Extrinsic Coagulation Activity Research Articles

Preclinical characterisation and first-in-human results on the tolerability and pharmacodynamic effect of REGN9933, a monoclonal antibody targeting the factor XI/activated factor XI apple 2 domain

Abstract Background/Introduction Standard-of-care anticoagulants are associated with increased bleeding risk. Genetic/pharmacological data suggest coagulation factor XI (FXI) inhibition can prevent thrombosis with minimal increased bleeding risk. We present preclinical and first-in-human (FIH) data on REGN9933, a FXI-binding monoclonal Ab. Purpose To characterise REGN9933 epitope binding sites, affinities for FXI and activated FXI (FXIa), and effect on high molecular weight kininogen (HMWK)’s interaction with FXI. Also, to assess safety, tolerability and pharmacodynamics in healthy human participants (pts). Methods Epitope binding sites mapped with cryogenic electron microscopy, binding affinities determined with plasmon resonance techniques, effect on thrombin generation assessed with a thrombin generation assay, and impact on HMWK:FXI interaction analysed using ELISAs. In the FIH, Ph 1, randomised, double-blind, single-centre study, pts received a single dose of REGN9933 intravenously (IV; 3–300 mg) or subcutaneously (SC; 100 mg & 300 mg), or placebo (PBO). Primary endpoint: incidence and severity of treatment-emergent AEs (TEAEs). Other endpoints: effect of REGN9933 on activated partial thromboplastin time (aPTT) and prothrombin time (PT) to assess intrinsic and extrinsic pathway-triggered coagulation, respectively; and FXI activity. Results REGN9933 was found to bind the FXI apple 2 domain, with subnanomolar binding affinities for FXI and FXIa at 37°C, resulting in reduced thrombin generation from the intrinsic (not extrinsic) pathway. REGN9933 competed with HMWK for FXI binding in a dose-related manner. In the Ph 1 study, 56 pts received REGN9933 (IV, n=30; SC, n=12) or PBO (n=14); 42.9% and 21.4% had ≥1 TEAE, respectively (most common with REGN9933: headache, 12%; oropharyngeal pain, 5%). No serious/severe TEAEs, or TEAEs of special interest (inc. moderate/severe bleeding) reported. Laboratory tests revealed no clinically meaningful differences in the number/type of treatment-emergent potentially clinically significant values with REGN9933 vs PBO. REGN9933 resulted in dose-dependent prolongation of aPTT; aPTT mean fold change from baseline was highest with IV 300 mg after 8 hrs (2.7) and remained >2 for 36 days (Fig 1). There was no detectable effect on PT or clinically meaningful effect on bleeding time. REGN9933 supressed FXI activity in a dose-dependent manner, with suppression to approx. the lower limit of assay quantification (5%) with IV ≥30 mg (Fig 2). Conclusion(s) REGN9933 binds the FXI apple 2 domain and inhibits HMWK:FXI interaction, preventing intrinsic pathway-triggered FXI activation. FIH data showed dose-dependent inhibition of the intrinsic coagulation pathway by REGN9933, without affecting the extrinsic or common pathways. Pharmacodynamic and safety findings support continued development of REGN9933 as an anticoagulant that may carry less bleeding risk than currently approved agents.

Increased Activated Protein C Response Rates to Thrombin Formation in Asymptomatic Factor V Leiden Carriers Are Driven By the Endothelium

Background The thrombophilic factor V Leiden (FVL) mutation shows a highly variable clinical expressivity. We have shown in vivo that asymptomatic FVL carriers respond to extrinsic coagulation activation with higher activated protein C (APC) formation rates than those with a history of venous thromboembolism (VTE). Recently we have established an ex vivo model for personalized assessment of the protein C (PC) pathway combining endothelial colony forming cells (ECFCs) and autologous plasma. Aim of this study was to investigate potential modulating factors of the APC response in the endothelium unraveling the enigma of the variable expressivity of the FVL phenotype. Methods ECFCs were isolated from FVL carriers with (FVL VTE+) or without (FVL VTE-) a history of unprovoked VTE and healthy controls (non-FVL) (n=7 each). Quality control of cultured cells included cobblestone morphology as examined by light microscopy, positive staining for CD31, CD309, CD201, and CD141, CD34, and negative staining for CD45 analyzed by flow cytometry. We either added autologous or pooled normal defibrinated plasma to the confluent cell culture and inititated thrombin formation by addition of tissue factor (1 pmol/L) and CaCl 2 (16.6 mmol/L) ( Fig. 1A). Thrombin and APC formation were measured over time using oligonucleotide-based enzyme capture assays (OECAs) and the ratio between the area under the curve (AUC) of APC formation and the AUC of thrombin formation was calculated as measure for APC response to thrombin. Moreover, we quantified thrombomodulin (TM) and endothelial protein C receptor (EPCR) expression in cell-based enzyme-linked immunosorbent assays (cell-ELISAs) and measured activation of purified PC on ECFCs in the APC-OECA. Results ECFC morphology in light microscopy and characteristic surface marker expression in flow cytometry did not differ between non-FVL, FVL VTE-, and FVL VTE+ subgroups. In the ECFC-based ex vivo model, the APC response in plasma on autologous cells was significantly higher in asymptomatic FVL VTE- carriers compared to non-FVL carriers (P = .0072) and compared to the FVL VTE+ subgroup (P = .0292) whereas it did not differ significantly between non-FVL and FVL VTE+ carriers. EPCR expression on ECFCs measured by cell-ELISA was higher in non-FVL carriers compared to FVL VTE+ subjects (P = .0374) but did not differ significantly when comparing non-FVL versus FVL VTE-, and FVL VTE+ versus FVL VTE-. Similarly, we showed that TM expression as well as activation of purified PC on ECFCs did not differ between cohorts. However, when the APC response was assessed on FVL VTE- and FVL VTE+ ECFCs using non-FVL plasma, the difference in the APC response between asymptomatic FVL carriers and FVL carriers with a history of VTE persisted (P = .0111) ( Fig. 1B). Conclusion Consistent with previous in vivo experiments, APC response rates to thrombin formation were increased on ECFCs obtained from asymptomatic FVL carriers compared to those from FVL carriers with a history of VTE in autologous plasma. Although TM and EPCR expression did not differ between both groups, we showed that the endothelial APC response in asymptomatic FVL carriers remained increased when using plasma from healthy controls. Our observations suggest that the increased APC response to thrombin in asymptomatic FVL carriers is driven by the endothelium. Further studies are warranted to elucidate the underlying mechanism for a better understanding of endogenous protective factors in FVL carriers. These findings may pave the way towards personalized, precision medicine approaches for managing the thrombotic risk in FVL and other disorders related to the PC pathway.

Complement component C1q initiates extrinsic coagulation via the receptor for the globular head of C1q in adventitial fibroblasts and vascular smooth muscle cells.

Vascular diseases are highly associated with inflammation and thrombosis. Elucidating links between these two processes may provide a clearer understanding of these diseases, allowing for the design of more effective treatments. The activation of complement component 1 (C1) is a crucial contributor to innate immunity and is associated with significant concentrations of circulating C1q. Many pathological pathways initiate when C1q interacts with gC1qR. This interaction plays a major role in inflammation observed during atherosclerosis and the initiation of intrinsic coagulation. However, the effects of C1 and the role of C1q/gC1qR on extrinsic coagulation, which is the more physiologically relevant coagulation arm, has not been studied. We hypothesized that C1q binding to gC1qR enhances the expression of tissue factor (TF) in adventitial fibroblastsand vascular smooth muscle cells, the primary TF bearing cells in the body. Using an enzyme-linked immunosorbent assay approach, TF expression and the role of gC1qR was observed. Cells were conditioned for 1 hwith C1q or a gC1qR blocker and C1q, to assess the role of gC1qR. Additionally, cell growth characteristics were monitored to assess changes in viability and metabolic activity. Our results indicate that the expression of TF increased significantly after incubation with C1q as compared with unconditioned cells. Cells conditioned with gC1qR blockers and C1q exhibited no change in TF expression when compared with cells conditioned with the blocking antibodies alone. Our results show no significant differences in metabolic activity or cell viability under these conditions. This indicates that gC1qR association with C1q induces TF expression and may initiate extrinsic coagulation. Overall, this data illustrates a role for C1q in the activation of extrinsic coagulation and that gC1qR activity may link inflammation and thrombosis.

C1q/gC1qR‐Mediated Activation of Extrinsic Coagulation

Vascular diseases can be characterized by and are highly associated with inflammation and thrombosis. Understanding the relationship between these two processes would allow us to treat vascular diseases more effectively. Activation of complement component 1 (C1) is an important contributor to innate immunity and drives the lysis of pathogens. Upon C1 activation, significant concentrations of circulating C1q are observed. C1q has an affinity for gC1qR, a cell surface receptor molecule that is commonly found on cell types associated with cardiovascular disease development, including vascular smooth muscle cells and adventitial fibroblasts. When C1q interacts with gC1qR, many pathological pathways initiate including the inflammation observed during atherosclerosis and the initiation of intrinsic coagulation. The intrinsic cascade plays a minor role in physiological and pathological thrombosis as compared with the extrinsic cascade; however, the effects of C1 and the role of C1q/gC1qR on extrinsic coagulation have not been studied. We hypothesized that C1q association with gC1qR would increase the amount of sub‐endothelial tissue factor expressed by both aortic adventitial fibroblasts (HAAFs) and coronary artery smooth muscle cells (HCASMCs). We investigated these cells as they are the primary TF bearing cells. Further, we hypothesized that the newly expressed TF would bind Factor VII (FVII), which is a crucial step in the initiation of the extrinsic coagulation cascade. The objective of this study was to elucidate the role of C1q/gC1qR‐mediated TF expression and its ability to bind FVII. Using a solid‐phase ELISA, TF expression was observed on both HAAFs and HCASMCs. Cells were conditioned for one hour with either platelet poor plasma (PPP), purified human C1q, LPS, or a combination of anti‐gC1qR antibodies (60.11 or 74.5.2 clones) and purified human C1q, PPP, or LPS. A capture ELISA approach was used to evaluate the binding of FVII to TF following exposure after the same conditions. Further, a capture ELISA was used to assess released TF. Finally, a live/dead assay to assess cell viability and cell density, as well as an MTT assay to assess cell metabolic activity were performed to monitor cell culture conditions. Our results indicate that HAAF and HCASMC expression of TF increased significantly by approximately 15% after incubation with C1q as compared with unconditioned cells. Cells conditioned with gC1qR blocking antibodies prior to conditioning with C1q exhibited no change in TF expression when compared to unconditioned cells. Additionally, a 20‐30% increase in bound FVII was observed in both cell types when conditioned with C1q as compared to unconditioned cells. Our results show no significant differences in metabolic activity or cell viability under these conditions; however, cell density decreased by approximately 25% when conditioned with C1q. Overall, this data illustrates a role for C1q in activation of extrinsic coagulation. Furthermore, our data suggests that gC1qR acts a mediator for both inflammation and thrombosis. This work serves as a meaningful step in identifying a convergence of inflammatory and thrombotic pathways that can be therapeutically targeted to mitigate vascular diseases. Future work will determine if the expressed TF and the binding of FVII can support downstream extrinsic coagulation activity. Thanks to NIH for supporting this research.

Evaluation of the Thrombophilic Response to rFVIIa-Induced Hemostasis Activation in Factor V Leiden Carriers By Monitoring of Activated Protein C (APC) In Vivo

Background: The majority of patients with unprovoked venous thromboembolism (VTE) does not exhibit laboratory risk factors. Monitoring a limited and standardized activation of the hemostatic system in these patients might help to identify prothrombotic phenotypes in VTE. Promising biomarkers to monitor subclinical changes of the hemostatic activation status are plasma levels of active enzymes including thrombin and APC. Aim of this study was to characterize the prothrombotic phenotype in factor V (FV) Leiden carriers as a prerequisite for the identification of novel prothrombotic mechanisms.Methods: Subclinical activation of extrinsic hemostasis was induced by i.v. injection of recombinant activated factor VII (rFVIIa, 15 µg/kg) into 14 asymptomatic FV Leiden carriers (thereof 12 heterozygous, five males) and 12 healthy FV wild type (WT) carriers (six males). rFVIIa-induced hemostatic activation was monitored by measuring plasma levels of free thrombin and APC at baseline and repeatedly during an eight-hour follow-up period using oligonucleotide-based enzyme capture assays (OECA). In addition, prothrombin fragment F1+2 (F1+2), thrombin-antithrombin complex, plasmin-α2-antiplasmin complex, soluble fibrin monomer, and D-dimer were determined.Results: Administration of rFVIIa was well tolerated in all subjects. Its pharmacokinetics showed an expected course with no differences between FV Leiden carriers and WT carriers (Figure 1A). At baseline, median (interquartile range, IQR) APC plasma levels were 0.081 (0.068-0.117) ng/ml in heterozygous FV Leiden carriers and 0.035 (0.028-0.067) ng/ml in WT carriers. Following rFVIIa injection, a significant (p<0.05) elevation of APC levels from baseline was observed between t=10 min and t=5 h (3 h for WT carriers). Peaks of 0.509 (0.431-0.721) ng/ml (p=10-4) in heterozygous FV Leiden carriers and of 0.164 (0.139-0.194) ng/ml (p=3·10-4) in WT carriers were observed at t=1 h (Figure 1B). In addition to higher peaks (p=2·10-4) in heterozygous FV Leiden carriers than in WT carriers, the area under the APC generation curve (AUC) was greater (p=4·10-6) with 2.40 (1.84-2.84) vs. 0.77 (0.63-0.97) ng/ml·h. Interestingly, the AUC of both homozygous FV Leiden carriers (2.82 and 2.67 ng/ml·h) and their APC peak levels (0.717 and 1.123 ng/ml) lay within the ranges of the heterozygous cohort. Among the other biomarkers, only F1+2 showed an increase from baseline (0.178, 0.122-0.197 nmol/l in heterozygous FV Leiden carriers; 0.125, 0.087-0.188 nmol/l in WT carriers) that became statistically significant at t=2 h (0.229, 0.187-0.264 nmol/l, p=0.001), t=3 h (0.230, 0.190-0.281 nmol/l, p=0.006), and t=5 h (0.230, 0.172-0.274 nmol/l, p=0.02) in heterozygous FV Leiden carriers, and at t=2 h (0.179, 0.111-0.200 ng/ml, p=0.03) in WT carriers. All other biomarkers remained unchanged, and no statistically significant differences were observed between FV carriers and WT carriers.Conclusion: Increased thrombin generation rates are counterbalanced by increased APC generation rates in clinically asymptomatic FV Leiden carriers after subclinical activation of extrinsic coagulation. Furthermore, the data presented demonstrate that subclinical activation of the hemostatic system by rFVIIa combined with serial APC testing is a new diagnostic tool to assess the functionality of the anticoagulant APC pathway in vivo. [Display omitted] DisclosuresRühl:Grifols: Research Funding; Sobi: Consultancy. Winterhagen:Sobi: Consultancy.

Complement Component C1q Initiates Extrinsic Coagulation via gC1qR

Vascular diseases can be characterized by and are highly associated with inflammation and thrombosis. Understanding the relationship between these two processes would allow us to treat vascular diseases more effectively. Activation of complement component 1 (C1) is an important contributor to innate immunity and drives the lysis of pathogens. Upon C1 activation, significant concentrations of circulating C1q are observed. When C1q interacts with gC1qR, many pathological pathways initiate and C1q/gC1qR plays a role in inflammation observed during atherosclerosis and the initiation of intrinsic coagulation. While this link between inflammation and thrombosis is important, the intrinsic cascade plays a minor role in physiological and pathological thrombosis. The effects of C1 and the role of C1q/gC1qR on extrinsic coagulation, which is more physiologically relevant, has not been studied. Extrinsic coagulation initiates upon vessel damage, exposing tissue factor (TF) to the blood. Adventitial fibroblasts and vascular smooth muscle cells are the primary TF bearing cells. We hypothesized that C1q binding to gC1qR enhances the expression of sub-endothelial TF in these cells. The objective of this study was to elucidate the role of gC1qR-mediated TF expression. Using a solid-phase ELISA, TF expression was observed on aortic adventitial fibroblasts (HAAFs) and coronary artery smooth muscle cells (HCASMCs). Cells were conditioned for one hour with either platelet poor plasma (PPP), purified human C1q, LPS, or a combination of anti-gC1qR (60.11 or 74.5.2 clones) and purified human C1q, PPP, or LPS. Further, a capture ELISA was used to assess released TF under these same conditions. Finally, a live/dead assay to assess cell viability and cell density, as well as an MTT assay to assess cell metabolic activity were performed to monitor cell culture conditions. Our results indicate that HAAF and HCASMC expression of TF increased significantly by approximately 35% after incubation with C1q as compared with unconditioned cells. Cells conditioned with gC1qR blocking antibodies exhibited a marked decrease in TF expression (by approximately 10-20%) when compared to cells conditioned with C1q alone. Our results show no significant differences in metabolic activity or cell viability under these conditions; however, cell density decreased by approximately 25% when conditioned with C1q. Overall, this data illustrates a role for C1q in activation of extrinsic coagulation. Furthermore, our data suggests that gC1qR acts a mediator for both inflammation and thrombosis. This work serves as a meaningful step in identifying common inflammatory and thrombotic pathways that can be therapeutically targeted to mitigate vascular diseases. Future work will determine if the expressed TF can support extrinsic coagulation activity.

Initiation of Extrinsic Coagulation via Complement

Thrombosis and inflammation are prevalent during and characteristic of vascular diseases. Understanding the interplay of these processes would likely allow us to treat vascular diseases more effectively. Activation of complement component 1 (C1) is an important contributor to innate immunity and ultimately drives the lysis of pathogens. Upon C1 activation, significant concentrations of circulating C1q are observed. C1q interacting with gC1qR, one of its cell‐surface receptors, initiates many pathological pathways. There is considerable evidence that C1q/gC1qR plays a role in inflammatory processes observed during atherosclerosis and in initiating intrinsic coagulation. While this link between inflammation and thrombosis is important, the intrinsic cascade plays a minor role in physiological and pathological thrombosis. The effects of C1 and the role of C1q on extrinsic coagulation, which is more physiologically relevant, has not been studied. Extrinsic coagulation initiates upon vessel damage, which exposes blood to sub‐endothelial constituents, especially tissue factor. Adventitial fibroblasts and vascular smooth muscle cells are the primary tissue factor bearing cells. We hypothesized that C1q binding to gC1qR initiates this pathway through the enhanced expression of sub‐endothelial tissue factor (TF) in these cells. The objective of this study was to elucidate the role of C1q‐mediated TF expression. Using a solid‐phase ELISA, TF expression was observed on aortic adventitial fibroblasts (HAAFs) and aortic smooth muscle cells (HASMCs) under various conditions. Cells were conditioned for one hour with either platelet poor plasma (PPP), purified human C1q, LPS, anti‐gC1qR (60.11), or a combination of 60.11 and purified human C1q. After the exposure conditions, cells were incubated for one hour with one of the following primary antibodies: anti‐human gC1qR (74.5.2), anti‐human TF, or anti‐human CD54 (ICAM‐1). Following primary antibody incubation, cells were incubated with an appropriate alkaline phosphatase conjugated secondary antibody for one hour. PNPP was added to the cells and allowed to develop for 30 minutes. Absorbance was documented at 405 nm. Our results suggest that HAAF and HASMC expression of TF increased significantly by approximately 35% after incubation with C1q as compared with unconditioned cells. We also observed increased ICAM expression, 20% above baseline, in HASMCs. Cells conditioned with gC1qR blocking antibodies exhibited a marked decrease in TF and ICAM expression (by approximately 10%) when compared to cells conditioned with C1q alone. Overall, this data illustrates a role for C1q in activation of extrinsic coagulation. Furthermore, our data suggests that gC1qR acts a mediator for both inflammatory and thrombotic reactions that may occur during vascular diseases. This work illustrates a step in identifying common inflammatory and thrombotic pathways that can be therapeutically targeted to mitigate vascular diseases and may provide new information regarding off‐target effects. Future work will determine if the expressed TF can support extrinsic coagulation activity and new therapeutics can be designed to mitigate vascular disease processes.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An in vitro screening study of food functions among vegetable cultivars consumed in Japan

Aim: Ingesting vegetables plays a major role in health maintenance and disease prevention. Therefore, this study aimed to identify hitherto unknown food functionalities among various vegetable cultivars consumed in Japan. Methods: Water-soluble vegetable extracts were screened for various types of functionality such as antioxidant action, the inhibition of platelet aggregation, blood coagulation and the enzymatic actions of a-glucosidase and pancreatic lipase. Antioxidant activity was measured as the ability to scavenge 1,1-diphenyl-2-picryl hydrazine (DPPH) radicals and the inhibitory activity of rat intestinal a-glucosidase was evaluated by measuring amounts of glucose produced from maltose. Lipase activities were measured using porcine pancreatic lipase and synthetic substrate. Platelet aggregation was measured using bovine whole blood stimulated with collagen. Plasma coagulation activities were measured using human plasma. Coagulation was initiated using intrinsic and extrinsic coagulation initiators and coagulation times were measured using a coagulometer. Results: All extracts from cabbage, onion, eggplant, tomato, green pepper, Japanese radish and Chinese cabbage and their cultivars exerted antioxidant activities. All vegetable extracts had weak a-glucosidase inhibitory activity, but one Chinese cabbage cultivar had significantly more of such activity. All vegetable extracts had very weak ability to inhibit pancreatic lipase and did not inhibit platelet aggregation or affect intrinsic and extrinsic blood coagulation activity. Conclusion: Although further investigations are needed, the present findings provide fundamental information about the functionality of some vegetables consumed in Japan.

Septic porcine blood does not further activate coagulation during in vitro membrane oxygenation.

For patients with a severe acute respiratory distress syndrome (ARDS), extracorporeal membrane oxygenation (ECMO) represents a life-saving measure. Frequently, patients with severe ARDS also show signs of severe sepsis. As blood contact with the membrane oxygenator surface leads to adverse effects due to insufficient biocompatibility partly caused by activation of platelets, coagulation factors and leucocytes, we hypothesized that these adverse effects would be amplified if septic blood in a preactivated state came into contact with the membrane oxygenator. In a previously established in vitro 12-h ECMO test system (mock loop), we used septic or healthy domestic pig blood to analyse coagulation and inflammatory parameters. Sepsis was induced by a caecal ligation and puncture model in pigs. At the beginning of the mock loop experiments, the septic blood showed significantly increased thrombin-antithrombin complexes (76.9 vs 27.7 µg/l), D-dimers (1.2 vs 0.3 mg/l) and fibrinogen concentration (1.8 vs 1.5 g/l), as well as elevated extrinsic coagulation activity (shorter EXTEM-CT: 44.2 vs 57 s) and higher lactate (3.4 vs 1.5 mmol/l) and cytokine levels (interleukin-6: 827 vs 31 pg/ml) when compared with the blood from healthy animals. Despite the preactivated status of the septic blood, no further increase of coagulation activity, inflammatory response or increased oxygenator resistance was observed in comparison to the control experiments. Septic porcine blood was not further activated due to the contact with an oxygenator, and no increased clot formation or biocompatibility problems were observed.

Thrombelastometric results and platelet function during pregnancy in women receiving low molecular weight heparin with a history of recurrent/late abortion--A retrospective analysis.

In women with a history of recurrent/late abortion and confirmed genetic/acquired thrombophilia, LMWH was given during subsequent pregnancy and serial coagulation testing was performed.In 82 consecutive pregnant women with recurrent (≥2) and/or late abortion (>12 GW) in the presence of single (n = 62; 75.6%) or combined (n = 20; 24.4%) genetic and/or acquired thrombophilia, Thromboelastometry (n = 50; ROTEM, TEM) and closure-time (n = 82; PFA-100; Siemens) underwent serial testing before and during pregnancy while receiving LMWH and puerperal.Throughout pregnancy, clotting-time (CT) after intrinsic and extrinsic induced coagulation activation in Thromboelastometry remained unchanged. TF-induced coagulation activation resulted in statistically significantly decreased mean clot-formation-times (CFT) (Trim I: 108.9 ± 5.2 S to Trim III; 81.7 ± 5.4 S; p = 0.001), whereas after contact activation (Intem-S: Trim I: 70.1 ± 4.0 S to Trim III: 65.4 ± 6.8; n.s.) CFT remained unchanged. Mean maximal-clot-firmness (MCF) continuously increased in the Intem-S and Extem-S during each trimester and decreased until 4th puerperal week (Extem-S: Trim I: 61.9 ± 1.0 S; Trim II: 65.4 ± 0.58 S; Trim III: 68.3 ± 1.1 S; p < 0.001; Intem-S: Trim I: 64.1 ± 0.6 S; Trim II: 66.8 ± 0.5 S; Trim III: 69.5 ± 1.2 S; p < 0.001). Mean Closure-times after Epinephrine/ADP/Collagen stimulation remained unchanged during pregnancy.In women with different thrombophilia receiving LMWH at prophylactic dose a significant increase in MCF was accompanied by barely unchanged CT after intrinsic and extrinsic coagulation activation and platelet mediated closure-times in the course of the pregnancy. Decrease in CFT was only seen after extrinsic coagulation activation, whereas unchanged CFT after intrinsic coagulation activation may be the result of LMWH given at low dose.

Circulating Procoagulant Microparticles with Coagulation Factors IXa and XIa Cooperatively Induce Spontaneous Clotting in Plasmas of Patients with Hematological Disorders

AbstractAbstract 3402 Background & Objective.Although blood of patients with hematological disorders is known to contain procoagulant material (activated clotting factors, tissue factor, microparticles), its functional significance is poorly understood. Using an in vitro experimental model of immobilized tissue factor-initiated clot growth in platelet-free plasma, we observed formation of activation-surface-independent isolated “spontaneous” clots (SC) throughout the plasma volume in patients with various disorders. The aim of this work was to identify the mechanisms of SC formation. Methods.Platelet-free plasma was prepared from citrated whole blood collected by venipuncture. It was supplemented with corn trypsin inhibitor (CTI), recalcified, placed into a thin flat chamber and brought into contact with a surface covered by immobilized tissue factor. Fibrin clot formation as a function of space and time was determined by imaging light scattering. with a CCD camera1 in a thrombodynamics analyzer device (Hemacore LLC, Moscow, Russia). Flow cytometry was used to monitor annexin V-labeled microparticles and platelets in plasma. Results.SC were reproducibly observed in plasmas of patients with various disoders including: 4 of 32 patients with ischemic cardiomyopathy, 11 of 29 patients with lymphogranulomatosis during chemotherary, 2 of 5 patients with acute leukemia. SC also appeared in 2 of 36 healthy donor plasmas.To determine contribution of intrinsic and extrinsic coagulation activation pathways, we blocked them with corn trypsin inhibitor (CTI) and active site-inhibited recombinant factor VIIa, respectively. Tissue factor-dependent activation was essential in 1 of 10 patients tested in this way. CTI inhibited SC in all samples, but never completely (Fig. 1).To test whether circulating active factors can induce SC, we supplemented normal plasma with factors Va, VIIa, IXa, or XIa known to have long lifetimes (more than 30 min). Addition of factors Va and VIIa led to uniform coagulation thoughout the plasma volume, which did not resemble typical SC patterns observed in the patient plasma (Fig. 2). In contrast, factors IXa and XIa induced isolated clotting centers very similar to those observed in the clinical samples.SC both in patient plasma and in normal plasma supplemented with factors IXa and XIa disappeared after centrifugation for 30 min at 16000 g, indicating also a contribution of microparticles. This was confirmed by inductions of SC in the bottom layers of normal plasma subjected to identical centrifugation. After phospholipid supplementation, SC reappeared in normal plasma supplemented with factors IXa or XIa, as well as in 3 out of 11 patient plasmas. Flow cytometry revealed an increase in procoagulant microparticles in patient plasmas with SC. Conclusions.Here we report observation of a SC phenomenon in vitro in plasma of hematology patients. Although its mechanisms are likely to be different in different illnesses, our data suggest that combination of circulating active factors (specifically, factors IXa and XIa) with circulating procoagulant microparticles is the predominant one. Possible association between the presence of spontaneous clots and clinical manifestations require further study, but prevalence of this phenomenon in the patient plasma indicates possible applicability in diagnostics and. [Display omitted] [Display omitted] Disclosures:Lipets:HemaCore LLC: Employment. Ataullakhanov:HemaCore LLC: Employment, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Panteleev:HemaCore LLC: Employment, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

Chronic idiopathic urticaria successfully treated by anticoagulant drugs

ejd.2012.1867 Auteur(s) : Philip Spring1 Philipp.spring@chuv.ch, Anne Angelillo-Scherrer2, Paul Bigliardi3 1 Dermatology unit, 2 Service and Central Laboratory of Hematology, CHU Vaudois, Beaumont 29, 1011 Lausanne, Switzerland 3 Department of Dermatology/Allergology, National University of Singapore, Singapore Chronic Idiopathic Urticaria (CIU) is a debilitating disease. Despite an extensive workup, the cause is often not identified. Intrinsic and extrinsic coagulation activation has been recently proposed [...]

Determination of the anti-F10a or anti-F2a generation action of rivaroxaban or dabigatran

Dabigatran and rivaroxaban are new oral inhibitors of the thrombin (F2a) and F10a, respectively. Both inhibitors decrease the extrinsic or intrinsic generation of F10a/F2a in plasma. An innovative test for extrinsic F10a/F2a generation is the extrinsic coagulation activity assay (EXCA). With the EXCA the action of all F10a/F2a generation inhibitors can be quantified. The present work aimed to establish the mean therapeutic ranges of these two new drugs, as determined in the EXCA. Forty microlitres reconstituted lyophilized commercial pooled normal plasma, that had been supplemented with 0-0.48 mg/l dabigatran or rivaroxaban, in high-quality polystyrene U-wells (Brand781600), were incubated with 4 μl 1 ng/ml human placental tissue factor in 5% human albumin, 250 mmol/l CaCl2 (EXCA-trigger) for 1 min (EXCA-1) or 2 min (EXCA-2) at 37°C. 80 μl 2.5 mol/l arginine, 0.16% Triton X 100, pH 8.6, were added. After 3 min, 20 μl 1 mmol/l CHG-Ala-Arg-pNA in 1.25 mol/l arginine was added and ΔA/t was determined (37°C). The mean therapeutic range (10-20% EXCA) of dabigatran is approximately 0.05-0.1 mg/l. The mean therapeutic range (10-20% EXCA) of rivaroxaban is approximately 0.13-0.22 mg/l. Mean prophylactic concentrations (20-40% EXCA) are approximately 0.02-0.05 mg/l dabigatran and approximately 0.05-0.13 mg/l rivaroxaban. These are the mean ranges. Individual plasma might well behave differently: either an individual patient could be more sensible or more resistant towards one or both of these drugs. It is suggested to perform EXCA tests for each individual patient to determine the real drug dosage he needs to reach 10-20% of normal EXCA for therapy or 20-40% of normal EXCA for prophylaxis.

FVII, FVIIa, and Downstream Markers of Extrinsic Pathway Activation Differ by EPCR Ser219Gly Variant in Healthy Men

The purpose of this study was to determine the effect of a variant in EPCR (Ser219Gly), previously shown to affect EPCR shedding, on plasma FVII, FVIIa, and downstream markers of activated coagulation. Statistical analysis was undertaken in approximately 2000 healthy middle aged men (NPHSII). Higher soluble EPCR levels were confirmed for Gly allele carriers (P<0.0001). Significantly higher levels of FVII, FVIIa, and downstream markers of activated coagulation in the extrinsic pathway (FIX activation pep [FIXpep]; FX activation pep [FXpep]), and prothrombin F1+2 (F1+2) were identified in baseline samples, in Gly carriers compared to Ser/Ser (P<or=0.04 for trend). In repeat samples collected for up to 5 years, levels of FVII and F1+2 were higher in Gly allele carriers compared to Ser/Ser by (FVII: 6.9% CI 5.5 to 8.4 in Ser/Gly; and 23.4% CI 16.3 to 30.8 in Gly/Gly, P<0.0001), (F1+2: 8.1% CI 5.2 to 11.1 in Ser/Gly; 25.2% CI 11.8 to 40.3 in Gly/Gly, P<0.04), confirming reproducibility of findings at baseline. Molar ratios for FIXpep, FXpep, and F1+2 to FVIIa were constant in Ser/Ser and Ser/Gly but tended to be higher in Gly/Gly, reaching statistical significance for FIXpep:FVIIa (P=0.04). These data suggest that higher levels of FVII and FVIIa circulate when EPCR shedding is greatest. Furthermore, these results suggest consequences for activation of extrinsic coagulation.

The Extrinsic Coagulation Activity Assay

A chromogenic assay for the tissue factor-mediated thrombin generation was developed, the extrinsic coagulation activity assay: 50 microL citrated plasma is incubated with 5 microL tissue factor in 6% albumin and 250 mM CaCl2. After 1-minute (37 degrees C) coagulation reaction time, (extrinsic coagulation activity assay with 1-minute coagulation reaction time; generating normally about 1 IU/mL thrombin) 100 microL 2.5 M arginine is added to stop hemostasis activation. Generated thrombin is then chromogenically quantified. The normal extrinsic coagulation activity assay range is 100% +/- 20%. Extrinsic coagulation activity assay in plasma of patients on heparin or coumarines is about 10-fold lower. Advantages of extrinsic coagulation activity assay: normal range of extrinsic hemostasis is truly represented, patients prone to hyper-activated extrinsic pathway are detected, anticoagulants result in respective test inhibition, fibrinogen/fibrin concentration does not artefactually alters the test result, plasma matrix is not changed significantly in the assay, and assay results are IU/mL thrombin or % of normal, which can be measured by every normal photometer.

Innovative Tests of Plasmatic Hemostasis

Up to this time, plasmatic hemostasis tests often did not reflect physiologic hemostasis: the final thrombin (IIa) activity is far above the physiological thrombin activity generated in recalcified clotting plasma, which is about 0.1 to 0.2 IU/mL thrombin. Using final supramolar concentrations of arginine and chromogenic substrate concentrations below 0.6 mM, an ultra-specific and -sensitive determination of thrombin activity in plasma becomes feasible. In this review, 4 recent plasmatic thrombin assays derived from this principle are presented: basal IIa activity (IIa test), recalcified coagulation activity assay (RECA), intrinsic coagulation activity assay (INCA), and extrinsic coagulation activity assay (EXCA). The EXCA is an especially suitable method for determining the anticoagulant power of any plasmatic anticoagulant that acts against

The efficiency of anti-activated factor X and anti-activated factor II anticoagulants

Six thrombin-generation inhibitors or thrombin inhibitors were compared in the extrinsic coagulation activity assay (EXCA), where the normal thrombin generation is about 1 IU/ml within 1 min (37 degrees C). Unfrozen pooled normal citrated plasma was supplemented on flat-bottom wells (23 degrees C) with increasing concentrations of dalteparin, danaparoid, heparin, fondaparinux, hirudin, or argatroban. To 50 microl plasma, 5 microl of 1.5 ng/ml tissue factor, 6% bovine serum albumin, and 250 mmol/l CaCl2 were added. After 1 and 2 min coagulation reaction time at 37 degrees C (EXCA-1 and EXCA-2), 100 microl of 2.5 mol/l arginine and 0.16% Triton X 100, pH 8.6, were added. After 3 min (23 degrees C), 25 microl of 1 mmol/l CHG-Ala-Arg-pNA in 1.25 mol/l arginine, pH 8.7, were added, and the linear increase in absorbance with time was determined at 405 nm. The 50% inhibitory concentrations of plasmatic anticoagulants tested in the EXCA-1 (37 degrees C) were 0.025 IU/ml dalteparin, 0.13 U/ml danaparoid, 0.12 IU/ml heparin, 1.3 microg/ml fondaparinux, 2.4 ng/ml hirudin, and 1 microg/ml argatroban. From the 50% inhibitory concentration of hirudin it can be concluded that inhibition of about 30 mIU/ml thrombin halves the normal EXCA-1 value (i.e. if about 0.1 IU/ml thrombin are inactivated, then thrombin cannot self-amplify its generation 10-fold). The efficiency of any clinically used plasmatic anticoagulant can be monitored in the EXCA.

The anticoagulant capacity of plasmatic unfractionated heparin decreases at 23°C

Unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) are important clinical anticoagulants. As polynegative molecules they are potential triggers of the contact phase of coagulation. An incubation temperature lower than the physiological 37 degrees C favours intrinsic haemostasis activation by the polynegative molecule SiO2. The efficiency of UFH and LMWH after a plasmatic preincubation at 37 or at 23 degrees C is therefore studied. Samples (150 mul) of unfrozen pooled normal plasma supplemented with 0, 0.01, 0.1, or 1 IU/ml heparin or dalteparin in 5-ml polystyrole tubes were incubated for 10-70 min at 37 or at 23 degrees C. The extrinsic coagulation activity assay (EXCA) was then performed. Preincubation at 37 degrees C of 0.1 IU/ml plasmatic UFH does not result in any thrombin generation in EXCA-1, whereas preincubation at 23 degrees C results in a thrombin generation of about 0.1 IU/ml thrombin. Plasmatic UFH (0.01 IU/ml) at 23 degrees C acts nearly half as efficiently as 0.01 IU/ml plasmatic LMWH. Polynegatively charged niches particularly in the larger UFH molecule might trigger the contact system of haemostasis, especially at 23 degrees C. In contrast, the anticoagulant capacity of LMWH does not change significantly with temperature.

Influence of coagulation factors on extrinsic thrombin generation

The extrinsic coagulation activity assay (EXCA) is a new thrombin generation test for the tissue factor pathway of coagulation. The EXCA was performed with 10 parts citrated plasma of different contents of fibrinogen. One part tissue factor, 250 mmol/l CaCl(2), generating about 1 IU/ml thrombin within 1 min (37 degrees C). After 0-30 min 2.5 mol/l arginine (pH 8.6) Generated thrombin was detected by addition of CHG-Ala-Arg-pNA and measurement of triangle upA/t. The EXCA is dependent on factors 10% of the factor VII norm in the sample achieves 70-80% of the thrombin generation norm. The EXCA is not dependent on factors VIII, IX, XI and XII. Even in antithrombin III-deficient plasma, a phase of thrombin inhibition appears after the thrombin peak. Supplemented purified fibrinogen resulted in decreased thrombin generation in the important. Fibrinogen seems to act as antithrombin I; thrombin might be entrapped in the nascent fibrin. The EXCA is suitable to diagnose the level of extrinsic factors in patient plasma.

The changing faces of tissue factor biology

The changing faces of tissue factor biology - A personal tribute to the understanding of the “extrinsic coagulation activation” -