene Responsible for Platelet-Type von Willebrand Disease

2014 ◽  
Vol 40 (02) ◽  
pp. 151-160 ◽  
Author(s):  
Adriana Woods ◽  
Analia Sanchez-Luceros ◽  
Emilse Bermejo ◽  
Juvenal Paiva ◽  
Maria Alberto ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Novel Mutation ◽  
In Silico Analysis ◽  
Von Willebrand Disease ◽  
Severe Bleeding ◽  
Low Concentrations ◽  
Healthy Control ◽  
Spontaneous Platelet Aggregation ◽  
Von Willebrand ◽  
Willebrand Factor

Platelet-type von Willebrand disease (PT-VWD) and type 2B von Willebrand disease (2B-VWD) are rare bleeding disorders characterized by increased ristocetin-induced platelet aggregation (RIPA) at low concentrations of ristocetin. Diagnosis of either condition is not easy and the differential diagnosis between the two entities is especially challenging as evidenced by high levels of misdiagnosis of both conditions, but particularly PT-VWD. Five mutations in the GP1BA gene related to PT-VWD and less than 50 patients are currently reported worldwide. We herein describe a patient with severe bleeding symptoms, macrothrombocytopenia, mild spontaneous platelet aggregation, positive RIPA at 0.3 and 0.4 mg/mL, von Willebrand factor ristocetin cofactor (VWF:RCo) to antigen (VWF:Ag) < 0.2, normal VWF propeptide/VWF:Ag ratio, and RIPA mixing tests and cryoprecipitate challenge positive for PT-VWD. GP1BA gene was studied in the patient, in his mother, and in 100 healthy control subjects. We identified a heterozygous substitution G > T located at nucleotide 3805 in the g.DNA of the patient's GP1BA gene, resulting in a Trp to Leu amino acid change at residue 246 (p.W246L). This mutation was absent in his unaffected mother and also in the 100 controls, and was predicted as damaging by in silico analysis. The residue W246 is located within the VWF-binding region and exists in a strongly conserved position in the phylogenetic tree, which is expected to be unable to tolerate substitutions without changing its functional characteristics. These findings argue strongly in favor of the view that this substitution does not represent a polymorphism and is therefore responsible for the PT-VWD phenotype of the patient.

EFFECT OF DIPYRIDAMOLE ON SPONTANEOUS PLATELET AGGREGATION IN WHOLE BLOOD DECREASES WITH THE TIME AFTER VENEPUNCTURE:EVIDENCE FOR THE ROLE OF ADP

1987 ◽  
Author(s):  
A R Saniabadi ◽  
G D O Lowe ◽  
C D Forbes
Keyword(s):  
Platelet Aggregation ◽  
Correlation Coefficient ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Blood Platelet ◽  
Inhibitory Effect ◽  
Time Interval ◽  
Adp Receptor ◽  
Spontaneous Platelet Aggregation

Spontaneous platelet aggregation (SPA) was studied in human whole blood at 3,5, 10, 20, 30, 40 and 60 minutes after venepuncture. Using a whole blood platelet counter (Ultra Flo 100), SPA was quantified by measuring the fall in single platelet count upon rollermixing aliquots of blood at 37°C. The extent of SPA increased with the time after venepuncture, with a correlation coefficient of 0.819. The inhibitory effect of dipyridamole (Dipy) on SPA was studied: (a) 10-5M at each time interval; (b) 0.5-100 x 10-6M at 3 and 30 minutes, and (c) 15 x 10-6M in combination with 2 x 10-4M adenosine (Ad), 8 x 10-6M 2-chloradenosine (2ClAd, a specific ADP receptor blocker) and 5 x 10-5M aspirin. There was a rapid decrease in the inhibitory effect of Dipy with the time after venepuncture; the correlation coefficient was -0.533. At all the concentrations studied, Dipy was more effective at 3 minutes than at 30 minutes after venepuncture. A combination of Dipy withAd, 2ClAd or aspirin was a more effective inhibitor of SPA than either drug alone. However, when an effective concentration of Dipy and an ineffective concentration of Ad (10-4M) were addedtogether, the inhibitory effect of Dipy was not increased, suggesting that Dipy inhibits platelet aggregation independent of Ad.The increase in SPA with the time after venepuncture was abolished when bloodwas taken directly into the anticoagulant containing 2ClAd (5 x 10-6M). We conclude that ADP released from the red blood cells is responsible for the increased platelet aggregability with the time after venepuncture, and makes a serious contribution to the artifacts ofin vitro platelet function studies. Furthermore, the decrease in the inhibitory action of Dipy with the time after venepuncture may explain why previously, it has not been possible to observe inhibition of platelet aggregation by Dipy in platelet rich plasma which requires time to prepare.

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