Abstract 613: In Vitro Microvessels to Study the Platelet-Endothelium Interface

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Jevgenia Zilberman-Rudenko ◽  
Andrew D Wong ◽  
Daniel E Sallee ◽  
Stephanie E Reitsma ◽  
Cristina Puy ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Thrombin Generation ◽  
Vascular Diseases ◽  
Coagulation Factors ◽  
Flow Chamber ◽  
Physical Parameters ◽  
Interstitial Flow ◽  
Disease States ◽  
Blood Flow Dynamics

Background: Under normal conditions, endothelial cells (ECs) govern blood flow dynamics including providing a barrier between blood and tissue and regulating platelet aggregation and thrombin generation in the bloodstream. In turn, blood components, primarily platelets and coagulation factors such as thrombin, regulate EC barrier integrity. The breakdown of EC barrier function is a hallmark of a variety of vascular diseases. In sepsis, for example, the dysfunction of vascular ECs has been correlated with poorer outcomes due to hemorrhage and multi-organ failure associated with consumption of platelets and coagulation factors into clots within the microcirculation, a condition termed disseminated intravascular coagulation (DIC). Aim: Develop an endothelialized flow chamber to study the platelet-endothelium interface. Methods and Results: We developed a 3D-chamber with a perfuseable cylindrical microvessel embedded in an extracellular matrix (ECM) material. This model allows for the study of the role of thrombin generation and platelet aggregation in endothelial barrier leak development and repair in healthy as well as inflamed microvessels. Incorporation of subendothelial matrix proteins in these 3D-microvessel devices expands the capacity of the microfluidic studies to investigate blood cell extravasation and enables the control of physical parameters such as transmural pressure and interstitial flow through the ECM. Conclusion: This model may provide insight into the pathophysiology of different disease states and serve as an expedient platform for therapy design and testing. The platelet-endothelium interface under shear flow. Diagram ( A ) and an experimental prototype ( B ) of a 3D-perfuseable device. Microvessel phenotype (following treatment with vehicle or 10 ng/mL TNFα) pre- and post- perfusion with recalcified whole blood for 33 min as visualized by differential interference contrast, DIC, ( C ) and fluorescence microscopy ( D ).

Impaired Haemostasis by Intravenous Administration of a Gelatin-based Plasma Expander in Human Subjects

1998 ◽  
Vol 79 (02) ◽  
pp. 286-290 ◽  
Author(s):  
M. Levi ◽  
F. Berends ◽  
A. E. van der Ende ◽  
J. W. ten Cate ◽  
C. P. Stoutenbeek ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Human Subjects ◽  
Fold Increase ◽  
Coagulation Factors ◽  
Platelet Glycoprotein ◽  
Plasma Expander ◽  
Plasma Substitute ◽  
Significant Impairment ◽  
Randomised Controlled

SummaryThe aim of this study was to investigate the effects of a gelatin-based plasma expander on blood coagulation and haemostasis in human subjects.Six healthy men were studied in a randomised, controlled cross-over study to investigate the effects of a 60 min intravenous infusion of either 1 l gelatin-based plasma substitute (Gelofusine) or 0.9% NaCl (control). The infusion of gelatin resulted in a 1.7 fold increase in bleeding time at 60 min and a 1.4 fold increase at 120 min, while saline had no effect (p <0.05). Aggregation studies revealed a significant impairment of ristocetin-induced platelet aggregation (p <0.05), associated with a substantial decrease of vWF:ag (–32% vs. –5%, p <0.05) and ristocetin co-factor (–29% vs. +1%, p <0.05) and without in vitro impairment of the platelet glycoprotein 1b receptor. Gelatin caused a decrease in thrombin-antithrombin complexes (–45% vs. –4%, p <0.05) and F1+2 (–40% vs. +1%, p <0.05). The decrease in circulating levels of vWF:ag, vWF R:Co, thrombin-antithrombin complexes and F1+ 2 was more than could be expected by the calculated plasma-dilution generated by Gelofusine.Our results demonstrated that the administration of a gelatin-based plasma substitute results in a significant impairment of primary haemostasis and thrombin generation. The defect in primary haemostasis appears to be related to a gelatin-induced reduction in von Willebrand factor, whereas the decreased thrombin generation may be due to the dilution of coagulation factors induced by Gelofusine.

scholarly journals Antiplatelet drugs and generation of thrombin in clotting blood

Blood ◽  
1992 ◽  
Vol 80 (8) ◽  
pp. 2006-2011 ◽  
Author(s):  
A Szczeklik ◽  
M Krzanowski ◽  
P Gora ◽  
J Radwan
Keyword(s):  
Oral Administration ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Coagulation Factors ◽  
Antiplatelet Drugs ◽  
Specific Marker ◽  
Rate Of Increase ◽  
Synthase Inhibitor ◽  
Thrombin Formation

Abstract Platelets participate in formation of thrombin through secretion of coagulation factors and by providing a catalytic surface on which prothrombinase complex is assembled. We studied the effects of four antiplatelet drugs on thrombin formation in healthy volunteers. Thrombin generation was monitored both in vitro--in recalcified plasma-- and ex vivo--in blood emerging from a standardized skin microvasculature injury, which also served to determine bleeding time. A mathematical model has been developed to describe the latter reaction. It is based on estimation of the rate of increase in fibrinopeptide A (FPA), a specific marker of thrombin activity, in blood emerging from skin incisions. Two hours after the ingestion of 500 mg of aspirin, thrombin formation became significantly impaired both in vitro and ex vivo. In contrast, 2 hours after the oral administration of placebo, indomethacin 50 mg, or OKY-046 (a thromboxane synthase inhibitor) 400 mg, thrombinogenesis remained unaltered. Ticlopidine, studied either 3 hours after 500 mg oral administration, or after 5 days of intake at a daily dose of 500 mg, had no effect on thrombin generation. Thus, aspirin, contrary to other antiplatelet drugs, depresses thrombin formation in clotting blood, a phenomenon that might be of clinical relevance. It is suggested that aspirin exerts this effect by acetylating prothrombin and/or macromolecules of platelet membrane.

Individual Variation in Hemostatic Alterations Caused By Tyrosine Kinase Inhibitors - a Way to Improve Personalized Cancer Therapy?

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1908-1908
Author(s):  
Kourosh Lotfi ◽  
Suryyani Deb ◽  
Clara Sjöström ◽  
Anjana Tharmakulanathan ◽  
Niklas Boknäs ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Side Effects ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Platelet Function ◽  
Thrombin Generation ◽  
Kinase Inhibitors ◽  
Thrombus Formation ◽  
Individual Variations

Abstract Introduction During the last two decades, Bcr-Abl tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myelogenous leukemia (CML), and are now considered standard treatment for this disease. However, TKIs can induce serious hemostatic side effects including cardiovascular disease and bleeding disorders. Blood platelet aggregation and formation of pro-coagulant platelets are important to allow a well-balanced hemostatic response. Therefore, a detailed understanding of what effect different TKIs exert on platelets and hemostasis could help to understand if there are differences of importance to minimize the risk of bleeding complications in treated patients. Aim To investigate how TKIs used in CML (imatinib, dasatinib, nilotinib, bosutinib, and ponatinib) affect platelet activation and hemostasis. Materials and Methods We have developed a multi-parameter six color flow cytometry protocol to study different aspects of platelet function upon activation, e.g. formation of aggregatory (PAC-1-positive) and pro-coagulant (phosphatidylserine-exposing) platelets, exocytosis of alpha- and lysosomal granules and mitochondrial membrane potential.This protocol was performed in presence or absence of TKIs in blood from normal donors and in treated patients. Whole blood aggregometry (Multiplate®), thrombin generation in platelet-rich plasma and in vitro thrombus formation by free oscillation rheometry (ReoRox G2) was further evaluated in some situations. Results At clinically relevant concentrations, dasatinib significantly decreased the formation of procoagulant platelets. Ponatinib induced a slight decrease in formation of procoagulant platelets, whereas bosutinib and nilotinib showed opposite tendencies (n=7). Dasatinib also decreased platelet aggregation (n=4-6) and in vitro thrombus formation (n=3). Thrombin generation was not significantly affected by therapeutic levels of TKIs, whereas higher doses of dasatinib, bosutinib, ponatinib and imatinib significantly changed one or several of the thrombin generation parameters (n=7-8). Interestingly, large differences in response to the drugs were observed among the healthy donors, especially for dasatinib and bosutinib. Major inter-individual variations were also observed in dasatinib-treated patients. Conclusions Different TKIs show varying potency to affect platelet-based hemostasis. In addition, we found large inter-individual variations in how some drugs affected platelet function. Therefore, we suggest that development of a clinically useful protocol for platelet function testing could help to identify patients more susceptible to adverse drug reactions. Such a protocol could potentially help clinicians to gain insight into the risk of side effects, which could help to choose the most suitable drug for each individual patient. Disclosures No relevant conflicts of interest to declare.

High-Vorticity with Periodic Flow Enhances in Vitro Biogenesis of Healthy Platelets from iPSC-Derived-Megakaryocytes

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2181-2181
Author(s):  
Yukitaka Ito ◽  
Sou Nakamura ◽  
Tomohiro Shigemori ◽  
Naoshi Sugimoto ◽  
Yoshikazu Kato ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Ex Vivo ◽  
Annexin V ◽  
Poor Quality ◽  
Flow Chamber ◽  
Physical Parameters ◽  
Platelet Production

Abstract Each transfusion requires 200-300 billion platelets in patients with thrombocytopenia. To continuously supply such a huge number of platelets by ex vivo generation, two distinct steps, megakaryopoiesis and platelet shedding, must be both considered. For the former, one approach is to increase the number of source cell, megakaryocytes. For example, the immortalized megakaryocyte cell line (imMKCL) system uses self-renewing megakaryocyte (MK) cell lines derived from induced pluripotent stem cells (iPSCs) (Nakamura et al., Cell Stem Cell, 2014). For the latter, there have been an idea of bioreactors whereby shedding of platelets from proplatelets could be promoted by flow-dependent shear force within the bone marrow in vivo (Junt et al., Science, 2007; Zhang et al., J Exp Med, 2012). Based upon this idea, we constructed a flow chamber type bioreactor recapitulating in vivo blood flow shear rate. However, this bioreactor failed to efficiently yield platelets, and moreover, the produced platelets had poor quality as indicated by high Annexin V levels (Exp Hematol, 2011 and unpublished result). Recently, we demonstrated two different kinetics of platelet biogenesis from bone marrow MKs, whereby either thrombopoietin (TPO) mostly regulates steady-state shedding of platelets from proplatelets, or interleukin-a (IL-1a) triggers inflammation-dependent rupture of MK cytoplasm contributing to a quick increase of platelet count at higher rate (Nishimura et al., J Cell Biol, 2015). However, the rupture type platelets revealed shorter half-life with relatively higher Annexin V levels. Therefore, to gain insights from platelet biogenesis in vivo, we focused on biophysical analysis of steady-state platelet biogenesis via proplatelets in bone marrow. Our observations strongly indicated that the presence of 'vorticity' defined by vortex turbulence in addition to shear-dependent 'stress' and 'strain' correlates with the efficient shedding of competent platelets. From this new finding, we developed an alternative bioreactor system, which enabled generation of 100 billion platelets from imMKCL in a 16L-scale liquid culture condition without any adherent machinery using two 10L-bioreactors. Furthermore, platelets generated via new bioreactors showed low Annexin V levels (<10-15%) and shortened bleeding time post transfusion into NOG mice and rabbits with thrombocytopenia, comparable to human blood product platelets. Regarding the platelet production using WAVE bag system (GE healthcare, UK), the system is already clinically available for cord blood cell expansion in most countries, but lacks adequate levels of vorticity and shear strain/stress. Accordingly, the produced platelets had high Annexin V levels (i.e., 50-65%) as well as diminished yield efficiency (P<0.001). In conclusion, our study has uncovered the novel biophysical aspect of platelet biogenesis. The application of the new set of physical parameters in constructing large sized bioreactors shall facilitate the industrialization of platelet production. Disclosures Eto: Megakaryon Co. Ltd.: Research Funding.

scholarly journals Prothrombin Membrane Binding and Gla-Dependent Function Are Not Required for Effective Hemostasis In Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 124-124
Author(s):  
Lindsey A. Greene ◽  
Nabil K. Thalji ◽  
Harlan Bradford ◽  
Sriram Krishnaswamy ◽  
Rodney M. Camire
Keyword(s):  
Research Funding ◽  
Thrombin Generation ◽  
Membrane Binding ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Full Length ◽  
Vessel Occlusion ◽  
Gla Domain

Abstract Prothrombin, like other vitamin K-dependent coagulation factors, undergoes γ-carboxylation of its Gla domain, a posttranslational modification critical for membrane binding. In patients on anticoagulant treatment with warfarin, the INR has historically been correlated with the degree of des-gamma-carboxy-prothrombin (DCP or PIVKA-II). PIVKA-II can be measured readily and used as a marker for vitamin K deficiency or warfarin therapy and is thought to be useful in detecting subclinical disease. Long-standing dogma suggests prothrombin γ-carboxylation is necessary for prothrombin membrane binding facilitating engagement with prothrombinase leading to rapid thrombin generation and effective hemostasis. However, recent studies indicate that despite an inability to bind membranes, uncarboxylated (desGla) full-length prothrombin demonstrated an unexpected modest decrease in the rate of thrombin generation (J Biol Chem 2013, 288:27789-800). Thus, it is possible loss of prothrombin γ-carboxylation, and thereby membrane binding, is far less significant for prothrombin activation than previously appreciated. Instead warfarin's effect on other coagulation factors (FX, FIX, and FVII) may be the primary causative determinant impairing hemostasis in these anticoagulated patients. To test these ideas, we first analyzed thrombin generation using recombinant full-length fully carboxylated and desGla prothrombin in vitro. Human prothrombin deficient plasma (Factor II activity <4%) was reconstituted to normal levels (100 μg/mL) with desGla or carboxylated prothrombin. DesGla prothrombin generated approximately half the amount of thrombin observed in carboxylated prothrombin plasma and normal human plasma controls. We next analyzed full-length desGla prothrombin's in vivo hemostatic function. A prothrombin anti-sense oligonucleotide (ASO) was administered to hemostatically normal mice to knock down endogenous murine prothrombin expression (<0.1-1μg/mL, 0.1-1%) and confirmed by ELISA analysis. Hemostasis was analyzed by the ferric chloride (FeCl3) carotid artery injury model. In mice treated with an ASO control, vessel occlusion occurred at approximately 8 minutes while mice treated with the prothrombin ASO did not clot during the 30-minute post injury observation period. In additional experiments two minutes following injury, prothrombin ASO treated mice were administered either carboxylated or desGla recombinant prothrombin to restore plasma concentrations to the normal range (100 μg/mL). Remarkably, administration of either desGla or carboxylated prothrombin restored vessel occlusion to ASO control findings, with minimal variability observed between desGla and carboxylated prothrombin treated mice (Figure 1). Warfarin treatment results in impaired prothrombin γ-carboxylation. However, if prothrombin γ-carboxylation, is, in fact, not necessary for prothrombin activation, fully carboxylated Factor Xa (FXa) should reverse the effects of warfarin by efficiently activating the un/under-carboxylated prothrombin thereby bypassing the other warfarin-affected factors. To study this, we used a "zymogen-like" factor Xa (FXaI16L) molecule previously developed by our group (Nat. Biotech 2011, 29:1028-33) that has a greater half-life than the wild-type protein. In thrombin generation assays, addition of 1nM FXaI16L to plasma from patients anticoagulated with warfarin, irrespective on INR (2.8, 4,4 7.1), resulted in thrombin generation comparable to that of normal human plasma. Importantly, similar results were obtained in vivo in warfarin-anticoagulated mice (INR 2-3). Administration of 3 mg/kg FXaI16L to 8 out of 8 warfarin mice corrected the time to carotid artery occlusion in the FeCl3 injury model. In two separate in vitro and in vivo model systems, we demonstrated that prothrombin membrane binding is not absolutely required for thrombin generation. Thrombin is unique among the coagulation serine proteases in that it does not have a Gla domain once fully processed by prothrombinase; thus, the absence of a Gla domain in the protease (thrombin) may explain the lack of a requirement for membrane binding by the zymogen (prothrombin) precursor. Our findings may also have clinical relevance, since they suggest that FXa (or a variant) could be used as a novel warfarin bypass strategy to rapidly achieve hemostasis in the setting of warfarin anticoagulation. Figure 1. Figure 1. Disclosures Greene: Baxter: Research Funding. Camire:Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Patents & Royalties, Research Funding; NovoNordisk: Research Funding.

Endothelial Microparticles Improve Primary and Secondary Hemostasis in Patients with Von-Willebrand Disease

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2287-2287
Author(s):  
Arne Trummer ◽  
Ingvild Birschmann ◽  
Sonja Werwitzke ◽  
Arnold Ganser ◽  
Andreas Tiede
Keyword(s):  
Platelet Aggregation ◽  
Human Plasma ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Von Willebrand Disease ◽  
Endothelial Microparticles ◽  
Circulating Microparticles ◽  
Von Willebrand

Abstract Abstract 2287 The bleeding phenotype in patients with von Willebrand disease (vWD) type 1 and 3 can usually be correlated to the amount and function of von Willebrand factor (vWF:Ag, vWF:RCo) but also to the impairment of functional coagulation assays: the ristocetin-induced platelet aggregation (RIPA) for primary hemostasis and the parameters of thrombin generation in a calibrated automated thrombogram (CAT, Thrombinoscope®) for secondary hemostasis. We therefore evaluated whether addition and depletion of endothelial microparticles (EMP) might influence these assays for samples of vWD patients. EMP were obtained from stimulated endothelial cell lines, isolated either by ultracentrifugation or magnetic beads selection for Annexin V and detected and quantified by flow cytometry after labelling for Annexin V and vWF. For CAT, EMP concentration was adjusted to 3×103/μl, for RIPA to 2×104/μl. vWD patient samples were diluted 3:1 or 2:1 with control buffer (HEPES/CaCl) or EMP solution. For thrombin generation, there was a significant shortening of lag time (6.8min vs 16.5min, p=.029) and time-to-peak (13.6min vs. 23.0min, p=.049) in vWD type 3 plasma (n=4) after addition of EMP using no reagent or MP reagent while there was no significant effect on these parameters in normal plasma. After addition of a tissue factor blocking antibody, the EMP effect could be largely reversed. For RIPA, we found a median maximum aggregation of 4% in vWD patients (n=6, vWF-Ag <40%). After addition of EMP, aggregation levels increased to 17.3%, which was significantly higher compared to control buffer (9.8%, p=.028), but lower compared to 250 IU/ml of a plasma-derived vWF product (Humate P®, 88.7%). To evaluate if significant amounts of vWF are bound to circulating microparticles in human plasma, we used magnetic bead selection to reduce MP counts in plasma of DDAVP stimulated vWD patients (n=10), because depletion of microparticles by 0.2μm filtration showed significant disruption of large vWF multimers. After MP reduction (1464/μl vs. 1863/μl, p=.036), both vWF:Ag (134.7% vs 161.1%, p=.005) and vWF:RCo (131.8% vs. 167.2%, p=.017) were significantly decreased. In summary, we could show that in vitro generated endothelial microparticles are able to improve thrombin generation and platelet aggregation in vWD patients and that significant amounts of vWF are bound to circulating microparticles in human plasma after DDAVP stimulation. Thus, in vitro generated EMP might have the potential to improve replacement therapy for vWD patients. Disclosures: No relevant conflicts of interest to declare.

The Effects Of An Anticoagulant Phospholipase A2 On The Procoagulant Activity Of Phospholipids And Platelets

1981 ◽  
Author(s):  
A C Cox
Keyword(s):  
Phospholipase A2 ◽  
Thrombin Generation ◽  
Coagulation Factors ◽  
Procoagulant Activity ◽  
Activation Process ◽  
Blood Coagulation Tests ◽  
Normal Platelet ◽  
Coagulation Tests ◽  
Lag Period

Although phospholipids readily substitute for platelets in many in vitro blood coagulation tests, their participation in normal platelet procoagulant activity is uncertain. Phospholipase A2 (PLA2) from Naja nigricollis nigricollis venom, a known anticoagulant, blocked the enhancement of the rate of prothrombin conversion to thrombin by both phospholipids and platelets. The rate of inhibition of the phospholipid procoagulant activity by PLA2 was reduced by indomethacin, an inhibitor of PLA2. At concentrations of PLA2 which inhibited phospholipid procoagulant activity, conversion of purified prothrombin by factors Xa and Va without phospholipid was unaffected.Unactivated, washed platelets combined with coagulation factors II, Va and Xa initially produce thrombin at a slow rate before the platelets become activated but after this lag period the rate of thrombin generation increases. PLA2 added at the same time as the coagulation factors increased the lag period and decreased the rate of thrombin generation during and after the lag. Preincubation of platelets with PLA2 further decreased only the lag rate and this inhibition was partially blocked by 300 uM indomethacin. At a concentration of about one ng/ml, PLA2 reduced the post-lag thrombin generation rate of 3 × 10-9 platelets/ml in half but had no effect on platelet aggregation induced by thrombin, ADP or collagen.These results combined with the known specificity of PLA2 support the theory that phospholipids are involved in platelet procoagulant activity. Furthermore, the ineffectiveness of preincubating PLA2 with platelets on the post-lag procoagulant activity suggests that the phospholipids involved in this post-activation process become accessible during the lag period.

scholarly journals Procoagulant activity induced by vascular injury determines contribution of elevated factor VIII to thrombosis and thrombus stability in mice

Blood ◽  
2011 ◽  
Vol 118 (14) ◽  
pp. 3960-3968 ◽  
Author(s):  
Kellie R. Machlus ◽  
Feng-Chang Lin ◽  
Alisa S. Wolberg
Keyword(s):  
Platelet Aggregation ◽  
Factor Viii ◽  
Tissue Factor ◽  
Vascular Injury ◽  
Thrombin Generation ◽  
Elevated Plasma ◽  
Vessel Occlusion ◽  
Short Treatment ◽  
Plasma Factor

Abstract Studies have correlated elevated plasma factor VIII (FVIII) with thrombosis; however, it is unclear whether elevated FVIII is a proinflammatory biomarker, causative agent, or both. We raised FVIII levels in mice and measured the time to vessel occlusion (TTO) after ferric chloride–induced injury. Compared with control (saline-infused) mice, elevated FVIII had no effect after longer (3-minute) carotid artery injury, but it shortened the TTO after shorter (2-minute) injury (P < .008). After injury, circulating thrombin-antithrombin (TAT) complexes were lower after short versus long injury (P < .04), suggesting short treatment produced less coagulation activation. TAT levels in FVIII-infused mice were higher than in controls after short, but not longer, injury. Accordingly, elevated FVIII had no effect on in vitro thrombin generation or platelet aggregation triggered by high tissue factor, but it increased thrombin generation rate and peak (2.4- and 1.5-fold, respectively), and it accelerated platelet aggregation (up to 1.6-fold) when initiated by low tissue factor. Compared with control mice, elevated FVIII stabilized thrombi (fewer emboli) after short injury, but it had no effect after longer injury. TTO and emboli correlated with TATs. These results demonstrate dependence of FVIII activity on extent of vascular injury. We propose elevated plasma FVIII is an etiologic, prothrombotic agent after moderate but not extensive vascular damage.

scholarly journals Thrombin Generation Capacity of Prothrombin Complex Concentrate in an in Vitro Dilutional Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4380-4380
Author(s):  
Henri M.H. Spronk ◽  
Rolf Rossaint ◽  
Henskens M.C. Yvonne ◽  
Rene van Oerle ◽  
Hugo Ten Cate ◽  
...  
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Coagulation Factors ◽  
Thrombin Generation Assay ◽  
Trauma Induced Coagulopathy ◽  
Healthy Donors ◽  
Ringer’S Lactate ◽  
Generation Capacity

Abstract Abstract 4380 Background: There is a growing use of prothrombin complex concentrates (PCCs) for the treatment of trauma-induced coagulopathy, which is addressed to their propensity to increase thrombin generation. Despite considerable differences in composition of commercially available PCCs, there is lack of data investigating the procoagulant capacity of different PCCs. Methods: The vitamin K-dependent coagulation factors, heparin, and antithrombin were assessed in five commercially available PCCs. The procoagulant potential of the PCCs was assessed in plasma and whole blood from 4 healthy donors by means of classical coagulation assays, thrombin generation assay and thromboelastometry. In order to reflect coagulopathy, whole blood was diluted with 20, 40, 60, and 80% Ringer's lactate solution. Results: The five different PCCs were characterised by comparable levels of factors II, VII, IX and X (all around 20–30 IU/mL), whereas the heparin (0 to 17.6 IU/mL) and antithrombin (0.06 to 1.29 IU/mL) levels were remarkably different between manufactures. In vitro dilution of blood induced a prolongation of the PT and aPTT, and attenuation of thrombin generation and ExTem induced thromboelastometry. Overall, non- or low-heparin containing PCCs restored the in vitro dilutional coagulopathy, whereas PCCs containing heparin has an anticoagulant effect. The thrombin generation assay showed to be the most sensitive method for assessment of PCC effects. Conclusions: This study shows that most available PCCs are not balanced regarding their pro-and anticoagulants. The effect of measured differences in thrombin generation among different PCCs require further investigations to elaborate the clinical meaning in the treatment of trauma induced coagulopathy. Disclosures: No relevant conflicts of interest to declare.

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