Thrombin Generation In Normal Plasma Research Articles

Protein S contributes to the paradoxical increase in thrombin generation by low-dose dabigatran in the presence of thrombomodulin.

Low dose of dabigatran paradoxically increased thrombin generation through inhibition of protein C activation. Protein S is a co-factor in the activation of protein C. However, the role of protein S in the enhancement of thrombin generation has not been addressed. Firstly, we measured thrombin generation by calibrated automated thrombinography (CAT) and prothrombin fragments 1+2 (F1+2) assays. Secondly, we assessed activated protein C (APC) formation in normal or protein S-deficient plasma spiking with dabigatran. Then, protein C activation was measured. Finally, heavy chain of factor Va (FVa) and its degradation products were detected by western blot. CAT assay showed that 70-141 ng/ml dabigatran paradoxically increased thrombin generation in normal plasma. However, higher concentrations of dabigatran (283 ng/ml) suppressed the level of ETP. F1+2 assay showed the similar results. In protein S-deficient or protein C-deficient plasma, the paradoxical increase in thrombin generation was absent. Level of generated APC was to a similar extent inhibited by dabigatran in normal and protein S-deficient plasma. Low-dose dabigatran inhibited the protein S-dependent inactivation of factor Va. Protein S participated in the paradoxical enhancement of thrombin generation in normal plasma spiking with low concentrations of dabigatran. Increased thrombin generation at low dabigatran can be explained by reduced thrombin-thrombomodulin mediated APC formation and subsequent reduced FVa inactivation that is protein S-dependent.

A novel anticoagulant peptide discovered from Crassostrea gigas by combining bioinformatics with the enzymolysis strategy: inhibitory kinetics and mechanisms.

A novel anticoagulant peptide (IEELEEELEAER) derived from oyster (Crassostrea gigas) was discovered by combining the emerging bioinformatics with the classical enzymolysis approach. The anticoagulant peptide drastically reduced the extrinsic clotting activity (49% residual PT activity) and impaired the intrinsic clotting activity (77% residual PT activity). Consistent with the clotting data, the thrombin peak height reduced to 88.7 from 123.4 nM, and the thrombin generation time delayed to 5.32 from 4.42 min when an extrinsic trigger was applied. The inhibitory kinetics of FXIa, FIXa, FXa, FIIa, and APC in a purified component system rationally explained the reduction of the extrinsic clotting activity and impairment of thrombin generation. Besides the inhibition of FXa and FIIa activity, the activation processes of FX and FII by an intrinsic/extrinsic tenase complex and prothrombinase were also damaged. The anticoagulant activity in the plasma system was the result of comprehensive inhibition of various factors. The research provided a frame for anticoagulant evaluation and inhibitory mechanism of bioactive peptides from food products.

Procoagulant Microparticles Derived from Myeloma Plasma Cells Have a Determinant Role in the Hypercoagulable State Associated with Multiple Myeloma. a Modelization in Vitro Study

Background: Multiple myeloma (MM) is a plasma cell malignancy characterized by bone marrow infiltration leading to multiple lytic bone lesions, renal failure, anemia and increased rate of thrombotic events. The cross-talk between cancer cells, plasma clotting mechanism, platelets and endothelial cells enhances hypercoagulability. The mechanisms by which myeloma plasma cells (MPCs) interfere with their microenvironment during the blood coagulation process have been poorly investigated. Several studies have established a link between levels of cancer cell derived procoagulant microparticles, cancer aggressiveness, response to anticancer treatments well as with the risk of cancer associated thrombosis., The mechanisms by which myeloma plasma cells interfere to blood coagulation process in their microenvironment have been poorly investigated. Aim: Set up an experimental model that allows the identification of the procoagulant fingerprint of MPCs and myeloma cell-derived microparticles (MC-dMPs) as well as their impact on thrombin generation of normal plasma. In addition, we evaluated the relative impact of TF-pathway and the intrinsic clotting pathway on thrombin generation triggered by myeloma plasma cells and MC-dMPs. Finally, we performed a simulation of thrombin generation process in the presence of myeloma plasma cells and MC-dMPs as well as in the presence of varying concentrations of TF and procoagulant phopsholipids, mimicking some of the conditions that potentially exist in MPCs. Methods. TF and annexin V expression by MPCs (MPC-dMPs) were analyzed by flow cytometry, TF activity (TFa) and TF gene expression was also determined. Thrombin Generation (TG) in the presence of MPCs or MPC-dMPs was assessed with the Calibrated Automated Thrombogram assay (CAT®) in normal human PPP. TG was also assessed in plasma spiked with MPCs and MPC-dMPs or variable concentrations of TF and procoagulant phospholipids. Results: MPC-dMPs expressed about 8-fold higher levels of TF as compared to MPCs. TFa expressed by MPC- dMPs was significantly higher as compared to that expressed by MPC cells. MPCs and PC- dMPs enhanced thrombin generation of human plasma. Thrombin generation was significantly higher with MPC-dMPs compared to MPCs. MPCs and MPC-dMPs, when concomitantly present in normal PPP, significantly amplified thrombin generation as compared to that observed in the presence of MPCs alone. Presence of TF and procoagulant phospholipids in the microenvironment resulted in significant amplification of thrombin generation induced by MPCs. Conclusion: This study support the concept that hypercoagulability induced by myeloma plasma cells is the result of the combination of the procoagulant properties of myeloma cells with the procoagulant elements of the plasma microenvironment which consist of procoagulant phospholipids and low TF concentration. Following this concept we assume that MPC-dMPs released by myeloma plasma cells into the microenvironment enhance the weak procoagulant activity of myeloma plasma cells. Whereas this combined effect of MPCs and MPC-dMPs on thrombin generation has an impact on the formation of local fibrin network their critical role into the hypercoagulablity, procoagulant MC-dMP could be a potential target for the evaluation of the aggressivity of MPCs and of their response to anti-myeloma treatment. Disclosures Dimopoulos: Sanofi Oncology: Research Funding. Mohty:Jazz Pharmaceuticals: Honoraria, Research Funding. Elalamy:Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; LEO Pharma: Honoraria, Research Funding; Aspen: Honoraria, Research Funding; Bayer Healthcare: Honoraria, Research Funding.

Procoagulant microparticles derived from cancer cells have determinant role in the hypercoagulable state associated with cancer.

Hypercoagulablity is a common alteration of blood coagulation in cancer patients. However, the procoagulant activity of cancer cells is not sufficient to induce hypercoagulability. The present study was aimed to identify the mechanism with which hypercoagulabilty is produced in the presence of cancer cells. We focused on the analysis of the procoagulant elements carried by cancer cell-derived microparticles (CaCe-dMP) and we evaluated the impact of microparticles associated with the cancer cells from which they stem on thrombin generation. CaCe-dMP from the cancer cells were isolated from the conditioned medium and analyzed for tissue factor (TF) and procoagulant phospholipid expression. Thrombin generation of normal plasma was assessed by the Thrombinoscope (CAT®) in the presence or absence of pancreas adeno-carcinoma cells (BXPC3) or breast cancer MCF7 cells supplemented with the respective CaCe-dMP. Both BXPC3 and MCF7 cells express abundant amounts of active TF. Phosphatidylserine was identified on the surface of CaCe-dMP, unlike the cancer cells themselves. The expression of TFa by the microparticles was significantly higher to that observed on the cancer cells. Culture of the cancer cells with their microparticles resulted in thrombin generation significantly higher as compared to the upper normal limit. In conclusion, cancer cells 'enrich' the microenvironment with procoagulant elements, especially procoagulant micro-particles which express TF and procoagulant phospholipids. The association of cancer cells with procoagulant microparticles is necessary for a state of hypercoagulability, at the level of the tumoral microenvironment. The intensity of the hypercoagulability depends on the histological type of the cancer cells.

Graft Product for Autologous Peripheral Blood Stem Cell Transplantation Enhances Thrombin Generation and Expresses Procoagulant Microparticles and Tissue Factor

The beneficial effect of autologous peripheral blood stem cell transplantation (APBSCT) may be compromised by acute vascular complications related to hypercoagulability. We studied the impact of graft product on thrombin generation of normal plasma and the expression of tissue factor (TF) and procoagulant platelet-derived procoagulant microparticles (Pd-MPs) in samples of graft products. Graft products from 10 patients eligible for APBSCT were mixed with platelet-poor plasma (PPP) or platelet-rich plasma (PRP) from healthy volunteers and assessed for in vitro thrombin generation. In control experiments, thrombin generation was assessed in (1) PPP and PRP without any exogenous TF and/or procoagulant phospholipids, (2) PPP with the addition of TF (5 pM) and procoagulant phospholipids (4 μM), (3) in PRP with the addition of TF (5 pM). Graft products were assessed with Western blot assay for TF expression, with a specific clotting assay for TF activity and with flow cytometry assay for Pd-MPs. The graft product enhanced thrombin generation and its procoagulant activity was related to the presence of Pd-MPs and TF. The concentration of Pd-MPs in the graft product was characterized by a significant interindividual variability. The present study reveals the need for a thorough quality control of the graft products regarding their procoagulant potential.

The impact of the endothelial protein C receptor on thrombin generation and clot lysis

IntroductionWhen thrombin is bound to thrombomodulin (TM), it becomes a potent activator of protein C (PC) and thrombin-activable fibrinolysis inhibitor (TAFI). Activation of PC is enhanced when PC is bound to the endothelial protein C receptor (EPCR). Activated protein C (APC) inhibits thrombin generation while activated TAFI (TAFIa) attenuates fibrinolysis. To determine the impact of diminished EPCR function on thrombin generation and fibrinolysis we generated cells that expressed TM and a variant of EPCR (R96C) that does not bind PC. MethodsTo determine the impact of EPCR on the generation of APC and TAFIa and how this affects thrombin generation and fibrinolysis we performed thrombin generation and clot lysis assays in the presence of cells expressing wild-type TM and EPCR (WT cells) or wild-type TM and the R96C variant of EPCR (R96C cells). ResultsIn the presence of R96C cells, thrombin generation in normal plasma is increased, as a result of impaired PC activation when compared to WT cells. In addition, clot lysis is delayed in normal plasma in the presence of R96C cells, despite no increase in TAFI activation. In PC deficient plasma, clot lysis is delayed in the presence of WT and R96C cells as a result of increased TAFI activation. ConclusionsWe demonstrate that impaired EPCR function can be detected by thrombin generation and clot lysis assays on cells expressing TM and EPCR. We also demonstrated that deficiency in EPCR has procoagulant effects that lead to a delay in clot lysis.

Inactivated antithrombins as fondaparinux antidotes: a promising alternative to haemostatic agents as assessed in vitro in a thrombin-generation assay.

In the absence of specific antidote to fondaparinux, two modified forms of antithrombin (AT), one recombinant inactive (ri-AT) and the other chemically inactivated (chi-AT), were designed to antagonise AT-mediated anticoagulants, e. g. heparins or fondaparinux. These inactive ATs were previously proven to effectively neutralise anticoagulant activity associated with heparin derivatives in vitro and in vivo, as assessed by direct measurement of anti-FXa activity. This study was undertaken to evaluate in vitro the effectivity of inactive ATs to reverse anticoagulation by heparin derivatives and to compare them with non-specific fondaparinux reversal agents, like recombinant-activated factor VII (rFVIIa) or activated prothrombin-complex concentrate (aPCC), in a thrombin-generation assay (TGA). Addition of fondaparinux (3 µg/ml) to normal plasma inhibited thrombin generation by prolonging lag time (LT) as much as 244 % and lowering endogenous thrombin potential (ETP) to 17 % of their control (normal plasma) values. Fondaparinux-anticoagulant activity was reversed by ri-AT and chi-AT, as reflected by the corrections of LT up to 117 % and 114 % of its control value, and ETP recovery to 78 % and 63 %, respectively. Unlike ri-AT that had no effect on thrombin generation in normal plasma, chi-AT retained anticoagulant activity that minimises its reversal capacity. However, both ATs were more effective than rFVIIa or aPCC at neutralising fondaparinux and, unlike non-specific antidotes, inactive ATs specifically reversed AT-mediated anticoagulant activities, as suggested by their absence of procoagulant activity in anticoagulant-free plasma.

Coagulation Factor XI and Factor XII in DNA-Induced Thrombin Generation

Components of the blood coagulation mechanism are activated during infections as part of the innate immune response to invading microorganisms. This process is crucial for limiting spread of infection; however, excessive activation of coagulation can contribute to thrombotic tissue ischemia and systemic inflammation. During a variety of conditions associated with leukocyte activation and tissue damage, including sepsis, levels of cell free DNA rise in plasma. Neutrophils are one source of free DNA, releasing chromatin upon activation to form neutrophil extracellular traps (NETs). DNA in NETs form a physical barrier that contains microorganisms, while histones and antimicrobial proteins associated with DNA have bactericidal properties. NETs trigger platelet activation and coagulation, and likely contribute to inflammation, thrombosis and disseminated intravascular coagulation in sepsis. In a murine venous thrombosis model, neutrophils and NETs appear to contribute to thrombus formation through a process involving factor XII (fXII) (von Bruhl et al. J Exp Med 2012;209:819). When plasma fXII binds to anionic surfaces it is converted to the protease fXIIa. The results with the mouse model suggest that DNA, an anionic polymer, contributes to thrombosis by enhancing fXII activation.FXIIa contributes to thrombin generation and thrombosis by converting zymogen factor XI (fXI), to the protease factor XIa (fXIa). FXI has been implicated in the pathology of sepsis and thrombosis in animal models. Mice lacking fXI have better survival than wild type mice after ligation and puncture of the cecum, and are more resistant to Listeria infection. Inhibition or deficiency of fXI/fXIa confers an antithrombotic phenotype in rodents, rabbits and primates. Furthermore, epidemiologic data support a role for fXI in arterial and venous thrombosis in humans. FXI is activated in plasma by fXIIa or thrombin; however, the reactions proceed slowly in the absence of anionic substances. This suggests that a cofactor with anionic properties is required for fXI activation in vivo. For example, polymers of inorganic phosphate (polyphosphate or polyP) released by platelets enhance fXI activation by thrombin 3000-fold and by fXIIa 30-fold. PolyP also induces fXI and fXII autoactivation. The backbone of DNA is a type of phosphate polymer. We examined the capacity of DNA to enhance fXI and fXII activation and to contribute to fXI/fXII-dependent plasma coagulation.We isolated DNA from human peripheral blood leukocytes. Like polyP, DNA induced fXII autoactivation. Similarly, fXI underwent autoactivated in the presence of DNA, with the rate of activation increasing as DNA concentration increased. For reactions with either fXII or fXI, the enhancing effect of DNA was neutralized by treatment with DNAse. DNA, but not DNAse treated DNA, enhanced fXI activation by fXIIa 4-fold and by thrombin 30-fold compared to reactions without DNA. FXI is a dimeric protein composed of two identical subunits. PolyP binds to each fXI subunit through two anion binding sites (ABS), one on the fXI apple 3 domain (ABS1) and the other on the fXI catalytic domain (ABS2). These interactions are required for polyP-mediated enhancement of fXI activation by thrombin and fXIIa. Using DNA as a cofactor, the rate of activation of fXI lacking ABS1 by thrombin or fXIIa was 4-fold slower than for wild type fXI, while DNA failed to enhance activation of fXI lacking ABS2. This indicates that binding of fXI to DNA through the ABSs is required for expression of the DNA cofactor effect. Addition of DNA (50 ug/ml) induced thrombin generation in normal plasma (Endogenous Thrombin Potential 356 nM.min) through a process that was blocked by a fXIIa inhibitor (corn trypsin inhibitor) or a monoclonal antibody to fXI. Thrombin generation is dependent on fXI in normal plasma triggered to clot with a low (0.2 pM) concentration of tissue factor. Addition of DNA (50 ug/ml) to this system increased peak thrombin generation and endogenous thrombin potential by ~50%, while shortening time to peak thrombin potential. This effect was completely neutralized by an antibody to fXI. In conclusion, fXII and fXI have a propensity to bind to, and become activated on, anionic polymers and surfaces. Our results suggest that DNA released from leukocytes or damaged tissues could enhance activation of these protease zymogens, facilitating their contribution to hypercoagulability and inflammation. DisclosuresGailani:Aronora: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Bayer: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Dyax: Consultancy, Research Funding; Instrument Laboratory: Consultancy, Research Funding; Isis: Consultancy; Merck: Consultancy; Novartis: Consultancy.

Platelet‐ and erythrocyte‐derived microparticles trigger thrombin generation via factor XIIa

The procoagulant properties of microparticles (MPs) are due to the of the presence of phosphatidylserine (PS) and tissue factor (TF) on their surface. The latter has been demonstrated especially on MPs derived from monocytes. To investigate the relative contribution of TF and factor (F)XII in initiating coagulation on MPs derived from monocytes, platelets and erythrocytes. Microparticles were isolated from calcium ionophore-stimulated platelets, erythrocytes and monocytic THP-1 cells. MPs were quantified, characterized for cell-specific antigens and analyzed for TF, PS exposure and their thrombin-generating potential. The MP number was not proportional to PS exposure and the majority of the MPs exposed PS. TF activity was undetectable on platelet- and erythrocyte-derived MPs (< 1 fM nM(-1) PS), whereas monocyte-derived MPs exposed TF (32 fM nM(-1) PS). Platelet-, erythrocyte- and monocyte-derived MPs, but not purified phospholipids, initiated thrombin generation in normal plasma in the absence of an external trigger (lag time < 11 min). Deficiency or inhibition of FVII had no effect on thrombin generation induced by platelet- and erythrocyte-derived MPs, but interfered with monocyte MP-triggered coagulation. Platelet- and erythrocyte-derived MPs completely failed to induce thrombin generation in FXII-deficient plasma. In contrast, monocyte-derived MPs induced similar thrombin generation in normal vs. FXII-deficient plasma. MPs from platelets and erythrocytes not only propagate coagulation by exposing PS but also initiate thrombin generation independently of TF in a FXII-dependent manner. In contrast, monocyte-derived MPs trigger coagulation predominantly via TF.

Evidence for a Factor IX-Independent Role for Factor XI in Thrombin Generation

Abstract 2244In the widely used activated partial thromboplastin time (aPTT) assay, fibrin formation is induced by a series of sequential activations of the plasma protease zymogens factor (f) XII, fXI, fIX, fX and prothrombin, in that order. Conversion of prothrombin to the protease α-thrombin results in fibrin formation. α-Thrombin also enhances its own generation through activation of the cofactors fV and fVIII. While the linear sequence of reactions in the aPTT implies that loss of any single protease should have a comparable deleterious effect on the system, it is recognized that complete deficiency of a protein near the start of the sequence (e.g. fXII or fXI) results in greater aPTT prolongation than deficiency of proteins further down the sequence (e.g. fIX). This implies that proteases activated early in the process have multiple plasma substrates. For example, fXIa was recently reported to activate fVIII and fV (JTH 8;1532:2010), in addition to its role in fIX activation. Here, we present evidence that fXIa contributes to α-thrombin generation in the absence of fIX through activation of fX and/or fV.We noted that an anti-fXI antibody (O1A6) prolonged the aPTT of plasma from a patient with severe hemophilia B (fIX antigen undetectable) or plasma immunodepleted of fIX. This observation held even when an anti-fIX antibody was added to neutralize potential traces of fIX. Addition of activated fXI (fXIa - 3 nM) directly to fIX-deficient recalcified plasmas induced clot formation, and the time to clot formation was prolonged by O1A6. To further exclude the possibility that traces of fIX were contributing to thrombin generation, we confirmed the results using plasma from mice with combined complete deficiencies of fXII, fXI, and fIX. We tested the capacity of fXIa to cleave/activate fX and fV, the protease zymogen and cofactor, respectively, immediately downstream of fIX in the coagulation cascade. FX, the zymogen of the protease fXa, is evolutionarily related to fIX. SDS-PAGE analysis confirmed that fXIa cleaves fX. FX cleaved by fXIa demonstrated fXa activity in a chromogenic substrate assay, and converted prothrombin to α-thrombin in the presence of fVa and phospholipid. As previously reported, fXIa readily cleaved fV. The cleavage pattern differed from that generated by α-thrombin, however, formation of the fVa light chain was clearly evident. In a plasma clotting assay designed to measure either fXa or fVa activity, fX or fV pre-incubated with fXIa significantly shortened the clotting time of fIX-deficient plasma, while fX or fV pre-incubated with vehicle did not.In thrombin generation assays, fXIa (1.25 to 15 nM) induced thrombin generation in fIX-deficient plasma supplemented with anti-fIX antibody in a concentration dependent manner. FXIa did not induce thrombin generation in plasma lacking fV, or in fIX-deficient plasma containing the fXa inhibitor apixaban. This indicates that fXIa is working at the level of fX/fV in this assay, and is not directly converting prothrombin to α-thrombin. A recombinant variant of fXIa lacking the major fIX-binding exosite (fXIaPKA3, J Biol Chem 1996;271:29023) demonstrated a marked defect, compared to wild type fXIa (fXIaWT), in its capacity to induce thrombin generation in normal plasma. However, in fIX-deficient plasma, fXIaPKA3 and fXIaWT are comparable in their ability to enhance thrombin generation, supporting the premise that fXIa is acting through activation of fX and/or fV in the absence of fIX.Previously, we observed that fXI deficient mice and fIX deficient mice are comparably resistant to carotid artery thrombosis induced by exposure of the vessel to ferric chloride, despite having very different propensities to bleed. The animals were uniformly resistant to thrombosis with 5% FeCl3, and some were resistant at 7.5% FeCl3. All experienced vessel occlusion with 10% FeCl3. This is consistent with fXIa contributing to thrombosis in this model through fIX activation. However, we observed that some mice with combined fIX and fXI deficiency were resistant to FeCl3 concentrations up to 12.5%, implying that fXIa was contributing to thrombosis in a fIX-independent manner, as well. These results are consistent with those from the in vitro assays described above, and support the hypothesis that fXIa contributes to thrombin generation through fIX-dependent and fIX-independent processes. Disclosures:Tucker:Aronora, LLC: Employment, Equity Ownership. Gruber:Aronora, LLC: Consultancy, Equity Ownership.

Syncytiotrophoblast Microvesicles Released from Pre-Eclampsia Placentae Exhibit Increased Tissue Factor Activity

BackgroundPre-eclampsia is a complication of pregnancy associated with activation of coagulation. It is caused by the placenta, which sheds increased amounts of syncytiotrophoblast microvesicles (STBM) into the maternal circulation. We hypothesized that STBM could contribute to the haemostatic activation observed in pre-eclampsia.Methodology/Principal FindingsSTBM were collected by perfusion of the maternal side of placentae from healthy pregnant women and women with pre-eclampsia at caesarean section. Calibrated automated thrombography was used to assess thrombin generation triggered by STBM-borne tissue factor in platelet poor plasma (PPP). No thrombin was detected in PPP alone but the addition of STBM initiated thrombin generation in 14/16 cases. Pre-eclampsia STBM significantly shortened the lag time (LagT, P = 0.01) and time to peak thrombin generation (TTP, P = 0.005) when compared to normal STBM. Blockade of tissue factor eliminated thrombin generation, while inhibition of tissue factor pathway inhibitor significantly shortened LagT (p = 0.01) and TTP (P<0.0001), with a concomitant increase in endogenous thrombin potential.Conclusions/SignificanceSTBM triggered thrombin generation in normal plasma in a tissue factor dependent manner, indicating that TF activity is expressed by STBM. This is more pronounced in STBM shed from pre-eclampsia placentae. As more STBM are shed in pre-eclampsia these observations give insight into the disordered haemostasis observed in this condition.

Increased thrombin generation measured in the presence of thrombomodulin in women with early pregnancy loss

Compared with 537 parous controls with no history of pregnancy loss, a lower thrombomodulin-related inhibition of the endogenous thrombin potential was measured in 264 cases with previous unexplained pregnancy loss, especially when losses occurred between 9 and 12 weeks of gestation. Adjusting age, protein S, factor VIII, factor V Leiden, and prothrombin G20210A did not change the results.

Characterization of the Procoagulant Activity of Two Different Microparticle Species.

AbstractAbstract 1143 Background:Microparticles (MP) exhibit procoagulant activity by exposure of tissue factor (TF) and consecutive factor (F) × activation via binding to activated FVII (FVIIa). We have shown that TF-negative MP derived from endothelial cells (EMP) after exposure to cisplatin induce thrombin generation in vitro in a TF-independent manner (Lechner et al., J. Thromb. Haemost. 2007). The procoagulant properties of TF-negative MP are not well characterized. We therefore aimed to investigate the mechanisms of coagulation activation by TF-negative EMP (obtained from human pulmonary microvascular endothelial cells - HMVEC-L) in comparison to TF-abundant MP derived from a cancer cell line (A431-MP). Methods:MP were obtained by ultracentrifugation (100,000 × g for 1 h) of cell culture supernatant. The procoagulant activity was measured in normal plasma and in plasmas deficient in coagulation factors VII, VIII, IX, X, XI, and XII, respectively, by an in vitro thrombin generation assay (Technothrombin TGA, Technoclone, Austria), by activated partial thromboplastin (aPTT), prothrombin time (PT) and a chromogenic FIX activation assay. Antibodies to TF and FVII (both American Diagnostica, USA) were used. Plasmas used in the thrombin generation assay were MP-depleted by ultracentrifugation before use. Results:EMP and A431-MP induced in vitro thrombin generation in normal plasma. In vitro thrombin generation induced by EMP was abolished in plasmas deficient in FVIII or FIX, and was markedly reduced in FXI-deficient plasma. In FVII-deficient plasma, normal thrombin generation after addition of EMP was seen. In contrast, A431-MP-triggered thrombin generation was abolished in FVII-deficient plasma, but was not influenced by depletion of FVIII, FIX or FXI. In FX-deficient plasma, thrombin generation could not be triggered by addition of either EMP or A431-MP. Thrombin generation was normal after addition of EMP or A431-MP to FXII-deficient plasma (Figure). In a modified aPTT system using kaolin as surface activator, EMP and A431-MP induced clot formation in normal plasma. In a modified PT system only A431-MP but not EMP induced clot formation. We then investigated whether MP can directly activate FIX. In a plasma-free environment FIX activation by A431-MP was much more pronounced than by EMP (14-fold vs. 2.4 fold increase compared to a control experiment with cell culture medium only). FIX activation by A431-MP was blocked by the addition of antibodies to TF and FVII, while no such effect was seen after addition to EMP. Conclusions:Our findings show that both TF-positive and TF-negative MP exhibit procoagulant activity. If TF is expressed (A431-MP), coagulation activation is triggered via FVII/FVIIa, which is abolished in FVII-deficient plasma or by the addition of antibodies to TF or FVII. TF-negative EMP induce thrombin generation via the intrinsic coagulation pathway by activating FXI and FIX, as shown by the generation of activated FIX in a FIX activation assay. EMP are also able to induce clot formation in an aPTT based system. We surmise that the procoagulatory effect of EMP may be due to their high phospholipid content or to a specific phospholipid surface composition. The results of our FIX activation assay also confirm a direct FIX activation by TF/FVIIa, which can be abolished by addition of antibodies to TF or FVII. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Monocyte IL-10 produced in response to lipopolysaccharide modulates thrombin generation by inhibiting tissue factor expression and release of active tissue factor-bound microparticles

Lipopolysaccharide (LPS)-stimulated monocytes are known to have a procoagulant effect. This property is currently explained by the fact that monocytes, in response to LPS, can express tissue factor (TF) and undergo a process of membrane microvesiculation. Interleukin-10 (IL-10) has been shown to downregulate TF expression and inhibit procoagulant activity (PCA). In order to further characterize the inhibitory effect of IL-10 on LPS-induced PCA, we used the integrated system of analysis of kinetics of thrombin generation in normal plasma (thrombinography). For this, we developed an original method of elutriation allowing to obtain a highly purified monocyte preparation, under endotoxin-free conditions. Thrombin generation was measured using a highly sensitive and specific fluorogenic method which we adapted to inhibit the contact factor pathway. Results show that recombinant human IL-10 decreased the kinetics of thrombin generation in a dose-dependent manner. Furthermore, the inhibition of endogenous IL-10 released by monocytes in response to LPS is associated with an increase in the kinetics of thrombin generation. We demonstrated that this effect was a consequence of the up-regulation of TF expression and TF-bound microparticle release. In conclusion, we report that IL-10 can regulate thrombin generation in conditions close to physiology as allowed by thrombinography, and that endogenous IL-10 regulates TF expression and release of active TF-bound microparticles by a negative feed back loop through IL-10 receptor alpha.

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.

Thrombin generation in normal plasma enriched with purified coagulation factors

During the intrinsic coagulation of normal platelet-rich plasma only 11% of the prothrombin is converted to thrombin. Complete conversion of prothrombin to thrombin occurs only via the extrinsic pathway (1). Addition of purified prothrombin to normal plasma to double or triple its concentration, doubled or tripled the amount of the generated thrombin as determined by the thrombin elution assay (1), so that the percentage of the proenzyme which was converted to thrombin remained the same. At the same time the activated partial thromboplastin time (APTT) was prolonged. Proportionality of the amount of the generated thrombin to the amount of prothrombin added and a delay in the appearance of thrombin activity was also observed with the thrombin generation test. Normalization of the APTT was observed when factor IX was added together with prothrombin. Addition of factor IX or X to normal plasma shortened the APTT but did not increase the amount of prothrombin which was converted to thrombin as determined by both the thrombin elution assay and the thrombin generation test. Further experiments indicated that (a) more factor X is activated per mg tissue factor than per mg of activated partial thromboplastin and (b) more thrombin is generated per unit of factor Xa in the presence of tissue factor than in the presence of activated partial thromboplastin. Thus, the two pathways differ not only by the mechanism of factor X activation but also by the extent to which prothrombin is activated by factor Xa.