Total Thrombus-formation Analysis System Research Articles

In vitro studies show synergistic effects of a procoagulant bispecific antibody and bypassing agents

Background Investigational non-factor products such as emicizumab offer a treatment option for patients with hemophilia and inhibitors. However, their mechanism of action raises questions regarding safety when they are combined with treatments for breakthrough bleeding. Objectives To evaluate in vitro thrombin generation (TG) and clot formation for combinations of activated prothrombin complex concentrate (aPCC), recombinant activated factor VII (rFVIIa), and a sequence-identical analog of emicizumab (SIA). Methods Therapeutic concentrations of SIA (20-600 nm) alone or with aPCC (0.05-1 U mL-1 ), isolated aPCC components or rFVIIa (0.88-5.25 μg mL-1 ) were tested for TG and compared with reference ranges for healthy donor plasma. Coagulation of FVIII-inhibited blood was determined with a widely established method, i.e. rotational thromboelastometry (ROTEM), and confirmed with the Total Thrombus-formation Analysis System. Results and conclusions SIA (600 nm) or aPCC (0.5 U mL-1 ) alone resulted in peak thrombin levels of 21.4 nm and 38.6 nm, respectively, both of which are lower than normal (83.7 ± 29.8 nm). SIA plus aPCC (0.5 U mL-1 ) increased the peak thrombin level 17-fold over SIA alone, exceeding the reference plasma value by 4.2-fold. This hypercoagulable effect occurred with 600 nmSIA combined with as little as 0.25 U mL-1 aPCC, confirmed by ROTEM. FIX was the main driver for enhanced TG. SIA plus rFVIIa (1.75 μg mL-1 ) induced a 1.8-fold increase in the peak thrombin level in platelet-rich plasma, but it did not reach the normal range. These in vitro experiments demonstrate excessive TG after administration of a combination of aPCC and SIA at clinically relevant doses. Careful judgement may be required when breakthrough bleeding is treated in patients receiving emicizumab.

Vascular Nitric Oxide–Superoxide Balance and Thrombus Formation after Acute Exercise

An acute bout of strenuous exercise in humans results in transient impairment of nitric oxide (NO)-dependent function, but it remains unknown whether this phenomenon is associated with increased risk of thrombotic events after exercise. This study aimed to evaluate effects of a single bout of exhaustive running in mice on the balance of vascular NO/reactive oxygen species production, and on thrombogenicity. At different time points (0, 2, and 4 h) after exercise and in sedentary C57BL/6 mice, the production of NO and superoxide (O2) in aorta was measured by electron paramagnetic resonance spin trapping and by dihydroethidium/high-performance liquid chromatography-based method, respectively, whereas collagen-induced thrombus formation was analyzed in a microchip-based flow-chamber system (total thrombus-formation analysis system). We also measured pre- and postexercise plasma concentration of nitrite/nitrate and 6-keto-PGF1α. An acute bout of exhaustive running in mice resulted in decreased production of NO and increased production of O2 in aorta, with maximum changes 2 h after completion of exercise when compared with sedentary mice. However, platelet thrombus formation was not changed by exercise as evidenced by unaltered time to start of thrombus formation, capillary occlusion time, and total thrombogenicity (area under the flow pressure curve) as measured in a flow-chamber system. Strenuous exercise increased the plasma concentration of nitrite but did not affect nitrate and 6-keto-PGF1α concentrations. An acute bout of strenuous exercise in mice reduced NO and in parallel increased O2 production in aorta. This response was most pronounced 2 h after exercise. Surprisingly, the reduced NO and increased O2 production in mice after exercise did not result in increased platelet-dependent thrombogenicity. These results show that transient reduction in NO bioavailability does not modify thromboresistance in healthy mice after exercise.

Microfluidics contrasted to thrombelastography: perplexities in defining hypercoagulability

BackgroundElevated clot strength (maximum amplitude [MA]) measured by thrombelastography (TEG) is associated with thrombotic complications. However, it remains unclear how MA translates to thrombotic risks, as this measurement is independent of time, blood flow, and clot degradation. We hypothesize that under flow conditions, increased clot strength correlates to time-dependent measurements of coagulation and resistance to fibrinolysis. Materials and methodsSurgical patients at high risk of thrombotic complications were analyzed with TEG and total thrombus-formation analysis system (T-TAS). TEG hypercoagulability was defined as an r <10.2 min, angle >59, MA >66 or LY30 <0.2% (based off of healthy control data, n = 141). The T-TAS AR and PL chips were used to measure clotting at arterial shear rates. T-TAS measurements include occlusion start time, occlusion time (OT), occlusion speed (OSp), and total clot generation (area under the curve). These measurements were correlated to TEG indices (R time, angle, MA, and LY30). Both T-TAS and TEG assays were challenged with tissue plasminogen activator (t-PA) to assess clot resistance to fibrinolysis. ResultsThirty subjects were analyzed, including five controls. TEG-defined hypercoagulability by MA was detected in 52% of the inflammatory bowel disease/cancer patients; 0% was detected in the controls. There were no TEG measurements that significantly correlated with T-TAS AR and PL chip. However, in the presence of t-PA, T-TAS AR determined OSp to have an inverse relationship with TEG angle (−0.477, P = 0.012) and LY30 (−0.449, P = 0.019), and a positive correlation with R time (0.441 P = 0.021). In hypercoagulability determined by TEG MA, T-TAS PL had a significantly reduced OT (4:07 versus 6:27 min, P = 0.043). In hypercoagulability defined by TEG LY30, T-TAS PL had discordant findings, with a significantly prolonged OT (6:36 versus 4:30 min, P = 0.044) and a slower OSp (10.5 versus 19.0 kPa/min, P = 0.030). ConclusionsMicrofluidic coagulation assessment with T-TAS has an overall poor correlation with most TEG measurements in a predominantly hypercoagulable patient population, except in the presence of t-PA. The one anticipated finding was an elevated MA having a shorter time to platelet-mediated microfluidic occlusion, supporting the role of platelets and hypercoagulability. However, hypercoagulability defined by LY30 had opposing results in which a low LY30 was associated with a longer PL time to occlusion and slower OSp. These discordant findings warrant ongoing investigation into the relationship between clot strength and fibrinolysis under different flow conditions.

Elucidating the Excessive Pro-Coagulant Effect of a Sequence Identical Analogue to ACE910 in Combination with Bypassing Agents

Introduction: Emicizumab (ACE910), an antibody to FIX(a) and FX(a), is currently under investigation for treatment of hemophilia with inhibitors. In a phase III trial, two thromboembolic complications and three cases of microangiopathy were reported in patients on ACE910 prophylaxis [Oldenburg et al. NEJM 2017], whose breakthrough bleeding was treated with activated prothrombin complex concentrate aPCC (FEIBA) or aPCC and rFVIIa. We generated a sequence identical analogue (SIA) to ACE910 and analyzed its synergistic interplay with bypassing agents.Aims: To monitor in vitro the pro-coagulant activity of SIA ACE910 in the presence of FEIBA and rFVIIa, and detect the source of excessive coagulation induced by SIA ACE910 combined with FEIBA.Methods: A sequence identical analogue (SIA) to ACE910 was expressed in HEK293 cells, purified as previously described [Sampei et al. PLoS One 2013], and analyzed in several global hemostatic assays at different concentrations and test conditions using plasma and whole blood assays. In thrombin generation (TG) experiments, platelet-poor plasma (PPP) from hemophilia A inhibitor patients and hemophilia A plasma reconstituted with platelets from 3 healthy donors (PRP) was used. A normal TG range was established in healthy donor plasma. Therapeutic concentrations of SIA ACE910 (20-600 nM) were tested alone and with FEIBA (0.05-1 U/mL) or rFVIIa (0.88-5.25 µg/mL). To measure FEIBA components' contribution to the synergistic effect with SIA ACE910, PPP was spiked with select FEIBA components at concentrations corresponding to 0.5 U/mL FEIBA in combination with the antibody. Thrombus formation was analyzed in FVIII-inhibited blood using rotational thromboelastometry (ROTEM) and Total Thrombus-formation Analysis System (T-TAS).Results: Normal peak thrombin was 47-144 nM for PPP and 88-231 nM for PRP. rFVIIa and FEIBA had an additive effect on TG in combination with SIA ACE910 in both plasma types. Combined with rFVIIa (0.88 µg/mL) or FEIBA (0.5 U/mL), SIA ACE910 (600 nM) induced a ~2- and ~16-fold increase over SIA ACE910 alone. SIA ACE910+rFVIIa did not reach the normal range, while SIA ACE910+FEIBA far exceeded it. Adding individual FEIBA components to PPP showed that FIX was, with a half-maximal effect, the main driver for enhanced TG, followed by FIXa. formation in FVIII-inhibited whole blood using ROTEM and T-TAS confirmed the excessive effect of SIA ACE910+FEIBA. In ROTEM, FEIBA and rFVIIa reduced clotting time to shorter than normal, whereas SIA ACE910 had only little effect. Moreover, adding SIA ACE910 to rFVIIa exerted no effect over rFVIIa alone.Conclusion: Combining SIA ACE910 at plasma concentrations observed in patients [Oldenburg et al. NEJM 2017] with FEIBA induced excessive thrombin generation and faster clot formation. In vitro, this effect is mainly mediated by FEIBA component FIX. ACE910 binds to FIX and FIXa to the same extent, and displays its pro-coagulant effect via an unregulated mechanism. Therefore, careful judgement is needed in treating breakthrough bleeds with FEIBA. DisclosuresHartmann:Shire: Employment. Feenstra:Shire: Employment. Knappe:Shire: Employment. Dockal:Baxalta: Patents & Royalties; Shire: Employment, Equity Ownership; Baxter: Equity Ownership, Patents & Royalties. Scheiflinger:Baxter: Equity Ownership; Shire: Employment, Equity Ownership.

Direct Oral Anticoagulants Form Thrombus Different From Warfarin in a Microchip Flow Chamber System

Direct oral anticoagulants (DOACs) have low risk of intracranial hemorrhage compared to warfarin. We sought to clarify the different mechanisms responsible for suppression of bleeding events using the Total Thrombus-formation Analysis System (T-TAS), a flow-microchip chamber with thrombogenic surfaces. Blood samples were obtained at Off- and On-anticoagulant (trough) from 120 consecutive patients with atrial fibrillation (warfarin; n = 29, dabigatran; n = 19, rivaroxaban; n = 47, apixaban; n = 25), which were used for T-TAS to compute the area under the curve (AUC) (AR10-AUC30) in the AR chip, and to measure plasma concentrations of DOACs at On-anticoagulant. In addition, the two-dimensional area covered by thrombi (%) in the capillary was analyzed every 3 minutes after sample applications. The AR10-AUC30 correlated weakly and negatively with plasma concentrations of DOACs, and the levels at On-anticoagulant were lower in all groups than at Off-anticoagulant. AR10-AUC30 levels at Off- and On-anticoagulant were identical among the groups. The thrombi areas in early phase were significantly larger in rivaroxaban and apixaban than warfarin and dabigatran groups. The findings suggested that visual analysis of the AR-chip can identify the differential inhibitory patterns of warfarin and DOACs on thrombus formation under flow condition.

Usefulness of the Total Thrombus-Formation Analysis System (T-TAS) in the diagnosis and characterization of von Willebrand disease.

The heterogeneity of von Willebrand disease (VWD) makes its diagnosis a difficult task. We report here on the usefulness of a microchip-based flow-chamber system, the total thrombus-formation analysis system (T-TAS), in the identification and characterization of VWD. Thirty VWD patients and 20 healthy subjects were studied with the T-TAS platelet (PL) and atherome (AR) microchips developed for the in vitro assessment of platelet thrombus formation and fibrin-rich platelet thrombus formation respectively. Samples from severe type 1 VWD, characterized by von Willebrand factor (VWF) levels below 10 U dL-1 , failed to occlude either the PL or the AR chip capillaries, while the occlusion times were normal in patients with mild type 1 VWD (VWF above 25 U dL-1 ). PL and/or AR chip occlusion occurred, but took longer than normal, for samples from type Vicenza and type 1 VWD patients, whose VWF levels ranged between 10 and 25 U dL-1 . No PL or AR chip capillary occlusion was seen for samples from patients with type 2A or 2B VWD featuring the absence of large VWF multimers, whereas no abnormalities emerged for type 2B patients with normal multimer patterns. The T-TAS appears to be sensitive mainly to plasma VWF concentration and the presence of large multimers. Failure of the PL and AR chips to become occluded points to a lack of large VWF multimers, or type 1 VWD with VWF levels below 10 U dL-1 . Although the T-TAS does not assure a precise VWD diagnosis, it does point us in the right direction, and thus seems a useful global preliminary test.

Efficacy Study of the COmbination of Edoxaban and Physiotherapy on the PRevention of Venous-Thromboembolism in patients after Total Knee Arthroplasty (ESCORT-TKA Trial): Study protocol for a randomized controlled trial

BackgroundDeep vein thrombosis (DVT) after total knee arthroplasty (TKA) often results in a fatal pulmonary thromboembolism (PTE). Edoxaban is an activated factor X inhibitor, which has been shown to prevent thromboembolic events in venous thromboembolism (VTE). Recently, the Total-Thrombus-formation Analysis System (T-TAS™), a microchip-based flow chamber system capable of evaluating thrombogenicity, was developed. In this study, utilizing the T-TAS™, we will examine the incidence of VTE after TKA and evaluate how thromboses form. Methods/designThis study will be a prospective, single-center, open-label, randomized, controlled clinical trial aimed at exploring the efficacy of edoxaban in reducing the incidence of VTE after TKA.A total of 80 patients who will undergo TKA will be randomly and evenly divided into groups receiving edoxaban plus physiotherapy or physiotherapy alone. The primary outcome measures will include the incidence rate of VTE as detected by ultrasonography 7days after TKA and the changes in T-TAS™ parameters. The secondary outcome measures will include the changes in prothrombin time and activated partial thromboplastin time, incidence of major/minor bleeding events and adverse effects of edoxaban. DiscussionThis study will provide clinical evidence on the combined efficacy and safety of edoxaban and physiotherapy compared with that of physiotherapy alone. This is will be the first prospective trial designed to explore how thrombus formation after TKA can be predicted by the T-TAS™.

Assessment of platelet‐derived thrombogenicity with the total thrombus‐formation analysis system in coronary artery disease patients receiving antiplatelet therapy

Accurate evaluation of thrombogenicity helps to prevent thrombosis and excessive bleeding. The total thrombus-formation analysis system (T-TAS) was developed for quantitative analysis of platelet thrombus formation by the use of microchips with thrombogenic surfaces (collagen, platelet chip [PL-chip]; collagen plus tissue factor, atherome chip [AR-chip]). We examined the utility of the T-TAS in the assessment of the efficacy of antiplatelet therapy in patients with coronary artery disease (CAD). In this cross-sectional study, 372 consecutive patients admitted to the cardiovascular department were divided into three groups: patients not receiving any antiplatelet therapy (control, n = 56), patients receiving aspirin only (n = 69), and patients receiving aspirin and clopidogrel (n = 149). Blood samples were used for the T-TAS to measure the platelet thrombus-formation area under the curve (AUC) at various shear rates (1500 s(-1) [PL18 -AUC10 ] and 2000 s(-1) [PL24 -AUC10 ] for the PL-chip; 300 s(-1) [AR10 -AUC30 ] for the AR-chip). The on-clopidogrel platelet aggregation was measured by the use of P2Y12 reaction units (PRUs) with the VerifyNow system. The mean PL24 -AUC10 levels were 358 ± 111 (± standard deviation) (95% confidence interval [CI] 328.9-387.1) in the control group, 256 ± 108 (95% CI 230.5-281.5) in the aspirin group, and 113 ± 91 (95% CI 98.4-127.6) in the aspirin/clopidogrel group. In the aspirin/clopidogrel group, the PL24 -AUC10 was higher in poor metabolizers (PMs) with cytochrome P450 2C19(CYP2C19) polymorphisms (152 ± 112, 95% CI 103.4-200.6) than in the non-PM group (87 ± 74, 95% CI 73.8-100.2). Our findings suggest that the PL24 -AUC10 level measured by the T-TAS is a potentially suitable index for the assessment of antiplatelet therapy in CAD patients.

Assessing the clinical severity of type 1 von Willebrand disease patients with a microchip flow‐chamber system

The clinical phenotype of von Willebrand disease (VWD) is heterogeneous, and von Willebrand factor ristocetin cofactor activity (VWF:RCo) does not always reflect clinical severity, especially in VWD type 1. We have reported the potential of a microchip flow-chamber system (Total-Thrombus Formation Analysis System [T-TAS®]) for assessing physiologic hemostasis in VWD. Aim To evaluate the relationship between T-TAS, bleeding score (BS) and laboratory test results in type 1 VWD patients. Microchips coated with collagen (platelet chip [PL-chip]) or collagen/thromboplastin (AR-chip) were used to assess platelet thrombus formation (PTF) at high shear rates or fibrin-rich PTF at low shear rates, respectively, in whole blood from 50 patients. The times needed for the flow pressure to increase by 10 kPa and 30 kPa (T10 and T30 ) from baseline were calculated from flow pressure curves. BS was determined by the use of a standardized questionnaire. PL-T10 values correlated with BS (R(2) ~ 0.45) better than VWF:RCo (R(2) ~ 0.36), irrespective of the flow rate, whereas AR-T10 showed only a weak correlation with BS (R(2) ~ 0.18). Patients with PL-T10 > 10 min or AR-T10 > 30 min had lower VWF levels and higher BS than those with PL-T10 ≤ 10 min or AR-T10 ≤ 30 min, and the greatest differences were observed with PL-T10. Clinical severity appeared to correlate best with PL-T10 > 8 min. BS was significantly higher in patients with VWF:RCo of < 10 IU dL(-1) than in those with VWF:RCo of 10 IU dL(-1) to < 25 IU dL(-1) and 25-40 IU dL(-1). In patients with VWF:RCo of < 10 IU dL(-1) , BS was significantly higher in those with PL-T10 > 8 min than in those with PL-T10 ≤ 8 min. T-TAS could be a useful technique for discriminating and predicting BS in VWD type 1 patients.

Evaluation of the Antithrombotic Effects of Rivaroxaban and Apixaban Using the Total Thrombus-Formation Analysis System®: In Vitro and Ex Vivo Studies.

BackgroundThe usefulness of the Total Thrombus-Formation Analysis System® (T-TAS®) for monitoring the anticoagulant effects of non-vitamin K oral anticoagulants (NOACs) in clinical practice has been poorly addressed.MethodsNOACs (rivaroxaban and apixaban) were added to whole blood from healthy subjects in an in vitro study, and their effects on thrombus formation were evaluated by the T-TAS®. We also evaluated antithrombotic effects using ex vivo samples of whole blood from patients given rivaroxaban or apixaban at the respective trough and peak drug concentrations.ResultsT-TAS® could determine anticoagulant effects in whole blood treated with rivaroxaban or apixaban in vitro. The increases in the anticoagulant effects of rivaroxaban and apixaban from the trough to peak concentrations in whole blood were successfully monitored by the T-TAS® using ex vivo samples. The antithrombotic effects of rivaroxaban and apixaban (in terms of factor Xa inhibition) at the peak were strongly linked to those at the trough.ConclusionT-TAS® could be a clinically useful tool for monitoring the anticoagulant effects of factor Xa inhibitors, and may represent an accurate quantitative analysis.

Abstract 13988: Novel Quantitative Assessment of Whole Blood Thrombogenicity in Patients Treated With Anticoagulants Undergoing Radiofrequency Catheter Ablation for Atrial Fibrillation

Introduction: Non-vitamin K antagonist oral anticoagulants (NOACs) are equivalent to warfarin in preventing thromboembolism in patients with atrial fibrillation (AF). Recently, Total Thrombus-formation Analysis System (T-TAS) was developed as an easy-to-use system for quantitative analysis of thrombus formation using microchips with thrombogenic surfaces (collagen, PL chip; collagen plus tissue factor, AR chip). Hypothesis: We evaluated the efficacy and safety of NOACs using the T-TAS in AF patients undergoing radiofrequency catheter ablation (RFCA). Methods: After exclusion of 19 from 121 consecutive patients undergoing RFCA for AF, the remaining 102 patients were divided into two groups; AF patients treated with warfarin (n=28), and NOACs (n=74). Blood samples obtained on the day (anticoagulant-free point), 3 days and 1 month after RFCA were used in T-TAS to compute the thrombus formation area under the curve (AUC) (PL24-AUC10, AUC for PL chip; AR10-AUC30, for AR chip). Results and Conclusion: The AR10-AUC30 and PL24-AUC10 levels were identical between the warfarin and NOACs groups on the day of RFCA (1708.5±162.1 vs 1757.5±138.0 in AR10-AUC30, p=0.13 and 389.1±95.5 vs 391.7±76.0 in PL24-AUC10, p=0.94, respectively). AR10-AUC30, but not PL24-AUC10 levels, was significantly lower in warfarin and NOACs groups at 3 days and 1 month after RFCA. Multiple logistic regression analysis identified AR10-AUC30 levels on the day of RFCA as a significant predictor of bleeding events in the period of three months after RFCA [odds ratio (OR) 20.6; 95% confidence interval (95%CI): 2.46 to 172.6; p=0.005]. Receiver-operating characteristic analysis showed that AR10-AUC30 levels on the day of RFCA significantly predicted bleeding events [AUC 0.849 (95%CI: 0.719-0.979; p&lt;0.001)]. The cut-off levels of AR10-AUC30 were 1647.6 for identification of bleeding events within three months after RFCA. These results suggested that AR10-AUC30 level determined by T-TAS is a potentially useful marker for assessment of the efficacy and safety of anti-coagulant therapy including warfarin and NOACs, in AF patients undergoing RFCA.

A novel quantitative assessment of whole blood thrombogenicity in patients treated with a non-vitamin K oral anticoagulant

Non-vitamin K oral anticoagulants(NOAC) negate complex patient management issues, such as frequent blood sampling, diet restriction, and drug interactions, that had to be dealt with during the warfarinonly era. However, there is no definite tool tomonitor the anticoagulant effects of NOACs, although some patients suffer from bleeding complications related to exceedingly high blood concentrations of NOACs. [1, 2] Routine tests, such as prothrombin time-international normalized ratio (PT-INR) and activated partial thromboplastin time (APTT), can be problematic formonitoring the anticoagulant effects of all NOACs because each individual NOAC has a different characteristic chemical structure and different pharmacokinetic profile (e.g., plasma half-life and tissue penetration rate) [3]. Recently, the Total Thrombus-formation Analysis System (T-TASTM) [4,5], a microchip-based flow chamber system capable of evaluating whole blood thrombogenicity, was developed as an easy-to-use system for quantitative analysis of thrombus formation (Fig. 1A,B). In the present study, we sought to examine whether the T-TASTM was useful for the quantitative analysis of thrombogenicity in patients treated with the NOAC edoxaban (Fig. 1C, D). We recruited 20 consecutive patients (male: n = 8 [40%] and female: n=12 [60%], average age: 75.2±8.7 years)whowere scheduled

Subcutaneous Preadipocytes Are Ideal Candidate To Produce A Large Number Of Functional Platelets In a Culture System

Platelet transfusions are widely used for patients with severe thrombocytopenia. There are, however, practical problems in the current donor-dependent platelet transfusions, such as the limited supply and risk of serious immune reactions. Thus, the development of new strategies for generating platelets for transfusion is crucial. Platelets have been differentiated from hematopoietic stem cells, fetal liver cells, embryonic stem cells, induced pluripotent stem cells, NF-E2-transduced fibroblasts, and preadipocytes. Here, among these cells preadipocytes, especially in the subcutaneous adipose tissue, could be ideal candidate cells for manufacturing megakaryocytes (MKs) and platelets, because (1) they are relatively easy to obtain large quantities and have ability to proliferate in vitro, (2) their differentiation does not require gene transfer, as they possess genes in relation to megakaryopoiesis and thrombopoiesis, such as p45NF-E2 and c-mpl, and (3) they differentiate into MKs and platelets using an endogenous thrombopoietin. Thus, to clarify the usefulness of preadipocytes as a donor-independent source for platelet transfusion, we compared both number and function between platelets derived from mouse subcutaneous preadipocytes and those from bone marrow mononuclear cells (BMMNCs), the established cell source for manufacturing platelets. First, BMMNCs were not feasible for their expansion in vitro and therefore the cells were directly seeded in MK lineage induction media. In contrast, preadipocytes were to be passaged 6 times without any morphological changes, and then cultured in MK lineage induction media for their differentiation into platelets. Thus, as assessed by CD41-positive platelet-sized cells, 106.2±5.0 ×105 or 3.9±1.0 ×105 platelets were obtained from 106 preadipocytes or 106 BMMNCs, respectively (p<0.01). To next analyze platelet function, the binding of Alexa Fluor 488-labaled fibrinogen to CD41-positive platelet-sized cells, as assessed by mean fluorescence, was analyzed in the presence of ADP/PAR4-activating peptide, and the binding was higher in preadipocyte-derived platelets (14.0±1.5) than in BMMNC-derived platelets (7.0±1.3, p=0.017). Furthermore, to examine the incorporation of these platelets into a thrombus under flow conditions, we performed the quantitative evaluation of the cells in the thrombus formation process using the Total Thrombus-formation Analysis System (T-TAS®) (Blood 2012; 120: 3812). We used platelets released from each preadipocyte- and BMMNC-derived MKs in vivo. Briefly, MK-sized cells were obtained by a 2-step BSA gradient, labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and then infused into irradiated thrombocytopenic mice (7 days after exposure to 2.0 Gy). After 90 min of infusion, whole blood samples were obtained from these mice and were shown to contain similar number of CFSE+/CD41+ platelet-sized cells; 1.8±0.4 ×104 for preadipocyte-derived cells and 1.9±0.2 ×104 for BMMNC-derived cells (p=0.9135). Subsequently, the samples were perfused on a collagen-coated chip, followed by assessment using T-TAS®, and the result showed that the frequency of incorporation of the cells into the thrombus formation was higher for preadipocyte-derived cells (77.8±5.1%) than for BMMNC-derived cells (52.5±5.3%, p=0.0257). And we also found that the frequencies were similar when 3-fold amounts of BMMNC-derived cells were used in this perfusion study. Here our microarray analyses showed that plasminogen activator inhibitor-1 (PAI-1), a well-known enhancer for platelet function, was secreted into the supernatant during MK differentiation process. Therefore, to elucidate the possibility that PAI-1 is associated with the findings as described above, the levels of PAI-1 in the supernatant were measured on Day 4 during MK differentiation, and were shown to be 3.3±0.1 ng/mL or 0.09±0.01 ng/mL for preadipocyte- or BMMNC-derived cells, respectively. Together, our findings provided the first evidence showing that preadipocytes produce a greater number of platelets than BMMNCs and their derived platelets also have more useful physiological function than BMMNC-derived platelets, allowing for the usage of fewer platelets in platelet transfusion. Disclosures:No relevant conflicts of interest to declare.

Studies of a microchip flow-chamber system to characterize whole blood thrombogenicity in healthy individuals

A whole blood flow-chamber system, the Total Thrombus-formation Analysis System (T-TAS), was developed for quantitative analysis of platelet thrombus formation (PTF) using microchips with thrombogenic surfaces (collagen, PL chip; collagen plus tissue thromboplastin, AR chip) under shear stress conditions. We evaluated the usefulness of the T-TAS for assessing individual thrombogenicity compared with other platelet function tests. Blood samples from 31 healthy volunteers were applied to the T-TAS to measure PTF starting time (T10: time to reach 10 kPa), occlusion time (T60 for PL chip; T80 for AR chip), and area under the curve (AUC10, area under curve until 10 min for PL chip; AUC30, 30 min for AR chip) under various shear rates (1000, 1500, 2000s(-1) for PL chip; 300 s(-1) for AR chip). Platelet functions were also tested using platelet aggregometry, the PFA-100 (collagen and epinephrine [C/EPI], collagen and adenosine diphosphate [C/ADP]), and the VerifyNow P2Y12 assay. Individual pressure waveforms, including PTF starting and ending points, varied among healthy subjects. In the PL chip, T10 and AUC10 showed a shear-dependent correlation with C/EPI or C/ADP. VerifyNow P2Y12 values were not significantly associated with the parameters of the T-TAS. Platelet counts were correlated with all AR measurements, and mostly with PL measurements. The results of the T-TAS were associated with those of the PFA-100 in many respects, indicating that its characteristics are related to shear-induced PTF. The T-TAS showed few correlations with platelet aggregometry and the VerifyNow P2Y12 assay. The T-TAS may allow for the measurement of comprehensive parameters of individual thrombogenicity under whole blood flow conditions.

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

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. [Display omitted] 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.