Correlation between plasma clot properties, thrombin generation and whole blood fibrinolytic assays in patients presenting with STEMI

Abstract Background Impaired endogenous fibrinolysis is a novel risk factor for recurrent adverse cardiovascular events in acute coronary syndrome (ACS) patients. This is independent of conventional cardiovascular risk factors and unaffected by dual antiplatelet therapy (DAPT). The mechanism underlying impaired endogenous fibrinolysis in ACS patients is currently unclear. Aim To identify the relationship between whole blood fibrinolysis, plasma fibrinolysis and thrombin generation in samples from STEMI patients. Methods In a large, prospective, observational study of 500 patients presenting with ST-segment elevation myocardial infarction (STEMI), blood samples were taken on arrival, after DAPT loading, and before administration of heparin or PPCI. Non-anticoagulated venous whole blood was analysed using the point-of-care Global Thrombosis Test, which assesses the time taken for occlusive thrombus formation under high shear (occlusion time, OT) and time required for spontaneous restart of flow as a measure of endogenous fibrinolysis (lysis time, LT). Patients were divided into 4 groups based on quartiles (Q) of whole blood LT (Q1: LT<1500s, Q2:1501–3000s, Q3:3001–4500s, Q4:>4500s). Plasma samples (20 per quartile) were examined in a thrombin generation assay using 1pM tissue factor to initiate and using a turbidity assay to determine the plasma clot lysis time (CLT). Results Clinical characteristics of patients were similar in the four groups. The whole blood LT in the 4 groups were Q1: 1194 (1125–1329) s, Q2: 1859 (1634–2157) s, Q3: 3638 (3252–3962) s, Q4: 6000 (5523–6000) s. As LT increased, there was a trend towards longer plasma CLT (50% CLT Q2: 88.5 [73.5–102] vs. Q4: 100 [85–128.5] min, p=0.088). As a continuous variable, there was no significant relationship between whole blood LT and plasma CLT, or between endogenous thrombin potential (ETP) and either whole blood LT or plasma CLT. There was a significant negative correlation between OT and velocity index (r=−0.425, p=0.0138), ETP (r=−0.519, p=0.002), peak thrombin generation (r=−0.390, p=0.0247) and a positive correlation with lag-time (r=0.427, p=0.013). There was positive correlation between CLT and white cell count (WCC, r=0.388, p=0.026), C-reactive protein (CRP, r=0.477, p=0.005) and maximum absorbance (MA, r=0.530, p=0.002). MA correlated with WCC (r=0.436, p=0.011) and platelet count (r=0.357, p=0.042). There was a negative correlation between OT and WCC (r=−0.537, p=0.001) and CRP (r=−0.381, p=0.029). Conclusion In patients with STEMI, increased platelet reactivity (shorter OT) correlated with increased thrombin generation (higher ETP, peak thrombin generation, velocity index and reduced lag time), demonstrating the key role of thrombin in occlusive thrombus formation. Fibrinolysis in whole blood was poorly related to plasma CLT or thrombin generation, suggesting that cellular components such as platelets, erythrocytes and neutrophil extracellular traps may significantly influence endogenous fibrinolysis. Funding Acknowledgement Type of funding source: None

Download Full-text

Evaluation of the REG1 Anticoagulant System Using Thrombelastography and Thrombin Generation Assay.

Blood ◽

10.1182/blood.v110.11.4010.4010 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4010-4010

Author(s):

Kenichi A. Tanaka ◽

Fania Szlam ◽

Christopher P. Rusconi ◽

Jerrold H. Levy

Keyword(s):

Whole Blood ◽

Thrombin Generation ◽

Thrombus Formation ◽

Point Of Care ◽

Phase 1 ◽

Lag Time ◽

Blood Draw ◽

Blood Samples ◽

Thrombin Generation Assay ◽

Anticoagulant System

Abstract Background: The REG1 system (Regado Biosciences, Inc. Durham, NC) is a novel anticoagulant system which comprises RB006 (drug) and RB007 (antidote). The aptamer, RB006 selectively binds to FIXa and blocks factor Xa generation. RB007 is a complementary ligand that selectively binds to RB006, and reverses its anticoagulant effect (1). Phase 1 testing demonstrated a clear pharmacodynamic dose response to RB006 in plasma coagulation assays (1), but further work is needed to understand the pharmacodynamic response to the REG1 system in whole blood assays. Therefore, we evaluated the effects of RB006 and RB007 alone and in combination using activated partial thromboplastin time (APTT), viscoelastic (TEG®, Haemoscope, IL) and thrombin generation assay (Thrombinoscope™, Synapse BV). Methods: After IRB approval, blood samples were collected from 4 consented healthy volunteers into 3.2% citrate tubes. For APTT and TEG testing blood was placed in thirteen 2 ml Eppendorf tubes. Tube one had 20 μl of saline added and served as a control. The remaining 12 tubes were divided into 3 groups. Group1: RB006 (final concentrations 3, 6, 12, 18 and 24 μg/ml); Group 2: RB007 (final concentrations 6, 12, 24, and 48 μg/ml) and Group 3: combination of both agents at a weight:weight ratio 2 to 1 for RB007:RB006 (final concentrations as above). All testing was performed within 3-hour of blood draw. APTT was done using Hemochron Jr® (ITC, NJ) instrument in recalcified whole blood. TEG was peformed in recalcified whole blood, 360 μl activated with 2 nM thrombin. For Thrombinoscope, whole blood was centrifuged at 2000 x g for 15 min to obtain platelet poor plasma (PPP). PPP samples were prepared to contain the same concentrations of RB006, RB007, and combination of both agents as described in whole blood samples. Thrombin generation analyses were performed using microplate format using diluted Actin (Dade Behring, Marburg, Germany) as a trigger (2). Results: RB006 dose dependently increased APTT (Table 1). Increasing concentrations of RB006 progressively prolonged onset, and decreased the rate of thrombus formation on TEG (Figure1A). On Thrombinoscope, RB006 dose dependently delayed lag time and decreased peak thrombin generation (Table 1, Figure1B). All the parameters of aPTT, TEG and thrombin generation returned back to control values when combination of aptamer-anti-aptamer was tested. RB007 alone had no effects on any of the tests performed. Conclusion: Along with conventional point of care APTT testing TEG and Thrombinoscope methodologies can be very useful in monitoring the anticoagulation effects of aptamer, RB006, and its reversal with anti-aptamer, RB007. Effects of Aptamer, RB006, on aPTT and Thrombinoscope lag time/peak thrombin RB006μg/ml APTT sec Lag time min Peak thrombin nM Data shown as mean ± SD 0 (control) 51.3 ± 1.0 10.8 ± 0.3 244 ± 16.2 3 95.3 ± 3.0 14.2 ± 0.9 173 ± 22.9 6 152 ± 10.0 18.8 ± 0.5 145 ± 23.0 12 183 ± 4.3 24.1 ± 1.1 105 ± 11.0 18 238 ± 15.3 26.3 ± 2.3 92.3 ± 7.3 24 257 ± 11.3 31.3 ± 3.8 88.2 ± 8.3 Figure 1 Figure 1.

Download Full-text

Abstract P683: Thrombin Generation in Plasma of Stroke Patients With Atrial Fibrillation

Stroke ◽

10.1161/str.52.suppl_1.p683 ◽

2021 ◽

Vol 52 (Suppl_1) ◽

Author(s):

Sarina Falcione ◽

Gina Sykes ◽

Joseph Kamtchum Tatuene ◽

Danielle Munsterman ◽

Twinkle Joy ◽

...

Keyword(s):

Atrial Fibrillation ◽

Ischemic Stroke ◽

Thrombin Generation ◽

Thrombus Formation ◽

Lag Time ◽

Thrombin Time ◽

Stroke Patients ◽

Time To Peak ◽

Generation Capacity

Background and Purpose: Thrombus formation is central to pathophysiology of stroke in patients with atrial fibrillation. Whether factors in plasma contribute to thrombus generation in patients with atrial fibrillation remains unclear. In this study we sought to determine whether plasma contributes to thrombin generation in patients with atrial fibrillation. Methods: There were 78 acute ischemic strokes with atrial fibrillation and 37 non-stroke controls. Plasma thrombin generation was measured by thrombin generation assay, resulting lag time, peak thrombin, time to peak and area under the curve was assessed. Thrombin generation capacity was compared in stroke patients with atrial fibrillation to non-stroke controls. The relationship to anticoagulation was assessed. In vitro, the effect of anticoagulation on plasma thrombin generation was determined. Results: Thrombin generation capacity was increased (shorter lag time and time to peak) in ischemic stroke patients with atrial fibrillation compared to non-stroke atrial-fibrillation controls (p<0.05 and p<0.01, respectively). Anticoagulation decreased plasma induced thrombin generation. Ischemic stroke patients with atrial fibrillation treated with anticoagulation (DOAC or warfarin) had lower plasma induced thrombin generation compared to atrial-fibrillation patients not on anticoagulation (p<0.05). Thrombin generation by plasma could be further reduced by DOAC in an in-vitro assay. Conclusions: Stroke patients with atrial fibrillation have a higher plasma induced thrombin generation compared to atrial fibrillation controls. Factors in plasma such as leukocyte derived tissue factor likely contribute to thrombus formation in patients with atrial fibrillation. As such, components in plasma may represent new targets to reduce thrombus formation and stroke risk in patients with atrial fibrillation.

Download Full-text

FEIBA® and Novoseven® Neutralize the Anticoagulant Effects of a Novel Small Molecule FXIa Inhibitor BMS-986177/JNJ-70033093 in Human Plasma and Whole Blood in Vitro

Blood ◽

10.1182/blood-2020-136378 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 10-11

Author(s):

Matthew W Bunce ◽

Zheng Huang Devine ◽

Madhu Chintala

Keyword(s):

Human Plasma ◽

Whole Blood ◽

Thrombin Generation ◽

Hemophilia A ◽

Lag Time ◽

Publicly Traded ◽

Routine Prophylaxis ◽

Control And Prevention ◽

Bleeding Episodes

Background: FXIa inhibition is a promising antithrombotic drug target. BMS-986177/JNJ-70033093 (BMS-177/JNJ-3093) is a novel small molecular inhibitor of FXIa currently in Phase II clinical trials with the potential for reduced bleeding risk as compared to the currently approved oral anticoagulantsHowever, reversal of anticoagulation may still be required in patients who have uncontrolled or life-threatening bleeding or need an urgent surgical procedure. Aim: To evaluate the ability of nonspecific reversal agents (NSRAs) FEIBA®, NovoSeven®, Kcentra®, Profilnine®, BeneFix®, Novoeight®, and Cyklokapron® to neutralize the anticoagulation of BMS-177/JNJ-3093 in the activated partial thromboplastin time (aPTT), thromboelastography (TEG) and thrombin generation assay (TGA) in vitro using human plasma or whole blood. Method: aPTT and TEG were performed in human plasma and whole blood, respectively, using standard assay procedures. TGA was performed in human plasma using diluted kaolin aPTT reagent (1:10,000). JNJ-3093 was evaluated at different concentrations (0.3 -10 µM) to cover the anticipated exposures in the Phase II clinical trials. The NSRAs were evaluated at the anticipated concentrations according to the dosing information in their respective labels. Results: BMS-177/JNJ-3093 produced concentration dependent increases in aPTT (up to 4.4x at 10 μM); prolongations of lag time in TEG (2.6X); prolongations of lag time (3X) as well as reductions in peak thrombin generation (70%) in TGA. FEIBA® effectively neutralized the anticoagulant effects of JNJ-3093 in aPTT, TEG and TGA. NovoSeven® neutralized the BMS-177/JNJ-3093-induced prolongations in aPTT, prolongations in lag time in TEG and TGA assays and partially restored the peak thrombin generation in TGA. In contrast, all other NSRAs tested had negligible effects or did not show neutralization of anticoagulation induced by BMS-177/JNJ-3093 in the referenced assays Conclusion: These results demonstrate that FEIBA® and NovoSeven® can effectively neutralize the anticoagulant effects of BMS-177/JNJ-3093 in vitro. A clinical study is required to determine if these agents can reverse the anticoagulant effects of BMS-177/JNJ-3093 in patients. Table Disclosures Bunce: Johnson & Johnson: Current Employment, Current equity holder in publicly-traded company. Huang Devine:Johnson & Johnson: Current Employment, Current equity holder in publicly-traded company. Chintala:Johnson & Johnson: Current Employment, Current equity holder in publicly-traded company. OffLabel Disclosure: FEIBA: hemophilia A and B patients with inhibitors for: control and prevention of bleeding episodes; use around the time of surgery; routine prophylaxis to prevent or reduce the frequency of bleeding episodes NovoSeven: Treatment of bleeding and prevention of bleeding for surgeries and procedures in adults and children with hemophilia A or B with inhibitors, congenital Factor VII (FVII) deficiency, and Glanzmanns thrombasthenia with a decreased or absent response to platelet transfusions; treatment of bleeding and prevention of bleeding for surgeries and procedures in adults with acquired hemophilia Kcentra: urgent reversal of acquired coagulation factor deficiency induced by vitamin K antagonist therapy in adult patients with need for urgent surgery/invasive procedure or acute major bleeding Profilnine: prevention and control of bleeding in patients with Factor IX deficiency due to hemophilia B BeneFix: control and prevention of bleeding episodes or peri-operative management in adult and pediatric patients with hemophilia B Novoeight: for use in adults and children with hemophilia A for control and prevention of bleeding, perioperative management, and routine prophylaxis to prevent or reduce the frequency of bleeding episodes Cyklokapron: patients with hemophilia for short-term use to reduce or prevent hemorrhage and reduce the need for replacement therapy during and following tooth extraction)

Download Full-text

Correlation between Percentage of Reticulated Platelets and Heart Score in Patients with Suspected Non-ST Elevation Acute Coronary Syndromes

INDONESIAN JOURNAL OF CLINICAL PATHOLOGY AND MEDICAL LABORATORY ◽

10.24293/ijcpml.v27i1.1633 ◽

2020 ◽

Vol 27 (1) ◽

Author(s):

Fransisca Mulyadi ◽

Delita Prihatni ◽

Coriejati Rita ◽

Dewi Kartika Turbawaty ◽

Astri Astuti

Keyword(s):

Acute Coronary Syndrome ◽

Thrombus Formation ◽

Cardiac Events ◽

Cross Sectional ◽

Positive Correlation ◽

Heart Score ◽

Coronary Syndrome ◽

Reticulated Platelets ◽

St Elevation ◽

Elevation Acute Coronary Syndrome

Thrombus formation in non-ST Elevation Acute Coronary Syndrome (NSTE-ACS) causes increased plateletconsumption, leading to a 20-fold increase of Reticulated Platelets (RP) release. Reticulated platelets have more granulesand proteins that make them quickly forming thrombus than mature platelets, potent to form bigger thrombus, andincrease the risk of Major Adverse Cardiac Events (MACE). HEART score is a risk stratification for possible NSTE-ACS, whichcan predict MACE. The study aimed to analyze the correlation between the percentage of reticulated platelets and HEARTscore. This research was a correlation observational cross-sectional study performed in Dr. Hasan Sadikin Hospital,Bandung, from August 2018 to May 2019. The subjects were patients suspected with NSTE-ACS by clinicians in theEmergency Department of Dr. Hasan Sadikin Hospital. These subjects were assessed for the HEART score andRP percentage. This study involved 52 subjects consisting of a higher number of males (76.9%) aged 45-64 years old (69.2%).HEART score stratification in this study was mostly high risk (69.2%), but none was low risk. Mean of platelet count, absolute3 3 RP, and RP percentage were 271±73 x103/mm , 9.3±4.3 x 103/mm , and 3.6±1.7%, respectively. The correlation testbetween RP percentage and HEART score with a 95% confidence interval using Spearman's correlation test showed asignificant positive correlation with moderate strength (p < 0.001 and r=0.475). The percentage of RP in this study was in thenormal range. However, there was a significant positive correlation with moderate strength between the percentage of RPand HEART scores in patients with suspected non-ST elevation acute coronary syndrome.

Download Full-text

Effect of New Synthetic Heparin Mimetics on Whole Blood Thrombin Generation In Vivo and In Vitro in Rats

Thrombosis and Haemostasis ◽

10.1055/s-0037-1612979 ◽

2002 ◽

Vol 87 (02) ◽

pp. 238-244 ◽

Cited By ~ 8

Author(s):

J.P. Hérault ◽

A. Bernat ◽

C. Gaich ◽

J.M. Herbert

Keyword(s):

Venous Thrombosis ◽

Charge Density ◽

Whole Blood ◽

Thrombin Generation ◽

Platelet Rich Plasma ◽

Lag Time ◽

Drug Candidate ◽

Platelet Factor

SummaryThe effect of new heparin mimetics (synthetic oligosaccharides) was studied in vitro with regard to thrombin generation (TG) in rat platelet rich plasma (PRP) and whole blood (WB) and in vivo on stasis-induced venous thrombosis in the rat.TG in PRP and in WB was highly dependent on platelet count and strongly influenced by the haematocrit. The peak of TG appeared to be significantly higher in WB than in PRP whereas the endogenous thrombin potential (ETP) was not significantly different under either condition.The effect of hirudin, the synthetic pentasaccharide SR90107/ Org31540 (SP) and heparin were measured on TG in PRP and WB. We then compared the effect of two new synthetic heparin mimetics (SR121903A and SanOrg123781) with potent and comparable antithrombin (AT) mediated activity against factor Xa and thrombin. These two compounds were made of a pentasaccharide with a high affinity to AT, prolonged at the non-reducing end by an oligosaccharide chain recognised by thrombin. In SR121903A, the charge density and charge distribution was analogous to that of heparin whereas in SanOrg123781 the charges were only located on the last 5 saccharides of the non-reducing end of the molecule. In PRP and in WB, SR121903A acted on the lag time and on the AUC whereas SanOrg123781 inhibited thrombin formation with no effect on the lag time. SanOrg123781 was more potent in inhibiting TG than SR121903A. This difference was due to the structures of the compounds that differed in their ability to be neutralised by platelet factor 4. The antithrombotic effect of the two compounds was examined in a venous thrombosis model in rats. We observed that SanOrg123781 was more active than SR121903A and heparin.Taken together, these results indicate that the activity of oligosaccharides is greatly influenced by the global charge density of the molecule and show that SanOrg123781 is a potent and promising antithrombotic drug candidate.

Download Full-text

The correlation of climate factors on dengue transmission in urban area: Bangkok and Singapore cases

10.7287/peerj.preprints.2322v1 ◽

2016 ◽

Author(s):

Sittisede Polwiang

Keyword(s):

Incidence Rate ◽

Negative Correlation ◽

Statistical Significance ◽

Time Lag ◽

Rank Correlation ◽

Lag Time ◽

Climate Factors ◽

Positive Correlation ◽

Spearman’S Rank Correlation ◽

Correlation Tests

The objective of this study is to find the correlation between climate factors and dengue incidence rate in Bangkok and Singapore during 2009-2015. Spearman's rank correlation tests with time-lag are performed to investigate the overall correlation between dengue incidence rates and climate factors , minimum, mean, and maximum temperatures, DTR, and rainfall. A Linear and Poisson regression analysis was performed. Spearman's rank correlation tests show that in Bangkok monthly rainfall (r=0.451, p<0.001), the number of rainy days (r=0.411, p<0.001) are positive correlation with 2 month lag time. DTR (r=-0.335, p<0.001) is negative correlation with 2 month lag time. Maximum (r=0.256, p<0.001), mean (r=0.304, p<0.001) and minimum (r=0.323, p<0.001) temperature are positive correlation with 4 month lag time. In Singapore, only minimum temperature (r=-0.299, p<0.001) with 1 month lag time is negative correlation and DTR (r=-0.289, p<0.001) with zero month lag time is positive correlation. The rest has no statically significance (p>0.05). This study concluded, climate factors play moderate role in dengue incidence in Bangkok. There is no statistical significance between rainfall and dengue incidence rate and temperature play a marginal role in Singapore.

Download Full-text

Assessment of endogenous fibrinolysis using a point-of-care assay to identify increased cardiovascular risk in patients with diabetes and ACS

European Heart Journal ◽

10.1093/ehjci/ehaa946.1540 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

R Kanji ◽

Y.X Gue ◽

D Dinarvand ◽

M.Q Farag ◽

D.A Gorog

Keyword(s):

Whole Blood ◽

Clinical Characteristics ◽

Thrombus Formation ◽

Point Of Care ◽

Occlusion Time ◽

Increased Risk ◽

Significant Difference ◽

Patients With Diabetes ◽

Endogenous Fibrinolysis ◽

Point Of Care Assay

Abstract Introduction Patients with diabetes mellitus (DM) are increased risk of myocardial infarction (MI) and following a MI, patients with DM have an increased risk of recurrent MI and cardiovascular (CV) death. Plasma turbidimetry studies show that hypofibrinolysis is a key abnormality in DM that may drive increased ischaemic risk. Such assays are cumbersome, require specialist expertise and do not provide information in a clinically-relevant timeframe. Assessment of fibrinolysis in whole blood, using a point-of-care assay, has revealed that in ACS patients, impaired fibrinolysis is predictive of adverse CV events. Whether this technique can identify residual risk in patients with DM, is unclear. Purpose It was our aim to compare thrombotic and endogenous fibrinolytic status between patients with and without DM, presenting with ACS. Methods We conducted a prospective, observational study of consecutive patients admitted with ACS. Venous blood was taken to assess thrombotic and thrombolytic status using the point-of-care Global Thrombosis Test, assessing time to occlusive thrombus formation under high shear (occlusion time, OT) and time taken for spontaneous lysis of the thrombus (lysis yime, LT). Blood was taken after dual antiplatelet therapy (DAPT) loading, but before administration of fondaparinux or low molecular weight heparin. Patients with renal or hepatic impairment, known bleeding diathesis, thrombocytopenia and those taking anticoagulation were excluded. Results A total of 775 patients were included, of whom 158 (20%) had DM. Patients with DM, compared to those without DM, more frequently had hypertension (70% vs. 39%, p<0.001), hyperlipidaemia (65% vs. 29%, p<0.001), higher BMI (28.6 [25.3–32.0] vs. 26.6 [23.7–29.8] kg/m2, p<0.001) and prior MI (28% vs 9%, p<0.001), but were less often smokers (23% vs. 34% p=0.007). In all other clinical characteristics DM and non-DM patients were matched. Time to occlusive thrombus formation was similar in patients with and without DM (OT 401 (284–519) s vs. 391 (289–514) s, p=0.603). There was a trend for longer LT in patients with DM compared to those without DM (LT 1634 (130–2321) s vs. 1562 (1247–2147) s, p=0.080). After propensity score matching to adjust for baseline differences in clinical characteristics, we observed a highly significant difference in LT between DM and non-DM patients (LT 1634 [1306–2321] s vs. 1387 [1109–1740] s, p<0.001). Patients with DM also had higher fibrinogen level (4.4 [3.5–5.4] vs. 4.1 [3.5–4.8] g/l, p=0.012) and higher C-reactive protein (5 [2–12] vs. 3 [1–8] mg/l, p=0.002). CRP correlated with LT (r=0.2, p=0.016), but no correlation was observed between fibrinogen and LT (r=0.069, p=0.424). Conclusions Amongst patients with ACS, those with DM exhibit markedly impaired endogenous fibrinolysis compared to those without DM, and this can be detected with a bedside assay using whole blood. This may explain the increased risk of secondary events in patients with ACS and DM. Funding Acknowledgement Type of funding source: None

Download Full-text

No difference in thrombotic profile of patients with ACS with obstructive CAD and MINOCA

European Heart Journal ◽

10.1093/ehjci/ehaa946.1541 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

R Kanji ◽

Y.X Gue ◽

D Dinarvand ◽

D.A Gorog

Keyword(s):

Myocardial Infarction ◽

Thrombus Formation ◽

Obstructive Coronary Artery Disease ◽

Venous Blood ◽

High Shear ◽

Occlusion Time ◽

Coronary Syndrome ◽

Heparin Administration ◽

Endogenous Fibrinolysis

Abstract Introduction Acute coronary syndrome (ACS) is caused by disruption of an atherosclerotic plaque with initiation of thrombosis, and outcome determined by the balance between prothrombotic drivers and the efficacy of endogenous fibrinolysis. Most patients have obstructive coronary artery disease (CAD), with high shear forces and turbulent flow across severe stenoses enhancing platelet activation. Recognition that some ACS patients have myocardial infarction (MI) with non-obstructive coronary arteries (MINOCA) has led to a search to identify drivers behind such presentations. Purpose To assess and compare the thrombotic status of patients with MINOCA and those with ACS due to obstructive CAD. Methods In a prospective observational study in patients with ACS, thrombotic and thrombolytic status was assessed from venous blood using the point-of-care Global Thrombosis Test, assessing time to in vitro occlusive thrombus formation under high shear (occlusion time,OT) and time taken for spontaneous lysis of the thrombus (lysis time,LT). Blood was taken after dual antiplatelet therapy loading, but before fondaparinux or heparin administration. Those with renal or hepatic impairment, bleeding diathesis, thrombocytopenia or on anticoagulation were excluded. MINOCA diagnosis was made according to the Fourth Universal Definition of MI, in the absence of obstructive CAD (no lesion ≥50%) and excluding patients with 1) other overt causes for elevated troponin, 2) overlooked obstructive CAD, and 3) nonischaemic causes for myocyte injury, according to the American Heart Association 2019 recommendation. Patients with Type 2, 4 and 5 MI were excluded. Results We assessed 746 patients, of whom 621 (83%) had ST-segment elevation MI (STEMI) and the rest non-STEMI. Of these, 706 (95%) had obstructive CAD and 40 (5%) had MINOCA. Apart from sex (78% obstructive CAD patients were male vs 50% MINOCA patients), cardiovascular risk factors were similar in MINOCA and obstructive CAD patients (smoking 28 vs 31%, p=0.615; hypertension 35 vs 47%, p=0.153; diabetes 20 vs 20%, p=0.948; hyperlipidaemia 30 vs 36%, p=0.475 and family history of premature CAD 35 vs 35%, p=1.000). There was no difference in time to form occlusive thrombus (OT 424 [371–471] vs 395 [287–512] s, p=0.093) or in endogenous fibrinolysis (LT 1450 [1082–2099] vs 1582 [1252–2130] s, p=0.178) between MINOCA and obstructive CAD patients. Even after propensity score matching with a ratio of 3:1 for clinical characteristics, there was no difference between patients with MINOCA and those with obstructive CAD, with respect to thrombus formation (OT 424 [371–471] vs 430 [300–538] s, p=0.602) or endogenous fibrinolysis (LT 1470 [1082- 2099] vs 1494 [1140–2074] s, p=0.625). Conclusion Amongst patients with ACS, those with MINOCA exhibit similar thrombotic profiles to patients with obstructive CAD with ACS. This represents a potential therapeutic target to modulate risk post myocardial infarction in patients with MINOCA and requires further research. Funding Acknowledgement Type of funding source: None

Download Full-text

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

Blood ◽

10.1182/blood.v120.21.1167.1167 ◽

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.

Download Full-text

Thrombin Generation in Zebrafish

Blood ◽

10.1182/blood.v124.21.4218.4218 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4218-4218

Author(s):

Evelien Schurgers ◽

Martijn Moorlag ◽

Hilde Kelchtermans ◽

Coenraad Hemker ◽

Bas De Laat

Keyword(s):

Whole Blood ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Genetic Research ◽

Model Organism ◽

Lag Time ◽

In Vivo Model ◽

Time To Peak ◽

Paper Disk

Abstract Introduction Recently, the whole blood calibrated automated thrombogram (CAT) was miniaturized enabling the measurement of thrombin generation (TG) in a limited volume (5 µl) of whole blood. Consequently, this approach may be used to determine TG in small lab animals. Zebrafish are readily available test animals for genetic research. However, their small size has been a hurdle in thrombosis and hemostasis research since most assays require large amounts of plasma. In this study we verified the possibility to measure TG in zebrafish using our newly developed miniaturized whole blood assay. Methods For TG, 5 µl of whole blood was mixed with 5 µl of buffer containing a rhodamin-based thrombin-sensitive P2Rho substrate (final concentration (fc) 300 µM). 5 µl of this mixture was put on a paper disk and covered with mineral oil to prevent evaporation. Calibration was done as described previously (Ninivaggi et al. Clin Chem 2012) by adding 5 µl of whole blood to 5 µl of a mixture containing P2Rho (fc 300 µM), a2M-thrombin calibrator (fc 100 nM) and citrate (fc 9,8 mM). Fluorescence was detected with a fluorometer (485/538 nm). Results Due to their limited blood volume, it is impossible to perform both a TG and calibrator measurement on the same fish. Since calibrator measurements performed on blood from different fish demonstrated acceptable variation (CV calibrator slopes < 15%), the average calibrator slope was used for calculations. The calculated TG parameters from 2 independent experiments are depicted in Table 1. TG measured in individual fish showed the same amount of inter-individual variation as in humans. Striking differences with human TG parameters were the short lag time, high peak and high velocity index. Moreover a further analysis of the fibrin network of the clot, by means of scanning electron microscopy (SEM), showed a much denser network composed of thinner fibers compared to humans. Table 1. Thrombin generation parameters and their coefficient of variance (CV) in zebrafish in two independent experiments. Experiment 1(n=4) Experiment 2(n=9) Mean CV Mean CV Peak ETP (nM.min) 575.68 19.69 689.71 24.91 Peak (nM) 1573.16 29.58 1668.05 14.46 Lag time (min) 0.27 0.00 0.49 10.92 Time to peak (min) 1.08 5.30 1.37 11.49 Velocity (nM/min) 4002.33 38.21 3826.35 17.04 Conclusion These results demonstrate the feasibility of measuring TG in whole blood collected from zebrafish. Consequently, zebrafish may be used as a in vivo model to test the effect of (novel) anticoagulant therapeutics on thrombin generation and serve as a model organism for mechanistical research in thrombosis and haemostasis. Disclosures No relevant conflicts of interest to declare.

Download Full-text