Total Thrombus-formation Analysis System Research Articles

Antiplatelet effects of hydroxychloroquine in patients with systemic lupus erythematosus evaluated by the total thrombus-formation analysis system (T-TAS)

ObjectivesHydroxychloroquine (HCQ) has been shown to reduce thrombotic events in patients with SLE. However, the antiplatelet effects of HCQ are only supported by the platelet aggregation assay, which is a...

New Findings Regarding the Effects of Selected Blue Food Colorants (Genipin, Patent Blue V, and Brilliant Blue FCF) on the Hemostatic Properties of Blood Components In Vitro.

Natural and synthetic colorants present in food can modulate hemostasis, which includes the coagulation process and blood platelet activation. Some colorants have cardioprotective activity as well. However, the effect of genipin (a natural blue colorant) and synthetic blue colorants (including patent blue V and brilliant blue FCF) on hemostasis is not clear. In this study, we aimed to investigate the effects of three blue colorants-genipin, patent blue V, and brilliant blue FCF-on selected parameters of hemostasis in vitro. The anti- or pro-coagulant potential was assessed in human plasma by measuring the following coagulation times: thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (APTT). Moreover, we used the Total Thrombus formation Analysis System (T-TAS, PL-chip) to evaluate the anti-platelet potential of the colorants in whole blood. We also measured their effect on the adhesion of washed blood platelets to fibrinogen and collagen. Lastly, the cytotoxicity of the colorants against blood platelets was assessed based on the activity of extracellular lactate dehydrogenase (LDH). We observed that genipin (at all concentrations (1-200 µM)) did not have a significant effect on the coagulation times (PT, APTT, and TT). However, genipin at the highest concentration (200 µM) and patent blue V at the concentrations of 1 and 10 µM significantly prolonged the time of occlusion measured using the T-TAS, which demonstrated their anti-platelet activity. We also observed that genipin decreased the adhesion of platelets to fibrinogen and collagen. Only patent blue V and brilliant blue FCF significantly shortened the APTT (at the concentration of 10 µM) and TT (at concentrations of 1 and 10 µM), demonstrating pro-coagulant activity. These synthetic blue colorants also modulated the process of human blood platelet adhesion, stimulating the adhesion to fibrinogen and inhibiting the adhesion to collagen. The results demonstrate that genipin is not toxic. In addition, because of its ability to reduce blood platelet activation, genipin holds promise as a novel and valuable agent that improves the health of the cardiovascular system and reduces the risk of cardiovascular diseases. However, the mechanism of its anti-platelet activity remains unclear and requires further studies. Its in vivo activity and interaction with various anti-coagulant and anti-thrombotic drugs, including aspirin and its derivatives, should be examined as well.

A novel haemodilution chip mounted on the total thrombus-formation analysis system, a flow chamber system, enables stable analysis of platelet products under low platelet concentration/high shear conditions.

The total thrombus-formation analysis system (T-TAS) can quantitatively analyse the contribution of platelets to haemostasis using reconstituted blood samples. However, it is unsuitable in cases with low platelet counts. We introduced a haemodilution (HD) chip with a shallow chamber depth, adapted to low platelet counts and high shear conditions (1500 s-1). Blood samples were prepared by mixing red blood cell products, standard human plasma and platelet products; the final platelet count was 50 × 103/μL. Aggregation tests were performed by using the aggregation inducers collagen, adenosine diphosphate (ADP) and ristocetin. Samples with 2-, 4- and 9-day-old platelet products (N = 10) were evaluated. The HD chip enabled the stable analysis of the haemostatic function of all samples at a platelet count of 50 × 103/μL. Haemostatic function was correlated with ADP aggregation (time to 10 kPa [T10]: r = -0.53; area under the curve for 30 min: r = 0.40) and storage period (T10: r = 0.44). The HD chip-mounted T-TAS can stably analyse haemostatic function under low platelet counts and high shear conditions; this approach is expected to serve as a bridge to in vivo haemostatic tests with experimental animals.

Factors that modulate platelet reactivity as measured by 5 assay platforms in 3429 individuals

BackgroundAssessment of platelet function is key in diagnosing bleeding disorders and evaluating antiplatelet drug efficacy. However, there is a prevailing “one-size-fits-all” approach in the interpretation of measures of platelet reactivity, with arbitrary cutoffs often derived from healthy volunteer responses. ObjectivesOur aim was to compare well-used platelet reactivity assays. MethodsBlood and platelet-rich plasma obtained from the Framingham Heart Study (N = 3429) were assayed using a range of agonists in 5 platelet assays: light transmission aggregometry, Optimul aggregometry, Multiplate impedance aggregometry (Roche Diagnostics), Total Thrombus-Formation Analysis System, and flow cytometry. Using linear mixed-effect models, we determined the contribution of preanalytical and technical factors that modulated platelet reactivity traits. ResultsA strong intra-assay correlation of platelet traits was seen in all assays, particularly Multiplate velocity (r = 0.740; ristocetin vs arachidonic acid). In contrast, only moderate interassay correlations were observed (r = 0.375; adenosine diphosphate Optimul Emax vs light transmission aggregometry large area under the curve). As expected, antiplatelet drugs strongly reduced platelet responses, with aspirin use primarily targeting arachidonic acid–induced aggregation, and explained substantial variance (β = −1.735; P = 4.59 × 10−780; variance proportion = 46.2%) and P2Y12 antagonists blocking adenosine diphosphate responses (β = −1.612; P = 6.75 × 10−27; variance proportion = 2.1%). Notably, female sex and older age were associated with enhanced platelet reactivity. Fasting status and deviations from standard venipuncture practices did not alter platelet reactivity significantly. Finally, the agonist batch, phlebotomist, and assay technician (more so for assays that require additional sample manipulation) had a moderate to large effect on measured platelet reactivity. ConclusionCaution must be exercised when extrapolating findings between assays, and the use of standard ranges must be medication-specific and sex-specific at a minimum. Researchers should also consider preanalytical and technical variables when designing experiments and interpreting platelet reactivity measures.

T-TAS® 01 as a new tool for the evaluation of hemostasis in thrombocytopenic patients after platelet transfusion.

Current laboratory tests fail to evaluate the hemostatic function of platelets in patients with thrombocytopenia. We investigated the use of the Total Thrombus-Formation Analysis System (T-TAS® 01 [Fujimori Kogyo Co, Tokyo, Japan]) to evaluate hemostasis under conditions of experimental thrombocytopenia, and in patients before and after platelet transfusion. Specific T-TAS 01 chips, for thrombocytopenic conditions, were used. The area under the curve (AUC) and occlusion time (OT, min) were measured in: (i) experimentally induced thrombocytopenia (183±15 to 6.3±1.2×103 platelets/μL) in blood samples from healthy donors (No.=13), and (ii) blood from oncohematological thrombocytopenic patients (No.=48), before and after platelet transfusion. The influences of hematocrit and number of transfusions were analyzed in these patients. Progressive reductions of AUC and prolongations of OT related significantly to decreasing platelet counts (p<0.05 for all) in experimental thrombocytopenia. In samples from thrombocytopenic patients, platelet counts, AUC and OT were, respectively, 10.8±0.6×103/μL, 175.2±59, and 27.2±1 min before transfusion; and 22±1.5×103/μL, 400.8±83 and 22.9±1.5 min after platelet transfusion (p<0.01 for all). A hematocrit below 25% or exposure to ten or more previous platelet transfusions had a negative impact on the T-TAS 01 performance in patients. In vitro correction of the hematocrit improved the hemostatic response in thrombocytopenic patients. T-TAS 01 measurements were sensitive to low platelet counts in the experimental setting. The technology was sensitive to evaluate the hemostatic capacity of platelet transfusions. Exposure to multiple medications, repeated platelet transfusions and lower hematocrits may interfere with the hemostatic performance in oncohematological patients with thrombocytopenia.

Elevated Leukocyte Count and Platelet-Derived Thrombogenicity Measured Using the Total Thrombus-Formation Analysis System in Patients with ST-Segment Elevation Myocardial Infarction.

High platelet-derived thrombogenicity during the acute phase of ST-segment elevation myocardial infarction (STEMI) is associated with poor outcomes; however, the associated factors remain unclear. This study aimed to examine whether acute inflammatory response after STEMI affects platelet-derived thrombogenicity. This retrospective observational single-center study included 150 patients with STEMI who were assessed for platelet-derived thrombogenicity during the acute phase. Platelet-derived thrombogenicity was assessed using the area under the flow-pressure curve for platelet chip (PL-AUC), which was measured using the total thrombus-formation analysis system (T-TAS). The peak leukocyte count was evaluated as an acute inflammatory response after STEMI. The patients were divided into two groups: the highest quartile of the peak leukocyte count and the other three quartiles combined. Patients with a high peak leukocyte count (＞15,222/mm3; n=37) had a higher PL-AUC upon admission (420 [386-457] vs. 385 [292-428], p=0.0018), higher PL-AUC during primary percutaneous coronary intervention (PPCI) (155 [76-229] vs. 96 [29-170], p=0.0065), a higher peak creatine kinase level (4200±2486 vs. 2373±1997, p＜0.0001), and higher PL-AUC 2 weeks after STEMI (119 [61-197] vs. 88 [46-122], p=0.048) than those with a low peak leukocyte count (≤ 15,222/mm3; n=113). The peak leukocyte count after STEMI positively correlated with PL-AUC during primary PPCI (r=0.37, p＜0.0001). A multivariable regression analysis showed the peak leukocyte count to be an independent factor for PL-AUC during PPCI (β=0.26, p=0.0065). An elevated leukocyte count is associated with high T-TAS-based platelet-derived thrombogenicity during the acute phase of STEMI.

BNP level predicts bleeding event in patients with heart failure after percutaneous coronary intervention

ObjectiveThis study aimed to investigate the association between heart failure (HF) severity measured based on brain natriuretic peptide (BNP) levels and future bleeding events after percutaneous coronary intervention (PCI).BackgroundThe Academic...

Pharmacodynamic and pharmacokinetic profiles associated with switching from cangrelor to prasugrel in patients undergoing percutaneous coronary intervention: the SWAP-6 study

Abstract Background Cangrelor is an intravenous P2Y12 inhibitor used in patients undergoing coronary intervention (PCI). Preliminary studies have shown the potential for a drug-drug interaction (DDI) when transitioning from cangrelor to oral P2Y12 inhibition with prasugrel. However, to date this has not been prospectively tested in patients undergoing PCI. Purpose To rule out a DDI when cangrelor and prasugrel are concomitantly administered in patients undergoing PCI. Methods SWAP-6 is a prospective, randomized, 3-arm, open-label, pharmacokinetic (PK) and pharmacodynamic (PD) study conducted in patients undergoing PCI (n=77). Patients were randomized to either a) prasugrel only administered at the start of PCI; b) cangrelor plus prasugrel concomitantly administered at the start of PCI; c) cangrelor administered at the start of PCI plus prasugrel administered at the end of the cangrelor infusion. Cangrelor (30 μg/kg bolus followed by 4 μg/kg/min infusion) was maintained for 2 hours and all patients were on a background of aspirin therapy. Prasugrel was administered as a 60 mg loading dose. PK analysis included plasma levels of prasugrel active metabolite, and PD assessments included VerifyNow PRU, total thrombus-formation analysis system (T-TAS), and vasodilator-stimulated phosphoprotein (VASP), at baseline and 6 time points after prasugrel/cangrelor administration. The primary end point was non-inferiority in PRU measured at 4 hours after prasugrel/cangrelor loading (2 hours after stopping cangrelor infusion) of cangrelor plus prasugrel concomitantly administered versus prasugrel only. The noninferiority margin was defined as 45 PRU. Results Cangrelor achieved lower PRU levels than prasugrel only as early as 30 minutes and up to 2 hours after bolus administration (Figure). At 3 hours after prasugrel/cangrelor loading, PRU levels were higher with cangrelor compared to prasugrel only. At 4 hours after loading (2 hours after stopping cangrelor infusion), PRU levels were significantly lower with prasugrel only compared to cangrelor and prasugrel concomitantly administered (LSM difference 130; 95% CI: 85-176), failing to meet the prespecified noninferiority primary end point (Figure). At this time point, cangrelor followed by prasugrel at the end of the infusion had higher PRU than prasugrel only, but lower than cangrelor and prasugrel concomitantly. Platelet reactivity by T-TAS and VASP showed consistent findings. PK analysis tracked PD results (data not shown). Conclusions In patients undergoing PCI, cangrelor is associated with more potent platelet inhibition than prasugrel alone. However, concomitant administration of prasugrel with cangrelor leads to a marked increase in platelet reactivity after stopping cangrelor infusion, supporting the presence of a DDI. This underscores the importance of administering prasugrel not until the end of cangrelor infusion, a strategy which mitigates the increase in platelet reactivity.

Abstract 12293: Subclinical Leaflet Thrombus on Transcatheter Heart Valve in Patients With Severe Aortic Stenosis and Atrial Fibrillation

Background: Subclinical leaflet thrombosis is one of transcatheter heart valve (THV) failure that should be avoided after transcatheter aortic valve implantation (TAVI). Although the effectiveness of direct oral anticoagulants (DOACs) on subclinical leaflet thrombosis has been demonstrated, there is insufficiently established evidence for post-TAVI antithrombotic therapy. The aim of this study was to clarify the formation process of subclinical leaflet thrombosis on THV and thrombogenicity during the perioperative period of TAVI. Methods: This multicenter, prospective, single-arm interventional study enrolled 26 patients with atrial fibrillation treated with edoxaban and severe aortic valve stenosis who underwent TAVI between September 2018 and September 2022. After exclusion, 24 patients were finally analyzed. We investigated changes in maximal leaflet thickness detected by contrast-enhanced computed tomography between 1 week and 3 months after TAVI and measured the thrombogenicity by Total Thrombus-formation Analysis System (T-TAS) and flow stagnation volume by computational fluid dynamics (CFD). Results: Of all, subclinical leaflet thrombosis was observed in 16.7% at 1 week, but decreased to 5.9% at 3 months after TAVI. There was no significant difference in maximum leaflet thickness between 1 week and 3 months, however, patients with subclinical leaflet thrombosis at 1 week had a significantly greater difference in that thickness compared to those without thrombosis. Thrombogenicity assessed by T-TAS and platelet count decreased markedly at 1 week and tended to improve at 3 months. Simple liner regression analysis showed that stagnation volume was positively associated with an increase in maximum leaflet thickness, and low-dose edoxaban was associated with a high risk of increased leaflet thickness at chronic phase. Conclusions: This study showed that course of leaflet thrombus formation in neo-sinus of THV and dynamic fluctuation of thrombogenicity in the acute phase after TAVI. These findings were merely hypotheses generation and thus necessitate future hypothesis testing with large sample sizes.

PB0132 Hemostatic Evaluation of Patients with Thrombocytopenia Receiving Platelet Transfusion using the Total Thrombus Formation Analysis System (T-TAS)

PB0132 Hemostatic Evaluation of Patients with Thrombocytopenia Receiving Platelet Transfusion using the Total Thrombus Formation Analysis System (T-TAS)

The oxylipin analog CS585 prevents platelet activation and thrombosis through activation of the prostacyclin receptor

AbstractCardiovascular disease remains the primary cause of morbidity and mortality globally. Platelet activation is critical for maintaining hemostasis and preventing the leakage of blood cells from the vessel. There has been a paucity in the development of new drugs to target platelet reactivity. Recently, the oxylipin 12(S)-hydroxy-eicosatrienoic acid (12-HETrE), which is produced in platelets, was shown to limit platelet reactivity by activating the prostacyclin receptor. Here, we demonstrated the synthesis of a novel analog of 12-HETrE, known as CS585. Human blood and mouse models of hemostasis and thrombosis were assessed for the ability of CS585 to attenuate platelet activation and thrombosis without increasing the risk of bleeding. Human platelet activation was assessed using aggregometry, flow cytometry, western blot analysis, total thrombus formation analysis system, microfluidic perfusion chamber, and thromboelastography. Hemostasis, thrombosis, and bleeding assays were performed in mice. CS585 was shown to potently target the prostacyclin receptor on the human platelet, resulting in a highly selective and effective mechanism for the prevention of platelet activation. Furthermore, CS585 was shown to inhibit platelet function in human whole blood ex vivo, prevent thrombosis in both small and large vessels in mouse models, and exhibit long-lasting prevention of clot formation. Finally, CS585 was not observed to perturb coagulation or increase the risk of bleeding in the mouse model. Hence, CS585 represents a new validated target for the treatment of thrombotic diseases without the risk of bleeding or off-target activation observed with other prostaglandin receptor agonists.

Hemostatic disorders in patients with infective endocarditis undergoing urgent surgical valve replacement – Rethinking current beliefs

BackgroundAlthough infective endocarditis (IE) represents a unique model of thrombo-inflammatory disease, the most frequent early complications of surgical valve replacement (SVR) in IE population are coagulopathy and bleeding. The hemostatic capacity and procedure-related coagulation disorders of IE patients undergoing SVR are unknown. The aims of this study were to test periprocedural hemostasis in IE patients undergoing urgent SVR, and to assess the association between disorders of hemostasis and early bleeding as well as with thromboembolic events. MethodsA prospective, two-center, hypothesis generating, observational study was performed between Dec 2017 and Jan 2020. Periprocedural hemostasis of IE patients was assessed using Total Thrombus-formation Analysis System (T-TAS Plus) within 24 h before and 72 h post SVR. ResultsOverall, 25 patients with active IE undergoing urgent SVR were tested. Hemostatic capacity of IE patients was significantly impaired pre-SVR as well as post-SVR compared to normal values, in most aspects of T-TAS assays under high and low shear forces, including prolonged activation of coagulation (T10), final clot formation (OT) and clot strength (AUC30). Post-SVR T-TAS results were significantly associated with early bleeding and with red blood cell, platelet, and fresh frozen plasma administration. No association with thrombo-embolic events was found. ConclusionsPatients with active IE undergoing urgent SVR have significantly reduced hemostatic capacity before and after SVR. Hemostatic insufficiency post-SVR is related to bleeding and blood products transfusion. T-TAS may be helpful in assessment of periprocedural hemostasis in patients with IE undergoing SVR.

#4203 PREDICTION OF BLEEDING AND THROMBOSIS IN PATIENTS ON CHRONIC HEMODIALYSIS WITH THE TOTAL THROMBUS-FORMATION ANALYSIS SYSTEM

Abstract Background End-stage renal disease patients receiving hemodialysis (HD) are at increased risk of both bleeding and thrombotic complications. There are, however, few tools to help clinicians with risk stratification of the individual patient. Aims and Method To study if the Total Thrombus-formation Analysis System (T-TAS01), which measures thrombus formation in flowing whole blood inside a microchip [1], can be used to predict bleeding and thrombotic complications in patients on HD. Odds-ratios were calculated using the median split procedure. ROC curve analyses were performed to calculate the ability of T-TAS measurements performed at baseline to distinguish between patients with and without future bleeding or thrombotic complications. Results 176 patients on chronic HD were included and followed for one year; 11.4% were on antiplatelet drugs and 8.0% on continuous anticoagulation. Venous blood samples were taken at inclusion before dialysis procedure, and platelet-thrombus formation (PL-AUC; higher value reflects increased thrombogenicity) and platelet-fibrin thrombus formation (AR-OT; higher value reflects reduced thrombogenicity) were registered. Fifty-seven patients (32%) developed a bleeding complication, whereof 12 (6.8%) were ISTH major bleedings. The Odds-Ratio (OR; 95% CI) to develop any bleeding for patients with T-TAS01 measurements below (PL-AUC) or above (AR-OT) median were 7.67 (3.59-16.36) and 8.94 (4.10-19.51), respectively. ROC analyses to predict bleeding with T-TAS01 showed AUCs of 0.85 for all bleeding and 0.83 for ISTH major bleed (PL-AUC and AR-OT data combined). Furthermore, thirty-one patients (17.6%) developed a thrombotic complication, whereof 13 (7.4%) were acute coronary syndrome (ACS) or ischemic stroke (IS). The Odds-Ratio (OR; 95% CI) to develop any thrombosis for patients with T-TAS01 measurements below (PL-AUC) or above (AR-OT) median were 0.14 (0.05-0.40) and 0.28 (0.12-0.67), respectively. ROC analyses to predict thrombosis with T-TAS01 showed AUCs of 0.78 for all thrombosis and 0.62 for ACS/IS (PL-AUC and AR-OT data combined). Conclusion T-TAS01 is a promising tool to aid in the prediction of bleeding and thrombosis in HD patients.

The changes of blood platelet reactivity in the presence of Elaeagnus rhamnoides (L.) A. Nelson leaves and twig extract in whole blood

Uncontrolled blood platelet activation is an important risk factor of cardiovascular disease (CVDs). Various studies on phenolic compounds indicate that they have a protective effect on the cardiovascular system through different mechanisms, including the reduction of blood platelet activation. One of the plants that is particularly rich in phenolic compounds is sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson). The aim of the present study in vitro was to determine the anti-platelet properties of crude extracts isolated from leaves and twigs of E. rhamnoides (L.) A. Nelson in whole blood using flow cytometric and total thrombus-formation analysis system (T-TAS). In addition, the aim of our study was also analyze of blood platelet proteomes in the presence of different sea buckthorn extracts. A significant new finding is a decrease surface exposition of P-selectin on blood platelets stimulated by 10 µM ADP and 10 µg/mL collagen, and a decrease surface exposition of GPIIb/IIIa active complex on non-activated platelets and platelets stimulated by 10 µM ADP and 10 µg/mL collagen in the presence of sea buckthorn leaf extract (especially at the concentration 50 µg/mL). The twig extract also displayed antiplatelet potential. However, this activity was higher in the leaf extract than in the twig extract in whole blood. In addition, our present findings clearly demonstrate that investigated plant extracts have anticoagulant properties (measured by T-TAS). Therefore, the two tested extracts may be promising candidates for the natural anti-platelet and anticoagulant supplements.

Japanese high bleeding risk criteria status predicts low thrombogenicity and bleeding events in patients undergoing percutaneous coronary intervention.

Although the Japanese high bleeding risk criteria (J-HBR) were established to predict bleeding risk in patients undergoing percutaneous coronary intervention (PCI), the thrombogenicity in the J-HBR status remains unknown. Here, we examined the relationships among J-HBR status, thrombogenicity and bleeding events. This study was a retrospective analysis of 300 consecutive patients who underwent PCI. Blood samples obtained on the day of PCI were used in the total thrombus-formation analysis system (T-TAS) to investigate the thrombus-formation area under the curve (AUC; PL18-AUC10 for platelet chip; AR10-AUC30 for atheroma chip). The J-HBR score was calculated by adding 1 point for any major criterion and 0.5 point for any minor criterion. We assigned patients to three groups based on J-HBR status: a J-HBR-negative group (n = 80), a low score J-HBR-positive group (positive/low, n = 109), and a high score J-HBR-positive group (positive/high, n = 111). The primary end point was the 1-year incidence of bleeding events defined by the Bleeding Academic Research Consortium types 2, 3, or 5. Both PL18-AUC10 and AR10-AUC30 levels were lower in the J-HBR-positive/high group than the negative group. Kaplan-Meier analysis showed worse 1-year bleeding event-free survival in the J-HBR-positive/high group compared with the negative group. In addition, both T-TAS levels in J-HBR positivity were lower in those with bleeding events than in those without bleeding events. In multivariate Cox regression analyses, the J-HBR-positive/high status was significantly associated with 1-year bleeding events. In conclusion, the J-HBR-positive/high status could reflect low thrombogenicity as measured by T-TAS and high bleeding risk in patients undergoing PCI.

A novel quantitative method to evaluate the contribution of platelet products to white thrombus formation in reconstituted blood under flow conditions.

Currently, the quality of platelet (PLT) products is evaluated using a series of in vitro tests, which only analyse PLTs as an inspection material. However, it would be ideal to assess the physiological functions of PLTs under conditions similar to the sequential blood haemostatic process. In this study, we attempted to establish an in vitro system where the thrombogenicity of PLT products was evaluated in the presence of red blood cells (RBCs) and plasma using a microchamber under constant shear stress (600/s). Blood samples were reconstituted by mixing PLT products, standard human plasma (SHP) and standard RBCs. Each component was serially diluted keeping the other two components fixed. The samples were applied onto a flow chamber system (Total Thrombus-formation Analysis System [T-TAS]), and white thrombus formation (WTF) was assessed under large arterial shear conditions. We observed a good correlation between the PLT numbers in the test samples and WTF. The WTF of samples containing ≦10% SHP was significantly lower than those containing ≧40% SHP, and no difference was observed in WTF among samples containing 40%-100% SHP. WTF significantly declined in the absence of RBCs, whereas no change in WTF was observed in the presence of RBCs, over haematocrit range of 12.5%-50%. The WTF assessed on the T-TAS using reconstituted blood may serve as a new physiological blood thrombus test to quantitatively determine the quality of PLT products.

Utility of the Total Thrombus-Formation Analysis System as a Tool for Evaluating Thrombogenicity and Monitoring Antithrombotic Therapy in Pediatric Fontan Patients.

There is no consensus regarding thromboprophylaxis after Fontan procedure, and novel tools to assess thrombogenicity are needed to establish optimal thromboprophylaxis. The Total Thrombus-formation Analysis System (T-TAS) was developed for the quantitative analysis of thrombus formation using microchips with thrombogenic surfaces. This prospective study evaluated the utility of T-TAS in the assessment of thrombogenicity in pediatric Fontan patients. The participants included 20 consecutive Fontan patients who underwent cardiac catheterization and 30 healthy controls. Blood samples collected without and with antithrombotic therapy (aspirin or aspirin and warfarin) were used for T-TAS to compute the area under the curve (AUC) in the atheroma (AR10-AUC30) and platelet (PL18-AUC10) chips. A higher AUC indicates higher thrombogenicity. T-TAS values showed that patients in the Fontan group without antithrombotic therapy had lower thrombogenicity than those in the control group [PL18-AUC10, median (interquartile range) 356 (313-394) vs. 408 (392-424); AR10-AUC30, median (interquartile range) 1270 (1178-1351) vs. 1382 (1338-1421)]. Aspirin and warfarin therapies significantly decreased PL18-AUC10 and AR10-AUC30, respectively, compared with those of patients without antithrombotic therapy (P < 0.001 for each comparison). Subgroup analysis divided by low (< 9mmHg) or high (≥ 9mmHg) central venous pressure (CVP) showed that CVP affects the reduction in AR10-AUC30 with antithrombotic therapy. T-TAS may be a useful tool for monitoring thrombogenicity and antithrombotic therapy in Fontan patients.

1156 CANGRELOR IN PATIENTS WITH CORONARY ARTERY DISEASE PRE-TREATED WITH TICAGRELOR: THE SWITCHING ANTIPLATELET (SWAP)-5 STUDY

Abstract Objectives To rule out a drug-drug interaction (DDI) among cangrelor-treated patients who have been pre-treated with ticagrelor. Background There are no studies specifically designed to rule out a DDI when cangrelor is used among patients who have been pre-treated with ticagrelor. Methods In this prospective, randomized, double-blind, placebo-controlled, cross-over, pharmacokinetic (PK) and pharmacodynamic (PD) study, patients with coronary artery disease (n = 20) were pre-treated with a 180-mg ticagrelor loading dose (LD) and after 1 hour randomized to placebo or cangrelor (bolus and infusion for 2 hours). Patients crossed over after 1-4 weeks of washout. PK analysis included ticagrelor plasma levels and its active metabolite. PD assessments included: VerifyNow P2Y12 reaction units (PRU), light transmittance aggregometry vasodilatorstimulated phosphoprotein, and Total Thrombus-Formation Analysis System. PK/PD assessments were performed at 7-time points. Results Compared to placebo, adding cangrelor to patients pre-treated with ticagrelor resulted in a significant reduction in PRU at 30 min and 1 hour after starting infusion. At 2 hours after stopping cangrelor/placebo infusion, PRU was low and similar in both groups (16.9 vs. 12.6; mean difference: 4.3; 95%CI: -28.6; 37.3), meeting the non-inferiority primary endpoint (predefined non-inferiority margin 45 PRU). Consistent findings were shown with all PD assays. PK tracked PD findings with no differences between groups in plasma levels of ticagrelor and its metabolite. Conclusions Compared with placebo, the use of cangrelor in patients pre-treated with ticagrelor results in enhanced platelet inhibition with no differences in PK/PD profiles after discontinuation of drug infusion indicating the absence of a DDI.

Hemostatic Ability of Thrombosomes® in Blood from Thrombocytopenic Patients Using the Total Thrombus Formation Analysis System (T-TAS) and Confocal Microscopy in Microfluidic Chambers

Hemostatic Ability of Thrombosomes® in Blood from Thrombocytopenic Patients Using the Total Thrombus Formation Analysis System (T-TAS) and Confocal Microscopy in Microfluidic Chambers

Follow-up and Evaluation of the Efficacy of the Hemostatic Treatment in Congenital FVII Deficiency during Development of Inhibitor

Introduction The development of inhibitors is an extremely rare complication in patients with factor (F)VII deficiency treated with replacement therapies such as recombinant activated FVII (rFVIIa), and represents a major therapeutic challenge in the treatment of this disorder. When inhibitors result in loss of response to high doses of rFVIIa, activated prothrombin complex concentrate (aPCC) is often used as an alternative treatment for these patients. The use of high-dose rFVIIa or aPCC might be accompanied by an increased risk of thromboembolic events, so it is important monitoring properly the effect of the hemostatic treatment Here we describe the therapeutic management and follow-up of a patient with elevated inhibitor titres, resistant to rFVIIa treatment who switched to prophylaxis with aPCC, using several global coagulation tests. Methods The coagulation status of the patient and the reponse to in vitro spiking of different doses of rFVIIa or aPCC were assessed with several global tests: Prothrombine time (PT) was tested using HemosIL®RecombiPlasTin 2G. Clot formation was evaluated with Rotational Thromboelastometry (ROTEM®) using blood with CTI (to inhibit contact activation) and activated with a low amount of Tissue Factor (TF) as trigger (dilution 1:50,000 of EXTEM reagent). The thrombin generation (TG) was tested with the Calibrated Automated Thrombogram (CAT) system using plasma collected with CTI and activated with 1 pM TF plus phospholipids (PPP-Low®, Stago). Thrombus formation under flow conditions was assessed with the Total Thrombus-formation Analysis System (T-TAS®) in AR-chips coated with collagen and thromboplastin. The Area under the flow pressure curve (AUC) was calculated over 30 min after starting. Results Three year-old girl affected by severe congenital FVII deficiency (FVII 9.1%) with significant severe bleeding history (spontaneous intracranial hemorrhage at 1st month of life). Since then she is in prophylaxis treatment with rFVIIa. At 6 months of life she developed an inhibitor (9.8 BU). Despite prophylaxis she presents frequent spontaneous mucocutaneous bleedings. When she was 2 years old she needed to be admitted at hospital because of port-A-Cath infection. Due to the administration of intensive treatment with rFVIIa during admission she developed thrombosis of peripheral venous access, so prophylaxis treatment was temporary suspended. After 5 days without prophylaxis treatment, and increasing inhibitor titres, she developed a new intracranial haemorrhage, which needed intensive treatment with continuous rFVIIa infusion (90 µg/Kg/12h), immunoglobulines and rituximab for lowering inhibitor titre. Hemostatic parameters improved during the administration of these treatments and inhibitor level was reduced. After restarting prophylaxis treatment with rFVIIa (50 µg/Kg/48h), inhibitors and a hypocoagulable state were observed with all global coagulation test showing extended PT, clotting time (CT), TG Lag-time (LT) and anomalous thrombus formation (AUC) but presented very good response to low dosis (2.5 IU/Kg) of aPCC in vitro (Table-1). At the age of three years she required admission at hospital because of port-A-Cath infection again, needing its replacement by a new one after administration of high doses of rFVIIa without bleeding complications. After this, inhibitor titre raised again (> 50 BU), presenting frequent bleeding episodes (spontaneous muscle bleedings and tonsillar bleeding), and due to the clinical worsening, treatment was modified to prophylaxis with aPCC (30 UI/Kg/48h). Despite the treatment change she continued presenting bleeding episodes, global tests continued showing an hypocoagulable state, and required 10 times higher doses in vitro of aPCC (25 IU/Kg) to restore all the monitored hemostatic parameters (Table-1), probably related to the important increase in inhibitor titre. This situation led to employing higher doses of aPCC prophylaxis (60 UI/Kg/48 h) to try to avoid bleeding episodes without increasing the risk of suffering thrombotic events. Conclusions Global coagulation tests used in this study were sufficiently sensitive methods to show an abnormal hemostatic profile in FVII-deficient patients with clinical hemorrhagic symptoms with inhibitor development, supporting their use to manage the risk of bleeding or thrombosis in this life-threatening condition. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal