Prolonged Expression of Procoagulant Activity of Human Platelets Degranulated by Thrombin

1995 ◽  
Vol 74 (03) ◽  
pp. 958-961 ◽  
Author(s):  
Raelene L Kinlough-Rathbone ◽  
Dennis W Perry
Keyword(s):  
Catalytic Activity ◽  
Thrombus Formation ◽  
Annexin V ◽  
Human Platelets ◽  
Procoagulant Activity ◽  
Prothrombinase Complex ◽  
Arterial Thrombus ◽  
Flowing Blood

SummaryPlatelets are exposed to thrombin when they take part in arterial thrombus formation, and they may return to the circulation when they are freed by fibrinolysis and dislodged by flowing blood. Thrombin causes the expression of procoagulant activity on platelets, and if this activity persists, the recirculating platelets may contribute to subsequent thrombosis. We have developed techniques to degranulate human platelets by treatment with thrombin, and recover them as single, discrete platelets that aggregate in response to both weak and strong agonists. In the present study we examined the duration of procoagulant activity on the surface of thrombin-degranulated platelets by two methods: a prothrombinase assay, and the binding of 125I-labeled annexin. Control platelets generated 0.9 ± 0.4 U thrombin per 107 platelets in 15 min. Suspensions of thrombin-degranulated platelets formed 5.4 ± 0.1 U thrombin per 107 platelets in this time. Binding of 125I-annexin V was also greater with thrombin-treated platelets than with control platelets (controls: 1.7 ±0.1 ng annexin/107 platelets; thrombin-degranulated platelets: 6.8 ± 0.2 ng annexin/107 platelets). With thrombin-degranulated platelets, increased procoagulant activity and annexin binding persisted for at least 4 h after degranulation and resuspension, indicating that the catalytic activity for the prothrombinase complex is not reversed during this time. These platelets maintained their ability to aggregate for 4 h, even in response to the weak agonist, ADP. Thus, platelets that have taken part in thrombus formation and returned to the circulation may contribute to the promotion of further thrombotic events because of the persistence of procoagulant activity on their surface.

A Humanin Analog Attenuates Platelet Responses to Vascular Injury

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1131-1131 ◽  
Author(s):  
Lijie Ren ◽  
Qiang Li ◽  
Zhao Zeng ◽  
Peipei Mou ◽  
Xiaohui Liu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Ischemia Reperfusion ◽  
Treated Group ◽  
Human Platelets ◽  
Control Group ◽  
Arterial Thrombus ◽  
Group 3

Abstract Abstract 1131 Humanin (HN), a 24-amino acid endogenous antiapoptotic peptide, was initially shown to protect against neuronal cell death by Alzheimer's disease-related insults. It has recently been found that an exogenous analog of HN (HNG) in which the 14th amino acid serine is replaced with glycine protected against cerebral and cardiac ischemia reperfusion (I/R) injury in cortical neurons and cardiomyocytes, respectively. Platelet activation and thrombus formation has been shown to play an important role during I/R injury by exacerbating the extent of the infarct size. However, it is presently unknown whether HNG affects platelet function and the subsequent arterial thrombus formation. We thus examined whether HNG affects platelet activation and thrombus formation both in vitro and in vivo. Human platelets were isolated from healthy adults. Preincubation of washed human platelets with HNG (4μM) reduced collagen- or convulxin-induced platelet aggregation by 56.8% (P<0.05) and 71.9% (P<0.001), respectively. Similarly, HNG significantly reduced ATP release stimulated by collagen or convulxin. Convulxin-induced P-selectin expression and fibrinogen binding on single platelet was inhibited by HNG, as measured by flow cytometry. Moreover, HNG reduced platelet spreading on the fibrinogen coated surface by 62.9 % (P <0.05). Western blot revealed a reduction of platelet AKT phosphorylation by HNG upon collagen stimulation, implying the involvement of PI3K pathway. In addition, MAPK P38 phosphorylation by collagen and convulxin was also reduced by HNG. HNG effects on thrombus formation were tested in vivo in a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of HNG (25μg/kg) to male C57BL6/J mice significantly extended the first occlusion time (7.3±0.4 min, N=10), when compared to the saline injected littermates (5.4±0.7 min, N=12) (P <0.05). Furthermore, the number of mice that formed stable thrombus was less in the HNG–treated group (3/13) than the control group (6/13), while the non-occlusion mouse number was more in the HNG-treated group (3/13) than the control group (1/13). Together, these data show that HNG inhibits platelet activation and arterial thrombus formation. This might suggest that the protective effects of HNG against ischemia reperfusion injury could be, in part, via attenuating platelet activation. Therefore, HNG could be a potential therapeutic agent in thrombotic and cardiovascular disorders. Disclosures: No relevant conflicts of interest to declare.

Kinetics of Thrombocyte and Thrombocyte-Microparticle Recruitment in Laser Induced Arterial Thrombus Formation in Zebrafish.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2622-2622 ◽  
Author(s):  
Pudur Jagadeeswaran ◽  
Matthew Cykowski ◽  
Bijoy Thattaliyath
Keyword(s):  
Transgenic Line ◽  
Genetic Model ◽  
Thrombus Formation ◽  
Annexin V ◽  
Arterial Injury ◽  
Red Cells ◽  
Red Cell ◽  
Arterial Thrombus ◽  
Transgenic Lines ◽  
Two Populations

Abstract We have been using zebrafish as a genetic model to study hemostasis and thrombosis. In this pursuit, we studied the initial events in thrombus formation in a laser induced arterial thrombus formation. First, we identified in zebrafish blood microparticles that have membrane proteins similar to those found in thrombocytes. We have then shown that in Weinstein transgenic line (where endothelial cells are labeled with GFP using fli-1 promoter) thrombocytes were also labeled with the GFP. In this line, we found two populations of GFP positive thrombocytes one that is more intense and larger in size than the other and others were of intermediate intensities and sizes. In Weinstein line as well as in Handin transgenic line which carries exclusively GFP labeled thrombocytes (driven under GpIIb promoter), we found GFP labeled microparticles. The GFP microparticles in both lines were similar in numbers suggesting that thrombocytes are generating more microparticles. They ranged in size between 0.2 to 0.8 microns. Thrombin and collagen treatment of thrombocytes increased the generation of microparticles. We also found that the microparticles agglutinated in a vWF dependent fashion. In Lin transgenic line (where mostly red cells are labeled with GFP using GATA-1 promoter), we found a small percentage of thrombocytes were also labeled with GFP (corresponding to less intense GFP thrombocytes in Weinstein line). By using the microparticles from Lin and Weinstein lines, we found that the agglutinates contained, thrombocyte microparticles, and to a larger extent red cell microparticles. By labeling the thrombocyte microparticles with DiI-C18 (a dye that selectively labels approximately 10% of thrombocytes at defined concentration), we found that microparticles accumulated first at the site of injury. Intravenous pan-caspase inhibitor (z-VAD-FMK) injections in zebrafish resulted in significant reduction of microparticles and prolongation of time to adherence in laser induced thrombosis assay. In Weinstein line we noted that less intense GFP thrombocytes were more intensely labeled with DiI-C18. We defined DiI-C18 +ve thrombocytes as young thrombocytes and found that expression of GPIb, and GPIIb/IIIa on native thrombocytes and P-selectin, annexin V and calcium levels after thrombocyte activation were higher in young thrombocytes compared to mature DiI-C18 -ve thrombocytes. We also found that in an aggregation reaction, young and mature thrombocytes formed independent clusters with a preference for formation of young clusters first. By using dye labeling methods as well as above transgenic lines we showed that on laser induced arterial injury young thrombocytes initiated arterial thrombus formation. To identify novel differences between the two populations of thrombocytes, we performed microarray analysis on thrombocyte RNA using a control red cell RNA and found approximately 100 five-fold upregulated genes in thrombocytes compared to red cells. These included genes such as xfo-6. We are currently studying the gene expression differences between the two thrombocyte populations. In summary, we found that microparticles adhere first to sub-endothelial matrix followed by young thrombocyte clustering and later by mature and young thrombocytes clusters in growing thrombus. The knowledge on the differences between the thrombocyte populations and their microparticles as well as their recruitment into thrombus, might provide insight into thrombus formation in mammals and may suggest novel antithrombotic targets.

“Young” Platelets Regulate Thrombus Formation by Expression of Increased Adhesive Properties and Procoagulant Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1527-1527
Author(s):  
Ammon M. Fager ◽  
Paula B. Tracy
Keyword(s):  
Thrombus Formation ◽  
Factor Xa ◽  
Fold Increase ◽  
Human Platelets ◽  
Membrane Properties ◽  
Adhesive Properties ◽  
Glycoprotein Vi ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Normal Hemostasis

Abstract Previous studies from our lab and others have demonstrated that thrombin mediated activation of platelets results in the formation of a distinct procoagulant subpopulation capable of assembling the components of both the prothrombinase complex and intrinsic tenase complex at its surface. A similar subpopulation has been identified following activation of platelets with the combination of thrombin and convulxin, a glycoprotein VI agonist, and has been found to be enriched in young platelets. In addition, young zebrafish thrombocytes were shown to have a higher density of adhesive receptors and greater functional activity than their more mature counterparts. Since the membrane properties that distinguish the platelet’s ability to assemble these procoagulant complexes are unknown, the purpose of this study was to elucidate these important differences. Following their isolation from whole, anticoagulated blood, human platelets were activated with 50 nM thrombin (2 min, ambient temperature) followed by the addition of hirudin (75 nM) to quench the reaction. Equimolar concentrations of factors Va and Xa were added (5 nM, 10 min) to saturate the functional Prothrombinase binding sites determined previously using kinetics of prothrombin activation, prior to platelet fixation with 2% v/v paraformaldehyde. Those platelets capable of binding factors Va and Xa were identified by flow cytometry using specific, non-inhibitory fluorophore-conjugated MoAbs directed against the required cofactor and serine protease, respectively. Analyses of the membrane density of well-known and characterized platelet receptors including glycoprotein IIb/IIIa and glycoprotein Ib-IX-V, were also assessed using specific MoAbs conjugated with the appropriate fluorophore to determine whether expression of any or all of these adhesive receptors defined the platelet subpopulation capable of assembling a competent Prothrombinase. Thiazole orange (TO), a dye frequently used to stain nucleic acid, and previously shown to identify the youngest platelets in circulation, was used to evaluate the effect of platelet age on these various parameters. Using this approach, we demonstrated a 7–8-fold increase in both CD62P (P-selectin) and CD61 (glycoprotein β3) expression in TO-positive platelets, while expression of the platelet marker CD42b (glycoprotein 1bα) increased as much as 14-fold. The subpopulation capable of assembling Prothrombinase likewise demonstrated a 3–4-fold increase in CD62P, CD61, and CD41 (glycoprotein IIb) expression, and a ~7-fold increase in CD42b expression. Dual-labeling studies demonstrated that ~60% of TO-positive platelets are able to bind factor Xa while ~85% of platelets able to bind Xa are TO-positive. Similar results were seen when platelets were evaluated for their ability to bind high levels of factor Va. These data suggest that it is predominantly the younger platelets with increased density of adhesive receptors that are capable of assembling Prothrombinase on their activated surface. Since platelet adhesion is the initiating step in both normal hemostasis and pathological thrombus formation, the observed increase in adhesive receptor density places this platelet subpopulation in a unique position of significance in the regulation of the hemostatic response, and indicates a potential role for these platelets, in the initiation of thrombus formation.

The Effects of Heparin and Annexin V on Fibrin Accretion after Injury in the Jugular Veins of Rabbits

1993 ◽  
Vol 69 (03) ◽  
pp. 227-230 ◽  
Author(s):  
J Van Ryn-McKenna ◽  
H Merk ◽  
T H Müller ◽  
M R Buchanan ◽  
W G Eisert
Keyword(s):  
Vessel Wall ◽  
Thrombin Generation ◽  
Antithrombin Iii ◽  
Specific Effect ◽  
Annexin V ◽  
Inhibitory Effect ◽  
Jugular Veins ◽  
Prothrombinase Complex ◽  
Wall Injury

SummaryWe compared the relative abilities of unfractionated heparin and annexin V to prevent fibrin accretion onto injured jugular veins in vivo. Heparin was used to accelerate the inhibition of thrombin by antithrombin III, and annexin V was used to inhibit the assembly of the prothrombinase complex on phospholipid surfaces, thereby blocking thrombin generation. Rabbit jugular veins were isolated in situ, a 2 cm segment was injured by perfusing it with air, and then blood flow was re-established. Five minutes later, each rabbit was injected with heparin (20 U/kg) or annexin V (0.3 mg/kg) and then with 125I-fibrinogen. The amount of 125I-fibrin accumulation onto each injured vessel wall segment was measured 4 h later. Each injured vessel was completely deendothelialized as a result of the air perfusion as demonstrated by electron microscopy. 125I-fibrin accretion onto the injured jugular veins was enhanced 2.4-fold as compared to the uninjured veins in sham-operated animals. Heparin treatment did not reduce fibrin accretion, whereas, annexin V treatment decreased fibrin accretion by 60%, p <0.05. This latter effect was achieved without sustained circulating anticoagulation. Additional experiments confirmed that the inhibitory effect of annexin V on fibrin accretion was associated with a surface specific effect, since more annexin V bound to the injured jugular vein segments as compared to the non-injured jugular veins. We conclude that, i) mild vessel wall injury (selective de-endothelialization) in veins results in a thrombogenic vessel wall; ii) the thrombogenecity of which is not inhibited by prophylactic doses of heparin; but iii) is inhibited by annexin V, which binds to injured vessel wall surface, and inhibits thrombin generation independently of antithrombin III.

Increased Procoagulant Activity of Red Blood Cells from Patients with Homozygous Sickle Cell Disease and β-Thalassemia

1996 ◽  
Vol 76 (03) ◽  
pp. 322-327 ◽  
Author(s):  
Dominique Helley ◽  
Amiram Eldor ◽  
Robert Girot ◽  
Rolande Ducrocq ◽  
Marie-Claude Guillin ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Annexin V ◽  
Mean Value ◽  
Procoagulant Activity ◽  
Dependent Manner ◽  
Cell Disease ◽  
Homozygous Sickle Cell Disease

SummaryIt has recently been proved that, in vitro, red blood cells (RBCs) from patients with homozygous β-thalassemia behave as procoagulant cells. The procoagulant activity of β-thalassemia RBCs might be the result of an increased exposure of procoagulant phospholipids (i. e. phosphatidylserine) in the outer leaflet of the membrane. In order to test this hypothesis, we compared the catalytic properties of RBCs of patients with β-thalassemia and homozygous sickle cell disease (SS-RBCs) with that of controls. The catalytic parameters (Km, kcat) of prothrombin activation by factor Xa were determined both in the absence and in the presence of RBCs. The turn-over number (kcat) of the reaction was not modified by normal, SS- or (3-thalassemia RBCs. The Km was lower in the presence of normal RBCs (mean value: 9.1 µM) than in the absence of cells (26 µM). The Km measured in the presence of either SS-RBCs (mean value: 1.6 µM) or β-thalassemia RBCs (mean value: 1.5 pM) was significantly lower compared to normal RBCs (p <0.001). No significant difference was observed between SS-RBCs and p-thalassemia RBCs. Annexin V, a protein with high affinity and specificity for anionic phospholipids, inhibited the procoagulant activity of both SS-RBCs and (3-thalassemia RBCs, in a dose-dependent manner. More than 95% inhibition was achieved at nanomolar concentrations of annexin V. These results indicate that the procoagulant activity of both β-thalassemia RBCs and SS-RBCs may be fully ascribed to an abnormal exposure of phosphatidylserine at the outer surface of the red cells.

scholarly journals Ectonucleotide Triphosphate Diphosphohydrolase-1 (CD39) Mediates Resistance to Occlusive Arterial Thrombus Formation after Vascular Injury in Mice

2012 ◽  
Vol 181 (1) ◽  
pp. 322-333 ◽  
Author(s):  
Zachary M. Huttinger ◽  
Michael W. Milks ◽  
Michael S. Nickoli ◽  
William L. Aurand ◽  
Lawrence C. Long ◽  
...  
Keyword(s):  
Vascular Injury ◽  
Thrombus Formation ◽  
Arterial Thrombus

scholarly journals Dietary α-Linolenic Acid Inhibits Arterial Thrombus Formation, Tissue Factor Expression, and Platelet Activation

2011 ◽  
Vol 31 (8) ◽  
pp. 1772-1780 ◽  
Author(s):  
Erik W. Holy ◽  
Marc Forestier ◽  
Eva K. Richter ◽  
Alexander Akhmedov ◽  
Florian Leiber ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Linolenic Acid ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Tissue Factor Expression ◽  
Arterial Thrombus ◽  
Factor Expression

Abstract 13598: The G-protein BetaGamma Subunits Regulate Platelet Function

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Ahmed Alarabi ◽  
Zubair Karim ◽  
Victoria Hinojos ◽  
Patricia A Lozano ◽  
Keziah Hernandez ◽  
...  
Keyword(s):  
G Protein ◽  
Platelet Function ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Inhibitory Effects ◽  
Clot Retraction ◽  
Betagamma Subunits

Platelet activation involves tightly regulated processes to ensure a proper hemostasis response, but when unbalanced, can lead to pathological consequences such as thrombus formation. G-protein coupled receptors (GPCRs) regulate platelet function by interacting with and mediating the response to various physiological agonists. To this end, an essential mediator of GPCR signaling is the G protein Gαβγ heterotrimers, in which the βγ subunits are central players in downstream signaling pathways. While much is known regarding the role of the Gα subunit in platelet function, that of the βγ remains poorly understood. Therefore, we investigated the role of Gβγ subunits in platelet function using a Gβγ (small molecule) inhibitor, namely gallein. We observed that gallein inhibits platelet aggregation and secretion in response to agonist stimulation, in both mouse and human platelets. Furthermore, gallein also exerted inhibitory effects on integrin αIIbβ3 activation and clot retraction. Finally, gallein’s inhibitory effects manifested in vivo , as documented by its ability to modulate physiological hemostasis and delay thrombus formation. Taken together, our findings demonstrate, for the first time, that Gβγ directly regulates GPCR-dependent platelet function, in vitro and in vivo . Moreover, these data highlight Gβγ as a novel therapeutic target for managing thrombotic disorders.

Circulating platelet-derived microparticles in systemic lupus erythematosus

2006 ◽  
Vol 95 (01) ◽  
pp. 94-99 ◽  
Author(s):  
Gino Alfaro ◽  
Manuela Goycoolea ◽  
Teresa Quiroga ◽  
Mauricio Ocqueteau ◽  
Loreto Massardo ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Lupus Erythematosus ◽  
Thrombin Generation ◽  
Thrombus Formation ◽  
Annexin V ◽  
Coagulation System ◽  
Systemic Lupus ◽  
Double Labeling ◽  
Cellular Source ◽  
Risk For Thrombosis

SummaryThe risk for thrombosis is significantly increased in systemic lupus erythematosus (SLE), affecting both venous and arterial vessels. Activated platelets are known to participate in thrombus formation and growth. A general feature of activated cells is the shedding of microparticles (MP) which support coagulation by exposure of negatively charged phospholipids and possibly tissue factor (TF). In this work we characterized circulating MP in patients with SLE and their relationship with a procoagulant state. Thirty patients with SLE (aged 15–72 years, mean age 38 years) and 20 healthy controls (aged 22–54 years, mean age 34 years) were studied; patients fulfilled 4 revised criteria for SLE. The number and cellular source of circulating MP were determined by flow cytometry using double labeling with specific monoclonal antibodies and annexin V. Thrombin generation was measured as the endogenous thrombin potential (ETP) without the addition of exogenous phospholipids and TF; under these conditions the generation of thrombin depended directly on the number of MP present in plasma. Thrombin anti-thrombin (TAT) and plasmin-antiplasmin (PAP) complexes were measured by ELISA. Compared to the controls, circulating MP were significantly elevated in the patient group (1218 ± 136 vs 653 ± 74 x103/ml plasma, p: 0.0007). In both groups, most of these MP were of platelet origin (927± 131 vs 517 ± 72 x103/ml plasma, p:0.009 ). ETP was higher among patients as compared to the controls (804± 64 vs 631 ± 37 nM thrombin, p: 0.025). Plasma levels of TAT in patients and controls were 3.4 ± 0.8 and 1.4 ± 0.5 µg/L, respectively (p:0.04), and of PAP complexes were 62.5 ± 14 and 24.05± 2.5 µg/ml, respectively (p: 0.014).The number of platelet-derived MP correlated significantly with thrombin generation (r: 0.42; p: 0.038) and TAT levels (r: 0.40; p: 0.035).We did not find an association of circulating MP with disease activity nor with the presence of antiphospholipid antibodies. The increased number of circulating platelet-derived microparticles and their association with high ETP and activation of the coagulation system suggest that these microparticles play an important role in the pathogenesis of the prothrombotic state in SLE patients.

scholarly journals Layer-by-layer coating of stainless steel plates mediated by surface priming treatment to improve antithrombogenic properties

2016 ◽  
Author(s):  
Irene Carmagnola ◽  
Tiziana Nardo ◽  
Francesca Boccafoschi ◽  
Valeria Chiono
Keyword(s):  
Stainless Steel ◽  
Surface Modification ◽  
Platelet Activation ◽  
Colorimetric Assay ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Steel Plates ◽  
Blue Dye ◽  
Layer By Layer

The stainless steel (SS) stents have been used in clinics since 1994. However, typical drawbacks are restenosis and thrombus formation due to limited endothelialisation and hemocompatibility. Surface modification is a smart strategy to enhance antithrombogenicity by promoting endothelialisation. In this work, the layer-by-layer (LbL) technique was applied for coating SS model substrates, after surface priming by functionalisation with 3-aminopropyl triethoxysilane (APTES). A LbL coating made of 14 layers of poly(styrene sulfonate)/poly(diallyldimethylammonium chloride) and heparin as last layer was deposited. FTIR-ATR analysis and contact angle measurements showed that LbL was an effective method to prepare nanostructured coatings. XPS analysis and colorimetric assay employing 1,9-dimethylmethylene blue dye to detect -COOH groups confirmed the successful polyelectrolyte deposition on the coated samples. Preliminary in vitro cell tests, using whole blood and human platelets, were performed to evaluate how surface modification affects platelet activation. Results showed that SS and SS-APTES surfaces induced platelet activation, as indicated by platelet spreading and filopodia formation. After surface modification by LbL coating, the platelets assumed a round shape and no fibrin nets were detected. Data demonstrated that LbL coating is a promising technique to fabricate antithrombogenic surface.

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