scholarly journals A microchip flow-chamber system for quantitative assessment of the platelet thrombus formation process

2012 ◽  
Vol 83 (2) ◽  
pp. 154-161 ◽  
Author(s):  
Kazuya Hosokawa ◽  
Tomoko Ohnishi ◽  
Masashi Fukasawa ◽  
Taro Kondo ◽  
Hisayo Sameshima ◽  
...  
Keyword(s):  
Formation Process ◽  
Quantitative Assessment ◽  
Thrombus Formation ◽  
Flow Chamber ◽  
Chamber System ◽  
Platelet Thrombus

scholarly journals Decreased Platelet Thrombus Size, Due to a Heightened Proteolysis of VWF By ADAMTS13, Is Quickly Restored after Valve Replacement in Aortic Stenosis Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2857-2857
Author(s):  
Hideo Yagi ◽  
Masaki Hayakawa ◽  
Naoko Yamaguchi ◽  
Keigo Yamashita ◽  
Shigeki Taniguchi ◽  
...  
Keyword(s):  
Shear Stress ◽  
Aortic Valve ◽  
Valve Replacement ◽  
Thrombus Formation ◽  
Flow Chamber ◽  
High Shear ◽  
Chamber System ◽  
High Shear Stress ◽  
Platelet Thrombus ◽  
Von Willebrand

Abstract Background: The patients with severe aortic-valve stenosis (AS) are often complicated with bleeding episodes. The association between AS and gastrointestinal bleeding due to angiodysplasia is reported as Heyde's syndrome, which is categorized as one of the acquired von Willebrand disease (AVWD) in cardiovascular disorders. An international survey has shown that Type 2A is the common subtype of AVWD in the patients with AS. AVWD Type 2A is characterized by impaired platelet-dependent VWF function caused by marked decrease or absence of the most hemostatically active HMW-VWFM. In the patients with AS, a significant correlation between the increased high shear stress and loss of HMW-VWFM in vivo. Further, the absence of HMW-VWFM and bleeding tendency are normalized after valve replacement. These results suggest that enhanced proteolysis of von Willebrand factor (VWF) as it passes through the stenotic valve may induce the loss of HMW-VWFM because high shear stress can induce structure changes in VWF, which is sensitive form to the VWF cleaving protease, termed ADAMTS13. Here, we performed investigation of plasma levels of VWF antigen (VWF:Ag), ADAMTS13 activity (ADAMTS13:AC), and platelet thrombus formation in the patients with AS by valve replacement to confirm the pathophysiological mechanism of this rare disease. Patients and Methods: Ten consecutive patients who underwent aortic valve replacement for AS in Nara Medical University Hospital were enrolled in this study. The severity of AS was judged by the American Heart Association guideline. All patients had no bleeding history and received bovine tissue valves replacement followed by administration of warfarin and/or anti-platelet agents for prevention of thrombosis a week after surgery. We collected a series of blood samples from these patients before and day1, 8, 15, 22 after valve replacement. Excluding one patient who developed critical cardiac failure just after valve replacement, 9 patients were eventually evaluated by analyses of VWF:Ag, VWF multimers, ADAMTS13:AC, and mural thrombus formation using flow chamber system. VWF:Ag was measured by sandwich ELISA using a rabbit anti-human VWF polyclonal antiserum. Analysis of VWF multimers was performed according to the method of Ruggeri and Zimmerman. ADAMTS13:AC was measured by a chromogenic ADAMTS13-act-ELISA. Platelet thrombus formation was evaluated by thrombus generation under a high shear stress in a parallel plate flow chamber system. Briefly, whole blood anti-coagulated with argatroban was incubated with the fluorescent dye DiOC6 (1uM), and these samples containing DiOC6 -labeled platelets were perfused for 7 min over a type I collagen-coated glass surface under a high shear rate (1500 s-1). The DiOC6 fluorescence corresponding to the platelets was examined at an excitation wavelength of 488 nm with a barrier filter at 500 nm. The percentage of the area covered by adhering platelets (surface coverage) and each thrombus volume were evaluated. Results: Plasma levels of VWF:Ag before surgery were 78.1 % (median) and those on day 1, 8, 15, 22 after surgery were 130, 224, 155, and 134 %, respectively (Fig 1). Conversely, these levels of ADAMTS13:AC were 50.5, 35.5, 25.5, 25.1, and 30.3 %, respectively (Fig 2). The ratio of VWF:Ag/ADAMTS13:AC at before and day 1, 8, 15, 22 after surgery were 1.6, 4.5, 8.1, 6.1, and 4.1, respectively. In VWF multimer analysis, we found the obvious defect of HMW-VWFM in 7 of 9 patients before surgery, who were diagnosed with severe AS. The remaining two patients had moderate AS with the slight defect of HMW-VWFM. These defects were improved within 14 days after surgery. In platelet thrombus formation, the amount of thrombus volumes significantly increased at day 8, 15, and 22 after compared with before surgery (Fig 3). Conclusion: The dramatic recovery of platelet thrombus formation was observed in the patients with AS by valve replacement. The rapid increment of VWF and normalization of VWFM pattern, together with reduction of ADAMTS13 after valve replacement suggested heightened proteolysis of VWF by ADAMTS13 under high shear stress would be a major cause of this unique bleeding complication. The highest ratio of VWF:Ag/ADAMTS13:AC at day 8 after surgery might imply the necessity of blockade of heightened VWF function with anti-platelet agents. Figure 1 Figure 1. Disclosures Matsumoto: Alfresa Pharma Corporation: Patents & Royalties. Fujimura:Alfresa Pharma Corporation: Patents & Royalties.

scholarly journals A modified microchip-based flow chamber system for evaluating thrombogenicity in patients with thrombocytopenia

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Bengo Atari ◽  
Takashi Ito ◽  
Tomoka Nagasato ◽  
Tomoko Ohnishi ◽  
Kazuya Hosokawa ◽  
...  
Keyword(s):  
Major Bleeding ◽  
Platelet Transfusion ◽  
Thrombus Formation ◽  
Flow Chamber ◽  
University Hospital ◽  
Minor Bleeding ◽  
Chamber System ◽  
Occlusion Time ◽  
Function Tests ◽  
Thrombogenic Potential

Abstract Background In the intensive care unit (ICU), patients with thrombocytopenia are at high risk for bleeding and should be assessed for their thrombogenic potential. However, the analytical conditions of conventional hemostatic tests are unsuitable for the evaluation of low-platelet samples. Here we aimed to establish suitable analytical conditions with the Total Thrombus-formation Analysis System (T-TAS) for quantitative assessment of thrombogenic potential in patients with thrombocytopenia and to investigate how T-TAS values relate to bleeding symptoms and the effects of platelet transfusion. Methods Modified chips with a different chamber depth were developed for the analysis of low-platelet samples in the T-TAS. We included 10 adult patients admitted to the ICU of Kagoshima University Hospital who required platelet transfusion. Patients were divided into major and minor bleeding groups according to their bleeding scale before platelet transfusion. The thrombogenic potential of these patients before and after platelet transfusion was assessed with hemostatic function tests, including rotational thromboelastometry, multiplate aggregometry, and the T-TAS. Results Analysis of low-platelet samples revealed that, compared with the conventional chip (80-μm-deep chamber), the modified chip (50-μm-deep chamber) achieved higher sensitivity in detecting elevation of flow pressure caused by growth of an occlusive thrombus in the T-TAS analytical chamber. All patients in the minor bleeding group retained thrombogenic potential that occluded the modified chip (occlusion time 16.3 ± 3.3 min), whereas most patients in the major bleeding group were unable to occlude the modified chip during the 30-min measurement (P <  0.01). The recovery of thrombogenic potential after platelet transfusion was confirmed with the T-TAS and correlated with the function, rather than the count, of transfused platelets. Among all evaluated parameters in hemostatic function tests, only the T-TAS showed significant differences in occlusion time and area under the curve both between the minor and major bleeding groups and between pre- and post-platelet transfusion. Conclusions We developed a modified microchip-based flow chamber system that reflects the hemostatic function of patients with thrombocytopenia.

FcγRIIa Enhances Thrombus Growth in Vitro and in Vivo

Blood ◽  
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.

Combined Dabigatran and Antiplatelet Agents Assessed by the Novel Microchip-Based Flow Chamber System

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1167-1167
Author(s):  
Kenichi Tanaka ◽  
Kazuya Hosokawa ◽  
Tomoko Ohnishi ◽  
Hisayo Sameshima ◽  
Takehiko Koide ◽  
...  
Keyword(s):  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Antiplatelet Agents ◽  
Lag Time ◽  
Flow Chamber ◽  
Chamber System ◽  
Shear Rates ◽  
Antithrombotic Effects ◽  
Flow Pressure

Abstract Abstract 1167 Evaluation of the overall antithrombotic activity of dabigatran in combination with antiplatelet agents is difficult because plasma-based clotting for dabigatran, and platelet aggregometry in anticoagulated blood are two separate tests which do not reflect physiological interactions between soluble factors and platelets. The use of a flow chamber could be more suitable in evaluating a flow-dependent platelet activation and coagulation responses. The aim of the current study was to comparatively evaluate antithrombotic effects of dabigatran in combination with dual antiplatelet therapy (aspirin plus P2Y12 blockade) using the microchip-based flow chamber (T-TAS, Fujimori Kogyo, Japan)(1), and thrombin generation (TG) assay (Thrombinoscope, Maastricht, the Netherlands)(2). After the local ethics committee approval, blood samples were obtained from consented 5 healthy volunteers in the tubes containing 3.2% sodium citrate. Whole blood samples were mixed with dabigatran (250, 500, 1000 nM), aspirin (100 nM) plus ARC-66096 (P2Y12 inhibitor, 1000 nM) at 25¡C for 10 min. Corn trypsin inhibitor (50 μg/ml) was used to prevent contact activation. The whole blood sample was perfused in the capillary pre-coated with collagen and thromboplastin at the shear rate of 240 or 600 s−1. The process of thrombus formation was monitored by flow pressure increases inside the capillary; (i) lag time before it reaches 10 kPa (T10), (ii) occlusion time (OT) is the lag time before it reaches 80 kPa as thrombus completely occludes the capillary, and (iii) AUC30 is an area under the flow pressure curve (under 80 kPa) after 30 min of perfusion. For TG assay, platelet-rich plasma (platelet count 150 × 103/μl) was prepared from citrated whole blood. TG was triggered by adding 20 μl of CaCl2-fluorogenic substrate buffer to 80 μl of the sample mixed with tissue factor (1 pM) in each well. The lag time (min), and peak thrombin concentration (nM) were evaluated. In the flow chamber, dabigatran inhibited white thrombus formation in a concentration dependent manner at shear rates of 240 and 600 s−1(Fig. 1). At 500 nM of dabigatran, OT was prolonged by ∼2-fold from the (non-treated) control at both shear rates. The combination of aspirin and AR-C66096 only weakly suppressed thrombus formation, but it enhanced the antithrombotic efficacy of dabigatran at both shear rates (Fig. 1). In TG measurements using platelet-rich plasma, dabigatran at 500 nM prolonged the by 3.17-fold, and reduced the peak by 57.6% compared to the untreated control (Table 1). Aspirin and AR-C66096 weakly prolonged the lag time without affecting the peak height. There were relatively small changes in these parameters when antiplatelet agents were combined with dabigatran (Table 1). Our results suggest that combined antithrombotic effects of dabigatran, aspirin, and P2Y12inhibition can be demonstrated in the whole blood using the flow chamber system compared without additional plasma preparation required for TG assay. The re-calcified whole blood was perfused at the shear rate of 240 s−1 or 600 s−1. Asp/AR-C=aspirin and AR-C66096 Table 1. Lag time (min) Peak (nM) Native Asp/AR-C Native Asp/AR-C Control 6.8 ± 0.8 9.4 ± 3.2 92.1 ± 23.7 91.2 ± 29.5 Dabi 250 nM 18.6 ± 5.4 21.1 ± 4.5 69.3 ± 20.6 52.2 ± 13.6 Dabi 500 nM 21.6 ± 5.3 26.2 ± 10.2 53.0 ± 5.8 47.8 ± 9.1 Dabi 1000 nM 30.2 ± 5.6 35.1 ± 6.3 23.0 ± 6.9 22.0 ± 8.4 Dabi=dabigatran, Native=no antiplatelet agents, Asp/AR-C=aspirin and AR-C66096 Disclosures: Hosokawa: Fujimori Kogyo: Employment. Ohnishi:Fujimori Kogyo: Employment. Sameshima:Fujimori Kogyo: Employment.

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

Blood ◽  
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.

scholarly journals Enhancement of thrombogenesis by plasma fibronectin cross-linked to fibrin and assembled in platelet thrombi

Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3555-3563 ◽  
Author(s):  
Jaehyung Cho ◽  
Deane F. Mosher
Keyword(s):  
Ex Vivo ◽  
Thrombus Formation ◽  
Flow Chamber ◽  
Plasma Fibronectin ◽  
Increased Risk ◽  
Platelet Thrombus ◽  
Platelet Thrombi ◽  
Artery Disease ◽  
Fibronectin Deposition ◽  
The Impact

To learn how plasma fibronectin stabilizes platelet-rich thrombi in injured mesenteric arterioles of mice, we studied the impact of plasma fibronectin on platelet thrombus formation ex vivo in a parallel flow chamber. Thrombi were greater on surfaces coated with fibrin cross-linked to fibronectin by activated factor XIII than on surfaces coated with fibrin lacking cross-linked fibronectin or with fibronectin alone. Platelet thrombi were even greater when plasma fibronectin was perfused with platelets, resulting in deposition of the perfused fibronectin in platelet thrombi. The effect of perfused fibronectin on thrombogenesis was lost if fibronectin deposition was blocked by coperfusion with the N-terminal 70-kDa fragment of fibronectin or a peptide based on the functional upstream domain of protein F1 of Streptococcus pyogenes. Increases in thrombus formation were dependent on a platelet activator such as lysophosphatidic acid, amount of fibronectin cross-linked to fibrin, and concentration of fibronectin in the perfusate. The dependency of fibronectin concentration extended into the range of fibronectin concentrations associated with increased risk of coronary artery disease. At such concentrations, the 2 mechanisms for insolubilization of plasma fibronectin—cross-linking to fibrin and assembly by adherent and aggregating platelets—synergize to result in many-fold enhancement of platelet thrombus formation.

scholarly journals Assessment of Platelet Thrombus Formation under Flow Conditions in Adult Patients with COVID-19: An Observational Study

2021 ◽  
Author(s):  
Andrea Artoni ◽  
Mauro Panigada ◽  
Stefano Ghirardello ◽  
Anna Lecchi ◽  
Stefano Aliberti ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Flow Chamber ◽  
Illness Severity ◽  
Pressure Curve ◽  
Start Time ◽  
Occlusion Time ◽  
Group A ◽  
Platelet Thrombus ◽  
Analysis System ◽  
Group B

Abstract Background Coronavirus disease 2019 (COVID-19) is associated with systemic inflammation, which may dysregulate platelet function. Total Thrombus-Formation Analysis System (T-TAS) is a flow-chamber device that analyses platelet-mediated thrombus formation in capillary channels through the following parameters: (1) the area under the flow-pressure curve (AUC), (2) occlusion start time (OST), time needed to reach OST, and (3) occlusion time (OT), time needed to reach the occlusion pressure. Methods and Findings Sixty-one COVID-19 patients admitted to intensive, subintensive, and low intensive care were prospectively enrolled according to the time of admission: group A (up to 8 days) (n = 18); group B (from 9 to 21 days) (n = 19), and group C ( > 21 days) (n = 24). T-TAS measurements were performed at enrolment and after 7 days. Median OST was similar among groups. AUC was lower in group A compared to B (p = 0.001) and C (p = 0.033). OT was longer in group A compared to B (p = 0.001) and C (p = 0.028). Platelet count (PC) was higher in group B compared to A (p = 0.024). The linear regression showed that OT and AUC were independent from PC in group A (OT: 0.149 [95% confidence interval [CI]: –0.326 to 0.624], p = 0.513 and AUC: 0.005 [95% CI: –0.008 to 0.017], p = 0,447). In contrast, in group B, PC was associated with OT (–0.019 [–0.028 to 0.008], p = 0.023) and AUC (0.749 [0.358–1.139], p = 0,015), similarly to group C. Conversely, patients with different illness severity had similar T-TAS parameters. Conclusion COVID-19 patients display an impaired platelet thrombus formation in the early phase of the disease compared to later stages and controls, independently from illness severity.

Sign in / Sign up

Export Citation Format

Share Document