The Total Thrombus Formation (T-TAS) platelet (PL) assay, a novel test that evaluates whole blood platelet thrombus formation under physiological conditions

SummaryThrombin plays a key role in platelet activation and thrombosis. Specific inhibition of thrombin appears to be one of the best approaches to prevent thrombus formation. We have studied the effects of a synthetic a-aminoboronic acid derivative - [Ac, (D) Phe-Pro-Boro-Arg-Hydrocloric acid] - on platelet deposition on severely damaged arterial wall. Platelet deposition was evaluated under well characterized rheological conditions in an original perfusion chamber and detected by autologous mIn-labeled platelets. The study was performed “in vivo” in a porcine model of arterial thrombosis triggered by severely damaged vessel wall at blood flow conditions mimicking mild stenosis (1690 s−1) and patent (212 s−1) vessels. In addition, ex-vivo platelet aggregation activity was evaluated by whole blood impedance aggregometry using collagen, ADP and thrombin as agonists. The synthetic a-aminoboronic peptide was intravenously administered as a bolus followed by continuous infusion. Ex vivo thrombin-induced whole blood platelet aggregation was totally abolished, while ADP- and Collagen-induced whole blood platelet aggregation was not modified. The effects of the synthetic antithrombin on platelet deposition were evaluated in native blood (non-anticoagulated) conditions and in combination with heparin. Under both experimental conditions, the synthetic peptide significantly inhibited platelet deposition at local flow conditions of both high (1690 s−1) and low (212s−1) shear rates. Our results suggest that specific inhibition of locally generated thrombin might be a good strategy to prevent platelet dependent arterial thrombus formation independently of the local flow shear rate of the area at risk.

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FcγRIIa Enhances Thrombus Growth in Vitro and in Vivo

Blood ◽

10.1182/blood.v118.21.191.191 ◽

2011 ◽

Vol 118 (21) ◽

pp. 191-191

Author(s):

Huiying Zhi ◽

Lubica Rauova ◽

Vincent M Hayes ◽

Jimmy Crockett ◽

Cunji Gao ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Platelet Function ◽

Whole Blood ◽

Thrombus Formation ◽

Flow Chamber ◽

Wild Type ◽

Integrin Αiibβ3 ◽

Platelet Thrombus

Abstract Abstract 191 Outside-in signal transduction is one of several autocrine amplification loops that platelets employ to stabilize and consolidate a platelet thrombus following their adhesion to each other or to components of the extracellular matrix. Binding of soluble fibrinogen to activated integrin αIIbβ3 on the platelet surface, or binding of αIIbβ3 to platelet-immobilized fibrinogen, initiates an outside-in signaling cascade that results in the activation of integrin β3-associated Src family kinases, which in turn phosphorylate tyrosine residues within the cytoplasmic domain of the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor protein, FcγRIIa. “Activation” of FcγRIIa sets off a cascade of events that result in the assembly and activation of other key signaling intermediates, including the tyrosine kinase Syk and phospholipase Cγ2(PLCγ2), through its lipase activity, generates lipid products that support a multitude of cellular activation responses, including cytoskeletal rearrangements leading to platelet shape change and spreading, secretion of platelet granules, and activation of additional cell surface integrins. We have previously shown that either antibody-mediated or genetic disruption of the functional interaction between integrin αIIbβ3 and FcγRIIa blocks tyrosine phosphorylation of FcγRIIa, Syk, and PLCγ2, and inhibits platelet spreading on immobilized fibrinogen. The physiological significance of FcγRIIa in supporting platelet thrombus formation, however, remains unknown. To further explore the importance of FcγRIIa in platelet function, we compared the relative ability of wild-type FcγRIIa-negative and transgenic FcγRIIa-positive (FcγRIIaTGN) murine platelets to support thrombosis and hemostasis in a number of well-accepted models of platelet function. FcγRIIaTGN platelets exhibited increased tyrosine phosphorylation of Syk and PLCγ2 and increased spreading upon interaction with immobilized fibrinogen. FcγRIIaTGN platelets also retracted a fibrin clot faster than did wild-type FcγRIIa-negative platelets. When anti-coagulated whole blood was perfused over a collagen-coated flow chamber under conditions of arterial shear, the rate and extent of adhesion, aggregation, and thrombus formation was significantly increased for FcγRIIaTGN platelets compared to their wild-type murine counterparts. Addition of Fab fragments specific for FcγRIIa to whole blood derived from either humans or FcγRIIaTGN mice strongly inhibited thrombus formation in the arterial in vitro flow chamber assay. Finally, to examine the in vivo relevance of FcγRIIa, mice were subjected to two models of vascular injury: electrolytic injury of the femoral vein and laser injury of cremaster arterioles. In both in vivo models, FcγRIIaTGN mice displayed increased thrombus formation compared with their wild-type, FcγRIIa-negative counterparts. Taken together, these data establish FcγRIIa as a physiologically-important functional conduit for αIIbβ3–mediated outside-in signaling, and suggest that modulating the activity of this novel integrin/ITAM pair might be effective in controlling thrombosis. Disclosures: No relevant conflicts of interest to declare.

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Characterization of a Microchip Flow-Chamber System for the Analysis of Platelet Thrombus Formation Using Whole Blood Samples - Studies On Healthy Subjects

Blood ◽

10.1182/blood.v120.21.1144.1144 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1144-1144

Author(s):

Yusuke Yamaguchi ◽

Takanori Moriki ◽

Atsuko Igari ◽

Yumiko Matsubara ◽

Tomoko Ohnishi ◽

...

Keyword(s):

Platelet Function ◽

Whole Blood ◽

Healthy Subjects ◽

Thrombus Formation ◽

Flow Chamber ◽

Advisory Committees ◽

Blood Samples ◽

Platelet Aggregometry ◽

Platelet Thrombus ◽

Whole Blood Samples

Abstract Abstract 1144 Introduction: A flow-chamber system was developed to evaluate the growth of platelet thrombus formation (PTF) quantitatively using whole blood under various shear stress conditions. This device, T-TAS (Total Thrombus-formation Analysis System, Fujimori Kogyo Co., Yokohama, Kanagawa), analyzes the process of PTF by monitoring the continuous pressure increase in the capillary of microchip where whole blood flows, using two kinds of thrombogenic surfaces (PL chip: coated with collagen, AR chip: coated with collagen plus tissue factor). In the current study, we characterized this system using whole blood samples from healthy subjects by comparing the measurements with those of other standard platelet function tests. Materials and Methods: Whole blood samples were collected from 32 healthy volunteers with hirudin (PL chip) or 3.2% sodium citrate (AR chip) as anticoagulants. For AR chip, CaCl2 with corn trypsin inhibitor was mixed immediately before the testing. The samples were individually applied on the system to measure the PTF starting time (T10: time to reach 10 kPa), occlusion time (OT: T60, time to reach 60 kPa for PL chip; T80, 80 kPa for AR chip), and AUC (area under the flow pressure curve: AUC10, until 10 min for PL chip; AUC30, until 30 min for AR chip) under various shear rates (1000, 1500, 2000 s−1 for PL chip; 300 s−1 for AR chip). Platelet function of the blood sample was also tested using platelet aggregometry (collagen, ADP, ristocetin, and epinephrine as agonists), PFA-100 (C/EPI-, C/ADP-CT: closure time) and VerifyNow P2Y12 assay (PRU). Results: In PL chip, T10 was correlated with C/EPI- and C/ADP-CT, and AUC10 was correlated with C/EPI-CT under all of the shear conditions. The correlation was enhanced in accordance with the increase of the shear rates. In addition, T60 and AUC10 were correlated with AUC of collagen-induced aggregation curve of platelet aggregometry. In AR chip, T10–80, reflecting the rate of thrombus growth, was likely correlated with C/ADP-CT. Measured values from VerifyNow P2Y12 assay was not significantly associated with those from this system. Interestingly, platelet numbers were significantly correlated with all of the measurements with AR chip, and partially with those with PL chip. Conclusion: In healthy subjects, PTF starting time and AUC with PL chip, and the growth rate of PTF with AR chip, seemed associated with PFA-100 measurements, indicating its characteristics related to shear induced PTF. However, the values from this system showed a rare correlation with those from platelet aggregometry and VerifyNow P2Y12 assay. This system may allow us to identify the parameters of individuals' thrombogenicity independent of those related to other platelet function tests, under whole blood flow conditions. Disclosures: Matsubara: Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees. Ohnishi:Fujimori Kogyo Co.: Employment. Hosokawa:Fujimori Kogyo Co.: Employment. Murata:Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees.

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Hemostatic Effects of Fibrinogen-γ Chain Dodecapeptide-Conjugated Polymerized Albumin Particles In Vitro and in Vivo.

Blood ◽

10.1182/blood.v104.11.3883.3883 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3883-3883

Author(s):

Yosuke Okamura ◽

Naohide Watanabe ◽

Shinji Takeoka ◽

Hidenori Suzuki ◽

Mitsuru Murata ◽

...

Keyword(s):

Bleeding Time ◽

Thrombus Formation ◽

Blood Platelet ◽

Becton Dickinson ◽

Platelet Surface ◽

Normal Platelet ◽

Platelet Thrombus ◽

Carboxy Terminal

Abstract We have studied a prototype of platelet substitutes focusing on a dodecapeptide, HHLGGAKQAGDV (H12)1), which is specific for fibrinogen γ-chain carboxy-terminal sequence (γ 400–411). In this study, we conjugated H12 to the surface of polymerized albumin particles (polyAlb) as biocompatible and biodegradable carriers to produce particles having hemostatic ability, and evaluated their in vitro and in vivo effects. H12 (H12; 9.6 x 103 /particles) containing cysteine to the N-terminal was conjugated to polyAlb (260 ± 60 nm), and the effect of H12-polyAlb on platelet thrombus formation was evaluated in vitro with thrombocytopenic whole blood ([platelet] = 2.0 x 104 /μL, anticoagulated with PPACK) under flow conditions (shear rate; 150 s−1). Thrombocytopenic rats were made by busulphan injection at a dose of 20 mg/kg, and a 2.5 mm length x 1.0 mm depth template-guided incision (QuikheelTM, Becton-Dickinson, San Jose, CA) was made 1 cm from the tip of tail. The tail was immersed in a 50 mL cylinder of saline and the time taken to stop bleeding was measured. When thrombocytopenic blood in the presence of H12-polyAlb ([rHSA]=0.14 mg/mL) was flowed on collagen-plate, the surface coverage of DiOC6-labeled platelets evaluated by fluorescence microscopy was increased to 3.9 ± 1.1 % (n=3) from 2.1 ± 0.4 % (n=3) obtained in the absence of H12-polyAlb. In the same experiments, rhodamine-labeled H12-polyAlb was found to be involved in platelet-platelet interactions by binding to activated platelet surface, thus enhancing platelet thrombus formation. Next, in vivo hemostatic effect was tested by measuring tail bleeding time of thrombocytopenic rats 5 minutes after the intravenous administration of H12-polyAlb. The bleeding times of normal ([platelet] = 8.1 ±0.9 x 105 /μL) and thrombocytopenic rats ([platelet] = 2.0 ±0.3 x 105 /μL) were 187 ± 51 s and 609 ± 153 s (n=6), respectively. H12-polyAlb administration at a dose of 4 mg/kg significantly shortened the bleeding time to 342 ± 73 s (n=10), whereas polyAlb was without effects (553 ± 104 s, n=6). These results indicate that H12-polyAlb can be a suitable candidate for an alternative to human platelet concentrates infused into thrombocytopenic patients.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645978 ◽

1987 ◽

Vol 58 (02) ◽

pp. 744-748 ◽

Cited By ~ 11

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.

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An Electron Microscopic Study of Platelet Thrombus Formation in the Rabbit with Particular Regard to 5-Hydroxytryptamine Release

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655068 ◽

1967 ◽

Vol 18 (03/04) ◽

pp. 592-604 ◽

Cited By ~ 15

Author(s):

H. R Baumgartner ◽

J. P Tranzer ◽

A Studer

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Vessel Wall ◽

Thrombus Formation ◽

Endothelial Damage ◽

Electron Microscopic ◽

Rabbit Ear ◽

Basement Lamina ◽

Platelet Thrombus

SummaryElectron microscopic and histologic examination of rabbit ear vein segments 4 and 30 min after slight endothelial damage have yielded the following findings :1. Platelets do not adhere to damaged endothelial cells.2. If the vessel wall is denuded of the whole endothelial cell, platelets adhere to the intimai basement lamina as do endothelial cells.3. The distance between adherent platelets as well as endothelial cells and intimai basement lamina measures 10 to 20 mµ, whereas the distance between aggregated platelets is 30 to 60 mµ.4. 5-hydroxytryptamine (5-HT) is released from platelets during viscous metamorphosis at least in part as 5-HT organelles.It should be noted that the presence of collagen fibers is not necessary for platelet thrombus formation in vivo.

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Platelet Aggregation in Whole Blood: The Role of Thromboxane A2 and Adenosine Diphosphate

Thrombosis and Haemostasis ◽

10.1055/s-0038-1660081 ◽

1985 ◽

Vol 54 (03) ◽

pp. 612-616 ◽

Cited By ~ 17

Author(s):

A J Carter ◽

S Heptinstall

Keyword(s):

Platelet Aggregation ◽

Whole Blood ◽

Adenosine Diphosphate ◽

Blood Platelet ◽

Thromboxane B2 ◽

Lipoxygenase Inhibitor ◽

Thromboxane Synthase Inhibitor ◽

Synthase Inhibitor

SummaryThe platelet aggregation that occurred in whole blood in response to several aggregating agents (collagen, arachidonic acid, adenosine diphosphate, adrenaline and thrombin) was measured using an Ultra-Flo 100 Whole Blood Platelet Counter. The amounts of thromboxane B2 produced were measured by radioimmunoassay. The effects of various inhibitors of thromboxane synthesis and the effects of apyrase, an enzyme that destroys adenosine diphosphate, were determined.Platelet aggregation was always accompanied by the production of thromboxane B2, and the amounts produced depended on the nature and concentration of the aggregating agent used. The various inhibitors of thromboxane synthesis - aspirin and flurbiprofen (cyclo-oxygenase inhibitors), BW755C (a cyclo-oxygenase and lipoxygenase inhibitor) and dazoxiben (a selective thromboxane synthase inhibitor) - did not markedly inhibit aggregation. Results obtained using apyrase showed that adenosine diphosphate contributed to the aggregation process, and that its role must be acknowledged when devising means of inhibiting platelet aggregation in vivo.

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The Effect of Agents which Modify Platelet Behaviour and of Magnesium Ions on Thrombus Formation In Vivo

Thrombosis and Haemostasis ◽

10.1055/s-0038-1666898 ◽

1979 ◽

Vol 42 (02) ◽

pp. 603-610 ◽

Cited By ~ 59

Author(s):

J H Adams ◽

J R A Mitchell

Keyword(s):

Thrombus Formation ◽

Cerebral Arteries ◽

Magnesium Ions ◽

Body Formation ◽

Inflammatory Agent ◽

Platelet Thrombus ◽

Magnesium Salts ◽

Higher Doses ◽

Do So

SummaryThe ability of potential anti-thrombotic agents to modify platelet-thrombus formation in injured cerebral arteries in the rabbit was tested. Low doses of heparin were without effect, while higher doses produced variable suppression of white body formation but at the expense of bleeding. Aspirin did not inhibit white body formation but another non-steroid anti-inflammatory agent, flurbiprofen was able to do so, as was the anti-gout agent, sulphinpyrazone. Magnesium salts both topically and parenterally, suppressed thrombus formation and increased the concentration of ADP which was required to initiate thrombus production at minor injury sites.

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