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Background Recent data propose a diagnostic and prognostic capacity for citrullinated histone H3 (H3Cit), a marker of neutrophil extracellular traps (NETs), in pathologic conditions such as cancer and thrombosis. However, current research is hampered by lack of standardized assays. Objectives We aimed to develop an assay to reliably quantify nucleosomal H3Cit in human plasma. Methods We assessed the common practice of in vitro enzymatically modified histone H3 as calibration standards and the specificity of available intrapeptidyl citrulline antibodies. Based on our findings, we developed and validated a novel assay to quantify nucleosomal H3Cit in human plasma. Results We show that enzymatically citrullinated H3 proteins are compromised by high enzyme-dependent lot variability as well as instability in plasma. We furthermore demonstrate that the majority of commercially available antibodies against intrapeptidyl citrulline display poor specificity for their reported target when tested against a panel of semi-synthetic nucleosomes containing distinct histone H3 citrullinations. Finally, we present a novel assay utilizing highly specific monoclonal antibodies and semi-synthetic nucleosomes containing citrulline in place of arginine at histone H3, arginine residues 2, 8, and 17 (H3R2,8,17Cit) as calibration standards. Rigorous validation of this assay shows its capacity to accurately and reliably quantify nucleosomal H3Cit levels in human plasma with clear elevations in cancer patients compared to healthy individuals. Conclusions Our novel approach using defined nucleosome controls enables reliable quantification of H3Cit in human plasma. This assay will be broadly applicable to study the role of histone citrullination in disease and its utility as a biomarker.

Significance DNA methylation and histone posttranslational modifications are key epigenetic marks that contribute to the fine-tuned regulation of gene expression and other chromatin-templated biological processes. Here, we build artificial chromatin templates and reveal key chromatin structural features and epigenetic marks that coordinately regulate the binding and enzymatic activity of the DNA methylation regulator UHRF1. Studying activities of epigenetic regulators in the context of defined chromatin templates, particularly for multidomain histone and DNA binding proteins such as UHRF1, is critical for understanding molecular mechanisms of epigenetic crosstalk and mechanics regulating epigenetic signaling, and for determining how epigenetic dysregulation contributes to human disease., Mitotic inheritance of DNA methylation patterns is facilitated by UHRF1, a DNA- and histone-binding E3 ubiquitin ligase that helps recruit the maintenance DNA methyltransferase DNMT1 to replicating chromatin. The DNA methylation maintenance function of UHRF1 is dependent on its ability to bind chromatin, where it facilitates monoubiquitination of histone H3 at lysines 18 and 23, a docking site for DNMT1. Because of technical limitations, this model of UHRF1-dependent DNA methylation inheritance has been constructed largely based on genetics and biochemical observations querying methylated DNA oligonucleotides, synthetic histone peptides, and heterogeneous chromatin extracted from cells. Here, we construct semisynthetic mononucleosomes harboring defined histone and DNA modifications and perform rigorous analysis of UHRF1 binding and enzymatic activity with these reagents. We show that multivalent engagement of nucleosomal linker DNA and dimethylated lysine 9 on histone H3 directs UHRF1 ubiquitin ligase activity toward histone substrates. Notably, we reveal a molecular switch, stimulated by recognition of hemimethylated DNA, which redirects UHRF1 ubiquitin ligase activity away from histones in favor of robust autoubiquitination. Our studies support a noncompetitive model for UHRF1 and DNMT1 chromatin recruitment to replicating chromatin and define a role for hemimethylated linker DNA as a regulator of UHRF1 ubiquitin ligase substrate selectivity.

Background The global supply of unfractionated heparin (UFH) and all commercially available low molecular weight heparins (LMWH) remain dependent on animal sources, such as porcine intestine or bovine lung. Recent experience has shown that contamination of the supply chain (with over-sulfated chondroitin sulfates) can result in lethal toxicity. Fondaparinux is currently the only commercially available synthetic analog of heparin. We recently described a new class of chemoenzymatically synthesized heparin analogs. One of these compounds (S12-mer) is a dodecasaccharide consisting of an antithrombin-binding moiety with repeating units of IdoA2S-GlcNS6S and two 3-O-sulfate groups that confer the ability to bind protamine. Objective/methods We sought to further characterize this new compound in vitro using biochemical and global coagulation assays and in vivo using thrombosis and hemostasis assays. Results The anticoagulant activities of the Super 12-mer (S12-mer) and Enoxaparin in anti-factor Xa and plasma-based thrombin generation assays were roughly equivalent with a 50% reduction in peak thrombin generation occurring at approximately 325nM. When protamine was titrated against a fixed concentration of S12-mer in plasma or blood, the S12-mer displayed a significant restitution of thrombin generation and clot formation. In vivo , S12-mer inhibited venous thrombosis to a similar extent as Enoxaparin, with similar bleeding profiles. Conclusions These data show that the S12-mer has almost identical efficacy to Enoxaparin in terms of FXa inhibition, while displaying significant reversibility with protamine. Taken together with the ability to ensure purity and homogeneity from batch to batch, the S12-mer is a promising new synthetic heparin analog with a potentially enhanced safety profile.

Expression of tissue factor (TF) antigen and activity in platelets is controversial and dependent upon the laboratory and reagents used. Two forms of TF were described: an oxidized functional form and a reduced nonfunctional form that is converted to the active form through the formation of an allosteric disulfide. This study tests the hypothesis that the discrepancies regarding platelet TF expression are due to differential expression of the two forms.Specific reagents that recognize both oxidized and reduced TF were used in flow cytometry of unactivated and activated platelets and western blotting of whole platelet lysates. TF-dependent activity measurements were used to confirm the results.Western blotting analyses of placental TF demonstrated that, in contrast to anti-TF#5, which is directed against the oxidized form of TF, a sheep anti-human TF polyclonal antibody recognizes both the reduced and oxidized forms. Flow cytometric analyses demonstrated that the sheep antibody did not react with the surface of unactivated platelets or platelets activated with thrombin receptor agonist peptide, PAR-1. This observation was confirmed using biotinylated active site-blocked factor (F)VIIa: no binding was observed. Likewise, neither form of TF was detected by western blotting of whole platelet lysates with sheep anti-hTF. Consistent with these observations, no FXa or FIXa generation by FVIIa was detected at the surface of these platelets. Similarly, no TF-related activity was observed in whole blood using thromboelastography.Platelets from healthy donors do not express either oxidized (functional) or reduced (nonfunctional) forms of TF.

Red blood cells have historically been viewed as innocent bystanders in the process of blood coagulation and thrombin generation; however a century of clinical evidence linking red blood cells to thrombosis suggests the contrary. In this brief review, the biochemical evidence for red blood cell involvement in thrombin generation is evaluated. It is concluded that in addition to platelets, red blood cells actively participate in thrombin generation. A sub-fraction of red blood cells express phosphatidylserine on their surface and unlike platelets, red blood cells produce thrombin through the meizothrombin pathway, which has interesting consequences in the context of clot formation and stabilization.

NETosis is a physiologic process in which neutrophils release their nuclear material in the form of neutrophil extracellular traps (NETs). NETs have been reported to directly promote thrombosis in animal models. Although the effects of purified NET components including DNA, histone proteins, and neutrophil enzymes on coagulation have been characterized, the mechanism by which intact NETs promote thrombosis is largely unknown. In this study, human neutrophils were stimulated to produce NETs in platelet-free plasma (PFP) or in buffer using phorbol myristate actetate or calcium ionophore. DNA and histone proteins were also separately purified from normal human neutrophils and used to reconstitute chromatin using a salt-gradient dialysis method. Neutrophil stimulation resulted in robust NET release. In recalcified PFP, purified DNA triggered contact-dependent thrombin generation (TG) and amplified TG initiated by low concentrations of tissue factor. Similarly, in a buffer milieu, DNA initiated the contact pathway and amplified thrombin-dependent factor XI activation. Recombinant human histones H3 and H4 triggered TG in recalcified human plasma in a platelet-dependent manner. In contrast, neither intact NETs, reconstituted chromatin, individual nucleosome particles, nor octameric core histones reproduced any of these procoagulant effects. We conclude that unlike DNA or individual histone proteins, human intact NETs do not directly initiate or amplify coagulation in vitro. This difference is likely explained by the complex histone-histone and histone-DNA interactions within the nucleosome unit and higher-order supercoiled chromatin leading to neutralization of the negative charges on polyanionic DNA and modification of the binding properties of individual histone proteins.

Introduction Cancer patients have a 4- to 7- fold increased risk of venous thromboembolism (VTE) compared with general population. Most tumor cells express tissue factor (TF) and constitutively release small membrane microvesicles called tumor microvesicles (TMVs). Clinical studies have shown that circulating MP-TF activity is associated with VTE in pancreatic cancer but not in other types of cancer. Thrombin is a potent platelet agonist and activates platelets via protease activated receptors (PARs). Aim To determine the contribution of the TF + TMV-thrombin-platelet pathway to cancer-associated thrombosis. Materials and Methods A human pancreatic adenocarcinoma cell line expressing high levels of TF (BxPc-3) was selected to study the effect of TF + TMVs on platelet activation and thrombosis. Results TF + TMVs induced platelet activation in vitro in a thrombin-dependent manner. The presence of orthotopically grown BxPc-3 tumors in mice was associated with increased levels of thrombin-antithrombin III complexes (TATc) and larger thrombi in an inferior vena cava stenosis model compared with control mice. Furthermore, injection of BxPc-3 TF + TMVs into mice triggered platelet activation and enhanced venous thrombosis in a TF-dependent manner. Importantly, BxPc-3 TF + TMV-enhanced thrombosis was reduced in Par4-deficient mice and wild-type mice treated with the platelet inhibitor clopidogrel, suggesting that platelet activation was required for the enhanced thrombosis. Conclusions These studies suggest that platelet inhibitors may reduce thrombosis in cancer patients with elevated levels of TF + TMVs.

Summary. Background: Replacement therapy for hemophilic patient treatment is costly, because of the high price of pharmacologic products, and is not affordable for the majority of patients in developing countries. Objective: To generate and evaluate low molecular weight agents that could be useful for hemophilia treatment. Methods: Potential agents were generated by synthesizing specific inhibitors [6-(Lys-Lys-Thr-[homo]Arg)amino-2-(Lys[carbobenzoxy]-Lys[carbobenzoxy]-O-benzyl)naphthalenesulfonamide] (PNASN-1)] for activated protein C (APC) and tested in plasma and fresh blood from hemophilia A patients. Results: In the activated partial thromboplastin time-based APC resistance assay, PNASN-1 partially neutralized the effect of APC. In calibrated automated thrombography, PNASN-1 neutralized the effect of APC on thrombin generation in normal and congenital factor VIII-deficient plasma (FVIII:C

Haemophilia A individuals displaying a similar genetic defect have heterogeneous clinical phenotypes. Our objective was to evaluate the underlying effect of exogenous factor (f)VIII on tissue factor (Tf)-initiated blood coagulation in severe haemophilia utilizing both empirical and computational models. We investigated twenty-five clinically severe haemophilia A patients. All individuals were on fVIII prophylaxis and had not received fVIII from 0.25 to 4 days prior to phlebotomy. Coagulation was initiated by the addition of Tf to contact-pathway inhibited whole blood ± an anti-fVIII antibody. Aliquots were quenched over 20 min and analyzed for thrombin generation and fibrin formation. Coagulation factor levels were obtained and used to computationally predict thrombin generation with fVIII set to either zero or its value at the time of the draw. As a result of prophylactic fVIII, at the time of the blood draw, the individuals had fVIII levels that ranged from

Essentials Sickle cell disease is increasingly being recognized as a chronic hypercoagulable state. Thrombin generation is elevated in the whole blood, but not the plasma of sickle cell patients. Whole blood thrombin generation inversely correlates to erythrocyte phosphatidylserine exposure. Acquired protein S deficiency is likely explained by binding of protein S to sickle red cells. Click to hear Dr Hillery discuss coagulation and vascular pathologies in mouse models of sickle cell disease. SummaryIntroduction Sickle cell disease (SCD) is a hypercoagulable state with chronic activation of coagulation and an increased incidence of thromboembolic events. However, although plasma pre-thrombotic markers such as thrombin-anithrombin complexes and D-dimer are elevated, there is no consensus on whether global assays of thrombin generation in plasma are abnormal in patients with SCD. Based on our recent observation that normal red blood cells (RBCs) contribute to thrombin generation in whole blood, we hypothesized that the cellular components in blood (notably phosphatidylserine-expressing erythrocytes) contribute to enhanced thrombin generation in SCD. Methods Whole blood and plasma thrombin generation assays were performed on blood samples from 25 SCD patients in a non-crisis ‘steady state’ and 25 healthy race-matched controls. Results Whole blood thrombin generation was significantly elevated in SCD, whereas plasma thrombin generation was paradoxically reduced compared with controls. Surprisingly, whole blood and plasma thrombin generation were both negatively correlated with phosphatidylserine exposure on RBCs. Plasma thrombin generation in the presence of exogenous activated protein C or soluble thrombomodulin revealed deficiencies in the protein C/S anticoagulant pathway in SCD. These global changes were associated with significantly lower plasma protein S activity in SCD that correlated inversely with RBC phosphatidylserine exposure. Conclusion Increased RBC phosphatidylserine exposure in SCD is associated with acquired protein S deficiency. In addition, these data suggest a cellular contribution to thrombin generation in SCD (other than RBC phosphatidylserine exposure) that explains the elevated thrombin generation in whole blood.

Essentials Cancer patients have a high rate of venous thrombosis (VT) but the underlying mechanisms are unknown. Tumor-derived, tissue factor-positive microvesicles in platelet activation in vitro and in vivo were studied. Tumor-derived, tissue factor-positive microvesicles enhanced VT in mice. Platelets may contribute to VT in some cancer patients, and this could be prevented with antiplatelet drugs. SummaryBackground Cancer patients have an approximately 4-fold increased risk of venous thromboembolism (VTE) compared with the general population, and cancer patients with VTE have reduced survival. Tumor cells constitutively release small membrane vesicles called microvesicles (MVs) that may contribute to thrombosis in cancer patients. Clinical studies have shown that levels of circulating tumor–derived, tissue factor–positive (TF+) MVs in pancreatic cancer patients are associated with VTE. Objectives We tested the hypothesis that TF+ tumor–derived MVs (TMVs) activate platelets in vitro and in mice. Materials and methods We selected two human pancreatic adenocarcinoma cell lines expressing high (BxPc-3) and low (L3.6pl) levels of TF as models to study the effect of TF+ TMVs on platelets and thrombosis. Results and conclusions We found that both types of TF+ TMVs activated human platelets and induced aggregation in vitro in a TF and thrombin-dependent manner. Further, injection of BxPc-3 TF+ TMVs triggered platelet activation in vivo and enhanced thrombosis in two mouse models of venous thrombosis in a TF-dependent manner. Importantly, BxPc-3 TF+ TMV-enhanced thrombosis was reduced in Par4-deficient mice and in wild-type mice treated with clopidogrel, suggesting that platelet activation was required for enhanced thrombosis. These studies suggest that TF+ TMV-induced platelet activation contributes to thrombosis in cancer patients.

Trauma is the leading cause of death in the younger population in the United States, frequently from the development of hemorrhagic shock. Controversy exists over the type of volume resuscitation for restoring hemodynamic stability that should be used in hemorrhagic shock. Little is known about how various resuscitative paradigms affect the coagulation cascade, which is essential to controlling hemorrhagic shock.We studied the effect of various resuscitative formulas on blood coagulation using a new model of whole blood in a controlled setting with corn trypsin inhibitor and a 5-pM stimulus of tissue factor. We investigated thrombin generation, fibrin formation, and platelet activation with four diluents: 0.9% NaCl (NS), lactated Ringer's solution (LR), 6% hydroxyethyl starch (HES), and 3% NaCl (HS), each from 0% to 75% blood dilution. Thrombin generation was measured periodically during a time course of 20 minutes in its complex with antithrombin III. Platelet activation and fibrinopeptide A (FPA) release were monitored in serum at a 20-minute time point. Fibrin clots were collected and weighed.The coagulation markers (thrombin generation, platelet activation, and FPA release) were significantly different by dilution (p0.001 in all) and diluent by dilution (p0.001 in all). Thrombin generation, platelet activation, and FPA release decreased the least with the diluents NS and LR. LR caused the least amount of variation in thrombin generation over the dilution course. HS produced the most dramatic change in all of the markers; no coagulation was seen between 30% to 75% dilution (p0.05). HES produced greater decreases in thrombin generation and FPA release than NS and LR. Fibrin clot mass decreased with a 10% to 20% dilution for NS and LR, whereas stable fibrin mass did not decrease with the diluents HES and HS at 10% to 20% dilutions. At30% dilutions, HS produced no stable clots and HES dramatically decreased clot formation by 61% and maintained this level.LR and NS had the least effect on thrombin generation, clot formation, and platelet activation at various concentrations compared with HES and HS. This observational data suggests that volume expanders such as HES and HS may be detrimental in treatment of hemorrhagic shock.

Homozygous sickle-cell disease (HbSS; SCD) is associated with vaso-occlusive manifestations of varying severity [1]. Pain crises are generally believed to result from obstruction of the microvasculature secondary to adhesion of red blood cells (RBCs) and other cellular elements, and decreased deformability of hypoxia-induced sickled RBCs, with ensuing activation of coagulation and inflammatory pathways [2, 3]. One of the proposed contributors to thrombosis in SCD is the loss of normal phospholipid asymmetry on sickled RBCs due to the repeated process of hypoxia-induced sickling and unsickling [4]. The exposure of anionic phosphatidylserine (PS) on the outer membrane supports the assembly of enzymatic clotting reactions, leading to a sub-population of RBCs with a prothrombotic phenotype [5, 6]. We recently demonstrated that in healthy individuals, a subpopulation (∼0.5%) of PS-expressing RBCs contribute a significant fraction (∼40%) of the total thrombin generating potential of blood [7]. Prothrombin activation requires factor Xa to perform two proteolytic cleavages at Arg 271 and Arg 320 to release the active α-thrombin (αIIa) product [8]. Depending on the order of proteolysis, prothrombin activation can occur via two possible intermediates; meizothrombin (mIIa), an active enzyme; or prethrombin-2 (pre2), a non-enzymatic intermediate [9]. Unlike platelets, which support thrombin generation exclusively through the pre2 intermediate [10], this subpopulation of procoagulant RBCs supports prothrombin activation via the mIIa intermediate in a manner similar to that on synthetic phospholipids [7]. mIIa is of interest because it exhibits the anticoagulant functions of αIIa towards protein C activation, while lacking any significant activity towards procoagulant substrates like fibrinogen, FV and platelets [11]. Due to the markedly enhanced PS expression by sickled RBCs, we therefore hypothesized that the total αIIa generation potential, as well as mIIa production, would be significantly increased in the whole blood of SCD patients compared to healthy controls. To test this hypothesis, we recruited 7 outpatients (4 female and 3 male, age 28-51) with HbSS, in their non-crisis, “steady states,” and 6 healthy African-American controls (3 female and 3 male age 24-38). None of the patients or controls was being treated currently with anticoagulant or anti-platelet therapy, and all SCD patients were at least 3 months remote from red cell transfusion or hospital admission for pain crisis. We utilized our immunoassays that are capable of selective quantitation of αIIa-antithrombin (αTAT) and mIIa-antithrombin (mTAT) to measure the relative production of the two species produced in SCD vs. control individuals after tissue factor (TF)-initiated coagulation. To examine the thrombin generation potential of the HbSS cohort vs. that of the control group, whole blood was subjected to a 5 pM TF stimulus in the presence of 0.1 mg/mL of corn trypsin inhibitor. Quenched time course samples were subsequently analyzed using αTAT and mTAT ELISAs. Figure 1A displays the time course data for αTAT generation in the HbSS and control cohorts. The control cohort clotted on average at 3.8±0.2 min (mean±SEM), generated αTAT at a rate of 63±3.9 nM/min and reached a maximum αTAT level of 502±14 nM. Unexpectedly, clot time (3.98±0.2 min) and the maximum level (515±49 nM) of αTAT generated in the HbSS cohort were similar to that observed in the controls while the rate of αTAT generation at 74.1±7.9 nM/min was only 15% higher (P>0.05). Figure 1 αTAT and mTAT ELISA analyses of HbSS and Control groups. Figure 1B displays the time course data for mTAT generation. As reported previously [7], the observed mTAT levels were significantly less than αTAT due to the lability of the mIIa intermediate. The control cohort generated mTAT at an average maximum rate of 1.02±0.13 nM/min and reached an average maximum level of 6.5±0.52 nM. The HbSS cohort generated mTAT 33% faster (1.51±0.12 nM/min) and reached a maximum level of mTAT (10.1±0.44 nM) that was 36% higher than that observed in the control group. Thus, unlike the case with αTAT, a significant difference (mTAT max level P

The Cdc42p GTPase regulates multiple signal transduction pathways through its interactions with downstream effectors. Specific functional domains within Cdc42p are required for guanine-nucleotide binding, interactions with downstream effectors, and membrane localization. However, little is known about how Cdc42p is clustered at polarized growth sites or is extracted from membranes by Rho guanine-nucleotide dissociation inhibitors (RhoGDIs) at specific times in the cell cycle. To address these points, localization studies were performed in Saccharomyces cerevisiae using green fluorescent protein (GFP)-tagged Cdc42p and the RhoGDI Rdi1p. GFP-Rdi1p localized to polarized growth sites at specific times of the cell cycle but not to other sites of Cdc42p localization. Overexpression of Rdi1p led to loss of GFP-Cdc42p from internal and plasma membranes. This effect was mediated through the Cdc42p Rho-insert domain, which was also implicated in interactions with the Bni1p scaffold protein. These data suggested that Rdi1p functions in cell cycle-specific Cdc42p membrane detachment. Additional genetic and time-lapse microscopy analyses implicated nucleotide binding in the clustering of Cdc42p. Taken together, these results provide insight into the complicated nature of the relationships between Cdc42p localization, nucleotide binding, and protein-protein interactions.

Prothrombin activation can proceed through the intermediates meizothrombin or prethrombin-2. To assess the contributions that these 2 intermediates make to prothrombin activation in tissue factor (Tf)–activated blood, immunoassays were developed that measure the meizothrombin antithrombin (mTAT) and α-thrombin antithrombin (αTAT) complexes. We determined that Tf-activated blood produced both αTAT and mTAT. The presence of mTAT suggested that nonplatelet surfaces were contributing to approximately 35% of prothrombin activation. Corn trypsin inhibitor–treated blood was fractionated to yield red blood cells (RBCs), platelet-rich plasma (PRP), platelet-poor plasma (PPP), and buffy coat. Compared with blood, PRP reconstituted with PPP to a physiologic platelet concentration showed a 2-fold prolongation in the initiation phase and a marked decrease in the rate and extent of αTAT formation. Only the addition of RBCs to PRP was capable of normalizing αTAT generation. FACS on glycophorin A–positive cells showed that approximately 0.6% of the RBC population expresses phosphatidylserine and binds prothrombinase (FITC Xa·factor Va). These data indicate that RBCs participate in thrombin generation in Tf-activated blood, producing a membrane that supports prothrombin activation through the meizothrombin pathway.

Background Despite trauma-induced hypothermic coagulopathy being familiar in the clinical setting, empirical experimentation concerning this phenomenon is lacking. In this study, we investigated the effects of hypothermia on thrombin generation, clot formation, and global hemostatic functions in an in vitro environment using a whole blood model and thromboelastography, which can recapitulate hypothermia. Methods Blood was collected from healthy individuals through venipuncture and treated with corn trypsin inhibitor, to block the contact pathway. Coagulation was initiated with 5pM tissue factor at temperatures 37°C, 32°C, and 27°C. Reactions were quenched over time, with soluble and insoluble components analyzed for thrombin generation, fibrinogen consumption, factor (f)XIII activation, and fibrin deposition. Global coagulation potential was evaluated through thromboelastography. Results Data showed that thrombin generation in samples at 37°C and 32°C had comparable rates, whereas 27°C had a much lower rate (39.2 ± 1.1 and 43 ± 2.4 nM/min vs 28.6 ± 4.4 nM/min, respectively). Fibrinogen consumption and fXIII activation were highest at 37°C, followed by 32°C and 27°C. Fibrin formation as seen through clot weights also followed this trend. Thromboelastography data showed that clot formation was fastest in samples at 37°C and lowest at 27°C. Maximum clot strength was similar for each temperature. Also, percent lysis of clots was highest at 37°C followed by 32°C and then 27°C. Conclusions Induced hypothermic conditions directly affect the rate of thrombin generation and clot formation, whereas global clot stability remains intact.

The prothrombotic nature of sickle cell disease (SCD) is evidenced by the chronically elevated levels of almost all coagulation activation biomarkers, and an increased incidence of certain thrombotic events, including venous thromboembolism. Numerous studies have attempted to define the extent and elucidate the mechanism of the observed increase in thrombin generation in SCD patients in vivo. In general, these studies were performed using thrombin generation assays in platelet poor or platelet rich plasma and showed little difference in endogenous thrombin potential between the SCD cohort and healthy matched controls. In SCD, erythrocytes and monocytes have been demonstrated to exhibit procoagulant characteristics. Thus, the absence of these cellular components in standard thrombin generation assays may fail to reflect global hypercoagulability in the whole blood of patients with SCD. We were therefore surprised to see no difference in net thrombin generation in tissue factor-initiated initiated clotting of whole blood from patients with SCD. However, we are continuing to reconcile these seemingly disparate observations by slight modifications of the whole blood model that include alternative coagulation triggers and a re-examination of the net thrombin generation when the protein/protein S system is simultaneously interrogated.

Summary. Background: In the extrinsic pathway, the essential procofactors factor (F) V and FVIII are activated to FVa and FVIIIa by thrombin. In the contact pathway and its clinical diagnostic test, the activated partial thromboplastin time (APTT) assay, the sources of procofactor activation are unknown. In the APTT assay, FXII is activated on a negatively charged surface and proceeds to activate FXI, which activates FIX upon the addition of Ca2+. FIXa feeds thrombin generation through activation of FX. FIXa is an extremely poor catalyst in the absence of its FVIIIa cofactor, which, in the intrinsic FXase complex, increases FXa generation by ∼ 107. One potential APTT procofactor activator in this setting is FXIa. Objective: To test the hypothesis that FXIa can activate FVIII and FV. Methods: Recombinant FVIII and plasma FV were treated with FXIa, and the activities and integrities of each procofactor were measured using commercial clotting assays and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Kinetic analyses of FXIa-catalyzed activation and inactivation of FV and FVIII are reported, and the the timing and sites of cleavage are defined. Conclusions: FXIa activates both procofactors at plasma protein concentrations, and computational modeling suggests that procofactor activation during the preincubation phase of the APTT assay is critical to the performance of the assay. As the APTT assay is the primary tool for the diagnosis and management of hemophilias A and B, as well as in the determination of FVIII inhibitors, these findings have potential implications in the clinical setting.

Haemophilia A displays phenotypic heterogeneity with respect to clinical severity. The aim of this study was to determine if tissue factor (TF)-initiated thrombin generation profiles in whole blood in the presence of corn trypsin inhibitor (CTI) are predictive of bleeding risk in haemophilia A. We studied factor(F) VIII deficient individuals (11 mild, 4 moderate and 12 severe) with a well-characterized 5-year bleeding history that included haemarthrosis, soft tissue haematoma and annual FVIII concentrate usage. This clinical information was used to generate a bleeding score. The bleeding scores (range 0-32) were separated into three groups (bleeding score groupings: 0, 0 andor = 9.6,9.6), with the higher bleeding tendency having a higher score. Whole blood collected by phlebotomy and contact pathway suppressed by 100 microg mL(-1) CTI was stimulated to react by the addition of 5 pM TF. Reactions were quenched at 20 min by inhibitors. Thrombin generation, determined by enzyme-linked immunosorbent assay for thrombin-antithrombin was evaluated in terms of clot time (CT), maximum level (MaxL) and maximum rate (MaxR) and compared to the bleeding score. Data are shown as the mean+/-SD. MaxL was significantly different (P0.001) between the groups: 504 +/- 114, 315 +/- 117 and 194 +/- 91 nM; with higher thrombin concentrations in the groups with lower bleeding scores. MaxR was higher in the groups with a lower bleeding score; 97 +/- 51, 86 +/- 60 and 39 +/- 16 nM min(-1) (P = 0.09). No significant difference was detected in CT among the groups, 5.6 +/- 1.3, 4.7 +/- 0.7 and 5.6 +/- 1.3 min. Our empirical study in CTI-inhibited whole blood shows that the MaxL of thrombin generation appears to correlate with the bleeding phenotype of haemophilia A.

Summary. Background: Sodium citrate has been used as an anticoagulant to stabilize blood and blood products for over 100 years, presumably by sequestering Ca++ ions in vitro. Anticoagulation of blood without chelation can be achieved by inhibition of the contact pathway by corn trypsin inhibitor (CTI). Objective: To evaluate the influence of citrate anticoagulation on the performance of blood, platelet-rich and platelet-poor plasma assays. Methods: Blood was anticoagulated in three ways: by collection into citrate, CTI and citrate with CTI. Plasma was prepared using each anticoagulation regimen. Functional analyses included calibrated automated thrombography, thromboelastography, plasma clotting, the synthetic coagulation proteome and platelet aggregation. Coagulation reactions were initiated with tissue factor–phospholipid and Ca++ (when indicated).Results: In all cases, citrate anticoagulation resulted in reaction dynamics significantly altered relative to blood or plasma stabilized with CTI alone. Subsequent experiments showed that calcium citrate itself impairs coagulation dynamics.Conclusion: Coagulation analyses using blood that has been exposed to citrate and recalcified do not yield reliable depictions of the natural dynamics of blood coagulation processes.

Introduction Netosis refers to the extracellular release of nuclear material from neutrophils (PMNs) in the form of a meshwork called neutrophil extracellular traps (NETs). NETs are composed of a scaffold of DNA decorated with histones and specific cytoplasmic granules of neutrophils. Beside their ability to trap bacteria, NETs have been shown to promote thrombosis in animal models. Purified NET components have been suggested to enhance coagulation. However, the exact mechanisms by which intact NETs affect coagulation remain unclear. Objective To assess the effect of intact NETs on the activation of the contact system of coagulation. Methods Human PMNs were isolated from whole blood collected in 3.2% citrate (1 vol citrate / 9 vol blood) from normal volunteers using a negative selection by magnetic beads coated with antibodies directed against specific antigens of other blood cell types. Purity of PMN isolates was ≥ 96% while viability (assessed using trypan blue staining) was ≥ 98%. Isolated PMNs were re-suspended in RPMI + 0.5% bovine serum albumin or in normal pooled platelet-poor plasma (PPP) at 10-15,000 cells/µl final concentration. Re-suspended PMNs were stimulated to form NETs using phorbol myristate acetate (PMA: 600 nM) or ionophore (A23187: 5µM) and incubated at 37ᵒC in 5% CO2 for 3 hours. The effect of NETs was assessed on a synthetic construct of the contact system in-vitro using purified proteins (FXII, FXI, Prekallikrein and High Molecular Weight Kininogen) at physiologic concentrations in Hepes buffered saline containing 2mM Ca2+ and 0.1% polyethylene glycol (8000MW). The reaction was quenched with an equal volume of antithrombin (7uM), corn trypsin inhibitor (CTI, 0.2mg/mL) and heparin (1U/mL) at time points up to 1 hour after mixing with NETs. FXIa-antithrombin complexes were then detected using an in-house sandwich ELISA method. Thrombin generation (TG) was also monitored during 1 hour in re-calcified PPP containing stimulated PMNs with added 4µM synthetic phospholipid vesicles (PC:PE:PS). Finally, DNA was purified from neutrophils and its effect on contact activation was assessed in both the synthetic contact and plasma systems. Kaolin was used as positive control in both test systems; buffer without surface was used as the negative control in the synthetic contact system, and re-calcified PPP without added neutrophils was used as negative control in the TG assay. Results Massive amounts of NETs were released from stimulated PMNs as seen on fluorescence microscopy after staining for DNA using sytox green (Fig. I A&C). Extracellular release of NETs was confirmed by digestion following treatment with DNAse 1 (Fig. I B&D). Extracted DNA triggered contact activation (measured as XIa-antithrombin complexes) in buffer and enhanced TF-independent TG in PPP in a concentration dependent manner (Fig. I E&G). The latter was attenuated by corn trypsin inhibitor (0.1mg/mL) and abolished in FXII- or FXI-deficient plasma (Figure IE) indicating the dependency on the contact pathway activation. In contrast, intact NETs did not trigger contact activation in buffer and failed to enhance TG in re-calcified PPP in the absence of TF (Fig. I E&H). Conclusions Unlike purified DNA, intact NETs failed to activate coagulation via contact activation. These findings need to be confirmed using other readouts of contact system activation. These results do not exclude an effect of intact Nets on contact system amplification. Disclosures Monroe: Novo Nordisk: Honoraria, Research Funding.

Introduction. Due to their increased half-life over unfractionated heparin (UFH) and marked decrease in the incidence of heparin induced thrombocytopenia (HIT), low molecular weight heparins (LMWH) are the most widely prescribed heparin in the US. However owing to their incomplete reversibility with protamine, LMWHs (such as Enoxaparin) carry the risk of bleeding. The synthetic pentasaccharide, Fondaparinux, also lacks a specific antidote. We recently published (Xu et al. Nat. Chem. Biol. 2014) on a new class of synthetic LMWH that is not renal-excreted and offers the benefit of reversal by protamine. The new compound, dubbed “Super 12-mer”, is a 3,483 Da dodecasaccharide consisting of an antithrombin (AT) binding moiety with repeating units of IdoA2S-GlcNS6S (S is sulfate) and two 3-O-sulfate groups which afford the ability to bind protamine. We sought to characterize this new compound in a series of biochemical and global coagulation assays to better characterize its efficacy as a new reversible anticoagulant. Methods. Factor (F) Xa-AT inhibition assays were performed in both purified and plasma-based systems. The Super 12-mer was further tested in a purified prothrombinase system, as well as by tissue factor-initiated thrombin generation assays in contact pathway inhibited citrated plasma. Results. In vitro FXa inhibition studies indicated the IC50 to be 2-fold higher (49 ng/mL, 24 nM) than was previously reported. Nevertheless, the Super 12-mer anti FXa activity was approximately 2-fold greater than Enoxaparin at identical concentrations. However, the anti FXa activities of the Super 12-mer and Enoxaparin in plasma-based systems were roughly equivalent. Prothrombinase experiments indicated that both the Super 12-mer and Enoxaparin were equivalent in their ability to inhibit FXa in complex with FVa. When the two heparinoids were compared in a plasma-based thrombin generation assay (TGA), their effects on thrombin generation were nearly identical with a 50% reduction in peak thrombin generation occurring at approximately 325 nM heparinoid. When protamine is titrated against a fixed concentration of Super 12-mer (625 nM), the Super 12-mer displays a complete reconstitution of thrombin generation. Conclusions. In plasma and purified systems, the Super 12-mer displayed virtually identical efficacy in FXa inhibition compared to Enoxaparin. In buffered systems, the Super 12-mer was approximately 2-fold more active than Enoxaparin against FXa suggesting the Super 12-mer may have other binding partners in plasma. Interestingly, FXa inhibition in prothrombinase was essentially identical between the two heparinoids. Unlike Enoxaparin however, the Super 12-mer displayed near complete reversibility with protamine in TGAs. A significant lag in thrombin generation was observed when protamine was added, consistent with a previous report (Ni Ainle et al. Blood 2009) that protamine itself can act as an anticoagulant by interfering with FV activation. These data show that the Super 12-mer has almost identical efficacy to Enoxaparin in terms of FXa inhibition, while displaying significant reversibility with protamine. Taken together with the fact that this compound can be safely used in renal-impaired patients, the Super 12-mer is a promising new heparanoid anticoagulant with a potentially enhanced safety profile. Disclosures No relevant conflicts of interest to declare.

Sickle cell disease (SCD) is considered to be a hypercoagulable state with chronic activation of coagulation and an increased incidence of thrombotic events. However, there is no consensus on whether global assays of thrombin generation in platelet poor (PPP) or platelet rich (PRP) plasma display an increased thrombin generation potential in SCD (reviewed in Lim et al. Curr. Opin. Hematol. 2013). Based on our recent observation that RBC contribute to thrombin generation in whole blood (Whelihan et al. Blood 2012), we hypothesized that the cellular components in blood (notably RBCs) contribute to enhanced thrombin generation in SCD. 25 SCD patients in a non-crisis, “steady state” and 25 healthy race–matched controls were recruited for study. Whole blood thrombin generation, thromboelastography (TEG) and plasma-based thrombin generation assays (TGA) were performed on contiguous blood samples from each individual. Complete blood counts, as well as quantification of phosphatidylserine (PS) exposure by RBCs (assayed by Annexin V binding in flow cytometry) were also performed. Whole blood thrombin generation was monitored by serial α-thrombin-antithrombin (TAT) complex formation following activation by both the extrinsic pathway (5 pM recombinant tissue factor (TF)) and intrinsic (native contact activation) pathways. Results. With extrinsic activation, controls clotted on average at 3.9±0.5 min (mean±SD), generated TAT at a rate of 38.8±22.2 nM/min and reached a maximum level of 261±58nM. In patients with SCD, clot time (4.4±0.8 min, ƿ=0.04) and the maximum rate of TAT generation (41.5±19.4 nM/min, ƿ=0.65) were similar to that observed in controls while the maximum level (369±123 nM) of TAT generated was significantly higher (ƿ=0.0026). With contact activation, there was no significant difference in clot time (7.2±1.2 min vs 6.4±1.0 min, ƿ=0.07) or the maximum rate of TAT generation (52.9.5±26.9 nM/min vs 42.3 ± 12.4 nM/min, ƿ=0.57) for the SCD and control cohorts, respectively. However, similar to what was observed with extrinsic activation, SCD patients generated a significantly higher (ƿ=0.024) maximum level of TAT (352±116 nM) than controls (276±21 nM). Interestingly, the SCD cohort showed a strong positive correlation (ƿ= We also examined extrinsic- and intrinsic-initiated clot formation using TEG. Re-calcification of citrated blood from SCD in the presence of TF and Corn Trypsin Inhibitor (CTI) displayed similar R (8.1±0.3 min vs 8.4±0.6 min, ƿ=0.7) and MA (68±2 mm vs 65±2 mm, ƿ=0.16) values to those exhibited by controls, respectively. The -angle however, was significantly (ƿ=0.035) higher in the SCD cohort (61±3°) compared to controls (53±3°). Contact activated blood also displayed no significant difference in R time between the two groups (14.5±3 min for SCD vs 16.5±4 min for controls). On the other hand, the MA and -angle were significantly increased (ƿ=0.04 and ƿ=0.012) in SCD (62±2 mm and 41±3°) compared to the controls (51±3 mm and 26±5°), suggesting a significant increase in the overall rate and extent of clot formation as a result of contact activation. To examine thrombin generation in the absence of cellular components, PPP was activated using an identical 5pM TF stimulus. In contrast to whole blood, TGA displayed no significant differences in the peak thrombin or maximum rate of thrombin generation between the two groups. Lag time was marginally longer and time to peak thrombin generation shorter in SCD (ƿ=0.036 and ƿ=0.03, respectively). Surprisingly, a weak negative correlation (ƿ=0.15) between RBC PS expression and total TAT was present in SCD patient samples. Conclusions: While plasma-based assays exhibit no major differences in thrombin generation potential between SCD and controls, corresponding whole blood samples showed a significant increase in overall thrombin generation and clotting potential, regardless of the initiating stimulus (extrinsic or intrinsic). Interestingly, there was no significant correlation between absolute cell counts (i.e. RBCs, Retics, Neuts, PLTs) or parameters and TAT levels. Collectively, these data make a strong case for cellular involvement in the hypercoagulability observed in SCD, but a direct role for RBC PS expression in net thrombin generation is not apparent. Disclosures No relevant conflicts of interest to declare.

Abstract 374 Introduction: The potential contributions of erythrocytes (RBCs) to coagulation biochemistry have been controversial. We evaluated the potential for RBCs to participate in thrombin generation using minimally altered whole blood. Methods: Platelet poor plasma (PPP), platelet rich plasma (PRP) adjusted to a physiologic concentration with PPP, washed RBCs (PLTs Disclosures: Mann: Haematologic Technologies: Chairman of the Board, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; corn trypsin inhibitor: Patents & Royalties; NIH, DOD, Baxter: Research Funding; Merck, Daiichi Sankyo, Baxter, GTI: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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 ( Disclosures: Mann: Haematologic Technologies: Chairman of the Board, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; corn trypsin inhibitor: Patents & Royalties; NIH, DOD, Baxter: Research Funding; Merck, Daiichi Sankyo, Baxter, GTI: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

The procofactors FV and FVIII are activated by thrombin, FXa and plasmin. During contact pathway-initiated thrombin generation, FXIa activates FIX thus feeding into the coagulation cascade; however, the procofactors FVIII and FV must be activated to achieve a robust level of thrombin generation. We tested the hypothesis that FXIa can activate FV and FVIII. FV (1uM) was subjected to FXIa (100nM) proteolysis. During the reaction the relative activity and integrity were measured at selected time points using a one stage PT clotting assay and SDS-PAGE. Over the 60 minute time course, FV showed a transient 50% activation followed by a reduction in activity to 20%. SDS-PAGE analyses showed that proteolysis of FV by FXIa initially generated fragments with mobilities similar to those produced by α-thrombin. This activation and inactivation pattern suggested that FXIa makes the required activation cleavages at R709, R1018 and R1545 coincidently with inactivating proteolyses. FV was activated with α-thrombin and the reaction quenched by the addition of hirudin prior to FXIa proteolysis. FVa’s cofactor activity was reduced by 50% after 30 min and 80% after 60 min. Analysis of the FXIa cleavage process by SDS-PAGE under reducing conditions showed no intact heavy chain and significant proteolysis of the light chain after 30 minutes. In addition to the Mr = 105000 (HC) and Mr = 75000 (LC), six new products were identified by SDS-PAGE under reducing conditions: Mr = 54000, 50000, 48000, 30000, 22000 and 20000. NH2-terminal sequence analysis indicated a single cleavage in the light chain at R1765 yielding the products Mr = 50000, 48000: (1766à2196) and the Mr = 30000 (1546à1764), also seen with plasmin and FXa. A cleavage in the heavy chain represented by the Mr = 22000, 20000 (R511à709) fragments is also observed. Sequence analysis determined that the Mr = 54000 fragment represented the NH2-terminus of the heavy chain. Western analysis using a heavy chain antibody showed a transient band Mr = 75000 lying under the light chain that is consistent with an initial cleavage R510. A subsequent cleavage, which is coincident with a decrease in the cofactor’s activity, results in a Mr = 30000 product which is consistent with a cleavage somewhere COOH-terminal to R306(R316?). Activity analyses suggest that the initial cleavage in the heavy chain at R510 leads to a FVaXla 1 molecule with similar activity (50%) to that seen in FVa cleaved by APC in the absence of phospholipid. The FVaXla 1 was treated with APC resulting in complete inactivation of the cofactor. We have also observed an analogous FXIa cleavage pattern in FVIII; however sequencing analysis has not yet been attempted. These data suggest that factor XIa may play a role in procofactor activation and inactivation of FV and FVIII in the context of contact pathway-initiated blood coagulation. Figure Figure

Sodium citrate has been used as an anticoagulant to prepare blood and fractions for over 100 years. Its use is sufficiently common that many investigators do not differentiate anticoagulated blood from blood per se. To evaluate the influence of citrate anticoagulation on the dynamics of the blood coagulation process, we compared the properties of blood, platelet rich plasma (PRP) and platelet poor plasma (PPP) prepared and stimulated using standard citrate/Ca++ reconstitution methodologies with matching samples in which transient anticoagulation without disruption of Ca++ homeostasis was achieved by blockade of the contact pathway (using corn trypsin inhibitor (CTI) or inhibitory antibodies to factor XIa). Phlebotomy blood was collected into citrate, CTI or CTI supplemented with citrate, the latter combination establishing conditions in which the effects of citrate anticoagulation in the absence of contact pathway contributions could be studied. Coagulation reactions in citrated milieus were initiated in typical fashion by Ca++ addition or following preincubation with Ca++ prior to tissue factor (Tf) addition in CTI citrate plasma; blood/products stabilized by CTI alone were initiated via Tf addition. Reaction dynamics were analysed using either calibrated automated thrombography (CAT) or thromboelastography (TEG). Platelet aggregation studies compared the reactivity of a panel of agonists. Significant differences were seen in the response of samples exposed to citrate. These results included: CTI plasma and blood displayed a more potent response to Tf when compared to their respective CTI citrate products. Specifically shorter lag phases (~120 s) and higher thrombin levels (1.5–2 fold higher maximum thrombin levels) are observed in CTI plasma with CAT and similarly shorter r (lag) times [(mean ± SD)CTI blood, r = 7.1 ± 0.9 min (n=21, 9 individuals) vs CTI citrate blood, r = 9.1 ± 1.5 min (n=10, 5 individuals)] with TEG. Reconstitution of prechelation Ca++ in CTI citrate plasma or blood prior to Tf addition only partially restored thrombin generation to that of its prechelation product. For example preincubation with Ca++ resulted in altered TEG profiles with prolonged “K” (rate) values (mean ± SD) for CTI citrate blood (7.5 ± 0.6 min (n=5)) compared to CTI blood (3.3 ± 0.5 min (n=5)) from the same individuals. Vitamin K dependent protein conformational transitions in response to chelation/Ca++ reconstitution were important contributors to the differences observed between CTI and citrated blood/plasmas, including the process of protein S polymerization and depolymerization Platelet function studies in citrate containing PRP showed significant enhancement in reactivity toward epinephrine and ristocetin with lesser effects with collagen; in 3 of 4 donors CTI PRP showed no sensitivity to epinephrine. The magnitude of alterations induced by citrate chelation were in the order epinephrine> ristocetin> ADP > collagen. Historically, the regimen of citrate chelation followed by Ca++ addition has proven useful in the interpretation of the integrity of the coagulation system and thereby in clinical diagnosis. However, our results suggest that the procedure of Ca++ chelation/recalcification results in artifactual representations of the dynamics and biology of the blood coagulation process.

Trauma is the leading cause of death in the younger population in the United States, frequently from the development of hemorrhagic shock. Controversy exists over the type of volume resuscitation to be used (dilutions ranging up to 66%) in hemorrhagic shock for restoring hemodynamic stability. Trauma results in massive exposure of tissue factor to the circulation and explosive amounts of thrombin being generated. We studied the effect of various resuscitative formulas to blood coagulation, specifically thrombin generation and fibrin formation, in a controlled setting using corn trypsin inhibited whole blood initiated with a 5pM stimulus of tissue factor. Thrombin generation measured as its complex with antithrombin III (TAT) was evaluated periodically over a time course of 20 min. Fibrin clots were collected and weighed. We investigated four diluents (0.9% NaCl (NS), lactated Ringer’s solution (LR), 6% hydroxyethyl starch (HES) and 3% NaCl (HS)) each at a 0,10, 20, 30, 40, 50 and 60% blood dilution. At a 10% dilution TAT generation was in the order of LR (−4%) < HES (−8%)< NS and HS (−12%). Diluting by 20% resulted in further decreases of TAT formation. The fibrin clot mass decreased dramatically with a 20% dilution for NS (−42%) and HES (−30%). Conversely, HS produced no change in fibrin mass but effected the largest change in thrombin generation rate (−56%). At a 50% dilution, comparable thrombin generation profiles were obtained for LR, HES and NS (~35% decrease). However, the fibrin masses decreased by 27% with LR, 46% with NS and 74% with HES. No clot formation or thrombin generation was seen with HS at > 20% dilution. This in vitro study shows that: 1) LR has the least effect on thrombin generation and gave higher than anticipated clot weights; 2) HES reduced the fibrin clot mass at higher dilutions; 3) HS abolishes coagulation after a 20% dilution. Overall, both the extent and nature of hemodilution cause profound alterations in the hemostatic mechanism.