Factor XIII Associates with the Guanine Exchange Factors Cool-1 and Cool-2 in Human Platelets.

Abstract Abstract 4015 Poster Board III-951 A major signaling pathway that regulates platelet shape change and reorganization of the cytoskeleton involves the Rho family of GTPases; CDC42, Rac1 and RhoA. These GTPases are converted from their inactive or GDP-loaded state to the active or GTP-loaded state by a class of enzymes called Guanine Exchange Factors (GEFs). GEFs are a family of multi-domain proteins that contain a GDP-GTP exchange domain (DH-PH) as well as other protein interacting domains that are regulated by the activation of receptors present on the platelet surface. We previously identified the presence of two homologous GEFs, Cool-1 and Cool-2, in human platelets. In nucleated cells, the activities and substrate specificities of the Cool GEFs are regulated by complex interactions with p21-activated kinases (PAK), Gb/g heterodimers, focal adhesion kinase, and the scaffolding proteins GIT-1 and GIT-2. The Cool GEFs are found at the sites of focal adhesions during spreading and migration in nucleated cells, however, little is known about the regulation or activity of Cool-1 or Cool-2 in platelets. Co-immunoprecipitation experiments were performed with Cool-1 and Cool-2 antibodies using lysates from resting and thrombin stimulated human platelets. We analyzed the precipitated proteins by Mass Spectroscopy and found a number of structural and scaffold proteins bound to the Cool GEFs in the thrombin activated lysates. These cytoskeletal proteins include talin, multimerin, tubulin, filamin A, actin and fibrinogen. Interestingly, a large number of Factor XIII also co-immunoprecipitated with Cool-1 and Cool-2 in the thrombin activated platelet lysates. Western analysis of the co-immunoprecipated platelet proteins from thrombin, TRAP1 and TRAP4 demonstrated that the association of factor XIII with the Cool GEFs is calcium dependent. Inhibition of transglutaminase activity and presence of RGD peptide did not affect the association of Factor XIII with Cool-1 or Cool-2. Factor XIII deficiency is commonly thought to result in bleeding due to the impaired crosslinking of fibrin. However, platelet factor XIII may also play an important role in cross linking platelet cytoskeleletal proteins such as actin and myosin. Consistent with that hypothesis is a recent report showing that platelet factor XIII deficiency results in decreased lamellapodia formation under static adhesive conditions. The association of Factor XIII with the Cool GEFs further supports investigating the potential for the transglutaminase to crosslink platelet cytoskeletal proteins. Disclosures: No relevant conflicts of interest to declare.

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Complex and Novel Associations of the Guanine Exchange Factors Cool- 1 and Cool-2 with the Scaffolding Proteins GIT1 and GIT2 in Human Platelets

Blood ◽

10.1182/blood.v112.11.2870.2870 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2870-2870

Author(s):

Daniel L. Greenberg ◽

Alex Spencer

Keyword(s):

Mass Spectroscopy ◽

Shape Change ◽

Focal Adhesions ◽

Scaffolding Proteins ◽

Ph Domain ◽

Platelet Surface ◽

Loaded State ◽

Membrane Bound ◽

Rho Family ◽

Guanine Exchange Factors

Abstract A major signaling pathway that regulates platelet shape change and reorganization of the cytoskeleton involves the Rho family of GTPases; CDC42 (fillapodia), Rac1 (lamellapodia) and RhoA (focal adhesions). These GTPases are converted from their inactive or GDP-loaded state to the active or GTP-loaded state by a class of enzymes called Guanine Exchange Factors (GEFs). GEFs are a family of multi-domain proteins that contain a GDP-GTP exchange domain (DH-PH) as well as other protein interacting domains that are regulated by the activation of receptors present on the platelet surface. We used an affinity binding technique followed by mass spectroscopy to identify novel Rho family binding GEFs in platelet lysates. Recombinant GST-Rho fusion proteins bound to agarose beads were prepared in the GTP, GDP and nucleotide-free states and incubated with clarified human platelet lysates. Platelet lysate proteins associated with the different GST-Rho preparations were eluted and run on SDS-PAGE. Gel slices were then cut out, trypsin digested and analyzed by Mass Spectroscopy. Platelet GEFs were identified by the presence of a DH-PH domain. Using this technique we found Cool-1 and Cool-2, two closely related GEFs that regulate Rac1 and CDC42 activation in nucleated cells. Cool-1 has not been previously described in platelets. Homodimeric Cool-1 specifically activates CDC42, whereas Cool-2 is capable of activating Rac1 as a homodimer or CDC42 as a monomer. The substrate specificity of Cool-2 is regulated by complex interactions with p21-activated kinases (PAK) and Gb/g proteins. In nucleated cells, Cool-1 and Cool-2 are localized to focal adhesions by the GPCR-kinase-interacting proteins 1 and 2 (GIT1 and GIT2). GIT binding to the Cool proteins has been shown to inhibit their GEF activity. Here we show that Cool-1 is present only in the membrane fraction of resting platelet lysates while Cool-2 is present in both the membrane and cytoplasmic fractions. Upon activation with thrombin, about half the membrane bound Cool-1 and all the membrane bound Cool- 2 migrate to the cytoplasm. Precipitation experiments in thrombin activated platelets with anti-GIT1 and anti-GIT2 antibodies show GIT-1 avidly binds Cool-2 whereas GIT2 binds a small amount of Cool-1. These data suggest a complex mechanism of CDC42 and Rac1 activation in platelets by the Cool proteins. Based on our findings and the current understanding of the regulation of Cool proteins in nucleated cells, we hypothesize that early after thrombin activation, the CDC42 and Rac1 GEF activities of Cool-1 and Cool-2 result in fillapodia (CDC42) and lamellapodia (Rac1) formation. As integrant mediated focal adhesions are formed, the GEF activities of Cool-1 and Cool-2 are inhibited by the scaffolding proteins GIT2 and GIT1, respectively. In this manner, Rac1 and CDC42 mediated fillapodia and lamellapodia formation may be regulated by the extent of integrin engagement and platelet spreading.

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Prevention Of Shwartzman’S Phenomenon In Factor XIII Deficiency

10.1055/s-0038-1652715 ◽

1981 ◽

Author(s):

Soo Young Lee ◽

Sung Keun Chang ◽

Soo II Chung

Keyword(s):

Factor Xiii ◽

Rabbit Platelet ◽

Factor Xiii Deficiency ◽

Platelet Factor ◽

Cortical Necrosis ◽

Rate Of Increase ◽

Shwartzman Reaction

Cross-linked clots formed in vitro are reported to be more resistant to fibrinolysis but the relevance of these observations to the situation in vivo is uncertain. The possible role of Factor XIII in the formation of diffuse intravascular fibrin deposition was examined in experimentally induced Factor XIII deficient rabbits. Factor XIII deficiency was induced by intravenous infusion of IgG isolated from goat anti-rabbit platelet Factor XIII. Control received normal goat IgG. The Shwartzman reaction was produced by two injections of bacterial endotoxin given 24 hours apart.The most striking histological differences were observed after 48 hours. A large number of glomerular loops were enlarged and engorged with red blood cells and platelet- fibrin thrombi; extensive bilateral cortical necrosis was observed in 8 out of 10 endotoxin injected control rabbits but none in the Factor XIII deficient group.Fibrinogen levels in control rabbits were increased 3-4 fold (1.1g/100 ml), at 24 hours and slightly decreased at 48 hours after endotoxin injection, whereas in Factor XIII deficient animals, the rate of increase was slower but reached similar levels at 48 hours. Fibrinolytic activity in vivo, studied by the degradation of infused 125I-fibrinogen, was significantly increased in both endotoxin injected groups, irrespective of Factor XIII levels.These results strongly suggest that cross-linked thrombi are more resistant to fibrinolysis in vivo as well as in vitro.

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Junctional Adhesion Molecule-A Is a Novel Csk-Binding Protein Which Recruits Csk to the Integrin aIIbb3 and Suppresses Outside-In Signaling In Platelets

Blood ◽

10.1182/blood.v116.21.2120.2120 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2120-2120

Author(s):

Meghna Ulhas Naik ◽

Ulhas P Naik

Keyword(s):

Adhesion Molecule ◽

Conflicts Of Interest ◽

Human Platelets ◽

Clot Retraction ◽

Negative Regulators ◽

Vessel Occlusion ◽

Platelet Surface ◽

Occlusion Time ◽

Functional Studies ◽

Signaling Events

Abstract Abstract 2120 Platelet activation is regulated by both positive and negative regulators present within the platelets so that unwanted activation is suppressed, but, when needed, occurs rapidly. A significant amount of effort has been devoted towards understanding the positive regulators of platelet function. However, very little is known about the negative regulators. Dysregulation of the endogenous negative regulators may aid the thrombotic complications seen in various diseases. Upon ligand binding to integrin aIIbb3, a cascade of signaling known as outside-in signaling is induced through the integrin that regulates platelet aggregation and clot retraction. How endogenous negative regulators suppress these events is not well understood. We have previously identified junctional adhesion molecule A (JAM-A), on the platelet surface. We found that Jam-a knockout mice show a prothrombotic phenotype as assessed by significantly (P<0.00001) shortened tail bleeding time, decreased carotid vessel occlusion time and increased pulmonary thromboembolism. Platelet functional studies revealed that Jam-a null platelets were hyperactive to physiological agonists. Surprisingly, inside-out signaling events were not affected in Jam-a null platelets. It is therefore possible that observed hyper-reactivity of Jam-a null platelets could be due to enhanced outside-in signaling. To test this, we performed a clot retraction assay. The clot retraction in wild type (Wt) occurred normally, which began after 1 h and about 50% was completed by 2 h. On the contrary, in Jam-a null platelets, the process of clot retraction was significantly enhanced (P<0.0001). It was initiated before 1h and was completed within 2 h. When analyzed for outside-in signaling events such as b3 tyrosine phosphorylation and c-Src phosphorylation, we found both significantly enhanced in Jam-a null platelets. To assess the mechanism by which JAM-A suppresses outside-in signaling, we analyzed the phosphorylation status of JAM-A. Interestingly, JAM-A was found to be phosphorylated on Y280 in unactivated platelets and rapidly dephosphorylated upon initiation of outside-in signaling. On the other hand, in resting platelets, a minimally phosphorylated S284 residue of JAM-A is rapidly phosphorylated, suggesting that there is a dephosphorylation/phosphorylation switch that may be involved in regulating outside-in signaling. Furthermore, we found that JAM-A associates with integrin aIIbb3 in unactivated human platelets, but this association was disrupted during outside-in signaling as determined by co-immunoprecipitation. We also found Csk, a C-terminal Src kinase, coimmunoprecipitating (IP) with JAM-A from resting, but not activated, platelet lysates, suggesting that JAM-A may be recruiting Csk to unactivated integrins and thus suppressing signaling. To test this, we analyzed association of Csk with integrin in Jam-a null platelets and found that in Wt platelets Csk was abundantly present in the integrin IP, but was completely absent in the integrin IP of the Jam-a null platelet lysates. These results clearly suggest that JAM-A recruits Csk to the integrin and thus suppresses outside-in signaling. Disclosures: No relevant conflicts of interest to declare.

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Specific Inhibition Of Ectodomain Shedding Of GPIba By Targeting Its Shedding Cleavage Site

Blood ◽

10.1182/blood.v122.21.21.21 ◽

2013 ◽

Vol 122 (21) ◽

pp. 21-21 ◽

Cited By ~ 8

Author(s):

Xin Liang ◽

Susan R. Russell ◽

Sandra Estelle ◽

Limei H. Jones ◽

Sungyun Cho ◽

...

Keyword(s):

Cleavage Site ◽

Conflicts Of Interest ◽

Human Platelets ◽

Supporting Evidence ◽

Ectodomain Shedding ◽

Complex Flow ◽

Fab Fragment ◽

Platelet Surface ◽

Strong Binding ◽

Platelet Aggregometry

Abstract Background Ectodomain shedding of GPIbα, a proteolytic event in which metalloprotease ADAM17 cleaves the Gly464-Val465 bond and releases glycocalicin to the plasma, is considered a critical step in mediating clearance of stored platelets. Supporting evidence has been obtained from animal studies using ADAM17 inhibitors. However, the definitive proof is lacking due to the broad substrate specificity of ADAM17. We report herein novel monoclonal antibodies (MAbs) that specifically inhibit shedding of human GPIbα in platelets and may be potentially developed into an additive to improve platelet storage. Methods A synthetic peptide that corresponds to a human GPIbα sequence containing the shedding cleavage site was used as the antigen for mouse immunization. Hybridoma clones obtained from immunized mice were screened in ELISA assays for binding activities to the shedding-site peptide, and purified human GPIb-IX complex. Flow cytometry-based assays and Western blotting were used to screen for direct binding to washed platelets and inhibition of GPIbα shedding in washed platelets. Platelet aggregometry using different agonists was performed on MAb-treated platelets. Results Six MAbs were obtained and characterized for their abilities to bind GPIbα and inhibit its shedding. The purified antibodies bind, with varying affinities, to immobilized monovalent shedding-site peptide, but all exhibit strong binding to ovalbumin-conjugated shedding-site peptide. Similarly, these antibodies bind with varying affinities to immobilized GPIb-IX, but all exhibit strong binding to human platelets. Considering the multivalent nature of ovalbumin-conjugated peptide and the abundance of GPIbα on the platelet surface, these results indicate the divalent binding of GPIbα to these MAbs on the platelet surface. The prototypic clone, designated 5G6, and its monomeric Fab fragment, bind specifically purified GPIb-IX complex, human platelets, and transgenic murine platelets expressing human GPIbα. It is specific to human GPIbα, as it does not bind mouse platelets. 5G6 exhibits similar inhibitory potency as the broad shedding inhibitor GM6001 in both constitutive and induced GPIbα shedding in human platelets. It does not inhibit shedding of other platelet receptors, such as GPVI and GPV. Treatment of washed human platelets with 5G6 or other MAb shedding inhibitors for an hour does not induce platelet activation or aggregation, and does not exhibit detectable effects on agonist-induced platelet aggregation. Consistently, infusion of 5G6 into human transgenic mice does not induce acute thrombocytopenia, unlike other MAbs targeting the N-terminal domain of GPIbα. Conclusion We have obtained, for the first time, reagents that specifically inhibit ectodomain shedding of human GPIbα with little effect on platelet functions. These reagents will be useful to define the functional significance of GPIbα shedding. The method of substrate-specific shedding inhibition by macromolecular binding of the shedding cleavage site can be applicable to many other transmembrane receptors undergoing ectodomain shedding. Disclosures: No relevant conflicts of interest to declare.

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Agonist-Induced Kindlin-3 Phsphorylation Regulates αIIbβ3 Integrin Activation In HEL Cells and Platelets

Blood ◽

10.1182/blood.v122.21.22.22 ◽

2013 ◽

Vol 122 (21) ◽

pp. 22-22

Author(s):

Katarzyna Bialkowska ◽

Eugene Podrez ◽

Tatiana V. Byzova ◽

Edward F. Plow

Keyword(s):

Conflicts Of Interest ◽

Focal Adhesions ◽

Human Platelets ◽

Phosphorylation Sites ◽

Integrin Activation ◽

Suspension Cells ◽

Integrin Αiibβ3 ◽

Hel Cells ◽

Β3 Integrin

Abstract The contributions of integrins to platelet responses depend upon the dynamic regulation of their activation status, which in turn depends on engagement of binding partners by their cytoplasmic tails. It is well-established that not only talin but also kindlin family members are essential for integrin activation, and both must present for optimal integrin function. Recent studies in humans have specifically emphasized the vital role of kindlin-3 in integrin functions in hematopoietic cells, including platelets, where kindlin-3 deficiency can lead to episodic bleeding, frequent infections and osteopetrosis, consequences of an inability to activate β1, β2 and β3 integrins. Despite this evidence, little is known about kindlin-3 structure-function relationship. Here, we used human platelets and human erythroleukemic HEL cell line that expresses integrin αIIbβ3 to investigate whether posttranslational modification(s) of kindlin-3 occurs and can influence its integrin activity. Non-stimulated HEL cells are suspension cells, and they do not adhere to fibrinogen or bind soluble fibrinogen and PAC-1 antibody (specific for activated αIIbβ3) readily. Thrombopoietin or PMA stimulation activated αIIbβ3 such that the cells adhered and spread on fibrinogen and increased their binding of PAC-1 and soluble fibrinogen. β3 integrin and kindlin-3 colocalized in focal adhesions in the adherent cells, and there was enhanced β3 integrin-kindlin-3 association as detected by coimmunoprecipitation. Kindlin-3 knockdown impaired agonist-stimulated adhesion and spreading on fibrinogen. Since, as we have shown previously, β3 integrin phosphorylation regulates kindlin and integrin interaction, we sought to determine whether kindlin-3 is also phosphorylated. Human platelets were stimulated with thrombin and HEL cells with PMA, and kindlin-3 was immunoprecipitated from lysates of control and stimulated cells. A kindlin-3 peptide showing significant increase in phosphorylation upon agonist stimulation was identified in both platelets and HEL cells by mass spectrometry. T482 or S484 were identified as phosphorylation sites in sequence that resides in the kindlin-3 variable region, which is not present either in kindlin-1 or kindlin-2 but is conserved across all species in which kindlin-3 has been sequenced. When expressed in HEL cells, TS/AA kindlin-3 mutant displayed decreased soluble fibrinogen binding and cell spreading on immobilized fibrinogen when compared to wild-type kindlin-3. Membrane-permeable, poly-arginine tagged kindlin-3 peptide containing the candidate phosphorylation sites kindlin-3 was introduced into HEL cells and platelets. HEL cell adhesion and spreading was blunted by the kindlin-3 peptide when compared to a scramble poly-arginine control peptide. Moreover, thrombin-induced platelet aggregation was inhibited by kindlin-3 peptide but not by the scramble peptide. Thus, our data emphasizes a role of previously unknown, agonist-induced kindlin-3 phosphorylation, in integrin αIIbβ3 activation in HEL cells and platelets and provides a basis for functional differences between kindlin-3 and its other two paralogs, kindlin-1 and kindlin-2. Disclosures: No relevant conflicts of interest to declare.

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Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets

Blood ◽

10.1182/blood.v96.13.4046.h8004046_4046_4054 ◽

2000 ◽

Vol 96 (13) ◽

pp. 4046-4054

Author(s):

Kenneth J. Clemetson ◽

Jeannine M. Clemetson ◽

Amanda E. I. Proudfoot ◽

Christine A. Power ◽

Marco Baggiolini ◽

...

Keyword(s):

Platelet Activation ◽

Messenger Rna ◽

Chemokine Receptors ◽

Polyclonal Antibodies ◽

Adenosine Diphosphate ◽

Functional Expression ◽

Human Platelets ◽

Platelet Factor ◽

Platelet Surface ◽

Stromal Cell Derived Factor

Platelets are known to contain platelet factor 4 and β-thromboglobulin, α-chemokines containing the CXC motif, but recent studies extended the range to the β-family characterized by the CC motif, including RANTES and Gro-α. There is also evidence for expression of chemokine receptors CCR4 and CXCR4 in platelets. This study shows that platelets have functional CCR1, CCR3, CCR4, and CXCR4 chemokine receptors. Polymerase chain reaction detected chemokine receptor messenger RNA in platelet RNA. CCR1, CCR3, and especially CCR4 gave strong signals; CXCR1 and CXCR4 were weakly positive. Flow cytometry with specific antibodies showed the presence of a clear signal for CXCR4 and weak signals for CCR1 and CCR3, whereas CXCR1, CXCR2, CXCR3, and CCR5 were all negative. Immunoprecipitation and Western blotting with polyclonal antibodies to cytoplasmic peptides clearly showed the presence of CCR1 and CCR4 in platelets in amounts comparable to monocytes and CCR4 transfected cells, respectively. Chemokines specific for these receptors, including monocyte chemotactic protein 1, macrophage inflammatory peptide 1α, eotaxin, RANTES, TARC, macrophage-derived chemokine, and stromal cell–derived factor 1, activate platelets to give Ca++ signals, aggregation, and release of granule contents. Platelet aggregation was dependent on release of adenosine diphosphate (ADP) and its interaction with platelet ADP receptors. Part, but not all, of the Ca++ signal was due to ADP release feeding back to its receptors. Platelet activation also involved heparan or chondroitin sulfate associated with the platelet surface and was inhibited by cleavage of these glycosaminoglycans or by heparin or low molecular weight heparin. These platelet receptors may be involved in inflammatory or allergic responses or in platelet activation in human immunodeficiency virus infection.

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Congenital Factor XIII Deficiency as Cause for Intracraneal Hemorrhage.

Blood ◽

10.1182/blood.v114.22.4208.4208 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4208-4208

Author(s):

Maria Angeles Dasi ◽

Bienvenida Argiles ◽

Ana R Cid ◽

M. Carmen Carreras ◽

J. Antonio Aznar ◽

...

Keyword(s):

Umbilical Cord ◽

Subdural Hematoma ◽

Factor Xiii ◽

Conflicts Of Interest ◽

Hemorrhagic Diathesis ◽

Minor Trauma ◽

Factor Xiii Deficiency ◽

Coagulation Tests ◽

Fxiii Deficiency ◽

Congenital Factor

Abstract Abstract 4208 Severe congenital factor XIII (FXIII) deficiency is an autosomal recessive disease of with a low prevalence in the general population (3-5 cases per million), associated with generally severe hemorrhagic diathesis, where the presence of intracraneal hemorrhage (ICH) is much higher than in other coagulopathologies such as hemophilia A or B. However, the basic coagulation tests are normal, which could delay the diagnosis. We present three cases of severe congenital FXIII deficiency (not diagnosed when referred to our center) with severe hemorrhagic pathology. These patients had normal basic coagulation tests and did not have a family bleeding history. The table show the results. Table Patient 1 Patient 2 Patient 3 Date of birth 1959 1978 2006 Sex Female Female Female Age at diagnosis 15 years old 12 years old 18 months old Cause of patient remission Ankle hemarthrosis Seizures Subdural hematoma Subdural hematoma Prior hemorrhagic history -umbilical cord bleeding-frontal hematoma requiring surgical management at 12 months-bleeding with dentition -umbilical cord bleeding-hematoma in buttock requiring RBC transfusion for anemia.-hemarthrosis in both knees -umbilical cord bleeding-growing cephalohematoma until 3rd week of life-hematoma by venipuncture lasting 2-3 weeks at 6 months.-delayed healing-Subdural hematoma after minor trauma 3 days prior. basic coagulation tests (PT, APTT, TT, fibrinogen) Normal Normal Normal Level of functional FXIII <1% < 5 % < 5 % Initial treatment Plasma Plasma FXIII concentrate Prophylaxis -Plasma: 2 Units every 6 weeks -FXIII concentrate every 4 weeks (since 1994) FXIII concentrate every 4 weeks FXIII concentrate every 3 weeks Evolution Major ICH at 30 years old. (had suspended prophylactic treatment in1989) Favorable evolution without consequences. Favorable evolution without consequences. COMMENTS Congenital Factor XIII deficiency, although infrequent, should be included in the differential diagnosis of hemorrhagic processes with normal coagulation tests, especially if triggered spontaneously or in a disproportionate manner (in quantity and/or duration). Bleeding of the umbilical cord in the first days or weeks after birth is characteristic of this deficiency (present in 80% of cases). The prophylactic administration of Factor XIII is fundamental due to the frequency of intracranial hemorrhaging. Disclosures: No relevant conflicts of interest to declare.

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The GPIIbIIIa Antagonist Drugs Eptifibatide and Tirofiban Inhibit Caspase-3 Activation in Thrombin- and Calcium Ionophore-Stimulated Human Platelets.

Blood ◽

10.1182/blood.v114.22.2998.2998 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2998-2998

Author(s):

Valery Leytin ◽

Asuman Mutlu ◽

Sergiy Mykhaylov ◽

David J. Allen ◽

Armen V. Gyulkhandanyan ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Calcium Ionophore ◽

Human Platelets ◽

Shear Stresses ◽

Ionophore A23187 ◽

Platelet Surface ◽

Apoptotic Effects

Abstract Abstract 2998 Poster Board II-976 Introduction: The platelet surface receptor glycoprotein (GP) IIbIIIa (integrin αaIIbβ3) mediates platelet aggregation and plays a key role in hemostasis and thrombosis. Numerous GPIIbIIIa antagonists have been designed and tested as inhibitors of platelet aggregation. Two of these antagonists, eptifibatide (Integrilin) and tirofiban (Aggrastat) have been approved by the U.S. Food and Drug Administration (FDA) and widely used for preventing and treating thrombotic complications in patients undergoing percutaneous coronary intervention and in patients with acute coronary syndromes. It has been reported, however, that some GPIIbIIIa antagonists, such as orbofiban and xemilofiban, promote apoptosis in cardiomyocytes by activation of the apoptosis executioner caspase-3, raising the possibility that platelets also may be susceptible to pro-apoptotic effects of eptifibatide and tirofiban. Over the past decade it has been well-documented that apoptosis occurs not only in nucleated cells but also in anucleated platelets stimulated with thrombin, calcium ionophores, very high shear stresses and platelet storage (Leytin et al, J Thromb Haemost 4: 2656, 2006; Mason et al, Cell 128: 1173, 2007). It has been further reported that platelet activation and apoptosis may be induced by different mechanisms and/or require different levels of triggering stumuli (Leytin et al, Br J Haematol 136: 762, 2007; Br J Haematol 142: 494, 2008). Recently, we have shown that injection of anti-GPIIb antibody induced caspase-3 activation in mouse platelets in vivo (Leytin et al, Br J Haematol 133: 78, 2006), suggesting that direct GPIIbIIIa-mediated pro-apoptotic signaling is able to trigger caspase-3 activation within platelets. Study Design and Methods: The current study aimed to examine, for the first time, the effect of eptifibatide and tirofiban on caspase-3 activation in human platelets. We studied the effects of eptifibatide and tirofiban on caspase-3 activation in resting platelets, which express GPIIbIIIa receptors in their non-active (“closed”) conformation, and in platelets stimulated with thrombin or calcium ionophore A23187, which induce transition of GPIIbIIIa receptors into active (“open”) conformation. Resting platelets were treated with control buffer, 0.48 μM eptifibatide or 0.48 μM tirofiban, and stimulated platelets were treated with 1 U/mL thrombin or 10 μM A23187, or preincubated with eptifibatide or tirofiban before treatment with thrombin or A23187. Caspase-3 activation was determined by flow cytometry using the cell-penetrating FAM-DEVD-FMK probe, which covalently binds to active caspase-3. Results and Discussion: We found that treatment of resting platelets with eptifibatide and tirofiban did not affect caspase-3 activation (P>0.05, n=7). In contrast, a 2.3-2.7-fold increase of caspase-3 activation was observed in platelets after thrombin or A23187 stimulation (P<0.01, n=7). However, when platelets were preincubated with eptifibatide and tirofiban before agonist treatment, these drugs significantly inhibited agonist-induced caspase-3 activation by an average of 44-50% (P<0.05, n=7). The fact that eptifibatide and tirofiban do not promote caspase-3 activation in unstimulated platelets suggests that these GPIIbIIIa antagonists do not induce transmission of pro-apoptotic transmembrane signals inside platelets through inactive GPIIbIIIa integrin. The inhibitory effect of eptifibatide and tirofiban on thrombin- and A23187-induced caspase-3 activation suggests a role of GPIIbIIIa integrin in caspase-3 activation induced by these platelet agonists. Conclusions: We have demonstrated a novel platelet-directed activity of two clinically used GPIIbIIIa antagonist drugs, eptifibatide (Integrilin) and tirofiban (Aggrastat), with ability to inhibit apoptosis executioner caspase-3 induced by potent platelet agonists, thrombin and A23187, and the absence of adverse pro-apoptotic effects on resting platelets. Taken together with earlier reported data (Leytin et al, Br J Haematol 133: 78, 2006), the current study indicates that, aside from their well-known participation in platelet activation and aggregation, GPIIbIIIa receptors are involved in the modulation of platelet apoptosis. This GPIIbIIIa-mediated mechanism of apoptosis modulation may be very efficient given the extremely large number of GPIIbIIIa copies (≈80,000) on the platelet surface. Disclosures: No relevant conflicts of interest to declare.

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Platelet α-Granule Secretion and Cytoskeletal Rearrangements Are Spatially Regulated At the Micro/Nanoscale.

Blood ◽

10.1182/blood.v120.21.2163.2163 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2163-2163

Author(s):

Yumiko Sakurai ◽

Yongzhi Qiu ◽

Byungwook Ahn ◽

Wilbur Lam

Keyword(s):

Platelet Adhesion ◽

Conflicts Of Interest ◽

Microcontact Printing ◽

Morphological Changes ◽

Human Platelets ◽

Cell Detection ◽

Platelet Surface ◽

Average Area ◽

Platelet Level ◽

Granule Secretion

Abstract Abstract 2163 Introduction At sites of vascular injury, activated platelets exhibit dramatic morphological changes and granule secretion to facilitate recruitment of other platelets and clot formation. In our previous work (Kita et al., 2011), we quantified the effect of the microenvironmental geometry on platelet adhesion using microcontact printing and showed that platelet adhesion and spreading is spatially regulated with microscale resolution. Here we demonstrate that platelet secretion of alpha granules, as indicated with P-selectin staining (a marker for a-granules), is also spatially regulated at the micro/nanoscale. Specifically, we show that fibrinogen micropatterns regulate and determine the spatial distribution of P-selectin at the single platelet level. We also show that tubulin, one of the major components of the platelet cytoskeleton, is also rearranged by the nanoscale geometry of the microenvironment. Methods Using microfabrication techniques we previously developed (Kita et al., 2011), patterned polydimethylsiloxane (PDMS) stamps were “inked” with fluorescently-labeled fibrinogen from human plasma. Fibrinogen patterns were then “microstamped” onto glass coverslips and the printed surface was blocked with 1 % BSA. 20 million/ml of washed human platelets were prepared in Tyrode's buffer and incubated onto protein micropatterned surfaces. Immunofluorescence staining was used to visualize P-selectin surface expression and intracellular distribution, and tubulin distribution of adhered platelets. Platelets were also counter-stained with a fluorescent membrane dye or phalloidin for cell detection or cytoskeleton arrangement, respectively. Images were taken via confocal microscopy with a 63x oil immersion objective. Results When washed platelets are incubated onto the micropatterned fibrinogen surface, they generally undergo activation and lamellipodia formation. On uniform, non-patterned fibrinogen glass surfaces, (Fig 1A), the average area of spread platelets is 24.86 μm2(n=30, standard error ± 2.27). When the fibrinogen micropattern was larger than the platelets' average area (Fig 1B), multiple platelets covered the micropattern and followed the microenvironmental geometry with high fidelity. P-selectin was detected on the entire platelet surface at slightly higher concentrations at the margins. However, when the features of the fibrinogen micropattern decreased to 5 μm in diameter (Fig 1C, D, and Fig2), platelet spreading was not constrained within the micropattern boundaries. Interestingly, the area of the platelets that spread beyond the fibrinogen micropattern exhibited much higher P-selectin expression than the areas atop the fibrinogen micropattern (Fig 2, arrows), indicating that expression and distribution of P-selectin are spatially regulated by the geometry of the fibrinogen substrate at the microscale. In addition, when platelets are incubated onto fibrinogen microstamps with densely spaced “holes” (0.4–1.0 μm in diameter) blocked with BSA, platelets fully spread and span over those holes. However, dense P-selectin expression was co-localized with these holes, indicating that P-selectin expression and a-granule release are spatially regulated by the underlying protein micropattern (Fig 3A). Interestingly, tubulin showed the opposite trend and only localized to areas directly above the fibrinogen micropattern (Fig 3B). These observations suggest that both platelet a-granule distribution/secretion and cytoskeleton arrangement are regulated at the single platelet level with nanoscale resolution (Fig 3C). Conclusions Using our platelet adhesion/microstamping technique, we demonstrated that platelets regulate the intracellular trafficking, distribution, and secretion of biomolecules at the nanoscale, responding to the geometry of microenvironment. We will continue to investigate how platelets spatially regulate other aspects of their physiology, including calcium signaling and distribution of receptors/ligands on different substrates such as vWF and collagen. Disclosures: No relevant conflicts of interest to declare.

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The PAI-I Gene 4G/5G Polymorphism and Central Nervous System Bleeding In Factor XIII Deficiency

Blood ◽

10.1182/blood.v122.21.4782.4782 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4782-4782 ◽

Cited By ~ 2

Author(s):

Majid Naderi ◽

Akbar Dorgalaleh ◽

Shaban Alizadeh ◽

Shadi Tabibian ◽

Bamedi Taregh ◽

...

Keyword(s):

Factor Xiii ◽

Conflicts Of Interest ◽

Clinical Manifestations ◽

Control Group ◽

Severe Bleeding ◽

Bleeding Tendency ◽

Intraparenchymal Hemorrhage ◽

Factor Xiii Deficiency ◽

History Of ◽

And Control

Background FXIII deficiency is one of the rare bleeding disorder (RBD) that has a highest incidence in Sistan and Baluchistan province around the world. This disorder represents with different clinical manifestations ranging from mild to severe bleeding tendency including CNS bleeding. The aim of this study is to evaluate the role of PAI-14G/5Gpolymorphism in central nervous bleeding (intra and extracranial hemorrhage) system in factor XIII deficiency. Methods In this case control study was studied 32 FXIII deficient patients with CNS bleeding and also 32 patients with factor XIII deficiency without history of CNS bleeding as control group. Initially both groups were evaluated for the previously reported polymorphism of factor XIII (Trp187Argpolymorphism) in order to confirm their disorder. Then all patients were assessed for PAI-14G/5G polymorphism. Eventually obtained data was analyzed by SPSS software. Results The result of this study revealed that all study patients were homozygote for Trp187Arg polymorphism. We also found that the equal numbers of patients (4 individuals) in case and control groups were heterozygote for PAI-14G/5G polymorphism and none of patients were homozygote for this polymorphism. All heterozygote patients had intracranial hemorrhage and patients with extracranial hemorrhage had no mutation of PAI-14G/5G. Intraparenchymal was the most common site of hemorrhage and was observed in 26 patients (92.8%).We also observed subdural and epidural hemorrhage in two patients (7.1%).Anatomic regions in patients with intraparenchymal hemorrhage, were temporal in nine (32.2%), occipital in eight (28.6%), diffused intraparenchymal hemorrhage in seven (25%), tempro-occipital in two (7.1%) and subdural with temporal in two (7.1%) patients. Conclusion: It seems that PAI-14G/5G polymorphism did not any effect on occurrence of intra and extracranial hemorrhage in patients with factor XIII deficiency. Disclosures: No relevant conflicts of interest to declare.

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