ALBERT

Description: Introduction The rapid coagulation response to vascular injury is mediated by the formation of three enzyme cofactor complexes (extrinsic tenase, intrinsic tenase and prothrombinase) on membrane surfaces. A common structural feature of these proteases is their GLA domains, each of which requires the binding of divalent metal ions at multiple sites to achieve the conformation necessary for optimal membrane and cofactor binding. Both Ca2+ and Mg2+ ions have been reported to bind to GLA domain sites. However almost all studies kinetically characterizing these complexes have been done in the presence of Ca2+ (2-5 mM) as the sole metal ion, despite the relatively equivalent availability in plasma of both free Ca2+ (∼1.1 mM) and Mg2+ (∼0.6 mM) (Ca2+/Mg2+). A recent study has systematically examined the effects of various Ca2+ concentrations with and without Mg2+ on the membrane binding of activated protein C (APC) and FVIIa and enzymatic activity of APC and the extrinsic tenase complex which were enhanced in Ca2+/Mg2+ relative to Ca2+ alone (Vadivel, K., et al, 2013 JMB). In the current study we compare the effects of plasma levels of Ca2+ and Mg2+ versus Ca2+alone on the catalytic performances of the extrinsic tenase, intrinsic tenase and prothrombinase complexes individually and collectively. Methods All experiments were conducted in Hepes buffered saline pH 7.4 containing 0.1% PEG and either 2 mM Ca2+ or 1.1 mM Ca2+/0.6 mM Mg2+ (Ca2+/Mg2+). In closed system experiments, enzyme-cofactor complexes were assembled on phospholipid vesicles composed of a 3:1 ratio of synthetic phosphatidylcholine and phosphatidylserine (PCPS), and zymogen activation monitored via sampling into assay mixtures containing the appropriate chromogenic substrate. In open system experiments complexes were preassembled on PCPS coated capillaries, the zymogen delivered in the flowing phase and the extent of zymogen activation monitored in the effluent as described previously (Haynes, LM., et al, 2011 Biophys J). The combined interaction of the procoagulant enzyme cofactor complexes under both metal ion conditions was studied in a synthetic coagulation proteome monitoring thrombin (IIa) generation as previously described (van’t Veer, C., and Mann, KG, 1997 JBC). Results Extrinsic tenase The extrinsic tenase complex had an approximately two-fold higher rate of FXa generation in the presence of Ca2+/Mg2+ (1.78 ±0.05 pM/s) versus Ca2+ alone (0.88 ± 0.02 pM/s) (N=3, p

Description: BACKGROUND: Trauma is the leading cause of death in individuals under the age of 44. Acute traumatic coagulopathy (ATC) occurs when severe injury is combined with shock and results in a hypocoagulable state resulting in increased bleeding, increased resuscitation requirements and worsened outcomes including a 4-fold increase in mortality. Our group has implicated protein C activation as a likely mechanism for ATC. We have evaluated whether activated protein C (APC) induces coagulopathy through cleavage of factor (f)V/Va by systematically evaluating 4 groups of increasing level of traumatic coagulopathy. METHODS: From a large cohort of plasma samples collected from trauma patients at time of admission to a major level 1 trauma sample, 80 plasma samples distributed equally between the following trauma severity groupings were selected: Group 1: ISS ≤ 15 and BD 〉 -6 (less injured, no shock); Group 2: ISS ≤ 15 and BD ≤ -6 (less injured, with shock); Group 3:ISS 〉 15 and BD 〉 -6 (more injured, no shock); Group 4:ISS 〉 15 and BD ≤ -6 (more injured, with shock). Each plasma sample was evaluated for plasma composition (functional activity of fII, fV, fVII, fVIII, fIX, fX, antithrombin, tissue factor pathway inhibitor, protein C), plasmin-antiplasmin complex (PAP), α-thrombin-antithrombin complex (TAT), and mathematically modeled thrombin generation. Quantitative Western blot analyses were performed using an anti-fV antibody directed at the fV heavy chain (residues 307-506). Intact fV levels were quantified via densitometry to determine whether levels were the within range characterizing healthy individuals and the presence of APC derived and other degradation products assessed via mobility comparison to purified standards. Demographic, resuscitation, and outcomes data were collected in parallel. RESULTS: All data were quantitated and evaluated for having concentrations above and/or below the normal range for each analyte. For PAP, Groups 3 (18/20) and 4 (17/20) had a greater percentage of individuals over Groups 1 (5/20) and 2 (6/20) that were above the normal range. Groups 3 (12/20) and 4 (12/20) also had a higher percentage of detectable α-TAT over Groups 1 (5/20) and 2 (2/20). For fV antigen, Group 4 had 16/20 individuals that were below the lower limit of normal. Groups 1, 2 and 3 were all similar with 5/20 below the limit of normal but had 3-5/20 above the upper limit of normal. Products associated with activated protein C were found (fV 30kD fragment) but also other cleavage products not associated with APC were observed. There was more fV fragmentation in Group 4, with the 30kD fragment being detected in 10 individuals. No detectable fV 30kD fragments were detected in either Groups 1 or 2. In terms of the overall plasma composition there is a large variation in functional activity, especially fVIII. Group 4 showed a pattern of all factors reduced except for TFPI versus Groups 1,2, 3. These pro and anticoagulant differences between individuals results in heterogenous modeled thrombin generation profiles with the majority all being procoagulant versus a normal control. Group 4 had a higher percentage of individuals that had a prolonged clot time and a pattern towards diminished thrombin generation. CONCLUSIONS: These data suggest that the combination of injury and shock were associated with altered PAP, TAT and functional activity changes of the plasma composition resulting in hemorrhagic thrombin generation profiles. In addition the most severely injured and shocked patients had less fVa activity, less intact fV and increased degradation of fV/fVa suggesting the presence of increased APC activity in mediating ATC. Disclosures No relevant conflicts of interest to declare.

Description: Tissue factor (TF) is an integral membrane protein, which is the key initiator of blood coagulation in vivo. Due to the limited availability of natural TF, recombinant proteins of various lengths and origins have been extensively used in research and clinical laboratories worldwide. Experimental results acquired with recombinant TF proteins are frequently used for the understanding of the coagulation processes occurring in vivo, although there is a lack of data confirming the structural and functional identity of natural TF proteins from various sources and recombinant ones. In the current study, human TF from cultured monocytes and purified from placenta were compared with three different species of recombinant TF: 1–218 (extracellular domain only), 1–242 (lacking cytoplasmic domain) and 1–263 (full-length). Anti-TF mAbs gave 93–98% inhibition of TF activity for all TF species tested, in both natural and relipidated preparations. It was established that purified placental TF has a higher affinity for factor VIIa (Kd 0.13 nM) than recombinant counterparts 1–242 and 1–263 (Kd 0.50–0.80 nM). Similarly, placental TF is more efficient in factor X activation by the extrinsic Xase than recombinant TF 1–242 (the second order rate constants are 3.0x107 and 0.7x107 M−1s−1, respectively). We explored the use of these TF species as well as monocyte TF (purified/relipidated and present on LPS-stimulated monocytes) for the initiation of thrombin generation in two in vitro models of blood coagulation. At equimolar concentrations (5 pM; determined by immunoassay), when evaluated in synthetic plasma reconstituted with 2x108/ml platelets, recombinant TF 1-263 provided an initiation phase of ~4 min. Placental TF and relipidated monocyte TF had similar profiles of thrombin generation with an initiation phase of ~3 min. In contrast, 0.5 pM TF on LPS-stimulated monocytes gave an initiation phase of ~1 min. Even at 0.05 pM concentration, monocyte TF was as active as any relipidated protein at 5.0 pM. A similar pattern of relative TF activity was observed in whole blood and plasma PT clotting assays. TF on stimulated monocytes gave the highest activity, exceeding that of any relipidated protein by 100–200-fold. Recombinant TF 1–242 was more active than recombinant TF 1–263 and placental TF in the PT assay but less active in synthetic plasma and whole blood. The lowest overall activity was observed for relipidated monocyte TF. Our data suggest that TF proteins from different sources are different with respect to their functional properties.

Description: Introduction: Effective treatment of HIV infection with antiretroviral therapy (ART) has prolonged survival and shifted causes of death to non-AIDS defining illnesses such as cardiovascular disease (CVD). Increased thrombin generation is evident from increased D-dimer levels in HIV; previously we have shown that inflammation and viral replication associate with imbalances in pro and anticoagulant factors resulting in increased thrombin generation profiles when mathematically modeled. We explore for the first time the hypothesis that factor compositional imbalance increases simulated thrombin generation and predicts mortality in HIV. Methods: In a nested case-control study of the SMART and ESPRIT participants randomized to continuous ART, we evaluated cases of all-cause mortality and controls (1:3) matched on enrollment date, age (± 5yrs), study, and country. Thrombin generation in response to a 5 pM tissue factor initiator for each individual was calculated by a mathematical model incorporating levels of factors II, V, VII, VIII, IX, X, antithrombin, tissue factor pathway inhibitor and protein C. Thrombin generation metrics included time to clot, maximum rate (MaxR), maximum level (MaxL) and area under the curve (AUC). Conditional logistic regression was used to compare parameters between cases and controls, adjusted for age, gender, and race/ethnicity. Results: Among cases (n=129) and controls (n=369), respectively, median CD4 count was 451 and 496 cells/mm3, and 65 and 77% had HIV viral suppression (

Description: Abstract 2239 Introduction. In in vitro models of tissue factor (TF)-initiated coagulation, FXI activation has been linked to increased thrombin generation. However the effects of FXI in experimental models of normal hemostasis have often been subtle, prompting ongoing investigations to define contributing cofactors, potential collaborating activators and/or reaction conditions (e.g. low TF concentrations). In this study, predictions from a computational model of TF-initiated thrombin generation that includes thrombin dependent FXI activation are used to direct an investigation of the role of FXI in two empirical models of TF-initiated coagulation. Methods. FXI activation and FXIa interactions were computationally modeled by adding the appropriate sets of equations describing thrombin activation of FXI, FXIa activation of FIX, antithrombin inhibition of FXIa, and high molecular weight kininogen binding to FXI to the existing framework of differential equations. The efficacy of FXIa in promoting thrombin generation (α- thrombin-antithrombin, αTAT) was assessed via titration in contact pathway inhibited whole blood and compared to activation by TF, FIXa, FXa and α-thrombin. TF-initiated reactions and their resupply were performed as described previously (Orfeo T et al. J. Biol. Chem., 2008) in either contact pathway inhibited blood ± an inhibitory anti-FXI antibody) or in synthetic coagulation proteome (SCP) mixtures (± FXI), allowing FXI effects on both the TF dependent phase and the resulting procoagulant pool of catalysts to be evaluated. Results. The computational model (± FXI pathway) was validated by showing congruence between computational thrombin generation profiles initiated with 5 pM TF and corresponding SCP reconstructions, which showed the lag phase shortened by 30 to 60 s and maximum thrombin levels increased when FXI was present. Similarly there was good correspondence between computational and SCP thrombin generation when FXIa (4 pM by active site) was the initiator. A computational analysis provided the following ranking of effectiveness in initiating thrombin generation: FXIa〉TF〉FIXa〉FXa〉α-thrombin. When tested in contact pathway inhibited blood, addition of 5 pM FXIa (by active site) resulted in clot times and 20 min αTAT levels similar to those observed with 5 pM TF, while similar outcomes required 25 pM FIXa, 100 pM FXa or between 10–100 nM α-thrombin. The computational modeling made clear two consequences of FXI feedback activation. The first was mechanistic, demonstrating that the amplification of thrombin generation was achieved by better coordinating the initial activations of FIX and FVIII; limited early initiation phase activation of FXI (

Description: There are several hypotheses which attempt to explain changes in tissue factor (TF) activity on cell surfaces upon treatment with various agents (“encryption-de-encryption”). However, these hypotheses are mostly based upon conjectural data interpretation. We evaluated the influence of lipopolysaccharide (LPS) stimulation on the surface and total expression of TF antigen on/in THP-1 monocytic cells and the corresponding TF activity. A cell-based immunoassay was used for the quantitation of TF antigen on the surface of monocytic cells, coupled with contact pathway-inhibited (corn trypsin inhibitor; CTI) plasma and blood dilute PT, extrinsic factor (F)Xase and synthetic coagulation proteome assays. Prior to stimulation, the concentration of TF was 240±60 molecules/cell on the cell surface and 510±180 total molecules/cell in cell lysates (n=8). Upon stimulation with LPS (1μg/ml), the TF antigen concentration steadily increased reaching a maximum at 4 hr of stimulation (2400±360 molecules/cell on the cell surface and 8400±360 molecules/cell total). Coincidently, the intact cell FXa generation rate increased from 4.2 pM/s to 46.3 pM/s. The clotting time of multi-donor plasma decreased from 192s to 80s corresponding to a 35.7±4.9-fold increase in TF activity. Thus all assays are consistent with increased surface expression of TF. Kinetic studies showed that the KM of the complex enzyme (TF/FVIIa) formed on the surface of LPS-stimulated monocytes for FX is 0.73±0.07 μM and the kcat 59.4±1.8 s−1. The kinetic properties for the FXase formed in lysates of LPS-stimulated THP-1 cells are similar to those observed for the cell surface TF (see Table 1). However, when LPS-stimulated monocyte TF was purified to homogeneity and relipidated using synthetic phospholipid vesicles (PCPS), the efficiency for FX activation was significantly lower due to both an increased KM and decreased kcat. As a consequence, the second order rate constant for FX activation by the extrinsic FXase on the surface of PCPS was only 8±1% of that observed on stimulated intact cells or lysates. A comparison of purified and relipidated monocyte TF with that cell-bound in situ in the synthetic coagulation proteome with platelets at mean physiologic concentration (2x108/ml) showed that thrombin generation profiles were similar for the reactions initiated with 5 pM relipidated TF and 0.05 pM TF expressed on the cell surface; a difference in specific activity of approximately 100-fold. Similar results were observed in CTI-inhibited blood. CTI blood, triggered with 5 pM relipidated monocyte TF clotted in 310 s, whereas initiation with 1.2 pM monocyte TF in situ led to a 40 s clotting time with maximum rates of thrombin generation of 1.3 and 〉7.0 nM/s, respectively. These data indicate that the increase in monocyte TF activity upon stimulation with LPS is related to an increased expression of TF protein on/in monocytic cells and that there are components of the cell membrane which substantially enhance TF activity and these accessories maintain their function(s) in cell lysates. These components are not represented by synthetic (PCPS) phospholipid vesicles. Our data emphasize the importance of cell membrane composition on TF activity and are in agreement with observations published by Pendurthi et al. (Blood2007;110:3900). Table 1. Monocytic TF in the extrinsic FXase Preparation of TF KM(μM) kcat(s−1) kcat/KM((μM•s−1) In situ 0.73±0.0 59.4±1.8 81.4 In lysates 0.41±0.0 44.6±0.1 108.8 On PCPS 1.54±0.14 12.0±0.4 7.8

Description: Several factor VIII products, recombinant and natural, have been used for hemophilia A treatment worldwide. Typically, two activity-based assays (factor Xase and aPTT) are used for the assessment of factor VIII concentration in these products. Frequently, the results are dependent upon the assay and its modifications in different laboratories. In this study, we evaluated five pharmacologic factor VIII products (three lots of each) in three activity-based assays and in two immunoassays for the concentration and activity of factor VIII protein. Two factor VIII products were plasma-derived (Immunate and Hemofil M) and three were recombinant; two of these contained full-length factor VIII (Recombinate and Kogenate) and one was B-domainless (ReFacto). Albumin-free full-length recombinant factor VIII was used as a standard in all assays. In the factor Xase assay, all recombinant factor VIII products and Immunate at 1U/ml (indicated by manufacturer) showed activity similar to that of 0.7nM (1U/ml) standard, whereas activity of Hemofil M was 64–68% of the standard. In the aPTT assay both full-length recombinant products and Hemofil M displayed activity similar to the standard, whereas Immunate had increased (142% of standard) and ReFacto decreased (83% of standard) activity. In synthetic plasma, all three recombinant products had standard-like activity, whereas Hemofil M and Immunate were slightly more active than standard. The ELISA immunoassay revealed that the factor VIII protein content in Recombinate, Kogenate and Hemofil M corresponded to the units assigned by manufacturers (1.4–1.6x1012U/mol vs1.4x1012U/mol calculated for standard), whereas the specific activity of Immunate was 50% of that expected (0.7x1012U/mol). In contrast, the specific activity of ReFacto was almost 3-fold that of full-length factor VIII (4.0x1012U/mol). The data of this study indicate that: 1) factor VIII activity estimated in different assays gives dissimilar results; 2) the specific activity of factor VIII in various factor VIII products is different and, as a consequence, administration of an equal factor VIII activity in U/ml means the administration of different amounts of factor VIII protein.