Thrombin Generation Experiments Research Articles

Assessing the individual roles of FII, FV, and FX activity in the thrombin generation process.

Thrombin generation (TG) is known as a physiological approach to assess the hemostatic function. Although it correlates well with thrombosis and bleeding, in the current setup it is not sensitive to the effects of fluctuations in single coagulation factors. We optimized the calibrated automated thrombinography (CAT) method to quantify FII, FV and FX activity within the coagulation system. The CAT assay was fine-tuned for the assessment of FII, FV and FX by diluting the samples in FII-, FV-, or FX-deficient plasma, respectively, and measuring TG. Plasma FII levels correlated linearly with the ETP up to a plasma concentration of 100% FII. FV and FX levels correlated linearly with the peak height up to a plasma level of 2.5% FV and 10% FX, respectively. Sensitized CAT protocols were designed by adding a fixed volume of a pre-diluted patient sample to FII, FV, and FX deficient plasma in TG experiments. This approach makes the TG measurement dependent on the activity of the respective coagulation factor. The ETP or peak height were quantified as readouts for the coagulation factor activity. The intra- and inter-assay variation coefficients varied from 5.0 to 8.6%, and from 3.5 to 5.9%, respectively. Reference values were determined in 120 healthy subjects and the assays were clinically validated in 60 patients undergoing coronary artery bypass grafting (CABG). The sensitized CAT assays revealed that the contribution of FII, FV, and FX to the TG process was reduced after CABG surgery, leading to reduced prothrombin conversion and subsequently, lower TG.

Patients With Multiple Myeloma Have a Disbalanced Whole Blood Thrombin Generation Profile.

BackgroundMultiple myeloma (MM) is associated with a high prevalence of bleeding and an increased risk of thrombo-embolism. MM patients have reduced platelet- and red blood cell (RBC) numbers in blood, which may indicate that the paradoxical hemostasis profile is a consequence of a disturbed platelet and RBC homeostasis.ObjectivesTo get better insight in the disbalanced hemostasis of MM patients.MethodsWe conducted a case-control study on the whole blood (WB) coagulation profiles of 21 MM patients and 21 controls. We measured thrombin generation (TG) in WB and platelet poor plasma (PPP) of MM patients and controls.ResultsIn WB-TG, we observed that the median time to the thrombin Peak was 52% longer in MM patients than in controls, while the median endogenous thrombin potential until the Peak (ETPp) was 39% higher in MM-patients than in controls. In line with these findings, the levels of platelets, RBCs, white blood cells and agonist induced platelet activation were decreased in MM patients compared to controls. The plasma TG experiments showed no differences between MM-patients and controls.ConclusionPatients with MM have a disturbed blood cell metabolism and a disbalanced WB-TG profile. This disbalance may explain the paradoxically high prevalence of bleeding symptoms in MM patients vs. an increased thrombosis risk. There was no disturbance observed in plasma TG, indicating that blood cells are the major determinants for the disbalanced hemostasis in MM patients.

VhH anti‐thrombomodulin clone 1 inhibits TAFI activation and enhances fibrinolysis in human whole blood under flow

BackgroundThrombomodulin on endothelial cells can form a complex with thrombin. This complex has both anticoagulant properties, by activating protein C, and clot‐protective properties, by activating thrombin‐activatable fibrinolysis inhibitor (TAFI). Activated TAFI (TAFIa) inhibits plasmin‐mediated fibrinolysis. ObjectivesTAFIa inhibition is considered a potential antithrombotic strategy. So far, this goal has been pursued by developing compounds that directly inhibit TAFIa. In contrast, we here describe variable domain of heavy‐chain‐only antibody (VhH) clone 1 that inhibits TAFI activation by targeting human thrombomodulin. MethodsTwo llamas (Lama Glama) were immunized, and phage display was used to select VhH anti‐thrombomodulin (TM) clone 1. Affinity was determined with surface plasmon resonance and binding to native TM was confirmed with flow cytometry. Clone 1 was functionally assessed by competition, clot lysis, and thrombin generation assays. Last, the effect of clone 1 on tPA‐mediated fibrinolysis in human whole blood was investigated in a microfluidic fibrinolysis model. ResultsVhH anti‐TM clone 1 bound recombinant TM with a binding affinity of 1.7 ± 0.4 nM and showed binding to native TM. Clone 1 competed with thrombin for binding to TM and attenuated TAFI activation in clot lysis assays and protein C activation in thrombin generation experiments. In a microfluidic fibrinolysis model, inhibition of TM with clone 1 fully prevented TAFI activation. DiscussionWe have developed VhH anti‐TM clone 1, which inhibits TAFI activation and enhances tPA‐mediated fibrinolysis under flow. Different from agents that directly target TAFIa, our strategy should preserve direct TAFI activation via thrombin.

Plasma FXIII Mediated Crosslinking Is Necessary for Thrombin Generation and Clot Retraction

IntroductionFXIII is a crucial transglutaminase enzyme which crosslinks fibrin polymers to increase the clot stiffness by 5-fold, thereby ensuring clot structure stabilization for efficient hemostasis. FXIII is activated by thrombin to form FXIIIa which in turn catalyzes the covalent bonds between fibrin alpha-gamma chains. FXIII has also been indicated to upregulate thrombin generation along with other coagulation factors. This interplay of FXIII and thrombin can have profound consequences on the resulting clot structure. Further, the role of FXIII in clot retraction is disputed. In this work, we investigated the effect of FXIII on thrombin generation and its effects on the structure of retracted clots by using FXIII inhibitors.MethodsPhlebotomized platelet rich plasma (PRP) and platelet poor plasma (PPP) were isolated from healthy donors. PRP and PPP samples were incubated with varying doses of FXIII inhibitors (T101, Zedira GmBH) at RT for 15mins prior to performing thrombin generation experiments (Calibrated Automated Thrombogram). Similarly, clot retraction experiments were carried out with 20mM Ca2+ in 1 mL PRP in glass test tubes and incubated at 37°C. After 2 h, the clots were weighed, preserved using liquid nitrogen and methyl butyrate, and embedded in OCT. These clots were further sectioned into 5 um sections and stained for fibrinogen and PLT CD61. The sections were imaged using confocal microscopy and Transmission Electron Microscopy to study the structure and PLT-Fibrin interaction in retracted clots. The exuded serum after retraction was used to measure PLT concentration and D-Dimer levels to understand the relative incorporation of PLTs and fibrinolysis respectively. The effect of FXIII inhibitors on platelet aggregation was quantified using multiplate aggregometry. FXIII deficient plasma was used as a control.ResultsInhibition of FXIII results in a drop in the total thrombin generation potential in PRP and PPP (Fig 1A), with a reduction in clot size and weight in the retracted clots (Fig 1B). FXIII inhibition does not affect platelet aggregation with ADP, platelet incorporation to clots and fibrinolysis (D-Dimers in exuded serum) under normal conditions. The structural studies reveal fibrin collapse, reduced clot size and weight, and increased plasma exclusion in FXIII inhibited clots.ConclusionOur data suggest that FXIII mediated crosslinking is important for thrombin generation and platelet-fibrin interaction for efficient clot retraction. The uncrosslinked fibrin collapse is due to higher local platelet contractile forces compared to the opposing fibrin tensile forces leading to abnormal retraction. The current work reveals the importance of force balances in clot structure and its resulting phenotypes associated with various pathologies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Thrombin-Fibrin(ogen) Interactions, Host Defense and Risk of Thrombosis.

Fibrinogen is a well-known risk factor for arterial and venous thrombosis. Its function is not restricted to clot formation, however, as it partakes in a complex interplay between thrombin, soluble plasma fibrinogen, and deposited fibrin matrices. Fibrinogen, like thrombin, participates predominantly in hemostasis to maintain vascular integrity, but executes some important pleiotropic effects: firstly, as observed in thrombin generation experiments, fibrin removes thrombin from free solution by adsorption. The adsorbed thrombin is protected from antithrombins, notably α2-macroglobulin, and remains physiologically active as it can activate factors V, VIII, and platelets. Secondly, immobilized fibrinogen or fibrin matrices activate monocytes/macrophages and neutrophils via Mac-1 interactions. Immobilized fibrin(ogen) thereby elicits a pro-inflammatory response with a reciprocal stimulating effect of the immune system on coagulation. In contrast, soluble fibrinogen prohibits recruitment of these immune cells. Thus, while fibrin matrices elicit a procoagulant response, both directly by protecting thrombin and indirectly through the immune system, high soluble fibrinogen levels might protect patients due to its immune diminutive function. The in vivo influence of the ‘protective’ plasma fibrinogen versus the ‘pro-thrombotic’ fibrin matrices on thrombosis should be explored in future research.

Efficacy of pro‐ and anticoagulant strategies in plasma of patients undergoing hepatobiliary surgery

BackgroundIn vitro efficacy of pro‐ and antihemostatic drugs is profoundly different in patients with compensated cirrhosis and in those who have cirrhosis and are critically ill. ObjectivesHere we assessed the efficacy of pro‐ and anticoagulant drugs in plasma of patients undergoing hepato‐pancreato‐biliary (HPB) surgery, which is associated with unique hemostatic changes. MethodsWe performed in vitro analyses on blood samples of 60 patients undergoing HPB surgery and liver transplantation: 20 orthotopic liver transplantations, 20 partial hepatectomies, and 20 pylorus‐preserving pancreaticoduodenectomies. We performed thrombin generation experiments before and after in vitro addition of fresh frozen plasma (FFP), prothrombin complex concentrate (PCC), recombinant factor VIIa (rFVIIa), low molecular weight heparin (LMWH), unfractionated heparin, dabigatran, and rivaroxaban. ResultsWe showed that patients undergoing HPB surgery are in a hypercoagulable state by thrombin generation testing. FFP and rFVIIa had minimal effects on thrombin generation, whereas PCC had a more pronounced procoagulant effect in patients compared with controls. Dabigatran showed a more pronounced anticoagulant effect in patients compared with controls, whereas rivaroxaban and LMWH had a decreased anticoagulant effect in patients. ConclusionWe demonstrate profoundly altered in vitro efficacy of commonly used anticoagulants, in patients undergoing HPB surgery compared with healthy controls, which may have implications for anticoagulant dosing in the early postoperative period. In the correction of perioperative bleeding complications, PCCs appear much more potent than FFP or rFVIIa, and PCCs may require conservative dosing and caution in use in patients undergoing HPB surgery.

Transmission of Procoagulant Properties to Daughter Generations of Endothelial Cells upon Exposure to Cancer Cell-Derived Microparticles. a New Implication of Cancer Derived Microparticles in the Pathogenesis of Cancer Associated Thrombosis

Introduction & Objectives: Endothelium activation by cancer cell derived MPs (CaCe-dMP) is essential in pathogenesis of cancer associated thrombosis (CAT). Recent studies demonstrated that exposed endothelial cells to CaCe-dMP acquire a new procoagulant phenotype characterized by endocytosis of tissue factor (TF) and enhancement of thrombin generation.The aim of the present study was to investigate whereas the procoagulant properties acquired upon exposure of endothelial cells to CaCe-dMP are transferred to daughter generations of the endothelial cells.Materials & Methods: Pancreas adenocarcinoma cells (BXPC3) and human breast cancer cells (MCF7) were cultured in 75 cm2 culture flasks at 37˚C in a 100% humidified atmosphere with 5% CO2 and were used for experiments at a confluence of 70%. Microparticles released in conditioned medium from BXPC3 and MCF7 cells were isolated by differential centrifugation. The pellets were washed twice and re-suspended in 1 ml endothelium cell basal medium (EBM-2). Human umbilical vein endothelial cells (HUVEC) were cultured for 72 h in the presence or absence of CaCe-dMP (mother generation : C0). Subsequently, they were washed and re-cultivated over three cycles to get daughter cell generations (Daughter Generations : C1, C2, C3). Tissue factor activity (TFa) was assessed with a chromogenic assay in the absence or presence of cells in normal PPP. Procoagulant phospholipid-dependent clotting time (Proag-PPL): was measured with STA®Procoag-PPL assay. Thrombin generation was studied as described elsewhere using Calibrated Automated Thrombogram®. All assays were from Diagnostica Stago (Asnières, France).Results: The HUVEC and their microparticles manifested faint amounts of TFa (0.18±0.03 pM and 1.2±1.0 pM respectively). At steady state, HUVEC did not induce any detectable thrombin generation. Abundant TFa was showed by BXPC3-dMP (92±50 pM). The levels of TFa were significantly lower at MCF7-dMP (12.1±2.2 pM; p<0.05). Following exposure to BXPC3-dMP, the HUVEC abundantly displayed TFa as compared to non-exposed cells (25±3 pM versus 0.18±0.03 respectively; p<0.001) and showed significantly shorter Procoag-PPL® as compared to non-exposed cells (56±2 sec versus 102±10 sec. respectively ; p<0.001). HUVEC exposed to MCF7-dMP showed significantly increased TFa as compared to non-exposed cells (0.75±0.2 pM versus 0.18±0.03 respectively; p<0.001) and reduced the Procoag-PPL® clotting time as compared to non-exposed cells (65±3 sec versus 102±10 sec respectively; p<0.001). HUVEC exposed to CaCe-dMP significantly increased thrombin generation as compared to non-exposed HUVEC. Data of thrombin generation experiments are summarized in Table 1.Daughter HUVEC from mother cells exposed to CaCe-dMP exhibited significantly higher TFa as compared to daughter generations from HUVEC non-exposed to CaCe-dMPs. The high TFa was conserved in three generations of cells derived from those exposed to CaCe-dMPs (Figure 1). Daughter generations of HUVEC exposed to CaCe-dMP induced significantly higher thrombin generation as compared to that induced by daughter generations of non exposed HUVEC (Table 1). The TFa of daughter cells stemming from HUVEC exposed to BXPC3 was significantly higher as compared to that of daughter cells stemming from HUVEC exposed to MCF7 (Figure 1). The Procoag-PPL® did not significantly change in daughter HUVEC exposed to microparticles derived either from BXPC3 or MCF7.Conclusion: Upon exposure to CaCe-dMP, endothelial cells acquire a procoagulant phenotype marked by abundant TFa and enhanced thrombin generation potential. The procoagulant properties are transferred to the daughter generations of cells. These results support the concept that CaCe-dMPs lead to a procoagulant shift of endothelial cells which, upon exposure, present high TF activity and enhance thrombin generation. The procoagulant shift of endothelial cells can be transferred to next generations of endothelial cells and could be an additional mechanism - together with cancer-induced blood hypercoagulability - in the pathogenesis of cancer associated thrombosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Haemostasis Impairment in Patients with Myelodysplastic Syndromes with Normal Platelet Counts

Background: Bleeding complications can have serious outcomes in patients with myelodysplastic syndromes (MDS) and are usually related to thrombocytopaenia. In a recent work we demonstrated that platelets from MDS have impaired capacity to respond to agonist stimulation and exposed more phosphatidylserine (PS) than those from healthy controls whichever their platelet count was (Martin et al, Thromb Haemost., 2013;109:909-19). The exposure of PS in the outer layer of cell membranes supports coagulation through enhanced formation of the tenase (factors IXa, VIIIa and X) and prothrombinase (factors Xa, Va and prothrombin) complexes and thrombin generation. So, the possibility exists that these processes are increased in these patients.Objectives: The aim of this work was to study whether thrombus formation and thrombin generation is impaired in MDS patients with normal platelet count employing global coagulation tests thromboelastrometry (ROTEM) and Calibrated Automated Thrombogram (CAT).Methods: Thirty-one MDS patients with normal platelet count and twenty healthy controls were included. Citrated blood was centrifuged at 152 g 10 min at 23°C for obtaining platelet rich plasma (PRP) and at 1,500 g for 15 min at 23°C for platelet-poor plasma (PPP). For ROTEM and CAT experiments, PRP was adjusted to a platelet count of 25 x 109/L. Aliquots for ROTEM assay were tested within the two hours after obtaining blood samples. For CAT experiments, adjusted PRP and PPP aliquots were stored at -70ºC until analysis.Kinetics of clot formation, non-activated ROTEM was performed on adjusted PRP. Clotting time (CT, time from start of measurement until initiation of clotting [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2-mm amplitude [in degrees]), and maximum clot firmness, which reflects the maximum tensile strength of the thrombus (MCF, [in mm]), were recorded.Thrombin generation was measured in adjusted PRP without any trigger and in PPP with 1 pM tissue factor and 4 µM phospholipids (PPP-Reagent LOW, Thrombinoscope BV, Maastricht, The Netherlands) as trigger. Endogenous thrombin potential (ETP, the total amount of thrombin generated over time); the lag time (the time to the beginning of the explosive burst of thrombin generation); the peak height of the curve (the maximum thrombin concentration produced); and the time to the peak were evaluated.Platelet activation was determined by PAC1 (BD, Madrid, Spain) binding after stimulation with 100 μM thrombin receptor-activating peptide 6 (TRAP, Bachem, Switzerland) and surface PS through Annexin-V binding and flow cytometry analyses.Results: Platelets from MDS patients had a reduced response to TRAP stimulation (control= 12016+6384 arbitrary units; MDS= 5829+3704 arbitrary units) and exposed more PS than controls (control= 362.1+80.5 MF; MDS= 378.5+173.5 MF).ROTEM experiments showed kinetic parameters corresponding to a hipocoagulable profile (CT: control= 550+95 sec, MDS= 922+216 sec, p<0.01; alpha angle control= 62.8+4.3, MDS= 47.5+6.0, p<0.05; A5: control= 29.8+3.2, MDS= 24.2+5.8, p<0.05 whereas MCF was similar to controls (control= 46.7+3.1mm, MDS= 47.5+4.3 mm).In order to evaluate whether the impaired clot formation was due to a reduction in plasma-associated thrombin generation, CAT experiments were performed in PPP samples. No differences were found between MDS patients and control group (ETP: control=1223.4+257.8 nMxmin, MDS= 1224.4+344 .1 nMxmin; peak: control= 279.5+54.7nM, MDS= 265.9+64.1 nM). On the other hand, when thrombin generation experiments were performed in adjusted PRP, ETP and peak values were lower than in control group (ETP: control=1574.8+430.5 nMxmin, MDS= 1167.4+354 nMxmin,p<0.05; peak: control= 225.6+82.9 nM, MDS= 266.9+92.2 nM, p<0.05).Conclusions: Platelet dysfunction might be resposible of bleeding complications observed in patients with MDS with normal platelet counts. Increment in PS exposure on platelet surface did not seem to compensate impairment in platelet function. DisclosuresNo relevant conflicts of interest to declare.

Predicting dosing advantages of factor VIIa variants with altered tissue factor‐dependent and lipid‐dependent activities

Recombinant factor VIIa (rFVIIa) is an FX-cleaving coagulation enzyme licensed for the treatment of bleeding episodes in hemophiliacs with inhibitory antibodies. Even though the optimal dosing and comparative dose efficacy of rFVIIa remain poorly understood, genetic or chemical modifications of rFVIIa have been proposed, with the goal of achieving faster and longer hemostatic action. No ongoing trial is currently comparing rFVIIa variants with each other. We used mathematical modeling to compare the pharmacokinetics, dose-response (pharmacodynamics) and dose-effect duration (pharmacokinetics/pharmacodynamics) of rFVIIa variants to predict their optimal doses. The pharmacodynamic (PD) model of FXa generation by FVIIa in complexes with tissue factor (TF) and procoagulant lipids (PLs) was validated against published ex vivo and in vitro thrombin generation (TG) experiments. To compare variants' safety profiles, the highest non-thrombogenic doses were estimated from the clinical evidence reported for the licensed rFVIIa product. The PD model correctly described the biphasic TF-dependent and PL-dependent dose response observed in TG experiments in vitro. The pharmacokinetic/PD simulations agreed with published ex vivo TG data for rFVIIa and the BAY 86-6150 variant, and explained the similar efficacies of a single dose of 270 μg kg(-1) (as reported in the literature) and repeated doses of 90 μg kg(-1) of unmodified rFVIIa. The duration of the simulated hemostatic effect after a single optimal dose was prolonged for rFVIIa variants with increased TF affinity or extended half-lives, but not for those with modulated PL activity. Some modifications of the rFVIIa molecule may not translate into a prolonged hemostatic effect.

Unifying the mechanism of recombinant FVIIa action: dose dependence is regulated differently by tissue factor and phospholipids

Recombinant factor VIIa (rFVIIa) is used for treatment of hemophilia patients with inhibitors, as well for off-label treatment of severe bleeding in trauma and surgery. Effective bleeding control requires supraphysiological doses of rFVIIa, posing both high expense and uncertain thrombotic risk. Two major competing theories offer different explanations for the supraphysiological rFVIIa dosing requirement: (1) the need to overcome competition between FVIIa and FVII zymogen for tissue factor (TF) binding, and (2) a high-dose-requiring phospholipid-related pathway of FVIIa action. In the present study, we found experimental conditions in which both mechanisms contribute simultaneously and independently to rFVIIa-driven thrombin generation in FVII-deficient human plasma. From mathematical simulations of our model of FX activation, which were confirmed by thrombin-generation experiments, we conclude that the action of rFVIIa at pharmacologic doses is dominated by the TF-dependent pathway with a minor contribution from a phospholipid-dependent mechanism. We established a dose-response curve for rFVIIa that is useful to explain dosing strategies. In the present study, we present a pathway to reconcile the 2 major mechanisms of rFVIIa action, a necessary step to understanding future dose optimization and evaluation of new rFVIIa analogs currently under development.

Peptides Inhibiting Tissue Factor Pathway Inhibitor Improve Hemostasis in Mice

Abstract 24Tissue factor pathway inhibitor (TFPI) is an activated (a) factor X (FXa)-dependent inhibitor of the extrinsic factor X (FX) activation complex and efficiently regulates the extrinsic pathway of coagulation. We are developing peptide inhibitors of TFPI with a view to improving hemostasis in hemophilia. The goal is to inhibit the interaction of TFPI with the factor Xa (FXa)-tissue factor (TF)-factor VIIa (FVIIa) complex, and thereby enhance thrombin formation to the extent that a stable clot is formed. Successful development of these peptides could allow treatment of hemophilia via a non-intravenous route of administration.By screening mRNA display libraries, we identified a de novo peptide which binds to and efficiently inhibit TFPI. The peptide was optimized by iterative amino acid substitution resulting in affinity-improved peptide with a well-characterized structure-activity relation. The affinity of the peptide was analyzed by Biacore and ELISA experiments. One of our optimized peptides bound to immobilized TFPI with an affinity below 1 nM. Kunitz domain 1 of TFPI was identified as the interaction site by NMR studies.We characterized the inhibitory activity of this TFPI binding peptide using in vitro model assay systems including inhibition of FXa and of the extrinsic tenase (TF-FVIIa-PL-Ca2+ complex). Inhibition of cell-associated TFPI was verified in a TF- and TFPI-dependent cell-based assay using TNFalpha-stimulated human umbilical vein endothelial cells (HUVECs) as a source of TF and TFPI. Inhibition of plasma TFPI was probed by thrombin generation experiments using FVIII-deficient patient plasma and ROTEM experiments using FVIII-inhibited whole blood. Furthermore, a cross-reactivity of the peptide to murine TFPI was demonstrated in thrombin generation experiments and in murine FXa inhibition model assays.Conjugation of a 40-kDa polyethyleneglycol (PEG) to the peptide base structure resulted in a peptide with significantly reduced renal clearance. The in vivo terminal half-life of this 40kD-PEG modified peptide was 21.5h and 19.8h after 1 mg/kg i.v and s.c. administration, respectively, with 73% bioavailability after s.c. administration.In a tail-tip bleeding model, administration of an anti-murine-TFPI antibody and 40kD-PEG-peptide in combination with sub-therapeutical doses of recombinant coagulation factors (10 IU/kg of rFVIII in FVIII ko mice and rFIX in FIX ko mice) led to a marked reduction of blood loss compared with buffer-treated or rFVIII/rFIX-treated mice.In addition, we established a murine nail-cut model using C57Bl6 wild-type mice for testing in vivo efficacy in a normal FVIII background as well. The TFPI sensitivity of the nail-cut model was verified by an anti-murine TFPI antibody. 40kD-PEG-peptide significantly reduced blood loss after i.v. and s.c. administration in all treatment groups compared with vehicle-treated animals. Furthermore, the i.v. administration of the peptide was well tolerated in all animals across all treatment groups without any signs of acute toxicity.In conclusion, we identified a low-molecular-weight peptide which efficiently inhibit all forms of naturally occurring TFPI proteins, including plasma TFPI and TFPI in vivo. Our results demonstrate that targeting TFPI efficiently improves hemostasis in hemophilia and provide an in vivo proof of concept for a non i.v. route of administration. Thus, the TFPI inhibitory peptides could be useful to prevent bleeding in hemophilia patients. Moreover, this new approach would probably allow treatment of inhibitor patients. Disclosures:Dockal:Baxter Innovations GmbH: Employment. Hartmann:Baxter Innovations GmbH: Employment. Polakowski:3B Pharmaceuticals GmbH: Employment. Frank:3B Pharmaceuticals GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Schiviz:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment. Reinecke:3B Pharmaceuticals GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Coagulation Cofactor Presentation and Complex Assembly on Platelets by Protease Activated Receptors (PARs): PAR4 Stimulation Leads to More FV and FVIII and More Thrombin Generation Than PAR1

Abstract 1135Protease activated receptor (PAR) stimulation induces procoagulant phenotypes on platelets leading to thrombin generation; however, the efficacy of PAR1 versus PAR4 in the presentation of coagulation cofactors and assembly of coagulation complexes on platelet membranes have not been compared. We used immunocytochemistry and flow cytometry techniques to measure PAR mediated binding of coagulation cofactors to the platelet surface. The maximal response for PAR4-activating peptide (PAR4-AP) mediated Factor (F) Va release and association with the platelet membrane was 1.61 fold greater than PAR1-AP (Fig. 1A, *p=0.0069). To monitor platelet provision of coagulation cofactors and assembly of individual coagulation complexes (prothrombinase, extrinsic Xase, intrinsic Xase), we developed a novel thrombin generation assay. Conditions were established in which the targeted cofactors were withheld and thrombin generation was triggered by platelet stimulation and provision of the missing cofactor. Thrombin generation targeting provision of FVa and assembly of the prothrombinase complex (FII+FXa+Antithrombin [AT]) yielded 1.5 fold higher peak thrombin levels for PAR4 versus PAR1 stimulation (Fig 1B, *p=0.0129). A similar difference between PAR1 and PAR4 was observed for FVIII binding, in which PAR4-stimulated platelets displayed 1.7 fold greater levels. Consistent with FVIII expression, thrombin generation targeting the intrinsic Xase complex (FII+FX+FIXa+AT) was 2.0 fold higher for PAR4 versus PAR1. Peak thrombin levels in thrombin generation experiments targeting intrinsic Xase complex assembly were significantly higher than experiments targeting the isolated prothrombinase complex, demonstrating the ability of the intrinsic Xase complex to amplify thrombin generation. We also observed a rapid mobilization of tissue factor (TF) to the cell surface following PAR stimulation, and activity in thrombin generation assays targeting the extrinsic Xase complex (FII+FX+FVIIa+TFPI+AT), but no significant difference between PAR1 and PAR4. Although thrombin generation was observed under conditions targeting extrinsic Xase complex assembly, peak thrombin levels were negligible compared to thrombin generation conducted under conditions targeting the prothrombinase and intrinsic Xase complexes. In conclusion, we have shown that platelets can assemble prothrombinase, intrinsic Xase, and extrinsic Xase complexes, but the prothrombinase and intrinsic Xase complexes are responsible for the majority of thrombin generated on the platelet. Moreover, PAR4 stimulation induces the provision of more FV and FVIII, consistently yielding higher peak thrombin levels than PAR1 stimulation. These data have important clinical impact in light of recent efforts to develop thrombin receptor antagonists as agents to treat thrombotic disorders, and suggest that a PAR4 antagonist would be a more effective strategy to control pathologic thrombosis. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Evaluation of Anticoagulant Drugs with TF-Induced Thrombin Generation: An In Vitrocomparative Study In Platelet-Rich Plasma (PRP) Versus Platelet-Poor Plasma (PPP)

AbstractAbstract 3335 Introduction:We previously reported that thrombin generation (TG) conducted in human PPP supplemented with synthetic phospholipids (PL) and triggered by 5 pM tissue factor (TF) was a very rapid, suitable and reliable pharmacological tool for screening thrombin and/or FXa inhibitors whatever their inhibition mode. However, a drawback of this work was that platelets, which are known to play a critical role in blood coagulation cascade by providing the catalytic surfaces for coagulation factors activation, were lacking in these experiments. Aims:The aim of this study was thus to compare the effects of various anticoagulant agents on TF-induced TG in platelet rich plasma (PRP)and platelet poor plasma (PPP) in order to define the most relevant and reliable assay to evaluate the potency and the behavior of drugs targeting thrombin and/or FXa. Methods:TG experiments were conducted with Calibrated Automated Thrombogram® (Thrombinoscope) in fresh PRP (150 000 platelets/μL) of 3 individual healthy donors with 1 pM TF + 16.7 mM CaCl2 and in normal pool frozen-thawed PPP of 32 healthy donors with 5 or 1 pM TF + 4 μM PL + 16.7 mM CaCl2. Five anticoagulants with various mode of action (argatroban, lepirudin, enoxaparin, fondaparinux and rivaroxaban) were spiked in the plasmas at increasing concentrations ranging from 10 nM to 5 μM. Results:In the absence of the drugs, the peak of active thrombin concentration was delayed (lag time increase), prolonged (Tmax increase) and reduced (Cmax decrease) when using PRP instead of PPP with 1 pM TF. These effects were still higher when comparing to PPP with 5 pM TF. However, the total amount of active thrombin (ETP) was similar in the three experimental designs. For all the parameters, the experimental CVs were <5% for PPP with 5 pM TF and < 15% for both PPP and PRP with 1 pM TF. In presence of the anticoagulant drugs, the peak of active thrombin concentration was concentration-dependently delayed, prolonged and reduced within all the assays. In PRP with 1 pM TF, all the drugs displayed close behaviors, excepting lepirudin. In PPP, and especially with 5 pM TF, more different profiles were found according to the mechanism of action of drugs. Regarding to the potency of the drugs, the inhibition parameters (i.e. 2x lag time, 2x Tmax, Cmax EC50 and Vmax EC50) were in the same range for argatroban and lepirudin whatever the chosen assay. For enoxaparin, rivaroxaban and fondaparinux, the drug effects varied among the assays but also following to the studied parameters. In all the assays, lepirudin was the most active drug to increase the lag time and the Tmax. The Cmax was mostly decreased by fondaparinux in PRP with 1 pM TF, by rivaroxaban in PPP with 5 pM TF and by both drugs in PPP with 1 pM TF. The ETP was mostly diminished by fondaparinux in PRP with 1 pM TF while the stronger effects were found with fondaparinux and enoxaparin using the two others inducers. Conclusions:The present study did not demonstrate a superiority of TF-induced thrombin generation assay conducted in PRP compared to PPP supplemented with synthetic PL. Although the potency of the drugs varied according to the studied parameter and the experimental design of the assay, experiments conducted in PL-supplemented PPP with 5 pM TF seemed to be the best suited to study the effects of anticoagulant drugs targeting thrombin and/or FXa due to their high reproducibility and their relevancy on drug mechanism of action. Moreover, the use of frozen-thawed normal pool plasma within these assays was more convenient regarding to the sample collection, storage and handling. It also excluded the inter-donor variability to be more relevant on the normal population hemostatic system. Disclosures:No relevant conflicts of interest to declare.

Thrombin Generation Is a Sensitive Measure of Low Molecular Weight Heparin Anticoagulant Activity.

AbstractAbstract 1099Current guidelines recommend monitoring of low molecular weight heparins (LMWHs) using the anti-Xa assay in high risk patients like those with renal impairment, pregnancy or overweight and in children. However, it is well accepted that this test does not accurately predict the anticoagulant effect of these drugs1. This is understandable since the various LMWHs available affect the coagulation cascade in different ways relative to their different molecular structure. Notoriously, the larger LMWHs like Tinzaparin have a higher anti-IIa to anti-Xa ratio that is not detected by the anti-Xa assay.We hypothesized that thrombin generation (TG) is a better, more sensitive way how to monitor LMWH anticoagulant activity since it is a measure of the interplay of all coagulation proteins.Blood samples from patients with acute thrombosis, pregnancy and other conditions and having LMWH monitoring were analysed using a chromogenic anti-Xa assay and a TG assay as per Hemker et al2 together with a chromogenic anti-IIa and FVIII clotting assay (FVIII:C). A tissue factor trigger of 10pM Innovin was used for the TG experiments. Patient samples were divided into 2 groups: those on Tinzaparin (n=45) or Enoxaparin (n=39).There was no difference between the FVIII:C levels of the Tinzaparin and Enoxaparin groups (mean 250 vs 327IU/ml P>0.05). A higher lower mean anti-Xa and a higher mean anti-IIa level was achieved with Tinzaparin (0.48 vs 0.63 U/ml P= >0.05 for anti-Xa and 0.3 vs 0.15U/ml P=0.005 for anti-IIa respectively). The endogenous thrombin potential (ETP) was significantly lower with Tinzaparin than Enoxaparin despite a higher anti-Xa (705 vs 1216nM.min P=0.006). Linear regression analysing TG with anti-Xa of the two LMWHs shows that 1.0U/ml antiXa activity for Enoxaparin is as potent as 0.7U/ml Tinzaparin in suppressing TG to similar levels. Significant inter-individual variation in TG suppression was noted with both LMWHs.This study demonstrates that anti-Xa results achieved for different LMWHs do not have the same anticoagulant significance. Using TG, we achieved similar therapeutic anti-Xa levels as achieved from the individual LMWH clinical trials3. It is clear that TG is a better test to predict LMWH anticoagulant activity. This needs to be proven in clinical studies.1. Baglin T et al British Journal of Haematol. 2006; 133(1): 19–34.2. Hemker HC et al Pathophysiol Haemost Thromb, 33, 4-15.Boneu B and de Moorloose P. Semin Thromb Hemost 2001; 27(5): 519–522. Disclosures:No relevant conflicts of interest to declare.

Real-time measurements of coagulation on bacterial cellulose and conventional vascular graft materials

The search for a functional, small diameter (<5mm) vascular graft has been ongoing for over 30 years, but yet there is no consistently reliable synthetic graft. The primary mechanisms of graft failure are intimal hyperplasia, poor blood flow and surface thrombogenicity. Bacterial cellulose (BC) became therefore a proposed new biosynthetic vascular graft material. Since conventional methods are not suited for coagulation measurements on BC, we have adapted the automated calibrated thrombin generation method for measurements of biomaterial-induced coagulation of BC as compared with clinically used graft materials i.e., expanded poly(tetrafluoroethylene) (ePTFE) and poly(ethyleneterephtalat) (PET). We have also visualized the coagulation propagation at the material surfaces. Thrombin generation experiments revealed dramatic differences between the materials tested. Both ePTFE and BC were found to generate longer lag times and ttpeak values than PET. Most importantly, BC was found to generate the lowest "peak", indicating a slower coagulation process at the surface. These results are also supported by the measurements of factor XIIa generation and analysis of surface coagulation times, which were detected in the following increasing order (mean + or - SD): PET (27 + or - 8 min)<BC (46 + or - 9 min)<ePTFE (61 + or - 21 min). Real-time measurement of coagulation seems to have the potential for becoming a powerful tool for evaluation of biomaterials for blood-contacting devices.

Circulating Erythrocyte-Derived Microparticles Are Associated with Coagulation Activation in Sickle Cell Disease

Backgound: Sickle cell disease (SCD) is characterized by a hypercoagulable state involving multiple factors, including chronic hemolysis and circulating cell-derived microparticles (MPs). There is still no consensus on the cellular origin of such MPs and the exact mechanism by which they may support coagulation activation in SCD.Aim: In the present study, we aimed to analyze the origin of circulating MPs and their pro-coagulant phenotype in 25 consecutive SCD patients during painful crises and steady state. conditionsMethods: MPs were identified by flowcytometry on basis of their size and density and on their ability to bind annexin V. Measurements were corrected for auto-fluorescence. Labeling with cell-specific monoclonal antibodies was corrected with isotype-matched control antibodies. For thrombin generation experiments patient derived MPs were reconstituted in defibrinated (reptilase-treated) normal pool (MP-free) plasma. For the inhibition experiments, the defibrinated plasma and the MPs were separately incubated with antibodies against coagulation factors VII or XI, or tissue factor pathway inhibitor (TFPI).Results: The majority of MPs originated from platelets (GPIIIa, CD61+: 86.1%) and erythrocytes (Glycophorin A, CD235+: 9.7%), and their numbers did not differ significantly between crisis and steady state. A distinct subset of transferrin receptor (CD71+)-exposing MPs was present, but neither MPs originating from monocytes (CD14+) nor endothelial cells (CD144+, CD146+, CD62E+) were detectable, and also no MPs exposing TF could be identified. The number of erythrocyte-derived MPs strongly correlated with plasma levels of hemolytic markers, i.e. hemoglobin (r=−0.58, P<0.001) and lactate dehydrogenase (r=0.59;P<0.001), von Willebrand factor as a marker of endothelial activation (r=0.44;P<0.001), and D-dimer and prothrombin fragment F1+2 (r=0.52;P<0.001 and r=0.59;P<0.001, respectively) as markers of fibrinolysis and coagulation activation. In the thrombin generation test, the total amount of thrombin generated (represented by the area under the curve (AUC) of the thrombin generation curve) correlated with the total number of circulating MPs (R=0.63, P<0.001). Thrombin generation was unaffected by pre-incubation with anti-human factor VII but increased slightly in the presence of anti-TFPI (16%; P=0.01) while the presence of anti-factor XI suppressed thrombin generation by about 50% (P<0.001). The extent of this inhibition (as a percentage of thrombin generation without antibody) was significantly correlated with the circulating number of erythrocyte-derived MPs (r=0.50, P=0.023), but not with platelet-derived MPs or reticulocyte-derived MPs. Also the absolute difference in thrombin generation between the experiments with and without factor XI antibody correlated with the absolute number of glycophorin A+ MPs (r=0.55, P=0.002).Conclusion: We conclude that the procoagulant state in SCD is, at least in part, due to the procoagulant effects of circulating erythrocyte-derived MPs. Their relation to the ability of anti-factor XI to block thrombin generation in SCD patients suggests an important role of factor XI-dependent thrombin generation in these patients.

Enhancement of the enzymatic activity of activated coagulation factor IX by anti-factor IX antibodies

Enhancement of the enzymatic activity of activated coagulation factor IX by anti-factor IX antibodies

Does activated protein C-resistant factor V contribute to thrombin generation in hemophilic plasma?

In this study we assessed the role of factor V (FV) inactivation in hemophilic plasma with particular reference to the activated protein C (APC)-resistant variants FV-R506Q (FV Leiden) and FV-R306T (FV Cambridge). Purified recombinant full-length FV carrying these single substitutions and FV-R306T/R506Q were used in thrombin generation experiments. Plasma was first immunodepleted of FV, and subsequently of factors VIII, IX, or combinations thereof. Thrombin generation was initiated by low concentrations of recombinant tissue factor. Recombinant soluble thrombomodulin (TM) was used to trigger the APC system. Surprisingly, TM concentrations that reduced thrombin generation in normal plasma by no more than 50% virtually abolished thrombin formation in plasma deficient in the factor VIII/IX complex. This was already apparent at TM levels as low as 0.1 nmol L(-1). By varying the concentrations of purified (activated) protein C to plasma that was additionally depleted of protein C, we confirmed that impaired thrombin generation indeed was the result of the action of APC. In contrast, this did not occur when FV-depleted plasma had been reconstituted with FV-R306T/R506Q. Addition of FV-R306T or FV-R506Q partially reduced prothrombin activation, demonstrating the involvement of both APC cleavage sites. FV inactivation also occurred on the surface of human microvascular endothelial cells. Apparently, these cells express sufficient TM to down-regulate thrombin production via the APC pathway. We further conclude that in hemophilic plasma this pathway can induce a secondary defect because of premature FV inactivation. It therefore seems conceivable that APC-resistant FV has the potential of alleviating hemophilic bleeding.

A549 Lung Epithelial Cells Synthesize Anticoagulant Molecules on the Cell Surface and Matrix and in Conditioned Media

To investigate mechanisms regulating intra-alveolar coagulation, we studied monolayers of the A549 human lung epithelial cell line. The surface of A549 cells delayed the onset of prothrombin-to-thrombin conversion and prevented total prothrombin consumption in normal plasma compared to plastic cell-free wells. Similar results were achieved with bovine pulmonary endothelial (CPAE) and rat intestinal epithelial (IEC-6) cell lines, whereas Madin-Darby canine kidney renal epithelial cell line accelerated thrombin formation. The A549 surface catalyzed antithrombin III-thrombin complex formation with no significant increase in thrombin inactivation from heparin cofactor II. The A549 cell surface effects were largely, but not completely, reversed to values obtained for plastic when protein C-deficient plasma was used. Pretreatment of the cell surface with chondroitinase ABC plus heparitinase prior to thrombin generation experiments had no effect on the total prothrombin consumed but decreased the initial delay. Heparan sulfate as well as dermatan sulfate and other chondroitin sulfates were detected on the A549 surface using alcian blue staining. Conditioned media from A549, CPAE, and IEC-6 cells delayed the clot time of recalcified plasma. Use of chondroitinase ABC and heparitinase were both required to obliterate the A549 conditioned media activity. After growing A549 cells in 35SO(2-)4-containing medium, the resultant conditioned medium was found to contain 2,000 kD and 300- to 1,000-kD proteoglycans that yielded chains of less than or equal to 100 kD on reductive elimination with base.(ABSTRACT TRUNCATED AT 250 WORDS)